Progress In NeurobIOlogy Vol 49, pp 363 to 380, 1996

Copynght ~, 1996 ElsevIer SCIence Ltd All nghts reserved

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WIRING AND VOLUME TRANSMISSION IN THE

CENTRAL NERVOUS SYSTEM:
THE CONCEPT OF CLOSED AND OPEN SYNAPSES

MICHELE ZOLI*tt and LUIGI F, AGNATI*t

• Department of Biomedical SCIences. SeclIon oj PhysIOlogy, Universay of Modena, Modena. Italy and
tInterun/rersity Center for the Study of Aging, Milan, Italy

(Recel1'ed 18 March 1996)

Abstract~Dunng the past two decades, several revtsions of the concepts underlying mterneuronal
communication in the central nervous system (CNS) have been advanced. Our group has proposed to
classify intercellular commumcatlOn m the CNS under two general frames: 'wiring' (WT) and 'volume'
transmissIOn (VT). WT is charactenzed by a smgle 'transmission channel' made by cellular (neuronal or
ghal) structures and with a regIOn of dlscontmuity not larger than a synaptic cleft. VT IS characterized
by the diffusIOn from a cell source (neuronal or glial) of chemical and electrical signals in the extracellular
fluid (ECF) for a distance larger than the synaptic cleft. Based on morphological and functional
characteristics, and in light of the distinction proposed, six mam modes of intercellular commumcatlOn
can be recogmzed m the CNS' gap-junction. membrane Juxtaposition, and closed synapse (which represent
WT-type modes of communication); open synapse, paracrine transmission and endocnne-like
transmissIOn (which represent VT-type modes of communication). Closed and open synapses are
distinguished on the basIs of the sealing of the signal Within or the leakage of the signal outside the synapse.
Intra-synaptic restrictIOn or extra-synaptic diffusion of transmitters are insured by a number of anatomical
arrangements (e.g. glial ensheathment of synapse, size of the synaptic cleft) and functional mechanisms
(e.g. denSity and locatIOn of transmitter re-uptake sites and metabolic enzymes). Some central synapses
can switch from closed to open state and vice versa, e.g. by changing the amount of transmitter released.
Fmally. a synapse contammg several transmitters can work as an open synapse for one transmitter and
as a closed synapse for another. Copynght (, 1996 Elsevier Science Ltd.

CONTENTS
I. IntroductIOn 364
2. The concept of wmng and volume transmiSSion 364
3. Closed and open synapses 365
3.1. The neuromuscular synapse 366
3.2 The sympathetic synapse 367
3.3. The hippocampal glutamaterglC synapse 367
3.4. The mesohmblc dopammerglc synapse 368
3.5. The peptidergic synapse 368
3.6. Types of synapses 368
4. Some baSIC differential features of WT and VT 370
4.1. WT and VT commumcatlOn channels 370
4.2. Monochemlcal vs plunchemlcal transmiSSIOn m WT and VT 371
4.3. WT and VT circuits 371
5. Basic elements composing VT-type intercellular commumcation 372
5.1. Types of cells acting as a source for VT signals 372
5 1.1. Release from neurons 372
5.1.2. Release from astrocytes 372
5.2. Features of the VT signals 373
5.2.1. Some general features of the VT signals 373
5.2.2. Types of VT signals 373
5.3 Characteristics of the commumcatlOn channels for VT 374
5.4. Targets of VT signals 374
6. VT and WT at work: some examples 375
6.1. Dynorphm m the hippocampus 375
6.2. Retrograde messengers and hippocampal LTP 375
6.3. Neuropeptides in the spinal cord 376
Acknowledgements 376
References 376

t Corresponding author: Dlpartimento dl Scienze Biomediche, Sezione di Fisiologia, Universita di Modena, via Campi
287, 41100 Modena, Italy.

