neuroscience Handbook of Neurochemistry and Molecular Neurobiology Neurotransmitter Systems (Springer, 2008)

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Abel Lajtha (Ed.)
Handbook of
Neurochemistry and
Volume Editor: E. Sylvester Vizi
With 96 Figures and 22 Tables
Molecular Neurobiology
Neurotransmitter Systems
4 Synaptic and Nonsynaptic Release of Transmitters . . . . . . . . . . . . . . . . . . . . 101
E. S. Vizi . B. Lendvai
5 Cholinergic Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
B. Lendvai
6 Molecular Genetics of Brain Noradrenergic Neurotransmission . . . . . . . . . . . 129
R. Meloni
7 Dopamine and the Dopaminergic Systems of the Brain . . . . . . . . . . . . . . . . . 149
Table of Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Structural Organization of Monoamine and Acetylcholine Neuron Systems
in the Rat CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
L. Descarries . N. Mechawar
2 Brain Neurons Partly Expressing Monoaminergic Phenotype: Distribution,
Development, and Functional Significance in Norm and Pathology . . . . . . . . 21
M. V. Ugrumov
3 In Vivo Imaging of Neurotransmitter Systems with PET . . . . . . . . . . . . . . . . . . 75
B. Gulya´s . C. Halldin . B. Mazie`re
L. G. Harsing Jr.
8 5‐Hydroxytryptamine in the Central Nervous System . . . . . . . . . . . . . . . . . . 171
A. C. Dutton . N. M. Barnes
9 GABA Neurotransmission: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
A. Schousboe . H. S. Waagepetersen
10 ATP‐Mediated Signaling in the Nervous System . . . . . . . . . . . . . . . . . . . . . . . 227
B. Sperla´gh
# 2008 Springer ScienceþBusiness Media, LLC.
11 Adenosine Neuromodulation and Neuroprotection . . . . . . . . . . . . . . . . . . . . 255
R. A. Cunha
12 Regulation of AMPA Receptors by Metabotropic Receptors and Receptor
Tyrosine Kinases: Mechanisms and Physiological Roles . . . . . . . . . . . . . . . . . 275
A. L. Carvalho . M. V. Caldeira . A. R. Gomes . A. P. Carvalho . C. B. Duarte
13 Taurine in Neurotransmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
viii
P. Saransaari . S. S. Oja
14 The Endocannabinoid System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
B. S. Basavarajappa . R. Yalamanchili . T. B. Cooper . B. L. Hungund
15 E Prostanoid Receptors in Brain Physiology and Disease . . . . . . . . . . . . . . . . 385
C. D. Keene . P. J. Cimino . R. M. Breyer . K. S. Montine . T. J. Montine
16 Nitric Oxide and other Diffusible Messengers . . . . . . . . . . . . . . . . . . . . . . . . 403
J. P. Kiss
17 Molecular Organization and Regulation of Glutamate Receptors in
Developing and Adult Mammalian Central Nervous Systems . . . . . . . . . . . . 415
E. Molna´r
18 Sympathetic and Peptidergic Innervation: Major Role at the Neural–Immune
Interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443
I. J. Elenkov . A. Tagliani
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
1 Structural Organization of
Monoamine and Acetylcholine
8.2 Noradrenaline Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
8.3 Serotonin Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
8.4 Acetylcholine Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
9 Concluding Remarks: A New Image of the Neuron . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
8
8.1
# 200
Developmental Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Dopamine Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7.4
 Spinal ACh Innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
7.3
 Neostriatal ACh Innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
7.2
 Septohippocampal ACh System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
7.1
 Basalocortical ACh System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
7
 Acetylcholine (ACH) Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6
 Histamine Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5.2
5.3
Rapheostriatal 5‐HT System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Rapheospinal 5‐HT System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5
5.1
Serotonin (5-HT) Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Rapheocortical 5‐HT System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4
 Adrenaline Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3.1
3.2
Coeruleocortical NA System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Myelencephalospinal NA System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3
 Noradrenaline (NA) Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.3
 Diencephalospinal DA System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.2
 Mesostriatal DA System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1
 Mesocortical DA System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2
 Dopamine (DA) Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1
 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
L. D
Neuron Systems in the Rat CNS
escarries. N. Mechawar
8 Springer ScienceþBusiness Media, LLC.
1 Introduction
2 Dopamine (DA) Neurons
2 1 Structural organization of monoamine and acetylcholine neuron systems in the rat CNS
The general organization of the dopamine (DA) system is rather compartmentalized compared with that of
the other monoamines (for a detailed description and bibliographical listing, see Bjo¨rklund and Lindvall,
1984). In the rat CNS, at least six DA projection subsystems have been described—mesostriatal, mesolim-
bocortical, diencephalospinal, periventricular, incertohypothalamic, and tuberohypophyseal, in addition to
DA interneurons in both the olfactory bulb and retina. The mesostriatal and mesolimbocortical DA systems
originate from three mesencephalic groups of cell bodies located in the retrorubral field, substantia nigra,
and ventral tegmental area, respectively designated as A8, A9, and A10 according to the nomenclature
proposed by Dahlstro¨m and Fuxe (1964) at the time of their initial description. The other cell groups giving
Nowadays, neuromodulation may be broadly defined as all actions of neuronally released compounds that
produce more than a direct, short‐lived effect on neuronal firing. In this sense, all known neurotransmitters,
including the neuropeptides, the purine nucleotides and nucleosides, as well as the gaseous compounds NO
and CO, presumably qualify as neuromodulators. In this chapter, however, a more conventional definition
is adopted, whereby neurotransmitters exert transient effects through receptors mostly confined to synaptic
junctions, whereas neuromodulators may act for longer periods of time through receptors that are mostly
located away from release sites. In this view, the major neuromodulatory systems are the catecholamine
(dopamine (DA), noradrenaline (NA), and adrenaline), serotonin (5‐HT), histamine, acetylcholine (ACh),
and neuropeptide systems, as opposed to the amino acid systems (glutamate, aspartate, glycine, and
GABA). Purines, NO, and CO need not be considered as forming systems on their own, since these
compounds appear to be always colocalized with modulators and transmitters. Such distinctions are merely
operational, however, since it is becoming increasingly clear that one or more of the modulators and/or
transmitters are most often coexistent in the same neurons.
