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347-470 (1996)
IInstitute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary and 2Centerfor Molecular and Behavioral Neuroscience, Rutgers University, Newark, New Jersey
KEY WORDS: GABA, GABAergic cells, nonpyramidal cells, nonprincipal cells, inhibition, inhibitory neurons
Much of our current knowledge about the neuronal organization of the cerebral cortex comes from studies of principal neurons. Only recent work points to the crucial role of inhibitory interneurons in regulating the complex interactions among principal cells, including population oscillations, plasticity, epileptic synchronization, hormonal effects, and cortical development. Perhaps the best illustration of this point is the pivotal role interneurons play in population oscillations (theta, gamma, 200-Hz ripples) and memory-related plasticity in the hippocampal formation. The very basics of neuronal cooperation is a subject of intensive research (integrateand-fire vs. coincidence detectors; rhythmic vs. stochastic activity; Gernstein and Mandelbrot, 1964; Shadlen and Newsome, 1994; Softky, 1995). Even if a complete identification of the molecular and biophysical properties of single cells eventually becomes possible, such knowledge is insufficient to predict the behavior of large neuronal aggregates in complex integrative areas of the brain, such as the hippocampal formation. Population interactions of neuronal ensembles underlying behavioral control cannot be revealed without a comprehensive understanding of the dialogue between interneuronal networks and principal cell populations. It has been known for over 100 years (Ram6n y Cajal, 1893, 1911) that neurons constituting any cortical area are far from being uniform with regard to their morphology and connectivity, suggesting that they possess the capacity to interact with each other in a complex and diverse fashion. Descriptions of increasing numbers of cell types with distinct dendritic morphology and axonal targets result in a multitude of possible interactions. Even if only a fraction of these connections are active at one time, a network with virtually endless numbers of possible activity patterns
The authors wish to dedicate this monograph to the memory of their late mentors, Janos Szentdgothai and Endre Grastyin.
.Accepted for publication July 1, 1996. Address correspondence and reprint requests to Tam& F. Freund, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, P.O. Box 67, H-1450, Hungary; e-mail: freund@koki.hu; or Gyorgy Buzsdki, Center for Molecular and Behavioral Neuroscience, Rutgers University, Newark, NJ 071 02; e-mail: buzsaki@axon.rutgers.edu.
emerge. Recent results provide evidence that electrophysiology of unitary cellular interactions and of large cell populations in conjunction with precise structural data on the synaptic architecture may eventually prove to be realistic experimental approaches to study the complex questions of interneuron function (Buzsiki et al., 1992; Gulyis et al., 1993a,b; Buhl et al., 1994a; Sik et al., 1995; Ylinen et al., 1995a,b; Miles et al., 1996). Significant steps have been made toward this goal in the past two decades (Schwartzkroin and Mathers, 1978), in which advantages of the marriage between structural and functional analyses of neuronal networks have been realized, and the painstaking labor involved in this approach has begun to provide critical information not accessible by other means. The multitude of recently aquired data representing several different levels of analysis still awaits synthesis. The outcome of such a synthesis may result in the generation of realistic hypotheses for the specific roles of hippocampal microcircuits and activity patterns and ultimately for the function of the hippocampal formation itself. The primary objective of this review is to provide a preliminary synthesis of much of the anatomical, cellular physiological, pharmacological, and systems physiological data focused on hippocampal interneurons and to propose specific unifying hypotheses for their roles in the control of network activity. Why do interneurons represent a key to the understanding of network operations? In contrast with the rather uniform population of principal cells in any of the hippocampal subfields, the afferent and efferent connectivity of interneurons shows great variation (Ram6n y Cajal, 1893, 1911; Lorente de N6, 1934), thereby enabling them to carry out multiple tasks (Sections VI-XIII). Inhibition is critical in shaping response properties in single cells and in assisting cooperativity in large cell populations (Lytton and Sejnowski, 1991; Buzsiki et al., 1992; Traub et al., 1996; Ylinen et al., 1995a,b). The basic cell types of the cerebral cortex were described in the pioneering Golgi impregnation studies of Ram6n y Cajal in 1893. For over half a century, neuroanatomists referred simply to two major cell types, pyramidal and nonpyramidal neurons, largely on the
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FREUND A N D BUZSAKI
basis of Ram6n y Cajals descriptions. A more detailed characterization of nonpyramidal cell types is found in the work of Lorente de N6 (1934), particularly for the hippocampal formation. It was only in 1966, however, when Colonnier (1966) brought attention to the large variety of interneurons with distinctive dendritic and axonal fields described first by Ram6n y Cajal (1893, 1911). The word inhibition was not mentioned in any of the early Golgi studies. The assumption that interneurons may be responsible for local inhibition arose from Grays (1959) description of two types of synapses, asymmetrical (type I) and symmetrical (type 11), which motivated Eccles 11964) promptly to pronounce type I as excitatory and type I1 as inhibitory (Andersen et al., 1963). An important step was again taken by Colonnier (1968), who demonstrated that the somata of pyramidal cells, where the basket type of interneurons terminate, were covered almost exclusively by type 11, presumably inhibitory, synapses. After Szentigothais (1962, 1965a,b) demonstration that specific basket types of axon arborizations remain intact in chronically isolated cortical slabs, the view of basket cells (and other types of nonpyramidal cells) as local inhibitory interneurons became well established. Further evidence for this conclusion has been provided by the selective interneuronal localization of glutamic acid decarboxylase (GAD; Ribak et al., L978), the synthesising enzyme of gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the cerebral cortex (Krnjevic and Schwartz, 1967). Debates over nomenclature have surfaced in more recent years, particularly when neurons with local axons were shown to establish asymmetrical (presumably excitatory) synapses. Examples of these types of cells include mossy cells of the dentate gyrus (Laurberg and Sorensen, 1981; Ribak et al., 1985) and spiny stellate cells of the neocortex (Somogyi, 1978). Confusion also exists concerning what to call interneurons with axon collaterals that project to distant brain areas (Seress and Ribak, 1983; Ribak et al., 1986; T6th and Freund, 1992; T6th et al., 1993). These findings clearly indicated that the correspondence among Golgis type 2 short axon cells, the nonpyramidalcells of Ramon y Cajal and Lorente de N6, and inhibitory interneurons of Eccles, Colonnier, and Szentigothai was not at all straightforward. For the hippocampal formation, the term nonprincipal cell was sufficiently simple and correct to designate neurons mostly involved in local synaptic circuits, but some of them, in addition to their local collaterals, may have an extrahippocampal or commissural projection. Neurons with well-established local excitatory output (e.g., mossy cells) are not considered interneurons in this review, even if their axon remains restricted to che hippocampal formation. Given that most, if not all, nonprincipal cells use GABA as a transmitter (Section 111.l ) , the definition GABAergic nonprincipal cells appears to be the most precise term for the subjects of this review. However, the use of the term interneurons should not be discouraged in spite of the debate related to its connotations because it carries an important and descriptive message about the major contribution of these cells to the wiring of local networks. Furthermore, the term interneuron is sufficiently simple, widely used, and will continue to be used in the future in spite of efforts to change it. Thus, we propose that the term
Abbreviations: 5-HT 5-hydroxy-tryptam in, serotonin trans-1-amino-cyclopentane-1,3-dicarboxylic acid tACPD amino-3-hydroxy-5-methyl-4-isoxazolepropiAMPA onic acid APV 2-amino-5-phosphonovaleric acid brain-derived neurotrophic factor BDNF fields of the hippocampus (Cornu ammonis) acCA1-3 cording to Lorente de N 6 calbindin D28k CB cholecystokinin CCK choline acetyltransferase ChAT 6-cyano-7-nitroquinoxal i ne-2,3-dione CNQX ca Iret in i n CR 3,3-diaminobenzidine 4HCI DAB ENK leu- and metenkephalin EPSP, EPSC excitatory postsynaptic potential, current gamma aminobutyric acid CABA glutamic acid decarboxylase GAD 40-1 00-Hz field oscillatory waves gamma osci I lations glutamate receptor CluR hilar neuron with axon distributed in the comHICAP cell missural/associational pathway termination zone hilar neuron with its axon distributed in the perHlPP cell forant path termination zone horseradish peroxidase HRP transient, rapidly inactivating current IA cyclic AMP-dependent potassium current ~AHP calcium-activated potassium current IC hyperpolarization-activated current IH sustained current (potassium) IK inhibitory postsynaptic potential, current IPSP, IPSC interneurons with cell bodies in stratum lacunoLM cell sum-moleculare long-term depression LTD long-term potentiation LTP metabotropic glutamate receptor mGluR molecular layer cell with axon arborizing in the MOPP cell perforant path termination zone nicotinamide adenine dinucleotide phosphate NADPH nerve growth factor NCF nickel-intensified 3,3-diaminobenzidine 4HCI Ni-DAB (chromogen for peroxidase reaction) N-methy I-D-aspartate NMDA nitric oxide NO neuropeptide Y N PY neurotrophin 3 NT3 interneuron with soma and dendrites in stratum 0 - L M cell orines, and axons in strata lacunosum-moleculare and oriens calcium or sodium permeability PCa, PNa fhaseolus vulgaris-leucoaggl uti nin PHAL PP perforant path PV parvalbumin ripple fast (120-200 Hz), transient field oscillation in association with sharp waves SBA soybean agglutinin somatostatin SOM substance P SP substance P receptor SPR SPW hippocampal or entorhinal sharp waves T channel low threshold calcium channel rhythmic slow wave activity (4-1 2 Hz) theta VIP vasoactive intestinal polypeptide VVA Vicia villosa agglutinin
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The types, characteristics, and connectivity of principal cells in the hippocampus (cornu ammonis, divided into three subfields, CA1-3, according to Lorente de No, 1934) and the dentate gyrus (fascia dentata and hilus) have been well known since the studies of Ramon y Cajal (1893) and Lorente de N6 (1934) and have been reviewed extensively (Amaral and Witter, 1989, 1995; Lopes da Silva et al., 1990). For sake of convenience, we will use the term hippocampal formation to refer to the hippocampus and dentate gyrus collectively, although it is acknowledged that the term is sometimes used to include the subicular complex and the entorhinal cortex (Amaral and Witter, 1995). The present overview is limited to the description and interpretation of data on interneurons in the hippocampus and the dentate gyrus (i.e., the hippocampal formation). The hippocampal formation therefore consists of a complex of three main subfields (Fig. l ) , the dentate gyrus, the CA3 region, and the CA1 region. The CA2 subfield is less well defined in the rat and appears to lack specific features regarding interneurons and will therefore be largely ignored here. The dentate gyrus is considered to be the first stage of the intrahippocampal trisynaptic loop. It is the target for the majority of entorhinal afferents (Fig. 1) by carrying sensory information of multiple modalities about the external world. Merents from the lateral entorhinal cortex terminate in the outer one-third and those from the medial entorhinal cortex terminate in the middle one-third of the molecular layer, where dendrites of dentate principal cells arborize. Principal cells of the dentate gyrus are the granule cells, which number almost 1 million in the rat and 5 million in the monkey (Claiborne et al., 1986, 1990; Seress, 1988) and the mossy cells of the hilus (Amaral, 1978). The relatively small cell bodies of granule cells (8-12 p m in diameter) form a
densely packed layer called stratum granulosum (or granule cell layer), which is 4-8 somata in thickness. These cells characteristically have two main radially oriented dendrites emitting several fine branches, which reach the pial surface or the hippocampal fissure. Except for the most proximal shafts, all dendrites are densely spiny. The entire dendritic tree of granule cells is confined to stratum moleculare, the layer adjacent to stratum granulosum. Basal dendrites in the rat are extremely rare and mainly appear in the ventral hippocampus (L. Seress, personal communication). The axons of granule cells, called mossy fibers, originate at the opposite pole of the soma and enter the hilus, where they give rise to several local collaterals that largely remain in the hilar region (Claiborne et al., 1986). Recurrent collaterals periodically enter the granule cell layer, climb along the cell bodies and dendrites of presumed basket cells, and form multiple synapses only on these interneurons in normal animals (Ribak and Peterson, 1991). Occasional mossy fiber collaterals may also contact granule cell dendrites, particularly in stratum moleculare of the ventral dentate gyrus, but the physiological role of monosynaptic recurrent excitation among granule cells is likely to be negligible. The main axon of the granule cells leaves the hilar region and courses adjacent to the pyramidal cell layer (in stratum lucidum, see below) of the CA3 subfield, where they form giant en passant boutons, the characteristic mossy terminals, on the proximal dendrites of pyramidal cells. The hilus, or polymorphic zone of the dentate gyrus, is located subjacent to the granule cell layer and is bordered on the other side by the dendritic layer of CA3c that lies between the upper (suprapyramidal) and lower (infrapyramidal) blades of the dentate gyrus. The principal and most numerous cell type of the hilus is the mossy cell, which has densely spiny dendrites and several thornlike excrescenses on both the cell body and proximal dendritic shafts. The dendrites of mossy cells are typically confined to the hilar region (Amaral, 1978), but some of them subsequently have been found to have a single dendritic branch penetrating stratum moleculare (Soltksz and Mody, 1994; Scharfman, 1995b). The axon of mossy cells innervates the inner one-third of the molecular layer of the dentate gyrus both ipsiand contralaterally and also emits collaterals within the hilus (Amaral, 1978; Laurberg and Sorensen, 1981; Rbak et al., 1985; Buckmaster et al., 1996). Although their primary postsynaptic targets are the dendrites of granule cells, mossy cell collaterals also terminate on unidentified dendritic shafts in the hilus and occasionally on dendrites of interneurons (Frotscher and Zimmer, 1983a,b; Frotscher et al., 1984; Ribak et al., 1985; Buckmaster et al., 1996). Whether mossy cells should be considered as modified displaced pyramidal cells of the hippocampus or whether they represent a unique and specific cell type of the dentate gyrus is still a debated question (Frotscher et al., 1991). The recent demonstration of a back projection of ventral hippocampal CA3c pyramidal cells to the inner one-third of the molecular layer of the dentate gyrus, similar in distribution and termination pattern to mossy cell projections, provides strong support for the former interpretation (Li et a]., 1994). Both cell types make asymmetrical, presumably glutamatergic, excitatory (Soriano and Frotscher, 1994; Scharfman, 1995b) connections. Moreover, no major differences between CA3c pyramidal cells and mossy cells are ap-
FIGURE 1. A: Main excitatory connections in the hippocampal formation. Layer I1 of the entorhinal cortex forms a longitudinally widespread projection to granule cells and CA3 pyramidal cells via the perforant path. (The direct entorhinal cortex to CA3 connection is not shown.) The next stage is the mossy fiber projection, organized in a lamellar fashion, from the granule cells to the CA3 pyramidal cells. CA3 pyramidal neurons are strongly interconnected by a longitudinally projecting recurrent associational system. The CA3-CAI associational projection (Schaffer collaterals) is, again, longitudinally widespread. The extent of CA3-CA3 and CA3-CA1 projections in the septotemporal direction is similar, but more callaterals are present in CAI than in CA3. In contrast with the divergent multisynaptic system, layer I11 pyramidal cells of the entorhinal cortex provide a direct and spatially restricted innervation (in parent in terms of their inputs or their electrophysiological or neurochemical features. For these reasons, mossy cells will be considered here as excitatory principal cells that provide long-range ipsilateral and commissural associational projections into the denlate gyrus (Amaral, 1978). The CA3 subfield represents the second stage of the trisynap-
the transverse dimension) of CAI pyramidal cells, which in turn project back to the same columns in the entorhinal cortex (deep layers). In essence, the entorhinal cortex is mapped onto the CAI region and the trisynaptic, intrahippocampal diffuse system is superimposed on this organized topography. Another major output from the CA1 region is to the subicular complex (not shown). Superimposed on the excitatory projections are the locally projecting and widely projecting inhibitory interneurons that form an interneuronal network (not shown). B: Coronal section through the dorsal hippocampus, immunostained for parvalbumin. 0, CA1 stratum oriens; p, stratum pyramidale; r, stratum radiatum; lm, stratum lacunosum-moleculare; m, dentate molecular layer (stratum moleculare); g, granule cell layer (stratum granulosum); h, hilus proper; M a - c , subregions of the CA3 field. From Buzsdki (1995).
tic loop (Fig. 1 ) . Pyramidal cells of this region are the principal targets of granule cell axons, the mossy fibers. According to Lorente de N6 (1334), the region is subdivided further: the area penetrating the dentate hilus is called CA3c, the segment adjacent to CA2 is called CA3a (the curved segment), and the area located between is called CA3b. CA3 pyramidal cells have char-
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a single, radially oriented apical dendrite that emits several processes in stratum radiatum (Ishizuka et al., 1995). These dendritic processes ultimately terminate in a tuft of thin branches in stratum lacunosum-moleculare, usually reaching the hippocampal fissure. Basal dendrites are numerous, arborize in stratum oriens, and often reach the alveus. The dendritic tree is densely covered with spines, but thorny excrescenses are absent in the CA1 region. The axon emerges from the region of the soma adjacent to the apical dendrite or occasionally from a basal dendrite before entering the alveus. Local collaterals and consequently the excitatory interactions among CAI pyramidal cells are relatively sparse (Lorente de NO, 1934; Amaral and Witter, 1989; Amaral et al., 1991; Radpour and Thomson, 1992) compared with the CA3 region. These collaterals travel parallel to the alveus in stratum oriens and remain restricted to this layer (Ram6n y Cajal, 1893; Lorente de N6, 1934; Tamamaki and Nojyo, 1990). The main extrinsic projections of CAI pyramidal cells are to the subiculum and entorhinal cortex (Fig. I), but other limbic cortical areas and the lateral septum, the nucleus accumbens, and the olfactory bulb are also among the targets of the CAI subfield (for a detailed description see, Van Groen and Wyss, 1990). The CA1 region (together with the subiculum) should therefore be considered as the major output structure of the hippocampus back to the entorhinal cortex and indirectly to neocortical areas. In addition to the ipsi- and contralateral Schaffer collaterals, the other excitatory inputs of CAI pyramidal cells include entorhinal afferents that terminate in stratum lacunosum-moleculare.
The possible functional significance of morphological differences of various hippocampal cell types was first addressed by Ram6n y Cajal (1893). He and, later his pupil, Lorente de N6 (1934) demonstrated that characteristic laminar distributions of dendritic trees predicted the possible sources of afferent input, whereas the pattern of axon arborization provided a strong indication of postsynaptic target selection. Their approach exemplified the utility of functional neuroanatomy, i.e., a method to predict function. A modern version of this approach, allowing direct conclusions to be made based on strict criteria, is the characterization of the input-output properties by combinations of tracing and multiple labeling techniques (for review, see Freund and Somogyi, 1989; Somogyi and Freund, 1989; Freund, 1993). Visualization of neurons with their dendritic and axonal processes can be achieved in different ways, including ( I ) classical Golgi impregnation methods, (2) intra- or extracellular dye injections, and (3) immunocytochemical staining for transmitters, their synthesizing enzymes, neuropeptides, calcium-binding proteins, cell surface markers, etc. The second approach, involving intracellular labeling, has the major advantage of combining direct physiological characterization and morphology. However, in immunocytochemical studies, most if not all cells that belong to the
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same neurochemically identified class, are simultaneously visualized. This finding may provide crucial information about the frequency of certain cell types and the consistency of morphological features found in single-cell labeling studies. Importantly, it may help decide whether a new morphological finding obtained by single-cell labeling techniques is a peculiarity (e.g., a pathological or developmental abnormality, fixation artifact, etc.) or may represent a consistent feature of the network. Obviously, once the physiological effects of substances (e.g., neuropeptides, calcium-binding proteins) are revealed, it will add a new dimension to the immunocytochemical data. A major aim of this review is to establish a unifying classification scheme that integrates morphological, neurochemical, and physiological features. The morphological characteristics, which are based on single-cell labeling studies, will be presented first. Additional cell types, described to date only by immunostaining, will also be summarized here.
Dentate gyms
Chandelier cells are located within or immediately adjacent to the granule cell layer. They have a dendritic tree with a tuft of several radially running branches in the molecular layer, most of which reach the outer one-third of the layer or even the hippocampal fissure (Soriano and Frotscher, 1989; Soriano et al., 1990; Han et al., 1993; Buhl et al., 1994a). Dendrites crossing the granule cell layer, directed toward the hilus, are small in number or occasionally even absent (Soriano et al., 1990). This arrangement of the dendritic wee suggests that the dominant excitatory input to these cells is feed-forward, originating from the perforant path and the commissural-associational projection. Nevertheless, the rather sparse basal dendrites appear to be suffcient to provide the cell with a feedback drive because antidromic activation of granule cells was shown to discharge the axo-axonic
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B
FIGURE 2. The axonal and dendritic arbors of an axo-axonic cell (A) and a basket cell (B) in the CA3 region intracellularly filled with biocytin in a 400-pm guinea pigslice in vitro and reconstructed from serial 60-pm Vibratome sections. Insets illustrate the extent and gross position of the axon arbors in CA3.The dendritic trees of both cell types are bitufted and span all layers, whereas the axon arbors are limited to strata pyramidale and proximal oriens. A small number of basket cell collaterals also penetrate stratum radiatum.
Note the numerous vertically oriented axon terminal segments in the case of the axo-axonic cell (A), which are rare in the basket cell arbor (B). From Gdy& et al. (1993a). Scale bars = 50 pm. Reproduced from GulyAs et al. (1993a) Precision and variability in postsynaptic target selection of hippocampal nonpyramidal cells. Eur. J .Neurosci., 51729-1751 by permission of Oxford University Press.
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Hippocampus
Chandelier cells of the hippocampus are very similar to those in the dentate gyrus. The cell bodies are located within or immediately adjacent to the pyramidal cell layer and possess radially oriented dendrites spanning all layers (Fig. 2). The dendrites are smooth, often varicose, and spines are only rarely present on a few branches. Three to six main dendritic trunks extend toward the hippocampal fissure, emitting few if any side branches in stratum radiatum. In stratum lacunosum-moleculare however, they form a tuft of fine, slender processes (Li et al., 1992; Gulyis et al., 1993a; Buhl et al., 1994b). There is a rich arbor of basal dendrites in stratum oriens, which extends up to, or occasionally penetrates, the alveus. Thus, according to the distribution of the dendritic tree, chandelier cells are in a position to receive excitatory input from all major sources of afferents in both the CA1 and CA3 subfields, with some preference for the perforant path input. The column occupied by the dendrites has a diameter of 200-300 pm. The axon, which originates from the soma or a primary dendrite, forms a dense arbor in strata pyramidale and proximal oriens and consists of vertical or oblique rows of boutons (Figs. 2, 3). The only axo-axonic cell with a complete axonal and dendritic tree intracellularly labeled in vivo was described by Li et al. (1992). They reported an axon arbor occupying an elliptical area of 600 by 850 p m , elongated in the septotemporal direction, within which 40-50-pm groupings of terminal segments could be observed in some sections. Three chandelier cells filled in the CA3 subfield of 400-pm-thick slices in the guinea pig had a transverse axonal spread of 800 p m , 1,300 p m , and 1,700 p m (Gulyis et al., 1993a), whereas one reported in CA1 extended for 950 p m (Buhl et al., 1994b). Each of these cells had axon collaterals in all sections cut from the 400-pm slice. The main axonal branches of axo-axonic cells run horizontally above the pyramidal cell layer and give rise to collaterals descending into stratum pyramidale, where they form characteristic bouton rows climbing on axon iniFIGURE 3. Light and electron micrographs of axo-axonic (A,C) and basket cell (B,D,E) axon collaterals. A: Axon collaterals (arrowheads) of a Golgi-impregnated axo-axonic cell in the monkey hippocampal CAI region climb along the axon initial segments of two unstained pyramidal cells (N, and N2). B: Basket cell collaterals (arrows) also form rows of boutons in strata pyramidale, proximal oriens, and radiatum, but these segments have no preferred orientation, unlike axo-axonic cells, which have numerous radially oriented terminal segments. Basket cell axons pass among the tightly packed pyramidal cell bodies and occasionallyfollow apical or basal dendrites into strata radiatum or oriens. The layers of the CA3 sub, . ] . field are indicated. s.o., stratum oriens; s . ~ .stratum pyramidale; s, stratum lucidum. C: Electron micrograph of an axon terminal of a Golgi-impregnated axo-axonic cell from the rat hippocampus, forming a symmetrical synapse (black arrow) on a spinelike appendage of an axon initial segment (ais). Ultrastructural characteristics of axon initial segments include an electron-dense membrane undercoating (arrowheads) and lamellar bodies (open arrows). D,E: Axon terminals of an intracellularly filled basket cell in the guinea pig hippocampal CA3 region form symmetrical synapses (arrows) on a cell body (s in D) and on a dendritic shaft (d in E). Data from Gulyds et al. (1993a) and Somogyi et al. (1983a, 19853. Scale bars = 10 pm for A,B, 0.2 pm for C-E.
tial segments. These rows are often obliquely oriented and follow the trajectory of axon initial segments. Each row consists of 2-15 axon terminals. The number of terminal segments closely corresponds to the number of innervated pyramidal cells, which was estimated to be approximately 1,200 for the completely reconstructed chandelier cell in CA1 (Li et al., 1992). This number may be even larger for CA3 cells, with an axon arbor twice as long in the coronal plane in the guinea pig (Gulyds et al., 1993a). Based on the average number of synapses on pyramidal cell axon initial segments and on the contribution of a single axo-axonic cell to these synapses, a convergence of 4-10 axo-axonic cells onto a single pyramidal cell has been estimated (Li et al., 1992). Electron microscopy of chandelier cell axon terminals confirmed the absolute selectivity of this cell type for termination on axon initial segments of pyramidal cells in rat (Fig. 3), cat, and monkey (Somogyi et al., 1983a,b, 1985a; Gulyis et al., 1993a; Buhl et al., 1994a,b). Ultrastructural features of chandelier cell axons, dendrites and somata are indistinguishable from those described for dentate chandelier cells.
Dentate gyms
Two types of basket cells can be distinguished on the basis of significant differences in the laminar distribution of dendritic arbors, which in turn appears to reflect differences in afferent excitatory drive. However, if variations in the location and shape of the cell bodies are also considered, which may eventually prove important, the types of basket cells in the dentate gyms increases to five or six (Ribak and Seress, 1983). Basket cells can be further subdivided by the presence of calcium-binding proteins and neuropeptides (Section IV). One of the two types to be discussed here is the so-called pyramidal-shaped basket cell (Lorente de N6, 1934), which, as suggested by its name, has a dendriric tree reminiscent of pyramidal cells in shape but is free of spines. Cell bodies of these neurons are located among deep granule cells at the hilar border but may
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occasionally be found just outside stratum granulosum either below or above the layer (Amaral, 1978; Seress and Pokorny, 198 1; Scharfman, 1995a). A prominent apical dendrite emerges from the soma, which may divide proximally, and runs across stratum moleculare to reach the pial surface or the hippocampal fissure (Seress and Pokorny, 1981; Ribak and Seress, 1983; Seress and Ribak, 1983, 1990a,b). Basal dendrites, which may vary in number from two to five, always enter the hilus for a considerable length. Thus, pyramidal basket cells are in a position to be activated in both a feed-forward manner by entorhinal cortex, the commissural-associational path, or even CA3 pyramidal cells and a feedback manner by mossy fiber collaterals (Ribak and Peterson, 1991; Kneisler and Dingledine, 1995a,b). Direct evidence for these afferent connections was provided by combining anterograde tracing and single-cell labeling techniques (Frotscher and Zimmer, 1983a,b; Ribak and Seress, 1983; Seress and Ribak, 1984; Zipp et al., 1989). Variations of this cell type include neurons with an upper and lower bouquet of dendrites, with larger or smaller, multipolar or fusiform cell bodies, or with a soma located in deep stratum moleculare or in the hilus. However, the relative distribution of dendrites in the different input layers of the dentate gyrus is similar for all these variations, which justifies pooling them into one type at this level of analysis. The distinguishing feature of the other type of basket cell is its dendritic tree, which is largely, if not completely, restricted to the hilus. This type of neuron was described by Ram6n y Cajal (1893) and Lorente de N6 (1934), but in recent Golgi and intracellular labeling studies basket cells with similar dendritic arbors have not been described. Neurons with similar dendritic trees in the hilus have been extensively reported, but their axons rami+ in stratum moleculare rather than in the granule cell layer, and therefore should not be called basket cells (Han et al., 1993; Buckmaster and Schwartzkroin, 1995a,b; Sik et al., submitted). A hilar dendritic arbor precludes feed-forward activation by entorhinal afferents. The major excitatory drive to these cells appears to be from mossy fiber collaterals and possibly also from CA3 pyramidal cells. The axonal arbors of the two basket cell types are indistinguishable. It originates from the soma or a primary dendrite and ascends through the granule cell layer, distributing long horizontal branches within deep stratum moleculare, or within stratum granulosum. These main axon trunks emit a large number of collaterals that descend into the granule cell layer, where they form dense pericellular arrays of synaptic boutons (Ribak and Seress, 1983; Seress and Ribak, 1990a,b; Han et al., 1993; Sik et al., submitted). Unlike in the cerebellum, axons of individual basket cells do not form typical baskets around the somata of their target cells; they only contribute to the pericellular array of boutons. The transverse extent of the axon of an intracellularly injected basket cell (within a 400-pm-thick slice) was estimated to be 900 p m ; the density of bouton-laden collaterals within this arbor is ex7 tremely high (Han et al., 1993). A basket cell intracellularly recorded and filled in the dentate hilus in vivo had an axon arbor that covered the entire suprapyramidal blade (longer than 1 mm transverse spread) and extended more than 1.5 mm along the septotemporal axis (Sik et al., submitted). More than 11,000 boutons were estimated to be present along the 44-mm-long axon,
Hippocampus
Axon arborizations of basket cells show similar features to those described in the dentate gyrus, but the laminar distribution and orientation of the dendritic tree is more heterogeneous, at least as can be seen on the drawings of Ram& y Cajal (1893) and Lorente de N 6 (1934). Recent Golgi and intracellular labeling studies visualized basket cells showing much less variation in soma location or dendritic arborization patterns (Figs. 2, 4. 6). However, this may be partly due to the biased sampling of neurons from strata pyramidale and oriens for intracellular recordings (Kawaguchi and Hama, 1987a,b, 1988; Gulyhs et al., 1993a; McBain et al., 1994; Buhl et al., 1994a; Sik et al., 1995). The predominant dendritic morphology of basket cells in these layers is pyramidal-shaped or bitufted, as in the dentate gyrus, hence
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The axon arbors of basket cells in the hippocampus also show some variation (Figs. 2, 4, 6). The horizontal extent and laminar distribution of the arbors are similar to those described for chandelier cells. This similarity has occasionally resulted in the misclassification of chandelier cells as basket cells in the literature. Basket cell axons fill the entire depth of stratum pyramidale and proximal stratum oriens, whereas chandelier cell axonal arbors show some preference for stratum oriens, reflecting the slight difference between the location of cell bodies and axon initial segments of pyramidal cells. An additional difference is that basket cells rarely form characteristic vertical bouton rows typical of axoaxonic cells, although some of the varicose collaterals around pyramidal cell bodies are similar to chandelier terminal segments (Fig. 3). The axon arbors of basket cells appear to respect the subfield boundaries (i.e., CA1-3), although in most intracellular labeling studies neurons close to these boundaries have not been sampled. There has been only one report of a basket cell (in the guinea pig) with an axon crossing from CA3 to the CAI subfield, providing almost equal number of boutons in both regions (Fig. 2; Gulyis et al., 1993a). The transverse extent of the axon arbor in a 450p m slice was between 900 and 1,300 p m (Gulyis et al. 1993a; Buhl et al., 1994a). The axon arbor was densest near the soma and became somewhat diffuse distally. The number of boutons generated by this cell was in the range of 3,000 to 10,000. The number of synapses established by a single basket cell on one of its targets varied between 2 and 10 (Figs. 4,5; Gulyis et al., 1993a; Buhl et al., 1994a; Miles et al., 1996). Thus, taking an average of six synapses per connection, one basket cell may innervate 500-1,600 postsynaptic neurons in a 400-pm slice (Miles et al., 1996). In a recent in vivo intracellular labeling study, five basket cells were completely reconstructed in the CA1 region. The total axon length for each cell was between 40 and 50 mm and had 9,000 to 12,000 boutons (Sik et al., 1995). The estimated number of target cells of a single basket cell is 1,500-2,500 pyramidal cells. A basket cell may also contact interneurons. Basket or chandelier cells visualized by immunostatining for parvalbumin (PV; Section IV.2a) were shown to be among the postsynaptic targets (about 1%). Atypical basket cells. In a recent account of interneurons in the CA3 subfield of the guinea pig hippocampus, Gulyis et al. (1993a) reported two neurons described as atypical basket cells. One had an unusually wide axon arborization (wide-axonal basket cell). Although the majority of the collaterals were still in stratum pyramidale, several axons penetrated deep into strata oriens and lucidum. The axon of the other type of atypical basket cell had a stronger bias for stratum lucidum, but again the pyramidal cell layer was heavily innervated (stratum lucidum basket cell). The dendritic trees of these neurons were indistinguishable from those of other basket cells.
Elechon microscopy of the synaptic contacts made by basket cell axon terminals confirmed the predictions of light microscopy, i.e., the majority of the postsynaptic elements were the somata and proximal dendrites of pyramidal cells (Figs. 3,5; Seress and Ribak, 1985, 1990a,b; Gulyis et al., 1993a; Buhl et al., 1994a; Miles et al., 1996). The proportion of postsynaptic dendrites and cell bod-
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FREUND AND S U Z S k l
FIGURE 5. Correlated light and electron micrographs of synaptic contacts between a basket cell and a pyramidal cell in the CA3 region of the guinea pig hippocampus, both recorded and filled intracellularly in vitro. By using paired intracellular recording, an action potential in the basket cell evoked, on average, a 1.67 mV IPSP in the postsynaptic pyramidal cell (see Fig. 4 for other pairs). A: An axon collateral of the basket cell climbs along the proximal basal
dendrite of the pyramidal cell and has several varicosities contacting it. Two of them (bl, b,) make symmetrical synapses on the dendritic shaft (asterisks in the low power micrographs) in B and D (b,) and in C and E (bz). Arrowhead in A and C indicates a small bouton of the same basket cell collateral, which is in contact with an unstained dendritic shaft. Scale bars = 5 p m in A, 0.5 p m in B,C, 0.2 pm in D,E.
FIGURE 4. A,B: Postsynaptic potentials evoked by interneurons that terminate in the perisomatic (A) or in the dendritic region (B) of pyramidal cells, as revealed by paired intracellular recording. An action potential (upper traces) triggered by intracellular current pulse in the interneurons evoked IPSPs in the postsynaptic pyramidal cells (lower traces). IPSPs at the somatic location had a larger amplitude and faster time to peak than did the dendritic IPSPs. The number and location of synaptic contacts responsible for the electrical interaction were identified by light and electron microscopy, following intracellular biocytin filling. C-E: Reconstructions of the
cell pairs (pyramidal cells are drawn in red, interneurons in black) made by a drawing tube. The basket cell in D that produced the IPSP shown in A formed three synaptic contacts on the postsynaptic pyramidal cell, as shown by arrows at higher magnification in C. The interneuron in E, responsible for the IPSP shown in B, arborized in stratum oriens and radiatum and is a bistratified cell. It formed two synapses on the apical and three on the basal dendrites of the pyramidal cell (arrows). All contacts identified at the light microscopic level were confirmed by correlated electron microscopy (see Figs. 5, 8). Scale bars in D,E = 50 pm.
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FIGURE 6. A: Camera lucida reconstructionof the axonal trees of two presynaptic pyramidal cells (black and blue) and a postsynaptic basket cell (red) simultaneouslyrecorded and filled with biocytin in the CA3 region of the hippocampus. The axon of both pyramidal cells established a single contact on a mid-distal dendrite of the basket cell in stratum radiatum (arrows). Correlated light and
electron micrographs of both contacts are shown in Figure 8. B: Synaptic transmission between one of the filled pyramidal cells (1) and the postsynapticbasket cell (2). Single pyramidal cell action potentials evoked variable amplitude EPSPs in the inhibitory cell and occasionally were associated with transmission failures (second trace). Data from Gulyds et al. (1993b). Scale bar = 200 pm.
ies varied considerably. Typically, somatic contacts accounted for 30-70% of the postsynaptic elements. The innervated dendrites were mostly proximal primary branches, which may be considered electrotonically equivalent to the soma. A small number of axon initial segments and spines have also been reported to receive input from basket cells in the hippocampus (Seress and Ribak, 1990a,b). The wide-zonal and the stratum lucidum basket cells in CA3 innervated a considerably larger proportion of dendritic shafts (60-80%), some of which were considered to be distal dendrites (Gulyis et al., 1993a). As in the dentate gyrus, the existence and proportion of nonpyramidal targets of basket cells in the hippocampus still need to be investigated. Ultrastructural features of basket cell bodies and dendrites are very similar to those described for chandelier cells and dentate basket cells. The large number of asymmetrical synapses on the dendrites and somata originate from local collaterals of pyramidal cells (Fig. 8; Gulyis et d., 1993b; Sik et al., 1993; Buhl et al., 1994a), from mossy fibers in the CA3 region (Frotscher, 1985), from Schaffer collaterals (Fig. 8), commissural fibers (Frotscher and Zimmer, 1983a; Seress and Ribak, 1985; Sik et al., 1993; Deller et al., 1994), and from entorhinal afferents (Kiss et al., 1996). Thus, basket cells in the hippocampus are activated in both feed-forward and feedback manners. Paired intracellular recordings and subsequent biocytin filling of the connected neurons showed that pyramidal cells form single synapses with each of their interneuron targets (Figs. 6, 8;
Gulyis et al., 1993b). This finding has been confirmed by in vivo intracellular labeling of a CA3 pyramidal cell (Fig. 7) reconstructed from serial Vibratome sections that were double stained for PV, a calcium-binding protein present in basket and axo-axonic cells. The intracellularly filled pyramidal cell contacted 220 PV-positive cells, 85% of them via a single bouton (Fig. 8). In 12% of the pyramidal cell-PV cell connections, two boutons were involved, and in 3% of the connections, three boutons were involved (Sik et al., 1993). These data suggest that the more than 2,000 asymmetrical synapses on the basket cell dendritic tree are likely to originate from more than 2,000 pyramidal cells. Thus, both convergence and divergence is extremely high in the pattern of efferent connectivity of pyramidal cells to interneurons (Gulyis et al., 1993b; Sik et al., 1993).
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III3.l.a. Interneurons with hihr dendrites and ascending axons (HIPP cellj). The most charactersitic type of cells innervating granule cell dendrites has a dendritic tree limited to the hilar region and an extensive axon arborization in the outer two-thirds of the molecular layer (Fig. 9; HIPP, hilar perforant path-associated cell of Han et al., 1993). The dendrites emerge from a hsiform cell body located subjacent to the granule cell layer and 111.3.2. Dentate gyms branch prohsely. They are covered with long thin spines, almost The dendritic arbors of practically all major cell types known to- as dense as mossy cells in this region (Sik et al., submitted). day from the category of dendritic inhibitory cells have been de- Thorny excrescenses never occur on the soma and dendrites of scribed by Rarn6n y Cajal (1893) and Amaral (1978). Their draw- the HIPP cell, thus providing another distinguishing feature from ings, based on Golgi impregnation, revealed only portions of the mossy cells (Amaral, 1978). Thus, the dendritic tree perfectly coaxonal arbors. Recent intracellular labeling studies, however, have incides with the distribution of mossy fiber collaterals and avoids provided a nearly complete picture of the processes (Han et al., 1993; the dentate molecular layer where entorhinal and commisBuckmaster and Schwartzkroin, 1995a,b; Sik et al., submitted). The sural-associational afferents terminate. following classification is based on the distinct dendritic and axonal The axon of HIPP cells originates from the soma, and several distributions presented in these more recent studies. main axon trunks cross stratum granulosum giving rise to long
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FIGURE 7 . A: The axond arborization of a Neurobiotin-labeled the plot, one in CA3 close to the soma, and two others in CAI,about CA3 pyramidal cell reconstructed from the whole rat hippocampus. 600-800 pm firom the soma. The broken lines indicate the approxThe axon spread through s t r a t u m radiatum and oriens of 32 (60 imate border region between the CAl and CA3 regions, and the pm) longitudinal sections, mostly arborizing in the C A I subfield. closed circle the position of the soma. C: Distribution of the conThe axon had over 15,000 boutons. The same sections were double tacts established by the labeled pyramidal cell on PV-immunoreacstriaeedfor PV. Of the 324 boutons in contact with PV-positive cells, tive interneurons, reconstructed and viewed as in B. Note that the 85% made single contacts (see Fig. 8E,F), 12% made double con- locations and peak densities coincide with those in B, i.e., with the . tacts, and 3% made triple contacts. B: Top view of the bouton & peaks in total bouton number. Scale bar = 200 pm in A Reproduced Complete ason arborization od a single CA3 tribution of the labeled pyramidal cell shown in A, obtained by com- from Sllr et al., (1993) puter reconstruction aod rotation of 32 sections. The darkness of pyramidal cell in the hippocampus. Eur J Neurosci, 51719-1728 rs. the rectangles are proportional to the number of boutons found by permission of Oxford University P e s within that area. Three patches of boutons appear to emerge from
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which corresponds to a total of 76,800 and 98,900 axon terminals, if one assumes an average density of 34 boutons per 100 p,m (Sik et al., submitted). Neuropeptide Y (NPY) was shown to be contained in one of these neurons (Sik et al., submitted), but on the basis of axonal and dendritic patterns, they are likely to contain somatostatin as well (Sections IV.3.a, IV.3.b). At the electron microscopic level, boutons of HIPP cells form symmetrical synapses, even at their nonvaricose segments, with spiny dendritic shafts and spines of presumed granule cells (Fig. 10; Halasy and Somogyi, 1993b). Occasionally, they also form symmetrical synapses on other interneurons (Sik et al., submitted). The same spines frequently receive an additional synapse of the asymmetrical type, most likely from entorhinal afferents. In the hilus, the dendrites and long thin spines of this interneuron type are covered by numerous asymmetrical synapses, most of which showed characteristic features of mossy fiber terminals (Halasy and Somogyi, 1993b). In summary, the HIPP cells are likely to mediate largely feedback inhibition of granule cells by terminating on distal granule cell dendrites and spines in conjunction with entorhinal afferents (Halasy and Somogyi, 1993b; Han et al., 1993; Sik et a]., submitted).