363
364 M. Zoli and L. F Agnatl
ABBREVIATIONS
ACh acetylcholIne
AChE acetylcholIne esterase
AMPA rx-amino-3-hydroxy-5-methyl-Isoxazole-4-
propionate
CGRP calcltonm-gene-related peptIde
CNS central nervous system
DA dopamme
ECF extracellular fluid
EM electron mIcroscopy
GABA y-aminobutinc acid
glr Caenorhabditis elegans glutamate receptor
LDCV large dense-cored vesicle
1. INTRODUCTION
The classical view of computation and communi-
cation in the central nervous system (CNS) has been
outlined in the neuron doctrine (Shepherd, 1991).
According to this doctrine, neuronal computation
takes place in the axon hillock where electrical signals
coming from a number of sites, the synapses on the
dendritic arborization and cell body, are integrated.
The synapses are structures specialized for communi-
cation consisting of the transmitting axon terminal of
the source neuron and the receptive postsynaptic
density of the target neuron.
This classical scheme has evolved greatly from both
the point of view of computation and of communi-
cation. Neuronal computation is clearly much more
complicated than was proposed in the neuron
doctrine. It is now largely accepted that the neuron
is endowed with multiple input-output sites for
electrical and chemical signals, present in dendrites,
cell body and axon. Furthermore, the neuronal
membrane has several excitable sites for local
integration of electrical signals (Shepherd, 1994).
The concept of intercellular communication in the
CNS has also evolved in recent years. Several authors
have proposed that other communication modes in
addition to synaptic transmission occur in the CNS
(Bach y Rita, 1993; Bach y Rita, 1994; Beaudet and
Descarries, 1976; Cuello, 1982; Guillemin, 1978
Herkenham, 1987; Nicholson, 1979; Schmitt, 1984;
Vizi, 1984; Vizi and Labos, 1991; see also Golgi, 1898
for a hypothesis about interneuronal communication
based on diffusion of electrical signals III the
extracellular fluid, ECF). Ten years ago, our group
suggested a dual classification of intercellular
communication in the brain (Agnati et al., 1985;
Agnati et al., 1986b; Agnati et al., 1990; Agnati et al.,
1995). The present paper reports the latest conceptual
developments in our original proposal in the context
of recent anatomical, neurochemical and physiologi-
cal data.
2. THE CONCEPT OF WIRING AND
VOLUME TRANSMISSION
As stated above, several authors have suggested
that the classical view of communication processes in
the CNS should be enlarged. The different names
chosen to define the alternative methods of
LTP long-term potentiation
mGluR metabotroplc glutamate receptor
NA noradrenalIne
NAAG N-acetylaspartylglutamate
NAALADase N-acetyl-rx-lInked acidic dIpeptidase
NMDA N -methyl-D-aspartate
NO nitric oxide
NOS nitnc OXide synthase
SP substance P
SV small vesicle
VT volume transmission
WT wiring transmissIOn
intercellular communication stress the differences in
the points of view of these authors. In fact, the
alternative mode of communication is usually
indicated by the terms 'non-synaptic' or 'para-synap-
tic' transmission. These terms imply a conceptual
restriction of these proposals to interneuronal
communication, as synaptic transmission is a defining
characteristic of neurons (Shepherd, 1994). As
discussed in the following, much evidence indicates
that neuron-glia and glia-glia communication is also
relevant for information handling in the CNS (Fuxe
and Agnati, 1991a; Raisman, 1991). Furthermore, the
focus on synaptic transmission overshadows the fact
that interneuronal communication also occurs via
gap-junctions and membrane juxtapositions (Shep-
herd, 1994). Finally, we think that a positive term
should be employed rather than a negative term (such
as non-synaptic transmission), as the latter term, on
the one hand does not give any hint as to what the
alternative modes of communication might be, and
on the other hand, implies that any difference
between other modes of transmission is negligible
when compared to the difference between all of them,
and synaptic transmission.
On the basis of these and other considerations
dIscussed below, ten years ago we suggested the terms
wiring transmissIOn (WT) and volume transmission
(VT) as the primary conceptual categories of a
systematization of intercellular communication in the
CNS (Agnati et al., 1985; Agnati et al., 1986b; Agnati
et al., 1992; Fuxe and Agnati, 1991a). The term WT
has been derived from the meaning of the word 'wire',
which also indicates "the telegram system, i.e. to send
a message by wire" (see Webster's New Collegiate
Dictionary, 1961). The wire is, in our opinion. a good
analogy for cellular circuits based on classical
synaptic transmission, as well as on other types of fast
intercellular communication (gap-junctions, mem-
brane juxtapositions), in which information flows in
constrained channels, corresponding to cell struc-
tures.
The term VT has been, inter alia, derived from the
name of a theory that describes the capability of the
brain ECF to conduct signals, namely the 'volume
conduction theory' (see, e.g. Martin. 1985). This
theory describes the flow of ionic currents generated
by nerve cells through ECF under various conditions
of cellular activity.
In order to begin to clarify these concepts, we may
consider three main features of intercellular trans-
 WIrIng and Volume TransmissIOn in the Central Nervous System
missIOn, namely the source/target ratio, the distance
between source and target and the source/target
delay, i.e. the time necessary for a signal released
from a given source to reach its target. We mean by
source/target ratio the relationship between the
number of source structures and the number of target
structures in the transmission. It must be noted that
this does not refer to the source celHarget cell link
but rather to the link between the subcellular
structure that releases the signal and that which
recognizes the signal, i.e. in the case of synaptic
transmiSSion, the presynaptic termmal--postsynaptic
density link.
On morphological and functIOnal grounds, five
main modes of intercellular communicatIOn have
been recognized in nervous tissue: gap-junction
(Baker and Llinas, 1971; Dermietzel and Spray, 1993:
Jefferys, 1995; Nedergard, 1994; Walker and Held,
1969: Wilkin et al., 1990); membrane juxtaposition or
ephapses (Jefferys, 1995; Shepherd, 1994); synapse
(Jessel et al., 1993; Shepherd, 1991): paracrine
transmission (Fuxe and Agnatl, 1991 a; Guillemin,
1978): and endocrine-like transmissIOn (Agnati et al.,
1992: Bjelke et al., 1995; Cserr and Ostrach, 1974:
Routtenberg et al., 1968; Ungerstedt et al., 1969).
Endocrine-like transmission is represented by the
convective transport of a slgnalm a flUid such as the
cerebrospinal fluid (CSF, see below). In addition, we
propose to distinguish closed from open synapses, on
the basis of the sealing within or leakage outside the
synapse of the signal (see below for extensive
discussion).
On the basIs of the critena listed m Table 1 it is
possible to provide the following definitIOns
1. Wiring Transmission (WT) IS the intercellular
communication characterized by a single (source/
target ratio = I) 'transmissIOn channel' made by
cellular (neuronal or glial) structures and with a
region of discontinuity not larger than a synaptic cleft
(Agnati et al., 1985: Agnatl et al., 1992: Fuxe and
Agnati. 1991b). Thus, WT is typified by the presence
of physically Identifiable communication channels
constituting the neuronal and/or glial cell network
wiring (Agnati et ai, 1992: Fuxe and Agnati, 1991a).
This morphofunctlOnal arrangement leads to a
relatively stable connection between the cellular
elements of the network, and it is suitable for the
rapid and safe conduction of mformation. According
Table 1. DIfferent Types of Intercellular Commumcallon m the Central Nervous System
TransmissIOn type
I. Gap-junctIOn
2. Membrane JuxtapOSlllOn (ephapsls)
3. Closed synaptic transmission
4. Paracnne transmiSSIOn
A. Open synaptIC transmiSSIOn
B Non-synaptIc source
5. Endocrine-like transmiSSIOn
A. Nerve-bundle-associated
B. Paravascular flUId CIrculation
C. Transmission in CSF
For further details on open and closed synaptic transmIssion. and endocnne-lIke transmission, see
SectIOns 3 and 53. respectively.
AbbreVIatIOns: CSF = cerebro-spmal fluid. SIT = source/target
365
to this definition, chemical synaptic, gap-junction and
membrane juxtaposltion- mediated transmission are
part of WT (see Table 1)
2. Volume Transmission (VT) is the intercellular
commUllicatlOn characterized by the diffusion of
chemical and electrical signals (in this latter case we
have 'volume conduction'; see above) in a three-di-
mensional fashion in the ECF. Thus, the 'trans-
miSSIOn channels' are multiple (source/target ratio
< 1) and, usually, not well-characterized (Agnati
et al., 1985; Agnati et al., 1992; Fuxe and Agnati,
1991 b). Volume transmission IS a relatively slow and
unsafe mode for intercellular communication, but it
allows Wider diffUSIOn of the signals and, in some
cases, long-lasting modification in the function of
large populations of target cells. However, it should
be underlined that relatively fast VT may also occur.
In fact, preferential pathways for convective trans-
port of substances in the CNS have been demon-
strated (endocrine-type VT, Table 1 and Section 5.3).
Thus, the discriminatIOn point between WT and
VT can be placed between modes 3 and 4 of Table 1.
It must be noted that the definitions ofWT and VT
focus on the modality of transmission and are neutral
with respect to source and target of the transmission,
as well as the type of informational substance
transmitted. Several examples of types of cells and