Owing to their progressive characterization by a variety of chemoanatomical techniques, and particu-
larly fluorescence histochemistry, uptake autoradiography, and immunocytochemistry, the morphological
features of the central monoamine and ACh systems can be currently described at three levels of morpho-
logical organization: (1) their overall anatomical distribution of constitutive cell bodies of origin and axonal
projections throughout the CNS; (2) their regional and intraregional (subnuclear or laminar) distribution
of axon terminals (or varicosities) in different territories of innervation; and (3) their ultrastructural
characteristics, intrinsic and relational, of putative release sites in the various brain regions.
This chapter examines the monoamine and ACh systems of rat brain from this triple standpoint,
including data on their development. It focuses mainly on rat, as knowledge in this species is the most
complete and detailed. The purpose is not to be exhaustive, but to illustrate some principles as well as
organizational features prevailing within and between these systems. In conjunction with the increasing
amount of data being currently accrued on the cellular and subcellular distribution of the multiple receptors
for each of the neuromodulators, it is thus expected to gain insights into their complementary modes of
action and functional properties.
Abstract: The anatomical, cytological, and ultrastructural features of monoamine (dopamine, noradrenaline,
adrenaline, serotonin, histamine) and acetylcholine neuron systems have been examined in many regions of
mammalian central nervous system, particularly in the rat. By considering these data with an emphasis on
innervation densities and ultrastructural relationships, including results obtained during postnatal develop-
ment, organizational principles and characteristics emerge for each of the modulatory systems.
List of Abbreviations: ChAT, choline acetyltransferase; DA, dopamine; DBH, dopamine‐b‐hydroxylase;
5‐HT, 5‐hydroxytryptamine; NA, noradrenaline; PNMT, phenylethanolamine N‐methyltransferase; TH,
tyrosine hydroxylase
the A17 group consists of a relatively sparse subpopulation of amacrine cells interspersed in the inner
from 0.6 to 1.7� 10 pg of DA for concentrations of 290–810 mg/g or 1.9–5.4� 10 M. Interestingly, the
figures for mediofrontal and cingulate cortex were very similar to those extrapolated for neostriatal DA
Structural organization of monoamine and acetylcholine neuron systems in the rat CNS 1 3
varicosities (Doucet et al., 1986).
All available observations suggest important regional and perhaps laminar differences in the frequency with
which DA axon varicosities in adult rat cerebral cortexmake synaptic specializations. Thus, in the anteromedial
and the occipital cortex, these axon terminals appear to be mostly if not entirely synaptic (Se´gue´la et al., 1988;
Papadopoulos et al., 1989), whereas in the suprarhinal cortex, only 56%display a junctional complex. Relatively
low synaptic incidences of 39% and 20% have also been reported for DA terminals in monkey prefrontal and
entorhinal cortex, respectively (Smiley and Goldman‐Rakic, 1993; Erickson et al., 2000). In terms of DA
function, the significance of such differences between cortical regions has yet to be investigated.
2.2 Mesostriatal DA System
Studies of anterogradely labeled axons after injection of biotin dextran in single nigrostriatal neurons have
shown that most of these axons travel directly to the striatum, in which they branch abundantly, whereas
others branch only sparsely in the striatum and arborize profusely in various extrastriatal structures,
portion of the inner nuclear layer. Thus, the DA systems are constituted by various cell types, from anaxonic
to long axon neurons. For a detailed mapping of these cell groups, see Ho¨kfelt et al. (1984b).
The number of DA cells in the mesencephalic tegmentum has been estimated at 15,000–20,000 on each side
of rat brain (Hedreen andChalmers, 1972; Guyenet and Crane, 1981; Swanson, 1982): some 9,000 in the ventral
tegmental area (Swanson, 1982) and the remainder in the zona compacta of the substantia nigra and retrorubral
field. These cells are at the origin of extensive mesotelencephalic projections. Neurons of the A10 group supply
the mesolimbocortical system innervating structures such as the amygdala, septum, olfactory tubercle, nucleus
accumbens and cerebral cortex, whereas those of the A8 and A9 groups are the main contributors of the
nigrostriatal system.
2.1 Mesocortical DA System
The DA projection to cerebral cortex is restricted in its distribution, at least in rat. This DA innervation was
initially described as confined to the anteromedial or prefrontal, anterior cingulate or pre‐and supragenual,
suprarhinal, perirhinal, piriform, and entorhinal cortex (for detailed references to the literature, see
Descarries et al., 1987). Additional DA terminal fields were later identified in the dorsomedial frontal
area, retrosplenial and adjacent occipital cortex, and in the deep layers of the frontal, parietal, temporal, and
occipital neocortex (Descarries et al., 1987). In each of these areas, there is a strong predilection of the DA
innervation for certain cortical layers. Moreover, axonal tracing studies have indicated that individual
DA neurons innervating the cerebral cortex have fairly circumscribed intracortical territories of projection
and do not collateralize extensively (Fallon and Loughlin,1982; Swanson, 1982; Albanese and Minciacchi,
1983; Loughlin and Fallon, 1984).
