From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience

USA

Preface

The past twenty years have witnessed an exponential increase in the understanding of the nervous system at all levels of analyses. Perhaps the most striking developments have been in the understanding of the cell and molecular biology of the neuron. The field has moved from treating the neuron as a simple black box that added up impinging synaptic input to fire an action potential to one in which the function of nerve cells involves a host of biochemical and biophysical processes that act synergistically to process, transmit and store information. In this book, we have attempted to provide a comprehensive summary of current knowledge of the morphological, biochemical, and biophysical properties of nerve cells. The book is intended for graduate students, advanced undergraduate students, and professionals. The chapters are highly referenced so that readers can pursue topics of interest in greater detail. We have also included material on mathematical modeling approaches to analyze the complex synergistic processes underlying the operation and regulation of nerve cells. These modeling approaches are becoming increasingly important to facilitate the understanding of membrane excitability, synaptic transmission, as well gene and protein networks. The final chapter in the book illustrates the ways in which the great strides in understanding the biochemical and biophysical properties of nerve cells have led to fundamental insights into an important aspect of cognition, memory.

We are extremely grateful to the many authors who have contributed to the book, and the support and encouragement during the two past years of Jasna Markovac and Johannes Menzel of Academic Press. We would also like to thank Evangelos Antzoulatos, Evyatar Av-Ron, Diasinou Fioravanti, Yoshihisa Kubota, Rong-Yu Liu, Fred Lorenzetii, Riccardo Mozzachiodi, Gregg Phares, Travis Rodkey, and Fredy Reyes for help with editing the chapters.

John H. Byrne, James L Roberts

Cellular Components of Nervous Tissue

Patrick R. Hof, Bruce D. Trapp, Jean de Vellis, Luz Claudio, David R. Colman

Several types of cellular elements are integrated to yield normally functioning brain tissue. The neuron is the communicating cell, and a wide variety of neuronal subtypes are connected to one another via complex circuitries usually involving multiple synaptic connections. Neuronal physiology is supported and maintained by the neuroglial cells, which have highly diverse and incompletely understood functions. These include myelination, secretion of trophic factors, maintenance of the extracellular milieu, and scavenging of molecular and cellular debris from it. Neuroglial cells also participate in the formation and maintenance of the blood–brain barrier, a multicomponent structure that is interposed between the circulatory system and the brain substance and that serves as the molecular gateway to the brain parenchyma.

THE NEURON

Neurons are highly polarized cells, meaning that they develop, in the course of maturation, distinct subcellular domains that subserve different functions. Morphologically, in a typical neuron, three major regions can be defined: (1) the cell body, or perikaryon, which contains the nucleus and the major cytoplasmic organelles; (2) a variable number of dendrites, which emanate from the perikaryon and ramify over a certain volume of gray matter and which differ in size and shape, depending on the neuronal type; and (3) a single axon, which extends in most cases much farther from the cell body than does the dendritic arbor (Fig. 1.1). The dendrites may be spiny (as in pyramidal cells) or nonspiny (as in most interneurons), whereas the axon is generally smooth and emits a variable number of branches (collaterals). In vertebrates, many axons are surrounded by an insulating myelin sheath, which facilitates rapid impulse conduction. The axon terminal region, where contacts with other cells are made, displays a wide range of morphological specializations, depending on its target area in the central or peripheral nervous system. Classically, two major morphological types of contacts, or synapses, may be recognized by electron microscopy: the asymmetric synapses, responsible for transmission of excitatory inputs, and the symmetric or inhibitory synapses.

FIGURE 1.1 Typical morphology of projection neurons. On the left is a Purkinje cell of the cerebellar cortex, and on the right, a pyramidal neuron of the neocortex. These neurons are highly polarized. Each has an extensively branched, spiny apical dendrite, shorter basal dendrites, and a single axon emerging from the basal pole of the cell.

The cell body and the dendrites are the two major domains of the cell that receive inputs, and the dendrites play a critically important role in providing a massive receptive area on the neuronal surface. In addition, there is a characteristic shape for each dendritic arbor, which is used to classify neurons into morphological types. Both the structure of the dendritic arbor and the distribution of axonal terminal ramifications confer a high level of subcellular specificity in the localization of particular synaptic contacts on a given neuron. The three-dimensional distribution of the dendritic arborization is also important with respect to the type of information transferred to the neuron. A neuron with a dendritic tree restricted to a particular cortical layer may receive a very limited pool of afferents, whereas the widely expanded dendritic arborizations of a large pyramidal neuron will receive highly diversified inputs within the different cortical layers in which segments of the dendritic tree are present (Fig. 1.2) (Mountcastle, 1978; Peters and Jones, 1984; Schmitt et al., 1981; Szentagothai and Arbib, 1974; Lund et al., 1995; Björklund et al., 1990). The structure of the dendritic tree is maintained by surface interactions between adhesion molecules and, intracellularly, by an array of cytoskeletal elements (microtubules, neurofilaments, and associated proteins), which also take part in the movement of organelles within the dendritic cytoplasm.

FIGURE 1.2 Schematic representation of four major excitatory inputs to pyramidal neurons. A pyramidal neuron in layer III is shown as an example. Note the preferential distribution of synaptic contacts on spines. Spines are labeled in red. Arrow shows a contact directly on the dendritic shaft.

An important specialization of the dendritic arbor of certain neurons is the presence of large numbers of dendritic spines, which are membrane-limited organelles that project from the surface of the dendrites. They are abundant in large pyramidal neurons and are much sparser on the dendrites of interneurons. Spines are more numerous on the apical shafts of the pyramidal neurons than on the basal dendrites. As many as 30,000 to 40,000 spines are present on the largest pyramidal neurons. Spines constitute the region of the dendritic arborization that receives most of the excitatory input. Each spine generally contains one asymmetric synapse; thus, the approximate density of excitatory input on a neuron can be inferred from an estimate of its number of spines. The cytoplasm within the spines is characterized by the presence of polyribosomes and a variety of filaments, including actin and α- and β-tubulin, as well as a spine apparatus comprising cisternae, membrane vesicles, and stacks of dense lamellar material (see Box 1.1) (see (Berkley, 1896; Gray, 1959; Ramón y Cajal, 1955; Coss and Perkel, 1985; Scheibel and Scheibel, 1968; Steward and Falk, 1986; Zhang and Benson, 2000; Nimchinsky et al., 2002).