signals involved in VT are given below and in Agnatl
et al. (1995).
In the next Section we Will deal With the critical
difference between closed and open synapses. ThiS
diSCUSSIOn Will also help us in clarifying the criteria
used to define VT and WT.
3. CLOSED AND OPEN SYNAPSES
For the purposes of our discussion, chemical
synapses can be diVided mto those assuring a
source/target ratIO of 1: 1 and those assuring a
source/target ratio of I:n with n > I.
In order to ensure a reliable I: I link, anatomical
arrangements and/or functional mechanisms limit
extra-synaptic transmitter diffUSion. The most obvi-
ous anatomical arrangement is the contiguity
between the source and the target structure. An
mcrease in the size of the synaptic cleft favours
extra-synaptic transmitter diffusion and makes other
restriction mechallisms less efficient. Other anatom-
SIT ratIO S'T distance SIT delay
II 2-3 nm msec
1:1 10 nm msec
l.l 20-50 nm msec
I:n. n >l-n»l 100 nm-mm msec-mm
I :n. II »1 Illm-mm sec-mm
hI. n»1 mm mm
I:n. II» I mm mm
I:n. II» I mm-{;m min
366 M. Zoli and L. F. Agnati
ical arrangements able to limit synaptic transmitter
diffusion are:
I. glial and/or extracellular matrix molecule
ensheathment of the synapse,
2. location of release sites apposed to the receptors
3. presence in the synaptic cleft of molecules able
to bind the transmitter (see below the transmitter
buffering sites in sympathetic synapses).
Astrocytes and extracellular matrix molecules can
constitute boundaries and barriers that segregate
neural elements at different spatial scales (from brain
regions to synapses) and in different physiopatholog-
ical states (e.g. during development and after lesion)
(Steindler, 1993). In several instances, astrocytic
processes and the extracellular matrix molecules
(such as tenascin and chondroitin sulfate proteogly-
can) constituting the so-called perineuronal nets
(Golgi, 1893; Celio and Bliimcke, 1994) have been
shown to ensheath central synapes (Steindler, 1993;
see Section 3.3).
Three types of release sites can be found in the
presynaptic terminals (Golding, 1994): synaptic,
para-synaptic and non- (or extra-) synaptic sites.
They correspond to three domains in the nerve
terminal. The synaptic domain of the terminal
membrane is identified by the presence of synaptic
thickenings. The para-synaptic domain corresponds
to apparently undifferentiated areas of the terminal
membrane lying adjacent to the postsynaptic cell. The
non-synaptic domain corresponds to areas of the
terminal membrane in contact with elements (e.g.
glia) that have no conventional synaptic relationship
with the terminal (Golding, 1994).
Functional mechanisms affecting synaptic trans-
mitter diffusion are related to the characteristics of
transmitter receptors (see buffered diffusion of
acetylcholine, ACh, discussed in Section 3.1),
re-uptake sites and metabolic enzymes. The efficiency
of these molecules in restricting transmitter diffusion
is dependent on both their biochemical features and
anatomical location (e.g. intra-synaptic vs extra-
synaptic; see below: dopamine, DA, re-uptake sites
discussed in Section 3.4).
These anatomical and/or functional features can
prevail differentially to generate a synapse that is
particularly suited to restrict (i.e. a closed synapse) or
favour (i.e. an open synapse) transmitter diffusion.
The fulfilment of chemical transmission ultimately
depends on the fact that the concentration of
transmitter in proximity to the receptor is high
enough to activate the receptor itself. Therefore, two
main determinants of (synaptic as well as extra-
synaptic) transmission are the amount of transmitter
released, and the affinity and density of the respective
receptor populations. As a consequence, the affinity
and density of intra- vs extra-synaptic receptor
populations (and their changes in different functional
states) will contribute to determining the prevalence
of intra- vs extra-synaptic transmission. The rel-
evance of the amount of transmitter released can be
grasped by considering that in different nerve activity
states, normally closed synapses can work as open
synapses, and vice versa. In fact, the concentration of
transmitter released at a typical closed synapse at
high discharge frequency can overwhelm the synaptic
buffering capabilities and cause the transmitter to
diffuse into the neighbouring ECF. Similarly, a
typically open synapse can restrict its transmission to
the closest target structure at low discharge
frequency.
The switch from closed to open synaptic trans-
mission (and vice versa) may be a basic feature of
some categories of central synapses (see Section
3.6). A simple mechanism for such a type of switch
is the just-mentioned impulse flow-related modifi-
cation in transmitter release. However, any
modification of the structural and/or functional
features of synapses mentioned above can, in
principle, change the functional state (closed/open)
of a synapse. For instance, it has recently been
shown that DA diffusion in striatum is mainly
regulated by the state of DA uptake sites (Giros
et al., 1996).
Finally, a synapse containing several transmitters
can work as an open synapse for one transmitter and
as a closed synapse for another (see below the case of
ATP and NA in the rat tail artery sympathetic nerve).
It is, therefore, more correct to speak of open and
closed synaptic transmission (and transmitters),
sometimes corresponding to an open or closed
synapse, but perhaps more often coexisting in the
same synapse.
These concepts can be analysed in the context of
some well-characterized synapses. Although some of
the following examples refer to peripheral synapses.
it is interesting to examine their features in view of
their detailed characterization and possible similarity
with central synapses.
3.1. The Neuromuscular Synapse
This is the prototypical closed synapse. A
source/target ratio of I: I at this synapse assures the
independent control of striated myofibres by the
nervous system, so that recruitment of myofibres can
only occur upon a central command. Many
mechanisms restrict the action of the transmitter,
ACh, to a single myofibre. The synapse is highly
specialized (for a review, see Duclert and Changeux,
1995; Hall and Sanes, 1993): the postsynaptic density
is directly apposed (50 nm) to the active zones (where
synaptic vesicles are concentrated) in the nerve
terminal, and is very rich in receptors (104hlm 2). The
metabolic enzyme ACh esterase (AChE) is highly
concentrated in the extracellular matrix (basal
lamina) of the synaptic cleft. The concentration of
receptors in the extra-synaptic membranes is more
than 1000-fold lower than in the postsynaptic density.
Moreover, the postsynaptic membrane is folded into
invaginations, directly apposed to the active zones of
the presynaptic terminal, which retard the diffusion
of ACh from the synaptic space. The result of this
morphofunctional arrangement is that ACh is
trapped (so-called 'buffered diffusion'; Armstrong
and Lester, 1979; Katz and Miledi, 1973) by the high
density of receptors and rapidly degraded by AChE.
Thus, ACh does not diffuse outside the synaptic cleft
at a relevant concentration.
 Wifing and Volume TransmIssIOn
3.2. The Sympathetic Synapse
This is classically considered the prototype of the
open synapse, and is, in fact, markedly less structured
than the neuromuscular junction. Recent evidence,
however, suggests the existence of a junctional
structure in autonomic synapses as well (for a review,
see Hirst et al., 1992). Serial electron microscopy
(EM) sections of several sympathetic and parasympa-
thetic synapses have shown that the synapse is
consistently present at the site where the basal
laminae of the presynaptic varicosity and the
postsynaptic membrane of the nearest muscle cell (the
key cell) are fused. At this level the synaptic cleft is
50-100 nm wide. The synaptic vesicles are relatively
dispersed in the varicosity, but tend to be
concentrated towards the membrane facing the key
cell. However, the presynaptic membrane is devoid of
an active zone and the postsynaptic membrane of a
postsynaptic density.
A detailed functional study of a sympathetic
synapse has been carried out by Stjiirne et al. (1994)
in the rat vas deferens noradrenergic nerve (for a
review, see Stjiirne and Stjiirne, 1995). This group has
identified two functional compartments:
• a junctional compartment, corresponding to the
above-described junctional structure, which contains
noradrenaline (NA) uptake sites, NA buffering sItes
(called'S', possibly a NA binding molecule present III
the synaptic space), and a relatively restricted number
of adrenergic receptors mainly of the ex2 type; and
• a non-junctional compartment. or surround,
which contains NA uptake sites, an effluent
responsible for NA washout, and a relatively large
number of adrenergic receptors mainly of the exl type.
The fate of released NA depends upon the
discharge rate (for thorough discussion, see Stjiirne
et al., 1994; Stjiirne and Stjiirne, 1995). After a single
pulse or a firing rate of ::::; 4 pulses at 20 Hz, NA
diffuses only within the synaptic cleft and activates
junctional ex2 receptors (Bao et aI, 1993). Extracellu-
lar NA is cleared by the uptake sites and by S (which
subsequently releases NA and causes a persistence of
its effect), thus restricting the NA effect to the
junctional area. At a firing rate of 2: 5 pulses at 20
Hz, the amount of released NA overwhelms
junctional uptake mechanisms and NA diffuses to
extrajunctional exl receptors.
The rat tail sympathetic nerve also releases ATP
which is responsible for the fast excitatory junctional
current elicited by stimulation of this nerve. However,
ATP is much more efficiently cleared than NA (within