The heterogeneous distribution of the mesocortical DA subsystem is substantiated by the available data
on the number of DA terminals in the different regions of rat cerebral cortex, ranging from 4 � 104 in layer
VI of the occipital cortex to 3.1 � 106 in layers II–III of the supragenual cingulate cortex (Descarries et al.,
1987). Basic neurochemical parameters have been deduced from these numbers. Assuming that cortical DA
is mostly concentrated within the varicosities as opposed to intervaricose axon segments, it has been
calculated that, depending on the cortical region, the average DA content of a single varicosity should range
�4 �3
rise to DA projections, A11 to A15, occupy various parts of the diencephalon. The most caudal, A11, is at
the origin of a long descending projection to the spinal cord, whereas the other groups give rise to short
projections to the diencephalon and circumscribed hypothalamic and hypophyseal areas. The DA neurons
in the olfactory bulb (group A16) are a subset of the periglomerular interneuron population, and thus
mostly found in the periglomerular area, with a few also scattered in the outer plexiform layer. In the retina,
extreme density of this DA innervation, it has also been hypothesized that the spontaneous and evoked
release from such a multitude of asynaptic as well as synaptic varicosities might permanently maintain a
(at thoracic level), more than two thirds are synaptic, whereas in the dorsal horn, two thirds at the cervical level
and one fourth at the thoracic level do not form conventional synapses (Ridet et al., 1992). Thus, as in the
4 1 Structural organization of monoamine and acetylcholine neuron systems in the rat CNS
cerebral cortex and neostriatum, the diencephalospinal DA system might operate at least partly by diffuse
transmission, depending on the region innervated. It is not yet known whether the synaptic and asynaptic DA
terminals in the different regions and/or at different levels of the cord arise from collaterals of the same cells.
3 Noradrenaline (NA) Neurons
The entire neuraxis, except for neostriatum, receives a NA innervation, issued from two major clusters of
brainstem neurons, one in the locus coeruleus (A6) and its dorsolateral extension (A4) and the other in a
series of smaller cell groups occupying the ventrolateral aspect of the medulla and pons (A1, A5, A7) (for a
detailed description, see Moore and Card, 1984; see also Ho¨kfelt et al., 1984b). After dopamine‐b‐hydroxylase
(DBH) immunostaining, the total number of these cells has been estimated at about 5,000 on each side of the
brainstem (Swanson and Hartman, 1975). Noradrenergic neurons are also present in the nucleus tractus
solitarii‐dorsal vagal complex and the area postrema (A2). Both the locus coeruleus and lateral tegmental NA
groups give rise to ascending and descending projections. The locus coeruleus projects principally to the
cerebral cortex, thalamus, cerebellum, and spinal cord, whereas the lateral tegmental groups projects princi-
pally to the basal forebrain, hypothalamus, brainstem, and spinal cord (Ungerstedt, 1971; Lindvall and
Bjo¨rklund, 1974a, b, 1978).
basal extracellular level of DA throughout the striatum Descarries et al., 1996. This should allow, among
other functions and in addition to the transsynaptic effects of DA, for a sustained regulation of widely
distributed high‐affinity receptors on neurons, glia, and microvascular elements.
2.3 Diencephalospinal DA System
The descending DA projection to the spinal cord originates from a few hundred cells (group A11) located in the
dorsal and posterior hypothalamus, paraventricular hypothalamic nucleus, zona incerta, and caudal thalamus
(Bjo¨rklund and Skagerberg, 1979; Ho¨kfelt et al., 1979; Swanson et al., 1981). Axons of these DA neurons have
been described as bifurcating into an ascending branch to the diencephalon and a descending branch to the
spinal cord (Lindvall and Bjo¨rklund, 1974a). In the cord, these descending axons travel partly within lamina I of
the dorsal horn and adjoining parts of the dorsal funiculus and partly around the central canal, spreading
scattered terminals to the spinal grey at all segmental levels, mainly in laminae III–IV of the dorsal horn, the
intermediolateral cell column, the periependymal region, and the ventral horn (Skagerberg et al., 1982; Shirouzu
et al., 1990;Ridet et al., 1992). In the intermediolateral cell column and ventral horn, these axon terminals appear
to be all synaptic, making junctions with cell bodies and dendrites (Ridet et al., 1992). Around the central canal
including globus pallidus and entopeduncular and subthalamic nuclei (Gauthier et al., 1999; see also Prensa
and Parent, 2001). In dorsal neostriatum, the density of DA varicosities or terminals, i.e., potential release
sites of DA, has been estimated at 1 � 108/mm3 in the striatal matrix and 1.7 � 108/mm3 in the striosomes
and subcallosal streak (Doucet et al., 1986), which is generally assumed to represent one‐tenth of all
terminals in the striatum. Since the volume of rat neostriatum is approximately 45 mm3, the total number
of DA varicosities in one striatum must be at least 4.5 � 109, which represents at least 1660 DA varicosities
per neostriatal neuron (for unbiased estimates of neuron number in striatum, see Oorschot, 1996). Then,
depending on whether the number of mesencephalic DA neurons projecting to neostriatum is considered to
be 3,500 (Ande´n et al., 1966) or 7,000 (Bjo¨rklund and Lindvall, 1984), these individual neurons must be
endowed, on average, with 1.3 or 0.6 � 106 axon varicosities in the striatum, emphasizing the bushy
character of their arborization. Furthermore, because only 30%–40% of these DAvaricosities are junctional,
and 60%–70% do not form synaptic membrane specializations (Descarries et al., 1996), it may be inferred
that this DA subsystem operates in large part by diffuse as well as synaptic transmission. Because of the
as the cerebellum or spinal cord (Ade`r et al., 1980; Nagai et al., 1981; Room et al., 1981; Steindler 1981).
Structural organization of monoamine and acetylcholine neuron systems in the rat CNS 1 5
The intracortical distribution of NA terminals has been studied in considerable detail in rat and
monkey. In rat neocortex, the NA axons and their varicosities have been shown to be rather uniformly
distributed between the various cytoarchitectonic areas, whereas in primates, there might be a greater
degree of regional and laminar heterogeneity (Levitt et al., 1984; Morrison et al., 1984). The average density
of NA innervation has been estimated at 1.2 � 106 varicosities/mm3 of tissue in rat neocortex, with no
statistically significant difference between the seven cortical areas examined in the anterior half of the brain
(Audet et al., 1988). In every region, the number of NA terminals was greatest in the molecular layer and
decreased progressively in the underlying cortex, with a two‐to threefold difference between the upper
and lower layers. These numerical data allowed to estimate the possible number of cortical NA varicosities
per locus coeruleus cell body of origin (at least 300, 000), their average number per cortical neuron (30–50),
their actual incidence among all terminals in the cortex (1/1,000), and the mean endogenous amine content
per varicosity (0.22 fg) (Audet et al., 1988).