BOX 1.1 SPINES

Spines are protrusions on the dendritic shafts of neurons and are the site of a large number of axonal contacts. The use of the silver impregnation techniques of Golgi or of the methylene blue used by Ehrlich in the late 19th century led to the discovery of spiny appendages on dendrites of a variety of neurons. The best known are those on pyramidal neurons and Purkinje cells, although spines occur on neuron types at all levels of the central nervous system. In 1896 Berkley observed that terminal boutons were closely apposed on spines (a fact that was later confirmed by Gray (1959) using electron microscopy) and suggested that spines may be involved in conducting impulses from neuron to neuron. In 1904, Santiago Ramón y Cajal suggested that spines could collect the electrical charge resulting from neuronal activity (Ramón y Cajal, 1955). He also noted that spines substantially increase the receptive surface of the dendritic arbor, which may represent an important factor in receiving the contacts made by the axonal terminals of other neurons. It has been calculated that the approximately 4000 spines of a pyramidal neuron account for more than 40% of its total surface area (Peters et al., 1991).

More recent analyses of spine electrical properties have demonstrated that spines are dynamic structures that can regulate many neurochemical events related to synaptic transmission and modulate synaptic efficacy (Coss et al., 1985) (see also Chapter 18). Spines are also known to undergo pathological alterations and have a reduced density in a number of experimental manipulations (such as deprivation of a sensory input) and in many developmental, neurological, and psychiatric conditions (such as dementing illnesses, chronic alcoholism, schizophrenia, and trisomy 21) (Scheibel and Scheibel, 1968). Morphologically, spines are characterized by a narrow portion emanating from the dendritic shaft, the neck, and an ovoid bulb or head. Spines have an average length of 2 μm despite considerable variability in morphology. At the ultrastructural level (Fig. 1.3), spines are characterized by the presence of asymmetric synapses and a few vesicles and contain fine and quite indistinct filaments. These filaments most likely consist of actin and α- and β-tubulins. The microtubules and neurofilaments present in the dendritic shafts do not penetrate the spines. Mitochondria and free ribosomes are infrequent, although many spines contain polyribosomes in their head and neck. Interestingly, most polyribosomes in dendrites are located at the bases of spines, where they are associated with endoplasmic reticulum, indicating that spines possess the machinery necessary for the local synthesis of proteins (Steward and Falk, 1986).

Another classic feature of the spine is the presence in the spine head of confluent tubular cisterns that represent an extension of the dendritic smooth endoplasmic reticulum. Those cisterns are referred to as the spine apparatus. The function of the spine apparatus is not fully understood but may be related to storage of calcium ions during synaptic transmission. For additional reviews on spines, see Zhang and Benson (2000) and Nimchinsky et al. (2002).

FIGURE 1.3 Ultrastructure of a single dendritic spine (Sp). Note the narrow neck emanating from the main dendritic shaft (DS) and the spine head containing filamentous material, the cisterns of the spine apparatus, and the postsynaptic density of an asymmetric synapse (arrows). AT, axon terminal.

The perikaryon contains the nucleus and a variety of cytoplasmic organelles. Stacks of rough endoplasmic reticulum are conspicuous in large neurons and, when interposed with arrays of free polyribosomes, are referred to as Nissl substance. Another feature of the perikaryal cytoplasm is the presence of a rich cytoskeleton composed primarily of neurofilaments and microtubules, discussed in detail in Chapter 2. These cytoskeletal elements are dispersed in bundles that extend into the axon and dendrites (Peters and Jones, 1984). Whereas the dendrites and the cell body can be characterized as the domains of the neuron that receive afferents, the axon, at the other pole of the neuron, is responsible for transmitting neural information. This information may be primary, in the case of a sensory receptor, or processed information that has already been modified through a series of integrative steps. The morphology of the axon and its course through the nervous system are correlated with the type of information processed by the particular neuron and by its connectivity patterns with other neurons. The axon leaves the cell body from a small swelling called the axon hillock. This structure is particularly apparent in large pyramidal neurons; in other cell types, the axon sometimes emerges from one of the main dendrites. At the axon hillock, microtubules are packed into bundles that enter the axon as parallel fascicles. The axon hillock is the part of the neuron from which the action potential is generated. The axon is generally unmyelinated in local-circuit neurons (such as inhibitory interneurons), but it is myelinated in neurons that furnish connections between different parts of the nervous system. Axons usually have larger numbers of neurofilaments than do dendrites, although this distinction can be difficult to make in small elements that contain fewer neurofilaments. In addition, the axon may be extremely ramified, as in certain local-circuit neurons; it may give out a large number of recurrent collaterals, as in neurons connecting different cortical regions; or it may be relatively straight in the case of projections to subcortical centers, as in cortical motor neurons that send their very long axons to the ventral horn of the spinal cord. At the interface of axon terminals with target cells are the synapses, which represent specialized zones of contact consisting of a presynaptic (axonal) element, a narrow synaptic cleft, and a postsynaptic element on a dendrite or perikaryon. We consider the fine structure of synapses later in this chapter. In the next section, we turn our attention to the principal morphological features of several neuronal types from the cerebral cortex, subcortical structures, and periphery as typical examples of the cellular diversity in the nervous system.

Pyramidal Cells Are the Main Excitatory Neurons in the Cerebral Cortex

All of the cortical output is mediated through pyramidal neurons, and the intrinsic activity of the neocortex can be viewed simply as a means of finely tuning their output. A pyramidal cell is a highly polarized neuron, with a major orientation axis perpendicular (or orthogonal) to the pial surface of the cerebral cortex. In cross section, the cell body is roughly triangular (Fig. 1.2), although a large variety of morphological types exist with elongate, horizontal, or vertical fusiform or inverted perikaryal shapes. A pyramidal neuron typically has a large number of dendrites that emanate from the apex and form the base of the cell body. The span of the dendritic tree depends on the laminar localization of the cell body, but it may, as in giant pyramidal neurons, spread over several millimeters. The cell body and dendritic arborization may be restricted to a few layers or, in some cases, may span the entire cortical thickness (Jones, 1984).

In most cases, the axon of a large pyramidal cell extends from the base of the perikaryon and courses toward the subcortical white matter, giving off several collateral branches that are directed to cortical domains generally located within the vicinity of the cell of origin (as explained later in this section). Typically, a pyramidal cell has a large nucleus, a cytoplasmic rim that contains, particularly in large pyramidal cells, a collection of granular material chiefly composed of lipofuscin. The deposition of lipofuscin increases with age and is considered a benign change. Although all pyramidal cells possess these general features, they can also be subdivided into numerous classes based on their morphology, laminar location, and connectivity (Fig. 1.4) (Jones, 1975). For instance, small pyramidal neurons in layers II and III of the neocortex have restricted dendritic trees and form vast arrays of axonal collaterals with neighboring cortical domains, whereas medium-to-large pyramidal cells in deep layer III and layer V have much more extensive dendritic trees and furnish long corticocortical connections. Layer V also contains very large pyramidal neurons arranged in clusters or as isolated, somewhat regularly spaced elements. These neurons project to subcortical centers such as the basal ganglia, brainstem, and spinal cord. Finally, layer VI pyramidal cells exhibit a greater morphological variability than do pyramidal cells in other layers and are involved in certain corticocortical as well as corticothalamic projections (Feldman, 1984; Hof et al., 1995a,b).