50-100 msec, i.e. ~ 30-fold faster) from the synaptic
cleft due to the action of an ecto-ATPase, so that it
cannot diffuse in relevant concentration outside the
junction (Stjiirne and Stjiirne, 1995).
Therefore, the sympathetic synapse works as a
closed synapse for ATP (and for NA at a low
discharge rate) and as an open synapse for NA at
higher discharge rates. The different fates of released
ATP and NA subserve distinct functions. While ATP
causes a fast and transient activation of junctional
receptors, NA causes a slow and prolonged activation
of junctional and extrajunctional receptors. Thus, the
two co-transmitters produce distinct patterns of
In the Central Nervous System 367
activation of the target smooth myocells (Milner and
Burnstock, 1995).
3.3. The Hippocampal Glutamatergic Synapse
Hippocampal glutamatergic synapses (for a review,
see Edwards, 1995b) are asymmetrical synapses with
a 20 nm wide synaptic cleft. These synapses are
largely ensheathed by astroglial processes which
probably limit glutamate diffusion from the synaptic
cleft (Clements et al., 1992; Mennerick and
Zorumski, 1995). Interestingly, the degree of glial
ensheathment varies in different functional states (e.g.
it increases after long-term potentiation, LTP).
Although the localization of the different classes of
glutamate receptors in the hippocampus is still
incompletely characterized, available data indicate
that:
• both N-methyl-D-aspartate (NMDA) (Petralia
et al., 1994) and ex-amino-3-hydroxy-5-methyl-isoxa-
zole-4-propionate (AMPA) (Hampson et al., 1992)
receptors are mainly localized in the postsynaptic
densitites. AMPA receptors are also present at
extra-synaptic dendritic membranes (Molnar et al..
1993);
• the different subtypes of metabotropic receptors
(mGluR) have distinct locations. mGluRl is concen-
trated at the periphery of the postsynaptic densities
(Baude et al., 1993), whereas mGluR5 is present in
synaptic densities of dendritic spines, in some
presynaptic terminals and, at a lower concentration,
in extra-synaptic dendritic membranes and, occasion-
ally, astrocytic processes (Romano et al., 1995); and
• the subcellular distribution of kainate receptors
in hippocampus is unknown. However, in monkey
neocortex GluR5-6-7 subunit immunoreactivity is
concentrated in postsynaptic densities (Huntley et al.,
1993).
It is possible that more precise EM studies will
show further extra-synaptic localization of glutamate
receptors in the hippocampus (see, e.g. immunogold
studies III cerebellum; Baude et al., 1994). In this
context, it must be remembered that glutamate
receptors can also be activated by transmitters
released by non-synaptic sources. For instance,
glutamate is released by astroglia through an
uncharacterized calcium-dependent mechanism and
can influence glutamate receptors of the NMDA type
in neurons (Parpura et al., 1994).
Glutamate clearance from the synaptic cleft is due
to diffusion and cellular re-uptake by specific carriers
that are concentrated in astroglia. Accordingly,
blockers of the glutamate transporter can alter
NMDA (Sarantis et al., 1993) and AMPA (Tong and
Jahr, 1994) currents in hippocampal neurons in vitro.
A recent study on hippocampal microislands showed
that diffusion is mainly responsible for glutamate
clearance after spontaneous miniature excitatory
potentials and at small quantal content, whereas
uptake becomes predominant at high quantal content
(Mennerick and Zorumski, 1995). In order to explain
the presence of prolonged excitatory postsynaptic
potentials in this latter situation, the authors propose
that glutamate can diffuse from several active
368 M. Zoli and L. F. Agnati
synapses and reach a concentration sufficiently high
to continually activate glutamate receptors. This
phenomenon, called 'spill-over' has also been
observed in other amino acidergic synapses (see, e.g.
glycine, Faber and Korn, 1988 and y-aminobutiric
acid, GABA, Isaacson et al., 1993).
In conclusion, the structural and functional
arrangements of glutamate synapses seem able to
assure restriction of transmission to synaptic and
perisynaptic receptors when a single quantum of
transmitter is released. When several quanta are
released from a single presynaptic site, or when
several neighbouring presynaptic sites are active,
glutamate concentration, after diffusion outside the
cleft, is still sufficiently high to influence receptor
populations located in proximity to the source
synapse(s).
3.4. The Mesolimbic Dopaminergic Synapse
The mesolimbic dopaminergic terminals form en
passant symmetric synapses (located in the thin
intervaricose portions, and more uncommonly in
varicosities, of the axons) on dendritic spines and
shafts of medium spiny neurons (Freund et al., 1984;
Groves et al., 1994). The postsynaptic density is
located about 15 nm from the presynaptic side, which
contains a recognizable active zone. DA receptors of
the DI and D2 subtype are present in postsynaptic
densities, or close to them, but are especially
concentrated in extra-synaptic membranes of den-
drites (Sesack et al., 1994; Yung et al., 1995).
Synaptic vesicles are also present, although at lower
concentration, in the intersynaptic segments of the
axon terminal.
DA is cleared fom the synapse primarily through
re-uptake, as enzymatic degradation of this transmit-
ter is relatively slow (Michael et al., 1985; Wightman
and Zimmerman, 1990). However, blockade of either
synaptic receptors or uptake sites does not markedly
influence diffusion of DA outside the synaptic cleft
(Garris et al., 1994). This implies that the
phenomenon of buffered diffusion due to receptor
occupancy (see above) is not effective in this type of
synapse. In addition, it indicates that DA uptake sites
are not concentrated within the synaptic cleft (see
also Cerruti et al., 1991 and Nirenberg et al., 1996)
and may preferentially regulate ECF rather than
synaptic DA concentration. The crucial role of DA
uptake sites in DA transmission and diffusion is
underlined by recent data showing a 100-fold increase
in the permanence of DA in the ECF in mice with
genetic inactivation of the DA transporter (Giros
et al., 1996). The result of this morphofunctional
arrangement is that DA may have synaptic effects but
can also efficiently diffuse into the extra-synaptic
space (more than 10 ~m from the source synapse
within I half-life of DA) to reach DA receptors
located in extra-synaptic membranes or other
synapses (for a thorough discussion, see Garris et al.,
1994).
In conclusion, the mesolimbic DA synapse is
organized to allow diffusion of the transmitter to the
ECF. Accordingly, much functional evidence has
accumulated for the non-synaptic actions of DA in
striatum, for instance, on striatal cholinergic inter-
neurons (for a review, see Yizi and Labos, 1991).
3.5. The Peptidergic Synapse
Peptidergic synapses are less characterized from a
functional standpoint than synapses that use classical
transmitters. However, several lines of evidence
indicate that in most instances many anatomical
arrangements and functional mechanisms are operat-
ive to ensure diffusion of the neuropeptide far from
the source synapse.
In varicosities of peptidergic nerve terminals,
neuropeptides are stored in large dense-cored vesicles
(LDCYs) typically located in para-synaptic or
non-synaptic positions (Golding, 1994; Matteoli
et al., 1991; Thureson-Klein and Klein, 1990; see also
below), from which they can easily reach the
extracellular space. In addition, neuropeptides, unlike
classical transmitters, are not taken up again into the
neuron via specific re-uptake mechanisms. Instead,
they diffuse in the ECF and undergo cleavage, by the
action of proteases and neuropeptidases, leading to
the formation of inactive or active fragments (Agnati
et al., 1983; Agnati et al., 1988; Agnati et al., 1990;
DeWied and Jolles, 1982;see below for further
discussion). A slow clearance of the neuropeptide
from the ECF may also occur at the target receptor
level through internalization of the neuropeptide/re-
ceptor complex (Alonso et al., 1994; Bjelke and Fuxe,
1993). Thus, neuropeptides, or their fragments, can
persist in the ECF for a long time. Also, neuropeptide
receptors, typically G-protein- linked receptors with
very high affinity, are mainly located outside synapses
(Dana et al., 1989; Hamel and Beaudet, 1984; Liu
et al., 1994; Pasquini et al., 1992; Shigemoto et al.,
1993).
The release of peptides as open synaptic transmit-
ters is consistent with their role as neuromodulators
that co-ordinate the functions of disparate systems.
Some well-characterized examples of neuropeptides
working as open synaptic transmitters in both
invertebrate (Arshavsky et al., 1988; Golding, 1994)
and vertebrate (Weisskopf et al., 1993 and below)
synapses are present in the literature.
3.6. Types of Synapses
Historically, the neuromuscular synapse has set a
standard for synaptic transmission. The data briefly
reviewed here suggest that this kind of synapse, a
prototype of closed synapse according to the present
classification, is, in fact, a rather peculiar instance of
synaptic transmission. A wide spectrum of synaptic
types likely exists in nervous tissue with different
morphological and functional organizations to
restrict or favour transmitter diffusion. To a first
approximation, we may distinguish five types of
synaptic transmission (Fig. 1).
Type 1: Closed (C) synaptic transmission. Closed
in all physiological states, exemplified by the
neuromuscular cholinergic synapse and the sympath-
etic purinergic synapse.
Type 2: Closed/open (C/O) synaptic trans-
mission. Closed at low but open at high discharge
 WIrIng and Volume TransmissIOn m the Central Nervous System 369