A similar quantitative study in the dorsal hippocampus revealed amore heterogeneous regional and laminar
distribution and a significantly higher density of NA innervation, averaging 2.1 � 106 varicosities/mm3
(Oleskevich et al., 1989). On the basis of a hippocampal volume of 56 mm3, and assuming an equal
share of hippocampal NA terminals per neuron, this should represent a further loadof 78,400 terminals per
locus coeruleus neuron. The number of NA varicosities per hippocampal neuron should range from 20 to
40 per granule cell in the dentate gyrus to 180 per pyramidal cell in CA3, and could represent one varicosity
per 880–1,500 synapses, in the DG and CA1, respectively. Similar to neocortex, the NA content per
varicosity should be in the order of 0.16–0.21 fg, for concentrations in the 10�2 M range.
In both neocortex and hippocampus, the synaptic incidence of NA varicosities has been shown to be
very low, with reported frequencies of 17% or 26% in the parietal cortex (Smiley et al., 1992; Se´gue´la et al.,
1990), 7% in the frontal cortex (Cohen et al., 1997), and 16% in the CA1 region of dorsal hippocampus
(Umbriaco et al., 1995). A single study in monkey provided a value of 18% for prefrontal cortex (Aoki et al.,
1998). Thus, there seems to be a principle of coherence at stake here, whereby a highly divergent projection
system such as the coeruleocortical NA system establishes rather loose interrelationships at the ultrastruc-
tural level as well, whereas more compartmentalized and more focused projections, such as the mesocortical
DA system, will establish more frequent, and perhaps more rigid, synaptic connections (Descarries et al.,
1988). Because of its widespread distribution as well as largely asynaptic character, this NA system appears
ideally built to act at a distance, on vast neuronal ensembles, and exert rather general, sustained and/or
indirect or mediated effects, as expected from a neuromodulator.
3.2 Myelencephalospinal NA System
The NA innervation of spinal cord provides further evidence of the extreme divergence of locus coeruleus
neurons. Numerous studies have indicated that, although the medullary NA cell groups contribute to
this innervation, its principal source is the locus coeruleus (A6) and the A5 and A7 lateral tegmental cell
groups (Westlund et al., 1981, 1982, 1983; Fritschy and Grzanna, 1990). The locus coeruleus and adjacent
subcoeruleus NA neurons supply input to both the dorsal and ventral horns at all segmental levels, whereas
the majority of the NA innervation of the intermediolateral cell column appears to arise from the A5 and
A7 groups. The density of this spinal innervation is much greater than that of its DA counterpart. Yet, as
3.1 Coeruleocortical NA System
The locus coeruleus (A6), comprising approximately 1,500 neuronal cell bodies on each side of the brainstem
(Descarries and Saucier, 1972; Swanson, 1976), is at the origin of the entire NA innervation of the cerebral
cortex, including hippocampus (Ungerstedt, 1971; for detailed references to the literature, see Audet et al.,
1988). A single coeruleocortical axon probably collateralizes from front to back throughout the cortex,
infiltrating its whole thickness (Morrison et al., 1981; Nagai et al., 1981; Loughlin et al., 1982). Moreover,
studies with retrogradely transported fluorescent dyes have convincingly demonstrated that at least some of
the NA neurons innervating the cerebral cortex can concomitantly innervate other distant CNS regions such
The serotonin (5‐hydroxytryptamine; 5‐HT) system is even more widespread than the NA system, as there
The 5‐HT innervations of cerebral cortex, hippocampus, and neostriatum have been the most thoroughly
6 1 Structural organization of monoamine and acetylcholine neuron systems in the rat CNS
examined. In seven cytoarchitectonic areas from the anterior half of the cerebral cortex, the density of
regional and laminar 5‐HT innervation was quantified after uptake labeling of the 5‐HT varicosities in
whole hemisphere sections incubated with tritiated 5‐HT in the presence of a monoamine oxidase inhibitor
6
is no single region of CNS without a 5‐HT innervation, including each of the circumventricular organs and
the cerebroventricular cavity, which is lined by the so‐called supra‐ependymal plexus of varicose 5‐HT fibers
(Steinbusch, 1981). Owing to the variety of methodological approaches applicable for the identification and
examination of central 5‐HT neurons, this is undoubtedly the transmitter‐defined system about which the
most is known in terms of distribution, cytological features, and particularly, ultrastructural relationships.
The 5‐HT neuronal cell bodies are mainly found near or in the midline or raphe region of the medulla,
pons, andmesencephalon, in nine groups designated B1–B9 according to Dahlstro¨m and Fuxe’s nomenclature
(1964). Themore caudal groups (B1, B2, andB3 in raphe pallidus, obscurus, andmagnus) projectmostly to the
medulla and spinal cord, whereas themost rostral (B5–B9, in raphemedianus, dorsalis, and the supralemniscal
region) provide extensive innervation to the diencephalon and telencephalon. 5‐HT cell bodies have also been
found in the area postrema, caudal locus coeruleus, and nucleus interpeduncularis. The nucleus raphe dorsalis
is the most prominent, with more than 11,000 5‐HT neurons, representing approximately one‐third of its
entire neuron population (Descarries et al., 1982). It is also the best characterized in terms of the cytological
features of its constituent neurons and afferent and efferent connectivity. Much as the locus coeruleus NA
neurons, it is likely that some of the nucleus raphe dorsalis 5‐HT neurons are highly collateralized and
simultaneously project to vast areas of forebrain distant from one another (Fallon and Loughlin, 1982).
5.1 Rapheocortical 5‐HT System
Neurons that are immunopositive not only for the biosynthetic enzymes tyrosine hydroxylase (TH) and
DBH but also for phenylethanolamineN‐methyltransferase (PNMT), and which presumably synthesize and
release adrenaline (Ho¨kfelt et al., 1974), also give rise to long and widely collateralized, albeit less abundant,
axonal projections distributed from the forebrain to the spinal cord (for detailed description, see Ho¨kfelt
et al., 1984a). The cell bodies of these neurons form three small groups, C1, C2, and C3, confined to medulla
oblongata and located near and within the A1 and A2 NA cell groups and on the midline, respectively,
within and dorsal to the medial longitudinal fasciculus. PNMT‐immunopositive fibers and nerve endings
have been described as concentrated along the ventricular system and most abundant in the bed nucleus
of the stria terminalis, various nuclei of hypothalamus, periaqueductal grey matter, brainstem nuclei of
visceral afferent and efferent systems, locus coeruleus, and intermediolateral cell column of the spinal cord.