FIGURE 1.4 Morphology and distribution of neocortical pyramidal neurons. Note the variability in cell size and dendritic arborization as well as the presence of axon collaterals, depending on the laminar localization (I–VI) of the neuron. Also, different types of pyramidal neurons with a precise laminar distribution project to different regions of the brain.

Adapted with permission, from Jones (1984).

The excitatory inputs to pyramidal neurons can be divided into intrinsic afferents, such as recurrent collaterals from other pyramidal cells and excitatory interneurons, and extrinsic afferents of thalamic and cortical origin. The neurotransmitters in these excitatory inputs are thought to be glutamate and possibly aspartate. Although this division may appear relatively simplistic, the complexity and heterogeneity of excitatory transmission in the neocortex may not be derived from the presynaptic side, but rather from the postsynaptic side of the synapse. In other words, at the molecular level, a variety of glutamate receptor subunit combinations may confer different functional capacities on a given glutamatergic synapse (see Chapter 11).

Pyramidal cells not only furnish the major excitatory output of the neocortex, but also act as a major intrinsic excitatory input through axonal collaterals. The collaterals of the main axonal branch that exits from the cortex are referred to as recurrent collaterals because they ascend back to superficial layers; thus, the collateral branches of a pyramidal cell synapse in layers superficial to their origin, although a deep or local system of branches is also present (see Fig. 1.4). Although many of these branches ascend in a radial, vertical pattern of arborization, there are a separate set of projections that travel horizontally over long distances (in some instances as much as 7–8 mm). One of the major functions of the vertically oriented component of the recurrent collaterals may be to interconnect layers III and V, the two major output layers of the neocortex. In layer III pyramidal cells, 95% of the synaptic targets of the recurrent cells are other pyramidal cells. This is true of both the vertical and the distant horizontal recurrent projections. In addition, the majority of these synapses are on dendritic spines and, to some degree, on dendritic shafts. It is possible that there are regional and laminar specificities to these synaptic arrangements, although such fine patterns are not yet fully elucidated (Schmitt et al., 1981; Szentagothai and Arbib, 1974; Lund et al., 1995; Kisvarday et al., 1986a,b). These recurrent projections function to set up local excitatory patterns and coordinate multineuronal assemblies into an excitatory output.

Spiny Stellate Cells Are Excitatory Interneurons

The other major excitatory input to pyramidal cells of cortical origin is provided by the interneuron class referred to as spiny stellate cells, small multipolar neurons with local dendritic and axonal arborizations. These neurons resemble pyramidal cells in that they are the only other cortical neurons with large numbers of dendritic spines, but they differ from pyramidal neurons in that they lack an apical dendrite. Although the dendritic arbor of these neurons tends to be local, it can vary from a primarily radial orientation to one that is more horizontal. The relatively restricted dendritic arbor of these neurons is presumably a manifestation of the fact that they are high-resolution neurons that gather afferents to a very restricted region of cortex. The dendrites rarely leave the layer in which the cell body resides. The spiny stellate cell also resembles the pyramidal cell in that it provides asymmetric synapses that are presumed to be excitatory, and, like pyramidal cells, these neurons are thought to use either glutamate or aspartate as their neurotransmitter.

Spiny stellate cells exhibit extensive regional and laminar specificities in their distribution. Spiny stellate cells are found in highest concentration in layers IVC and IVA of the primary visual cortex, where they constitute the predominant neuronal type. They are also found in large numbers in layer IV of other primary sensory areas. However, several cortical regions have relatively few of these neurons, and even in areas in which these neurons are well represented, they are vastly outnumbered by aspiny interneurons (Peters and Jones, 1984; Lund et al., 1995).

The axons of spiny stellate neurons are primarily intrinsic in their targets and radial in orientation and appear to play an important role in forming links between layer IV, the major thalamorecipient layer, and layers III, V, and VI, the major projection layers (Fig. 1.5). In some respects, the axonal arbor of spiny stellate cells mirrors the vertical plexuses of recurrent collaterals; however, they are more restricted than recurrent collaterals. Given its axonal distribution, the spiny stellate neuron appears to function as a high-fidelity translator of thalamic inputs, maintaining strict topographic organization and setting up initial vertical links of information transfer within sensory areas. Presumably, both pyramidal cells and aspiny nonpyramidal cells receive these radially limited inputs of the spiny stellate neuron, suggesting that this interneuron plays a key role in setting up the excitatory component of a functional cortical domain (Peters and Jones, 1984).

FIGURE 1.5 Drawing of Golgi-impregnated spiny stellate neurons in layer IV of the primary somatosensory cortex. The insets show the cortical localization of each neuron. Coarse branches represent the dendrites and fine branches represent the axonal plexus. Note that the axon is organized vertically.

Adapted with permission, from Jones (1975).

Basket, Chandelier, and Double Bouquet Cells Are Inhibitory Interneurons

A large variety of inhibitory interneuron types are present in the cerebral cortex and in subcortical structures. These neurons contain the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and exert strong local inhibitory effects. Three major subtypes of cortical interneurons are discussed in this section as examples. In all three cases, the dendritic and axonal arborizations offer important clues to their role in the regulation of pyramidal cell function (Cobb et al., 1995; Sik et al., 1995). In addition, for several GABAergic interneurons, a subtype of a given morphological class can be further defined by a particular set of neurochemical characteristics (Somogyi et al., 1984). Although the following examples are taken from neurons prevalent in the neocortex and hippocampus of primates, inhibitory interneurons are present throughout the cerebral gray matter and exhibit a rich variety of morphologies, depending on the brain region as well as on the species studied (see Freund and Buzsaki, 1996).

Basket Cells

This class of GABAergic interneurons takes its name from the fact that its axonal endings form a basket of terminals surrounding a pyramidal cell soma (see Fig. 1.6) (Somogyi et al., 1983). Basket cells can be divided into large and small cells. This cell class provides most of the inhibitory GABAergic synapses to the somas and proximal dendrites of pyramidal cells, although the basket cells also synapse on the shaft of the apical dendrite. One basket cell may contact numerous pyramidal cells, and, in turn, several basket cells can contribute to the pericellular basket of one pyramidal cell. The basket cells have relatively large somas and multipolar morphology, with dendrites extending in all directions for several hundred micrometers such that the vertically oriented dendrites cross several layers. The axonal pattern is the defining characteristic of this cell. The axon rises vertically, quickly bifurcates, and travels long distances (1–2 mm), forming multiple pericellular arrays as it spreads horizontally. The basket cells predominate in layers III and V in the neocortex and preferentially innervate the pyramidal cells within these layers, although they do not synapse exclusively on pyramidal cells. They are also numerous amid pyramidal neurons in the hippocampus. Thus, the basket cell is the primary source of horizontally directed inhibitory inputs to the soma, proximal dendrites, and apical shaft of a pyramidal neuron. Interestingly, these cells are also characterized by certain biochemical features in that the majority of them contain the calcium-binding protein parvalbumin, and cholecystokinin appears to be the most likely neuropeptide in the large basket cells.