AG

NT
CLOSED
SYNAPTIC
TRANSMISSION

\ AG ""
~

AG ASTROGLIA
ES EXTRA-SYNAPTIC TARGET
NT NERVE TERMINAL
BIMODAL
PS POST-SYNAPTIC TARGET
SYNAPTIC •
SC SYNAPTIC CLEFT
TRANSMISSION
o RECEPTOR
_ UPTAKE SITE
~ CATABOLIC ENZVME
• TRANSMITTER
o CATABOLITE

NT SC
OPEN
SYNAPTIC
TRANSMISSION
•
•

FIg. 1. SchematIc representation of the different types of synaptIc transmiSSIOn accordmg to the
classification proposed in the text Some of the structural arrangements and functIOnal mechamsms
limiting or faVOUrIng transmitter diffusion outside the synaptic cleft are indicated.

rates, exemplified by the hippocampal glutamaterglc
synapse.
Type 3: Bimodal (B) synaptic transmission.
Equally suited for closed and open transmission,
exemplified by the mesolimbic dopaminergic synapse.
Type 4: Open/closed (O/C) synaptic trans-
mission. Open in most physiological states but with
a preferential target at low discharge rates, exem-
plified by the sympathetic noradrenergic synapse.
Type 5: Open (0) synaptic transmission. Open in
all physiological states, exemplified by the typical
peptidergic synapse.
Note that chemical signals leaking from an open
synapse diffuse in the tissue ECF in a way
indistinguishable from that of paracrine signals