IIL3.I.b. Hilar border neurons with ascending axom and dendrites ( H I W cells). Han et al. (1993) named this cell type hi-
lar commissural-associational pathway related (HICAP), indicating their major distinguishing features, i.e., the predominant innervation of the inner molecular layer (Fig. 9). The triangular cell body of these neurons is located in the polymorphic zone of the hilus or within stratum granulosum at the hilar border (Han et al., 1993; Soriano and Frotscher, 1993a; Buckmaster and Schwartzkroin, 199513; Sik et al., submitted). The primary dendrites have a multipolar origin and give rise to several smooth or sparsely spiny branches that invade both the hilus and the rnolecular layer, with a small bias for the molecular layer in terms of total dendritic length. The main axon runs in the hilus, and gives rise to ascending collaterals that branch in a Y-shaped manner in stratum moleculare, just above the granule cell layer. The dense axon arbor possesses a large number of boutons that are generally confined to FIGURE 8. Correlated light and electron micrographs of synap- the inner one-third of the molecular layer (Han et al., 1993; tic contacts established by two presynaptic pyramidal cells onto a Buckmaster and Schwartzkroin, 1995b). However, the HICAP basket cell. The cells were intracellularly recorded, the electrical in- cell reported by Sfk et al. (submitted) had a significant number teraction characterized, and the cells were subsequently filled with biocytin (see Fig. 6). A spiny dendrite of the pyramidal cell is visi- of collaterals within stratum granulosum (22.6%), near the parble in A (on the right), and the basket cell dendrite (left) is smooth. ent cell body. In a frontal section through the center of the arEach pyramidal cell established a single contact on the dendrites of bor, the axon occupies approximately half of the dentate gyrus, the basket cell in stratum radiatum (A,C), and at the electron mi- either the upper or the lower blade. The axon of the cell injected croscopic level both contacts (bl, bz) were found to be conventional in vivo in the gerbil had the richest arbor near the soma and spread synapses (large arrows in B and D). The pre- and postsynapticmembranes are indicated by white arrows in B and D and show a char- over 1,500 ,urn in the longitudinal direction, reaching 22% of acteristic widening of the synaptic cleft. E,F: An intracellularly filled the total length of the hippocampus (Buckmaster and CX3 pyramidal cell (the reconstruction is shown in Fig. 7) estab- Schwartzkroin, 1995b). The axon of the other cell (from the rat) lished mostly single synapses on PV-containinginterneurons visual- spanned 2.6 mrn in the septotemporal direction (Sik et al., subized in the same sections. At the light microscopiclevel (in E) a vari- mitted) and was particularly dense around the parent cell body case pyramidal cell axon formed one bouton (b) on a PV-positive dendrite (dz). This contact w s shown to be an asymmetrical synapse and gradually diminished toward the mediolateral and the sepa (arrowin F) in the electron microscope. Scale bars = 5 pm in A,C,E, totemporal edges of the arbor. The total two-dimensional axonal length of this cell was 91 rnrn, which corresponded to 26,300 0.5 pm in B,D, 0.2 pm in F.
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torhinal afferents, and innervates granule cell dendrites in conjunction with the same pathway (Halasy and Somogyi, 199313).
III.3.l.c. Neurons with axons and dendrites in stratum moIecuh r e (MOM cells). The axonal and dendritic trees of this cell type are largely limited to the outer two-thirds of the dentate molecular layer, and thus was named molecular layer perforant pathassociated cell (MOPP cell) by Han et al. (1993). Smooth dendrites that originate from a soma in deep stratum moleculare ascend to reach the hippocampal fissure and span an area over 800 p m in transverse length. The axon of the MOPP cell is even more extensive. The majority of collaterals run perpendicular to granule cell dendrites and arborize profusely in a terminal cloud that extends beyond the upper blade of the dentate gyrus (Fig. 9). The number of varicosities along the fine terminal branches of the MOPP cell is somewhat fewer than that for the two types of hilar interneurons described above. Electron microscopy demonstrated that axon terminals of this cell type form symmetrical synapses mostly with spiny distal dendrites of granule cells (Fig. 10; Halasy and Somogyi, 1993b). The dendrites of these interneurons were densely covered by both symmetrical and asymmetrical synapses. Thus, this interneuron type is likely to be driven in a feed-fonvard manner, largely by enCostratification of the axons of three distinct types of GABAergic neuron with the major glutamatergic pathways to the
1II.3.1.d. H i h r neurons with aprojection to the h+pocampus and subiculum. Two cells with these characteristics have been described recently, one in the rat (Sik et al., submitted) and another in the gerbil hippocampus (Buckmaster and Schwartzkroin, 1995b) by intracellular injection. The soma of both cells are located at the border of the hilus and stratum radiatum of the CA3c subfield. Their dendrites leave the soma in all directions in a stellatelike fashion to penetrate the deep hilus and stratum moleculare of the dentate gyrus and stratum radiatum of the CA3c region. Apart from a few collaterals that enter the hilus and stratum moleculare, the majority of axon collaterals follows the path of mossy fibers to subsequently innervate strata radiatum, lucidum, oriens, and pyramidale of the CA3 subfield. Thus, because of their dendritic distribution and afferent input, it is difficult to determine whether these neurons should be considered a part of the dentate gyrus or of the hippocampus. Their output, however, is clearly associated with the CA3 region. The total axon length of this cell in the rat is about 100 mm, with 28,000 boutons, and has a septotemporal extension of 4.3 mm (Sik et al., submitted). In this cell, one main branch could be followed to the subiculum, but varicose collaterals were not seen to originate at this distant site. The neuron labeled in the gerbil also had numerous collaterals in stratum radiatum of the CA1 region (Buckmaster and Schwartzkroin, 1995b). Electron microscopic studies have not been reported; therefore, the types of synapses and postsynaptic elements of this neuron type are still unknown. According to the laminar distribution of its axon, this cell type may be considered a CA3c-specific variety of bistratified or trilaminar cell, described elsewhere in the hippocampus (Section III.3.2.b). III.3.1.e. H i h r neurons with unknown projection. There are interneurons in the hilus with a great variability in dendritic morphology as revealed by Golgi and immunocytochemical studies (Amaral, 1978; Gulyis et al., 1992). Due to the lack of axonal staining, these cells cannot yet be reliably classified because, as shown above, cells with different dendritic morphology may belong to the same type if classified on the basis of postsynaptic elements (e.g., basket cells). Nevertheless, there is a cell type with unique dendritic features, which deserves mention. Its dendrites are limited to fields densely innervated by mossy fibers, i.e., to the hilus of the dentate gyrus and to stratum lucidum of CA3 (Section IV.2.c). They have been previously described in Golgi preparations (Amaral, 1978; Soriano and Frotscher, 1993b) and visualized in large numbers by immunostaining for the calciumbinding protein, calretinin ( C R GulyPs et al., 1992) and heat shock protein after ischemia (Hsu and Buzsiki, 1993). Amaral (1978) named these cells long-spined multipolar neurons according to the characteristic hairlike shape of the spines covering their dendrites and cell bodies. The entire dendritic tree receives abundant asymmetrical synapses from terminals or thin pretermind segments of axons displaying ultrastructural features of mossy fibers (Gulyds et al., 1992; Soriano and Frotscher, 1993b). The dendritic spines of these cells penetrate into bundles of mossy
FIGURE 9.
dentate gyrus. The hilar neuron, which has its axon distributed in the perforant path termination zone (HIPP cell), and a molecular layer cell, which also has its axon arborizing in the perforant path termination zone (MOPP cell), have terminals that avoid the inner one-thirdof the molecular layer. In contrast, the hilar neuron, which has its axon distributed in the commissurallassociational pathway termination zone (HICAP cell), has terminals that avoid the outer two-thirds of the molecular layer. GABA immunoreactivity was demonstrated in the terminals of each of these three cell types. These cells were recorded intracellularly in vitro and visualized with bio+n. Results are based on Han et al. (1993), Halasy and Somogyi (1993, 1996a,b), and Halasy et al. (1996). This figure was kindly prepared by Peter Somogyi. Scale bar = 100 pm.
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FIGURE 10. Immunocytochemical demonstration of GABA in boutons of dentate gyrus interneurons that innervate stratum moleculare. The electron micrographs show biocytin-filled boutons of intracellularly recorded HIPP (A-C), MOPP (D,E), and HICAP (F,G) cells. A, D, and F show that the boutons establish type two synapses (arrowheads) with the dendritic s h a h (d) of granule cells. C, E, and G show boutons in sections that were immunoreacted for GABA by the postembedding silver-intensified immunogold method. The high density of electron-dense immunoparticles over the bio-
cytin-labeled boutons demonstrates their high GABA content. B and C are adjacent sections. Open arrows point at boutons immunonegative for GABA, and unidentified GABA-immunoreactive boutons are marked by asterisks. The peroxidase reaction product is less electron dense in the immunoreacted sections due to the removal of osmium. Data based on Halasy and Somogyi (1993a,b) and on unpublished results for the HIPP cell. This figure was kindly prepared by Katalin Halasy. Scale bars = 0.2 pm for A-G. B,C and D-G are at the same magnification.
fibers, where each spine may receive as many as six synapses from small-caliber preterminal mossy fibers (Gulyis et al., 1992). The hilar variety of these cells are similar to neurons that project to the outer molecular layer (HIPP cells), which also have spiny dendrites limited to the hilus (Han et al., 1993; Buckmaster and Schwartzkroin, 1995a,b). However, the axons oflong-spined mul-
tipolar cells have never been visualized by Golgi or immunostaining techniques, probably due to myelination. This suggests that these long-spined multipolar neurons either belong to a novel class of hilar interneurons or, according to combined immunocytochemical and tracing studies (Seress and Ribak, 198.3; Bakst et al., 1986; Miettinen et al., 1992; Deller et al., 1995b; Sections
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FIGURE 11. Reconstructions of 0-LM cells intracellularly recorded and filled in vivo in the CAI region (A) and in vitro in the C43 region (B). The axon of this cell type arborizes primarily in stratum lacunosum-moleculare in their respective regions and also have a minor projection to stratum oriens. However, the dendritic trees show a different laminar specificity. Dendrites of the CA1 0-
LM cell are horizontallyoriented and are restricted to stratum oriens, whereas the dendrites of the 0-LM cell in CA3 span all layers except stratum lacunosum-moleculare. The laminar distribution of the dendrites in both subfields overlaps that of recurrent collaterals of local pyramidal cells. The nonvaricose main axon trunks are not drawn for the CAI cell. Scale bars = 100 pm in A, 50 p m in B.
IV and VI for details), may represent a commissurally projecting variety of HIPP neurons. The CA3 variety of long-spined multipolar cells may send its axon to stratum radiatum (M. Frotscher, personal communication) or to stratum lacunosum-moleculare of the CA3 region and back to the outer stratum moleculare of the dentate gyrus (C. McBain, personal communication). For more details, see Section IV.2.c.
111.3.2. Hippocampus
With respect to the relative distribution of dendritic and axonal arbors as compared with the laminar pattern of major excitatory afferent pathways, the cell types innervating principal cell dendrites in the hippocampus are very similar to those described in the dentate gyrus. The corresponding types are therefore described in the same sequential order.
III.3.2.a. Cells terminating in conjunction with entorhinal afferents (0-LM ceZls). The major distinguishing features of these
neurons, as described by Ram6n y Cajal (1893) and Lorente de N6 (1934), are a dense axon arbor restricted to stratum lacunosum-moleculare and a dendritic tree localized to layers occupied by recurrent collaterals of local principal cells. In this respect, they .Ire similar to the HIPP cells of the dentate gyrus (see above), whose s o n collaterals also terminate in conjunction with perforant path afferents and whose dendrites, which are located exclusivelywithin the hilus, are innervated by mossy fiber collaterals. Further similarity is found in their colocalization of neuropeptides because both HIPP and 0 - L M cells contain somatostatin, and some of them
also contain NPY (Sections IV.3.a, IV.3.b). The axons of 0 - L M cells ascend from the soma directly to stratum lacunosum-molecdare to form a dense cloud of fine varicose collaterals (Fig. 11; Gulyh et al., 1993a,b; McBain et al., 1994; Sik et al., 1995). Occasional branches are also directed toward stratum oriens. The axon arbor in stratum lacunosum-moleculare is highly focused, having a limited transverse and longitudinal spread. In 400-pmthick slices, the arbor extends 300%400 p m in contrast with the same cell type in the dentate gyrus, which covers more than twothirds of the entire transverse length of the molecular layer in slices of similar thickness (Han et al., 1993). The axons of HIPP cells intracellularly filled in vivo were shown to innervate the entire upper and lower blades of the dentate gyrus (Sik et al., submitted). There is a single example in the literature of an in vivo filled 0LM cell with a completely reconstructed axonal field. The dendrites of this cell were confined to the stratum oriens-alveus border. Its axon gave rise to a large dense cloud in stratum lacunosum-moleculare (91 S%),whereas a smaller arbor was distributed within stratum oriens (7%) (Fig. 11; Sik et al., 1995). Importantly, the size of the axon arbor was not significantly larger than those reconstructed from 400-pm-thick slices. The axon arbor had a central core of approximately 500 p m in diameter, which gradually diminished for another 100-200 p m in the septa1 and temporal directions. The total two-dimensional axon length was 63.4 mm, and the calculated number of boutons, based on a density of 26.6 boutons per 100 p m , was 16,800. The dendritic tree of 0-LM cells located in the CA1 and CA3 regions is different, perhaps reflecting the difference in the par-
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tent of entorhinal afferents and the axon arbors of single 0-LM and HIPP cells in the hippocampus and the dentate gyrus. Anterograde labeling of a small group of entorhinal cells (Tamamalu and Nojyo, 1995) or a single-layer I1 neuron (Tamamaki and Nojyo, 1993) gives rise to axonal staining throughout the dorsal and ventral blades of the dentate gyrus. In contrast, the diameter of the terminal cluster in stratum lacunosum-moleculare of the CA1 region is smaller than 1 mm. Thus, it appears that the spatial extent of the axon arbors of single 0 - L M neurons in the CA1 region and of HIPP cells in the dentate gyrus closely resemble the size of the terminal fields of perforant path afferents in these regions. The functional significance of this hypothesized match is still unknown but may be related to inhibitory flanking in the CA1 region, carving out slabs of pyramidal cells with similar representations of entorhinal-cortex-mediated neocortical information (Cohen and Eichenbaum, 1993; Buzsiki et al., 1995).
lII.3.2.b. Interneurons innervating pyramidal cell dendrites in strata radiatum and oriens (bistratiJied and trihminar cells). The first descriptions of bistratified and horizontal trilaminar cells were given by Buhl et al. (19943 and Sik et al. (1995). Cell bodies of these neurons are localized within or near to stratum pyramidale or at the stratum oriens-alveus border (Fig. 12). The dendritic trees of bistratified cells are spine free, often varicose, and multipolar. Several primary dendrites bifurcate close to the soma. These dendrites have a predominantly radial orientation but frequently also emit horizontal branches. In contrast with the dendritic tree of basket and chandelier cells, the vertical branches of bistratified cells do not reach stratum lacunosum-moleculare (Miles et al., 1996; Halasy et al., 1996). The dendrites of the horizontal trilaminar cell run horizontally at the stratum oriens-alveus border, extending over several hundred micrometers in the septotemporal and transverse directions. This cell has a large soma, with prominent primary dendrites, and occasional spines on more distal segments (Sik et al., 1995). Radial trilaminar cells are very similar to bistratified cells with regard to their axonal and dendritic trees, but there are also some consistent differences. In contrast with bistratified cells, dendrites of radial trilaminar cells often penetrate stratum lacunosum-moleculare (A.I. Gulyk and R. Miles, unpublished observations). This is especially true for those cells located in distal stratum radiatum (i.e., portion of stratum radiatum closer to stratum lacunosum-moleculare) . The axon of bistratified cells forms a dense arbor of fine varicose collaterals both above and below the pyramidal cell layer, occupying the entire width of stratum oriens and a zone of 200-300 p m of proximal stratum radiatum (i.e., toward the pyramidal cell layer) in the CA1 and CA3 regions (Buhl et al., 1994a; Sik et al., 1995; Miles et al., 1996). The transverse spread of the axon was found to be between 800 and 1,100 p m in cells filled in vitro, whereas in the only example of a bistratified cell filled in vivo the axonal arbor was approximately 2 mm in diameter in the transverse and septotemporal directions (Sik et al., 1995). The two-dimensional axon length of this cell was 79 mm, carrying a total of 16,600 synaptic varicosities. Immunocytochemical double-staining techniques revealed that this cell type contained the calcium-binding protein calbindin (see Section IV.2.b).
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Horizontal trilaminar neurons have a similar axon arbor, but unlike the axon arbor of bistratified cells, a considerable number of varicose collaterals also distribute within stratum pyramidale (16.7%). In addition, the distribution of the trilaminar cell axon is more strongly biased toward proximal stratum radiatum (68.4%). Another important difference between bistratified and trilaminar cells is that trilaminar cells can be antidromically activated by stimulating the fimbria (Sik et al., 1995, submitted). This activation demonstrates that trilaminar cells project outside of the hippocampus, possibly to the medial septum (T6th and Freund, 1992). The axon extended 2.6 mm in the septotemporal and 2.45 mm in the mediolateral direction. The total two-dimensional axon length amounted to 56 mm and possessed a calculated 15,800 axon terminals. The axon arbor of radial trilaminar cells is sparse and consists mainly of long, radially running collaterals, which are uniformly varicose along their entire length, regardless of the layer they cross (A.I. Gulyis and R. Miles, unpublished observations). This fact suggests that a considerable proportion of the postsynaptic elements are likely to be cell bodies in stratum pyramidale, unlike bistratified cells. Electron microscopy of a large sample of axon terminals demonstrated that bistratified cells established symmetrical synaptic contacts with proximal dendrites (79%) and dendritic spines (17%) of pyramidal cells (Halasy et al., 1996). In contrast, cell bodies (4%), mostly of pyramidal neurons, were rarely innervated. Axon terminals of bistratified cells were found to be significantly smaller than basket cell boutons in two independent studies, one measuring cross-sectional area at the synaptic junction in the electron microscope (Halasy et al., 1996), the other using camera lucida at the light microscopic level (Miles et al., 1996). The absolute values calculated in the two studies are different, but both demonstrate that basket cell boutons, on average, are more than two times larger in cross-sectional area than boutons of bistratified cells. The latter often lack mitochondria, whereas the basket cell boutons usually contain one or even two mitochondria and appear to have somewhat longer synaptic active zones (Miles et al., 1996). These differences suggest that the probability of release is higher at perisomatic synapses, which is supported by findings that most, if not all, spontaneous inhibitory postsynaptic potentials (IPSPs) recorded from CA1 pyramidal cells in vitro are of perisomatic origin (Miles et al., 1996). In paired intracellular recording and labeling experiments in vitro, the three bistratified cells reported to date were shown to establish five, six, and nine electron microscopically verified synaptic contacts with their target pyramidal cell (Buhl et al., 1994a; Miles et al., 1996). With an average of 6.7 contacts per target cell, a bistratified neuron (with 16,600 boutons calculated by reconstructing an in vivo injected cell; Sik et al., 1995) may innervate approximately 2,500 pyramidal cells. Interestingly, the contacts were distributed on different branches of the postsynaptic pyramidal cell dendritic tree, which suggests that these synapses are not designed to amputate any dendritic branch (Miles et al., 1996). The trilaminar cell also made symmetrical synapses predominantly with dendritic shafts of unidentified origin (Sik et al., 1995). The laminar distribution of the dendritic tree of bistratified
111.3.2.c. Neurons with axon and dendrites in stratum radiaturn. Somata of this cell type may be located in strata radiatum and pyramidale, from which several smooth and varicose dendrites ascend and descend radially to form a tree largely confined to stratum radiatum (Kawaguchi and Hama, 1988; Gulyds et al., 1993a; Kauer and McMahon, 1995; Miles et al., 1996). Some of the cells may have a multipolar stellatelike appearance, The dendrites, even the radially running branches, rarely enter stratum lacunosum-moleculare. The axon branches in close proximity to the soma and forms a rather sparse arbor that extends throughout the entire width of stratum radiatum. Only a small number of collaterals enter strata pyramidale or oriens. Relatively small en passant varicosities are evenly distributed along the axon. The transverse extent of the axon cloud is smaller than that of perisomatic (basket and chandelier) cells, never exceeding 600 p m in a 400-pm-thick slice. Some of the radially running collaterals, which enter stratum pyramidale, may have been cut during the slicing procedure, and the possibility that some of these neurons are partially visualized bistratified cells cannot be excluded.
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or on shafts of nonpyramidal cells were less common. Axon collaterals in stratum moleculare of the dentate gyrus made symmetrical synapses with dendritic shafts, which showed the ultrastructural features of granule cell dendrites (Kunkel et al., 1988).
most striking examples of interneurons with a considerable proportion of their axons in more than one hippocampal subfield are the back-projection neurons of Sik et al. (1774, 1775). Only two such cells have been visualized to date by intracellular injections in vivo, but their regular occurrence has been confirmed by NADPH-diaphorase histochemistry and anterograde tracing (Sik et al., 1994). These cells typically occur at the stratum oriensalveus border of the CA1 subfield and have horizontal sparsely spiny dendrites, which largely remain in stratum oriens (Fig. 12). The axon forms a local arbor in stratum oriens but also extends into strata pyramidale and radiatum of the CAI region. A main branch of the axon travels to stratum radiatum of CA3, giving rise to a large axon cloud in this layer and also in stratum oriens. Other main axons cross the hippocampal fissure and arborize in stratum radiatum of CA3c and the subjacent hilus of the dentate gyrus. One of the cells had a collateral that became myelinated and disappeared as it entered the fornix, thus suggesting an exIIL3.2.d. Interneurons in stratum hnosum-molecuhre ( f )trahippocampal or commissural projection. The cell that had a U,. Cell bodies of these neurons are within stratum lacunosum-mol- completely filled axon extended for 3.1 mm in the septotempoeculare or at the border of this layer with stratum radiatum. These ral direction. The two-dimensional axon length was 101 mm, and cells were first described by Ram6n y Cajal (1873, 1711) and the total number of boutons was approximately 25,000. The maLorente de N6 (1934) and were recently studied by intracellular jority of the boutons were in CA3 (61.5%), whereas the CA1 subrecording and subsequent visualization at the light and electron mi- field (24.3%) and the hilar-CA3c region (14.2%) received a croscopic levels (Kunkel et al., 1788; Lacaille and Schwartzkroin, smaller proportion of the synapses. Electron microscopy has been done on a limited sample of 1988a; Williams et al., 1794). The dendritic tree is typically bitufted with a predominantly horizontal orientation as opposed to similar axon terminals, and all of them were found to establish symmetdendritic trees of neurons in stratum pyramidale. Some branches, rical synaptic contacts with spiny dendritic shafts and cell bodies however, often descend to stratum pyramidale, whereas others of presumed pyramidal cells. Interestingly, axospinous contacts may travel across the hippocampal fissure and terminate in stra- have not been observed (Sik et al., 1974). Several boutons were tum moleculare of the dentate gyrus. All dendrites are spine free found in the immediate vicinity of capillaries but never came in and often varicose. The axon originates from the soma or a main direct contact with the endothelium. proximal dendrite. Most of the collaterals are also horizontally With the exception of the CA3 projection, these cells are reoriented and arborize predominantly in stratum lacunosum-mol- markably similar to the trilaminar neurons, particularly in regard eculare or in the bordering region of stratum radiatum. Similar to their dendritic morphology, laminar distribution of local (CAI) to dendrites, some axon branches may also cross the hippocam- axons, and a projection through the fimbria, which may prove to pal fissure and terminate in the dentate gyrus. All published ex- terminate in the medial septum (T6th and Freund. 1972). As to amples of this cell type have only partially recovered axons; thus, their neurochemical identity, they appear to correspond to cells that colocalize calbindin-D28k (CB), somatostatin (SOM), NPY, the true extent of the axon arbors remains to be determined. The ultrastructural features of interneurons in stratum la- and nitric oxide synthase (NOS) (Sections IV.3.a, IV.3.b), but dicunosum-moleculare are similar to those of other nonpyramidal rect evidence is not currently available. According to the laminar cell types, i.e., infolded nuclei, cytoplasm rich in organelles, and specificity of their dendritic tree, they are likely to be driven prisynaptic input of both asymmetrical and symmetrical type cov- marily by the local collaterals of CA1 pyramidal cells in a feedering the dendrites and at a lower density also the soma (Kunkel back manner. They exert their inhibitory effects on the dendritic et al., 1988). With anterograde degeneration, at least some of the tree of pyramidal cells in the CA1 region and most notably in area asymmetrical, presumed excitatory, synapses were shown to orig- CA3. The inhibition mediated by back-projection cells, therefore, inate from the ipsi- and contralateral CA3 region and from the is in a direction opposite to the excitatory dentate gyrus-CA3-CAl ipsilateral entorhinal cortex. Axon terminals of these interneurons axis. A cross-regional timing of action potentials by these inwere shown to make symmetrical synapses with spiny dendritic terneurons may be important to secure population synchrony of shafts of presumed pyramidal cells in strata lacunosum-molecu- principal cells in distributed networks and may allow a coordilare and radiatum. Contacts on dendritic spines of pyramidal cells nated induction of synaptic plasticity (Sik et al., 1994).
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FIGURE 13. A: Camera lucida drawings of three types (IS-1, IS2, IS-3) of interneuron that selectively innervate other interneurons (interneuron-selectivecells) in the hippocampus (somata and dendrites in black, axons in red). IS-1 (cells 3, 4, 6): CR-containing interneurons with axonal arbors in stratum radiatum have dendrites located primarily in the same layer and often form axodendritic and dendrodendritic contacts with other CR-positive neurons. For example, the axon of cell 3 forms multiple contacts (open arrows) with the dendrite (dotted outline) of cell 5. The sites and extent of dendrodendritic contacts are labeled with arrowheads and dotted outlines for cells 3, 4, and 6 (see B). One of the dotted dendrites is labeled as cell 5, and it is also indicated in the cluster shown in B. Arrows label those points where CR-positive axon terminals contact the dendrites of cells 3,4, and 6. IS-2 neurons are VIP immunoreactive (cell 1).The dendritic arbor is confined to stratum lacunosum-moleculare,where entorhinal afferents terminate. The axons of these neurons arborize mainly in stratum radiatum. IS-3: B i d e d neuron containing VIP (cell 2). Its axon innervates the stratum oriens-alveus border. B: Schematic drawing of a cluster of CR-immunoreactive interneurons (15 cells) forming dendrodendritic contacts with each other. Parallel red lines and red crosses indicate long (50-200 pm) and short (1-5 pm) dendritic appositions, respectively. Cells 3 and 5 are also shown in A. Because the duster was reconstructed only from seven consecutive sections, the true size of the cluster is likely to be larger. C-F: Multiple contacts (arrows, arrowheads) are formed by axons of CR-containing (C,D) or VIP-containing (E,F) interneurons on the soma and dendrites of CBimmunoreactive (C,E,F) dendritic inhibitory cells and on a VIP-positive basket cell (D). The presynaptic processes are visualized by NiDAB (in black) and the postsynaptic cells by DAB (in brown) in each micrograph. From Ascidy L, G6rc.s TJ, and Freund TF (1996) Different populations of VIP-immunoreactive interneurons are specialized to control pyramidal cells of interneurons in the hippocampus. Neuroscience 73:317-334 by permission of Elsevier Science Ltd and from Gulyh AI, Hajos N, Freund TF (1996) Interneurons containing calretinin are specialized to control other interneurons in the rat hippocampus. J Neurosci 16:3397-3411 by permission of the Society of Neuroscience. Scale bars = 100 p m in A,B, 10 pm C-F.
111.4. Morphological Differences Between Interneurons Terminating in the Dendritic or Perisomatic Region of Principal Cells
The most striking difference between these two major classes of interneurons lies in the laminar distribution of their axon (Figs. 17 and 18) and the resulting difference in postsynaptic elements, i.e., the dendrites or the perisomatic region (soma, axon initial segment, proximal dendrites) of principal cells. However, in addition to this obvious distinguishing feature, there are numerous, more subtle but consistent, differences. Some of these were briefly mentioned in the preceding sections, and they will be collectively described here. Perisomatic interneurons have large cell bodies with an abundant cytoplasm and larger and more numerous mitochondria than do interneurons innervating principal cell dendrites. This fact suggests that the former have a higher metabolic rate, and it may also explain why their dendrites are frequently studded with large varicosities containing clumps of mitochondria. In contrast, interneurons that distribute their axon arbor to dendritic layers have smooth dendrites with occasional flat varicosities. The main orientation of the dendritic tree of perisomatic cells is radial. Dendrites cross several (in most cases all) layers of the subfield, thus allowing these cells to sample excitatory input from the majority of local and extrahippocampal sources. In contrast, interneurons innervating principal cell dendrites frequently have horizontal or oblique dendritic trees limited to one or two laminae. These cell types specifically receive excitatory input from certain sources and avoid others. Accordingly, interneurons activated selectively in a feedback or feed-forward manner are often found among dendritic cells, whereas perisomatic interneuron types can be driven both ways (Section X.l). Important differences also exist in the axonal branching pattern. The perisomatic interneurons usually have long, tangentially running, main axon trunks that are mostly myelinated. These main axon branches emit regular unmyelinared collaterals that arborize densely and form multiple synapses in the perisomatic region of principal cells. Interneurons with an axon arborizing in the dendritic layers often have unmyelinated main axons, which typically divide into branches of similar diameter, particularly upon reaching their target laminae. These morphological characteristics may point to differences in the reliability and velocity of action potential conduction to the terminals. Members of both interneuron classes usually form multiple contacts with their target principal cells (unlike the pyramial cell-interneuron connection, which is mediated by single synapses; Section 111.2).However, whereas boutons of perisomatic cells are concentrated in a relatively small area of the postsynaptic cell membrane, the dendritic interneurons typically distribute single boutons to different branches. This distribution suggests that even single perisomatic cells may be able to influence the firing of target principal cells, whereas activity of dendritic inhibitory cells may have to be synchronized to modulate dendritic conductance efficiently (Miles et al., 1996; Section XIII.4). Another consistent difference is observed in the size and mitochondrion content of axon terminals of perisomatic and dendritic interneurons, as described in Section III.3.2.b (Miles et al.,
1996; Halasy et al., 1996). Axon terminals of perisomatic interneurons are larger, usually possess a mitochondrion, and have on average larger synaptic active zones. Interneurons innervating principal cell dendrites have smaller boutons, which frequently lack mitochondria and form synapses with small active zones. In the case of perisomatic interneurons, these differences may underlie a higher tonic firing rate and more reliable transmission. This notion is indeed supported by the findings that spontaneous IPSPs recorded from pyramidal or granule cells have a perisomatic origin (Solttsz et al., 1995; Miles et al., 1996). Although these differences exist between the typical types of the two main classes, one should be aware of cells that show transitory features in one or several of the characteristics listed above. Such examples include the wide-zonal basket cells, trilaminar cells, and HICAP cells. Members of the third main class of interneurons, which will be described in Section 111.5 do not innervate principal cells but only other interneurons.
served in early Golgi studies, but their selectivetermination on other types of interneurons was not recognized. Hippocampal microcircuits have been considered for over a century as a neuronal network built up from two basic components: a profusely interconnected ensemble of excitatory principal cells and the different subsets of interneurons that govern their activity. A third component should be added to this scheme. A systematic analysis with double-immunostaining techniques identified specialized interneurons that innervate specific subsets of interneurons. GABAergic interneurons belonging to this component of hippocampal microcir-
cuits constitute at least three types (IS-1, IS-2, IS-3) based on their connectivity and neurochemical characteristics.
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the hilus or stratum granulosum of the dentate gyrus. They have a smooth dendritic tree that arborizes most extensively in stratum radiatum but may also invade other layers (Fig. 13). The dendrites of IS-1 neurons in the dentate gyrus occur in all layers. The most characteristic feature of these dendrites is that they form long dendrodendritic junctions with each other, in which typically two to three dendrites are intermingled for more than 100 p m (Fig. 14). Varicose axon collaterals of other IS-I cells are also often involved in these braids. Reconstructions from five serial Vibratome sections reveal that IS-I cells form clusters of 10-15 dendrodendritically connected neurons spanning an area of 500-800 ,um in the transverse direction. Although the axon arbors of these cells can only be partially reconstructed from immunostained sections, basic axonal characteristics can nevertheless be identified. The main axons ramify in stratum radiatum, where they emit several collaterals that course in all directions. These collaterals carry both en passant and drumsticklike axon terminals, which are distributed unevenly along the axon. These collaterals frequently become varicose upon encountering an interneuron soma or dendrite in stratum radiatum. The striking association of the CR-positive axons with interneuron dendrites and somata becomes even more apparent in sections double stained for calbindin, which labels interneurons terminating on pyramidal cell dendrites (Section IV). In addition to the large number of multiple climbing fiberlike contacts on calbindin cells, the CRpositive axons often form similar connections with other CR-positive neurons (Acsidy et al., 1996b; Gulyh et al., 1996). Electron microscopy confirmed that all boutons of IS-I cells established multiple symmetrical synapses with the dendrites and somata of other interneurons, which were identified with postembedding immunogold staining for GABA. The CR-positive neurons and axon terminals were also GABA-positive. The postsynaptic GABAergic neurons were mostly calbindin-containing interneurons (Section IV.2.b), other CR-positive IS- 1 cells, and less frequently the vasoactive intestinal polypeptide (VIP)-positive basket cells (Section IV.3.d). PV-containing interneurons were not innervated. Interestingly, one subset of basket cells containing VIP was innervated by IS-1 CR-positive cells, whereas the other subset containing PV was avoided. This behavior may suggest that functional differences exist between the two neurochemically distinct basket cell types (Sections IV.2.a, IV.3.d). Although numerous zonula adherencia were found between adjacent CR-positive dendrites that were attached for several hundred micrometers, the presence of gap junctions, although very likely, could not be unequivocally demonstrated. The dendrites of IS-1 cells are covered by an unusually high density of asymmetrical and symmetrical synapses. The major excitatory input to these neurons likely originates from commissural-associational fibers (Schaffer collaterals) and perhaps from the entorhinal cortex. Their activity may be synchronized by the multiple dendrodendritic and axodendritic contacts they form with each other. In turn, they innervate mostly interneurons terminating on principal cell dendrites (Fig. 16; Acsidy et al., 1996b; Gulyis et al., 1996). Synchrony of dendritic inhibition appears to be essential for an efficient control of electrogenesis and plasticity in principal cell dendrites (Miles et al., 1996; Section XIII.4).
FIGURE 14. A,B: Two light micrographs demonstrate the extent of dendrodendritic contacts between CR-immunoreactive dendrites in the C41 area. Pairs of radially oriented dendrites often cross several laminar boundaries and run attached for more than 200 pm. Besides the extensive dendrodendritic contacts (arrowheads), axodendritic contacts (arrows in B) were also observed. C: Zonula adherentia (arrowheads) are formed between contacting CR-immunoreactive dendrites. One of the CR-positive dendrites receives an additional axodendritic contact (white arrow, symmetrical
synapse), adjacent to the punctum adherens, from CR-immunoreactive axon terminals. D,E: CR-immunoreactive axons form symmetrical synapses (arrow) on GABA-immunoreactive dendrites (d-A) both in the CA1 area (D) and in the dentate gyms (E). As demonstrated in D, in several cases multiple axon terminals innervated the same postsynaptic dendrite. F: A CR-immunoreactive axon terminal (bCR) forms a symmetrical synapse (arrow) on the soma of a VIP-positive basket cell (&p), weakly labeled by the DAB precipitate. Scale bars = 10 p m in A,B, 0.25 p m in C, 0.5 p m in D-F.
FIGURE 15. A,B: The plexus of VIP-immunoreactive axons originating from IS-3 cells (A) shows a perfect overlap with dendrites of mGluR1-positive neurons (B) at the border of strata oriens and alveus in the CA1 region. C: Electron micrograph shows two VIPpositive boutons in synaptic contact (white arrow: the other synapse is visible only in adjacent sections) with an mGluR1-immunoreactive dendrite, which also receives an unlabeled synapse. D: A VIPimmunoreactive bouton forms a symmetrical synaptic contact (arrow) with a GABA-immunoreactive soma (note the accumulation of colloidal gold particles) in stratum radiatum of the CAI region. The
GABA labeling of the VIP-positive bouton is poor in this section due to the dense DAB precipitate; however, it is evident in consecutive sections (not shown), where mitochondria in the bouton are larger. E,F: Correlated light and electron micrographs of VIP-positive boutons forming multiple contacts with a CB-positive interneuron in CA1 stratum oriens. One of the VIP-positive boutons (b,) was shown at the electron microscopic level in F to establish a conventional synaptic contact (arrow) with the CB-positive dendritic shaft (d). Scale bars = 30 pm in A,B, 0.5 p m in C,D,F, 10 pm in
E.
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vides an ideal tool to classify and study the connectivity of these neurons further. Greater value is likely to be gained by these chemical neuroanatomical data as the roles of calcium-binding proteins become elucidated. Highly specific antibodies against neurotransmitter receptors and subunits are now available for immunocytochemical analysis at the light and electron microscopic levels. Studies with these antibodies reveal striking differences between principal cells and interneurons and among morphologically distinct types of interneurons. They provide further evidence for a functional specialization of interneurons with different connectivities and also allows predictions to be made about the action of different transmitters on GABAergic cell types. The presence of neurotrophic factors (nerve growth factor, NGF; neuotropin 3, NT3; brain-derived neurotrophic factor, BDNF), either synthesized or accumulated, and the presence of nitric oxide (NO)-synthase activity in interneurons of the cerebral cortex implies that interneurons are also engaged in functions other than those derived from synaptic connectivity. This engagement may involve the regulation of synaptic plasticity, sprouting and local blood flow through diffusible messengers, and trophic factors. However, even these roles appear to be coupled to classical functions as suggested by correlation to input-output features. Here we describe the neurochemical characteristics of interneurons according to the questions outlined above and focus on the correspondence between morphological and neurochemical classification schemes (Table 1.). The functional implications, if available, of the presence or absence of these markers in particular sets of neurons will be described in Sections VII-XVI.
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INHIBITORY CELLS
PRINCIPAL CELLS
INTERNEURON-SELECTIVE
INHIBITORY CELLS
INHIBITORY CELLS
PRINCIPAL CELLS
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tation as immunostaining when GABAergic cell types are to be identified. The proportion of GABAergic neurons in the total neuron population of the hippocampus has been studied by immunostaining for GABA and was found to be between 7% (Aika et al., 1994) and 11% (Woodson et al., 1989). These measurements may prove to be underestimates due to false-negative staining, particularly of those GABAergic cells with distant projection (Section VI). Even the GABAergic projection neurons appear to be visualized by in situ hybridization for GAD (Houser and Esclapez, 1994; Esclapez and Houser, 1995), but a quantitative analyzis of this materal has not been reported to date. O n the basis of the presently available information, we tentatively suggest that 10% of all neurons in the hippocampus and dentate gyrus are GABAergic interneurons, with the assertion that this number differs in the different subregions and along the longitudinal axis of the hippocampal formation.
Badet and chandelier cells. The first indirect evidence for the GABAergic nature of basket and chandelier cells derives from immunostaining experiments using antisera against GAD. Somata (Ribak et al., 1978) and axon initial segments (Somogyi et al., 1983b) of hippocampal and neocortical (Freund et al., 1983) pyramidal cells are ensheathed by GAD-positive axon terminals making symmetrical synapses. At tissue levels well penetrated by antisera, practically all synapses that contact somatic and axon initial segment membranes are GAD positive, thus suggesting that interneurons known to innervate these regions of pyramidal cells are GABAergic. The first direct evidence that supports this observation is the demonstration of GABA immunoreactivity in the somata of Golgi-impregnated chandelier cells by using a postembedding immunostaining procedure developed for osmiumtreated semithin sections (Fig. 19; Somogyi et al. 1985a). Subsequently, these results have been confirmed for both basket and chandelier cells by a combination of intracellular labeling and postembedding immunogold staining for GABA in the dentate gyrus (Halasy and Somogyi, 1993b; Soriano and Frotscher, 1989; Halasy et al., 1996). Interneurons innervating principal cell dendrites. It is clear from the early GAD-immunostaining experiments in the hippocampus and dentate gyrus that the dendritic layers (i.e., strata radiatum, oriens, moleculare, and lacunosum-moleculare), where basket and axo-axonic cells do not terminate, also contain a high density of GABAergic axon terminals (Ribak et al., 1978; Somogyi et al., 1983b; Katsumaru et al., 1988b; Halasy and Somogyi, 1993a). The implications of these findings (i.e., that interneurons arborizing in these layers are also GABAergic) has been confirmed by the demonstration of GABA immunoreactivity in neuropeptide or calcium-binding-protein-containing cell types known to terminate on principal cell dendrites (Somogyi et al., 1984; Kosaka et al., 1985, 1988b; T6th and Freund, 1992; for further details, see Sections IV.2, IV.3). Intracellular labeling combined with postembedding immunogold staining for GABA confirmed these findings for additional dendritic interneuron types (bis-
Schematic diagrams of circuits involving the three types of GABAergic interneurons that selectively innervate other interneurons (iterneuron-selective cells IS-1,IS-2,IS-3).A: IS-1cells (in red) contain CR, are in multiple dendrodendritic and axodendritic contact with each other, and innervate CB- and VIP-CCKcontaining interneurons (in blue) responsible for dendritic and perisomatic inhibition of pyramidal cells, respectively (i.e., bistratified and basket cells). B: IS-2cells contain VIP and ihnervate other VIPpositive cells and the CB-containing interneurons (bistratified cells) that synapse on pyramidal cell dendrites in stratum radiatum. IS-3 cells are also VIP immunoreactive and selectively target the SOMcontaining horizontal cells of the stratum oriens (0-LM cells). In conjunction with entorhinal afferents, 0-LM cells are responsible for the innervation of the most distal dendritic segments of pyramidal cells in stratum lacunosum-moleculare. IS-1,IS-2,and IS-3 nenrons may have a role in synchronizinginhibitory cells that convecge onto a particular group of pyramidal cells. Alternatively, they may disinhibit pyramidal cell dendrites targeted by particular excitatory inputs, e.g., IS-3cells are likely to reduce feedback inhibition exerted by 0-LM cells in the entorhinal termination zone.