FIGURE 1.6 Drawing of a Golgi-impregnated basket cell from layer IVA of the primary visual cortex. Note the widely ramified dendritic tree and the wide horizontal spread of the axon that makes contact with many local neuronal perikarya. Cortical layers are indicated by Roman numerals.

Adapted with permission, from Somogyi et al. (1983).

Chandelier Cells

The chandelier cell generally has a bitufted or multipolar dendritic tree, but the dendritic tree of this neuron is quite variable (Fig. 1.7) (Freund et al., 1983). The defining characteristic of this cell class is the very striking appearance of its axonal endings. In Golgi or immunohistochemical preparations, the axon terminals appear as vertically oriented cartridges, each consisting of a series of axonal boutons, or swellings, linked together by thin connecting pieces. These axonal specializations look like old-style chandeliers, which explains why this cell type is so named. The most salient characteristic of the chandelier cell is the extraordinary specificity of its synaptic target. These neurons synapse exclusively on the axon initial segment of pyramidal cells. This characteristic is responsible for their alternate name, axoaxonic cells (Somogyi et al., 1982). Most of the chandelier cells are located in layer III, and their primary target appears to be layer III pyramidal cells, although they also synapse to a lesser extent on pyramidal cells in the deep layers. One pyramidal cell may receive inputs from multiple chandelier cells, and one chandelier cell may innervate more than one pyramidal cell. Because of the high density of chandelier cell axon endings in layer III, this particular neuron may be highly involved in controlling corticocortical circuits. In addition, because the strength of the synaptic input is correlated directly with its proximity to the axon initial segment, there can be no more powerful inhibitory input to a pyramidal cell than that of the chandelier cell. Presumably, this interneuron is in a position that enables it to completely shut down the firing of a pyramidal cell (Hof et al., 1995a; Somogyi et al., 1982, 1983; Freund et al., 1983; DeFelipe and Jones, 1992).

FIGURE 1.7 Drawing of an axoaxonic chandelier neuron from layer II of the primary visual cortex. The dendritic spread of this neuron is quite limited. Note the typical axon terminal specializations (arrow).

Adapted, from Freund et al. (1983).

Double Bouquet Cells

The cell bodies of double bouquet cells are most prevalent in layers II and III, and are present in layer V of the neocortex as well. These interneurons are characterized by a vertical bitufted dendritic tree and a tight bundle of vertically oriented varicose axon collaterals that traverse layers II through V (Fig. 1.8) (Somogyi and Cowey, 1981) and are therefore entirely different from those of chandelier and basket cells.

FIGURE 1.8 Drawing of a double bouquet cell in layer III of the primary visual cortex. The axonal tree (A) has been broken into three segments contiguous at X–X′ and Y–Y′, to display its entire radial extent. The arrow in (B) corresponds to the arrow in (A). This neuron has very long radial axonal extensions, but very limited horizontal spread. Its location is shown in the inset (C).

Adapted from Somogyi and Cowey (1981).

Of the inhibitory interneurons, the double bouquet cell serves as perhaps the best example of the emerging concept of cell typology in which connectivity, location, morphology, and neurochemical phenotype are all features that are considered in typing a given cell (DeFelipe and Jones, 1992). It is clear that neurochemical phenotype subdivides the double bouquet cell into multiple classes. For example, a GABA–calbindin–somatostatin double bouquet cell appears to be localized primarily in layers II and III and has 40% of its synapses on spines and the remaining synapses primarily on distal shafts of pyramidal and nonpyramidal cells. Large numbers of this particular subtype of double bouquet cell are present in association cortices, with fewer in primary sensory cortices. Its regional, laminar, and synaptic organization suggests that it plays a crucial role in the regulation of pyramidal cells that furnish corticocortical projections. A different subclass of double bouquet cell contains calbindin and tachykinins as peptide neuromodulators. This subclass appears to have similar synaptic targets but is present primarily in layer V and, thus, presumably regulates the activity of a different group of pyramidal cells.

Other Interneuron Subtypes

Several other subtypes of interneurons can be distinguished on the basis of their morphology and neurochemical characteristics. A particularly interesting neuron is the poorly understood clutch cell, which is driven primarily by thalamocortical inputs and, in turn, targets the spiny stellate cell of layer IV. Thus, this inhibitory interneuron is in essence situated so that it can regulate the firing rate of the spiny stellate cell in a fashion similar to how the three GABAergic neurons (basket, chandelier, and double bouquet) regulate the firing rate of pyramidal cells. Another important interneuron type is the bipolar neuron, which is characterized by elongated apical and basal dendrites and a locally ramifying axonal plexus, presumably making contacts with the apical dendrites of neighboring pyramidal cells. This cell is highly prevalent in the neocortex of rodents and has a modulatory role in the integration of cortical activity with noradrenergic projections from the brainstem. In the rat brain, some of these bipolar neurons may also contain the calcium-binding protein calretinin, but their homolog, if any, in the primate cortex remains to be determined.

Noncortical Neurons Have Distinct Morphological Characteristics

This section reviews characteristics of four neuronal types found in subcortical structures: the medium-sized spiny cells of the basal ganglia, the dopaminergic neurons of the pars compacta of the substantia nigra, the Purkinje cell of the cerebellum, and the alpha motor neuron of the ventral horn of the spinal cord. The rationale for choosing these particular neurons as representative is that each plays a determinant role in the pathogenic mechanisms of severe neurological disorders that affect humans. Thus, degeneration of the medium-sized spiny neurons is a central feature of Huntington’s disease; the death of dopaminergic neurons is the neuropathological signature of Parkinson’s disease; the loss of Purkinje cells is seen in familial cerebellar cortical degeneration; and degeneration of spinal cord motor neurons is the hallmark of lower motor neuron disease, a form of amyotrophic lateral sclerosis.