JPN 49/4---F
370 M. Zoh and L. F. Agnatl
Table 2. DifferentIal PropertIes of WT and VT CommunIcation Channels
1. Type of signal
2. Chemical signal
concentration at receIver
3. ReceIver affinity for chemIcal sIgnal
4. Transmission code
5. Transmission delay
6. Transmission safety
For further details, see Section 4.1
released from non-synaptic sources (see the Section
below on VT signal release). We propose, therefore,
to include open synapses in the class of paracrine
transmission (Table I).
4. SOME BASIC DIFFERENTIAL FEATURES
OF WT AND VT
Some of the differential features ofWT and VT are
summarized in Tables 2 and 3. It becomes apparent,
when regarding these features, that WT and VT are
complementary modes for intercellular communi-
cation, each suited to carrying out different tasks in
the CNS.
4.1. WT and VT Communication Channels
In general, for both wiring and volume chemical
transmissions, there must be a relationship between
extracellular concentration of the transmitter, volume
of the compartment in which the transmitter diffuses
to reach its target, and affinity of the biochemical
sensor for the transmitter. Therefore, although some
exceptions might be found, a distinctive characteristic
of VT vs WT receptors is their high (low nanomolar
to picomolar vs high nanomolar to micromolar)
affinity for the parent transmitter (Table 2). This
becomes obvious when we consider the difference
(several orders of magnitude) between the volume of
a synaptic cleft and that of even a small portion of
the extra-synaptic microenvironment. Conversely, a
high affinity receptor will be easily saturated in a
synapse.
Some data are available on ECF concentration of
transmitters. Accurate measurements are available
for DA in the retina (Witkovsky et al.. 1993) and
stnatum. In the latter case, ECF DA levels are in the
low nanomolar range in basal conditions (Kawagoe
et al., 1992) but can reach a concentration of 250 nM
when a maximal stimulus is administered (Garris
et al., 1994). This range of ECF concentrations spans
Table 3. DifferentIal Properties of WT and VT Circuits
1. Cell composition Usually only neurons or only astrocytes
2. DIvergence
3. Type of connectivity Preferentially senal
4. Time-scale
5. Space filling
6. Energy cost
For further details, see Section 4.3
WT VT
ChemICal and electncal Chemical and electrical
Usually hIgh Usually low
Usually low Usually high
Rate and temporal code Rate code
Low HIgh
HIgh Low
the known high affinity states of DA receptors.
Recent data also indicate that for some peptides, the
ECF levels in basal conditions (e.g. cholecystokinin
in the medial posterior nucleus accumbens, Maid-
ment et al., 1991) or after stimulus (e.g. substance P,
SP, and calcitonin-gene-related peptide, CGRP, in
spinal cord, Duggan et al., 1990; Duggan et al., 1992;
Schaible et al., 1993) are sufficient to activate the
respective high affinity receptors.
The issue of the safety of transmission in WT and
VT deserves clarification (Table 2). We refer, with
this term, to two characteristics of transmission: the
reliability of the transmission, i.e. the likeliness that
a signal once delivered reaches its target not being
altered by noise, and the persistence over time of the
transmission channel. It is obvious that transmission
modes in which the channel is mainly formed by
cellular structures are less easily perturbed than
transmission modes mainly based on diffusion in the
ECF. In this sense, while changes in release or
degradation rate of the signal may modulate the
efficacy of a synapse, they can result in the
disappearance of a paracrine transmission, since, in
the latter case, a target cell may not be reached by the
signal. In addition, establishment or loss of a synapse
(or a gap-junction, etc.) is a relatively long process,
while diffusion in the ECF pathways can be easily
modified by chemical changes of the extracellular
milieu (e.g. changes in pH or ion composition) as well
as by changes in the geometry of cellular structures
(e.g. transmitter-induced astrocyte swelling, Kimel-
berg et al., 1990; Martin, 1992).
Another differential aspect of WT and VT regards
the code used to carry information from source to
target (Table 2). The issue of how neurons encode
information is still largely unsolved (Ferster and
Spruston, 1995; Softki, 1995). Classical theories of
neuronal code are based on the rate code model. This
model holds that changes in firing rate signal an event
(a train of impulses), whereas the average rate of
impulses in the train codes the strength of the
WT VT
Any cell type
Low to high High to very high
Preferentially parallel
msec to sec sec to min
High Low
High Low
 WIrIng and Volume TransmIssIOn in the Central Nervous System
stimulus. Alternatively, temporal codes have been
proposed (Hopfield, 1995; Softki, 1995). According
to these models, information is encoded by the precise
occurrence of spikes over time, and not by their
average in a tram. In both cases, transmission of
informatIon depends on the, more or less noisy,
timing of spike occurrence, which is, in turn,
dependent on pulses of transmitter released m
chemical synapses or transfer of electrical currents m
gap-junctions. These temporal features of neuronal
communication cannot be maintained in VT,
especially in paracrine and endocrine-like types of
VT. As in the case of systemic hormones, the
occurrence of the communicatIon event may be
encoded by the arrival of a transmitter at the receptor
at suprathreshold concentration, whereas the
strength of the stimulus would be encoded by the
transmitter concentration. This code can be equated
to the rate code of neuronal communication
described above, although at a much longer time
scale. It seems unlikely, instead, that a temporal code
exists for VT. Therefore, VT signals appear suited for
causing prolonged changes in electrical and/or
metabolic features of potentially very large ensembles
of target cells.
4.2. Monochemical Vs Plurichemical Transmission
in WT and VT
In an interesting paper by Mayer and Baldi (1991),
chemical transmission IS dIscussed within the
framework of mformation theory. Intercellular
communicatIOn based on a single symbol/molecule
(monochemlcal transmission) can encode different
messages through the changes in transmitter concen-
tration at the receptor level (see above). An increased
channel capacity can be obtained by increasing the
number of receptor subtypes, provided that they
differ in their affinity for the transmitter and signal
transduction features. When source and target are
linked by more symbols/molecules (plurichemlcal
transmission), the informatIOn content of the message
is increased (Agnati et al., 1986b). An elegant
demonstration of this phenomenon has been recently
obtained in the synapses of the sensory neurons
transmitting mechanic and osmotic information in
Caenorhabditis elegans (Hart eI aI, 1995; Maricq
et al., 1995). In mutants devoid of a glutamate
receptor subtype (glr-I) usually present in these
synapses, mechanic sensitivity IS absent, whereas
osmotic sensitivity is preserved. It has therefore been
proposed that the differential release of two
co-transmitters (glutamate and a yet unknown
neuropeptide) codes for the two sensory modalitIes.
Further encoding strategies are available in
plurichemical transmission, including modulation of
the ratio between the concentration of the different
molecules. Interestingly, it has been shown m several
central and peripheral systems that release rates of
classical and peptidic co-transmitters by a nerve
terminal vary with the frequency of nerve stimulation
(De Vries and Baylor, 1993; Weisskopf et al., 1993;
Whim et al., 1993). Therefore, the ratio of released
transmitters can vary in different functional states
thus increasing the information content of the
message transmitted by the nerve terminal
371
This analysis of plurichemical transmission can be
applied to WT (i.e. co-transmission at a single
synapse, Hokfelt et al., 1980; Hokfelt, 1987) as well
as to VT. However, in this latter case, plurichemical
transmission acquires further functional meaning. In
fact, while in WT the source/target link is primarily
identified by the specificity of the anatomical
connection, in VT the target cell recognizes and
decodes symbols/molecules coming through the ECF
from multiple sources. The diversity of symbols is
therefore the baSIS for the identification of the
different sources. At the same time it allows a source
to send specific messages to multiple targets.
A second feature of VT signals can be discussed in
the light of information theory. To a first approxi-
mation, a transmitter molecule can be considered as
a single symbol, i.e. a basic unit of information.
However, as discussed below, especially in the case of
peptides (see Section 5.2.2), a single VT signal can be
cleaved into active fragments (syndromic signal) or
influence a catabolic enzyme that induces the
cleavage of another VT signal (enzyme modulatory
signal), thus generating multiple VT signals. Persist-
ence of the molecule in the ECF allows the higher
informational content of complex molecules such as
peptides to be unveiled. In this way, VT monochem-
lcal transmission can acquire the wider communi-
cation features of a plurichemical transmission
(Agnati et al., 1983; Agnati et al., 1988; Agnati et al.,
1990; DeWied and Jolles, 1982). In addition, as the
type of cleavage can be specific for different regions
reached by the VT signal diffusing in the ECF, further
complexity can be added to VT peptidergic
transmission.
4.3. WT and VT Circuits
Although it is conceivable that most neural systems
utilize both WT and VT, it is interesting to discuss the
main features of pure WT and VT circuits (Table 3).
A WT circuit IS composed of a chain of cells in
quasi-continuity. In fact, as the WT-type link (e.g.
closed synapse or gap-junction) ensures a I: I
source/target ratIo, the morphological contiguity
between neurons corresponds to a functional
continuity. In thIS sense, a good analogy for a WT
circuit is a wire. It is classically believed that cell
networks in the CNS are made of neurons connected
through chemical synapses and more infrequently
gap-junctions. More recently, astrocyte networks
based on gap-junctions have also been shown
(Cornell-Bell et al., 1990; Dani et al., 1992;
Dermietzel and Spray, 1993; Jefferys, 1995). WT links
between neuronal and astrocytic networks, i.e.
gap-junctions (Nedergard, 1994) and neuro-astro-
cytic synapses (Aoki, 1992; Mudnck-Donnon et al.,
1993), seem to be relatively uncommon. A VT circuit
is composed of any cell releasing signals into, or
recogmzing signals from, a certain portion of brain
ECF. Together with the cellular components, the
ECF matrix and ECF chemico-physical composition
are essential components of a VT circuit, as they can
markedly alter VT-type intercellular communication.
Further differences between WT and VT become
apparent when we consider the pattern of connec-
tivity in WT and VT circuits (Table 3). Owing to the
372 M. Zoh and L. F. Agnatl
I: I source/target ratIO at WT communication
channels, the number of targets that can be reached
by a single cell (i.e. divergence) is relatively limited.
In contrast, signals released in the ECF by a single
cell can, in principle, reach any cell in the brain. This
dichotomy between WT and VT can be magnified in
real neural systems. For instance, in cortical
monoaminergic systems, the prevalence of non-
synaptic vs synaptic varicosities is directly correlated
with the degree of divergence of the nerve terminal
arborization (Descarries et al., 1991)-the more
restricted (as far as regional and laminar patterns of
distribution are concerned) dopaminergic system has
a high incidence of synaptic varicosities; on the other
hand, the noradrenergic and serotoninergic, widely
collateralized projections to the entire neocortex have
a high to very high incidence of non-synaptic
varicosities.
A difference in the general architecture of WT and
VT circuits stems from the difference in single-cell
divergence just discussed. In fact, WT circuits appear
more suited for serial treatment of information,
whereas VT circuits are better suited for parallel
treatment of information.
When we consider the time-scale on which WT and
VT circuits operate, the advantage of VT vs WT in
terms of number of possible targets is contrasted with
the fact that these targets can be reached more slowly
in VT than in WT.
WT and VT circuits also differ in terms of space
filling and energy cost. In fact, essential components
of WT circuits are cellular processes that maintain
electrical polarization and convey electrical signals in