FIGURE 1 . 6
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Hippocampus
LAYER-SPECIFIC INPUT
ho-axonic cell
Basket cell
Bistratified cell
0-LM
cell
Dentate gyrus
LAYER-SPECIFIC INPUT:
4
entorhid afferents
s.m.
commissurat/
associational
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S.Q.
hilus
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Axo-axonic cell Basket cell HICAP cell MOPP cell
+cotlaterals
Mossy fiber
HlPP cell
IAJTERNEURONSOF T H E HIPPOCAMPUS
FIGURE 17. Summary diagram of the morphological classification scheme. Interneuron types are identitied according to dendritic
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symmetrical synapses. Their nuclei display several deep infoldings. The soma membrane is also engaged in synaptic contacts, and axonal arborization patterns in the hippocampus (top) and in the dentate gyrus (bottom). Filled circles mark the cell body loca- including asymmetrical synapses, although at a much lower fretion. The cell bodies give rise to thick horizontal and/or vertical lines quency than the dendritic shafts (Frotscher et al., 1986). indicating the predominant orientation and laminar distribution of In the neocortex, ChAT-positive cells were found to contain the dendritic tree. The hatched boxes cover those laminae where the VIP (Eckenstein and Baughman, 1984; Chedotal et al., 1994). axon of each interneuron typically arborizes. The transverse exten- Because VIP is present in GABAergic bipolar cells (DeFelipe, sion of the axons or dendrites are not indicated. Principal cells in the background provide an idea of which membrane domains (so- 1993), acetylcholine and GABA could coexist in this cell type. matic, proximal, or distal dendritic regions) are innervated by the Evidence for the colocalization of ChAT and GABA in a small different interneuron types. The laminar distribution of different ex- number of cortical neurons has been provided (Kosaka et al., citatory aEerents (indicated on the right margin) oftenshows a per- 1988a). In the hippocampus, most of the ChAT-positive cells also fect match with that of the axon arbor of an interneuron type. show morphological characteristics of VIP-positive GABAergic interneurons (Section IV.3.d), but a direct demonstration of coexistence of ChAT with both GABA and VIP is yet to be shown, Whether acetylcholine is synthesized and released from axon tertratified, HICAP, HIPP, and MOPP cells) at the single-cell level minals of these neurons together with GABA remains uncertain. (Fig 10; Halasy and Somogyi, 1993b; Halasy et al., 1996). The lack of ChAT in the same cell types of the cat cerebral cortex implies that acetylcholine may not be vital for the normal opIV.1.b. Are there cholinergic interneurons in erations of these neurons.
hippocampus?
Interneurons immunoreactive for ChAT, the synthesizing enzyme of acetylcholine, have been reported in different areas of the rat cerebral cortex by several investigators (Eckenstein and Thoenen, 1983; k e y et al., 1984; Houser et al., 1985; Frotscher et al., 1986), although they may not exist in all mammalian species (Stichel and Singer, 1985). The staining intensity of these clearly nonpyramidal cell types was weaker than that of cholinergic septal or striatal neurons, but cells with the same morphology consistently occurred in specific locations from animal to animal. The most detailed description of ChAT-positive cells in the hippocampus has been provided by Frotscher et al. (1986). In this report, Frotscher et al. counted approximately 50 ChAT-positive cells in 63 Vibratome sections, which on average corresponds to fewer than 1 cell per section. Our recent findings (N. Hijos, K. T6th, Zs. Horvith, and T.F. Freund, unpublished observations) stemming from experiments using a more sensitive antiserum against ChAT (Cozzari et a]., 1990) show that these cells are much more frequently encountered. Up to 20 ChAT-positive cells were found in a single 60-pm-thick Vibratome section. Moreover, the morphology and location of ChAT-positive cells in this study agrees well with the description provided by Frotscher et al. (1 986). The largest number of cells occur in stratum lacunosummoleculare of the CAI subfield, but they are also found in strata radiatum and pyramidale of CAI and in strata granulosum and deep moleculare of the dentate gyrus. These cells are rarely present in the CA3 region, and they always have small cell bodies (average diameter about 10 pm), which are mostly fusiform or round. Those in stratum lacunosum-moleculare of CAI have two to three horizontal dendrites and at least one long radially oriented dendrite that extends up to the stratum pyrmidale. All dendritic branches are free of spines. ChAT-positive cells in other layers have primarily vertical dendritic trees similar to those in the neocortex. Electron microscopy provided further evidence that ChATimmunoreactive neurons in the hippocampus are interneurons. Their spine-free dendrites receive numerous asymmetrical and
IV.2. Calcium-Binding Proteins Are Present in Largely Nonoverlapping Subsets of GABAergic Interneurons
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spiny segments can also be detected, particularly in stratum radiarum of CA1 and CA3. The axon initial segment originates either from the soma or from a proximal primary dendrite and may be directed toward the apical or basal dendrites. The axons cannot be followed beyond the initial segment due to the thick myelin sheat that begins very close to the soma (30-50 pm). Axon terminal fields immunoreactive for PV also have a characteristic laminar distribution (Fig. 20). They are extremely dense in the cell body layers and proximal stratum oriens. In contrast, practically no PV-positive axon terminal staining can be detected in the dendritic layers fi.e., in stratum moleculare of the dentate gyrus and strata radiatum and lacunosum-moleculare of the CAI and CA3 regions). Although the majority of PV-positive varicosities surround the somata and axon initial segments of principal cells (Fig. 20), several boutons are also found to contact PV-positive cell bodies. This characteristic laminar distribution of PV-positive axon terminal fields overlaps with that of basket and chandelier cells, suggesting that one or both of these morphologically identified cell types selectively contain this calcium-binding protein Kawaguchi et al., 1987. Direct evidence for this notion has been provided by Katsumaru et al. (1988b), who demonstrated that the majority of PV-positive axonal varicosities formed symmetrical synapses with the somata, proximal dendrites, and axon initial segments of pyrathe apical dendritic layers is negligible (Kosaka et al., 1987; Aika midal neurons in the CA1 and C.43 regions (Fig. 20). Similar observations were made by Soriano et al. (1990) in the dentate gyrus. et al., 1994). An interesting observation in several PV-GABA colocalization In a recent in vivo study, intracellularly recorded and filled basket studies is the weak GABA immunoreactivity of PV-positive so- cells were shown to be immunoreactive for PV but not for calmata compared with other GABAergic cell bodies lacking this bindin or CR (Fig. 21; Sik et al., 1995). calcium-binding protein (Kosaka et al., 1987; Katsumaru et al., Thus, taken together, all PV-positive neurons can be classified 1988b; GulyAs et al., 1991b; Aika et al., 1994). The reason for as basket or chandelier cells, but the question arises as to whether this is still unknown. It may be due to high spontaneous firing all basket and chandelier cells contain PV. The answer is no. Ribak and an extensive release of transmitter, which requires a rapid et al. (1990) demonstrated that PV-positive boutons do not actransport of the synthesizing enzyme to the terminals and allows count for all symmetrical synapses on the perisomatic and axon little transport of GABA back to the soma. initial segment membrane of granule and pyramidal cells. Recent The dendrites of PV-positive cells span all layers and are ori- light and electron microscopic studies of neuropeptide-containented largely in the radial direction parallel to the principal cell ing cells (Nunzi et al., 1985; Acsidy et al., 1996b) and their colodendrites (Kosaka et al., 1987; Sloviter, 1989; Celio, 1990; Nitsch calization with calcium-binding proteins (Gulyh et al., 1991b; et al., 1990b; GulyAs et al., 1991b). The general appearance of Acsidy et al., 1996a) have provided further evidence for the exthe dendritic tree of most cells is bitufted, with frequent branch- istence of basket cells that do not contain PV. This different subing close to the soma, and a few additional branches at more dis- set of basket cells are visualized with immunostaining for CCK tal segments (Fig. 20). In the hippocampus, dendrites become rel- and VIP (Sections IV.3.c, IV.3.d). atively sparse in stratum lacunosum-moleculare, although a small The ultrastructural features of PV-positive somata and dennumber of cells still emit weakly stained branches in this layer. drites have also been studied by several laboratories (Kosaka et al., They have a rather even density in stratum moleculare of the den- 1987; Katsumaru et al., 1988b; Sik et al., 1993), and the results tate gyrus, and most of the branches terminate near the hip- agree with those obtained for identified basket and chandelier cells pocampal fissure or the pial surface. The dentate hilus and stra- in single cell labeling studies (Sections 111.1, 111.2.) and will not tum oriens of the CAI and CA3 regions also contain a small be repeated here. One feature that has not been emphasized in number of multipolar PV-positive neurons. They have oblique Section 111 is the occurrence of gap junctions between PV-posidendrites that may not reach the layers containing the most dis- tive dendrites andlor somata and between PV-positive dendrites tal dendrites of principal cells. However, PV-positive neurons hav- and shafts or spines of unknown origin (Katsumaru et al., 1988a). ing a horizontal dendritic tree confined to stratum oriens or the The overall frequency and selectivity of gap junctions in this neuhilus have not been observed. The majority of PV-immunoreac- rochemically characterized group of GABAergic neurons remains tive dendrites are varicose, especially regions near more distal seg- to be established (see Section III.5.a for CR-containing neurons ments. Large dendritic varicosities are often a sign of accumu- with dendrodendritic junctions), but in general, this membrane lation of numerous mitochondria, which may suggest a high meta- specialization appears to be common among interneurons bolic rate. They are generally spine free, but occasional sparsely (Kosaka, 1983b,c; Kosaka and Hama, 1985).
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SR
so
FIGURE 19. Demonstration of GABA immunoreactivity in a Golgi-impregnated axo-axonic (chandelier) cell in the cat hippocampus. A Low power light micrograph of the axo-axonic cell showing numerous vertically running rows of boutons (arrows),each wrapping around the axon initial segment of a pyramidal cell. B: Camera lucida drawing of the same cell, with the axon confined to stratum pyramidale and proximal oriens and a radially oriented dendritic tree spanning all layers. C: High power light micrograph of the cell body (open arrow) as seen in the 80-pm-thick Golgi section. D,E: Serial semithin (1 pm) sections cut from the soma of the
Golgi-impregnated axo-axonic cell, one immunostained for GABA (D) and the other with an anti-GABA serum preadsorbed with GABA (E). A small capillary (c) and a GABA-positive but not Golgi-impregnated soma (s) serve as landmarks to identify the axo-axonic cell in each figure. The axo-axonic cell (arrow) is positive for GABA in D; in the control section (E), only the silver deposit deriving from Golgi impregnation is visible as an outline of the cytoplasm. Pyramidal cells (P) are GABA-negative and indicate background level. Scale bars = 50 pm in A, 100 p m in B, 10 p m in C-E.
The major excitatory synaptic inputs of PV-containing neurons can be predicted from their dendritic distribution, and from earlier tracing studies of morphologically identified basket and axo-axonic cells (Section 111). Intracellular labeling studies have
visualized several chandelier cells with a dendritic tuft in stratum lacunosum-moleculare, whereas basket cells described in the same studies had shorter dendrites in this layer (Li et al., 1992; Gulyb et al., 1993a; Han et al., 1993; Buhl et al., 1994a). This finding
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rich and cell-poor segments of stratum radiatum alternate often in an unpredictable fashion, making quantification of these cells very difficult. Their dendrites and somata always stain stronger than those of pyramidal cells and can therefore be distinguished even if they are located in stratum pyramidale (Fig. 20). Their dendrites can be followed for a considerable length. The reconstruction of even partially stained dendritic trees reveals diverse morphologies. Dendrites may originate in any direction but often turn radial at some point; thus, the predominant dendritic trajectory remains radial. Exceptions to this rule are found in strata oriens and lacunosum-moleculare, where CB-positive neurons often have a horizontally oriented dendritic tree. Dendrites of the former type may extend for more than a millimeter without leaving a narrow zone at the oriens-alveus border. The majority of these cells contain the neuropeptide somatostatin (Section IV.3.a). The horizontal cells at the stratum radiatum-lacunosum-moleculare border often have a considerable proportion of their dendritic tree in stratum lacunosum-moleculare. In contrast, dendrites of radially oriented bitufted cells in stratum radiatum appear to avoid this layer. Their ascending branches course horizontally at the stratum lacunosum-moleculare border and may run in this direction for a considerable distance without penetrating the perforant path termination zone (Section III.3.2.b). Axons of CB-positive interneurons are difficult to visualize. In those few animals with successful axonal staining, the arbors were found in the dendritic layers of pyramidal cells, most notably in stratum radiatum, but also in strata oriens and lacunosum-moleculare (Figs. 20, 26; Gulyis and Freund, in press). Axons frequently crossed the stratum pyramidale without giving rise to varicose collaterals in this layer (but see the CA3 subfield below). The axonal branches in strata radiatum and oriens were evenly covered with en passant varicosities; drumsticklike terminals, however, were rarely observed. CB-positive axons were rarely seen in stratum lacunosum-moleculare. CB-positive neurons are more numerous and more easily visualized in the CA3 subfieId than in the CA1 region. The dendritic orientation here is truly multipolar, and most cells have a stellatelike appearance in all layers. The largest number of CBpositive somata is found in stratum radiatum of CA3a-b, but cells are also common in stratum oriens. They gradually diminish in number in CA3c and in the hilus of the dentate gyrus. Only short axonal segments can be reconstructed in this subfield, but those were highly varicose and arborized in stratum radiatum. Large neurons with triangular somata had axon initial segments that became myelinated close to the soma and were seen to enter the white matter. These cells are likely to be among those projecting to the medial septum (T6th and Freund, 1992). The dentate gyrus contains the smallest number of CB-positive interneurons, and even those few cells are barely visible due to the strong immunoreactivity of granule cells. In stratum moleculare, their number varies from 3 to 20 per single coronal section (60 p m , dorsal hippocampus), and their major dendritic orientation may be either horizontal or vertical. Interneurons in stratum granulosum cannot be distinguished, whereas those in the hilus number 3-10 per section. Axons of CB-positive interneurons in the dentate gyrus have not yet been reconstructed. Electron microscopy of CB-positive axon terminals, originat-
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the CA3c stratum oriens-hilus border and a bistratified cell in the CAI region, both of which arborize in strata radiatum and oriens of the same subfields, have been shown to contain CB (Fig. 21; Sik et al., 1995, submitted). Horizontal 0-LM cells, the trilaminar cells and the back-projection neurons in the CA1 region were found to be immunonegative for CB (Sik et al., 1995). Importantly, however, such negative findings, especially when immunostaining of intracellularly injected cells is attempted, should be taken with great caution. Basket and chandelier cells are clearly not among the CB-positive neurons because of the complementary laminar distribution of axon arbors (perisomatic vs. dendritic innervation of pyramidal cells).
IV.2.c. CR-containing interneurons form spiny and spine-free subpopulations with distinct connectivities
Immunostaining for CR reveals a large number of interneurons in all layers and subfields of the hippocampus and dentate gyrus (Figs. 23, 26; Jacobowitz and Winsky, 1991; Gulyis et al., 1992; Miettinen et al., 1992; Resibois and Rogers, 1992; Rogers, 1992). Two major types can be distinguished on the basis ofdendritic morphology and distribution.
ing from somata in stratum radiatum, has been done in combination with postembedding immunogold staining for GABA (Fig. 20). The axon terminals in stratum radiatum establish symmetrical synapses and are all GABA positive. Their postsynaptic elements, which include dendritic shafts and spines, are negative for GABA. This result confirms that CB-positive interneurons are involved in the GABAergic innervation of pyramidal cell dendrites in strata radiatum and oriens. The ultrastructural data on somata and dendrites of CB-positive neurons is rather limited (Danos et al., 1991; Gulyis and Freund, in press). Their indented nuclei and asymmetrical synaptic input on somata are characteristic of interneurons, but the relatively sparse afferent synapses along the dendritic shafts is a feature that distinguishes them from other interneurons. According to the dendritic and z o n a l distribution and location, CB-positive neurons correspond to cell types described in Section 111.3.2 as those innervating pyramidal cell dendrites. They appear to include the bistratified cells, other interneurons with dendrites and axons arborizing in stratum radiatum (Sections 1II.3.2.b, II1.3.2.c), and the horizontal cells located at the border (ofstrata radiatum and lacunosum-moleculare (Section III.3.2.d). 4n intracellularly injected trilaminar or bistratified cell located at
Spiny CR-immunoreactive neurons. The spiny variety is present mostly in regions where mossy fibers have a high density, i.e., in the hilus of the dentate gyms and in stratum lucidum of the CA3 subfield. Their dendrites and frequently the somata possess numerous long hairlike spines that penetrate into bundles of mossy fibers. These thin preterminai mossy fibers establish numerous (3-6) synaptic contacts on these dendrites. In the dentate gyrus, the dendrites are restricted to the hilus and never enter stratum granulosum. In CA3 stratum lucidum, the dendrites run parallel with the pyramidal cell layer, precisely follow its curve, and rarely enter strata pyramidale or radiatum. The axon of these neurons cannot be visualized by immunostaining, probably because of myelination. However, neurons of this type located in stratum lucidum of CA3 have been intracellularly injected with biocytin in vitro. Their axons arborized in stratum lacunosum-moleculare of the CA3 region, and a large arbor also formed in the outer molecular layer of the dentate gyms (C. McBain, personal communication). In another unpublished study, spiny cells with the same location and dendritic morphology sent axon collaterals to stratum radiatum (M. Frotscher, personal communication). These cells apparently form symmetrical synapses with presumed pyramidal cell dendrites; however, the types of synaptic contacts are difficult to identify in sections from the surface of immersionfixed slices due to poor ultrastructural preservation (M. Frotscher, personal communication). Thus, these neurons are likely to be among the interneurons that innervate principal cell dendrites but have the distinguishing feature in that they receive a predominant mossy fiber input (also Section III.3.1.e). It is interesting to note here that pyramidal cells in CA3c of the ventral hippocampus show transitional features of mossy cells and CA3 pyramidal cells in that ventral CA3c pyramids may project to the inner molecular layer of the dentate gyrus and to the CA1 region (Li et al.,
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INTERNEURONS OF THE H I P P O C M P U S
Neurochemical identification of intracellularly recorded and biocytin-filled interneurons. A: Basket cells are PV immunoreactive and arborize mostly in the pyramidal cell layer. Photomicrograph shows the biocytin-labeledbasket cell and the fluorescent immunostaining for PV in the same section. B: Trilaminar interneuron. Reconstruction of the dendritic arbor and partial reconstruction of the axon collaterals (from 7 of 53 60-pm-thick sections, centered at the cell body). Photographs depict the CB immunoreactivity of the biocytin-filled cell, as in A. C: Hilar interneuron with axon terminals associated with the perforant path (HIPP cell). Reconstruction of the dendritic arbor and partial reconstruction of the axon collaterals (from 7 of 39 80-pm-thick sections, centered at the cell body). The dendrites remained in the hilus, and the axon collaterals densely innervated the outer molecular layer of dentate gyms. The light microscopic pictures show the fluorescent NPY immunoreactivity (FITC) and biocytin-labelingof the cell body in the same section. Arrows in the photographs point to the biocytin-filled cell bodies. p, pyramidal layer; r, stratum radiatum; Im, stratum lacunosum-moleculare; g, granule cell layer; f, hippocampal fissure: h, hilus; m, molecular layer. Data are from Sik et al., 1996, submitted.
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FIGURE 21.
1994). Spiny CR cells may represent a similar transitional cell type showing characteristics of HIPP cells of the dentate hilus and of 0 - L M cells of the CA3 region, in which they innervate principal cell dendrites in the entorhinal termination zone in CA3 and in the dentate gyrus. However, inhibition produced by this cell type in CA3 stratum lacunosum-moleculare is of the feed-forward type, which is in contrast with its effect in the dentate gyrus or with the inhibition likely exerted by conventional 0 - L M and HIPP cells. The GABAergic nature of the spiny CR-positive cells remains controversial. Neurons of this type, visualized by either CR immunostaining or Golgi impregnation, are negative for GABA or stain close to background level (Miettinen et al., 1992; Soriano and Frotscher, 1993b). However, negative somatic staining for GABA does not necessarily mean that the neuron is not GABAergic (see Section IV.2.b). For example, although some earlier immunostaining studies have suggested that a large proportion of the SOM-positive hilar cells are negative for GABA or GAD, all hilar SOM-synthesizing neurons were shown by in situ hybridization to colocalize GAD65 mRNA (Esclapez and Houser, 1995). Spiny CR cells may have an extensive projection, which would explain the low levels of GABA in the soma. A small number of spiny CR-positive neurons in the hilus have been found to project commissurally, and a single cell has been retrogradely labeled from the medial septum (Miettinen et d., 1992). Glutamate immunoreactivity has been observed in the soma of a Golgi-impregnated cell of this type (Soriano and Frotscher, 1993b), but whether it should be considered as glutamate dedicated to transmitter or merely as a reflection of the low level of GAD activity in the soma (which would convert all glutarnate into GABA) is unknown. Nevertheless, the symmetrical synapses they appear to form on dendritic shafts in stratum radiatum may argue against the glutamatergic nature of these neurons (see above).
Spine-fee CR-immunoreactive neurons. The majority of these neurons belong to the class of cells described as interneurons specialized to innervate other interneurons (Section III.5.a). The CR-positive axons with a clustered distribution of varicosities
(both en passant and drumsticklike) in stratum radiatum and axons forming a horizontal plexus at the stratum oriens-alveus border are those shown to selectively innervate other interneurons (Gulyis et al., 1996). These axons originate from spine-free CRpositive cells with primarily radial (bipolar or bitufted) dendritic trees, several of which form multiple dendrodendritic junctions with other CR-positive cells of the same type, as described in Section 111.5. Additional CR-immunoreactive interneurons not included among those described in Section III.5.a are horizontal neurons located near the hippocampal fissure. The soma of these neurons are located on either side of the fissure. Their axons could not be visualized. According to developmental studies of calcium-binding-protein-containing cells, these neurons appear to correspond to Cajal-Retzius cells (Soriano et al., 1994). Main axon trunks and fine varicose fibers with a predominant horizontal orientation and unidentified origin are also found in stratum lacunosum-moleculare. Electron microscopy showed that most of the varicosities along these axons establish asymmetrical synapses with GABA-negative dendrites or spines, and they themselves were also GABA negative. These findings suggest that most of the CR-positive axon terminals in stratum lacunosum-rnoleculare have an extrahippocampal origin, probably from the nucleus reuniens. This thalamic nucleus was shown to send a projection specifically to this layer (Wouterlood et al., 1990) and has CR-irnmunoreactive neurons in large numbers (Jacobowitz and Winsky, 1991).
IV.3. Neuropeptides Are Contained in Subsets of GABAergic Interneurons With Distinct Connectivities IV.3.a. SOM-immunoreactive neurons
Prosomatostatin is a 28-amino-acid-long peptide that is cleaved to produce a leader peptide (SOMI-12) and the biologically active peptide (SOM14-28). Antisera raised against either of these peptides have been used in immunocytochemical studies and were found to visualize similar cell populations. We will not distinguish between data obtained by antisera directed against one or the other peptides in the present review. Immunostaining for SOM visualizes a large number of neurons in all subfields of the hippocampus, with characteristic laminar distribution (Figs. 24, 26; Kohler and Chan-Palay, 1982; Morrison et al., 1982; Johansson et al., 1984; Roberts et al., 1984; Somogyi et al., 1984; Bakst et al., 1986; Kohler et al., 1987; Sloviter and Nilaver, 1987; Kosaka et al., 1988b; Kunkel and Schwartzkroin, 1988; Milner and Bacon, 1989b; Leranth et al., 1990; Nitsch et al., 1990a; Finsen et al., 1992; Buckmaster et al., 1994). All SOM-positive neurons have been characterized as interneurons on the basis of morphological features, frequency of occurrence, and distribution. Colocalization studies have demonstrated that practically all SOM-positive neurons in the hippocampus and dentate gyrus are immunoreactive, or contain the mRNA, for GAD (Somogyi et al., 1984; Kosaka et al., 1988b; Esclapez and Houser, 1995). According to Kosaka et al. (1988b), SOM-positive cells account
FIGURE 22. Photomicrographs of fluorescent confocal images of sections double-immunostained for calretinin (A,C,E) and NO synthase (B,D,F) in the C A I region of the rat hippocampus. The same areas are shown in pairs of photographs in A-B, C-D, and
E-F. There are numerous double-labeled cells (arrows), but neurons immunoreactive for NO synthase (arrowheads) or CR (open arrows) alone are also frequently seen. This figure was kindly prepared by Toshio Kosaka. Scale bars = 100 p m in A,B, 20 p m in C-F.
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to run parallel to the granule cell layer. Those cells located deeper in the hilus may have multipolar dendritic trees. The spiny or spine-free nature of dendrites is impossible to establish from SOM immunostaining alone. However, when visualized by immunostaining for SP or mGluRl receptors, all probable SOM-containing dendrites are covered by numerous long spines (Sections IV.6.a, IV.6.f). In summary, the dendritic tree of SOM-positive neurons in the dentate gyms is typically restricted to the hilus and is covered by numerous long spines. The density of axon terminal staining differs considerably from animal to animal and with the type of antisera and staining procedure used. However, the laminar distribution of SOM-positive axons is consistent. There is a terminal plexus in the outer twothirds of the molecular layer, the outer one-third being more dense than the middle one-third. The inner molecular layer and the granule cell layer have a small number of radially running axons, which are mostly nonvaricose. Main axon collaterals are often seen to cross the hippocampal fissure. Varicose collaterals in the hilus are sparse but occur consistently. The majority of axon collater-
Parvalbumin
Calretinin
Calbindin
NADPH-diaphorase
/.--~----.-
+ .
.. .
.
FIGURE 23. Camera lucida drawings illustrate the distribution aphorase, to visualize neuronal NO-synthase-containingcells. Each of neurons in the hippocampus that contain one of the calcium- dot represents one cell. The relative distribution and numerical denbinding proteins or NO synthase. Drawings represent single 60-pm- sity of PV-, CB-, CR-, and NADPH-diaphorase-positive neurons can
thick Vibratome sections, cut from approximately the same anteroposterior level of the hippocampus,immunostainedfor the different calcium-bindingproteins or histochemicallystained for NADPH-di-
l be compared in a l subfields and layers. Straight dashed lines indicate the CA1-CA2 border.
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VIP
Somatostatin
CCK
NPY
FIGURE 24. Camera lucida drawings illustrate the distribution of neuropeptide containing neurons. Single 60-pm-thick Vibratome sections cut from approximately the same anteroposterior level of the hippocampus as that shown in Figure 23 were immunostained
for different neuropeptides. Same conventions as in Figure 23. The relative distribution and numerical density of VIP-, CCK-, SOM-, and NPY-immunoreactive neurons can be compared in all subfields and layers.
als in the molecular layer originate from ipsilateral hilar neurons, as demonstrated by the disappearance of most SOM-fiber labeling from this layer after hilar kainate injections (Bakst et al., 1986). Hilar SOM-positive neurons can be retrogradely labeled by tracer injections into the contralateral dentate gyrus, suggesting that a smaller proportion of axons originates from the contralateral hilus (Bakst et al., 1986; Deller et al., 199Sb). Finally collaterals may also derive from ipsilateral CA1 neurons because axons passing through the hippocampal fissure are also found (Bakst et al., 1986). Electron microscopy of SOM-immunoreactive axon terminals show that the majority of postsynaptic elements in the outer molecular layer are spines and spiny dendritic shafts, most likely belonging to granule cells (Leranth et al., 1990). The synapses are always symmetrical and are accompanied by an unstained asymmetrical synapse when terminating on a spine. Occasional synapses are formed on deep hilar neurons and on granule cell bodies. At the electron microscopic level, SOM-positive dendrites were found to be spiny and received numerous asymmetrical synapses from mossy fiber terminals both along the shaft and onto
the spines (Leranth and Frotscher, 1987). The cell body and somatic spines were also contacted by mossy terminals, although at a lower frequency. Only a negligible number of dendrites penetrate the stratum moleculare, where they were shown to receive input from the entorhinal cortex (Leranth et al., 1990). The cell bodies showed typical features of interneurons, i.e., invaginated nuclei, asymmetric synaptic input, intranuclear rods, and numerous cytoplasmic organelles. The cytoplasm, however, was not always as abundant as in some other types of interneurons (C;ulyis et al., 1990; Leranth et al., 1990). According to the characteristic soma location and axonal and dendritic distribution, hilar SOM-positive cells are likely to be identical to HIPP cells of the morphological classification scheme (Section III.3.1.a). The correlation of morphology and neurochemical character for this cell type is perhaps the most straightforward of all such attempts; thus, SOM cells and HIPP cells in the dentate gyrus can be considered as synonymous.
FIGURE 25. Light micrographs show the colocalization in the rat DG of GluR subunits and calcium-binding proteins or SOM. By applying the mirror technique with paired surfaces of adjacent Vibratome sections, the colocalization of GluR2/3 (al) with PV (az), GluR2/3 (b,) with CB (bz), and GluR4 (c1) with SOM (cz) is demonstrated. Immunoreactivity for GluR2/3 is associated with granule cells and neurons (most likely mossy cells) in the deep hilus. None of the PV-positive and only a small number of hilar CB-positive cells (probably misplaced granule cells, numbered 1-3 in bl and bz), contain GluFW3 immunoreactivity. Large GluRW3-positivecells (probably mossy cells) in bl (long arrows) are CB-negative, as shown in
the adjacent section. The silhouettes of the negative somata are visible in bz. Immunostaining for the GluR4 subunit in the dentate gyrus w s limited to hilar neurons, with occasional cells stained in a stratum granulosum. AU SOM-immunoreactive cells in the hilus (numbered arrowheads in c2) were also immunoreactive for GluR4 (arrowheads in c1). From Leranth C, Szeidemannz, Hsu M, Bum& G (1996) AMPA receptors in the rat and primate hippocampus: a possible absence of Glur 213 subunits in most interneurons. Neuroscience 70:631-652 by permission of Elsevier Science, Ltd. This figure was kindly prepared by Csaba Leranth. Scale bars = 50 Pn.
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FREUND AND S U Z S k I
similarly straightforward for most of the SOM-containing cells, as in the dentate gyrus. In the CA1 region SOM cells correspond to the horizontal 0-LM cells, whereas in CA3 SOM cells correspond to the LM-projecting cells with multipolar dendritic tree. This correspondence was confirmed by the identical laminar distribution of dendritic and axonal arbors (Section III.3.2.a). The vast majority (87%) of calbindin-containing neurons located at the stratum oriens-alveus border also contain SOM; this corresponds to 32% of SOM cells in stratum oriens (Katona et al., 1996). This observation suggests that at least some of these SOMpositive cells project to the medial septum (T6th and Freund, 1992; T6th et al., 1993; Section VI.2).
number of cells in stratum pyramidale, and practically none in strata radiatum and lacunosum-moleculare (Fig. 20). They are more widespread in the CA3 region and frequently occur in strata pyramidale, lucidum, and proximal radiatum. Some cells can be found embedded in the myelinated axon bundles of the alveus in both regions but especially in CAI. As in the dentate hilus, SOMimmunostaining alone visualizes only the somata and the most proximal dendrites. However, SOM-positive cells are immunoreactive for the metabotropic glutamate receptor mGluRl, which stains their dendritic tree in great detail (Baude et al., 1993; BIasco-Ibanez and Freund, 1995). The dendrites of SOM-positive neurons in the CA1 region run horizontally and are restricted to a narrow zone at the border of stratum oriens and the alveus. In the CA3 subfield, SOM-positive cells have a multipolar dendritic tree that penetrates all layers except stratum lacunosummoleculare. Thus, there is a remarkable correlation between the laminar distribution of SOM cell dendrites and the local axon collaterals of principal cells both in the hippocampus and in the dentate gyrus. Pyramidal cells in the CAI region have horizontally running local collaterals, which distribute to the border between stratum oriens and alveus, whereas collaterals of CA3 pyramidal cells arborize extensively throughout strata oriens, radiatum, and pyramidale (Section 11). Local collaterals of granule cells in the dentate gyrus (i.e., mossy fiber collaterals) in normal animals are known to arborize predominantly in the hilar region in a manner similar to the dendrites of SOM-positive neurons. These data predict that the major excitatory input of SOM cells will derive from local principal cells, i.e., they are likely to mediate feedback inhibition (Section X. 1). Direct evidence for this hypothesis has been observed in the CA1 region, where degenerating boutons of pyramidal neurons selectively damaged by ischemia were shown to account for more than 80% of all asymmetrical synaptic input along the dendrites of SOM cells in stratum oriens (Blasco-Ibanez and Freund, 1995). As in the dentate gyrus, the laminar distribution of SOM-positive axon terminal fields shows a perfect overlap with the termination zone of entorhinal afferents, densely covering stratum lacunosum-moleculare in both CAl and CA3 (Fig. 20). The axons are highly varicose in this layer, whereas only sparse nonvaricose main axons are found in stratum radiatum. Occasionally, beaded collaterals are observed in stratum oriens. Electron microscopic data on SOM-immunoreactive neurons in the hippocampus are scarce (Kunkel and Schwartzkroin, 1988; Gulyas et al., 1990; Danos et a]., 1991; Baude et al., 1993; BlascoIbanez and Freund, 1995). The available data suggest that SOM cells have no distinguishing ultrastructural features as compared with other interneurons. Their dendrites receive both asymmetrical and GABAergic symmetrical synapses, most of the former originating from CA1 pyramidal cell collaterals and the latter from VIP-containing interneurons (Section III.5.c and below). SOMpositive axon terminals in stratum lacunosum-moleculare make symmetrical synapses, largely with spines and spiny dendritic shafts of presumed pyramidal cells, in conjunction with asymmetrical synapses derived from the entorhinal cortex (Fig. 20; Sik et al., 1995; Katona et a]., 1996). The correlation with morphologically identified cell types is
395
rise to the hilar NPY terminals. Perhaps these neurons represent a basket cell type with a predominant innervation of hilar mossy cells (see Section 1V.3.c) because NPY-positive terminal density in the hilus is higher than in the granule cell layer (but see Section V.4 about NPY-containing noradrenergic afferents).
396
and radiatum of both the CA1 and CA3 regions (Sik et al., 1995) and could be a source of NPY-positive axon arbors in these layers.
Hippocampus. The distribution of CCK-immunoreactive neurons in the CA1 and CA3 regions is complementary to that of SOM- and NPY-positive cells. They occur in large numbers in stratum radiatum, less frequently in strata pyramidale and oriens, and only seldom in stratum lacunosum-moleculare. Most of the CCK-positive neurons, regardless of the laminar position, have a bitufted dendritic tree with a predominant radial orientation. Several of them have a pyramidal-shaped soma in stratum pyramidale, with an apical dendrite extending through stratum radiatum. Many others in stratum radiatum show an inverted pyramidal morpholgy, in which a major dendritic trunk runs toward stratum pyramidale, and a tuft of dendrites emerge in the opposite direction. Multipolar cells with no obvious orientation are frequently observed in strata radiatum and lucidum of the CA3 region. Axon terminal fields of CCK-positive neurons are largely limited to strata pyramidale and proximal radiatum in both the CA1 and CA3 regions, where they surround the somata and proximal dendrites of pyramidal cells. Apart from a few radially running collaterals in stratum radiatum, most of them having no varicosities, the other layers contain only negligible numbers of CCK-positive axon terminals. Thus, apparently all CCK-positive cells, whether located distantly near the stratum radiatum-lacunosum-moleculare border or within stratum pyramidale send their axon collaterals into stratum pyramidale to form perisomatic arrays of boutons that primarily surround pyramidal cell somata. Electron microscopy of cell bodies and dendrites revealed no unique ultrastructural features that might distinguish CCK-positive cells from other types of interneurons in the hippocampus. CCK-positive axon terminals make symmetrical synapses with pyramidal cell bodies and occasionally with somata and dendrites of other interneurons (e.g., other CCK-positive cells), as shown in the rat and cat hippocampus (Harris et al., 1985; Nunzi et al.,
Dentate g m The majority of CCK-positive neurons in the y . dentate gyrus are located within or just below the row of granule cell bodies bordering the hilus. They have a pyramidal-shaped soma, a prominent apical dendrite running toward the pial surface, and basal dendrites penetrating the hilus. The apical dendrites often branch when they reach the stratum moleculare but still remain radial in orientation. CCK-positive neurons are extremely rare in stratum moleculare and the deep hilar region. However, cells with a pyramidal-like or bitufted morphology can often be found in the subgranular polymorphic zone. Some cells in this region may have an initially horizontal orientation of the main dendrites, but they eventually turn at a right angle and penetrate strata granulosum and moleculare. Independent of their location, practically all CCK-positive neurons in the dentate gyrus have a bitufted dendritic tree, i.e., one tuft of dendrites (apical) in stratum moleculare, and another (basal) in the hilus, thus allowing them to receive inputs both from the perforant path and from the local mossy fiber collaterals. This neurochemically characterized cell type also has a characteristic distribution of axon terminals. There is a dense band of terminals in the upper one-third of the granule cell layer and the adjacent narrow (20-50 p m thick) zone of the molecular layer. Lower parts of the granule cell layer and the polymorphic zone are relatively poor in CCK-positive varicosities. The deep hilus also contains a larger number of terminals, most of which surround large unstained somata. With the exception of the narrow proximal zone, the molecular layer contains practically no CCK-immunoreactive axon terminals. Electron microscopy of CCK-containing cell bodies and dendrites revealed no unique ultrastructural features that would distinguish them from other interneuron types. Their dendrites in the hilus receive numerous synapses from mossy fiber terminals, whereas those in stratum moleculare are contacted both by asymmetric and symmetric synapses (Leranth and Frotscher, 1986). CCK-positive boutons in stratum granulosum and in the adja-
1985).
According to the known axon termination site for CCK-positive terminals, all CCK-immunoreactive neurons can be identified as basket cells. This implies that basket cells are present also in distal stratum radiatum and in stratum oriens (for cat data, see Nunzi et al., 1985). Stratum radiatum contains no 1V-positive
397
Dentate g m . VIP-positive cells are present in all layers of the ys dentate gyrus, with a slightly higher frequency within or close to the granule cell layer. Smaller number of cells are found in the outer molecular layer and the deep hilus. The morphology and laminar distribution of dendrites are highly variable. The most frequent rype of VIP-positive cell has a bitufted dendritic tree, with a predominant radial orientation originating from somata in the inner molecular layer. These neurons always have a prominent upper dendritic tuft that extends toward the pial surface; the lower tuft, however, is sometimes missing or is limited to a single or a few long slender branches. Cells within or just below the granule cell layer may have similar morphologies or more often have a pyramidal-shaped soma with a rich basal dendritic arbor and a sparse apical dendritic tuft. In the deep hilus, a small number of cells are found with multipolar dendrites that may be very long and often cross stratum granulosum to enter the molecular layer. The largest number of VIP-positive axon terminals is found in the hilus. Their density is higher in the ventral than in the dorsl hippocampus. The size of the boutons is relatively small, and a they are diffusely distributed in the neuropil without an obvious association with cell bodies. Another type of VIP-positive axon is observed in stratum granulosum; it is studded with large varicosities and appears in clusters of varying diameters (100-200 pin). There are often long segments of granule cell layer without this type of axon arbor, but a cluster is always present at the apex of the dentate gyrus. A smaller number of VIP-positive varicose collaterals are observed in the molecular layer, where they often contact the somata or proximal dendrites of VIP-positive or other interneuron types (Hijos et al., 1996). Reconstruction of VIP-
positive neurons shows that the hilar terminal fields originate from bitufted cells located in the inner molecular layer. These cells have a main descending axon. Some VIP cells with soma and dendrites in the hilus also appear to contribute to the hilar axon collaterals. Several neurons that give rise to the clusters of large varicosities in stratum granulosum may have a soma-dendritic morphology and location similar to the cells with hilar projection. The ultrastructure of cell bodies and dendrites of VIP-positive neurons is similar to that of most other interneuron types. The axon terminals immunoreactive for VIP always make symmetrical synapses and are positive for GABA. The postsynaptic targets of the large varicose axon type are granule cell bodies and dendrites in stratum granulosum (GABA-immunonegative profiles). The neurons that project to the hilus innervate mostly GABAergic interneurons, as was described in Section 111.5. The majority of targets of VIP-positive cells that project to stratum moleculare also appear to be other interneurons, although only short axon segments have been visualized in this layer. For VIP-positive neurons, there is a clear correlation between input-output features and neurochemical characteristics in the dentate gyrus. The basket type of VIP cells contains CCK but not CR, whereas those projecting to the hilus are immunoreactive for CR but not for CCK (Hijos et al., 1996). The VIP-positive cells that project to the hilus have a target selectivity (Section III.5.c) complementary to that of some CCK-containing basket cells. Hilar collaterals of these CCK-positive basket cells predominantly innervate the mossy cells (Leranth and Frotscher, 1986; L. Acsidy and T.F. Freund, unublished observations), whereas the VIP-CR-positive cells with hilar projection innervate mostly GABAergic interneurons (specifically cells immunoreactive for S P R Section IV.6.0 in the same region. VIP-containing basket cells account for only a small proportion of CCK-positive basket cells in this region, and both the CCK- and the VIP-CCKcontaining neurons represent basket cell populations that show no overlap with the PV-containing neurons (Gulyis et al., 1991b; Acsidy et al., 1996a).