Medium-Sized Spiny Cells

These neurons are unique to the striatum, a part of the basal ganglia that comprises the caudate nucleus and putamen, where they are present in large numbers (as many as 10⁸ in humans). Medium-sized spiny cells are scattered throughout the caudate nucleus and putamen and are recognized by their relatively large size, compared with other cellular elements of the basal ganglia, and by the fact that they are generally isolated neurons (Braak and Braak, 1982). These neurons differ from all others in the striatum in that they have a highly ramified dendritic arborization radiating in all directions and densely covered with spines (Fig. 1.9) (Carpenter and Sutin, 1983). Medium-sized spiny neurons are central to the function of the basal ganglia because they furnish a major output from the caudate nucleus and putamen and receive a highly diverse input from, among other sources, the cerebral cortex, thalamus, and certain dopaminergic neurons of the substantia nigra. They have long axons that leave the basal ganglia and also form a large array of recurrent collaterals that innervate neighboring medium-sized spiny cells. These neurons are neurochemically quite heterogeneous, contain GABA, and may contain several neuropeptides such as enkephalin, dynorphin, substance P, and the calcium-binding protein calbindin. In Huntington’s disease, a neurodegenerative disorder of the striatum characterized by involuntary movements and progressive dementia, an early and dramatic loss of medium-sized spiny cells occurs. Interestingly, medium-sized spiny neurons that contain somatostatin appear to be relatively resistant to the degenerative process.

FIGURE 1.9 Drawing of a medium-size spiny neuron from the striatum. Note the highly ramified dendritic arborization radiating in all directions and the very high density of spines.

Adapted, from Carpenter and Sutin (1983).

Dopaminergic Neurons of the Substantia Nigra

The substantia nigra is characterized by a rich diversity of neuronal types that exhibit differential distributions among the various functional compartments. Of these neurons, the most conspicuous are the large dopaminergic neurons that reside mostly within the pars compacta of the substantia nigra and in the ventral tegmental area. A distinctive feature of these cells is the presence of a pigment, neuromelanin, in compact granules in the cytoplasm. These neurons are medium-sized to large, fusiform, and frequently elongated; they have several large radiating dendrites. The axon emerges from the cell body or from one of the dendrites and projects to large expanses of cerebral cortex and to the basal ganglia. These neurons contain the catecholamine-synthesizing enzyme tyrosine hydroxylase, as well as the monoamine dopamine as their neurotransmitters; some of them colocalize calbindin and calretinin. These neurons are severely and selectively affected in Parkinson’s disease, a movement disorder different from Huntington’s disease and characterized by resting tremor and rigidity, and their specific loss is the neuropathological hallmark of this disorder (van Domburg and ten Donkelaar, 1991).

Purkinje Cells

The structure of the cerebellar cortex, in contrast with that of the cerebral cortex, is basically identical all over; it is composed of three layers that contain very distinct neuronal types. One of these layers contains the Purkinje cells, which are the most salient cellular elements of the cerebellar cortex. They are arranged in a single row throughout the entire cerebellar cortex between the molecular (outer) layer and the granular (inner) layer. They are the largest cerebellar neurons and have a round perikaryon with a highly branched dendritic tree shaped like a candelabra and extending into the molecular layer where they are contacted by incoming systems of afferent, parallel fibers from the granule neurons as well as other afferents from the brainstem. The apical dendrites of Purkinje cells have an enormous number of spines (more than 80,000 per cell). A particular feature of the dendritic tree of the Purkinje cell is that it is distributed in one plane, perpendicular to the longitudinal axes of the cerebellar folds, and each dendritic arbor determines a separate domain of cerebellar cortex (Fig. 1.1). The axons of Purkinje neurons course through the cerebellar white matter and contact the deep cerebellar nuclei or the vestibular nuclei. They also furnish recurrent collaterals, mostly within the granular layer. Humans have approximately 15 million Purkinje cells (Palay and Chan-Palay, 1974). These neurons contain the inhibitory neurotransmitter GABA and the calcium-binding protein calbindin. A severe disorder combining ataxic gait and impairment of fine hand movements, accompanied by dysarthria and tremor, has been documented in some families and is related directly to Purkinje cell degeneration.

Spinal Motor Neurons

The motor cells of the ventral horns of the spinal cord, also called alpha motor neurons, have their cell bodies within the spinal cord and send their axons outside the central nervous system to innervate the muscles. Different types of motor neurons are distinguished by their targets. The alpha motor neurons innervate skeletal muscles, but smaller motor neurons (the gamma motor neurons, forming about 30% of the motor neurons) innervate the spindle organs of the muscles. The alpha motor neurons are some of the largest neurons in the entire central nervous system and are characterized by a multipolar perikaryon and a very rich cytoplasm that renders them very conspicuous on histological preparations. They have a large number of spiny dendrites that arborize locally within the ventral horn. The alpha motor neuron axon leaves the central nervous system through the ventral root of the peripheral nerves. The cell bodies are arranged in a nonrandom fashion in the ventral horn so that they are grouped in functional vertical columns that span a certain number of spinal segments. This disposition corresponds to a somatotopic representation of the muscle groups of the limbs and axial musculature (Brodal, 1981). The spinal motor neurons use acetylcholine as their neurotransmitter. Large motor neurons are severely affected in lower motor neuron disease (a form of amyotrophic lateral sclerosis), a neurodegenerative disorder characterized by progressive muscular weakness that affects, at first, one or two limbs and that can be initially asymmetric. As the disease progresses, it becomes symmetric and affects more and more of the body musculature, which shows signs of wasting as a result of denervation. Neuropathologically, a massive loss of ventral horn motor neurons occurs, and the remaining motor neurons appear shrunken and pyknotic.

Retinal Photoreceptors and Cochlear Hair Cells Are Examples of Specialized Sensory Receptors

Retinal photoreceptors and cochlear hair cells are modified neuroepithelial cells that are specialized in the initial transduction of visual and acoustic stimuli respectively. Comparable specialized neuronal types exist for other sensory modalities, that is, olfactory, gustatory, and vestibular inputs. In contrast, somatosensory inputs are transmitted by peripheral nerve cells whose endings are associated with a variety of sensory structures in the peripheral tissues. Receptor neurons are extremely polarized cells, with one uniquely diversified end that is responsible for the reception of the sensory stimulus. This morphology is particularly well demonstrated in retinal photoreceptors. Photoreceptor cells are of two types, the rod and the cone, which are specialized for scotopic (light/dark) and color vision, respectively. The rods are slender cells, with an elongated cylindrical outer portion, whereas the cones are smaller elements, with shorter, conical outer portions (Fig. 1.10) (Krebs and Krebs, 1991). Each cell type consists of an outer segment and an inner segment. The inner segments of both rods and cones contain the metabolic machinery necessary for protein and lipid synthesis and oxidative metabolism. In rods, the outer segment is composed of a very large number of parallel lamellae stacked perpendicularly to the main axis of the cylinder. These lamellae are closed, flattened membranous disks that appear in thin-section electron microscopy as pairs of parallel membranes. In cones, these lamellar stacks are less numerous. These structures are responsible for the mechanisms of phototransduction and contain several visual pigments, located inside the membranous disks, that are necessary for the absorption of light.