their membranes.
5. BASIC ELEMENTS COMPOSING VT-TYPE
INTERCELLULAR COMMUNICATION
VT is basically composed of four elements:
I. A cell source of the VT signal, thus capable of
releasing a substance in the ECF outside the synaptic
cleft or inside the cleft, but able to diffuse out of it.
2. A signal diffusing in the ECF for a distance
larger than the synaptic cleft (VT signal).
3. A communication channel in the ECF, which
may consist of specialized portions of ECF (ECF
pathways)
4. A cell target of the VT signal, i.e. a cell
possessing biochemical sensors capable of detecting
the VT signal.
5.1. Types of Cells Acting as a Source for
VT Signals
As already mentioned, WT and VT refer to a
transmission mode and are therefore independent of
the type of cell that is source or target of the
transmission. For instance, WT also includes direct
cell--eell signalling between glial cells, such as the
astrocyte-astrocyte (Cornell-Bell et al., 1990; Dani
et al., 1992; Dermietzel and Spray, 1993; Jefferys,
1995) or astrocyte-neuron (Nedergard, 1994) com-
munication through gap-junctions. Accordingly, any
cell present in the neural tissue (neurons, astroglia,
microglia, ependyma, tanycytes, etc.) can be a source
of VT signals. The following discussion will be
focused on release of VT chemical signals from
neurons and astrocytes, as much less is known about
signal release from other cell types.
5.1.1. Release from Neurons
As already discussed, diffusion of the transmitter
from synapses is a feature common to many central
neuronal systems (open synapses). In addition, a
large body of literature has shown that release sites
exist outside synapses.
In the last twenty years, release sites for LDCVs
have been demonstrated in both invertebrates and
vertebrates (Buma, 1989; Buma and Nieuwenhuys,
1988; Buma et al., 1989). Three types of release sites
have been recognized: synaptic, para-synaptic and
non-synaptic sites (Golding, 1994). Their proportion
is variable in different systems. However, it is
interesting to note that in most cases para- and
non-synaptic LDCV fusion events are much more
frequent than synaptic events.
In the case of small vesicles (SVs), the standard
view holds that a synaptic active zone is required for
exocytosis (De Camilli and Jahn, 1990). However.
several instances of varicosities containing
monoaminergic or amino acidergic SVs and lacking
synaptic specializations have been shown. In this
case, it is presumable that the same, or similar,
biochemical mechanisms are operating, although the
molecules are not sufficiently concentrated, or
regularly organized, to form an active zone
recognizable in EM analysis. The most classical
example is certainly the autonomic nervous system
(see above). Some evidence for this phenomenon has
also accumulated for the CNS (Beaudet and
Descarries, 1976; Descarries et al., 1975; Descarries
et al., 1991).
In addition to exocytosis of LDCVs and SVs, VT
signals can be released in the ECF by non-vesicular
carrier-mediated release (Adam-Vizi, 1992; Attwell
et al., 1993). The molecular mechanism underlying
this type of release is the reverse functioning of
transmitter uptake carriers. These molecules can in
fact uptake specific transmitters from the ECF or
release them into the ECF according to the direction
of transmembrane ion and voltage gradients (Adam-
Vizi, 1992; Attwell et al., 1993). Several instances in
which this type of release mechanism may be
physiologically relevant are reported in Attwell et al.
(1993).
5.1.2. Release from Astrocytes
More than 20 neuroactive substances (gliotrans-
mitters), such as neuropeptides, amino acids,
eicosanoids, steroids and growth factors, can be
synthesized and released by glial cells (Martin, 1992).
The mechanisms assuring release of these substances
are not well known. In the case of peptides, there is
no clear evidence for the presence of LDCVs in
astrocytes. It is therefore possible that peptide release
occurs through the so-called constitutive pathway for
protein release (Martin, 1992). In the case of small
molecules, such as glutamate, D-serine, glycine,
 Winng and Volume TransmissIOn
GABA and taurine, the release can occur via the
reverse functioning of membrane carriers (Adam-
Vizi, 1992; Attwell et al., 1993). As these carriers
couple transmembrane transport of a substrate with
that of one or more ions, membrane polarization
state influences the direction of the substrate
transport. Therefore, depolarization of glial cell
membranes, e.g. via activation of lonotropic recep-
tors, can induce release of VT signals by glial cells
(Martin, 1992). Small molecules. such as glutamate
and taurine, can also be released by astrocytes as a
result of changes in osmolarity. Interestingly. some
molecules are released by increase and others by
decrease in osmolarity. The mechanisms underlying
this type of release are thought to be activation of
stretch-activated or stretch-inactivated ion channels.
Accordingly, any stimulus able to change cell swelling
can release these compounds (tension-controlled
release) (Kimelberg et al., 1990; Martlll, 1992).
Substance release through the constitutive pathway
and reverse functioning of transmembrane carriers or
tension-controlled release are Ca' ~ -independent
mechanisms. It has been recently shown that a
Ca 2 + -dependent release of glutamate also occurs in
astrocytes through a not yet characterized mechanism
(Parpura et al., 1994).
5.2. Features of the VT Signals
5.2.1. Some General Features of the VT Signals
The chemico-physlcal characteristics of the VT
signal, especially its hydrophilic or hydrophobic
character, are essential in defining the extent of signal
diffusion. Hydrophilic substances have a diffusion
space mainly limited to the ECF, while hydrophobic
substances can easily cross biological membranes,
diffuse into cells and also leave the CNS. Therefore,
it may be surmised that hydrophilic VT signals (e.g..
neuropeptides) usually diffuse III an anisotropic
fashion following the irregular spaces of the ECF
and specialized ECF pathways (Fuxe and Agnati,
1991a. b and see below). In contrast, lipophilic VT
signals (e.g. nitric oxide, NO. neurosteroids) invade
the entire tissue III an isotropic way.
VT signals can be placed Illto two general classes
(Agnati et al., 1994):
• private signals, which are delivered and recog-
nized only by a restricted number of cells. These are
exemplified by chemical neurotransmitters. which are
both released and recognized by a specific group of
cells; and
• accessible signals, such as electncal signals,
which are released and decoded by almost every CNS
cell. For instance, all neuronal cells produce electrical
potentials in their membranes, which, via ion fluxes
in the ECF, may theoretically affect any cell Within
a hundred micrometres.
It has recently been proposed that some general
physico-chemical features of CNS tissue related to
cell metabolism and function can also be considered
to be accessible signals. In fact, the efficiency of both
WT and VT in entire networks can be modified by the
action of 'global regulatory signals' (Agnati et al.,
In the Central Nervous System 373
1994). The proposal holds that carbon dioxide,
hydrogen ion concentration, temperature gradients
and pressure waves can reset the efficacy of both WT
and VT in entire brain regions.
Carbon dioxide IS a gas of substantial interest for
communication in the brain. It not only plays an
important role in pH control in the CNS, but can also
influence intercellular signalling. For instance, CO 2
leads to the formation of ex-carbamates from L-amino
acids present in the ECF. Some of the carbamates
have been characterized as powerful agonists of
NMDA receptors (Max, 1991; Olney et al., 1990).
More recently, it has been proposed that. at high
discharge rates, CO 2 diffusion from ECF to
cytoplasm and the consequent regeneration of
HCO)- concentration in this latter compartment, are
responsible for the SWitch of GABA transmission
from inhibition to excitation (Staley et al., 1995). It
has also been shown that changes in ECF pH modify
the sensitivity of NMDA and GABA A receptors
(Pasternack et al.. 1992; Traynelis and Cull-Candy.
1990). Thus. CO 2 and related signals, such as
ex-carbamates. can affect both WT and VT. These
Signals related to cell metabolism may be of
particular relevance for the maintenance of the
spontaneous activity in CNS networks (Agnati et al..
1994; Gram!, 1977; Llinas, 1988).
5.2.2. Types of VT Signals
Every informational molecule (classical transmit-
ters, monoamines. peptides, gas transmitters, neuros-
teroids, neuropeptides) can be a VT chemical signal
(for a review. see Agnati et al., 1995). Among them,
several features make neuropeptides a special type of
VT signal. They are, in fact, complex molecules which
can encode in their structure more information than
classical transmitters (Agnati et al., 1983; Agnati
et al., 1990; Fuxe et al., 1991; Herbert, 1993; Mayer
and Baldi. 1991). It is possible to distinguish. in
principle. four types of peptide signals:
I. the simple Signal. i.e. a peptide that is active on
its own;
2. the pre-signal, I.e. a peptide that needs some sort
of structural modification to become active;
3. the syndromic signal, i.e a complex peptide
contallllllg two or more active fragments; and
4. the en::yme modulatory signal. I.e. a peptide that
affects the metabolism of another signal.
Let us now examine some examples of t.hese
different types of peptide Signals.
The pre-signal peptide is secreted in an inactive
form but, upon the action of a peptidase, is cleaved
Illto an active fragment. An interesting example is
that of N-acetylaspartylglutamate (NAAG). This
dipeptide is heterogeneously distributed in the rat
brain (Anderson et al., 1986). is localized in synaptic
vesicles, and released upon neuronal stimulation
(Stauch Slusher et al., 1992; Tsai et al., 1990). A
membrane-bound metalloprotease, the N-acetyl-ex-
linked acidic dipeptidase (NAALADase), catabolizes
NAAG to N-acetylaspartate and glutamate. which
may represent the active signal (Stauch Slusher et al.,
1992). In this way. the activity of NAALADase can
374 M. Zoli and L. F. Agnatl
influence glutamate transmission to neuronal and
glial networks.
The syndromic signal (Agnati et al., 1990; DeWied
and Jolles, 1982; Fuxe et al., 1991) is a large peptide
molecule (which may already be active in the secreted
form) from which peptidases can detach one or more
active fragments with multiple related biological
actions. In this way a complex response can be
triggered by a single release event (syndromic
response). ~-endorphin (DeWied and Jolles, 1982)
and neuropeptide Y (Fuxe et al., 1991; Martire et al.,
1993; Yang et al., 1994) are examples of syndromic
signals, since it has been shown that their fragments
can trigger responses that are functionally related to
the parent peptide action.
The enzyme modulatory signal is a peptide that
modulates the activity of an enzyme involved in the
catabolism of another signal (e.g. another VT signal).
A peptidase inhibitor can increase the response of the
neuropeptides that escape inactivation and diffuse
into a larger volume of nervous tissue. An example
of this type of VT signal is CGRP in the spinal cord
(see Schaible et al., 1993 and below).
5.3. Characteristics of the Communication
Channels for VT
Diffusion of VT signals in the brain ECF can be
described by two main parameters, the interstitial
space size (interstitial volume fraction, about 20% of
the brain volume) and the tortuosity (diffusion path
length in relation to free medium) (Nicholson, 1979;
Nicholson, 1980). It is important to note that volume
fraction and tortuosity are not fixed parameters, as
they can change during, e.g. increased nervous
activity, hypercapnia and brain ischemia (Lundbaek
and Hansen, 1992).
As stated above, lipophilic substances can diffuse
in the ECF in an isotropic fashion. An example of
this type of VT signals is NO. A growing body of
evidence indicates that gas transmitters exist in the
CNS as well as in the periphery. NO is the prototype
of this new class of transmitters, which also includes
carbon monoxide and hydrogen sulfide (Abe and
Kimura, 1996; Dawson and Snyder, 1994). Owing to
the fact that it is small, neutral and relatively
hydrophobic, NO, once formed upon activation of
NO synthase (NOS), directly diffuses outside the
source cell and enters the target cell where it can
activate NO-sensitive enzymes. Therefore, NO
represents a rapidly diffusible signal for VT, which
can easily cross cell membranes and link the activities
of neurons in a restricted brain volume by activation