Hippocampus. VIP-positive neurons are present in all layers of the CA1 and CA3 subfields, with higher frequency in strata pyramidale, lacunosum-moleculare, and the bordering radiatum. The somata are relatively small and fusiform, and the predominant dendritic orientation is vertical. The dendritic trees of cells in strata pyramidale, radiatum, and oriens are mostly bipolar or bitufted, and the majority appear to span all layers. The ascending branches often form a profuse tuft upon entering stratum lacunosum-moleculare, but those of another type with a larger soma in stratum pyramidale branch proximally and do not reach stratum lacunosum-moleculare. VIP-positive neurons in strata lacunosum-moleculare or distal radiatum usually have only an upper tuft, which is confined to the stratum lacunosum-moleculare. Three major types of axon collaterals can be observed in the hippocampus, each originating from different VIP-positive neurons (Acsidy et al., 1996a,b). The first type innervates the horizontal 0-LM cells at the stratum oriens-alveus border, whereas the second type forms multiple contacts on interneurons in stratum radiatum (Section 111.5). Both of these types are known to
398
Hippocampus
LAYER-SPEC1FIC INPUT
entorhinal afferents
PV
CCK
CB
SOM "PY)
VIP
CR
Dentate gyrus
LAYER-SPECIFIC INPUT:
4
entorhinal
affwentS
s.m.
t
cornrnissural/ asscciational afferents
S.Q.
hilus
399
contain the calcium-binding protein CR and lack CCK. The third type gives rise to axon terminals in stratum pyramidale, which surround the cell bodies of pyramidal neurons. These cells contain CCK but not CR and have a proximally branching bitufted dendritic tree that does not reach stratum lacunosum-moleculare. Electron microscopy confirms that VIP-positive boutons in stratum pyramidale are positive for GABA and establish symmetrical synapses with somata and proximal dendrites of GABA-negative, presumed pyramidal cells (Acsidy et al., 1996a,b). Thus, VIP-positive neurons in the hippocampus are of three morphologically and neurochemically distinct types, one of which is a basket cell that also contains CCK but not PV. This observation confirms that there are at least two neurochemically different types of basket cells: one contains CCK and VIP, and the other contains the calcium-binding protein PV. The remaining two types of VIP-positive cells do not contact pyramidal cells. Instead, they selectively innervate well-defined classes of other interneurons, either at the stratum oriens-alveus border (0-LM cells) or in stratum radiatum (Section 111.5).
largely parallel with the pyramidal cell dendrites. Another group of ENK-positive interneurons is present at the border of strata radiatum and lacunosum-moleculare and have horizontal or oblique dendrites running primarily in stratum lacunosum-moleculare. Axonal staining is observed in stratum lacunosum-moleculare of the CAI and CA3 regions and in the outer one-third of the dentate molecular layer. The origin of the latter is likely to be the entorhinal cortex. The axons of local ENK-positive cells are difficult to visualize. They ramify primarily in stratum radiatum, where their boutons are arranged in clusters around nonprincipal cell bodies and dendrites. In a recent study in the guinea pig, hamster and rat, all ENK-positive neurons in the CA1 region were also shown to contain GABA and VIP. However, only a subset of VIP-positive cells (i.e., those innervating interneurons in stratum radiatum) was also imunoreactive for ENK U.M. BlascoIbanez, F.J. Martinez-Guijarro, and T.F. Freund, unpublished observations). Electron microscopic and postembedding immunogold staining confirm that ENK-positive boutons are GABAergic, and make symmetrical synaptic contacts primarily on GABA-positive dendrites or somata U.M. Blasco-Ibanez, F.J. MartinezGuijarro, and T.F. Freund, unpublished observations). Thus, we conclude that ENK-containing neurons represent a subset of VIP-positive cells, which selectively innervates other interneurons in strata radiatum and oriens and has a dendritic tree that either spans all layers or arborizes predominantly in stratum lacunosum-moleculare.
400
at the base
shaped somata are often found to be immunoreactive for NKB of the granule cell layer, and interneurons with short dendritic segments can also be visualized in strata pyramidale, radiatum, and lacunosum-moleculare of the CAI subfield. Faint NKB immunoreactivity is also present in mossy fibers, which can be upregulated by seizure activity (Marksteiner et al., 1992).
IV.4. Neuronal NO Synthase (or NADPHdiaphorase) Is Selectively Present in Interneurons in the Hippocampal Formation
N O represents a new class of neuronal messengers. It is a water- and lipid-soluble gas, which freely diffuses across cellular membranes and the extracellular space. It is likely to have both short- and long-term modulatory effects on neurons mostly by influencing cGMP formation (Garthwaite, 1991; Snyder, 1992). The neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) forms of N O synthase, the enzymes synthesizing NO from arginine, are present in the brain (Paakkari and Lindsberg, 1995). In the hippocampal formation, the endothelial form is localized in pyramidal cells (Dinerman et al., 1994), whereas nNOS immunoreactivity appears to be restricted to interneurons, which accounts for practically all staining obtained by NADPH-diaphorase enzyme histochemistry (Hope et al., 1991; Vincent and Kimura, 1992; Valtschanoff et al., 1993; Dun et al., 1994). All neurons showing NADPH-diaphorase reactivity were shown to be immunoreactive for GABA, thus providing direct evidence that they represent interneurons (Valtschanoff et al., 1993).
Dentate gyrus. Interneurons immunoreactive for nNOS (or staining for NADPH-diaphorase) in the dentate gyrus are most abundant in the hilus (Fig. 23). Numerous cells are also present in stratum granulosum, where they are concentrated near the hilar border (Valtschanoff et al., 1993). Cells in the deep hilus have generally large cell bodies and bitufted or multipolar dendritic trees, which are moderately spiny, and rarely enter the granule cell layer. Large hilar cells at the border of the granule cell layer display the typical morphological features of basket cells known from Golgi and immunocytochemical studies (CCK, PV). They have prominent apical dendrites crossing stratum moleculare and several basal dendrites in the hilus. Cells in stratum granulosum have smaller, fusiform, or pyramidal-shaped somata and a radially running apical dendrite, which forms a tuft that often reaches the fissure or the pial surface. Basal dendrites also emerge from the soma and run in the subgranular polymorph zone, emitting only a small number of branches. Multipolar or radially oriented bitufted cells are also present in stratum moleculare. Dendrites of the multipolar and radial bitufted cells are varicose and spine free. The staining intensity of small neurons is generally weaker than that of large cells. Axonal staining is remarkably dense in stratum moleculare, especially in the outer two-thirds, whereas the inner one-third displays a strong homogeneous background (fine granular) staining. Thick main axons originating from nNOS-positive cells in CA1 often cross the hippocampal fissure to terminate in the dentate hilus (Sik et al., 1994). The hilus contains a smaller number of stained axons. Collaterals in all layers are densely varicose.
Colocalization with other markers. In the dentate gyrus, the majority (95%) of hilar SOM-containing neurons were also shown to be immunoreactive for NOS. In the same region, only a negligible number (5%) of PV-positive cells contained NOS immunoreactivity (Dun et al., 1994; Divila et al., 1995). NOS-positive cells located at the base of the granule cell layer have pyramidal-shaped somata and prominent apical dendrites that penetrate stratum moleculare. These cells are never immunoreactive for SOM. Although they have morphological features typical of pyramidal basket cells, they lack PV. In the hippocampus, the correspondence between NOS-containing and other neurochemically defined cell groups is even less clear. The majority of SOM- and PV-positive cells in stratum oriens of the CA1 and CA3 regions appears to be negative for NOS (Dun et al., 1994; Divila et al., 1995). Because there are no data available on the coexistence of NOS with other neuropeptides and calcium-binding proteins, one can only speculate
401
association with various interneuron subpopulations) will be described. These data should not be taken as evidence for particular transmitter actions on those interneuron types because several examples of transmitter-receptor mismatch can be quoted from the literature, and there is no guarantee that those receptors are utilized under physiological conditions. Nevertheless, these immunocytochemical descriptions can be used in physiological and pharmacological studies to predict interactions between transmitters via given receptors and may also serve as a guide for using some of these receptor antisera as markers of specific interneuron dendrites for connectivity studies.
Metabotropicglutamate receptors. The mGluRl a subunit of the metabotropic glutamate receptors is particularly enriched in a well-defined type of GABAergic interneuron in the hippocampus. Several immunocytochemical studies show a striking staining of horizontally running dendrites at the stratum oriens-alveus border (Martin et al., 1792; Gorcs et al., 1993), but the types of immunoreactive structures remain unidentified. Subsequently, Baude et al. (1993) provided direct evidence that all mGluRlapositive neurons in this location were also immunoreactive for somatostatin, the marker of horizontal 0 - L M cells (Section III.3.2.a). The staining of the dendritic tree of mGluRla-positive cells is nearly complete and can be used to study the synaptic input of this particular cell type (Blasco-Ibanez and Freund, 1995), which has been impossible previously due to the lack of dendritic staining with SOM antisera. At the electron microscopic level, mGluRla receptors are localized perisynaptically around the edges of the postsynaptic density of asymmetrical (presumed glutamatergic) synapses (Baude et al., 1993). The ultrastructural lo-
402
calization of mGluR5 is similarly perisynaptic, but this receptor is present on both pyramidal and interneuron dendrites and dendritic spines (Lujan et al., 1996).
5HT-3 receptors. Both immunocytochemical and in situ hybridization data are available for the localization of 5HT-3 receptors in the hippocampal formation. The first description was provided by Tecott et al. (1993), who reported on the clistribution of 5HT-3 mRNA. In the hippocampus, labeling appears to be clearly confined to interneurons, which have a distribution complementary to that of 5HT-2-containing interneurons. 5HT3-positive cells are more frequent in the ventral than in the dorsal hippocampus, and they occur predominantly in strata lacunosum-moleculare and radiatum and less frequently in strata oriens or pyramidale. In the dentate gyrus, the predominant cell type is the interneuron at the granule cell layer-hilar border, which has a large, often pyramidal-shaped, soma. Immunohistochemical studies to date have been reported only in abstract form (Battenberg et al., 1994; Morales et al., 1994). They confirm the distribution data derived from in situ hybridization and provide evidence for the colocalization with GABA. Furthermore, 5HT-3 mRNA is not expressed in SOMimmunoreactive neurons in the hippocampus. This finding supports the idea of complementary distribution of 5HT-2 and 5HT3 receptors among interneurons because 5HT-2 receptors are likely to be localized in SOM-NPY-positive cells. O n the basis of cell distribution and morphology, the 5HT-3-containing interneurons may include the CB-, VIP-, CCK-, and CR-immunoreactive subpopulations. Double-labeling studies are re-quired to establish which of these cell types indeed express 5HT-2 or 5HT-3 receptors. The striking correlation of receptor localization with electrophysiological data is described in Section IX.5.
5-bydroxy-tryptamin-2 receptors (5HT-2). An antiserum against an N-terminal epitope of the 5HT-2 receptor has been developed
403
laminar distribution of those few m2-positive axon arbors, which could be traced for a reasonable distance in spite of the heavy myelination, the m2-positive horizontal cells at the stratum oriens-alveus border are likely to correspond to the horizontal trilaminar cells or back-projection neurons (Sections III.3.2.b, III.3.2.e). Nicotinic receptors. Hippocampal interneurons are preferentially labeled by imrnunostaining against the beta2 subunit of nicotinic receptors and by radiolabeled alpha-bungarotoxin, one of the selective ligands of nicotinic receptors (Freedman et al., 1993; Hill et al., 1993). The general distribution of interneurons expressing the receptor was similar in the two studies. They are numerous at the stratum oriens-alveus border and in strata radiatum, lucidum, and lacunosum-moleculare of the hippocampus. In the dentate gyrus, most labeled cells are found at the hilar and molecular layer borders of stratum granulosum and less frequently in the hilus. As expected from their location, most of these neurons are immunoreactive for GABA and constitute approximately one-fifth of all GABA-immunoreative neurons in the dentate gyrus (Freedman et al., 1993). Colocalization of alphabungarotoxin binding and different neuropeptides demonstrate that no single neurochemically identified subpopulation can account for the nicotinic receptor-positive neurons because CCK, SOM, and NPY are all present in some radiolabeled cells (Freedman et al., 1993).
W.6.f. SPRs
A specific antibody against SPR has been developed and has been reported to label interneurons in the cerebral cortex of the rat (Kaneko et al., 1994; Nakaya et al., 1994). In the hippocampus, immunostaining for SPR labels numerous morphologically heterogeneous interneurons in all subfields (Nakaya et al., 1994). Principal cells (granule, mossy, and pyramidal cells) are consistently devoid of SPR immunoreaction. However, pyramidal cells of the ventral subiculum show intense SPR immunostaining. The immunoprecipitate is membrane bound and outlines cell bodies and proximal and distal dendrites in a Golgi-like manner. In spite of the detailed visualization of dendritic morphology, the classification of SPR-positive interneurons is very difficult due to the lack of axonal staining. However, colocalization with neurochemical markers that label interneuron types with known affer-
TABLE 1.
CR
non-
VIP
basket
1%6
GABA
NOS
-9%6
spiny
NPY
13Yo2
spiny
-11yO3
22.4Y04
++47 10.5%' 10%~~
CB
14Y07 100%2O o%l4
PV
CCK
VIPICS
SOM
SPR
mGlu
M2
11.1~~3
S.1%16
0Yo 0%
+21
- <1 ~ ~ 4 7
1 1 ~ ~ ~
+ 1'
5.6%19
20
+ +46 +23
~
+ + +24
S%47
++29 0 ~ ~ 3 0
+22 +31
92%
+ +22 + + + / +25
100%~~
o w 4
0% 0%
0% 0%
SYP
7%
100~4
++34 ++35
0Y o 0%
0Yo 32Y03'
+33
+ + +33 +39
3%
100%~~
+ +44
3%
+45
GABA CR spiny12 CR non-spiny12 CB PV NOS CCK VIP-ICS2' VIP basket2' SOM NPY SPRZ3 mGlu
25%
6%
4%
+ +3'
3%
+ +34 + +40
100~~36 2%
+ +41
M2I1
20~~43
'Values in each row indicate what portion of neurons containing a given marker (indicated at the beginning of the row) are also immunoreactive for the markers indicated at the top of each column. The degree of overlap is given in percentages, where accurate data are available. However, in several cases, only a semiquantitative estimate can be provided, where 0% means no overlap; + minimal overlap; + +, medium overlap; + + +, strong overlap; 100%, total overlap. The symbol means estimated values from the ratio of coexistence with other markers. If the percentage of overlap considerably differs among hippocampal subfileds, this is indicated in the notes. 'Miettinen et al. (1992). 3Estimated from CB/PV ratio based on Gulyas et al. (1991b). "Osaka et al. (1987). "Osaka et al. (1985). "Estimated from Kosaka et al. (1985) and Acsidy et al. (1996a); VIP/CCK cells are basket cells. 7Kosaka et al. (1988b);30% of GAD cells in the hilus and CA3-1 oriens are SOM, 510% in CA1 and CA3 stratum pyramidale, and 2% in the granule cell layer. 8Deduced from data on SOM-NPY colocalization, i.e., SOM-NPY cells represent the same proportion of SOM cells and of NPY cells, which means that their ratio is -1:1. 9Estimated from CR/SPR ratio from Acsady et al. (submitted). "That is, mGlu cells perfectly overlap with SOM cells (Baude et al., 1993). 'lH&jos et al. (submitted). lLMiettinen et al. (1992); the 5% overlap of CB and CR might arise from the slight cross reactivity of the CR antibody with CB used in this study. I3The lack of GABA immunoreactivity in these cells may be due to the rather extensive axon abrobization of these cells (many of them also have commissural and/or cross-regional projection).
1 -
''Acs6d> et al. (in press). 15Valuesonly for regions that do not contain spiny cells. l6Va1ue is for spiny/nonspiny cells cumulatively. 17T6th and Freund (1992). 1sGuly5set al. (1991b); 0% except in CA1 stratum oriens, where 21% of CB cells contain PV. 19Gulyaset al. (1991b); there are small differences between areas and layers. 2oKatonaet al. (1996); 87% in CA1 stratum oriens and 0% in other areas. 21Gulyaset al. (1991b);O"/o except in CA1 stratum oriens, where 9.6% of PV cells contain CB. 22Dunet al. (1994). 23A~siidy al. (1996; SPR), 90% in DG and 5% in other areas. et 24Valtschanoffet al. (1993); 100% except for some cells in stratum pyramidale, which make up 5% of all NOS cells. 25Dunet al. (1994); hilus/other areas. 26Gulyaset al. (1991b). 27Kosakaet al. (1985), Acsady et al. (1996a); 100% for VIP basket cells and + for other VIP cells. 28Acsadyet al. (1996a). 2yVIPtype 3: loo%, VIP type 2a: 41%, VIP type 2b: 0%. 300ne percent in CA1 and 0% in CA3 for both VIP populations. 31VIPtype 3: O%, VIP type 2a: 1370,VIP type 2b: 0%. 32Katonaet al. (in press). 33Dunet al. (1994). 34Kohleret al. (1987); a ratio of approximately 1 can be deduced for SOM/NPY. "Acsady et al. (submitted); 73% in hilus and 8% in CA1. 36Baudeet al. (1993). 37Theremaining 11% were spiny cells in hilus, possibly GABAergic neurons with distant projection, which have low somatic GABA levels. 38Fifty-ninepercent in DG and 5% in CA3-I. 3yFourto 19%;percentage differs among areas. 400nehundred percent in hilus and + in CA3. 41Twenty-eightpercent in DG, 92% in hilus and lucidum, and 36% elsewhere. 42Deducedfrom value for SOM. 43Fifteenpercent in CA1, 36% in CA3 stratum oriens, 16% in CA3 stratum radiatum, and 19'4~ in the hilus of DG. 4 4 0 n e percent and 2% in CA3 and CA1 and 0% in DG and hilus. 45Acsddyet al. (submitted); 80% in DG and hilus and 51% and 55% in CA1 and CA3, respectively. 4hAcsadyet al. (submitted); 10% and 14% in CA1 and CA3; no data from DG or hilus. 47L. Seress (personal communication).
406
ent and efferent connections has been successfully used to identify SPR-positive neurons (Table 1; Acsidy et al., submitted). O n the basis of location and dendritic morphology, four different cell types may be distinguished in the dentate gyrus and the hippocampus: (I) pyramidal-shaped or fusiform cells, with the soma located in stratum granulosum of the dentate gyrus and with radially oriented aspiny dendrites spanning all layers; (2) fusiform cells in the hilus and stratum lucidum of the CA3 region, which have spiny dendrites running parallel to laminar boundaries and which remain confined to the same layers; (3) aspiny multipolar cells in strata pyramidale, proximal radiatum, and oriens of the CA1-CA3 regions, which have thin, radially running primary denlarge, more robust asdrites branching close to the soma; and (4) piny or sparsely spiny multipolar cells with thick, distally branching dendrites. These cell types are found in the same layers and strata of the hippocampus as are the type 3. Several SPR-positive cells show transitional features of cell types 3 and 4. Markers for perisomatic inhibitory cells, PV, and CCK, colocalize with SPR in pyramidal-like basket cells in the dentate gyrus (type 1 cells, described above) and in large multipolar cells of the hippocampus (type 4 cells). The dense meshwork of SPR-immunoreactive spiny dendrites in the dentate hilus and in stratum lucidum of the CA3 region belong to inhibitory cells that innervate the distal dendritic region, as suggested by their SOM and NPY content. In addition, in the hippocampus, SPR and NPY colocalize in numerous small multipolar interneurons, with dendrites branching close to the soma. There is a 25% overlap between the SPR-immunoreactive cells and CR-positive interneurons, suggesting that interneurons specialized to contact other GABAergic cells (Section III.5.a) also contain SPR. Thus, on the basis of the known termination pattern of the colocalized markers, SPR-positive interneurons may be functionally heterogeneous and participate in different inhibitory circuits, i.e., in perisomatic inhibition of principal cells (CCK-containing cells in all subfields and PV-positive cells in the dentate gyrus), in feedback dendritic inhibition in the entorhinal termination zone (SOM- and NPYcontaining cells), and in the innervation of other interneurons (CR-containing cells).
NT3
Interneurons expressing this neurotrophin are less numerous. Colocalization of N T 3 mRNA with calcium-binding proteins reveal that only a small number of PV- and CR-immunoreactive neurons show hybridization (Rocamora et al., 1996). According to their laminar distribution, the majority of NT3-positive cells are likely to contain one or several neuropeptides. Immunostaining studies with antisera against NT3 protein have not been published.
BDNF
In situ hybridization studies have shown that interneurons do not express BDNF mRNA under normal conditions, in spite of heavy labeling in the principal cell somata (Hofer et al., 1990; Isackson et al., 1991; Berzaghi et al., submitted; Rocamora et al., 1996). In contrast with NGF immunoreactivity, immuno-staining for BDNF protein is weaker in the neuropil, but a small population of cell bodies is faintly labeled (Berzaghi et al., submitted). Immunocytochemical double labeling reveals that most if not all BDNF-positive somata belong to PV-containing interneurons in both the hippocampal formation and neocortex. The lack of BDNF mRNA in all these cells suggests that IV-containing neurons do not synthesize but accumulate this neurotrophic factor. Interestingly, the level of BDNF protein can be remarkably enhanced afier seizure activity for several days (Nawa et al., 1995). In a recent study by Marty et al. (1996), GABAergic stimulation was shown to increase the size and NPY content of interneurons in dissociated hippocampal cultures at a developmental stage when GABA is still enhancing calcium influx. This trophic effect of GABA was found to be mediated by an enhancement of synthesis and release of BDNF from nearby neurons. GABAergic drugs had no such effects in cultures prepared from BDNF knockout embryos. At a later stage, when GABA is inhibitory and reduces BDNF synthesis, GABA agonists induce a reduction in size and NPY immunoreactivity of neurons.
NGF
Immunocytochemical studies have shown no association of NGF immunoreactivity with any interneuron types. There is a homogeneous intense staining of the neuropil, which is particularly dense in areas innervated by mossy fibers (Conner et al., 1992). However, a recent study has demonstrated that NGF mRNA is expressed in GABAergic interneurons in the hippocampal formation (Lauterborn et al., 1993), indicating that interneurons are the major source of NGF production. The neurochemical identification of the NGF-producing interneuron subpopulations has been done in a study combining in situ hybridization for NGF mRNA and immunostaining for calciumbinding proteins PV, CB, and CR (Rocamora et al., 1996). The majority of PV-positive neurons (82%) express NGF mRNA,
Numerous small subcortical nuclei, with neurons containing different neurotransmitters, send a diffuse innervation to the majority of neo- and archicortical areas. These pathways include GABAergic and cholinergic projections from the basal forebrain,
407
FIGURE 27. Photomicrographs show septohippocampal (A,C) and raphe hippocampal (B) aEerents labeled with the anterograde tracer PHAL (black, Ni-DAB) and their postsynaptic targets. Postsynaptic targets were identified by immunostaining for PV and CB by using DAB as the chromogen (brown). A: Multiple contacts (arrows) are formed by septal fierents on a PV-positive interneuron (S,) in a climbing fiber-like manner in stratum oriens of the CA3 region. Somata of additional interneurons &) are also contacted. C: A PHAGlabeIed septal axon that traversed stratum moleculare of the
dentate gyrus forms synaptic varicosities exclusively on PV-positive dendrites of interneurons (arrows). B: Varicose axons from the median raphe establish multiple contacts (arrows) on the soma and proximal dendrites of a CB-positive interneuron in stratum radiatum of the CAl subfield. Varicosities of both septal and raphe origin that made multiple contacts with interneurons were synaptic boutons in all examined cases (see Figs. 28, 29). Data from Freund and Antal (1988) and Freund et al., (1990~). Scale bars = 10 pm.
INTERNEURONS OF T H E HIPPOCAMPUS
gyrus, GABAergic interneurons receive multiple innervation from median raphe fibers (Halasy et al., 1992). The target cells include mostly pyramidal-shaped neurons at the granule cell layer-hilar border and fusiform cells in the hilus. CB-positive interneurons are hardly visible due to the heavy staining of granule cells. Nevertheless, some fusiform CB-containing interneurons in the subgranular zone are surrounded by PHAL-labeled fibers. PVpositive neurons were not seen to receive multiple contacts. All PHAL-labeled boutons in close apposition with interneurons form conventional synaptic contacts (Fig. 29; Acsidy et al., 1993). Serial ultrathin sections cut from PHAL-labeled raphehippocampal axons, immunostained for GABA with the postembedding immunogold procedure, reveal that the majority (over 70%) of the synaptic varicosities terminate on GABA-immunoreactive dendrites or somata (Halasy et al., 1992). The PHAL-labeled boutons themselves are always negative for GABA. The results obtained with anrerograde tracing have been confirmed by double immunostaining for serotonin and the interneuThe ron markers (Freund et al., 1990~). same types of interneurons as in the PHAL study were shown to be innervated, demonstrating that the nonserotonergic component of the raphe-hippocampal projection (Kohler and Steinbush, 1982) has a negligible, if any, contribution to this innervation pattern (Freund et al., 1990c; Halasy et al., 1992; Acsidy et al., 1993). In sections with relatively good quality staining for serotonin, approximately 70% of CB-positive neurons in the CAl region and 50% in the CA3 region receive multiple innervation. Contacts on PV-positive cells are sparse and limited to 1-3 varicosities per connection. Electron microscopy of serotonin-immunoreactive axons confirm, in accordance with earlier findings (Oleskevits et al., 1991), that only about 25% of the varicosities make synaptic contacts. These synapses are established mostly with GABA-positive dendrites or somata in the hippocampus and in the dentate gyms (Freund, 1992). In conclusion, the two types of serotonergic afferents may have different mechanisms of action in the hippocampus. More than 70% of the varicosities (probably those originating from thin dorsal raphe fibers; Kosofsky and Molliver, 1987) release serotonin at nonsynaptic sites, which may diffuse to different target cells having 5-HT 1-2 receptors, to exert a slow, tonic, G-protein-mediated action. The other fiber type with large boutons (mostly of median raphe origin; Kosofsky and Molliver, 1987) always forms synaptic contacts. The postsynaptic elements are specific interneuron types, in particular those exerting dendritic inhibition (Gulyis and Freund, in press; Miles et al., 1996). The receptor at these synapses is likely to be of the 5-HT3 type, which allows a fast excitation of these GABAergic cells, and an enhanced GABA-A receptor-mediated inhibition (Ropert and Guy, 1991; Section IX.5).
409
lucidum of the CA3 subfield. Stratum lacunosum-moleculare of the CA1 region is also heavily innervated, but other regions and layers receive relatively sparse input (Loy et al., 1980; Morrison et al., 1979; Oleskevich et al., 1989; Moudy et al., 1993). The majority of TH-positive (mostly noradrenergic) varicosities do not make conventional synaptic contacts. However, those that form synapses often terminate on dendritic shafts and somata of putative interneurons (Frotscher and Leranth, 1988; Milner and Bacon, 1989a). These synapses are symmetrical, whereas others terminating on spines of presumed pyramidal cells are asymmetrical (Frotscher and Leranrh, 1988). The postsynaptic elements have been confirmed to be GABAergic for a small sample in strata lucidum and radiatum of the CA3 subfield and in the hilus of the dentate gyrus by immunostaining for GAD (Frotscher and Leranth, 1988) or for GABA (Milner and Bacon, 1989a). The types and frequency of GABAergic neurons innervated in these and other regions remain to be established.
410
411
GABAergic projection neurons should not be referred to as interneurons or local circuit neurons (but see Section I). The most common are those that project from the hilus to the contralateral dentate gyrus and those in stratum oriens of the hippocampus that innervate the medial septum. They most likely have local collaterals as well, by which they contribute to (ipsilateral) intrahippocampal inhibitory networks (Sections III.3.2.b, IV.2.b, IV.3.a, IV.3.b). The GABAergic nature of these interneurons that have extrahippocampal or commissural projections is difficult to establish. It has been shown in several brain areas that GABAergic neurons with distant projections (e.g., the striatonigral, nigrothalamic, nigrocollicular, pallidonigral, septohippocampal neurons, the Purkinje cells) have very low levels of GABA in their cell bodies, levels that do not reach the immunocytochernical detection threshold. However, anterograde tracing combined with postembedding immunogold staining for GABA has shown that axon terminals of GABAergic cells with distant projections are immunoreactive for GABA (Figs. 28, 30; Freund and Antal, 1988; T6th et al., 1993).
terneurons that give rise to a comrnissural projection belong to the SOM-containing subpopulation of GABAergic neurons (Zimmer et al., 1983; Bakst et al., 1986; Leranth and Frotscher, 1987). As one might expect from double-labeling studies, NPYcontaining cells (Deller and Leranth, 1990) and spiny CR-positive neurons (Miettinen et al., 1992) are also involved in these commissural connections (Sections IV.2.c, IV.3.a, IV.3.b). There appears to be a discrepancy between the results of retrograde and anterograde labeling studies. SOM-positive cells definitely contribute to the commissural projection according to the retrograde labeling data, and numerous immunostaining studies have shown that SOM-containing axons arborize mostly in the outer two-thirds of the molecular layer of the dentate gyrus (Section IV.3.a). However, anterograde tracing techniques using radioactive tracers, degeneration, or HRP visualized axons only in the inner one-third of the molecular layer of the contralateral dentate gyms (Blackstad, 1956; Gottlieb and Cowan, 1973; Laurberg, 1979). A recent experiment has solved this apparent conflict of data by demonstrating that hilar commissural fibers also terminate in the outer molecular layer (Deller et al., 1995b), although these axons still appear sparse compared with the relatively large number of SOM-containing neurons participating in the projection. These data suggest that there is a component of direct inhibition in the feed-forward inhibitory response evoked in the dentate gyrus by commissural stimulation (Buzsdci and Eidelberg, 1981; Buzsiki, 1984), but its specific function remains to be established.
412
413
The wealth of anatomical knowledge about interneurons discussed above may be contrasted with the paucity of information available about their distinctive physiological properties (Tables 2-4). A rational taxonomy of interneurons should be based on the premise that the anatomically/chemically distinguishable classes have different functions, whereas neurons of the same class should have the same properties. Prior to the widespread application of intracellular staining border are also immunoreactive for SOM (Katona et al., in press). methods, researchers had already recognized that spontaneous acThe GABAergic nature of these projection neurons was equivo- tivity, evoked responses, and passive membrane properties of neucal because GABA immunoreactivity of their cell bodies did not rons outside the pyramidal and granule cell layers are distinct from exceed background level (T6th and Freund, 1992). However, an- those of principal cells, and they used those criteria for interneuterograde tracing with PHAL combined with postembedding im- ron identification (Andersen et al., 1963, 1964, 1969; Ranck, munogold staining for GABA confirmed that axon terminals of 1973; Fox and Ranck, 1981; Buzsiki and Eidelberg, 1981, 1982; these neurons in the medial septum are GABA positive and form Ashwood et al., 1984; Lacaille et al., 1987). In the intact hipsymmetrical synaptic contacts (Fig. 30). The postsynaptic targets pocampus, pyramidal cells spontaneously discharge bursts of 2-1 0 have been examined by double immunostaining for PHAL and action potentials of decreasing amplitude and increasing duration markers of cholinergic (ChAT) and GABAergic (PV) septal neu- (complex-spike bursts). Under several circumstances, these fearons. In some experiments, additional retrograde tracer injections tures alone may distinguish them from interneurons of the hipinto the hippocampus allowed the identification of cholinergic and pocampal formation (Ranck, 1973). However, under most conGABAergic septohippocampal neurons in the same material. The ditions, a set of criteria are needed for the positive identification predominant targets of hippocamposeptal GABAergic fierents are of interneurons. These typically include their high spontaneous the PV-containing GABAergic neurons in the medial septum and firing rate (5-80 Hz), short duration action potentials, short lathe vertical limb of the diagonal band of Broca (Fig. 31), whereas tency, and repetitive discharge in response to afferent inputs. cholinergic neurons are seldom innervated (T6th et al., 1993). The Interneurons, in general, have a significantly lower threshold for targets also include numerous retrogradely labeled GABAergic and action potential generation in response to afferent stimulation than do principal cells (Buzs&i and Eidelberg, 1982; Ashwood a small number of cholinergic septohippocarnpal neurons. It is important to note here that the cells of origin of this et al., 1984; Lacaille, 1991). Several possible reasons for this low GABAergic hippocamposeptal feedback projection are interneu- threshold include their larger size shaft synapses as opposed to rons, which appear to be driven primarily by recurrent collater- spine synapses in principal cells, different leak conductances, acals of local pyramidal cells, as suggested by the stereotyped dis- tivity and density of Na+-K+ pumps, and hypothesized dendritic tribution of their dendritic trees (Sections 1 1 IV.2.b, IV.3.a; T6th generation of fast action potentials (Section IX. 1). Furthermore, 1, and Freund, 1992; Baude et al., 1993; Blasco-Ibanez and Freund, the absence of G l u m and enhanced expression of GluR4 sub1995). The dendritic trees of CB- and/or SOM-positive cells of units ofAMPA-gated channels in interneurons (Jonas et al., 1994; this type in CAI stratum oriens have a horizontal orientation and Baude et al., 1995; Geiger et al., 1995; Jonas and Burnashev, are restricted to the stratum oriens-alveus border in a manner 1995; Leranth et al., 1996) may be responsible for the low threshsimilar to the local collaterals of CA1 pyramidal cells. These col- old of spike generation in interneurons. Recombinant AMPA laterals were shown to account for more than 80% of the excita- channels that contain the GluR4 but lack the GluR2 subunit tory input to the dendrites of horizontal cells (Blasco-Ibanez and when inserted into oocytes possess the fastest kinetics (Hollmann Freund, 1995). In the CA3 subfield, the dendrites of CB- and/or and Heinemann, 1994; Seeburg, 1993). The passive electrical properties of most hippocampal inSOM-containing neurons are not restricted to stratum oriens. They often penetrate stratum radiatum and overlap in distribu- terneurons are characteristically different from those of principal tion with recurrent collaterals of local pyramidal cells. Because cells. Early in vitro experiments with anatomically characterized each pyramidal cell is likely to establish only a single synapse with cells listed a number of criteria for the physiological verification of interneurons (Gulyh et al., 1993b), the activity of these neurons interneurons, including (1) a relatively high spontaneous firing rate, (2) short duration spikes (<1.2 ms), (3) large spike afterhyi s likely to reflect synchrony in the activity of large local pyramidal cell populations (T6th et al., 1993). The functional implica- perpolarization, (4) weak spike frequency accommodation in retions of such a synchrony-dependent GABAergic feedback directly sponse to depolarizing current injection, and ( 5 ) high input resis-
414
F R E W D AND BUZSAKI
FIGURE 30. Photomicrographs and line drawing show the distribution and termination of the hippocamposeptal projection labeled with PHAL. A: PHAL injection site in stratum oriens of the CAI region of the hippocampus. In addition to numerous pyramidal cells, interneurons with horizontal dendrites (white arrows) at the stratum oriens-alveus border also accumulated the lectin. B: Drawing of the distribution of PHAL-labeled hippocampal afferents in the septal region. In addition to the well-known pyramidal cell projection to dorsal parts of the lateral septum (LS), numerous afferents to the medial septum (MS), and the vertical limb of the diagonal band of Broca (VDB) were also visualized. LV, lateral ventricle. C-E: Electron micrographs of three serial sections of a PHAL-labeled hippocampal afferent bouton (b,) in the MS. C: In
contrast to pyramidal cell boutons in the LS, hippocampal afferent terminals in the MS and VDB regions (e.g., as bl, shown here) form symmetrical synapses (arrows in C-E). D,E: Postembedding immunogold staining of adjacent sections for GABA show that hippocampal afferents (bl) in the MS and VDB regions are GABAergic. Other GABA-positive boutons (small asterisks) and GABA-negative profiles (large asterisks) are also indicated. From T6th K, Borhegyi Z, F r e u d TF (1993) Postsynaptic targets of GABAergic hippocampal neurons in the medial septum-diagonal band of broca complex. J Neurosci 13:3712-3724 by permission of Society of Neuroscience. Scale bars = 50 p m in A, 0.5 p m in C,D, 0.75 pm in E.
415
activated K+ current, Ic. However, the firing rate of the interneuron may be limited by the slow component of spike afterhyperpolarization (Zhang and McBain, 1995a). Unique combinations of the above physiological features or explicit absence of some of them have been used in attempts to group interneurons into subclasses. Although the exact relationship between physiological and anatomical features of interneurons has yet to be worked out, the available physiological information already indicates that the different anatomical subgroups may have distinct physiological properties (Schwartzkroin and Mathers, 1978; Kawaguchi and Hama, 1987a,b; LacaiIle et al., 1987; Lacaille and Schwartzkroin, 1988a,b; Lacaille 1991; Buhl et al., 1994a,b, 1995; Sik et al., 1995, submitted; Khazipov et al., 1995a). CAI interneurons in the pyramidal layer (putative basket cells and chandelier cells) display some spike frequency adaptation in response to depolarizing current injection and have smaller spike afterhyperpolarization. Whole-cell recordings indicate that basket cells have a membrane resistivity of 7-66 k a cm2 and specific cytoplasmic resistivity of 52-484 cm2 (Thurbon et al., 1994). Basket and chandelier cells are less responsive to mGluR agonists than are oriens-alveus interneurons (McBain et al., 1994). In addition, basket and chandelier cells can be monosynaptically activated by both CA3 and CA1 pyramidal cells, whereas the majority of neurons in the horizontal group are innervated by CAI but not CA3 pyramidal neurons (Blasco-Ibanez and Freund, 1995; Maccaferri and McBain, 1995; Sik et al., 1994, 1995). Chandelier cells were suggested to fire repetitive doublet action potentials in response to depolarizing current pulses (Buhl et al., 1994b). The dendritic tree of basket cells and chandelier cells are somewhat distinct, suggesting that at least some of their afferents are nonoverlapping (Sections 111.1, 111.2). Nevertheless, probably both cell types may be discharged by the direct entorhinal afferents (Fig. 33). O n the basis of the anatomical variability of interneurons in the oriens-alveus region (Sections 111.1-II1.3), one might expect further physiological differences among them. Large inward current responses to mGLuR activation and an expressed H-current (depolarizing sag; McCormick and Pape, 1990) in response to hyperpolarizing current pulses appear to be characteristic features of 0 - L M cells but not of all interneurons with similar horizontal dendritic trees (McBain 1994; McBain et al., 1994; Sik et al., 1994). These neurons contain the peptide SOM and have an exceptionally high density of mGluRla receptors (Sections IV.3.a, IV.6.a; Baude et al., 1993). The sag current, however, is weak in back-projection cells, with dendritic morphology similar to 0 - L M cells (Sik et al., 1994). Back-projection neurons are likely NPY immunoreactive (Section IV.3.b), and immunocytochemically labeled NPY neurons in the hilar region also displayed a weak sag current (Sik et al., submitted). Another common feature of the NPY-SOM-immunoreactive family is a very prominent spike afterhyperpolarization and the selective presence of the Kv3.2 potassium channel subunit (Weiser et al., 1994). The trilaminar interneuron type can be distinguished from the above cells by the absence of the late IPSP and by antidromic activation from the fimbria-fornix (Sik et al., 1995, submitted). In contrast to 0-LM, trilaminar, and backprojection cells, bistratified interneurons in the CA1 region can be monosynaprically activated by stimulation of the Schaffer collaterals (Buhl et al., 1994a; Sik et al., 1995).
k a
416
FREUND AND B U Z S k I
417
Interneurons with cell bodies in CAI stratum lacunosum-moleculare (LM cells) have been treated as a relatively homogeneous population by physiologists; some LM cells have characteristics similar to CAI pyramidal cells and other properties more like other interneurons (Kawaguchi and Hama, 1987a,b; Lacaille and Schwartzkroin, 1988a,b; Khazipov et a!., 1995a,b). Some of these LM cells have extensive local axon collaterals. Similarities with CA1 pyramidal cells include broad and slowly decaying action potentials (2.0 ms), a relatively slow time constant (8-9 ms), no or very little spontaneous firing, and little evidence of spontaneous synaptic potentials. The lack of spontaneous EPSPs and action potentials in vitro, however, may simply reflect the lack of afferent drive because the somata giving rise to afferents to these interneurons are lost when using the slice procedure. The precise inputs to these LM interneurons are not known, but they likely include the perforant path, intrahippocampal, thalamic, and subcortical afferents, most of which are lost during slicing of the brain. Stimulation of dendritic layers often induces a short lasting oscillation (20-40 Hz) of the membrane potential of LM cells. As with other interneurons, cells in stratum-lacunosum moleculare have a high input resistance (40-100 M a , with sharp electrodes), a prominent afterhyperpolarization, and only limited spike frequency accommodation in response to depolarizing pulses (Kawaguchi and Hama, 1987a; Lacaille and Schwartzkroin,
1988a,b; Kunkel et al., 1988). LM cells usually display anodal break excitation and burst firing when the membrane is released from hyperpolarization (Lacaille and Schwartzkroin, 1988a; Fraser and MacVicar, 1991), indicating the presence of lowthreshold calcium channels (T channels; Jahnsen and Llinas, 1984). Such features, however, have also been observed in some interneurons of the CAI stratum oriens-alveus (Lacaille and Williams, 1990; Zhang and McBain, 1995a). The low-frequency discharges and the relatively wide action potentials of LM interneurons suggest that they are endowed with currents distinct from other interneurons. LM interneurons conspicuously lack potassium channels of the Kv3 family (Weiser et al., 1994). However, tissue culture experiments indicate that LM cells possess delayed rectifier currents different from those described in 0-LM cells and basket cells (Chickwendu and McBain, in press). Classification of interneuron subgroups by physiological means in the CA3 region (Gulyis et al., 1993a; Arancio et al., 1994; Miles et al., 1996; Poncer et al., 1995) and the dentate gyrus (Han et al., 1993; Scharfman 1995a; Sik et al., submitted) have also progressed recently. For example, McBain and Dingledine (1993) discriminated two major classes of CA3 stratum radiatum interneurons on the basis of their current-voltage relations to kainate application and the kinetic properties of spontaneous mEPSCs. They hypothesized that the two groups may have different GluR subunit compositions in their AMPA receptors. In the dentate gyrus, Scharfman (1995a) described heterogeneous physiological properties of interneurons located in the granule cell layer and subgranular layer. Pyramidalshaped interneurons in the granule cell layer have significantlylonger action potentials (1.2 ms) and stronger spike frequency adaptation than those of the hilar group. Cells with similar dendritic morphology often have different axonal arbors. Dentate gyrus interneurons, recorded in vivo in the gerbil, can be classified into fastspiking and slow-spikinggroups (Buckmaster and Schwartzkroin, 1995b). Fast-spiking neurons displayed a low threshold to perforant path stimulation, whereas slow-spiking interneurons responded with predominantly inhibitory potentials. Neurons in the fast-spiking group were not labeled in that study, but one of the slow-spiking interneurons had morphological features similar to NPY and/or SOM cells and spiny CR-immunoreactive cells. This short summary of the passive and evoked properties of interneurons suggests that some of their morphological characteristics may be predicted from their physiological properties. To date, however, most correlative studies lack rigorous criteria at either the physiological or anatomical level. Although it is not expected that all morphological classes of interneurons have distinct functional features, it may well be that a given physiological property is associated with a unique set of morphological features, and a blend of these features may explain the often subtle differences in the physiological domain.