In most of these CNS regions, the fine structural features and relationships as well as functional properties
of the adrenergic nerve terminals remain to be characterized. In view of its anatomical distribution, this
system is assumed to play a significant role in neuroendocrine mechanisms and blood pressure control.
In adult rat spinal cord, PNMT‐immunopositive axon terminals have been shown to establish axosomatic
and axodendritic synaptic contacts with the preganglionic neurons of the intermediolateral cell column
(Milner et al., 1988).
5 Serotonin (5-HT) Neurons
in the case of the DA system, only 25%–29% of the NA terminals in the dorsal horn would display
junctional specializations, whereas a much greater proportion (87% and 85%) is synaptic in the inter-
mediolateral cell column and ventral horn, respectively (Rajaofetra et al., 1992; Ridet et al., 1993).
4 Adrenaline Neurons
(Audet et al., 1989). The mean regional density of cortical 5‐HT innervation was thus estimated at 5.8� 10
their incidence among all cortical axon terminals 1 in 200, and their mean endogenous amine content 0.045 fg
for a concentration in the order of 3� 10�3 M. A similar study carried out in the subiculum, Ammon’s horn,
varicosities ranging between 12% and 24% have also been reported for CA1, CA3, and the dentate gyrusof
hippocampus (Oleskevich et al., 1991; Cohen et al., 1995; Umbriaco et al., 1995).
been estimated at 10%–13%. In the rostral striatum, for example, this small proportion should correspond
to some 4.8 � 105 synapses/mm3, i.e., approximately 1 in every 2000 striatal synapses.
Structural organization of monoamine and acetylcholine neuron systems in the rat CNS 7
5.3 Rapheospinal 5‐HT System
The 5‐HT innervation of spinal cord is relatively dense, with particular regions of the spinal grey standing
out because of their strong 5‐HT innervation: the dorsal horn, particularly lamina I and to a lesser extent
lamina II, the ventral horn motor nuclei (laminae VIII and IX), and the intermediolateral cell column in the
thoracic cord (Bowker et al., 1982; Skagerberg and Bjo¨rklund, 1985). As in the case of the DA and the
NA innervations of the dorsal horn, the 5‐HT innervation in this area has been shown to be only partly
synaptic (37%) (Ridet et al., 1993), with little variation between the different laminae of the dorsal horn
or at different spinal cord levels (Marlier et al., 1991). In the intermediolateral cell column and anterior
horn, however, the 5‐HT varicosities are presumably mostly if not entirely synaptic (Poulat et al., 1992;
Ridet et al., 1993), as also reported for DA and NA axon varicosities.
6 Histamine Neurons
Immunocytochemical studies with antibodies against histidine decarboxylase or histamine itself have
revealed the existence in the rat brain of a widely distributed network of fine, varicose, unmyelinated
5.2 Rapheostriatal 5‐HT System
In neostriatum, the 5‐HT innervation appears rather uniformly distributed, without any suggestion of a
patch and matrix pattern. Its density increases from rostral to caudal, however, and is always higher
ventrally than dorsally. It ranges from 4.8 � 106 varicosities/mm3 rostrally to 6.3 � 106 caudally, for an
average of 5.6 � 106 (Mrini et al., 1995), almost equal to that in cerebral cortex.
Such a density corresponds to approximately 90 5‐HT varicosities per neostriatal neuron, thus roughly
18 times less the number for DA terminals. It predicts a value in the order of 0.09 fg for the mean 5‐HT
content per neostriatal 5‐HT varicosity, compared to 0.045 fg and 0.06 fg in the cerebral cortex and
hippocampus, respectively. The proportion of these 5‐HT varicosities engaged in synaptic contact has
and dentate gyrus of the dorsal hippocampus yielded values of 2.7 � 106 varicosities/mm3 for the average
density, but with the values in subiculum> Ammon’s horn> dentate gyrus, and a marked heterogeneity in
laminar distribution (Oleskevich and Descarries, 1990). The average number of hippocampal 5‐HT
varicosities per cell body of origin could thus be evaluated at 150,000, the number of 5‐HT varicosities
per target neuron at 20–130, and the mean endogenous amine content per hippocampal 5‐HT varicosity at
0.05–0.07 fg, a value similar to that in cerebral cortex.
In rat neocortex, a detailed study by Se´gue´la and coworkers (1989) has estimated the synaptic incidence
of 5‐HT axon varicosities at 36% and 28% in the superficial and deep layers of the frontal cortex, 46% in
the parietal cortex, and 37% in the occipital cortex. Even lower frequencies of synaptic specialization were
reported for the sensorimotor and prefrontal cortex of monkey (DeFelipe and Jones, 1988; Smiley and
Goldman‐Rakic, 1996) and for the cat auditory cortex (DeFelipe et al., 1991). Frequencies of synaptic 5‐HT
varicosities/mm3 of tissue, with significant variations between areas. The highest laminar density was always
that of layer I, except in piriform cortex, but each region showed a distinct laminar pattern of 5‐HT
innervation. On the basis of these figures, the average number of cortical 5‐HT varicosities per cell body of
origin could be calculated to be at least 500,000, their average number per cortical neuron from 145 to 230,
1
axons, originating from small subgroups of nerve cell bodies located in the tuberomammillary nucleus
chemical description of these nerve fibers and axon varicosities has shown that in cerebral cortex and
ACh innervations. The ACh innervation intrinsic to the spinal cord will also be described, as it allows for
8 1 Structural organization of monoamine and acetylcholine neuron systems in the rat CNS
meaningful comparisons with the monoaminergic innervations of this region.