FIGURE 1.10 Drawing of a cone (left) and a rod (right) from the monkey retina. Note the differences in shape and size of these cells. They are composed of an outer segment (OS), an inner segment (IS), a perikaryon (P), and the inner fiber (IF). The outer segment is connected to the inner segment by a thin connecting cilium (C). The outer fiber (OF) is thin and well visible in rods, whereas the perikarya of both cell types are comparable in appearance. In cones the inner fiber is thicker and ends as a large cone pedicle, whereas in rods the inner fiber is rather thin and terminates in a unique spherule. Cone pedicles (CP) and rod spherules (S) are specialized synaptic endings where the photoreceptors make contact with specific subtypes of retinal relay neurons. DM, membranous disks (lamellae); ELM, external limiting membrane; G, Golgi apparatus; M, mitochondria, MV, microvilli of pigment epithelium; N, nucleus; PC, calycoid process; PE, pigment epithelium; RER (SER), rough (smooth) endoplasmic reticulum; RF, rootlet fibers.

Adapted with permission, from Krebs and Krebs (1991).

Cochlear (and vestibular) hair cells also are highly polarized and present striking apical differentiation specialized in the detection of endolymphatic movements in the inner ear. In the cochlea, receptor hair cells that detect stimuli produced by sound are short, goblet-like cells embedded in supporting cells (the phalangeal cells of Deiters). Their apical domain contains a U-shaped row of stereocilia (hairs) that are in contact with the tectorial membrane of the organ of Corti. The vibrations of this membrane, generated by sound waves in the endolymph, displace the hairs and initiate the transduction of the acoustic stimulus. The other pole of the hair cell contains the nucleus and a dense population of mitochondria and receives synaptic contacts from afferent and efferent fibers from the cochlear nerve, which spreads around the lower third of the receptor cell (Fig. 1.11) (Hudspeth, 1983).

FIGURE 1.11 Schematic drawing of an electron micrograph of an outer hair cell and its relationships to supporting (outer phalangeal) cell and cochlear nerve endings. Note the apical domain containing stereocilia. The other pole of the hair cell contains the nucleus and a dense population of mitochondria and receives synaptic contacts from the cochlear nerve, which spread around the lower third of the receptor cell.

Enteric Motor Neurons Form an Independent Neural Plexus in the Gut Wall

The enteric nervous system constitutes a part of the autonomic nervous system that innervates the gastrointestinal tract, as do the sympathetic and parasympathetic systems. Although all three systems take part in the regulation of intestinal function, the enteric system is by far the most important and has the unique feature that it can function relatively independently of the control of higher centers (Furness and Costa, 1980). The enteric system consists of an extremely rich plexus of nerve fibers and neurons disseminated among all of the layers that form the wall of the intestinal tract (Fig. 1.12). It contains nerve cells arranged in ganglia interconnected by complex bundles of fibers that extend from the lower third of the esophagus to the internal anal sphincter. In humans, this system contains about 10⁷ to 10⁸ neurons.

FIGURE 1.12 Scanning electron micrograph of the myenteric plexus in the intestine. Note the dense axonal bundles and synaptic boutons (pseudocolored green) and the network of large multipolar neurons with relatively short extensions contacting neighboring cells (pseudocolored red).

The principal enteric plexus is located between the circular and longitudinal layers of the muscularis and is known as the myenteric plexus of Auerbach. It is composed of ganglia, each containing from 3 to 50 neurons linked by unmyelinated fibers and forming a continuous network. The cells in this plexus are of two main morphological types. One is a large multipolar neuron with short dendrites in direct contact with similar nearby cells and a long axon that contacts different cell types in neighboring ganglia. These cells are thought to be association interneurons. The other cell type, considered an enteric motor neuron, is by far more dominant and demonstrates more variable morphology. These cells make extensive contacts with neurons of either type within the same ganglia or with distant cells. Other ganglia are found within the submucosal plexus of Meissner. Their relatively large multipolar neurons form a network interconnecting the outer nerve bundles with the submucosal tissue.

The neurochemistry of the enteric system is extremely complex and still poorly understood. A large number of classic neurotransmitters, such as acetylcholine, GABA, and noradrenaline (see also Chapter 9), have been identified in enteric nerve fibers and ganglionic neurons. In addition, enteric neurons in both the myenteric and the submucosal plexuses contain a variety of neuropeptides. Neurons in the submucosal plexus are enriched in somatostatin, substance P, and vasoactive intestinal peptide but do not seem to contain Leu-enkephalin, which is observed in the myenteric nerve cells (see also Chapter 10). It is not clear, however, whether these neuropeptides are the principal transmitters of subclasses of enteric neurons or are colocalized compounds that act as local neuromodulators (Furness and Costa, 1980).

Summary

The neuron is one of the more highly specialized cell types and is the critical cellular element in the brain. All neurological processes are dependent on complex cell–cell interactions between single neurons and/or groups of related neurons. Neurons can be described according to their size, shape, neurochemical characteristics, location, and connectivity.

A neuron’s size, shape, and neurochemistry are important determinants of that neuron’s particular functional role in the brain. In this respect, there are three general classes of neurons: the inhibitory GABAergic interneurons that make local contacts, the local excitatory spiny stellate cells in the cerebral cortex, and the excitatory glutamatergic efferent neurons, exemplified by the cortical pyramidal neurons. Within these general classes, the structural variation of neurons is systematic, and careful analyses of the anatomic features of neurons have led to various categorizations and to the development of the concept of cell type. The grouping of neurons into descriptive cell types (such as chandelier, double bouquet, and bipolar cells) allows the analysis of populations of neurons and the linking of specified cellular characteristics with certain functional roles. The relevant characteristics may include morphology, location, connectivity, and biochemistry.

Also, neurons form circuits, and these circuits constitute the structural basis for brain function. Macrocircuits involve a population of neurons projecting from one brain region to a distant region, and microcircuits reflect the local cell–cell interactions within a brain region. The detailed analysis of these macro- and microcircuits is an essential step in understanding the neuronal basis of a given cortical function in the healthy and the diseased brain. Thus, these cellular characteristics allow us to appreciate the special structural and biochemical qualities of that neuron in relation to its neighbors and to place it in the context of a specific neuronal subset, circuit, or function.