The dendrites, cell bodies, and the axon initial segment of every principal cell in cortical structures are innervated by inhibitory
TABLE 2.
Dentate Gyt-usl
Afferent input Intrahippocampal (excitatory) Extrahippocampal CBPs/NOS Neuropeptides C/A, mossy C/A, mossy ec, ms PV C/A, mossy mossy CR ( 2 ) NOS (?) C/A, mossy ec, ms, mr(?) ec, ms ms, mr ec, ms Receptors etc.
Neurochemical markers
Cell type
Dendrites
Axon
Axo-axonic r.s, pd
PV
Basket 1
all layers s g . , h
HICAP
SPR2, Gal, W A SPR2, Gal, VVA CCK, VIP( 2 ) SPR SOM, NPY mGluR1, SPR CCK (?) SPR (?)
narrow AP, large AHP, slow spiking, gamma rhythmicity gamma rhytmicity, activated during dentate EEG spikes
MOPP Spiny CR
IS-1
s.m. ( 0 ) s.m. ( 0 ) r.dd h, (CA3c) s.m. (0) r.dd(?) all layers all layers i.s, i.pd, i.dd i.s, i.pd, i.dd C/A, mossy ec, ms CR
CB (?) CR CR
(VIP)
'Tables 2 4 provide a list of morphological, neurochemical, and physiological features of identified intemeurons. Those types at the bottom of the tables, indicated in boldface type, are known only from immunocytochemical studies; therefore, physiological and detailed anatomical data are not available for these types. AHP, spike afterhyperpolarization; AP, action potential; C/A, commissural-associational path; dd, distal dendrites; ec, entorhinal cortex; Gal, GABA-A receptor alpha 1 subunit; g.is, granule cell axon initial segment; h., hilus; IH, hyperpolarization-activated current; i.dd, interneuron distal dendrite; i.pd, interneuron proximal dendrite; is, interneuron soma; 15-1, type l interneuron-selective cells (CR); IS-2, type 2 interneuron-selective cells (VIP); 15-3, type 3 interneuron-selective cells (VIP); lc, local collaterals of pyramidal cells; mr, median raphe; ms, medial septum; pd, proximal dendrite; pis, pyramidal cell axon initial segment; r., random (i,e., contacts both granule cells and interneurons in proportion of their occurrence); s., soma; s.g., stratum granulosum; s.m. (i), inner stratum rnoleculare; s.m. (o),outer stratum moleculare; s.l., stratum lucidum; s.1-m., stratum lacunosum-moleculare; s.o., stratum oriens; s.P., stratum pyramidale; s.r., stratum radiatum; VVA, Vicin villosa agglutinin. 20nly in the dentate gyrus 3Only on axon terminals.
TABLE 3.
CA1 Region
Neurochemical markers Receptors etc. VVA Physiological features spike doublets (?), accommodation
Axon PV PV
m22,Gal,
Axo-axonic r.s,pd
Basket 1
relatively wide AP, accommodation, low-amplitude AHP, theta, gamma, 200-Hz rhythms, A/C activation
Basket 2 CB(t)
(4, ms
CCK, VIP(?)
0-LM
all layers s.P., s.0, r.s,pd (s.r.) s.o., a h . s.1-m., r.dd (s.o., s.r.)
r.pd,s,dd
C/A, lc.
ms, mr
5-HT-3(?)
large IH, low-threshold Ca2+ spikes, intrinsic oscillation at theta frequency, lack of C/A activation, discharge by CA1 pyramidal cells C/A activation
Horizontal trilaminar r.pd,s,dd C/A, Ic. ms, mr (ec) ms(?), mr(?) ms, mr, (ec) ec, ms NOS(?) CR CR( 5 ) CB(?)
S.O.
SOM(?), NPY(?)
s.o., s.r.,
s.r., s.P.,
mW, large AHP, prominent late mGluRl(?) depolarizing potential to afferent activation SPR(?) -
lc., C/A(?)
C/A, lc. C/A, lc.
Radial trilaminar Backprojection IS-1 (smooth CR) IS-2 (VIP) C/A, Ic. ec, ms CR
s.P., s.1-m. S.O. s.o., a h . s.r.,l S.O.,] r.pd,dd s.p., h.l all layers s.r., s.P., i.s,i.pd, S.O. i.dd all layers s.r., s.P., i.s,i.pd, S.O. i.dd
m2(?), small IH, C/A inputs hyperpolarize mGluRl(?) SPR(?), Gal( ?) Gal(+) 3slow firing, wide AP, widespread inhibitory inputs, low-threshold Ca2+ spikes, oscillation at theta frequency
IS-3
VIP
(?)
(VIP)
i.s,i.pd, i.dd
'Also in other subfields; i.s. in the CA3 region and in the dentate gyrus. 20nly on axon terminals. 3Characteristicsof LM cells, which may include IS-2 VIP cells.
TABLE 4.
CA3 Region
Cell type p.is r.s, pd r.s, pd r.dd r.pd, s, d d r.pd,s,dd C/A, mossy ms, mr, (ec) C/A, mossy ms, mr C/A, mossy ms, mr C/A, mossy C/A, mossy C/A, mossy PV PV
Dendrites
Axon
Axo-axonic Basket 1
Basket 2
all layers
ec, ms
0-LM
Bistratified
s.o., s.r., S.P., s.1. s.o., s.r., S.I., s.p. all layers
S.P., S.O. S.P., S.O., (s.r.) S.P., s.0, (s.r.) s.1-m., (s.o., s.r.) s.r., s.o., 6.P.) s.r., s.P.,
S.O.
s.1.
ms
S.O.
all layers
'Also in other subfields; i s . in the CA3 region and in the dentate gyrus. 20nly on axon terminals.
(VIP)
m22, VVA m22, Gal may be discharged by a single VVA pyramidal axon terminal CB(2) CCK, VIP(2) Gal, m22(?), SPR(?) CB(?) SOM, NPY mGluR1 may be discharged by a single pyramidal axon terminal 5-HT-3(?), CB, NOS(?) NPY(? ?) GluR2(B)(+) CB(5 ) NPY(?), large AHP, theta, gamma, SPR(?), CCK(?) rhythmicity activated GluR2(B) during dentate EEG spikes CR SOM, NPY(?) SPR, mGluRl(?) CR VIP(?) SPR(t), Gal( ?) VIP CR(?)
421
PYRAMIDAL CELL
P l
INTERNEURON O/A
-58
-64
-58
30 ms
FIGURE 32. Action potentials and afterhyperpolarizationsof interneurons at the CAI stratum oriens-radiatum border (A&) and pyramidal cells (B). A: A1 and A2. Averaged action potential at different time scales. Broken lime indicates resting membrane potential. Note lilige amplitude spike afterhyperpolarization (arrow). B: B1 and B2. Average action potential at fast and slow time scales. The spike afterhyperpolarization is small amplitude (arrow), followed by spike depolarization (double arrow) and a medium duration afterhyperpo-
larization (triple arrow). C: Depolarizing pulse evoked a train of action potentials (Cl) and a long afterhyperpolarization (arrowin C2). D: Responses to long depolarizing current pulses of increasing intensity (0.125-1.0 nA). Note progressive shortening of the intervals between action potentials. Reprinted from Lacaille J-C and Williams S (1990) "Membrane properties of interneurons in stratum oriensalveus of the CAI region of the rat hippocampus in vitro." Neuroscience 36:349-359 by permission of Elsevier Science Ltd.
interneurons. The terminals of interneurons release the inhibitory substance GABA and may also release peptidcs that colocalize with GABA in many types of interneurons (Section IV.3). The complex issue of GABA-mediated transmission has recently been reviewed (Mody et al., 1994), so only the major aspects will be briefly summarized here.
terneuron-mediated inhibition of principal cells and among interneurons themselves. Are separate groups of inhibitory cells rcsponsible for activating postsynaptic GABAA and CABAS receptors (Alger and Nicoll, 1982a; Segal, 1990a; Muller and Misgeld 1991; Williams and Lacaille, 1990; Samulack and Lacaille, 1993; Williams et al., 1993)? If so, why are spontaneous GABAB-mediated synaptic events not visible (Staley and Mody, 1992)?What is the spatial relationship between the two receptor families? An early hypothesis suggested that dendritic inhibition is mediated by GABAH receptors and perhaps by a separate group of interneurons (Alger and Nicoll, 1982a; Segal, 1990a; Williams and Lacaille, 1992). In support of this hypothesis, IPSPs with similar kinetics to the GABAB-receptor-mediated responses are produced by activation of interneurons in stratum lacunosum-moleculare (Lacaille and Schwartzkroin, 1988b). However, the slow rise time o f somatic IPSPs in postsynaptic pyramidal cells may simply reflect elcctrotonic filtering by the dendrites (Soltisz and Mody, 1994). Furthermore, other experiments that have involved phar-
422
' a
5 mV
in
PP
' u
FIGURE 33. Feed-forward inhibition of CAI pyramidal cells by the perforant path input. A: Low threshold and monosynatic (6-8 ms) activation of an interneuron in the C A I pyramidal layer (putative basket or chandelier cell) by single-pulse stimulation of the perforant path (PP). Top three traces indicate stimulation with increasing intensity. Asterisk indicates volume-conducted population spike from the dentate gyrus. Bottom trace indicates frequency potentiation (4 Hz, 2 s. Triangle indicates CAI population spike. B: ) Inhibition of a CAI pyramidal cell. Simultaneous intrasomatic recording (intra) and extracellular field response in the stratum radiatum (extra). Black square indicates trisynaptically evoked Schaffer
collateral response. Arrow indicates early inhibition of the pyramidal cell at a latency comparable to the discharge of the putative baskedchandelier cell in A. C: Schematic interpretation of the physiological observations. The entorhinal afferents (PP) excite both pyramidal neurons (p) and chandelier (c) and/or basket (b) cells. The discharging interneurons in turn induce a strong perisomatic inhibition in the pyramidal cell. D: Degenerating perforant path terminals form synapses (arrows) on the distal dendrite of a PV-immunoreactive (putative basket or chandelier) interneuron in stratum lacunosum-moleculare. Data from Bum& and Eidelberg (I 982), Bum& et al. (1995), and Leranth et al. (1996).
423
bistratified cells, or they may have other functions than just action potential regulation (Section XIII.4). Local application of GABA in the cell body layer in vitro typically produces hyperpolarization in pyramidal cells. However, when GABA is applied to the dendrites, depolarization may be observed. Strong electrical stimulation in the dendritic layers also evokes fast depolarization in pyramidal cells. Both the hyperpolarizing and depolarizing fast responses are mediated by GABAA receptors because they are abolished by GABAA receptor blockers and are enhanced by barbiturates (Alger and Nicoll, 1979, 1982b; Andersen et al., 1980; Perreault and Avoli 1989; Thalmann 1988). These experiments favor the hypotheses that (1) the hyperpolarizing and depolarizing responses are mediated by different receptor subtypes having different ion selectivity and/or (2) the distribution of chloride in somatic and dendritic regions is different (Alger and Nicoll, 1982b). Other observations, however, argue against such a suggestion (Gaiarsa et al., 1995). First, dendritic application of a low concentration of GABA during intrasomatic or intradendritic recording in pyramidal cells produces monophasic hyperpolarizing responses (Wong and Watkins, 1982; Newberry and Nicoll, 1985). Second, GABAA receptors are present on the axon initial segment, soma, and proximal and distal dendrites of principal cells (Houser et al., 1988; Gao and Fritschy, 1994; Nusser et al., 1995). Third, current source density analysis of population IPSPs in the presence of excitatory amino acid receptor antagonists shows that depolarizing IPSPs are evoked only when the amount of released GABA is potentiated by 4-aminopyridine (Lambert et d., 1991). Fourth, paired recordings from interneuron-pyramidal cell pairs reveal hyperpolarizing GABAA IPSPs even when the interneuron innervates only the dendrites of the postsynaptic pyramidal cell. The rise time is slower and IPSP duration is longer when evoked by the interneurons innervating pyramidal cell dendrites as opposed to basket cells (Buhl et al., 1994a; Miles et al., 1996). Because the number of boutons established by presynaptic bistratified cells (n = 6) is similar to that of the basket cells, the slower kinetics of the IPSPs evoked by bistratified interneurons may reflect electrotonic attenuation due to the large distance between the dendritic synapses and the recording site. Another interpretation of the different kinetics of the interneuron-mediated responses is that they activate different subtypes of GABAA receptors at the soma and dendrites (Nusser et al., 1995). The fast GABAA-mediated current enters at or near the soma, decays rapidly (3-8 ms), is blocked by furosemide, and rapidly curtails the excitatory response. The slower GABAA-mediated current enters the dendrites, decays slowly (30-70 ms), and, importantly, is not blocked by furosemide (Pearce 1993). Subunit composition of GABAA receptors may also change as a function of development (Gaiarsa et al., 1995). Recent experiments reveal that spontaneous inhibitory events (miniature IPSCs), resulting from action potential-independent release of GABA, arise mostly around the perisomatic region of principal cells (Solt6sz et al., 1995; Miles et a]., 1996), i.e., through fast GABAA receptors. An important practical implication of this finding is that experimental comparison of tonic inhibition due to spontaneous release of GABA from inhibitory terminals around the soma and evoked synaptic inhibition may be
424
assessing functions of overlapping but different interneuronal populations. A possible conclusion from these experiments is that when only a small amount of GABA is released by the presynaptic terminals, the postsynaptic membrane of the pyramidal cell is hyperpolarized. In contrast, larger amounts of synaptically released or locally applied GABA may result in postsynaptic depolarization (depolarizing IPSP). These dose-dependent differences may be explained by the hypothesis that, at a low concentration of the transmitter, the GABA effect is primarily mediated by C1- ions. During intense GABAA receptor activation, however, the electrochemical gradient for CI- is diminished and the charges are carried mostly by the efflux of bicarbonate anions (Grover et al., 1993; Staley et al., 1995). Therefore, the hyperpolarizing or depolarizing nature of GABA may depend on the amount of transmitter released. The routinely used 95% 0 2 - 5 % COZ perfusion in slice experiments may facilitate the bicarbonate gradient and produce a more positive reversal potential for GABA. When bicarbonate is reduced by Hepes buffer and the slices are perfused by 100% 0 2 , GABA agonists or tetanic stimulation produces only hyperpolarizing responses (Staley et al., 1995). Because the interstitial concentration of CO2 in vivo varies from virtually zero to a relatively high value, depending on the energy consumption of the locally active neurons, the overall activity of surrounding neurons may affect the hyperpolarizing or depolarizing nature of GABA.
VIII.2.b. NPY
In many respects, the effects of NPY appear very similar to that of SOM. Bath application of the peptide suppresses glutamatergic excitation of CA1 pyramidal cells but has no effect on the passive or active properties of the presynaptic CA3 cells (Colmers et al., 1987, 1988; Haas et al., 1987). It has been suggested that NPY also potentiates NMDA-mediated excitatory responses in CA3 pyramidal cells by an action at a sigma or phencyclidine binding site. The peptides NPY (which is active at Yl-Y3 receptors), [Leu31, Pro341NPY (a selective Y1 agonist), and NPYl3-36 (which mimics the effects of NPY in Y2 models) all enhance NMDA-induced activation of the pyramidal neurons in a dose-dependent manner but do not alter the activation of the same neurons by quisqualate (Monnet et al., 1992; de Montigny et al., 1993). A patch-clamp study, however, failed to see these effects in the same hippocampal region (Colmers, 1992). Although the spatial overlap between excitatory inputs and NPY release sites is greatest in the outer molecular layer of the dentate gyrus, no effect of the peptide has been observed on synaptic inputs to the granule cells or on their passive electrical properties (Klapstein and Colmers, 1993). Recent experiments, however, have demonstrated that NPY suppresses depolarization-induced increases in intracellular Ca2+ concentrations in granule cells by inhibiting an N-type calcium current (McQuiston et al., 1996). An interesting implication of this finding is that N-type calcium channels are also involved in the release of dynorphines from the dendrites of granule cells (Simmons et al., 1995), and therefore NPY may be involved in the regulation dynorphin release.
WII.2.a. SOM
Early studies on the effects of SOM reported both excitation and inhibition of pyramidal cells, depending on the dose and the site of application (Dodd and Kelly, 1978; Scharfman and Schwartzkroin, 1988). Perisomatic application of SOM consistently depolarizes pyramidal cells. The physiological relevance of such action is questionable, however, because SOM terminals are absent in the pyramidal cell layer, and it is, therefore, unlikely that interneuron-released peptide would diffuse from release sites at the distal dendrites to the soma. Extracellular studies on evoked field responses failed to observe any effect of SOM application to the apical dendritic layers (H.L. Haas, personal communication). At least part of the intrasomatically observed inhibitory effect is
425
VIII.2.d. V I P
The first attempts at investigating the physiological effects of VIP on hippocampal function failed to demonstrate any changes in pyramidal cell activity (Warnick and Pellmar, 1986). A later study, however, found that submicromolar concentrations of VIP, added to the perfusion medium, enhanced the excitability of CA3 and CA1 pyramidal cells, mostly by blocking the Ca2+ and cyclic AMP-dependent potassium current (IAHP)and spike frequency accommodation (Haas and Gahwiler, 1992). These effects persist in tetrodotoxin-containing medium, suggesting a direct effect on pyramidal cells. GABAergic IPSPs are also enhanced, likely reflecting excitation of some interneurons. The calcium regulatory effects of VIP has also been investigated in cultured rat hippocampal neurons by using the calcium-sensitive fluorescent dye fura-2. VIP increases [Ca2+]i but only at micromolar concentrations (Tatsuno et al., 1992).
rons and converted to changes in firing rate. O n the output side, axon terminals of interneurons are often found in the proximity of blood vessels, and several peptides have been associated with local regulation of metabolic activity and blood flow (Magistretti, 1990). In future research on peptidergic function, it is essential that experiments more directly assess the physiological effects of peptides. A potentially effective and convincing approach in the assessment of peptide function is to record from identified interneuron-principal cells pairs. The physiological effect of peptide release may then be specifically tested with the aid of specific peptide blockers when such compounds are available.
VIII.2.e. SP
Although both hippocampopetal afferents and hippocampal interneurons may release SP (Section IV.3.0, the only physiological study in the hippocampal formation to date has concluded that it has no effect on pyramidal cells (Dodd and Kelly, 1981).
All intrahippocampal and hippocampopetal afferents that terminate on principal cells also innervate interneurons (Buzsdki, 1984). However, very little is known about the way different interneuronal types respond to neurotransmitters and neuromodulators. Neurotransmitter actions on interneurons may be split into two major categories: fast synaptic excitation and inhibition and slow synaptic actions mediated by G proteins. Two neurotransmitters, glutamate and GABA, appear to exert both fast and slow actions via different sets of receptors. T o date, most of the available information is indirectly inferred from studying the evoked or spontaneous GABAergic responses in principal cells. Although such indirect information is important, it typically cannot ident i k the interneuron types responsible for the action, nor can it reveal whether the different types of actions are mediated by the same interneuron or by distinct classes. Direct recordings from identified interneurons and the selective activation of their presynaptic inputs are needed to dismiss such ambiguities. When considering the effects of transmitter-neuromodulator action, it is important to recognize the typical dual actions of several neurotransmitters on both the somadendritic surface and presynaptic terminal. In many cases, the apparent paradoxical and opposing dose-dependent effects of locally or bath-applied neurotransmitters and modulators may be explained by their distinct actions on interneuronal firing and inhibitory presynaptic terminals. GABA may be released into the synaptic cleft spontaneously (i.e., without a preceding action potential in the presynaptic terminal) or be triggered by the action potential-induced depolarization of the presynaptic bouton. Because the size of the action potential-independent IPSPs is much smaller, they are typically referred to as spontaneous miniature potentials (Fatt and Katz, 1952). Both the miniature IPSPs and action potentialevoked IPSPs are attenuated by GABA blockers but tetrodotoxin, which blocks sodium-dependent action potentials, can distinguish the two forms because it has no impact on the miniature IPSPs. Given this scenario, neuroactive compounds and clinically used drugs may modulate inhibition of principal cells by affecting both spontaneous and evoked release of GABA (Mody et al., 1994). To date, numerous studies performed with tissue cultures and brain slices have demonstrated that bath or local applications of
426
neurotransmitters and drugs exert a measurable effect on synaptic release without, or in concert with, modulation of presynaptic K+ and Ca2+ currents. An obvious question in this context, however, is whether and how a given neurotransmitter or neuromodulator affects GABA release in the intact brain because the transmitter-modulator release sites are often spatially distant and the physiologically relevant concentrations are difficult to predict or measure. Because in vivo preparations often do not have the necessary analytical resolution, methods should include combined slice preparations in which afferent neurons can be selectively activated (T6th et al., submitted).
Electrically excitable dendrites in interneurons? The anatomical evidence for a single release site between pyramidal cells and basket cells is difficult to reconcile with the faithful and rapid transmission of action potentials across this synaptic junction (Gulyis et al., 1993b; Arancio et al., 1994; Miles et al., 1996). This paradox may be explained, however, if the interneurori dendrites are electrically excitable (Spencer and Kandel, 1961; Llinas and Nicholson, 1971; Traub and Llinas, 1977; Poolos and Kocsis, 1990; Traub, 1995). Several observations support the possibility of dendritic fast spike initiation in interneurons. First, the amplitude variability of extracellularly recorded interneurons sometimes exceeds SO%, an observation compatible with multiple site generation of action potentials throughout the dendritic arbor. Occasionally, a bimodal distribution of action potentials is observed (Fig. 34). Second, hyperpolarization of hilar interneurons by somatically placed electrodes reduces the amplitude but not the number of population burst-induced action potentials (Michelson and Wong, 199 1). Third, action potentials equally often emanate from the decaying and rising phases of spontaneous EPSPs without a fixed threshold in CA1 interneurons (Fig. 34; Sik et al., 1995), indicating a dissociation of the locally measured membrane potential and the
FIGURE 34. Possible dendritic generation of fast spikes in interneurons. A: Spontaneous activity of a CAI trilaminar neuron in vivo (urethane anesthesia). The boxed area is shown at a higher resolution. Open arrows indicate spike initiation. The spikes are not necessarily triggered at most depolarized levels of the membrane. The dissociation between local depolarizingpotentials and the variability of the spike threshold suggest that fast spikes were initiated at location(s) other than the recording site. B: Four-site recording (tetrode) from a stratum oriens-interneuronin the CAI region in the awake rat. Small (a, arrowheads) and large (b) spikes are present. C: Spikes a and b at a faster scale. Note the double positivegoing spikes in b. D: Spatial clustering of spike parameters revealed two possible units (a and b). E: However, cross correlation between spikes a and b revealed a reliable refractory period, suggesting that both spikes were emitted by the same neuron. An alternative possibility is that spikes a and b were generated by interneurons coupled by gap junctions. From K. Moore, Z. Nidasdy, and G. BuzsAki (unpublished findings).
427
intracellular
extracellular
a versus b
... .I
-6.4
ms
6.4
428
occurrence of action potentials. Fifth, the critical site of the sodium spike generation in pyramidal cells, granule cells, and mossy cells is the axon initial segment (Spruston et al., 1995; Magee et al., 1995b), which is under the control of densely packed GABAergic terminals of chandelier cells (Somogyi et al., 1983a,b, 1985a; Li et al., 1992; Han et al., 1993; Halasy and Somogyi, 1993b). In contrast, the axon initial segment of interneurons is free of synapses (Halasy and Somogyi, 1993b; Ribak et al., 1993; Buzsiki and Sik, unpublished observations), suggesting that the axon initial segment of interneurons may not be as critical as in principal cells. Sixth, a recent computer model of the synaptic transmission between pyramidal cells and interneurons failed to evoke a short-latency action potentials in the interneuron having branching passive dendrites. However, dendritic EPSPs regularly triggered dendritic action potentials when the density of sodium channels in the dendrites was about half of the somatic region (Traub, 1995; Traub and Miles, 1995). Although these observation are compatible with the idea of active dendritic spike generation, determination of the sodium channel distribution along the somadendritic surface and simultaneous patch-clamp recordings are necessary for providing more direct evidence. Activation of interneurons by principal cells is mediated by excitatory amino acids via AMPA, NMDA, and metabotropic receptors. These actions will be discussed below.
FIGURE 35. GluR-mediated effects on interneurons. A: Current-voltage relationship of AMPA receptor and spontaneous mIPCSs in a type 1 interneuron of CA3 stratum radiatum. Ai: Kainate activation of the AMPA receptors show a modest outward rectification. Kainate currents reversed at approximately 0 mV. Aii: Spontaneous mEPSCs recorded in the presence of bicuculline and tetrodotoxin. At a holding potential of -70 mV, mEPSCs are dominated by an AMPA-receptor-mediatedcomponent. At a holding potential of +50 mV, a two-component EPSC was present. P r of the at response was blocked by D-2-amino-5-phosphonopentanoic acid, demonstrating that at positive potentials both AMPA and NMDA receptors are activated in the interneuron. B: Type 2 interneurons of CA3 stratum radiatum possess an inwardly rectifying I-V relationship in response to kainate (Bi). Bii: Averaged spontaneous EPSCs in a type 2 interneuron show only NMDA-receptor-mediated responses at positive potentials. D-APV completely blocked the slow kinetic response at +50 mV, but mEPSCs recorded at -70 mV were not altered. These observations suggest that AMPA receptors possess inwardly rectifying properties at positive potentials. C: 0-LM interneurons in the CA1 region are modulated by presynaptic mGluRs. Ci: Averaged evoked EPSCs are markedly potentiated in the presence of a metabotropic receptor agonist (ACPD). Cii: In addition, ACPD applicationmay induce a large inward current with prolonged oscillatory episodes. Such responses were not observed in CAI basket cells. This figure was kindly prepared by Chris McBain.
429
Ai
6oo
Aii
-600
-900
-80
-40
40
Bi
600
Bii
300
b
+50mV Control
-1500
-80
-40
40
Ci
Cii
50pM ACPD
'
1 0 0 , u M ACPD
430
1990; McBain and Dingledine, 1992; Perouansky and Yaari, 1993; Koh et al., 1995). The time course of the non-NMDA component of EPSCs is similar to that recorded in pyramidal cells. The magnitude of NMDA currents on average is somewhat smaller in interneurons, but the individual variability is high. The smaller size of the NMDA current may be important because it would explain why topical application of NMDA in hippocampal cultures evokes a smaller calcium influx in GAD-positive neurons than in pyramidal cells (Segal and Greenberger, 1992). Nevertheless, these findings demonstrate that interneurons also possess NMDA receptors, and despite the different anatomical structure of the synapses formed by the Schaffer collaterals on the two cell populations (spine versus shaft), their physiological and receptor-pharmacological properties are similar. Owing to their slower kinetics, NMDA channels have a higher probability for synaptic summation than do the fast AMPAreceptor-mediated events, a property that may be particularly important for the maintenance of population synchrony. Interneurons may be activated by NMDA receptors under baseline conditions because the magnitude of recurrently evoked IPSPs in CA1 pyramidal cells is attenuated by NMDA blockade (Grunze et a]., 1996).
431
al., 1995, submitted). However, even pyramidal cells are dye coupled in the epileptic hippocampus both in vitro and in vivo (PerezVelazquez et al., 1994; Penttonen et al., 1995), although the existence of gap junctions among principal cells has yet to be demonstrated. The possible physiological role of interneuronal interactions will be considered further in Section XIV.
IX.3. Acetylcholine
Cholinergic activation also exerts a dual effect on the GABA interneuronal system. Although acetylcholine can directly modulate intrinsic ionic conductances and calcium channels of pyramidal cells and affect presynaptic terminals of principal cells (Kmjevic et al., 1971; Benardo and Prince, 1982; Cole and Nicoll, 1984; Madison et al., 1987; Gahwiler and Brown, 1987), similar effects on interneurons make cholinergic regulation particularly complex. As discussed earlier, interneurons in the stratum oriens-alveus region possess particularly dense cholinergic receptors (Section IV.6). Muscarinic agonists or direct stimulation of cholinergic fibers induce a prominent and sustained increase in the frequency and amplitude of spontaneous IPSCs in principal cells, even when fast excitatory neurotransmission is blocked pharmacologically (Pitler and Alger, 1992; Behrends and Ten Bruggencate, 1993). At least part of the effect is related to excitation of interneurons by modulation of Kt conductances because cholinergic agonists induce a fast muscarinic excitation of interneurons in CA1 alveus-oriens and the pyramidal layer (Recce and Schwartzkroin, 1991). In these same preparations, however, muscarinic receptor activation decreased the frequency of miniature IPSCs, suggesting a cholinergic suppression of GABA release at inhibitory terminals. This latter observation is supported by recent morphological evidence showing high density of muscarinic-2 receptors on the terminals of interneurons that innervate the cell bodies and axon initial segment of pyramidal cells (Section IV.6.d). It remains to be determined whether suppression of GABA release is affected at all terminals of the interneuron or occurs selectively on GABAergic terminals contacting the principal cells. In urethane-anesthetized rats, iontophoreretic application of atropine on putative interneurons reduced their firing rates during theta rhythm, but did not substantially change the discharge rate of successively recorded pyramidal neurons (Stewart et al., 1992). Firing rate changes of putative interneurons have also been reported in aged rats having possible cholinergic deficit (Mizumori et al., 1992). Given the laminar distribution of the cholinergic terminals and the somadendritic segregation of the terminals of the various interneuronal classes, it is unlikely that acetylcholine in the intact brain equally affects all interneuron types. Such discriminative action of acetylcholine is also evident in its presynaptic action because the terminals of basket cells and chandelier cells have an especially high density of muscarinic-2 receptors (Section IV.6.d). These principles are, of course, also valid for the evaluation of other neurotransmitters acting on principal cells, interneurons, and presynaptic terminals, and only direct recordings from interneurons will reveal whether and how the different interneuronal classes are involved in each action of the neurotransmitter.
432
FREUND A N D BUZSAKI
clamp recording from interneurons in the CA1 strata radiatum and lacunosum-rnoleculare revealed fast and large inward currents In vivo studies have suggested that the overall effect of norep- that were attenuated by a 5-HT3 antagonist. Importantly, the efinephrine release in the hippocampus is to suppress pyramidal cell fects of serotonin on interneurons is not blocked by bicucullin or discharge (Segal and Bloom, 1974). The suppressive action of nor- pharmacological blockade of fast excitatory neurotransmission, epinephrine may be mediated disynaptically by direct excitation suggesting a direct action of the neurotransmitter (Kauer and of interneurons. Local application of norepinephrine suppresses McMahon, 1995). the activity of pyramidal cells but excites putative interneurons in Similar effects have been observed in the dentate gyrus. Local vivo. The suppression of pyramidal cells is dominantly mediated application of serotonin elicits and evokes a transient burst of by a 1 receptors. However, a 2 and P-adrenergic agonists excite IPSPs that are blocked by tetrodotoxin or bicuculline and the 5both pyramidal cells and putative interneurons. These findings HT3 receptor antagonist dolasetron (Piguet and Galvan, 1994). raise the possibility that a large part of the norepinephrine effect Focal application of serotonin or a selective 5-HT3 receptor agis mediated by interneurons and that the differential responses onist on visually identified basket cells in the granule cell layer arise from the activation of distinct populations of noradrenergic induced a train of action potentials superimposed on a baseline receptors (Pang and Rose, 1987). These suggestions are supported membrane depolarization. Under voltage-clamp conditions, seroby recent in vitro experiments. The norepinephrine-induced extonin evoked an inward current that was accompanied by a mulcitation of biocytin-filled CAI interneurons is mimicked by an titude of small inward currents of short duration (<I00 ms) a1-adrenoceptor agonist (phenylephrine), persists in the presence caused by serotoninergic excitation of nearby GABAergic presyof an cr2-adrenoceptor agonist (atipamezole), and can be blocked naptic neurons innervating the recorded principal cell. The seroby a selective al-adrenoceptor antagonist (Bergles et al., 1996). tonin-induced effects in presumed basket cells are blocked by spcA P-adrenoceptor-dependent depolarization also occurs in those cific blockers of the 5-HT3 receptor subtype. Kawa (1994) CA1 interneurons that display time-dependent inward rectifica- concluded that the excitatory action of serotonin occurred exclution. The axon arborizations of the filled cells indicate that the sively on GABAergic interneurons. majority of the recorded cells are basket cells. The P-adrenocepIntraperitoneal injection of a 5-HT3 antagonist, ondasetron, tor agonist isoproterenol also increases the spontaneous discharge in the awake rat facilitates the induction of long-term potentiarate of putative hilar interneurons and the frequency of GABAA tion in the CA1 region (Staubli and Xu, 1995). The results supIPSPs in granule cells (Bijak and Misgeld, 1995). Interestingly, port the hypothesis that the drug removes the excitatory effects in the presence of AMPA and NMDA blockers, norepinephrine of serotonin on a subset of hippocampal interneurons. reduces the discharge rate of putative hilar interneurons, indicatOther experiments have suggested that 5-HT1A receptors may ing a complex interaction between interneurons and granule cells also be involved in the interneuron-mediated actions of serotonin or a possible differential effect of norepinephrine on different on pyramidal cells (Segal, 1990b; Ghadimi et al., 1994; Schmitz types of interneurons. Overall, the norepinephrine-induced in- et al., 1995). Evoked fast and slow IPSPs in CAI pyramidal cells crease in the firing frequency of inhibitory interneurons and the are reduced by the 5-HTlA-receptor agonist 8-OH-DPAT, and resulting increase in both the frequency and amplitude of IPSPs this effect is blocked by the 5-HTlA-receptor antagonist NANin their target principal cells may account for the decrease in spon190. These drugs have no effect, however, on GABA-mediated taneous activity of pyramidal neurons following activation of the currents evoked by GABA application to the dendritic or somatic locus coeruleus in vivo. layers, suggesting the involvement of interneurons. Minimal concentrations of serotonin appear to reduce selectively the late, IX.5. Serotonin GABA-B receptor-mediated component of evoked IPSPs in both Actions of serotonin are mediated by second-messenger-linked the CA1 (Segal, 1990b) and CA3 regions (Oleskevich and 5-HT1, 5HT2, and 5-HT4 receptors and membrane-ion- Lacaille, 1992) and in the dentate gyrus (Ghadimi et al., 1994). channel-linked 5-HT3 receptors (Bobker and Williams, 1990). Importantly, putative interneurons impaled in the pyramidal layer Serotonin treatment leads to an enhancement of evoked popula- were hyperpolarized and the evoked EPSPs were substantially retion spikes both in vivo (Klancnik et al., 1989; Richter-Levin and duced by both serotonin and 8-OH-DPAT (Schmitz et al., 1995). Segal, 1990) and in vitro (Ropert 1988), yet it hyperpolarizes These results were interpreted by assuming that 5-HTIA receppyramidal cells (Andrade and Nicoll, 1987; Colino and Halliwell, tors directly inhibit interneurons or reduce glutamate release from 1987) and granule cells (Ghadimi et al. 1994). The hyperpolar- the presynaptic axon terminals onto interneurons. Overall, these physiological data suggest that at least part of izing effects of serotonin may be direct on principal cells or may the action of serotonin on principal cells is mediated by be mediated by excitation of interneurons. It has been inferred from studies on principal cells that a subset of interneurons may GABAergic interneurons in two different ways: serotonin (1) exbe selectively conveying the serotonin-mediated suppression of cites, via 5-HT3 receptors, a subset of hippocampal interneurons, pyramidal cell activity. Serotonin increases both the frequency and thereby producing GABA-A receptor-mediated IPSPs and (2) amplitude of GABA-A receptor-mediated unitary IPSPs in pyra- may reduce GABA release by activating presynaptic 5-HTlA remidal cells. These effects persist when fast neurotransmission is ceptors of hitherto unidentified interneurons. Anatomical eviblocked pharmacologically but is reduced by bicuculline and by dence for a specific serotonergic innervation of different in5-HT3 antagonists (Ropert and Guy, 1991). Whole-cell voltage- terneuron types and for the selective interneuronal expression of
IX.4. Norepinephrine
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IX.6. Opioids
Opiates and the opioid peptide enkephalin may disinhibit hippocampal pyramidal neurons by reducing inhibitory synaptic transmission mediated by GABAergic interneurons (Zieglgiinsberger et al., 1979; Nicoll et al., 1980; Siggins and Zieglgansberger, 1981). Application of met-ENK and opioid agonists decreases the firing rates of putative interneurons in vivo (Zieglgksberger et al., 1979; Raggenbass et al., 1985; Pang and Rose 1989) and hyperpolarizes them in vitro (Madison and Nicoll, 1988). Recent experiments, however, have pointed to the presynaptic effects of opiates (Lambert et al., 1991; Lambert and Wilson, 1993). Both in hippocampal slice cultures and in hippocampal slices, preceptor agonists decreases the frequency of miniature IPSCs without causing a change in their amplitude. At the same time, the amplitude of action potential-dependent GABA release is reduced (Cohen et al. 1992; Reckling, 1993). This reduction suggests a direct action of opioids on the inhibitory presynaptic terminals. Such a presynaptic mechanism may also contribute to the action potential-evoked GABA release by decreasing synaptic inhibition. These presynaptic effects of p opioids do not exclude the possibility that delta or kappa receptors are involved, although iontophoretic and systemic application of a selective kappa-receptor agonist (U50488H) has little effect on either granule cells or putative hilar interneurons in vivo (Mayer et al., 1994). Future experiments should reveal the types of interneurons affected and whether different receptor subtypes are involved in the somadendritic and presynaptic effects of opioids.
feed-forward inhibition. Groups of interneurons tonically or phasically hyperpolarize and/or increase membrane conductance (shunting) in the perisomatic and/or dendritic regions of neurons and thereby decrease the efficacy of excitatory afferents in discharging their principal cell targets. Activation of hippocampal interneurons may be brought about by extrahippocampal inputs, by intrahippocampal inputs afferent to interneurons (both feed-forward), or by principal cells of the same hippocampal region (recurrent or feedback). In the feed-forward regulatory system, afferent volleys directly activate the inhibitory neuron (first went) that in turn reduces the probability of firing of the principal cells (second event). In the feedback system, an excitatory input discharges the principal cells, whose excitatory output is fed hack to the inhibitory cell(s) through recurrent axon collaterals (Andersen et al., 1964). The inhibitory interneuron(s) then may discharge and inhibit a group of principal cells, including those that initially activated the interneuron(s). In short, the directions
of firing rate changes of local principal cells and inhibitory interneurons are the same in the feedback systems but opposite in the feed-forward scheme (Buzsiki, 1984). It has been hypothesized that all extrahippocampal and intrahippocampal pathways that terminate on principal cells also innervate hippocampal interneurons (Buzsiki, 1984). Some interneuron subtypes are innervated exclusively by extrahippocampal afferents (e.g., MOPP cells, LM cells; Section 111.3) and are therefore part of the feed-forward mechanism only (Fig. 36). Most of these interneurons are located in the stratum lacunosum-moleculare and the dentate molecular layer. However, the majority of interneurons in strata pyramidale, granulosum, oriens, and hilar regions are innervated by both intraregional and extraregional and by extrahippocampal inputs and are therefore part of both feedback and feed-forward mechanisms (Buzsiki, 1984; Lacaille et al., 1987). Basket cells are regarded as the arche type of feedback (recurrent) interneurons, even though other inrerneuron types (e.g., trilaminar interneuron, 0 - L M cells, HIPP cells) may better fit such requirements (Blasco-Ibanez and Freund, 1995; Sik et al., 1995, submitted). Putative and anatomically identified basket cells are bidirectionally connected to principal cells (Lacaille et al., 1987; Buhl et al., 1994a). Because the time required for the onset of recurrent inhibition in pyramidal cells is shorter than the latency of the action potential-induced depolarizing afterpotential, repetitive spiking can be prevented altogether (Miles et a]., 1996). Feed-forward inhibition is particularly strong in the hippocampus, although this is often not appreciated when electrical stimulation is used. An obvious problem with artificial stimulation is that recruitment of inhibition and excitation are nonlinearly coupled and the relevant physiological patterns are not mimicked properly by stimulation. Low-intensity stimulation as a rule evokes only feed-forward inhibition in all pathways of the hippocampal formation due to the lower discharge threshold of interneurons (Buzsiki, 1984). Additional excitation may or may not override the inhibition, depending on the afferents involved. For example, stimulation of the entorhinal-CA1 path (Bragin and Otmakhov, 1979; Doller and Weight, 1982; Yeckel and Berger, 1990; Colbert and Levy, 1932; Soltksz et al., 1993; Buzsiki et al., 1995), commissural-dentate gyms path (Deadwyler et al., 1975; McNaughton et al., 1978; Buzsiki and Czeh, 1981; Buzsiki and Eidelberg, 1981, 1982), and the dentate associational path (Scharfman, 1394, 1995b) typically evokes hyperpolarization in the target principal cells due to the strong feed-forward inhibition parallel with direct excitation (Fig. 33). Nevertheless, activation of these same pathways by natural stimuli may excite and discharge target cells, probably by suppressing feed-forward activation of the interneurons (Buzsiki et al., 1994, 1995).