7.1 Basalocortical ACh System
It is currently estimated that, in the rat brain, there are 7,000–9,000 Ch4 neurons projecting to the
cortex (Rye et al., 1984; Gritti et al., 1993). Although the great majority of ACh axons in cerebral cortex
neostriatum the majority of histamine varicosities do not form synaptic specializations (Takagi et al., 1986).
7 Acetylcholine (ACH) Neurons
Our present knowledge on the structural basis of ACh transmission in the CNS has been largely acquired
from the detailed examination of neurons immunostained for choline acetyltransferase (ChAT), the rate‐
limiting enzyme for ACh synthesis. All regions of the CNS are pervaded by dense networks of ChAT‐
immunostained axons originating either from projection neurons located in the basal forebrain or
midbrain, and/or from local interneurons (Armstrong et al., 1983; Woolf, 1991). The latter have been
shown to contribute either a small fraction (neocortex), almost all (neostriatum), or the totality (spinal
cord) of respective regional ACh innervations. Groups of ACh projection neurons and their targets have
been extensively described from investigations combining tract‐tracing methods and ChAT immunocyto-
chemistry (Rye et al., 1984; Saper, 1984) and are now commonly referred to as Ch1–Ch8 on the basis of
nuclear localization (Mesulam et al., 1983; Mesulam, 1988). The basal forebrain groups Ch1–Ch4 provide
for the rich and widespread innervations of neocortex, hippocampus, olfactory bulb, and amygdala. The
medial septum and nucleus of the vertical limb of the diagonal band of Broca, Ch1 and Ch2, respectively,
send dense ACh projections to the hippocampal formation, while the lateral portion of the horizontal limb
of the diagonal band of Broca (group Ch3) innervates mainly the olfactory bulb. Together, groups Ch1–Ch3
also contribute a limited fraction of the total cortical ACh innervation, with axons restricted to limbic areas
(cingulate, entorhinal, orbitofrontal and piriform cortex). In contrast, the whole cortical mantle (including
these limbic areas) and amygdala receive a rich axon network stemming from the Ch4 neurons in the
nucleus basalis magnocellularis of Meynert, which is spread over the substantia innominata and globus
pallidus. The pontomesencephalic ACh system (Ch5–Ch6) is the principal projection pathway outside the
basal forebrain and has most of its efferents reaching the thalamus and basal forebrain. ACh neurons in
the habenula constitute the Ch7 system, which extends projections exclusively to the interpeduncular
nucleus via the fasciculus retroflexus pathway. Finally, the parabigeminal nucleus group Ch8, also limited in
its scope of innervation, targets the majority of its axons to the superior colliculus (tectum).
The availability of a highly sensitive monoclonal antibody with high affinity for whole rat brain ChAT
(Cozzari et al., 1990) has allowed the development of experimental conditions leading to the integral
staining of ACh axon networks through the full thickness of perfusion‐fixed brain sections. This made it
possible to conduct thorough and unbiased electron microscopic descriptions of ChAT‐immunostained
axon varicosities (Umbriaco et al., 1994), and led to the development of a semicomputerized light
microscopic method to quantify the length of axons and related number of varicosities from ChAT‐
immunostained axon networks (Mechawar et al., 2000). In consequence, our group has produced quanti-
tative descriptionsof the cortical, hippocampal, and neostriatal ACh innervations, both in terms of
quantified distribution and ultrastructural features (Umbriaco et al., 1994, 1995; Contant et al., 1996;
Mechawar et al., 2000; Aznavour et al., 2002). In this section, we mainly discuss these systems (basalocor-
tical, septohippocampal, and neostriatal), as they have been the most thoroughly scrutinized of all central
(Watanabe et al., 1984; Inagaki et al., 1988; Panula et al., 1989). This system comprises long projections to
regions such as the olfactory bulb, cerebral cortex, caudate‐putamen, and thalamus, a relatively dense
innervation of numerous areas of hypothalamus, and descending projections to the upper and lower
brainstem, cerebellum, and spinal cord. To date, the only light and electron microscopic immunocyto-
Structural organization of monoamine and acetylcholine neuron systems in the rat CNS 1 9
originate from the basal forebrain, a 20%–30% portion is contributed by intrinsic bipolar interneurons
scattered throughout layers II–VI (Johnston et al., 1981; Eckenstein and Thoenen, 1983; Eckenstein and
Baughman, 1984; Levey et al., 1984). In a detailed light microscopic description of ChAT‐immunostained
axons distributed in the rat cerebral cortex, 13 different patterns of ACh innervation were identified that
generally corresponded to functionally similar cortical areas (Lysakowski et al., 1989). This patterning has
recently been associated with modality‐and region‐specific ACh release in neocortex (Fournier et al., 2004),
indicating at least a regional control in Ch4 output activity.
The laminar and regional densities of the cortical ChAT immunoreactive axon network have been
estimated in transverse sections from the frontal (Fr1), parietal (Par1), and occipital (Oc1) cortical areas
(Mechawar et al., 2000). The number of varicosities per unit length of axon was counted directly at the
microscope and found to be constant throughout these areas (average of 4 varicosities/10 mm of axon). In
consequence, the actual number of varicosities in the network could be directly derived frommeasurements
of its length, and the laminar and regional densities of ACh innervation expressed in meters of axon
and millions of varicosities per mm3 of tissue. The densest ACh innervation was thus measured in
the frontal, followed by the occipital and the parietal cortex, with respective values of 5.4, 4.6, and 3.8 �
106 varicosities/mm3. In the three areas, layers I and V were the most densely innervated, with respective
interareal means of 5.3 and 5.0 � 106 varicosities/mm3. The least densely innervated were layers IV and VI
of the primary sensory areas, with interareal means (Par1 and Oc1) of 3.4 and 3.8 � 106 varicosities/mm3,
respectively. As expected, the laminar distributions were area specific, and characterized by uniformly high
densities throughout the frontal cortex and lower densities in layers II/III, IV, and VI of the parietal cortex,
as well as in layers IV and VI of the occipital cortex. Compared to monoaminergic innervation densities
previously measured in the same areas, the mean density of 4.6 � 106 ACh varicosities/mm3 for these
regions represented the densest of all neuromodulatory inputs to the neocortex.