of guanylate cyclase (Bredt and Snyder, 1992;
Dawson and Snyder, 1994; Gaily et al., 1990;
Garthwaite et al., 1988).
Models of NO diffusion in brain tissue are reported
in two recent papers (Lancaster, 1994; Wood and
Garthwaite, 1994). According to these models, and
contrary to what is usually believed, the sphere of
influence of NO from a point source can be rather
wide (up to a diameter of 100-200 11m). This is due
to the fact that NO diffusion is much faster than its
(still rapid) inactivation rate, and the scavenging
action of blood vessels is relatively long range.
Accordingly, tissues containing a high density of NO
sources could achieve high steady-state levels of NO
(Wood and Garthwaite, 1994).
Migration of VT signals in the brain does not
always follow a simple, isotropic pattern of diffusion.
On the contrary, preferential pathways have been
described in the CNS. Studies with dye-coupled
dextrans and imaging techniques have shown the
existence of paravascular (Greitz, 1993; Rennels
et al., 1985; Rennels et al., 1990) and nerve
bundle-associated pathways in the CNS (Bjelke et al.,
1995; Cserr and Ostrach, 1974; Le Bihan et al., 1993;
Routtenberg et al., 1968; see also Agnati et al., 1995).
These pathways are suited for long-distance transport
of VT signals and are based on convection, a faster
type of solute transport than simple diffusion. A
further case of VT signal convection in the CNS is
transmission through the CSF. Nerve bundle-associ-
ated transmission, paravascular fluid circulation and
transmission in the CSF can be considered as
endocrine-like types of transmission in the brain (see
Table 1). In fact, in all of them, the signal once
released by the source cell diffuses in the neighbour-
ing ECF, enters a specialized fluid compartment
(CSF or ECF pathways) in which it is moved by
convective forces, and finally diffuses into the ECF
around the target cell. Endocrine-like types of VT
transmission allow rapid transport of VT signals
between distantly located brain areas.
5.4. Targets of VT Signals
As stated above, any cell present in the neural
tissue (neurons, astroglia, microglia, ependyma,
tanycytes etc.) can be a target of VT, provided that
it contains a biochemical sensor with the appropriate
affinity for the VT signal present in the ECF. For
instance, non-synaptic varicosities of monoaminergic
diffuse systems are often in proximity to astrocytic
processes (Maxwell et al., 1983; Ridet et al., 1993).
The astroglia are known to contain high affinity
G-protein-coup1ed receptors for monoamines (for
reviews, see Hansson, 1991; Hasli and Hash, 1993;
Shao et al., 1994). The activation of these receptors
can lead to, e.g. changes in cell shape (Narumi et al.,
1978), regulation of energy metabolism (Hasli and
Hasli, 1993; Magistretti et al., 1993; Stone and
Ariano, 1989), stimulation of astroglial transmitter
release and uptake (Hansson, 1991; Stone and
Ariano, 1989). Thus, monoamines can indirectly
influence neuronal function via informational, meta-
bolic and trophic signals originating in the astroglia.
A defining requirement of VT is that a distance
larger than the synaptic cleft exists between release
sites and receptors for a VT signal. In fact, in recent
years, much morphological evidence has accumulated
for transmitter release sites located far from their
putative target receptors both at the light and
electron microscopic levels. This phenomenon has
sometimes been called transmitter-receptor mismatch
(Agnati et al., 1986a; Cuello, 1982; Fuxe and Agnati,
1991b; Herkenham, 1987; Kuhar, 1985; for a recent
discussion of this subject, see Agnati et al., 1995).
More generally, when referring to VT, this phenom-
enon should be defined as signal source/signal target
mismatch.
 WIrIng and Volume TransmissIOn
6. VT AND WT AT WORK: snl\1~ -;:;:AAivh'~~'"
Several instances of VT have already been given III
previous chapters. We discuss here a few further
examples, especially focusing on the Illterplay
between VT-type and WT-type transmissions.
6.1. Dynorphin in the Hippocampus
An example of interplay between WT and VT III
the brain is represented by dynorphin and glutamate
transmission in the hippocampus (Morns and
Johnston, 1995). Dynorphin and glutamate are
co-stored in mossy fibres which project from granular
neurons of the dentate gyrus to apical dendrites of
pyramidal neurons of the CA3 field (Morris and
Johnston, 1995). Release of glutamate from these
fibres causes a fast excitatory postsynaptic potential,
through the activation of AMPA receptors. Release
of dynorphin activates kappa 1 receptors located on
mossy fibre terminals and causes a slow-onset and
long-lasting 00-60 min) inhibition of synaptic
responses by decreasing glutamate release (Simmons
et at., 1994; Weisskopf et at., 1993). Recent findings
indicate the possibility that dynorphin at IlM
concentrations can directly inhibit NMDA (Chen
et at., 1995) and non-NMDA (Kolaj et at .. 1995)
receptor function. Notably, dynorphin effects are
only detected at high discharge rate (Welsskopf et at.,
1993). This finding indicates that, as shown in other
synapses containing a classical transmitter and a
neuropeptide (De Vries and Baylor. 1993. Whim
et at., 1993), dynorphin is only released after strong
stimulation of the terminal.
In this system dynorphin can work as a VT signal.
In fact, it has been shown that when a mossy fibre is
tetanized, a second, inactive, mossy fibre is depressed
for several minutes (Weisskopf ct at., 1993).
Therefore, dynorphin inhibition of glutamate func-
tion is also present at distant synapses (so-called
heterosynaptic depression). Accordingly, the long
duration of dynorphin action IS not due to
intracellular effects in target cells (as dynorphin
action is immediately blocked by admimstration of
an opiate antagonist), but rather to permanence of
dynorphin in the ECF. Dynorphill-illduced de-
pression of glutamate function IS increased when the
pathway has been previously potentiated (Wagner
et at., 1993; Weisskopf et at.. 1993), suggesting that
dynorphin can modulate plastic changes occurnng in
this hippocampal circuit involved III learning and
memory (Morris and Johnston, 1995).
In conclusion, a complex interplay between an
excitatory WT signal (glutamate) and an Illhlbltory
VT signal (dynorphin) co-stored in the same nerve
terminal takes place in the mossy fibre system.
Thanks to this multiple transmitter system, a strongly
active pathway may inhibit normal transmission and
development of LTP in close-by pathways dunng a
certain time (a few minutes) after the discharge.
6.2. Retrograde Messengers and Hippocampal LTP
Much research has recently focused on the possible
influence of diffusible retrograde Signals on the
function of hippocampal glutamate synapses, namely
In the Central Nervous System 375
~ .. ~he development of LTP. Several studies have
shown that some retrograde signals (NO, carbon
monoxide, arachidonic acid, platelet activating
factor) contribute to the establishment of, at least
some forms of. hippocampal LTP (Arancio et at"
1995; Edwards, 1995a; Larkman and Jack, 1995;
O'Dell et at., 1994; Williams et at., 1993). Among
them, NO has received particular attention.
NO is generated from L-argimne by NOS, which
IS highly enriched in nervous tissue. The prevalent
forms of NOS in the neural cells are types I and III
(so-called neuronal and endothelial NOS, respect-
ively), which are Ca' + -calmodulin-dependent (Ker-
win and Heller, 1994). A main stimulus for these
enzymes IS calcium increase induced by neuronal
eXCitatIOn, in particular by NMDA receptor acti-
vation. Once formed upon activation of NOS, NO
diffuses outside the source cell and enters the target
cell where it can activate a soluble form of guanylate
cyclase (Bredt and Snyder, 1992; Dawson and
Snyder, 1994; Gaily et at., 1990; Garthwaite et at.,
1988). Owing to these properties, NO can work as a
retrograde signal in glutamatergic synapses where it
increases the release of glutamate from the presyn-
aptic termillal (Segovia et at., 1994).
This evidence has prompted theoretical research on
the role of diffusible signals such as NO in neuronal
computation (Gaily et at., 1990). A recent theory of
such a type has been termed 'volume learning'
(Montague and Sejinovski, 1994). A diffusible signal,
through its diffusion in a local volume of tissue, forms
a transient domain in which synaptic strengths can be
modified. Interestingly, the rules for synaptic strength
modification III the transient domain may be different
than standard hebbian rules. Hebbian rules hold that
a synapse is strengthened when there is a temporal
coinCidence between presynaptic and postsynaptic
activity. Montague and Sejinovski (1994) propose a
more complex scheme based on predictive learning
rules that are sensitive to the temporal order of input
activities: synapses are strengthened if active during
phases of high local concentration of a diffusible
substance, but weakened if active during phases of
low concentration.
Synaptic learning related to diffusible signals may
further differ from classic hebbian learning. In fact,
synaptic specifiCity can also be lost as the molecule
diffuses from active to inactive target cells, and may
influence ~ynaptic strengths therein. Some eVidence
for thiS phenomenon has been obtained for
NO-dependent LTP (Schuman and Madison, 1994).
In these expenments, LTP was elicited in CAl
pyramidal neurons by pairing Schaeffer collateral
activation with depolarization of the target (paired)
neuron and blocked by injecting an NOS inhibitor in
the paired neuron. Intracellular recording of non-
paired neurons located around 150 (but not 500) Ilm
from a paired neuron, showed that LTP was also
present in synapses contacting non-paired neurons.
As already mentioned, the same theory can be
applied to any VT signal. The chemical features of
each VT signal (hydrophilicity, charge, molecular
weight, etc.) as well as the presence and character-
istics of appropriate biochemical sensors would put
peculiar spatial and temporal constraints on the
domain influenced by the signal. For instance, VT
376 M. Zoli and L. F. Agnati
signal diffusion may occur through preferential
pathways and be non-isotropic (see above).
6.3. Neuropeptides in the Spinal Cord
Neurokinin A, CGRP and SP are stored in primary
nociceptive afferents in the substantia gelatinosa of
the spinal cord. Duggan et al. (1990) have shown that
a noxious stimulus causes release and diffusion of
neurokinin A throughout the dorsal horn and even in
the white matter of the spinal cord, while the
diffusion of SP is restricted to the substantia
gelatinosa. Interestingly, electrical recording of
neurons of the laminae I and II during SP
superfusion, showed increased background electrical
activity, enlargement of cutaneous receptive fields
and increased response to mechanic noxious stimuli
(Liu and Sandkiihler, 1995). Overall, such findings
may help explain long-term changes in the excitability
of spinal cord neurons after activation of unmyeli-
nated primary afferents of the dorsal roots (Mendell,
1966), and present compelling experimental evidence
for the existence of VT in nerve terminal networks of
the dorsal horn under such experimental conditions.
Duggan et al. (1992) also demonstrated that
CGRP, which is co-stored with SP, can work as a
peptidase modulatory signal for SP (Schaible et al.•
1993). After microinjection of CGRP into the dorsal
horn, nerve stimulation caused diffusion of immuno-
reactive SP into the entire dorsal horn, a pattern
similar to that found after application of peptidase
inhibitors (Duggan et al., 1992). These results
indicate that CGRP can reduce the degradation of
substance P via inhibition of endopeptidases, thus
increasing VT for substance P. The mixture and
relative content of co-stored peptides can therefore be
critical for their VT-dependent effects.
These results indicate that the nociceptive afferents
to the substantia gelatinosa of the spinal cord operate
not only via their release of a WT transmitter, namely
glutamate (Dougherty et al., 1992; Rustioni and
Weinberg, 1989), mediating fast synaptic trans-
mission, but also via release of various VT signals, i.e.
neuropeptides (Levine et al., 1993; Rustioni and
Weinberg, 1989), which can lead to long-term effects
on nociception. In addition, the neuropeptides can
have different metabolic patterns, some being more
suited for short-distance diffusion and others for
long-distance diffusion.
Acknowledgements-We thank Dr. Marina R. Picciotto for
critical reading of the manuscnpt. The work has been
supported by MURST and CNR grants.
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