Interpretations
When interneurons are serially connected, it is assumed that increased activity of the primary interneuron will lead to increased firing of the target of the secondary interneuron through a process generally referred to as disinhibition (Fig. 37). As an example,
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submitted). Because the in vitro experiments were done in the presence of atropine and AMPA and NMDA blockers, it is very likely that septal GARAergic afferents innervating hippocampal interneurons were responsible for the effect (Section V. 1). The suppression of hippocampal interneurons by septal GABAergic cells may be responsible for the enhancement of granule cell discharge (Fantie and Goddard, 1982; Bilkey and Goddard, 1985). Whereas such stimulation studies will continue to provide important information about connectivity, they often fail to explain firing patterns of populations of neurons. For example, both puCAI pyr tative septal GABAergic neurons and hippocampal iqterneurons increase their firing frequency during hippocampal theta activity, and this relationship holds after pharmacological blockade of the septohippocampal cholinergic afferents (Buzsjki et al., 1983; Stewart and Fox, 1989, 1990). As discussed in Section VI.2, CBimmunoreactive interneurons in CA1-CA3 stratum oriens project back to septal GABAergic cells, and such a feed-back loop may substantially modify the firing relationship of neurons during normal physiological operations (Fig. 37). Another type of observation that is not easily compatible with simple schemes of CAI rad inhibition-disinhibition is that when networks are entrained into oscillations of various frequencies, interneurons and principal cells often discharge with virtually no time lag (Fox and Ranck, 1986; Bragin et al., 1995b; Ylinen et al., 1995a). O n the basis of Boolean logic (Fig. 37), one might predict that interneurons should discharge with some delay after the principal cells. In interconnected networks of interneurons, even the direction of frequency changes of individual neurons is hard to predict. These examples aim to illustrate that, although inhibition and CAI rad disynaptic disinhibition continue to be useful concepts in the description of physiological effects of interneurons, application of Boolean logic often fails to provide correct predictions in systems where interneurons are interconnected with each other (Fig. 37, Moser, 1996). In networks of interneurons, independent of whether members are connected unidirectionally or mutually, 0scillatory activity often emerges (Perkel and Mulloney, 1974; Wang and Rinzel, 1993; Bragin et al., 1995a; Whittington et al., 1995; Wang and Buzsriki, in press; Traub et al., 1996), and the rules that 100 0 msec 100 govern the timing of action potentials and the frequency changes FIGURE 36. Interneuronal activity during sharp wave-related of the participating cells can no longer be inferred from the Boolean ripples in the CAl region. Averaged field activity reveals a short- logic of inhibition and disinhibition (Sections XIV. 1). lived 200-Hz field oscillation (trace). Top histogram: Auto-correla-
CAI pyr
. I
tion of spikes from a putative basket cell in the pyramidal layer. Note increased activity during field ripple and phase modulation of the cell discharge with individual ripple waves. Middle and bottom histograms: Autocorrelations from unidentitied fast fuing interneurons in the stratum radiatum. Interneurons that decrease their discharge frequency or remain unaffected during ripples are very rare.
In Vivo
What is the physiological implication of the powerful connectivity between principal cells and interneurons? The in vitro data indicate that the discharge of single interneurons can modify the phase of intrinsic oscillations in its target pyramidal cells and/or prevent their firing altogether (Cobb et al., 1995; Miles et al., 1996). Conversely, a discharging pyramidal cell may induce firing in many of its target interneurons (Gulyk et al., 193313). Because a single pyramidal cell innervates hundreds of interneurons and interneurons in turn innervate 1,000-3,000 pyramidal cells (Li et al., 1992; Buhl et al., 1994a; Sik et al., 1995), a discharging single pyramidal neuron is expected to result in inhibition of tens of thousands of pyramidal cells. Such reasoning may
stimulation of the medial septum enhances population discharges of granule cells in response to perforant path activation (Fig. 38; Alvarez-Leefmans and Gardner-Medwin, 1975). Tetanic stimulation of the medial septal area reduces the frequency of ongoing IPSPs in pyramidal cells, which is associated with an increase of input resistance (Krnjevic et al., 1988). Similarly, stimulation of the septal area in a combined septdhippocampal slice suppresses spontaneous IPSCs in pyramidal neurons (Fig. 38; T6th et al.,
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Inhibitory circuits may modify the long-term excitability of principal cells in several ways. It has been long recognized that inhibition plays a critical role in electrically induced synaptic plasticity because bath application of GABAA antagonists enhances postsynaptic depolarization brought about by the afferent tetanus and consequently facilitate the induction of long-term potentiation (Bliss and Lomo, 1973; Wigstrom and Gustafsson, 1985;
1
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FIGURE 37. Inhibition, disinhihition, and problems of interpretation in interneuronal networks. A: The Boolean interpretation of inhibition and disinhibition in a simple serial circuitry. Activation of a primary GABAergic interneuron (i) inhibits the secondary interneuron. As a result, the tonic background inhibition onto the principal cell (p) is removed, i.e., the principal cell is disinhibited. B,C: Ambiguity of firing changes in interconnected networks. B: Simple case. Secondary interneurons are unidirectionally connected (il-i2). Excitation of the primary interneuron (i) inhibits secondary interneurons (il, 2 ) . In turn, the targets of i l become disinhibited. However, the discharge rate changes of interneuron i2 depend on the relative strengths of i-i2 and il-i2 connections. As a result, targets of i2 may be either disinhibited or more inhibited. In the example, dendritic disinhibition may be coupled with increased or decreased somatic inhibition. Such circuitries exist in the hippocampus (see D). C: With increased interneuronal connectivity, firing changes of principal cells are more complex. If the synaptic strengths are different (e.g., arrow), subpopulations of principal cells (pl, p2) may show differential or even opposite changes. D: Similar interneuron types may be reciprocally connected (e.g., basket cells; open arrows), whereas some interneuron types are unidirectionally connected to other interneurons (e.g., NPY-immunoreactive cells in hilus; filled arrows). Both interneuron types may be innervated by a common inhibitory input (e.g., septal GABAergic afferents). Such master-slave configurations allow for an immediate synchronization and phase locking of a large number of principal cells (gray triangles), but the direction of their firing changes is ambiguous.
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postsynaptic sensitivity to GABA. It is also important to stress that enhancement or decrease of inhibition in LTP-LTD protocols is not necessarily associated with direct modification of the inhibitory circuitry (Maccaferri and McBain, 1995).
Abraham et al., 1986, 1787; Bliss and Collingridge, 1993). Conventional wisdom held that long-term alteration of synaptic transmission took place only at excitatory synapses, and inhibitory interneurons at best only modulated such changes. This assumption is based on the theoretical expectation that modification of synaptic strength among principal cells is the basis of specific associative memory and on the generally accepted view that both long-term potentiation (LTP) and long-term depression (LTD) depend on activity-dependent Ca2 increase in the postsynaptic neurons (Bliss and Collingridge, 1993; Lisman and Harris, 1993; Singer, 1975). However, recent experiments have suggested that GABAergic synapses on principal cells and interneurons may also undergo long-term modifications (cf. Marty and Llano, 1995). The interneuron circuitry may be modified in a number of ways, including (1) presynaptic changes of excitatory terminals on interneurons, (2) modification of the postsynaptic sites on inpresyterneurons, (3) excitability changes of interneurons, (4) naptic modification of GABA release, and (5) changes in
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oriens, which failed to support LTP by stratum radiatum stimulation. Obviously, many technical issues must first be clarified before we can confidently state which interneuron types, if any, display reliable synaptic plasticity. Much less is known about the conditions that may induce LTD of synaptic excitability in hippocampal interneurons When tetanic stimulation is applied to CA1 stratum radiatum, EPSCs in interneurons recorded in the same layer are depressed. This depression is coupled to the enhancement of evoked responses in pyramidal neurons. A second, unstimulated pathway onto the same interneuron is also depressed, suggesting a postsynaptic locus of depression (McMahon and Kauer, 1995). These observations support previous findings in the CA3 region that showed tetanus-induced depression of IPSPs in simultaneously recorded pyramidal cells (Miles and Wong, 1987b). When using the lowfrequency stimulation paradigm (1 Hz, 10 min) and whole-cell or perforated-patch techniques, Maccaferri and McBain (1995) failed to induce LTD in CA1 stratum oriens interneurons, some of which were identified as 0 - L M cells. Although they regularly observed reduction of EPSPs in response to stratum radiatum stimulation, such a change most likely reflected primary changes in the pyramidal cells, and the decreased EPSPs in the interneurons simply passively reflected LTD of the principal cells. In some cases, high-frequency stimulation was also used and failed to induce LTP in 0 - L M cells. Because Schaffer collaterals do not innervate 0-LM cells (Blasco-Ibanez and Freund, 1995; Maccaferri and McBain, 1995; Sik et al., 1995), the lack of LTP in response to stratum radiatum stimulation is not surprising. However, LTD of 0 - L M cells also failed when low-frequency stimulation was applied to the stratum oriens (Maccaferri and McBain, 1995, 1996). It is noteworthy that the prolonged low-frequency activation paradigm is not effective in producing LTD of pyramidal cells in vivo (Thiels et al., 1994). A possible source of the contradiction may be the differences of the inhibitory circuits in vivo and in vitro. Another explanation may be an age difference of the subjects used in these studies. Recent in vitro experiments suggest that LTD is very difficult to induce with low-frequency stimulation in slices derived from adult animals, whereas it is quite robust in slices from juvenile rats (T.C. Foster and R.F. Thomson, personnal communications). The findings that induction of LTP in interneurons follows the same cellular mechanisms as those in principal cells, including NMDA-receptor activation and intracellular calcium elevation, may have important implications for understanding its underlying mechanisms. Dendritic spines have been suggested to play a critical role in the LTP of pyramidal cells (Lisman and Harris, 1993), yet spines are rare in most interneuron types. However, postsynaptic receptors and numerous postsynaptic mechanisms similar to those of principal cells may account for LTP in at least certain subclasses of interneurons.
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Importantly, the activity of all recorded pyramidal cells were also suppressed on entering the new environment; thus, it could not be excluded that the decreased interneuronal activity was a consequence of their reduced recurrent activation. During hippocampal sharp waves (Section XI11.4), population bursts of principal cells coincide with the most rapid firing of the majority of interneurons. Some interneurons in stratum radiatum may transiently discharge up to 800 Hz (Buzsiki et al., 1983), whereas others may not be affected or even decrease their activity (Fig. 36). What might be the advantage of such intense collective activity in both principal cells and interneurons? As discussed above, potentiation of interneurons may be associated with diminished inhibition of principal cells in LTP paradigms, and the large amounts of GABA may lead to depolarization in target principal cells. O n the basis of in vitro evidence, we may speculate that intensely discharging interneurons during hippocampal or entorhinal sharp waves (SPWs) may in fact induce a hyperpolarizing-depolarizing sequence in pyramidal cells. Hyperpolarization followed by depolarization is the appropriate sequence for de-inactivation of low-threshold calcium channels in pyramidal cell dendrites (Magee and Johnston, 1995b). Therefore, intense recurrent excitation via AMPA receptors and GABA-mediated depolarization during SPW bursts may potentiate the activation of NMDA channels and lead to the cooperation of voltage-dependent and NMDA-dependent calcium influx into the dendrites. Testing the reality of this scenario will require anatomical identification of fast-firing interneuron types, voltage-clamp monitoring of pyramidal cell dendrites during hippocampal SPWs, and paired recordings from the interneuron type(s) in question and pyramidal cells. From this perspective, activation of interneurons with dendritic targets could either prevent or facilitate synaptic plasticity, depending on their cooperative synchrony and firing frequency (Lambert and Grover 1995; Miles et al., 1996).
Different population patterns, as reflected by spontaneous field potentials and rhythms, are present in the hippocampal formation, including theta activity and associated gamma patterns (40-100 Hz oscillation), hippocampal SPWs, and associated high-frequency (200 Hz) oscillation (ripple) and dentate spikes (cf. OKeefe and Nadel, 1978; Buzsiki et al., 1983, 1994; Bland, 1990; Bragin et al., 1995b). Interneurons appear critically involved in the induction and maintenance of network oscillations in the theta, gamma (40-100 Hz), and ultrafast (200 Hz) frequency ranges and regulate the recruitment of principal cells during SPW bursts (Buzsiki et al., 1983, 1992; Fraser and MacVicar, 1991; Soltksz and Deschenes, 1993; Bragin et al., 1995a; Minen et al., 1995a,b).
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(Buzsriki and Eidelberg, 1983; Buzsiki et al., 1983). Phasing of hippocampal interneurons at the theta frequency may derive from (1) rhythmic excitation by the septal cholinergic input, (2) rhythmic excitation coupled with rhythmic inhibition of the same interneurons by the septal GABAergic input, (3) tonic cholinergic and/or glutamatergic activation coupled with rhythmic modula-
extracellular
FIGURE 39. Rhythmic bursts of basket cells hyperpolarize pyramidal cells during theta. A: Simultaneous recording of extracellular theta activity in the CA1 pyramidal layer (extracellular) and intracellular activity of a basket interneuron. Action potentials are clipped. An epoch with poor extracellular theta w s chosen to aca centuate the very regular intracellular rhythm. Note rhythmic 30-60Hz bursts of action potentials with every theta cycle. B: Camera lucida drawing of the dendritic tree and axon collaterals of the basket cell in the pyramidal layer. C: Evoked field (upper) and intracellu-
lar responses to commissural (c) stimulation. The basket cell fired earlier than the peak of the population spike (arrow) and emitted three fast spikes. D: Simultaneous recording of extracellular electroencephagraphic activity in the CA1 pyramidal layer (extracellular) and intracellular activity of an identified pyramidal cell. Note hyperpolarization of the pyramidal cell membrane at the onset of spontaneous theta activity (arrow). Dotted line, -60-mV resting potential. From Ylinen et al. (1995b).
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FIGURE 40. Relationship between theta phase and interneuronal activity in the behaving rat. A: Sinusoid wave represents a single theta wave. B: Distribution of maximal-firing phases of interneurons in the CAI region. Most cells were recorded from stratum oriens (data adopted from Buzs& et al., 1983). C: Distribution of maximal-firing phases of interneurons in the CAI region. Most cells were recorded from the pyramidal layer (data adopted from Skaggs et al., 1996). Note bimodal distribution of phase relationshipin both studies. Although individual interneurons are strongly modulated and their phase-locking to theta waves is quite constant, the phase relationship may vary among the interneuron groups.
Deschenes, 1993; Lee et a!., 1994). In the intact rat, the combination of the pacemaker inputs from the septum (mechanisms 1-3) and the resonant intrinsic and network properties of hippocampal interneurons and principal cells (mechanisms 4 and 5 ) contributes to the prominent entrainment of hippocampal neurons during theta behaviors (Buzsiki et al., 1983) and to the production of field theta activity. Although nearly all interneurons express rhythmic discharges during theta, their relationship to the phase of the theta cycle shows a large variability (Buzsiki et al., 1983; Fox and Ranck, 1986). Identified basket cells in the CAI region discharge on the phase opposite to the maximal activity of the pyramidal cells in the urethane-anesthetized animal (Fig. 39). The basket cells are rhythmically hyperpolarized by either the septa1 GABAergic input (Freund and Antal, 1988) or by other hippocampal interneurons w i n e n et al., 1995b). In turn, they hyperpolarize their target pyramidal cells at the theta frequency (Fig. 39). In the awake rat, interneurons in the CAI oriens and pyramidal layer show a bimodal distribution with respect to the phase of theta (Fig. 40),
and it is logical to assume that these subgroups represent morphologically and functionally separate sets of interneurons, although their anatomical identity has yet to be revealed. The discrepancy of the phase relationship of interneurons to the theta cycle in the anesthetized and drug-free animals (Fox and Ranck, 1986) may be explained by the suggestion that recurrent excitation of interneurons is very sensitive to blockade of NMDA receptors (Grunze et al., 1996). Therefore anesthetics, such as ketamine and urethane, may attenuate principal cell-interneuron feedback. Experiments on the regulation of theta activity have indicated a complex interaction between interneurons and target principal cells. Rhythmic activation of identified basket or chandelier cells at the theta frequency in vitro instantly phase lock simultaneously recorded postsynaptic CA1 pyramidal cells (Cobb et al., 1995). During the entrainment, the interneurons and pyramidal cells fire on alternate phases, as in the urethane-anesthetized rat (Buzsiki and Eidelberg, 1983; Ylinen et al., 1995b). Subthreshold membrane oscillations, induced by depolarization of the pyramidal cell, are also entrained. Furthermore, pacing of hippocampal interneurons by rhythmic stimulation of the septohippocampal GABAergic pathway in a combined septum-hippocampus slice preparation also induced theta oscillations in pyramidal cells (T6th et al., submitted). These observations indicate that regulation of intrinsic conductances of their targets is as fundamental a function in the repertoire of interneurons a s their membrane polarizing and shunting effects (Cobb et al., 1995). Resonant properties of some interneuron types may further enhance the hippocampal network to respond to rhythmic subcortical inputs. 0 - L M cells have been shown to fire very regularly at theta frequency even in the absence of fast synaptic transmission (McBain, 1994). A possible candidate for such intrinsic, pacemaker conductances is the hyperpolarization-activated current, IH (Maccaferri and McBain, in press). Rhythmic cooperative discharge patterns of interneurons, evidenced by field theta waves, show a predictive relationship with behavior. They also display some limited correlation with the animals spatial position, suggesting that they receive a disproportionally stronger excitation from spatially firing pyramidal cells (OKeefe and Nadel, 1978) than from other inputs (Barnes et al. 1991). These observations offer the possibility that local interactions between interneurons and principal cells may allow substantial deviation from average population behavior. From this perspective, interneurons are not only responsible for a global control of the principal cells but may also be involved in finely tuned local regulation of their principal cell targets.
442
tum radiatum, hilus) and physiological correlates. In the absence of theta waves, antitheta neurons fire very regularly in a "clocklike" manner, typically 15-40 Hz (Fig. 41). Their firing rate becomes especially regular after inactivation of the medial septum, suggesting that subcortical inputs tonically suppress these neutons. Alternatively, antitheta cells may be inhibited by other hippocampal interneurons. At least some antitheta cells exhibited spatial selectivity (Mizumori et al., 1990).Although small in number, given their peculiar relationship to network activity, expected distinguishing intrinsic properties, and differential subcortical and hippocampal interneuronal innervation, it would be especially rewarding to reveal the anatomical identity of antitheta interneurons.
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FIGURE 41. Interactions among interneurons during theta attivity. Simultaneous recordings of three interneurons (1-3) in the hilar region. Interneurons 1 and 2 fired rhythmically during wakingrelated theta activity, whereas interneuron 3 (antithetacell) was completely suppressed. Cessation of discharge of interneuron 1 (open arrow) coincided with the discharge of the antitheta cell. Interneuron 2 continued its periodic discharge beyond interneuron 1 (dots be-
low trace 2). When interneurons 1 and 2 stopped discharging (filled arrow), the antitheta cell fired regularly at 18 Hz. Such a reciprocal relationship may be brought about by common inputs to the two cell types acting on different receptors or theta-related interneurons may be innervating antitheta cells. Antitheta cells constitute fewer than 1% of all interneurons (Buzsiiki et ai., 1983; Colom and Bland, 1987; Mizumori et al., 1990).
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FIGURE 42. Theta modulation of gamma activity. a: Gamma activity in the hilar region during exploration. Upper trace: Wide band recording. Middle and lower traces: Gamma activity and unit firing (500 Hz-5 kHz), respectively. Note both theta- and gammarelated modulation of the isolated neuron. b: Gamma-triggered average of the wide band activity. c: Cross correlogram of unit firing and peaks of the gamma waves. Note the relationship between av-
eraged field (b) and unit discharges (c). d: Coherence of gamma activity in the longitudinal axis of the hippocampus. Electrodes 1-7 are indicated by black dots. e: Superimposed traces of simultaneously recorded signals from the hilus in the longitudinal axis (electrodes 1-7 in d). Note near-zero phase shift of both theta and gamma in the longitudinal axis.
stratum oriens interneurons are not innervated by basket cells, chandelier cells, or CA3 pyramidal cells, the delay may reflect their recruitment into the population oscillation by the CAI pyramidal cells. Interneuronal communication through gap junctions has been suggested to be critical for providing coherent synchrony of ripples throughout the CAI region and subiculum (Ylinen et al., 1995b; Traub 1995; Chrobak and Buzsiki, 1996). The majority, but not all, interneurons are activated during SPW-associated population bursts. A subgroup of cells in CA1 stratum radiatum does not change its discharge rate during the population bursts, although the group is phase modulated by the theta rhythm (Fig. 36). Obviously, these interneurons are not innervated by the axon collaterals of either CA1 or CA3 pyramidal cells. Yet another type of cell in CA1 stratum radiatum specifically decreases its discharge frequency during SPW bursts (Urioste et al., 1992). It is tempting to speculate that suppression of activity in this cell type is brought about by the discharging bistratified, trilaminar, and back-projection neurons and VIP- or CR-immunoreactive interneurons (Acsidy et al., 1996a,b; Gulyh et al., 1996) because axon collaterals of these cells course in stratum radiatum and terminate on other interneurons. Another unexplored mechanism for the participation of the interneuronal
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FIGURE 43. Coherent discharge of interneurons time the occurrence of pyramidal cells action potentials. a: Single trace of wide band recording from the C A I pyramidal layer. Putative basket cell recorded in the awake rat. Note rhythmic discharge of the interneuron phase locked to the field oscillation. b: Intracellular recording from a CAl basket cell during ripple activity. Note phaselocked discharge of the interneuron to the extracellular field. Arrows indicate spike failures and rhythmic membrane oscillation (urethane anesthesia). c: Relationship between high-frequency field oscillation and neuronal spike activity. Simultaneous recording from 40 pyra-
midal cells and 2 putative interneurons (i at electrodes 4,5). Averaged field event (bottom) and superimposed spike events in the CAI pyramidal layer. d: Cross correlogram between field oscillation and summed pyramidal cell activity (PYR) and interneuronal firing (INT). Continuous line indicates Gabor function curves. Modified from Ylinen A, Sik A, Bragin A, NBdasdy Z, Jand6 G, Szab6 I, Buzsdki G, (1995) "Sharp wave-associated high-frequency oscillation (200 Hz) in the intact hippocampus: Network and intracellular mechanisms." J Neurosci 1530-46 by permission of Journal of Neuroscience.
types in SPW bursts is the hippocampal interneuron-medial septum CABAergic loop (Sections IV.2.b, V). Clearly, the precise relationship between population rhythms and interneuronal cell types awaits further experimentation.
The first significant interaction between systems neuroscience and cellular neuroscience has occurred in the field of neuronal oscillations. The recognition that neurons are not simply "integratcand-fire" devices but are endowed with different intrinsic ligandand voltage-dependent mechanisms (cf. Llinas, 1988) has begun to exert an impact on hypotheses of network activity. The "pace-
maker-follower'' schemes are gradually being replaced by models based on interactions between the emerging network properties and resonant behavior of the participating neurons. At the very least, this new effort provides an alternative approach to such very complex issues, such as perception, cognition, movement initiation, and memory (Murthy and Fetz, 1992; Buzsiki et al., 1994; Gray, 1994; Llinas et al., 1994; Singer, 1994). Although the neuronal mechanisms of communication between neocortical and hippocampal representations could not be realistically addressed by any previous physiological theory, coupling via network oscillations is a novel and unexplored possibility. A hypothesis that was advanced in connection with these issues suggests that networks of inhibitory interneurons impose a coordinated oscillatory "context" for the "content" carried by networks of principal cells. The hypothesis implies that GABAergic neuronal "super networks" may cooperatively entrain large populations of
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larization, so that the synaptic action always produces hyperpolarizations. The frequency of gamma oscillation can be regulated by several factors, including the synaptic decay time constant, the eficacy of GABAAsynapses (i.e., the amplitude of IPSCs), and the driving currents to the interneurons. In contrast with the monotonically varying frequencies of single interneuron firing rates (0-400 Hz) brought about by these variables, the degree of network synchronization shows a relatively narrow peak in the gamma frequency range (20-70 Hz). In the slice experiment, thiopental was used to prolong the GABAA time constant, the GABAA conductance was varied by bicuculline and diazepam, and the driving current was increased by increasing doses of L-glutamate. In general, there was a good fit between the experiments and the model. In vivo, field gamma tails, associated with phase-locked interneuron discharges, are observed in the absence of principal cell firing between epileptic spikes (Traub et al., 1996). The gamma frequency of the interneuronal network can be modulated by slow driving currents, enabling the interneurons to express gamma and theta rhythms at the same time, as occurs in the intact animal (Buzsiki et al., 1983; Bragin et al., 1995a; Ylinen et al., 1995b). Although modeling studies assumed, based on preliminary physiological observations (Whittington et al., 1995; Ylinen et al., 1995b), that basket cells are involved in gamma oscillations, the contribution and importance of other interneuron types, particulary those involved in selective innervation of other interneurons (Section I11.4), have yet to be examined. In addition, in the modeling studies the connectivity among the interneuronal pool is statistically homogeneous and random, whereas in the hippocampus the different interneuronal types are rarely interconnected symmetrically. For example, basket cells and chandelier cells may not innervate other interneuron types, but they themselves are heavily interconnected and are innervated by other interneuron types (Fig. 37). The ubiquitous nature of gamma oscillation in numerous central structures (cf. Gray, 1994) may be explained by the assumption that physiological properties of interneurons and their connectivity and effects on target cells have been preserved during phylogeny.
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GABAergic neurons in the basal forbrain are reciprocally connected to hippocampal interneurons (Sections V. 1, VI.2). Moreover, these basal forebrain GABAergic neurons also innervate inhibitory neurons of the neocortex (Freund and GulyPs, 1991; Freund and Meskenaite, 1992) and the GABAergic neurons in the reticular nucleus of the thalamus (Asanuma and Porter, 1992). In such widespread interneuron-controlled networks, timing of principal cell action potentials within and outside the hippocampal formation may be determined by a combination of the oscillatory phase (i.e., the level of inhibition) and the strength of the excitatory drive. Stronger excitation produces a larger phase advance of the occurrence of the action potentials. In other words, the information about the integration of excitatory inputs to a given cell is expressed by the timing of the action potentials with A respect to average population oscillation (Fig. 44). formal computation model building on similar ideas has recently been published (Hopfield, 1995). Phase precession of principal cell discharge relative to the population theta rhythm has been observed experimentally (OKeefe and Recce, 1993; Skaggs et al., 1996). The spikes of granule cells and CA1 pyramidal cells advance to earlier phases of theta as the rat passes through the cells place field (i.e., region of spatially restricted firing), and the magnitude of phase advance is a function of the animals position within the place field. Simultaneously
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Oscillation of inhibitory networks provides a clock signal for timing of action potentials of principal cells. The hypothesized distributed clock leads to a zero-phase lag of inhibitory subroups (gray ticks indicate action potentials of interneurons). As a result, the membrane potential of spatially distant principal cells are rhythmically hyperpolarized in a highly coherent fashion (sinusoid wave indicates rhythmic hyperpolarization of principal cell membrane potential). Tonic or ramplike excitatory inputs may therefore produce rhythmic discharges of principal cells (black ticks correspond to discharging principal cells highlighted in black in the embedded network); the stronger the excitation, the earlier the phase advance in discharge (top four cells). Phasic inputs discharge the principal cells irregularly, but, again, timing is determined by a combination of the oscillatory phase (i.e., the level of inhibition) and the strength of the
principal cell
FIGURE 44.
excitatory drive (bottom four cells). In this scenario, coherent membrane oscillations, mediated by the interconnected inhibitory network, is a necessary but not sufficient condition for representing information. Information is assumed to be embedded in the temporal relationship of action potentials of principal cells during a given cycle. Oscillations of two or more different frequencies (e.g., theta and gamma) can co-occur and may represent different time scales for different representations. The frequency of the distributed clock is determined by the excitatory (local and subcortical) inputs impinging on the interneurons, the inhibitory inputs from other interneurons, and the intrinsic oscillatory properties of interneurons. Reproduced Buzsiki G, Chrobak JJ (1995) Temporal structure in spatially organized neuronal ensembles: a role for interneuronal networks. Curr Opin Neurobiol5:504-510 by permission of Current Biology Limited.
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XIII.4. Physiological Roles of Interneurons: Separation of Fast Spike Transmission From Plasticity
Different inhibitory cell types target precisely specified areas of pyramidal cell membrane (Han et al., 1993; Gulyis ec al., 1993b;
Buhl et al., 1994a; Sik et al. 1995). Perisomatic connections typically involve multiple, closely spaced contacts, whereas a dendritic axon generally contacts multiple dendritic branches of a target pyramidal cell. Membrane channels controlling pyramidal cell excitability are also expressed in a segregated fashion. Sodium channels have a high density in axon initial segment and soma, whereas calcium channel expression is primarily dendritic (Hamill et al. 1991). Thus, there is a striking cosegregation of specialized interneuron groups and the generations sices of the two kinds of action potentials in pyramidal cells. A hypothesis has been submitted that dendritic inhibitory cells control calcium-dependent electrogenesis, whereas somatic inhibitory cells regulate generation of sodium-spikes (Traub et al., 1994; Miles et al., 1996). Paired recordings from presynaptic basket cells and postsynaptic CA3 pyramidal cells reveal that the recurrent inhibitory circuitry is typically faster than the peak of the depolarizing afterpotential following a spike. The result of this well-tuned timing mechanism is that afferent excitation typically evokes only a single spike in the pyramidal cell because the fast somatic IPSP (4-10 ms after the spike), brought about by the recurrent interneurons, suppresses further firing (Fox and Ranck, 1981; Buzsiki and Eidelberg, 1982; Miles 1991; Miles et al., 1996). Thus, a main objective of perisomatic inhibition is timing and suppression of sodium spikes generated in the axon initial segment. What then is the function in spike genesis of interneurons with dendritic targets? It is generally assumed that dendritic shunting can locally filter or divide the efficacy of local excitatory inputs. Such a function may be brought about by cooperative action of several interneurons because, in contrast to basket cells and chandelier cells, interneurons with dendritic targets typically do not innervate the same dendrites with multiple synapses. In addition, such cooperative action may also affect calcium spike genesis (Miles et al., 1996) because this is where calcium spikes are initiated (Wong et al., 1979; Spruston et al., 1995). Intradendritic recordings from CA1 pyramidal cells in vivo reveal that calcium spikes can be delayed, prevented, or aborted by commissural activation of interneurons, without affecting sodium spike genesis (Fig. 45; Buzsiki et al., 1996). The studies in vitro have used minimal stimulation of inhibitory fibers in the presand 2-amino-5ence of 6-cyano-7-nitroquinoxaline-2,3-dione phosphonovaleric acid to block excitatory transmission. Surgical cuts in the slice assured that perisomatic inhibition was not activated by dendritic layer stimulation. Single pulses delivered to somatic layers delay or suppress repetitive discharge of fast sodium spikes. In contrast, single stimuli in dendritic zones (s. radiatum) suppress the discharge of calcium-dependent spikes initiated by current injection in dendritic recordings (Fig. 46; Miles et al., 1996). In addition, stimulation in the distal dendritic layers reduce the spike-associated [CaZf]i changes in the distal dendrites but have little effect on the changes in the cell body (Tsubokawa and Ross, in press). A possible explanation for these observations is that activity of certain incerneurons that target principal cell dendrites can selectively interfere with dendritic calcium spike genesis while leaving sodium spike generation-propagation unaffected (Traub et al., 1994; Miles et al., 1996). An alternative explanation is based on demonstrations that the dendritic mem-
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brane actively supports propagation of action potentials in dendritic compartments, and back propagation of sodiums spike bursts are associated with a large voltage-dependent calcium influx (Jaffe et al., 1992; Stuart and Sakmann, 1994; Magee and Johnston, 19951.3; Spruston et al., 1995). The success or failure of dendritic invasion of a spike series depends on the level of membrane polarization, which is preset by the inhibitory interneurons. It follows that activation of interneurons with dendritic terminals can also prevent the active regeneration of the back-propagating sodium spikes. In the absence of the regenerative process, the passively propagating sodium spikes fail to trigger dendritic calcium spikes (Bus& et al., 1996). Dendritic inhibition could also selectively regulate the efficacy of NMDA receptors and prevent plasticity simply due to the dendritic localization of NMDA receptors. However, plastic changes could be largely independent of the regulation of neuronal excitability at the somatic level, as has been demonstrated in the pyriform cortex (Kanter et al., 1996). An important implication of these experiments is that by selec-
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FIGURE 45. Inhibition-induced changes in the active dendritic propagation of Na+ spikes. Dendritic recording from a CAI pyramidal cell in vivo. Aa: Depolarization of the dendrite by current injection evoked fast spikes and a slow (calcium) spike. Ab: Weak commissural stimulation (arrow)-inducedinhibition, paired with dendritic depolarization, abolished the slow spike without much affecting the Na+ spikes. Ac: Repetitive (2 Hz), strong commissurd stimulation led to repetitive spiking. The amplitude oc the fast spikes is substantially decreased (long arrows). B: Hypothesis: dendritic inhibition hyperpolarizes and/or shunts the membrane and attenuates active backpropagation of Na+ spikes from soma to dendrites. i, inhibitory interneuron with dendritic targets. After Bum& G, Penttonen M, Nadady Z, Bragin A (1996) Pattern and inhibition-dependent invasion of pyramidal cell dendrites by fast spikes in the hippocampus in vivo. Prco Natl Acad Sci (USA) 939921-3925 by . permission of . Proceedings from the National Academy of Science.
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FIGURE 46. Pyramidal cell activity is differentially controlled by somatic and dendritic inhibition. A: A perisomatic IPSP can suppress repetitive firing of fast action potentials. Three action potentials were elicited by current injection into a pyramidal cell (top). Field IPSP was evoked by focal stimulation in the somatic region (middle). When the IPSP was initiated just after the first spike, subsequent spikes were suppressed (lower). B: A cut was made in the slice (shown in the diagram) to prevent activation of apical dendritic inhibitory synapses. Dendritic generation of Ca-dependent spikes are more effectively suppressed by dendritic IPSPs than by perisomatic IPSPs. Current injection into a CA3 pyramidal cell dendrite elicited a complex burst consisting of small fast spikes followed by a slower depolarization (top). A dendritic IPSP suppressed the slow Ca-dependent component of the potential in 40 of 53 trials. A perisomatic IPSP, of similar amplitude in the dendritic recording, was effective in only 13 of 51 trials. Five superimposed traces are shown. The slice preparation and electrode placement for selective activation of somatic and apical dendritic inhibitory synapses is shown in the diagram. In A and B, excitatory synapses were blocked with CNQX (20 pM) and APV (100 pM). C-F: Control of repetitive pyramidal cell firing by single perisomatic IPSPs. C: A single inhibitory cell (1) could block repetitive firing of Na spikes in a target pyramidal cell (2). Two action potentials elicited by depolarization of cell 2 (dotted trace). An IPSP initiated during the pyramidal cell depolarizing afterpotential suppressed the second spike. The second spike was suppressed on 73 of 115 trials, and in the other trials was delayed by 5-32 ms beyond its mean control latency. D: Light and electron microscopy showed the inhibitory cell (1) made three synaptic contacts on the soma of the pyramidal cell (2) at the sites indicated by black dots. E The difference in pyramidal cell : membrane repolarization after a single spike in the absence (dotted) and the presence (solid line) of an IPSP initiated by cell 1 (arrow) is shown. F: The IPSP initiated by cell 1 in the absence of activity in cell 2 is shown. Reproduced from Miles et d. (1996) Distinct functional roles for somatic and dendritic inhibition in the hippocampus. Neuron, 16:815-823 by permission of Cell Press. This figure was kindly prepared by Richard Miles.
the anatomical identity of interneurons dominantly involved in the control of sodium spikes and calcium spikes and characterize the conditions necessary for the activation of these cells.
prevalent hypotheses proposed to explain the selective vulnerability of hippocampal neurons is the excitotoxic hypothesis of Olney (1978), which postulates that an increase in the release of excitatory amino acids leads to neuronal death by allowing a lethal
Given the widespread involvement of hippocampal interneurons in the control of principal cell networks, interneuronal impairment is expected to exert far-reaching consequences on hippocampal function. The consistent observation that blockade of GABAergic transmission precipitates seizures led to the assumption that loss of inhibitory neurons or impairment of GABAergic transmission may be causally related to epilepsy (Prince, 1978; Ben-Ari et al., 1979; Traub and Wong, 1982; Dichter and Ayala, 1987; McNamara, 1994; During et al., 1995). Many studies have been stimulated by these findings, but the results are rather controversial. The following anatomical and physiological descriptions will be largely limited to phenomena in which the involvement of interneurons is apparent. Epileptic and/or ischemic challenges may induce long-term changes in the hippocampal circuitry. T o date, one of the most
abnormal degree of cation and water influx into the cells, which may be calcium independent, and (2) the delayed apoptotic-like cell death (Pollard et al., 1994; Nitatori et al., 1995), which is calcium dependent (for review, see Schmidt-Kastner and Freund, 199 1). Besides calcium dependence, the two mechanisms also differ in reversibility and selectivity: the former affects all neurons that are predisposed to a toxic level of glutamatergic input and have glutamate receptors (not necessarily calcium permeable). Depending on the magnitude of the excitotoxic impact, these cells may recover to a normal ion and water homeostasis, unless the plasma membrane has been ruptured. Thus, there is also differential vulnerability in acute mechanism, but it depends largely on the level of excitotoxic input. In contrast with the acute mechanism, delayed cell death affects only the population of selectively vulnerable neurons, which in the hippocampus are the CA1 pyramidal cells. The delayed mechanism affects primarily the CA1
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pyramidal neurons both in ischemia and epilepsy, whereas interneurons appear to be destroyed acutely (Freund and Magloczky, 1993; Hsu and Buzsiki, 1993; Magl6czky and Freund, 1993, 1995; Freund et al., 1990a,b, 1992). Accordingly, the crucial factors determining vulnerability via the acute mechanism-relevant from the point of interneurons-are the types and density of glutamatergic inputs and receptors, whereas the factors determining vulnerability via the delayed mechanism (primarily affecting pyramidal cells) include the calcium buffering capacity and/or the presence of the necessary biochemical machinery to activate apoptosis (Pollard et al., 1994; Nitatori et al., 1995). Based on this logic, the calcium-binding protein content of neurons might predict vulnerability, if at all, only in delayed calcium-dependent cell death. Several studies have attempted to correlate the vulnerability of neurons with the presence or absence of the calcium-binding proteins PV, CB, and CR (Mudrick and Baimbridge, 1989; Nitsch et al., 1989a,b; Sloviter, 1989; Freund et al., 1990a,b, 1992; Baimbridge et al., 1992; Freund and Magloczky, 1993; Johansen et al., 1990). Intracellular injection of the calcium chelator BAPTA effectively protects neurons from stimulation-induced cell death in vitro (Scharfman and Schwartzkroin, 1989). Furthermore, intracellular injection of PV or CB was shown to reduce the rate of rise of calcium and altered the kinetics of its decay in dorsal root ganglion cells (Chard et al., 1993). Additional support for the calcium-buffer hypothesis derives from experiments showing that a subpopulation of SOM-containing interneurons, but not PV- and CB-immunoreactive interneurons, degenerate in ischemia and status epilepticus (Johansen et al., 1987, 1990; Sloviter 1989). However, studies that systematically examined the relationship between the distribution of calciumbinding proteins and neuronal vulnerability failed to demonstrate such a relationship in either forebrain ischemia (Freund et al., 1990a; Johansen et al., 1990; Freund and Magloczky, 1993; Magloczky and Freund, 1993) or status epilepticus (Freund et al., 1992). PV-immunoreactive neurons in all hippocampal regions are resistant. CA1 interneurons survive ischemic insults (Johansen et al., 1983; Nitsch et al., 1989a,b) and even persist for several months, despite a total loss of the surrounding pyramidal cells (Murdick and Baimbridge, 1989; Hsu et al., 1994). However, neurons in the reticular nucleus of the thalamus containing both PV and CR are especially vulnerable (Freund et al, 1990a). The spiny class of CR-positive cells in the hilus and the CA3 region is damaged shortly after ischemia, whereas the aspiny neurons containing the same calcium-binding protein are fairly resistant (Hsu and Buzsiki, 1993; Freund and Magloczky, 1993). The content of neuropeptides (NPY, CCK, or VIP) also fails to predict susceptibility to an ischemic insult (Grimaldi et al., 1990; Yanagihara et al., 1785). Some SOM-positive cells in the hilar region are vulnerable, whereas others in the CA3-CA1 regions are resistant. The lack of correlation with calcium-binding protein content is expected if interneurons are indeed damaged by an acute ion and water influx, which may be calcium independent. The correlation between calcium-binding protein content and vulnerability or resistence does not hold for the delayed death of pyramidal cells either because those in the CA3 region are far
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here, not only because of the often false cross-species interpretation and correlation of anatomical and physiological data but also because variability of some features or phylogenetical conservation of others may indicate major differences in the relative importance of various characteristics for basic network operations in the archicortex. Such comparisons make sense only if archicortical structures are compared because the neocortex has a different origin and parallel evolution. Furthermore, such comparative data may also demonstrate whether the function of a cell type or any of its features was already present when archicortex was first formed in various reptilian species or it appeared during later stages of archicortical evolution. The cerebral cortex of the lizard shows remarkable similarities to the mammalian hippocampus and may be considered to be the most ancient archicortical structure in phylogenesis, which already possesses the cell types, connections, and lamination typical of the mammalian hippocampus. The primate cerebral cortex possesses major quantitative and qualitative changes compared with rodents, whereas the hippocampus as an archicortical area is a more conserved structure. Here we aim to draw attention to these issues by providing pro and contra examples for the cross-species difference and consistency of different cell types and connections. However, it would be very difficult to give a complete account of the innumerable species differences that occur even if considering only interneurons.