When broken down to individual cells, these figures allow to calculate that, on average, each ACh
neuron projecting to the cerebral cortex must be endowed with an axonal arborization totalling at least
0.5 m in length and bearing more than 200,000 varicosities. Moreover, this is likely to be an underestimate,
since Ch4 neurons have also been shown to extend several axon collaterals within the basal forebrain, which
make synapse onto dendrites of surrounding (unspecified) neurons (Zaborszky and Duque, 2000). This
latter observation leads to the conclusion that the activity of basalocortical ACh neurons may itself be
subjected to a dual cholinergic modulation: one intrinsic, from its own recurrent axon collaterals; and one
extrinsic, from its ACh afferents of the mesopontine tegmentum.
The relational features of cortical ACh axon terminals (varicosities) were first described in the primary
somatosensory cortex of adult rat (Umbriaco et al., 1994). In all layers of Par1, only a small fraction of these
ChAT immunoreactive varicosities were found to form a synaptic contact (junctional complex), i.e., 10%,
14%, 11%, 21%, and 14% in layers I, II/III, IV, V, and VI, respectively, for an interlayer mean of 14%. In
general, cortical ACh varicosities were relatively small, and those bearing a synaptic junctionwere slightly but
significantly larger than their nonsynaptic counterparts, i.e., 0.67 versus 0.57 mm in diameter, respectively.
The junctional complexes formed by these terminals were single, occupied a small fraction of the total
surface of varicosities (3%), and were almost always symmetrical (99%). The relatively few synapses made by
ACh varicosities were always axodendritic, either on branches (75%) or on spines (25%). Subsequent
investigations in other laboratories have confirmed that the vast majority of ACh varicosities in rat cortex
are asynaptic, with reported estimates of 14% and 9% for the frontoparietal and entorhinal region,
respectively (Che´dotal et al., 1994; Vaucher and Hamel, 1995). The value of 66% recently reported by Turrini
and coworkers (2001) for layer Vof rat parietal cortex after labeling with the vesicular ACh transporter was
presumably the result of a sampling bias, as suggested by a significantly larger size of the profiles examined in
that particular study.
In the prefrontal cortex of rhesus monkey, Mrzljak et al. (1995) reported that 44 of 100 serially sectioned
ChAT-immunoreactive axon varicosities at the border of layers II and III made synaptic contact, mostly
onto small dendritic shafts. In a similar study of two samples of human anterior temporal lobe removed at
surgery for epilepsy, Smiley et al. (1997) found 28 of 42 ACh varicosities from layer I and II endowed with
small but identifiable synaptic specializations. Whether such variations of synaptic incidence reflect
sampling biases, regional differences or species differences remains to be determined. In any event, these
tion of muscarinic ACh receptors in hippocampus or cerebral cortex (reviewed in Volpicelli and Levey, 2004),
the above results strongly suggest that the modulatory effects of ACh on cortical function, so well documented
juxtaposition of ChAT‐immunostained varicosities was also observed. From this and a subsequent study in
10 1 Structural organization of monoamine and acetylcholine neuron systems in the rat CNS
the developing brain (Aznavour et al., 2003), it was concluded that the diffuse mode of transmission was an
inherent characteristic of ACh neurons, both as interneurons (neostriatum) and projection neurons (cerebral
cortex).
in hippocampus, are largely conveyed by diffuse (or volume) transmission in addition to synaptic transmis-
sion. In both regions in which this innervation is relatively dense, many of these effects could also depend on
the existence of a low ambient level of ACh, permanently maintained in the extracellular space in spite of the
presence of acetylcholinesterase (for further discussion, see Descarries et al., 1997).
7.3 Neostriatal ACh Innervation
The neostriatum receives by far the densest ACh innervation in the mammalian brain, as manifested by the
high measures of cholinergic markers expressed throughout this region. Among others, values of ACh content
(Cheney et al., 1975; Hoover et al., 1978), ChAT activity (Hoover et al., 1978), and choline uptake (Rea and
Simon, 1981) are particularly elevated. These parameters reflect the profuse ACh axon network originating
from cholinergic aspiny interneurons, which are estimated to account for less than 2% of all neostriatalneurons (Woolf and Butcher, 1981; Phelps et al., 1985). ACh interneurons in the caudate and putamen are
large cells resembling their basal forebrain counterparts (Armstrong et al., 1983), and also give rise to fine
unmyelinated axons periodically adorned with small, round, or ovoid varicosities. Although this local cell
population provides the neostriatum with most of its cholinergic innervation, a minor fraction of ACh axons
has been found to originate from the Ch5–Ch6 system (Woolf and Butcher, 1981, 1986).
The ultrastructural features of these putative release sites were first described by Contant and coworkers
(1996), after ChAT‐immunostaining in single thin sections for electron microscopy. As previously found
in cerebral cortex and hippocampus, neostriatal ACh varicosities were seldom engaged in synaptic contact.
Their frequency of junction of 3% in single sections amounted to 9% when extrapolated to the whole volume
of varicosities. The very few ACh synapses were made with synaptic branches (6/10) or spines (4/10).
Other axon terminals, unlabeled, were often directly apposed to neostriatal ACh varicosities. Occasional
data allow the conclusion that the actions of ACh depend on both diffuse and synaptic transmission in the
neocortex of primates as well as of rat (further discussion in Descarries et al., 2002).