THE NEUROGLIA

The term neuroglia, or nerve glue, was coined in 1859 by Rudolph Virchow, who conceived of the neuroglia as an inactive connective tissue holding neurons together in the central nervous system. The metallic staining techniques developed by Santiago Ramón y Cajal and Pio del Rio-Hortega allowed these two great pioneers to distinguish, in addition to the ependyma lining the ventricles and central canal, three types of supporting cells in the central nervous system (CNS): oligodendrocytes, astrocytes, and microglia. In the peripheral nervous system, the Schwann cell is the major neuroglial component.

Oligodendrocytes and Schwann Cells Synthesize Myelin

The more complex the brain, the more interconnections must be formed and maintained. As shown in depth later, there is a practical limit to how fast an individual bare axon can conduct an action potential (Chapters 4, 5). Thus, neurons and their associated processes cannot communicate with each other extremely rapidly through the action potential without some help. Organisms have developed two kinds of solutions for enhancing rapid communication between neurons and their effector organs. In invertebrates, the diameters of individual axons that must conduct rapidly are enlarged. In vertebrates, the myelin sheath (Fig. 1.13) has evolved to permit rapid nerve conduction.

FIGURE 1.13 Electron micrograph of a transverse section through part of a myelinated axon from the sciatic nerve of a rat. The tightly compacted multilayer myelin sheath (My) surrounds and insulates the axon (Ax). Mit, mitochondria. Bar = 75 nm.

Axon enlargement greatly accelerates the rate of conduction of the action potential, which increases with axonal diameter (Chapter 4). The net effect, therefore, is that small axons conduct at a much slower rate than larger ones. The largest axon in the invertebrate kingdom is the squid giant axon, which is about the thickness of a mechanical pencil lead. It conducts the action potential extremely rapidly, and the axon itself mediates an escape reflex, which must be rapid if the animal is to survive. An obvious tradeoff in a nervous system with 10 billion neurons, as in the human brain, is that all axons cannot be as thick as pencil lead, or each human head would be very large indeed.

Thus, along the invertebrate evolutionary line, there is a natural, insurmountable limit—a constraint imposed by axonal size—to increasing the processing capacity of the nervous system beyond a certain point. Vertebrates, however, devised a way to get around this problem through the evolution of the myelin sheath, allowing the tremendous evolutionary advantage of increased rapidity of conduction of the nerve impulse along axons with fairly minute diameters (Morell and Norton, 1980). As we know, neurons interact in complex ways with the other cell types that exist within the nervous system. Virtually all axons, for example, are wrapped or ensheathed by cells that subserve what is vaguely termed a supportive or trophic function. This is true in invertebrate as well as vertebrate nervous systems. Along the vertebrate lineage, however, certain ensheathing cells have become highly specialized to generate vast quantities of plasma membrane that is compacted to form the myelin sheath, which supports rapid nerve conduction.

Not all axons in central or peripheral nervous systems are myelinated, and one of the puzzles is to determine why some are selected for myelination and others remain unmyelinated. It is believed that early in the nervous system development of an organism, signals relayed between the axon and the myelinating cell determine whether the myelination program is triggered in that cell. These signals have not yet been identified.

In the central nervous system, the myelin sheath (Fig. 1.14) is elaborated by oligodendrocytes, nonneuronal glial cells that, during brain development, send out a few cytoplasmic processes that engage adjacent axons, some of which go on to become myelinated (Bunge, 1968). Myelin itself consists of a single sheet of oligodendrocyte plasma membrane, which is wrapped tightly around an axonal segment (Bunge et al., 1962). Each myelinated segment of an axon is termed an internode because, at the end of each segment, there is a bare portion of the axon, the node of Ranvier, that is flanked by another internode. Physiologically, myelin has insulating properties such that the action potential can leap from node to node and therefore does not have to be regenerated continually along the axonal segment that is covered by the myelin membrane sheath. This leaping of the action potential from node to node allows axons with fairly small diameters to conduct extremely rapidly (Ritchie, 1984). The jumping of the action potential from node to node along a given axon is called saltatory conduction (see Chapters 4 and 5 for additional details on the propagation of nerve impulses).

FIGURE 1.14 An oligodendrocyte (OL) in the central nervous system is depicted myelinating several axon segments. A cutaway view of the myelin sheath is shown (M). Note that the internode of myelin terminates in paranodal loops that flank the node of Ranvier (N). Inset: Enlargement of compact myelin with alternating dark and light electron-dense lines that represent the intracellular (major dense lines) and extracellular (intraperiod line) plasma membrane appositions, respectively.

The evolution of a system in which a single oligodendrocyte cell body is responsible for the construction and maintenance of several myelin sheaths (Fig. 1.14) and the removal of the cytoplasm between each turn of the myelin lamellae so that only the thinnest layer of plasma membrane is left has resulted in saving a huge amount of intracranial space. Brain volume is thus reserved for further expansion of neuronal populations (Colman et al., 1996).

Conservation of space in the peripheral nervous system (PNS) does not seem to have presented such a pressing problem. Myelin in the PNS is generated by Schwann cells (Fig. 1.15), each of which wraps only a single axonal segment. The biochemical composition of the myelin derived in the CNS and the composition of that derived in the PNS differ somewhat, although there are proteins common to the two nervous system subdivisions. Myelin has a high lipid-to-protein ratio, and the lipids are specialized. The myelin sheath has become an excellent model system for studying the generation or formation of specialized plasma membrane and membrane adhesion, because each layer of the multilayered myelin sheath must adhere to the adjacent layers. This adhesion is largely accomplished by protein–protein interactions, which have been best studied in the PNS.

FIGURE 1.15 An unrolled Schwann cell in the PNS is illustrated in relation to the single axon segment that it myelinates. The broad stippled region is compact myelin surrounded by cytoplasmic channels that remain open even after compact myelin has formed, allowing exchange of materials among the myelin sheath, the Schwann cell cytoplasm, and perhaps the axon as well.