As pointed out in the Introduction, the present review largely focused on rodents, particularly rats, but one should be aware of the difference among species with regard to various properties of interneurons. For example, considerable differences exist in the nrurochemical characteristics of interneurons between rodent species as close as the rat and the mouse. However, there are basic rules in connectivity, which are similar even between rats and primates. Species difference is an important issue to be addressed
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found in the mammalian hippocampus. Therefore, they are likely to be phylogenetically ancient features of cortical architecture, which have been highly conserved in the archicortex during evolution. However, when studying more complex connectivity features and neurochemical properties, numerous differences become apparent. For example, there is no evidence for the existence of interneurons that specifically innervate other interneurons in the lizard cortex as the CR- and some of the VIP-containing cell do in the rat hippocampus (see Section 111.4). In lizards, CR coexists with the other two calcium-binding proteins, PV and CB, and is present in GABAergic basket cells innervating the perisomatic region of pyramidal neurons (Martinez-Guijarro and Freund, 1992a). It is interesting to note here that these three calcium-binding proteins, or at least two of them, often occur in the same cells during early cortical development in rodents (Alcantara et al., 1996) and even in monkey (Yan et al., 1995), although in adults they are present in interneuron types with different connectivity and function (see Section IV.2). Neuropeptides, including SOM and NPY, are also present in GABAergic interneurons in the lizard cortex. They show a minor overlap with the interneurons that contain calcium-binding proteins and are located in the inner or outer plexiform layers. They are likely to be involved in dendritic inhibition because they form an axon plexus in the outer one-third of the outer plexiform layer (Divila et al., 1991, 1993; Martinez-Guijarro et al., 1993) in a fashion remarkably similar to the SOM-NPY-containing 0-LM cells in the mammalian hippocampus (Sections III.3.2.a, IV.3.a, IV.3.b). Thus, the segregation of interneuron types responsible for the selective innervation of the perisomatic versus the distal dendritic regions of principal cells appears to have taken place very early in archicortical evolution. This notion further suggests that the differential control of synaptic plasticity and principal cell spike transmission by separate classes of interneurons (Miles et al., 1996) is a fundamental and indispensable component of network operations in the archicortex. The selective innervation of these separate interneuron classes by different subcortical pathways, which may subserve a behavior-dependent modulation of network events, is also a phylogenetically well-preserved organizational principle (Freund, 1992; Martinez-Guijarro and Freund, 1992b; Martinez-Guijarro et al., 1994).
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1995). Cells in this location are also frequent in the guinea pig but scarce in the rat (see above). Similar differences between rats and primates are apparent in the number of CR-positive cells in the dentate molecular layer. The radially oriented CR-positive cells occur in all layers of the hippocampus in rats, whereas these cells are distributed more toward the distal stratum radiatum and extremely few cells are visible in stratum oriens in primates (Nitsch and Leranth, 1993; Seress et al., 1993b; Nitsch and Ohm, 1995). The overall distribution of axonal and dendritic arbors of interneurons immunoreactive for NPY and/or SOM is highly conserved throughout phylogeny. A similar population of cells in the hilar polymorphic area is immunoreactive for these peptides in the rat, monkey, and human dentate gyrus and give rise to a dense axon terminal field in the outer two-thirds of the molecular layer (Bakst et al., 1985, 1986; Chan-Palay et al., 1986; Kohler et al., 1986, 1987; Chan-Palay, 1987; Amaral et al., 1988; Nitsch and Leranth, 1991). The same cell population in primates also contains SP (Seress and Leranth, 1996), whereas there is a much smaller degree of colocalization in rats, although SP immunostaining in this species is highly variable and strongly depends on the type of fixative used (Z. Borhegyi, L. Seress, and C. Leranth, personal communication). In the hippocampus, the density of NPY- and SOM-containing fibers is highest in stratum lacunosum-moleculare, and the parent cell bodies are located predominantly in stratum oriens in both primates and rodents. Thus, GABAergic interneurons specialized to innervate the most distal dendrites of principal cells appear to be similar throughout the phylogenetic scale from lizards through rodents up to the monkey and human archicortex, not only in their efferent connections but also in neuropeptide content and in afferent drive (mostly feedback; see Section IV.3.a). However, there are qualitative and quantitative differences in cell types located (and probably also terminating) in the dendritic layers of both the dentate gyrus and the hippocampus. The CB- and CR-containing neurons in these locations are more numerous in primates (see above), suggesting that feed-forward dendritic inhibition, involved in the control of afferent synaptic plasticity, may be more complex in these species.
The rich interconnectivity of interneurons and their hypothesized role in synaptic plasticity provide the possibility that the different interneuronal types may selectively influence the efficacy of afferent inputs. For example, during exploration-associated theta-gamma oscillations, VIP-positive interneurons with dendritic targets may suppress other interneurons innervating the distal apical dendrites of principal cells (0-LM and HIPP cells). As a result, the direct entorhinal-CA1 transmission is facilitated, whereas the efficacy of the intrahippocampal associational connections are suppressed. Obviously, working hypotheses like these will be continuously generated and modified in the process of revealing the precise connectivity and physiological properties of in-
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terneurons. Clarifying the relation between the chemical content of the neuron and its axonal targets is the first logical step in the classification process. A further level of analysis is required to establish a correlation between electrophysiological properties and morphology/immunocytochemistry. Intracellular recordings from interneurons in vitro and their subsequent visualization are a direct approach for studying the functional properties of interneurons that provides the essential bridge between physiological features and their structural substrate. In turn, intracellular recordings from anatomically identified interneurons in whole animals enables us to relate interneuron activity to network patterns. The differential involvement of the interneuronal types in the various oscillations and intermittent bursts indicates that examination of their associations with population patterns is the correct strategy for comparing their functional and anatomical features. Because some of these population patterns can also be induced in the anesthetized animal, the neurons involved can be labeled and anatomically characterized. The location, firing pattern, and afferent responses of the subtypes can be examined in the behaving animal. Extracellular labeling of the physiologically and behaviorally characterized interneurons in the awake animal is a possibility that should be explored. Understanding the complex interactions between interneurons and principal cells will benefit from computational modeling. Overall, in vivo and in vitro studies will complement each other and provide a precise knowledge about physiological properties, connectivity, and immunocytochemical identification of interneurons. Such knowledge is a prerequisite for understanding of the operational principles of the hippocampal network and may serve as a model for exploring neocortical circuitries. Caveats and expected difficulties in the classification process should also be ackowledged. Precise biophysical, pharmacological, and network analyses of interneuron-mediated effects depend on a reliable taxonomy of interneuron types. To date, interneuron classes cannot always be unanimously defined even when multiple criteria are used. For example, typical basket cells also have their variants innervating the proximal dendrites, and at least two subsets exist that contain different neurochemical markers. However, even within the same subclasses, the dendritic and axonal features may have large variability (Gulyis et al., 1993a; Sik et al., 1995). Such observations raise the intriguing possibility that axon length and even the targets of interneurons may vary not only as part of the ontogenetic development but also in the adult in a use-dependent manner. Such plasticity of interneurons would especially be significant for shaping the topographical representations of information in hippocampal networks. The available evidence along these lines is in support of the uniquely plastic properties of CABAergic axons. When a sciatic nerve is implanted into the thalamus, the overwhelming majority of cells that grow axons into the nerve graft are GABAergic cells of the reticular nucleus (David and Aguayo, 1981). Although neurons of the reticular nucleus normally innervate only thalamic nuclei, their newly generated axon collaterals into the peripheral nerve grow longer than 50 mm. NPY-immunoreactive interneurons of the hilar region also display a remarkable sprouting of their axon collaterals (Deller et al., 1995a). Obviously, the task is to determine how much variability is reasonable in the classification process and to examine
Acknowledgments
This work was supported by the NIH (NS34994), Human Frontier Science Program Organization, Howard Hughes Medical Institute, OTKA (T 16942), Hungary, and the Whitehall Foundation. We thank Richard Miles and Roger Traub for their constant encouragement, discussions, and friendship. We are grateful to our immediate colleagues, LhzM Acsidy, Attila Gulyis, Norbert Hijos, Isrvan Katona, Zoltan Nadasdy, Attila Sik, and Katalin Toth, for their help with suggestions to several versions of this manuscript and with the preparation of figures. We are grateful to Katalin Halasy, Toshio Kosaka, Jean-Claude Lacaille, Csaba Leranth, Chris McBain, Richard Miles, and Peter Somogyi for their contribution of valuable figures to the review. Consultation with several individuals helped shape the final version of the manuscript, including David G. Amaral, Zsolt Borhegyi, Eberhard Buhl, James J. Chrobak, Cynthia Dolorfo, Michael Frotscher, Toshio Kosaka, Brian Leonard, Csaba Leranth, Zsofia Magloczky, Francisco Martinez-Guijarro, Chris McBain, Isrvin Mody, Charles Ribak, Philip A. Schwartzkroin, Liszl6 Seress, IvLn Solttsz, Peter Somogyi, Armin Stelzer, James Tepper, and Xiao-Jing Wang. The excellent technical assistance of Erzsebet Borok, Agnes Miiller, Andrea Zoldi, Ggbor Terstyinszky, and Isrvin Csap6 with the preparation of the illustrations and the text is gratefully acknowledged.
Abraham WC, Gustafsson B, Wigstrom H (1986) Single high strength afferent volleys can produce long-term potentiation in the hippocampus in vitro. Neurosci Lett 70:217-222. Abraham WC, Gustafsson B, Wigstrom H (1987) Long-term potentiation involves enhanced synaptic excitation relative to synaptic inhibition in guinea-pig. J Physiol (Lond) 394:367-380. Acsddy L, Halasy K, Freund T F (1993) Calretinin is present in nonpyramidal cells of the rat hippocampus-111. Their inputs from the median raphe and medial septa1 nuclei. Neuroscience 52:829-841. Acsddy L, Papp E, Hdjos N, Freund T F (1994) Subcortical innervation of VIP-containing interneurons in the hippocampus. Eur J Neurosci (Suppl) 7:209. Acsddy L, Arabadzisz D, Freund T F (19964 Correlated morphological and neurochemical features indentify different subsets of VIP-immunoreactive interneurons in rat hippocampus. Neuroscience 73: 299-3 15. Acsddy L, Gorcs TJ, Freund TF (1996b) Different populations of VIPimmunoreactive interneurons are specialized to control pyramidal cells or interneurons in the hippocampus. Neuroscience 73:317-334. Acsidy L, Katona I, Arabadzisz D, Gulyds AI, Shigemoto R, Freund TF (submitted) Substance P receptor labels GABAergic cells with distinct termination pattern in the hippocampus. J Comp Neurol.
~~
455
Aika Y, Ren JQ, Kosaka K, Kosaka T (1994) Quantitative analysis of GABA-like-immunoreactive and parvalbumin-containing neurons in the CAI region of the rat hippocampus using a stereological method, the disector. Exp Brain Res 99:267-276. Aitken PG, Jaffe DB, Nadler JV (1991) Cholecystokinin blocks some effects of kainic acid in CA3 region of hippocampal slices. Peptides 12: 127-129. Alcantara S, de Lecea L, Del Rio JA, Ferrer I, Soriano E (1996) Transient co-localization of parvalbumin and calbindin D28K in the postnatal cerebral cortex: evidence for a phenotypic shift in developing nonpyramidal neurons. Eur J Neurosci 8: 1329-1339. Alger B, Nicoll RA (1979) GABA-mediated biphasic inhibitory responses in hippocampus. Nature 28 1:3 15-3 17. Alger B, Nicoll RA (1982a) Feed-forward dendritic inhibition in rat hippocampal pyramidal cells studied in vitro. J Physiol (Lond) 328: 105-1 23. Alger B, Nicoll RA (1982b) Pharmacological evidence for two kinds of GABA receptor on rat hippocampal pyramidal cells studied in vitro. J Physiol (Lond) 328:125-141. Alger BE, P i t h TA, Williamson A (1990) A prolonged post-tetanic hyperpolarizations in rat hippocampal pyramidal cells in vitro. Brain Res 521:118-124. Alonso A, Kohler C (1982) Evidence for separate projections of hippocampal pyramidal and non-pyramidal neurons to different parts of the septum in the rat brain. Neurosci Lett 31:209-214. Alonso A, Khateb A, Fort P, Jones BE, Muhlethaler M (in press) Differential oscillatory properties of cholinergic and non-cholinergic nucleus basalis neurons in guinea big brain slices. J Neurophysiol. Alvarez-Leefmans FJ, Gardner-Medwin AR (1975) Influences of the septum on the hippocampal dentate area which are unaccomplanied by field potentials. J Physiol (Lond) 249: 14P-16P. Amaral DG (1978) A Golgi study of cell types in the hilar region of the hippocampus in the rat. J Comp Neurol 182:851-914. Amaral DG, Witter MP (1989) The three-dimensional organization of the hippocampal formation: a review of anatomical data. Neuroscience 31:571-591. Amaral DG, Witter MP (1995) Hippocampal formation. In: The rat nervous system, 2nd ed (Paxinos G, ed), pp 443-494. New York: Academic Press. Amaral DG, Insausti R, Campbell MJ (1988) Distribution of somatostatin immunoreactivity in the human dentate gyrus. J Neurosci 8: 3306-3316. Amaral DG, Dolorfo C, Alvarez-Roy0 P (1991) Organization of CA1 projections to the subiculum: a PHA-L analysis in the rat. Hippocampus 4:415-436. Andersen P, Eccles J C (1962) Inhibitory phasing of neuronal discharge. Nature (Lond) 196:645-647. Andersen P, Eccles JC, Loyning Y (1963) Recurrent inhibition in the hippocampus with identification of the inhibitory cell and its synapse. Nature 198:540-542. Andersen P, Eccles JC, Loyning Y (1964) Location of synaptic inhibitory synapses on hippocampal pyramids. J Neurophysiol 27:592-607. Andersen P,Gross GN, Lomo T, Sveen 0 (1 969) Participation of inhibitory and excitatory interneurons in the control of hippocampal cortical output. MIT Press, Boston. In: The interneuron (Brazier M, ed), pp 415-432. Andersen P,Dingledine R, Gjerstad L, Langmoen LA,Mosfeldt-Laursen A (1980) Two different responses of hippocampal pyramidal cells to application of gamma-aminobutyric acid. J Physiol (Lond) 305: 279-296. Andrade R, Nicoll RA (1987) Pharmacologically distinct actions of serotonin on single pyramidal neurons of the rat hippocampus recorded in vitro. J Physiol (Lond) 39499-124. Arancio 0, Korn H , Gulyas A, Freund TF, Miles R (1994) Excitatory synaptic connections onto rat hippocampal inhibitory cells may involve a single transmitter release site. J Physiol (Lond) 481(2): 395-405. Arimatsu Y, Naegele JR, Barnstable CJ (1987) Molecular markers of neu-
ronal subpopulations in layers 4, 5, and 6 of cat primary visual cortex. J Neurosci 7:1250-1263. Asanuma C, Porter LL (1992) Light and electron microscopic evidence for a GABAergic projection from the caudal basal forebrain to the thalamic reticular nucleus in rats. J Comp Neurol 302:159-172. Ashwood TJ, Lancaster B, Wheal H V (1984) In vivo and in vitro studies on putative interneurones in the rat hippocampus: possible mediators of feed-forward inhibition. Brain Res 293:279-29 1. Babb TL, Pretorius JK, Kupfer WR, Crandall PH (1989) Glutamate decarboxylase-immunoreactive neurons are preserved in human epileptic hippocampus. J Neurosci 9:2562-2574. Baimbridge KG, Miller JJ (1982) Immunohistochemical localization of calcium-binding protein in the cerebellum, hippocampal formation and olfactory bulb of the rat. Brain Res 245:223-229. Baimbridge KG, Miller JJ, Parkes CO (1982) Calcium-binding protein distribution in the rat brain. Brain Res 239:519-525. Baimbridge KG, Celio MR, Rogers J H (1992) Calcium-binding proteins in the nervous system. Trends Neurosci 15:303-308. Bakst I, Morrison JH, Amaral D G (1985) The distribution of somatostatin-like immunoreactivity in the monkey hippocampal formation. J Comp Neurol 236:423-442. Bakst I, Avendano C, Morrison JH, Amaral DG (1986) An experimental analysis of the origins of somatoscatin-like immunoreactivity in the dentate gyrus of the rat. J Neurosci 6:1452-1462. Barnes CA, Foster TC, Rao G, McNaughton BL (1991) Specificity of functional changes during normal brain aging. Ann NY Acad Sci 640:80-85. Battenberg E, Morales M, de Lecea L, Johnson DS, Bloom FE (1994) Immunocytochemical characterization of neurons expressing the 5TH3 receptor. SOCNeurosci Abstr 20: 1156. Baude A, Nusser Z, Roberts JD, Mulvihill E, McIlhinney RA, Somogyi P (1993) The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron 11:771-787. Baude A, Nusser Z, Molnar E, McIlhinney RAJ, Somogyi P (1995) Highresolution immunogold localization of ampa type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus. Neuroscience 69: 1031-1055. Behrends JC, Ten Bruggencate G (1 993) Cholinergic modulation of synaptic inhibition in the guinea pig hippocampus in virro: excitation of GABAergic interneurons and inhibition of GABA release. J Neurophysiol 69:626-629. Bekenstein JW, Lothman EW (1993) Dormancy of inhibitory interneurons in a model of temporal lobe epilepsy. Science 25997-100. Ben-Ari Y (1994) Kainate-induced apoptotic cell death in hippocampal neurons. Neuroscience 63:7-18. Ben-Ari Y, Krnjevic K, Reinhardt W (1979) Hippocampal seizures and failure of inhibition. Can J Physiol Pharmacol 57: 1462-1466. Benardo LS, Prince DA (1982a) Cholinergic excitation of mammalian hippocampal pyramidal cells. Brain Res 249:315-33 1. Benardo LS,Prince DA (1 982b) Ionic mechanisms of cholinergic excitation in mammalian hippocampal pyramidal cells. Brain Res 249:333-344. Bergles DE, Doze VA, Madison DV, Smith SJ (1996) Excitatory actions of norepinephrine on multiple classes of hippocampal CAI interneurons. J Neurosci 16:572-585. Bernabeu A, Martinez-Guijarro FJ, Luis de la Iglesia JA, Lopez-Garcia C (1994) An axosomatic and axodendritic multipolar neuron in the lizard cerebral cortex. J Anat 184:567-582. Berzaghi M, Freund TF, Papp E, Lindholm D, Zirrgiebel U, Castren E, Thoenen H (submitted) Brain-derived neurotropic factor (BDNF) in the rat cerebral cortex: mismatch between site of synthesis and accumulation. J Neurosci. Bialek W, Rieke F, van Steveninck RR (1991) Reading a neural code. Science 252:1854-1857. Bijak M, Misgeld U (1995) Adrenergic modulation of hilar neuron activity and granule cell inhibition in the guinea-pig hippocampal slice. Neuroscience 67:541-550. Bilkey DK, Goddard GV (1985) Medial septa1 facilitation of hip-
456
pocampal granule cell activity is mediated by inhibition of inhibitory interneurons. Brain Res 361:99-106. Blackstad T W (1956) Commissural connections of the hippocampal region of the rat, with special reference to their mode of termination. J Comp Neurol 105:417-537. Blackstad TW, Kjaerheim A (1961) Special axo-dendritic synapses in the hippocampal cortex: electron and light microscopic studies on the layer of mossy fibers. J Comp Neurol 117:113-I 59. Bland BH (1990) Physiology and pharmacology of hippocampal formation theta rhythms. Prog Neurobiol 26: 1-54. Blasco-Ibanez JM, Freund T F (1995) Synaptic input of horizontal interneurons in stratum oriens of the hippocampal CAI subfield: structural basis of feed-back activation. Eur J Neutosci 7:2170-2180. Bliss TVP, Collingridge GL (1993) A synaptic model of memory: longterm potentiation in the hippocampus. Nature 361 :31-39. Bliss rVP, Lomo T (1973) Long-lasting potentiation of synaptic transmission in the dentate area of the anaesthetized rabbit following stimulation of the perforant path. J Physiol (Lond) 232:331-356. Bobker D H , Williams JT (1990) Ion conductances affected by 5-HT receptor subtypes in mammalian neurons. Trends Neurosci 10:502-509. Bradley SR, Levey AI, Hersch SM, Conn PJ (1996) Immunocyto-chemical localization of group I11 metabotropic glutamate receptors in the hippocampus with subtype-specific antibodies. J Neurosci 16:20442056. Bragin AG, Otmakhov NA (1979) Comparison of direct influences of the perforant path on hippocampal CA1 and CA3 neurons in vitro [in Russian]. Neurophysiology 11:220-225. Bragin A, Jand6 G, NadBsdy Z, Hedke J, Wise K, Buzsdki G (1995a) 40-100Hz, Oscillation in the Hippocampus of the behaving rat. J Neurosci 15:47-60. Bragin A, Jand6 G, Nadisdy Z, van Landeghem M, and Buzsiki G (1995b) Dentate EEG spikes and associated population bursts in the hippocampal hilar region of the rat. J Neurophysiol 73:1691-1705. Bragin A, Csicsvary J, Penttonen M, Buzsdki G (in press) Epileptic afterdischarge in the hippocampo-entorhinal system: current source density and unic studies. Neuroscience. Buckmaster PS, Schwartzkroin PA (1 9 9 5 4 Interneurons and inhibition in the dentate gyrus of the rat in vivo. J Neurosci 15:774-789. Buckmasrer PS, Schwarzkroin PA (1995b) Physiological and morphological heterogeneity of dentate gyrus-hilus interneurons in the gerbil hippocampus in vivo. Eur J Neurosci 7:1393-1402. Buckmaster PS, Strowbridge BW, Kunkel DD, Schmiege DL, Schwartzkroin PA (1992b) Mossy cell axonal projections to the dentate gyms molecular layer in the rat hippocampal slice. Hippocampus 2:349-362. Buckmaster PS, Kunkel DD, Robbins RJ, Schwartzkroin PA (1994) Somatostatin immunoreactivity in the hippocampus of mouse, rat, guinea pig and rabbit. Hippocampus 4:167-180. Buckmaster PS, Wentzel HJ, Kunkel DD, Schwartzkroin PA (1996) Axon arbors and synaptic connections of hippocampal mossy cells in the rat in vivo. J Comp Neurol 366:270-292. Buhl EH, Halasy K, Somogyi P (1994a) Diverse sources of hippocampal unitary inhibitory postsynaptic potentials and the number of synaptic release sites. Nature 368:823-828. Buhl EH, Han ZS, Lorinczi Z, Stezhka W, Karnup SV, Somogyi P (1994b) Physiological properties of anatomically identified axo-axonic cells in the rat hippocampus. J Neurophysiol 71:1289-1307. Buhl EH, Cobb SR, Halasy K, Somogyi P (1995) Properties of unitary IPSPs evoked by anatomically identified basket cells in the rat hippocampus. Eur J Neurosci 7: 1989-2004. Buhl EH, Otis TS, Mody I (1996) Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model. Science 271 :369-373. Burgess N, Recce M, OKeefe J (1994) A model of hippocampal function. Neural Networks 7: 1065-1081. Burnashev N, Khodorova A, Jonas P,Helm PJ, Wisden W, Monyer H,
457
Cozzari C, Howard J, Hartman B (1990) Analysis of epitopes on choline acetyltransferase ( C U T ) using monoclonal antibodies. SOC Neurosci Abstr 16(1):200. Danos P, Frotscher M, Freund T F (1991) Non-pyramidal cells in the CA3 region of the rat hippocampus: relationships of fine structure, synaptic input and chemical characteristics. Brain Res 546: 195-202. David S, Aguayo AJ (1981) Axonal elongation into PNS bridges after CNS injury in adult rats. Science 214:931-933. Davies CH, Starkey SJ, Pozza MF, Collingridge GL (1991) GABABautoreceptors regulate the induction of LTP. Nature 349:609-611. Davies S, Kohler C (1985) The substance P innervation of the rat hippocampal region. Anat Embryo1 173:45-52. Divila JC, de la Calle A, Gutierrez A, Megias M, Andreu MJ, Guirado S (1991) Distribution of neuropeptide Y (NPY) in the cerebral cortex of the lizards Psummodromus ulgirus and Iudarcis hipanicu: co-localization of NPY, somatostatin and GABA. J Comp Neurol 308:397-408. Divila JC, Megias M, de la Calle A, Guirado S (1993) Subpopulation of GABA neurons containing somatostatin, neuropeptide Y, and parvalbumin in the dorsomedial cortex of the lizard Psummodromus algirzls. J Cornp Neurol 336161-173. Divila JC, Megias M, Andreu MJ, Real MA, Guiradi S (1995) NADPH diaphorase-positive neurons in the lizard hippocampus: a distinct subpopulation of GABAergic interneurons. Hippocampus 5:60-70. de Montigny C, Monnet FP, Fournier A, Debonnel G (1993) Sigma ligands and neuropeptide Y selectively potentiate the NMDA response in the rat CA3 dorsal hippocampus: in vivo electrophysiological studies. Clin Neuropharmacol (Suppl) 15:145A-l46A. de Quidt ME, Emson PC (1986) Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system-11. Immunohistochemical analysis. Neuroscience 18:545-618. Deadwyler SA, West JR, Cotman CW, Lynch G (1975) Physiological studies of the reciprocal connections between the hippocampus and entorhinal cortex. Exp Neurol 49:35-57. DeFelipe J (1993) Neocortical neuronal diversity: chemical heterogeneity revealed by colocalization studies of classic neurotransmitters, neuropeptides, calcium-binding proteins, and cell surface molecules. Cereb Cortex 3:273-289. Deller T , Leranth C (1990) Synaptic connections of neuropeptide-Y (NPY) immunoreactive neurons in the hilar area of the rat hippocampus. J Comp Neurol 300:433-447. Deller T , Nitsch R, Frotscher M (1994) Associational and commissural afferents of parvalbumin-immunoreactive neurons in the rat hippocampus: a combined immunocytochemical and PHA-L study. J Comp Neurol 350:612-622. Deller T, Frotscher M, Nitsch R (1995a) Morphological evidence for the sprouting of inhibitory commissural fibers in response to the lesion of the excitatory entorhinal input to the rat dentate gyrus. J Neurosci 15:6868-6878. Deller T, Nitsch R, Frotscher M (1995b) Phaseulus vulgaris-leucoagglutinin tracing of commissural fibers to the rat dentate gyrus: evidence for a previously unknown commissural projection to the outer molecular layer. J Comp Neurol 352:55-68. Dent AJ, Galvin NJ, Stanfield BB, Cowan W M (1983) The mode of termination of the hypothalamic projection to the dentate gyrus: an EM autoradiographic study. Brain Res 258:l-10. Dichter MA, Ayala GF (1987) Cellular mechanisms of epilepsy: a status report, Science 237:157-163. Dinerman JL, Dawson T M , Schell MJ, Snowman A, Snyder SH (1994) Endothelial nitric oxide syiithase localized to hippocampal pyramidal cells: implications for synaptic plasticity. Proc Natl Acad Sci USA 9 1:42 14-4218. Dodd J, Kelly JS (1978) Is somatostatin an excitatory transmitter in the hippocampus? Nature 273:674. Dodd J, Kelly JS (1981) The actions of cholecystokinin and related peptides on pyramidal neurones of the mammalian hippocampus. Brain Res 338:97-113.
458
Doller HJ, Weight FF (1982) Perforant pathway activation of hippocampal CAI stratum pyramidale neurons: electrophysiological evidence for a direct pathway. Brain Res 237:l-13. Douglas RJ, Martin KAC (1991) Opening the grey box. TINS 14: 286-293. Drake CT, Mulligan KA, Wimpey TL, Hendrickson A, Chavkin C (199 1) Characterization of Vicia villosa agglutinin-labeled GABAergic interneurons in the hippocampal formation and in acutely dissociated hippocampus. Brain Res 554:176-185. Du J, Zhang L, Weiser M, Rudy B, McBain CJ (1996) Developmental expression and functional characterization of the potassium-channel subunit Kv3.1b in parvalbumin-containing interneurons of the rat hippocampus. J Neurosci 16:506-518. Dudek SM, Bear MF (1992) Homosynaptic long-term depression in area CA1 of the hippocampus and the effects of NMDA receptor blockade. Proc Natl Acad Sci USA 89:4363-4367. Dun NJ, Dun SL, Wong RK, Forstermann U (1994) Colocalization of nitric oxide synthase and somatostatin immunoreactivity in rat dentate hilar neurons. Proc Natl Acad Sci USA 91:2955-2959. Dunwiddie TV, Haas HL (1985) Adenosine increases synaptic facilitation in the in vitro rat hippocampus: evidence for a presynaptic site of action. J Physiol 69:365-77. During MJ, Ryder KM, Spencer D D (1995) Hippocampal GABA transporter function in temporal-lobe epilepsy. Nature 376: 174-177. Dutar P, Nicoll RA (1 988) Classification of muscarinic responses in hippocampus in terms of receptor subtypes and second-messenger systems: electrophysiological studies in vitro. J Neurosci 8:4212-4224. Eccles JC (1 964) The physiology of synapses. Berlin: Springer-Verlag. Eckenstein F, Baughman RW (1984) Two types of cholinergic innervation in cortex, one co-localized with vasoactive intestinal polypeptide. Nature 309:153-155. Eckenstein F, Thoenen H (1983) Chlonergic neurons in the rat cerebral cortex demonstrated by immunohistochemical localization of choline acetyltransferase. Neurosci Lett 36:211-215. Edwards FA, Konnerth A, Sakmann B (1990) Quanta1 analysis of inhibitory synaptic transmission in the dentate gyrus of rat hippocampal slices: a patch-clamp study. J Physiol (Lond) 430:213-249. Esclapez M, Houser CR (1995) Somatostatin neurons are a subpopulation of GABA neurons in the rat dentate gyrus: evidence from colocalization of pre-prosomatostatin and glutamate decarboxylase messenger RNAs. Neuroscience 64:339-355. Esclapez M, Chang DK, Houser CR (1996) Subpopulations of GABA neurons in the dentate gyrus express high levels of the alpha1 subunit of the GABA-A receptor. Hippocampus 6:225-238. Fantie BD, Goddard GV (1982) Septa1 modulation of the population spike in the fascia dentata produced by perforant path stimulation in the rat. Brain Res 252:227-37. Fatt P, Katz B (1952) Spontaneous subthreshold activity at motor nerve endings. J Physiol (Lond) 117:109-128. Finsen BR, Tonder N, Augood S, Zimmer J (1992) Somatostatin and neuropeptide Y in organotypic slice cultures of the rat hippocampus: an immunocytochemical and in situ hybridization study. Neuroscience 47:105-113. Fox SE (1989) Membrane potential and impedance changes in hippocampal pyramidal cells during theta rhythm. Exp Brain Res 77:283-294. Fox SE, Ranck JB J r (1981) Electrophysiological characteristics of hippocampal complex-spike cells and theta cells. Exp Brain Res 4 1:299-3 13. Fox SE, Ranck JB Jr (1986) Hippocampal theta rhythm and the firing of neurons in walking and urethane anesthetized rats. Exp Brain Res 62495-508. Fraser DD, MacVicar BA (199 1) Low-threshold transient calcium current in rat hippocampal lacunosum-moleculare interneurons: kinetics and modulation by neurotransmitters. J Neurosci 1 1:2812-2820. Freedman R, Wetmore C, Stromberg I, Leonard S, Olson L (1993) Alpha-bungarotoxin binding to hippocampal interneurons: immunocytochemical characterization and effects on growth factor expression. J Neurosci 13:1965-1975.
459
Grover LM, Lambert NA, Schwartzkroin PA, Teyler TJ (1993) Role of HC03-ion in deporarizing GABAA receptor-mediated responses in pyramidal cells of rat hippocampus. J Neurophysiol 69:1541-1555. Grunze HCR, Rainnie DG, Hasselmo ME, Barkai E, Hearn EF, McCarley, Greene RW (1 996) NMDA-dependent modulation of CA1 local circuit inhibition. J Neurosci 16:2034-2063. Gulyis AI, Freund T F (in press) Calbindin-containing interneurons innervate pyramidal cell dendrires in the hippocampus. Hippocampus. Gulyis AI, Gorcs TJ, Freund T F (1990) Innervation of different peptide-containing neurons in the hippocampus by GABAergic septa1 afferents. Neuroscience 37:31 4 4 . Gulyis AI, Seress L, T6th K, Acsidy L, Antal M, Freund T F (1991a) Septa1 GABAergic neurons innervate inhibitory interneurons in the hippocampus of the macaque monkey. Neuroscience 41 :38 1-390. Gulyis AI, T6th K, Danos P, Freund T F (1991b) Subpopulations of GABAergic neurons containing parvalbumin, calbindin D28k, and cholecystokinin in the rat hippocampus. J Comp Neurol 3 12:371378. Gulyis AI, Miettinen R, Jacobowitz DM, Freund T F (1992) Calretinin is present in non-pyramidal cells of the rat hippocampus-I. A new type of neuron specifically associated with the mossy fibre system. Neuroscience 48: 1-27. Gulyis AI,Miles R, Hijos N, Freund T F (1993a) Precision and variability in postsynaptic target selection of inhibitory cells in the hippocampal CA3 region. Eur J Neurosci 5:1729-1751. Gulyis AI, Miles R, Sik A, Toth K, Tamamaki N, Freund T F (1 993b) Hippocampal pyramidal cells excite inhibitory neurons through a single release site. Nature 366:683-687. Gulyis AI, Hijos N, Freund T F (1996) Interneurons containing calretinin are specialized to control other interneurons in the rat hippocampus. J Neurosci 16:3397-3411. Haas HL, Gahwiler BH (1992) Vasoactive intestinal polypeptide modulates neuronal excitability in hippocampal slices of the rat. Neuroscience 47:273-277. Haas HL, Hermann A, Greene RW, Chan-Palay V (1987) Action and on location of neuropeptide tyrosine (Y) hippocampal neurons of the rat in slice preparation. J Comp Neurol 257:208-217. Haas HL, Rose G (1982) Long-term potentiation of excitatory synaptic transmission in the rat hippocampus: the role of inhibitory processes. J. Physiol (Lond) 329:541-552. Haas HL, Rose G (1984) The role of inhibitory mechanisms in longterm potentiation. Neurosci Lett 47:303-308. Haglund L, Swanson LW, Kohler C (1984) The projection of the supramammillary nucleus to the hippocampal formation: an immunohistochemical and anterograde transport study with the lectin PHA-L in the rat. J Comp Neurol 229:171-185. Hijos N, Acsidy L, Freund T F (1996) Target selectivity and neurochemical characteristics of VIP-immunoreactive interneurons in the rat dentate gyrus. Eur J Neurosci 8:1415-1431. Hijos N, Cs. Papp E, Acsidy L, Levey AI, Freund T F (submitted) Distinct interneuron types express m2 muscarinic receptor immunoreactivity on their dendrites and/or axons in the rat hippocampus. Neuroscience. Halasy K, Somogyi P (1993a) Distribution of GABAergic synapses and their targets in the dentate gyrus of rat: a quantitative immunoelectron microscopic analysis. J Hirnforsch 34:299-308. Halasy K, Somogyi P (1993b) Subdivisions in the multiple GABAergic innervation of granule cells in the dentate gyrus of the rat hippocampus. Eur J Neurosci 5:411-429. Halasy K, Miettinen R, Szabat E, Freund T F (1992) GABAergic interneurons are the major postsynaptic targets of median Raphe afferents in the rat dentate gyrus. Eur J Neurosci 4:144-153. Halasy K, Buhl EH, Lorinczi Z, Tamas G, Somogyi P (1996) Synaptic target selectivity and input of GABAergic basket and bistratified interneurons in the CA1 area of the rat hippocampus. Hippocampus 6:306-329. Hamill OP, Huguenard JR, Prince DA (1991) Patch-clamp studies of
460
voltage-gated currents in identified neurons of the rat cerebral cortex. Cereb Cortex 1:48-61. Han ZS, Buhl EH, Lorinczi Z, Somogyi P (1993) A high degree of spatial selectivity in the axonal and dendritic domains of physiologically identified local-circuit neurons in the dentate gyrus of the rat hippocampus. Eur J Neurosci 5:395-410. Handelmann G, Meyer DK, Beinfeld MC, Oertel WH (1981) CCKcontaining terminals in the hippocampus are derived from intrinsic neurons: an immunohistochemical and radioimmunological study. Brain Res 224:180-184. Harris KM, Marshall PE, Landis DM (1985) Ultrastructural study of cholecystokinin-immunoreactive cells and processes in area CA1 of the rat hippocampus. J Comp Neurol 233:147-158. Hasselmo ME, Bower JM (1993) Acetylcholine and memory. TINS 16~218-222. Heiligenberg WF (1991) Neural nets in electric fish. Cambridge, MA: MIT Press. Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, Prystay W, Gilbert MM, Snutch TP, Catterall WA (1993) Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits. J Cell Biol 123:949-962. Hestrin S (1 993) Different glutamate receptor channels mediate fast excitatory synaptic currents in inhibitory and excitatory cortical neurons. Neuron 11:1083-1091. Hill JA, Zoli M, Bourgeois J-P, Chngeux J-P (1993) Immunocytochemical localization of a neuronal nicotinic receptor: the alpha2-subunit. J Neurosci 13:1551-1558. Hofer M, Pagliusi SR, Hohn A, Leibrock J, Barde YA (1990) Regional distribution of brain-derived neurotrophic factor mKNA in the adult mouse brain. EMBO J 92459-2464. Hokfelt T, Johansson 0, Ljungdahl A, Lundberg JM, Schultzberg M (1980) Peptidergic neurones. Nature 284:5 15-522. Hollmann M, Heinemann S (1994) Cloned glutamate receptors. Annu Rev Neurosci 17:31-108. Hollmann M, Hartley M, Heinemann S (1991) Ca2+ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition. Science 2522351-853. Hope BT, Michael GJ, Knigge KM, Vincent SR (1991) Neuronal NADPH diaphorase is a nitric oxide synthase. Proc Natl Acad Sci USA 88:2811-2814. Hopfield JJ (1995) Pattern recognition computation using action potential timing for stimulus representation. Nature 376:33-36. Hounsgaard J (1978) Presynaptic inhibitory action of acetylcholine in area CAI of the hippocampus. Exp Neurol 62:787-797. Houser CR, Esclapez M (1 994) Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation. Hippocampus 4:530-545. Houser CR, Crawford GD, Salvaterra PM, Vaughn JE (1985) Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses. J Comp Neurol 234:17-34. Houser CR, Olsen RW, Richard JG, Mohler H (1988) Immunocytochemical localization of benzodiazepine/GABAA receptors in the human hippocanipal formation. J Neurosci 8: 1370-1 383. Hsu M , Buzsiki G (1993) Vulnerability of mossy fiber targets in the rat hippocampus to forebrain ischemia. J Neurosci 13:3964-3979. Hsu M, Sik A, Gallyas F, Horvath Z, Buzsaki G (1994) Short-term and long-term changes in the postischemic hippocampus. Ann NY Acad Sci 743:121-140. Huerta PT, Lisman JE (1995) Bidirectional synaptic plasticity induced by a single burst during cholinergic theta oscillation in CA1 in virro. Neuron 15:1053-1 063. Iritani S, Fujii M, Satoh K (1989) The distribution of substance P in the cerebral cortex and hippocampal formation: an immunohistochemical study in the monkey and rat. Brain Res Bull 22:295-303. Isackson PJ, Huntsman MM, Murray KD, Gall CM (1991) BDNF
461
pocampal slices: relation to synaptic responses of dentate neurons. Brain Res Bull 18:25-27. Kosaka T (1983a) Axon initial segments of the granule cell in the rat dentate gyrus: synaptic contacts on bundles of axon initial segments. Brain Res 274:129-134. Kosaka T (1983b) Gap junctions between non-pyramidal cell dendrites in the rat hippocampus (CA1 and CA3 regions). Brain Res 271:157161. ) Kosaka T (1 9 8 3 ~ Neuronal gap junctions in the polymorph layer of the rat dentate gyrus. Brain Res 277347-351. Kosaka T, Hama K (1985) Gap junctions between non-pyramidal cell dendrites in the rat hippocampus (CA1 and CA3 regions): a combined Golgi-electron microscopy study. J Comp Neurol 231:150-161. Kosaka T , Heizmann C W (1989) Selective staining of a population of parvalbumin-containing GABAergic neurons in the rat cerebral cortex by lectins with specific affinity for terminal N-acetylgalactosamine. Brain Res 483:158-163. Kosaka T, Kosaka K, Tateishi K, Hamaoka Y, Yanaihara N, Wu JY, Hama K (1985) GABAergic neurons containing CCK-8-like and/or VIP-like immunoreactivities in the rat hippocampus and dentate gyrus. J Comp Neurol 239:420-430. Kosaka T , Katsumaru H, Hama K, Wu JY, Heizmann C W (1987) GABAergic neurons containing the Ca2+-binding protein parvalbumin in the rat hippocampus and dentate gyrus. Brain Res 419:119-130. Kosaka T, Tauchi M, Dahl JL (1988a) Cholinergic neurons containing GABA-like and/or glutamic acid decarboxylase-like immunoreactivities in various brain regions of the rat. Exp Brain Res 70:605-617. Kosaka T , Wu JY, Benoit R (1988b) GABAergic neurons containing somatostatin-like immunoreactivity in the rat hippocampus and dentate gyrus. Exp Brain Res 71:388-398. Kosofsky BE, Molliver ME (1987) The serotoninergic innervation of cerebral cortex: different classes of axon terminals arise from dorsal and medial raphe nuclei. Synapse 1: 153-168. Krnjevic K, Schwartz S (1967) The action of gamma-aminobutyric acid on cortical neurons. Exp Brain Res 3:320-336. Krnjevic K, Pumain R, Renaud L (1971) The mechanism of excitation by acetylcholine in the cerebral cortex. J Physiol (Lond) 215:247-68. Krnjevic K, Ropert N, Casullo J (1988) Septohippocampal disinhibition. Brain Res 438:182-192. Kuffler SW (1960) Excitation and inhibition in single nerve cells. Harvey Lect 54:176. Kunkel DD, Schwartzkroin PA (1988) Ultrastructural characterization and GAD co-localization of somatostatin-like immunoreactive neurons in CA1 of rabbit hippocampus. Synapse 2:371-381. Kunkel DD, Lacaille JC, Schwartzkroin PA (1 988) Ultrastructure of stratum lacunosum-moleculare interneurons of hippocampal CA1 region. Synapse 2:382-394. Lacaille J-C (1 991) Postsynaptic potentials mediated by excitatory and inhibitory amino acids in interneurons of stratum pyramidale of the CA1 region of rat hippocampus slices in vitro. J Neurophysiol 66:1441-1454. Lacaille J-C, Schwartzkroin PA (1988a) Stratum lacunosum-moleculare interneurons of hippocampal CAI region. I. Intracellular response characteristics, synaptic responses, and morphology. J Neurosci 8: 1400-14 10. Lacaille J-C, Schwartzkroin PA (1988b) Stratum laculosum-moleculare interneurons of hippocampal CA1 region. 11. intrasomatic and intradendritic recordings of local circuit synaptic interactions. J Neurosci 8:1411-1424. Lacaille J-C, Williams S (1990) Membrane properties of interneurons in stratum oriens-alveus of the CAI region of rat hippocampus in vitro. Neuroscience 36:349-359. Lacaille J-C, Mueller A, Kunkel DD, Schwartzkroin PA (1987) Local circuit interactions between oriens/alveus interneurons and CA1 pyramidal cells in hippocampal slices: electrophysiology and morphology. J Neurosci 7:1979-1993.
462
Lambert N, Grover L (1995) The mechanism of biphasic GABA responses. Science 269:928-929. Lambert NA, Wilson WA (1993) Heterogeneity in presynaptic regulation of GABA release from hippocampal inhibitory neurons. Neuron 11~1057-1067. Lambert NA, Borroni AM, Grover LM, Teyler TJ (1991) Hyperpolarizing and depolarizing GABAA receptor-mediated dendric inhibition in area CAI of the rat hippocampus. J Neurophysiol66:1538-1548. Laurberg S (1979) Commissural and intrinsic connections of the rat hippocampus. J Comp Neurol 184:685-708. Laurberg S, Sorensen KE (198 1) Associational and commissural collaterals of neurons in the hippocampal formation (hilus fasciae dentatae and subfield CA3). Brain Res 212:287-300. Laurerborn IC, Tran TMD, Isackson PJ, Gall C M (1 993) Nerve growth factor mRNA is expressed by GABAergic neurons in rat hippocampus. NeuroReport 5:273-276. Lee MG, Chrobak JJ, Sik A, Wiley RG, Buzsriki G (1994) Hippocampal theta activity following selective lesion of the septa1 cholinergic system. Neuroscience 62:1033-1047. Leranth C, Frotscher M (1 986) Synaptic connections of cholecystokininimmunoreactive neurons and terminals in the rat fascia dentata: a combined light and electron microscopic study. J Comp Neurol 254:51-64. Leranth C, Frotscher M (1987) Cholinegic innervation of hippocampal GAD- and somatostatin-immunoreactive commissural neurons. J Comp Neurol 261:33-47. Leranth C, Ribak CE (1991) Calcium-binding proteins are concentrated in the CA2 field of the monkey hippocampus: a possible key to this regions resistance to epileptic damage. Exp Brain Res 85: 129-136. Leranth C, Frotscher M, Tombol T, Palkovits M (1984) Ultrastructure and synaptic connections of vasoactive intestinal polypeptide-like immunoreactive non-pyramidal neurons and axon terminals in the rat hippocampus. Neuroscience 12:53 1-542. Leranth C, Malcolm AJ, Frotscher M (1 990) Afferent and efferent synaptic connections of somatostatin-immunoreactive neurons in the rat fascia dentata. J Comp Neurol 295:lll-122. Leranth C, Szeidemann Z, Hsu M, Buzsiki G (1996) AMPA receptors in the rat and primate hippocampus: a possible absence of Glur213 subunits in most interneurons. Neuroscience 70631-652. Leung L-WS, Yim C-YC (1991) Intrinsic membrane potential oscillations in hippocampal neurons in vitro. Brain Res 553:261-274. Leung LS (1992) Fast (beta) rhythms in the hippocampus: a review. Hippocampus 293-98. Levey AI, Wainer BH, Rye DB, Mufson EJ, Mesulam M M (1984) Choline acetyltransferase-immunoreactive neurons intrinsic to rodent cortex and distinction from acetylcholinesterase-positive neurons. Neuroscience 13:341-353. Levey AI, Edmunds SM, Koliatsos V, Wiley RG, Heilman CJ (1995) Expression of ml-m4 muscarinic acetylcholine receptor proteins in rat hippocampus and regulation by cholinergic innervation. J Neurosci 15:4077-4092. Li X-G, Somogyi P, Tepper JM, Buzsriki G (1992) Axonal and dendritic arborization of an intracellularly labeled chandelier cell in the CA1 region of rat hippocampus. Exp Brain Res 90:519-525. Li X-G, Somogyi P, Minen A, Buzsiki G (1994) The hippocampal CA3 network: an in vivo intracellular labeling study. J Comp Neurol 339: 181-208. Lidov HG, Grzanna R, Molliver ME (1980) The seroronin innervation of the cerebral cortex in the rat-an immunohistochemical analysis. Neuroscience 5:207-227. Lisman JE, Harris KM (1993) Quanta1 analysis and synapric anatomyintegrating two views of hippocampai plasticity. TINS 16:141-147. Lisman JE, Idiart MAP (1995) A mechanism for storing 7 -+ 2 shortterm memories in oscillatory subcycles. Science 267:1512-1514. Liu Y, Fujise N, Kosaka T (1996) Distribution of calretinin immunoreactivity in the mouse dentate gyrus. Exp Brain Res 108:389403.
463
synaptic currents in rat hippocampal CA3 pyramidal neurons. J Neurophysiol 68: 16-27. McBain CJ (I 994) Hippocampal inhibitory neuron activity in the elevated potassium model of epilepsy. J Neurophysiol 72: 1-1 1. McBain CJ, DiChiara TJ, Kauer JA (1994) Activation of metabotropic glutamate receptors differentially affects two classes of hippocampal interneurons and potentiates excitatory synaptic transmission. J Neurosci 14:4433-4445. McBain CJ, Dingledine R (1993) Heterogeneity of synaptic glutamace receptors on CA3 stratum radiatum interneurones of rat hippocampus. J Physiol 462373-392. McCormick DA, Pape H-C (1 990) Properties of a hyperpolarization-activated cation current and its role in rhythmic oscillation in thalamic relay neurones. J Physiol (Lond) 431:291-318. McMahon LL, Kauer JA (1995) Tetanus-induced LTP in hippocampal pyramidal cells is accompanied by synaptic depression in neighboring interneurons. SOCNeurosci Abstr 21: 1102. McNamara J O (1994) Cellular and molecular basis of epilepsy. J Neurosci 14:34 13-3425. McNaughton BL, Douglas RM, Goddard G V (1978) Synaptic enhancement in fascia dentata: cooperativity among coative afferents. Brain Res 157:277-293. McQuiston AR, Petrozzino JJ, Connor JA, Colmers WF (1996) Neuropeptide Y1 receptors inhibit N-type calcium currents and reduce transient calcium increases in rat dentate granule cells. J Neurosci 15:1422-1429. Medina I, Fillipova Barbin G, Ben-Ari Y, Bregestovski P (1994) Kainateinduced inactivation of NMDA currents via an elevation of intracellular Ca2+ in hippocampal neurons. J Neurophysiol 7 2 4 5 6 4 6 5 . Michelson HB, Wong RKS (1991) Excitatory synaptic responses mediated by GABA receptors in the hippocampus. Science 253:14201423. Michelson HB, Wong RKS (1994) Synchronization of inhibitory neurones in the guinea-pig hippocampus in vitro. J Physiol 4 7 7 3 4 5 . Miettinen R, Freund T F (1992a) Convergence and segregation of septal and median raphe inputs onto different subsets of hippocampal inhibitory interneurons. Brain Res 594263-272. Miettinen R, Freund T F (1992b) Neuropeptide-Y-containing interneurons in the hippocampus receive synaptic input from median raphe and GABAergic septal afferents. Neuropeptides 22: 185-1 93. Miettinen R, Gulyis AI, Baimbridge KG, Jacobowitz DM, Freund T F (1992) Calretinin is present in non-pyramidal cells of the rat hippocampus-11. Co-existence with other calcium binding proteins and GABA. Neuroscience 4829-43. Migaud M, Roques BP, Durieux C (1994) Effects of cholecystokinin octapeptide and BC 264, a potent and selective CCK-B agonist on aspartate and glutamate release from rat hippocampal slices. Neuropharmacology 33:737-743. Miles R (1 99 1) Tetanic stimuli induce a short-term enhancement of recurrent inhibition in the CA3 region of guinea-pig hippocampus in vitro. J Physiol (Lond) 443:669-682. Miles R, Poncer J-C (1993) Metabotropic glutamate receptors mediate a post-tetanic excitation of guinea-pig hippocampal inhibitory neurones. J Physiol (Lond) 463:461-473. Miles R, Wong RKS (1987a) Inhibitory control of local excitatory circuits in the guinea-pig hippocampus. J Physiol (Lond) 388:611-629. Miles R, Wong RKS (1787b) Latent synaptic pathways revealed after tetanic stimulation in the hippocampus. Nature 329:724-726. Miles R, Tbth K, Gulyis AI, Hijos N, Freund T F (1996) Differences between somatic and dendritic inhibition in the hippocampus. Neuron 16415-823. Milner TA, Bacon CE (1989a) GABAergic neurons in the rat hippocampal formation: ultrastructure and synaptic relationships with catecholaminergic terminals. J Neurosci 9 3 4 10-3427. Milner TA, Bacon CE (1989b) Ultrastructural localization of somatostatin-like immunoreactivity in the rat dentate gyrus. J Comp Neurol 290:544-560.
464
Milner TA, Veznedaroglu E (1992) Ultrastructural localization of neuropeptide-Y-like immunoreactivity in the rat hippocampal formation. Hippocampus 2: 107-1 26. Misgeld U, Frotscher M (1986) Postsynaptic-GABAergic inhibition of non-pyramidal neurons in the guinea-pig hippocampus. Neuroscience 19:193-206. Misgeld U, Nee MR (1984) Long-term potentiation and inhibition in CA3 neurons. Exp Brain Res 9:325-332. Misgeld U, Muller W, Brunner H (1989) Effects of (-)badofen on inhibitory neurons in the guinea pig hippocampal slice. Eur J Physiol 414:139-1 44. Mizumori SJY, Barnes CA, McNaughton BL ( I 990) Behavioral correlates of theta-on and theta-off cells recorded from hippocampal formation of mature young and aged rats. Exp Brain Res 80:365-373. Mizumori SJY, Barnes CA, McNaughton BL (1992) Differential effects of age on subpopulations of hippocampal theta cells. Neurobiol Aging 13:673-679. Mody I, Heinemann U (1987) NMDA receptors of dentate gyrus granule cells participate in synaptic transmission following kindling. Nature 326:701-704. Mody I, De Koninck Y, Otis TS, SoltCsz I (1994) Bridging the cleft at GABA synapses in the brain. TINS 17:517-525. Molnar E, Baude A, Richmond SA, Pate1 PB, Somogyi P, McIlhinney RA (1 993) Biochemical and immunocytochemical characterization of antipeptide antibodies to a cloned GluRl glutamate receptor subunit: cellular and subcellular distribution in the rat forebrain. Neuroscience 53:307-326. Monnet FP, Fournier A, Debonnel G, de Montigny C (1992) Neuropeptide Y potentiates selectively the N-methyl-D-aspartate response in the rat CA3 dorsal hippocampus. I. Involvement of an atypical neuropeptide Y receptor. J Pharmacol Exp Ther 263: 12121218. Morales M, Battenberg E, de Lecea L, Sanna P, Bloom FE (1994) Development and characterization of antibodies against the 5TH3 receptor. SOCNeurosci Abstr 20:1156. Morilak DA, Garlow SJ, Ciaranello RD (1993) Immunocytochemical localization and description of neurons expressing serotonin2 receptors in the rat brain. Neuroscience 54:701-717. Morrison JH, Molliver ME, Grzanna R (1979) Noradrenergic innervation of cerebral cortex: widespread effects of local cortical lesions. Science 205:313-316. Morrison JH, Benoit R, Magistretti PJ, Ling N, Bloom FE (1982) Immunohistochemical distribution of pro-somatostatin-related peptides in hippocampus. Neurosci Lett 34: 137-142. Moser EI (1996) Altered inhibition of dentate granule cells during spatial learning and an exploration task. J Neurosci 16:1247-1259. Motr DD, Lewis DV (1991) Facilitation of the induction of long-term potentiation by GABA-B receptors. Science 252:1718-1720. Moudy AM, Kunkel DD, Schwartzkroin PA (1993) Development of dopamine-beta-hydroxylase-positive fiber innervation of the rat hippocampus. Synapse 15:307-318. Mudrich LA, Baimbridge KG (1989) Long-term structural changes in the rat hippocampal formation following cerebral ischemia. Brain Res 493: 179-1 84. Mulkey KM, Malenka RC (1992) Mechanisms underlying induction of homosynaptic long-term depression in area CAI of the hippocampus. Neuron 9:967-975. Muller W, Misgeld U (199 1) Picrotoxin- and 4-aminopyridine-induced activity in hilar neurons in the guinea pig hippocampal slice. J Neurophysiol 65:141-147. Murthy VN, Fetz EE (1992) Coherent 25- to 3 5 - H ~ oscillations in the sensorimotor cortex of awake behaving monkeys. Proc Natl Acad Sci USA 89:5670-5674. Naegele JR, Katz LC (1990) Cell surface molecules containing N-acetylgalactosamine are associated with basket cells and neurogliaform cells in cat visual cortex. J Neurosci 10:540-557. Naegele JR, Arimatsu Y, Schwartz P, Barnstable CJ (1988) Selective
465
GABAA responses in hippocampal pyramidal cells. J Neurosci 12: 41224132. Pitler TA, Alger BE (1994) Depolarization-induced suppression of GABAergic inhibition in rat hippocampal pyramidal cells: G protein involvement in a presynaptic mechanism. Neuron 13:1447-1455. Pollard H , Charriaut-Marlangue C, Cantagrel S, Represa A, Robain 0, Moreau J, Ben-Ari Y (1994) Kainate-induced apoptotic cel death in hippocampal neurons. Neuroscience 63:7-18. Poncer J-C, Shinozaki H , Miles R (1995) Distinct metabotropic glutamate receptors modulate synaptic inhibition in the rat hippocampus. J Physiol (Lond) 485:121-134. Poncer J-C, Shinozaki H , Miles R (1995) Dual modulation of synaptic inhibition by distinct metabotropic gluatamate receptors in the rat hippocampus. J Physiol (Lond) 485:124-154. Poolos N, KocsisJ (1990) Dendritic action potential activated by NMDA receptor-mediated EPSPs in CAI hippocampal cells. Brain Res 524:342-346. Prince DA (1978) Neurophysiology of epilepsy. Annu Rev Neurosci 1:359-4 15. Radpour S, Thomson AM (1992) Synaptic enhancement induced by NMDA and Qp receptors and presynaptic activity. Neurosci Lett 1381119-122. Raggenbass M, Wuarin JP, Ghwiler BH, Dreifuss JJ (1985) Opposing effects of oxytocin and a p-receptor agonistic opioid peptide on the same class of non-pyramidal neurons in the rat hippocampus. Brain Res 344:392-396. Ramon y Cajal S (1893) Estructura del asfa de Ammon y fascia dentata. Ann SOC Esp Hist Nat 22. Ramon y Cajal S (191 1) Histologie de systeme nerveux de IHomme et des vertebres tomme 11. Paris: Maloine. Ranck JB Jr (1973) Studies on single neurons in dorsal hippocampal formation and septum in unrestrained rats. I. Behavioral correlates and firing repertoires. Exp Neurol 42:461-531. Recce LJ, Schwartzkroin PA (1991) Effects of cholinergic agonists on two non-pyramidal cell types in rat hippocampal slices. Brain Res 566: 115-1 26. Reckling J C (1993) Effects of met-enkephalin on GABAergic spontaneous miniture IPSPs in organotypic slice cultures of the rat hippocampus. J Neurosci 13:1954-1964. Regidor J, Martin-Trujillo JM, Lopez-Garcia C, Marin-Guiron F (1974) Estudio citoarquitectonico de la corteza cerebral de reptiles. 11. Tipologia dendritica y distribucion neuronal en areas corticales de Lacerta galloti. Trab Inst Cajal Inv Biol 66:l-32. Resibois A, Rogers J H (1992) Calretinin in rat brain-an immunohistochemical study. Neuroscience 46: 101-1 34. Ribak CE, Peterson GM (1991) Intragranular mossy fibers in rats and gerbils form synapses with the somata and proximal dendrites of basket cells in the dentate gyrus. Hippocampus 1:355-364. Ribak CE, Seress L (1983) Five types of basket cell in the hippocampal dentate gyrus: a combined Golgi and electron microscopic study. J Neurocytol 12:577-597. Ribak CE, Vaughn JE, Saito K (1978) Immunocytochemical localization of glutamic acid decarboxylase in neuronal somata following colchicine inhibition of axonal transport. Brain Res 140:315-332. Ribak CE, Vaughn JE, Barber RP (1981) Immunoqtochemical localization of GABAergic neurons at the electron microscopical level. Histochemistry 13:555-582. Ribak CE, Seress L, Amaral DG (1985) The development, ultrastructure and synaptic connections of the mossy cells of the dentate gyrus. J Neurocytol 148355857, Ribak CE, Seress L, Peterson GM, Seroogy KB, Fallon JH, Schmued LC (1986) A GABAergic inhibitory component within the hippocampal commissural pathway. J Neurosci 63432-3498. Ribak CE, Nitsch R, Seress L (1990) Proportion of parvalbumin-positive basket cells in the GABAergic innervation of pyramidal and granule cells of the rat hippocampal formation. J Comp Neurol300449461. Ribak CE, Seress L, Leranth C (1993) Electron microscopic immuno-
466
FREUND A N D BUZSAKI
Schmitz D, Empson RM, Heinemann U (1995) Serotonin reduces inhibition via 5-HTlA receptors in area CAI of rat hippocampal slices in vitro. J Neurosci 15:7217-7225. Schwartzkroin PA, Kunkel DD (1985) Morphology of identified interneurons in the CA1 regions of guinea pig hippocampus. J Comp Neurol 232:205-218. Schwartzkroin PA, Mathers LH (1978) Physiological and morphological identification of a nonpyramidal hippocampal cell type. Brain Res 157: 1-10. Schwerdtfeger WK, Lopez Garcia C (1986) GABAergic neurons in the cerebral cortex of the brain of a lizard (Podarcis hirpanicu). Neurosci Lett 68:117-121. Scotti AL, Nitsch C (1991) The perforant path in the seizure sensitive gerbil contains the Ca(2+)-binding protein parvalbumin. Exp Brain Res 85:137-143. Seay-Lowe SI., Claiborne RJ (1 992) Morphology of intracellularly labeled interneurons in the dentate gyrus ofthe immature rat. J Comp Neurol 32423-36. Seeburg PH (1993) The TINS/TIPS lecture. The molecular biology of mammalian glutamate receptor channels. TINS 16:359-365. Segal M (1990a) A subset of local interneurons generate slow inhibitory postsynaptic potentials in hippocampal neurons. Brain Res 5 11: 163-164. Segal M (1990b) Serotonin attenuates a slow inhibitory postsynaptic potential in rat hippocampal neurons. Neuroscience 36:63 1-641. Segal M, Bloom FE (I 974) The action of norepinephrine in the rat hippocampus. 11. Activation of the input pathway. Brain Res 7299-114. Segal M, Greenberger V (1992) Acidic amino acids evoke a smaller [Ca2+]; in GABAergic than non-GABAergic hippocampal neurons. Neurosci Lett 140:243-246. Seress L (1988) Interspecies comparison of the hippocampal formation shows increased emphasis on the regio superior in the Ammons horn of the human brain. J Hirnforsch 29:335-340. Seress L (1992) Morphological variability and developmental aspects of monkey and human granule cells: differences between the rodent and primate dentate gyrus. Epilepsy Res (Suppl) 7:3-28. Seress L, Frotscher M (199 1) Basket cells in the monkey fascia dentataa Golgi/electron microscopic study. J Neurocytol 20:9 15-928. Seress L, Leranth C (1996) Distribution of substance P-immunoreactive neurons and fibers in the monkey hippocampal formation. Neuroscience 71:633-650. Seress L, Pokorny J (1981) Structure of the granular layer of the rat dentate gyrus: a light microscopic and Golgi study. J Anat 133: 18 1-195. Seress L, Ribak CE (1983) GABAergic cells in the dentate gyrus appear to be local circuit and projection neurons. Exp Brain Res 50:173-182. Seress L, Ribak CE (1984) Direct commissural connections to the basket cells of the hippocampal dentate gyrus: anatomical evidence for feed-fonvard inhibition. J Neurocytol 13:2 15-225. Seress L, Ribak C E (1985) A combined Golgi-electron microscopic study of non-pyramidal neurons in the CA1 area of the hippocampus. J Neurocytol 1 4 717-730. Seress L, Ribak CE (1990a) Postnatal development of the light and electron microscopic features of basket cells in the hippocampal dentate gyrus of the rat. Anat Embryo1 181:547-565. Seress L, Ribak C E (1990b) The synaptic connections of basket cell axons in the developing rat hippocampal formation. Exp Brain Res 8 1~500-508. Seress L, Ribak CE (1992) Ultrastructural features of primate granule cell bodies show important differences from those of rats-axosomatic synapses, somatic spines and infolded nuclei. Brain Res 569: 353-357. Seress L, Gulyis AI, Freund TF (1991) Parvalbumin- and calbindin D28k-immunoreactive neurons in thc hippocampal formation of the macaque monkey. J Comp Neurol 313:162-177. Seress L, Gulyis AI, Ferrer I, Tunon T, Soriano E, Freund TF (1993a) Distribution, morphological features, and synaptic connections of
cytochemical study of the distribution of parvalbumin-containing neurons and axon terminals in the primate dentate gyrus and Ammons horn. J Comp Neurol 327:298-321. Richards JG, Schoch P, Haring P, Takacs B, Mohler H (1987) Resolving GABAA/benzodiazepine receptors: cellular and subcellular localization in the CNS with monoclonal antibodies. J Neurosci 7:18661886. Richmond BJ, Optican LM (1990) Temporal encoding of two-dimensional patterns by single units in primate primary visual cortes: 11. Information trasrnission. J Neurophysiol 64:370-380. Richter-Levin G, Segal M (1990) Effects of serotonin releasers on dentate granule cell excitability in the rat. Exp Brain Res 82:199-207. Roberts GW, Woodhams PL, Polak JM, Crow TJ (1984) Distribution of neuropeptides in the limbic system of the rat: the hippocampus. Neuroscience 11:35-77. Rocamora N, Pascual M, Acsidy L, de Leces L, Freund TF, Soriano E ( 1 996) Expression of NGF and N T 3 mRNA in hippocampal interneurons iiiiiervated by the GABAergic septohippocampal pathway. J Neurosci 16:3991-4004. Rogers J H (1992) Irnmunohistochemical markers in rat cortex-co-localization of calretinin and calbindin-d28k with neuropeptides and GABA. Brain Res 587:147-157. Ropert N (1988) Inhibitory action of serotonin in CA1 pyramidal neurons in vitro. Neuroscience 36:631-641. Ropert N , Guy N (1991) Serotonin facilitates GABAergic transmission in the CA1 region of rat hippocampus in vitro. J Physiol (Lond) 44 1 :12 1-1 36. Rose G, Schubert P (1977) Release and transfer of [3H]adenosine derivatives in the cholinergic septa1 system. Brain Res 121:353-357. Rosene DL, Van Horsen GW (1987) The hippocampal formation of the primate brain. A review of some comparative aspects of cytoarchitecture and connections. In: Cerebral cortex, vol 6: further aspects of cortical function, including hippocampus (Jones EG, Peters A, eds), pp 345-356. New York: Plenum Press. Sah P, Hestrin S, Nicoll RA (1990) Properties of excitatory postsynaptic currents recorded in vitro from rat hippocampal interneurones. J Physiol (Lond) 430:605-616. Saint DA, Buckett KJ ( I 99 1) Modulation of the transient potassium current in rat hippocampal neurons by cholecystokinin. Neuropeptides 20: 151-1 57. Samulack DD, Lacaille J C (1 993) Hyperpolarizing synaptic potentials evoked in CAI pyramidal cells by glutamate stimulation of interneurons from the orienslalveus border of rat hippocampal slices. 11. Sensitivity ro GABA antagonists. Hippocampus 3:345-358. Schaffer K (1 892) Beitrag zur histologie der Amnionshornformation. Arch Mikrosk Anat 39:611-632. Scharfman HE (1 994) EPSPs of dentate gyrus granule cells during epileptiform bursts of dentate hilar mossy cells and area CA3 pyramidal cells in disinhibited rat hippocampal slices. J Neurosci 14:604 1-6057. Scharfman H E (1995a) Electophisiological diversity of pyramidal-shaped neurons at the granule cell laydhilus border of the rat dentate gyms recorded in vitro. Hippocampus 5:287-305. Scharfman HE (1 995b) Electrophysiological evidence that dentate hilar mossy cells are excitatoty and innervate both granule cells and interneurons. J Neurophysiol 74: 179-1 94. Scharfman HE, Sarvey JM (1985) Gamma-aminobutyrate sensitivity does not change during long-term potentiation in rat hippocampal slices. Neuroscience 15:695-702. Scharfman HE, Schwartzkroin PA (1988) Further studies of the effects of somatostatin and related peptides in area CAI of rabbit hippocampus. Cell Mol Neurobiol 8:411-429. Scharfman HE, Schwartzkroin PA (1989) Selective depression of GABAmediated IPSPs by somatostatin in area CA1 of rabbit hippocampal slices. Brain Res 493:205-211. Schmidt-Kastner R, Freund T F (1991) Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 40:599-636.
467
SoltCsz I, Deschenes M (1993) Low- and high-frequency membrane potential oscillations during theta activity in CA1 and CA3 pyramidal neurons of the rat hippocampus under ketamine-xylazine anesthesia. J Neurophysiol 70:97-116. Solttsz I, Mody I (1994) Patch-clamp recordings reveal powerful GABAergic inhibition in dentate hilar neurons. J Neurosci 14:23652376. Solttsz I, Smetters DK, Mody I (1995) Tonic inhibition originates from synapses close to the soma. Neuron 14:1273-1283. Somogyi P (1977) A specific axo-axonal interneuron in the visual cortex of the rat. Brain Res 136:345-350. Somogyi P (1978) The study of Golgi stained cells and of experimental degeneration under the electron microscope: a direct method for the identification in the visual cortex of three successive links in a neuron chain. Neuroscience 3: 167-180. Somogyi P, Freund T F (1989) Immunocytochemistry and synaptic relationships of physiologically characterized, HRP-filled neurons. In: Neuroanatomical tract-tracing methods, vol 2: recent progress (Heimer L, Zaborszky L, eds), pp 239-264. New York: Plenum. Somogyi P, Freund TF, Halasz N, Kisvarday ZF (1981) Selectivity of neuronal [3H]GABA accumulation in the visual cortex as revealed by Golgi staining of the labeled neurons. Brain Res 225:431-436. Somogyi P, Nunzi MG, Gorio A, Smith AD (1983a) A new type of specific interneuron in the monkey hippocampus forming synapses exclusively with the axon initial segments of pyramidal cells. Brain Res 259:137-142. Somogyi P, Smith AD, Nunzi MG, Gorio A, Takagi H , Wu JY (1 983b) Glutamate decarboxylase immunoreactivity in the hippocampus of the cat: distribution of immunoreactive synaptic terminals wiht special reference to the axon initial segment of pyramidal neurons. J Neurosci 3: 1450-1468. Somogyi P, Hodgson AJ, Smith AD, Nunzi MG, Gorio A, Wu JY (1984) Different populations of GABAergic neurons in the visual cortex and hippocampus of cat contain somatostatin- or cholecystokinin-inimunoreactive material. J Neurosci 4:2590-2603. Somogyi P, Freund TF, Hodgson AJ, Somogyi J, Beroukas D, Chubb I W (1985a) Identified axo-axonic cells are immunoreactive for GABA in the hippocampus and visual cortex of the cat. Brain Res 332: 143-149. Somogyi P, Hodgson AJ, Chubb IW, Penke B, Erdei A (1 985b) Antisera to gamma-aminobutyric acid. J Histochem Cytochem 33:240-248. Soriano E, Frotscher M (1989) A GABAergic axo-axonic cell in the fascia dentata controls the main excitatory hippocampal pathway. Brain Res 503:170-174. Soriano E, Frotscher M (1993a) GABAergic innervation of the rat fascia dentata: a novel type of interneuron in the granule cell layer with extensive axonal arborization in the molecular layer. J Comp Neurol 334:385-396. Soriano E, Frotscher M (1993b) Spiny nonpyramidal neurons in the CA3 region of the rat hippocampus are glutamate-like immunoreactive and receive convergent mossy fiber input. J Comp Neurol 333:435448. Soriano E, Frotscher M (1994) Mossy cells of the rat fascia dentata are glutamate-immunoreactive. Hippocampus 4:65-70. Soriano E, Nitsch R, Frotscher M (1990) Axo-axonic chandelier cells in the rat fascia dentata: Golgi-electron microscopy and immunocytochemical studies. J Comp Neurol 293:l-25. Soriano E, Del Rio JA, Martinez A, Super H (1994) Organization of the embryonic and early postnatal murine hippocampus. I. Immunocytochemical characterization of neuronal populations in the subplate and marginal zone. J Comp Neurol 342:571-595. Spencer WA, Kandel ER (1961) Electrophysiology of hippocatnpal neurons: IV. Fast prepotentials. J Neurophysiol 24260-271. Spruston N, Schiller Y, Stuart G, Sakmann B (1995) Activity-dependent action potential invasion and calcium influx into hippocampal CAI dendrites. Science 268:297-300.
468
Staley KJ, Mody I (1992) Shunting of excitatory input to dentate gyms granule cells by a depolarizing GABAA receptor-mediated postsynaptic conductance. J Neurophysiol 68: 197-212. Staley KJ, Soldo BL, Proctor WR (1995) Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors. Science 269:977-8 I . Stanfield BB, Cowan WM (1984) An EM autoradiographic study of the hypothalamo-hippocampal projection. Brain Res 309:299-307. Stanton PK, Sejnowski TJ (1989) Associative long-term depression in the hippocampus induced by hebbian covariance. Nature 339:2 15227. Staubli U, Xu FB (1995) Effects of 50HT3 receptor antagonism on hippocampal theta rhythm, memory and LTP induction in the freely moving rat. J Neurosci 15:2445-2452. Stelzer A, Slaw NT, Bruggencate G (1987) Activation of NMDA receptors blocks GABAergic inhibition in an in vitro model of epilepsy. Nature 326:698-701. Stelzer A, Simon G, Kovacs G, Rai A (1994) Synaptic disinhibition during maintenance of long-term potentiation in the CAI hippocampal subfield. Proc Natl Acad Sci USA 91:3058-3062. Stevens CF, Wang Y (1995) Facilitation and depression at single central synapses. Neuron 14795-802. Stewart M, Fox SE (1989) Two populations of rhythmically bursting neurons in rat medial septum are revealed by atropine. J Neurophysiol 61 :982-993. Stewart M, Fox SE (1990) Do septal neurons pace the hippocampal theta rhythm? TINS 13:163-168. Stewart M, Luo Y, Fox SE (1992) Effects of atropine on hippocampal theta cells and complex-spike cells. Brain Kes 591 :122-128. Stichel CC, Singer W (1985) Organization and morphological characteristics of choline acetyltransferase-containing fibers in the visual thalamus and striate cortex of the cat. Neurosci Lett 53:155-160. Storm-Mathisen J, Leknes AK, Bore AT, Vaaland JL, Edminson P, Haug FMS, Ottersen OP (1983) First visualization of glutamate and GABA in neurones by immunocynochemistry. Nature 301 :5 17-520. Stuart G, Sakmann B (1994) Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Science 367:69-72. Stumpf C (1965) Drug action on the electrical activity of the hippocampus. Int Rev Neurobiol 8:77-138. Sutula T, He XX, Cavazos J, Scott G (1988) Synaptic reorganization in the hippocampus induced by abnormal functional activity. Science 239:1147-1150. Suzuki SS, Smith GK (1987) Spontaneous EEG spikes in the normal hippocampus: 111. Relations to evoked potentials. Electroencephalogr Clin Neurophysiol 69:541-549. Swanson LW (1983) Neuropeptides-new vistas on synaptic transmission. Trends Neurosci 6:3-19. Swanson I,W, Sawchenko PE, Cowan W M (1980) Evidence that the commissural, associational and septal projections of the regio inferior of the hippocampus arise from the same neurons. Brain Res 197: 207-212. Szentigothai J (1 962) O n the synaptology of the cerebral cortex. In: Structure and function of the nervous system (Sarkissov SA, ed), pp 6-14. Moscow: Medgiz. Szentigothai J (1965a) The synapse of short local neurons in the cerebral cortex. In: Modern trends in neuromorphology (Szentigothai J, ed), pp 251-276. Budapest: Akademiai Kiado. Szentigothai J (1965b) The use of degeneration methodsin the investigation of short neuronal connections. In: Degeneration patterns in the nervous system (Singer M, Schade JP, eds), pp 1-32. Amsterdam: Elsevier. Szentigothai J, Arbib MA (1974) Conceptual models of neural organization. Neurosci Res Prog Bull 12:307-510. Tamamaki N, Nojyo Y (1990) Disposition of the slab-like modules formed by axon branches originating from single CAI pyramidal neurons in the rat hippocampus. J Comp Neurol 291:509-519. Tamamaki N, Nojyo Y (1993) Projection of the entorhinal layer-I1 neu-
469
Wilcox BJ, Unnerstall JR (1990) Identification of a subpopulation of neuropeptide Y-containing locus coeruleus neurons that project to the entorhinal cortex. Synapse 6:284-291. Williams S, Lacaille J-C (1990) Bicuculline- and phaclofen-resisant hyperpolarizations evoked by glutamate applications to stratum lacunosum-moleculare in CAI pyramidal cells of the rat hippocampus in vitro. Eur J Neurosci 2:993-1003. Williams S, Vachon P, Lacaille J-C (1993) Monosynaptic GABA-mediated inhibitory postsynaptic potentials in CA1 pyramidal cells of hyperexcitable hippocampal slices from kainic acid-treated rats. Neuroscience 52:54 1-5 54. Williams S, Samulack DD, Beaulieu C, Lacaille J C (1994) Membrane properties and synaptic responses of interneurons located near the stratum lacunosum-moleculare/radiatum border of area CAI in whole-cell recordings from rat hippocampal slices. J Neurophysiol 71 :22 17-2235. Wilson MA, McNaughton BL (1993) Dynamics of the hippocampal ensemble code for space. Science 261:1055-1058. Wong RKS, Watkins DJ (1982) Cellular factors influencing GABAA response in hippocampal pyramidal cells. J Neurophysiol48:938-954. Wong RKS, Prince DA, Basbaum A1 (1979) Intradendritic recordings from hippocampal neurons. Proc Natl Acad Sci USA 76:986-990. Woodson W, Nitecka L, Ben-Ari Y (1989) Organization of the GABAergic system in the rat hippocampal formation: a quantitative immunocytochemical study. J. Comp Neurol 280:254-271. Woodward MP, Young WW, Bloodgood RA (1985) Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods 78: 143-1 53. Wouterlood FG, Saldana E, Witter MP (1990) Projection from the nucleus reuniens thalami to the hippocampal region: light and electron microscopic tracing study in the rat with the anterograde tracer Pbaseohs vulgaris-leucoagglutinin. J Comp Neurol 296: 179-203. W u L-G, Saggau P (1994) Pharmacological identification of two types of presynaptic voltage-dependent calcium channels at CA3-CA1 synapses of the hippocampus. J Neurosci 14:5613-5622. Xie Z, Sastry BR (1992) Actions of somatostatin on GABA-ergic synaptic transmission in the CA1 area of the hippocampus. Brain Res 59 1:239-247. Yamamoto C, Chujo T (1978) Long-term potentiation in thin hippocampal sections studied by intracellular and extracellular recordings. Exp Neurol 58:242-250. Yamamoto C, Marshall P, Hemmendinger LM, Boyer AB, Caviness VS Jr. (1988) Distribution of glucuronic acid-sulfate-containing glycoproteins in the central nervous system of the adult mouse. Neurosci Res 5:273-298. Yan YH, Van Brederode JF, Hendrickson AE (1995) Transient co-localization of calretinin, parvalbumin and calbindin-D28k in developing visual cortex of monkey. J Neurocytol 24:825-837. Yanagihara T, Yoshimine T, Morimoro K, Yamamoto K, Homburger HA (1985) Immunohistochemical investigation of cerebral ischemia in gerbils. J Neuropathol Exp Neurol 44:204-215. Yeckel MF, Berger TW (1990) Feedfonvard excitation of the hippocampus by afferents from the entorhinal cortex: redefinition of the role of the trisynaptic pathway. Proc Natl Acad Sci USA 87:58325836. Minen A, Bragin A, Nadasdy Z, Janda G, Szabo I, Sik A, Buzsiki G (19 9 5 3 Sharp-wave-associated high-frequency oscillation (200 Hz) in the intact hippocampus: network and intracellular mechanisms. J Neurosci 14:30-46. Minen A, Solttsz I, Bragin A, Penttonen M, Sik A, Buzsiki G (1995b) Intracellular correlates of hippocampal theta rhythm in identified pyramidal cells, granule cells and basket cells. Hippocampus 5:78-90. Yoon KW, Rothman SM (1991) Adenosine inhibits excitatory by not inhibitory synaptic transmission in the hippocampus. J Neurosci 11:2217-2235. Zhang L, McBain CJ (1995a) Potassium conductances underlying ac-
470
FREUND A N D BUZSAKI
excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons. Science 205:415417. Zimmer J, Laurberg S, Sunde N (1983) Neurobiology of the hippocampus. In: Neuroanatomical aspects of normal and transplanted hippocampal tissue (Seifert W, ed), pp 39-64. New York: Academic Press. Zimprich F, Zezula J, Sieghart W, Lassmann H (1991) Immunohisto-chemical localization of the alpha 1, alpha 2 and alpha 3 subunit of the GABA-A receptor in the rat brain. Neurosci Lett 127:125-128. Zipp F, Nitsch R, Soriano E, Frotscher M (1989) Entorhinal fibers form synaptic contacts on pawalbumin-immunoreactive neurons in the rat fascia dentata. Brain Res 495:161-166.
tion potential repolarization and afterhyperpolarization in rat CA1 hippocampal interneurons. J Physiol (Lond) 488:661-672. Zhang L, McBain CJ (1995b) Voltage-gated potassium currents in stratum oriens-alveus inhibitory neurones of the rat CA1 hippocampus. J Physiol (Lond) 488:647-660. Zhang DX, Levy W 3 (1993) Bicucullin permits the induction of longE term depression by heterosynaptic, translaminar conditioning in the hippocampal dentate gyrus. Brain Res 613:309-312. Zhu PJ, Krnjevic K (1994) Endogenous adenosine deaminase does not modulate synaptic transmission in rat hippocampal slices under normoxic or hypoxic conditions. Neuroscience 63:489-97. Zieglgansberger W, French ED, Bloom FE (1979) Opioid peptides may