The major integral membrane protein of peripheral nerve myelin is protein zero (P0), a member of a very large family of proteins termed the immunoglobulin gene superfamily. These proteins have in common recognition or adhesion functions or both, and, although the primary amino acid sequences differ among the members of this family, all members are related to one another by certain common structural motifs. Members of the immunoglobulin (Ig) gene superfamily have one or more Ig-like domains that contain cysteines placed about 100 amino acids or so apart. These cysteines are linked to one another by disulfide bridges. Most of these Ig domains are displayed on the extracellular surfaces of cells, where they can act as ligands or receptors. Protein zero is relatively simple in primary structure, consisting of a single Ig-like domain, a transmembrane segment, and a highly charged (basic) cytoplasmic domain (Lemke and Axel, 1985). This protein makes up about 80% of the protein complement of peripheral nerve myelin. The interactions between the extracellular domains of P0 molecules expressed on one layer of the myelin sheath and those of the apposing layer yield a characteristic regular periodicity that can be seen by thin-section electron microscopy (Fig. 1.13). This zone, called the intraperiod line, represents the extracellular apposition of the myelin bilayer as it wraps around itself. On the other side of the bilayer, the cytoplasmic side, the highly charged P0 cytoplasmic domain probably functions to neutralize the negative charges on the polar head groups of the phospholipids that make up the plasma membrane itself, allowing the membranes of the myelin sheath to come into close apposition with one another. In electron microscopy, this cytoplasmic apposition is a bit darker than the intraperiod line and is termed the major dense line. In peripheral nerves, although other molecules are present in small quantities in compact myelin and may have important functions, compaction (i.e., the close apposition of membrane surfaces without intevening cytoplasm) is accomplished solely by P0–P0 interactions at both extracellular and intracellular (cytoplasmic) surfaces (Giese et al., 1992).

Protein zero is a perfect plasma membrane compactor (Shapiro et al., 1995), allowing the close apposition of adjacent bilayers such that the space between them effectively prevents the passage of anything but small ions and water along the compacted bilayer surfaces. It is in effect a streamlined Ig superfamily molecule that probably arose de novo with the development of the myelin sheath (Colman et al., 1996). Curiously, P0 is not present in all myelin sheaths in the central nervous system of every species, an evolutionary paradox that has attracted much attention. In fish, P0 is present in both the central and peripheral nervous systems, where it performs its compaction function, as the major integral membrane protein. However, in terrestrial vertebrates (reptiles, birds, and mammals), P0 is limited to the PNS, and so is not found in the CNS. Instead, the compaction function is probably subserved by totally unrelated molecules, the DM20 protein and its insertion isoform, the myelin proteolipid protein (PLP) (Folch-Pi and Lees, 1951). These two proteins are generated from the same gene and are identical to each other with the exception that the proteolipid protein has, in addition, a positively charged segment exposed on the cytoplasmic aspect of the bilayer (Milner et al., 1985; Nave et al., 1987). Both PLP and DM20 are extremely hydrophobic and traverse the bilayer four times, and so have hydrophilic segments exposed on both cytoplasmic and extracellular surfaces of the bilayer. In this respect, the topology of these molecules is very similar to that of connexins and other polypeptides that are known to function in channel or pore formation (e.g., Chapter 15).

A large number of naturally occurring neurological mutations can affect the proteins specific to the myelin sheath. These mutations have been named according to the phenotype that is produced: the shiverer mouse, the shaking pup, the rumpshaker mouse, the jimpy mouse, the myelin-deficient rat, the quaking mouse, and so forth. Many of these mutations have been well characterized, and their analyses have allowed us to begin to understand at a molecular level what the proteins affected by each mutation actually do in the formation and maintenance of the myelin sheath (see Box 1.2) (Nave, 1994).

BOX 1.2 INHERITED PERIPHERAL NEUROPATHIES

Ueli Suter

The peripheral myelin protein-22 (PMP22) is a very hydrophobic glycoprotein and is highly expressed in compact PNS myelin. It has been mapped to the previously defined Tr locus on mouse chromosome 11. Comparison of marker genes on mouse chromosome 11 and human chromosome 17 revealed that PMP22 was also a candidate gene for the most common form of autosomal-dominant demyelinating hereditary peripheral neuropathy in humans, Charcot–Marie–Tooth disease type 1A (CMT1A). Indeed, the entire PMP22 gene is contained within a 1.5-Mb intrachromosomal duplication on chromosome 17p11.2, a genetic abnormality that had been linked to CMT1A by human molecular genetics. Consistent with these results, PMP22 is overexpressed in CMT1A patients who carry the characteristic duplication. The crucial role of PMP22 in the etiology of CMT1A was confirmed by generating transgenic mice and rats with increased PMP22 gene dosage, which resulted in severe PNS myelin deficits.

CMT is one of the more frequent hereditary diseases of the nervous system, with an overall prevalence of approximately 1 in 4000, and the CMT1A duplication accounts for around 70% of all cases. Why is this chromosomal abnormality so common? Detailed analysis of the CMT1A locus suggests that the duplication is due to crossing over involving repetitive sequences that flank the monomeric region. If correct, such a mechanism should also generate an allele carrying the reciprocal deletion of the same region. Indeed, the expected deletion is associated with the relatively mild recurrent neuropathy with liability to pressure palsy (HNPP). Thus, overexpression and underexpression of the myelin protein PMP22 are associated with myelin deficiencies in distinct human diseases. Although one might speculate from these data that correct stochiometry of myelin protein expression is crucial for a myelinating Schwann cell, the exact disease mechanism remains to be clarified.

Interestingly, the finding that a myelin protein was responsible for CMT1A led to the discovery that two other components of PNS myelin are mutated in rare forms of CMT1. The adhesion protein P0, which is largely responsible for PNS myelin compaction, is affected in CMT1B, and an X-linked form of CMT (CMTX) has been linked to mutations in the gap junction protein connexin-32. In contrast to PMP22 and P0, connexin-32 is located in uncompacted lamellae of PNS myelin, where it is thought to facilitate the exchange of small molecules via reflexive gap junctions between adaxonal and abaxonal aspects of myelinating Schwann cells.

Finally, there is a striking correlation between the role of PMP22 in the PNS and that of PLP/DM20 in the CNS with respect to biology and involvement in disease; both genes can be affected by various genetic mechanisms, including gene duplication and gene deletion. However, despite our vast knowledge derived from human molecular genetics, the molecular functions of both proteins are largely unknown. Given the recent findings that PMP22 and PLP/DM20 are members of extended gene families and may be involved in the control of cell proliferation and cell death, these proteins may have broader functions than simply being stabilizing building blocks of compact myelin.

In summary, the combination of basic and clinical sciences has led to substantial progress in the current understanding of common hereditary neuropathies. Using clinical, genetic, and cell biology approaches in concert, we will continue to learn more about disease mechanisms involved in neuropathies to the benefit of the clinic as much as to the understanding of myelin biology.

The first neurological mutation that was studied in this respect was the shiverer mouse, in which the gene that encodes a major set of peripheral membrane proteins, the myelin basic proteins (MBPs), is functionally deleted. Normally, these proteins serve to seal the cytoplasmic aspects of the myelin bilayer, possibly by charge neutralization similar in function to the cytoplasmic tail of P0. When gene expression of these proteins is completely compromised, as in the shiverer, the cytoplasmic aspects fail to appose and do not fuse, and the mouse exhibits tremors and convulsions (shivers) as it walks. This is a naturally occurring mutation and was the first neurological mutation whose effects were cured by gene transfer. This was accomplished