7.2 Septohippocampal ACh System
The vast majority of ACh axons in the hippocampus originate from the estimated 7,150 ACh Ch1–Ch2
neurons in each septum (Cadete‐Leite et al., 2003). Indeed, very few bipolar ACh interneurons have been
described in the hippocampus, most of which were observed in the stratum lacunosum moleculare of CA1
(Aznavour et al., 2002). As previously found for the neocortical innervation, the periodicity of varicosities
along ACh axons remained constant at 4 per 10 mm throughout the dorsal hippocampus, suggesting that this
is an intrinsic feature of ACh innervations. The densest laminar ACh innervations were measured in the
stratum lacunosum moleculare of CA3, stratum pyramidale of CA1 and CA3, and stratum moleculare
of the dentate gyrus, with respective values of 7.5, 8.2, 6.8, and 7.8 million varicosities/mm3 (Aznavour
et al., 2002). Regional values were similarly high, ranging from 4.9 � 106 varicosities/mm3 in CA1 to
6.2 � 106 varicosities/mm3 in CA3, for an average of 5.9 � 106 varicosities/mm3 in hippocampus, a value
28% greater than for neocortex and higher than any neuromodulatory input to cortex described so far.
Ultrastructural analysis of this innervation in the stratum radiatum of CA1 revealed additional
common features with the basalocortical ACh system (Umbriaco et al., 1995). The ACh varicosities in
this hippocampal region measured 0.6 mm on average, and only 7% were synaptic. Their few synaptic
contacts were symmetrical and made either with dendritic branches or spines.
Together with immunoelectron microscopic data demonstrating a predominant extrasynaptic localiza-
Structural organization of monoamine and acetylcholine neuron systems in the rat CNS 1 11
7.4 Spinal ACh Innervation
Unlike the DA, NA, and 5‐HTsystems, there are no descending ACh projections from the brain to the spinal
cord (Sherriff et al., 1991). Therefore, the spinal ACh innervation originates exclusively from the different
categories of spinal ACh neurons, which have been thoroughly described by Barber and collaborators (1984).
The preponderant type of ACh neuron in this region is the ventral horn somatic motor neuron, regarded as
the canonical ACh neuron since Langley’s seminal work on neuromuscular activation one century ago. The
only other ACh neurons with projections leaving the spinal cord are the preganglionic autonomic neurons,
located in the intermediate grey matter at thoracolumbar and lumbosacral levels. The three other types of
ACh cells in the spinal cord are the small laminae III–V neurons of the dorsal horn, lamina VII partition
neurons at the border between the dorsal and ventral horns, and lamina X neurons in the central grey
surrounding the central canal. Transverse sections of adult rat spinal cord immunostained for ChAT reveal
that somatic motor neurons are organized in central, medial, and lateral motor columns, and that the medial
and lateral columns can be further divided into five subcolumns (Barber et al., 1984). There are apparently
widespread intra‐ and intercolumnar interactions, as suggested by extent of intermingling longitudinal and
transverse motor dendrite bundles. Likewise, at autonomic levels, the somata and dendritic arborizations of
partition cells and central canal ACh neurons are heavily mixed with those of autonomic neurons. These
extensive interconnections between morphologically diverse ACh neurons have been described as the basis of
a cholinergic propriospinal system (Sherriff and Henderson, 1994; Huang et al., 2000).
A rich ACh axon network pervades all layers and regions of the spinal cord, including the ependymal
cell layer (Phelps et al., 1984; Scha¨fer et al., 1998). These varicose axons originate in various (undetermined)
proportions from the different types of ACh neurons mentioned above. Initial reports have shown ChAT‐
immunoreactive terminals to contact both ACh and non‐ACh elements in the spinal cord. The current
prevalent view is that these terminals are mostly, if not exclusively, synaptic in nature. This view is likely
biased by the fact that most studies on the subject have focused specifically on contacts made on the cell
bodies and proximal dendrites of motoneurons, Renshaw cells, and sympathetic preganglionic neurons
(Markham and Vaughn, 1990; Alvarez et al., 1999). Of these descriptions, the ones concerning the ACh
innervation on motoneuron somatodendrites are of particular interest. This innervation, which is thought
to arise from the canal cluster cells, consists of C‐type cholinergic terminals characterized by periodic
postsynaptic specializations called subsurface cisterns (Nagy et al., 1993).
8 Developmental Aspects
The early development of monoamine and ACh neurons in mammalian CNS has led to the notion that
these modulatory systems play significant roles in the installment and refinement of neuronal connectivity
during brain maturation. In rat, for example, dopaminergic fibers enter the cortical plate just before birth
(Kalsbeek et al., 1988); noradrenergic fibers reach the anterior parietal cortex around E20, and occipital cortex
at birth (Verney et al., 1982); serotoninergic fibers and terminals (Seiger and Olson, 1973), as well as
pioneering cholinergic fibers (Mechawar and Descarries, 2001), are already seen in the cortical plate at
birth (for reviews, see Semba, 1992, 2004). Yet, only two laboratories have examined the morphological
features of these systems at electron as well as light microscopic levels during development: the University of
Thessaloniki group, in Greece, which has focused on the monoamine systems, and our own laboratory, in
Montreal, which has mainly studied the developing cholinergic system.
8.1 Dopamine Neurons
The ultrastructural features of DA neurons have been examined in two brain regions during development:
lateral septum and striatum. The DA innervation of the lateral septum was reported to undergo a marked
reorganization during the first two postnatal weeks, when it acquired features comparable to the adult
(Antonopoulos et al., 1997). The ultrastructural analysis suggested that there might be two different
DA inputs to this region: the first developing earlier in life, affecting remote parts of neurons through
weeks. At the ultrastructural level, however, much more diversity was apparent. In the lateral septum, 5‐HT
12 1 Structural organization of monoamine and acetylcholine neuron systems in the rat CNS
varicosities were described as almost always synaptic and showingsymmetrical synapses, whether on somata,
dendritic shafts, or spines. In the dorsal portion of the lateral septum, they formed characteristic pericellular
basket‐like arrangements around cell somata and their primary dendrites, as previously described for DA
terminals (Descarries and Beaudet, 1983). In the lateral ventricles, the 5‐HT varicosities were located close to
the ventricular surface of the ependymal lining, but never made synapses on ependymal cells, even when
morphologically mature (shape, size, and content). In the lateral geniculate and basal forebrain, the synaptic
frequency of the 5‐HT varicosities displayed a biphasic temporal profile. The proportion of varicosities forming
synapses was reported to increase from birth to the end of the second postnatal week, and then declinemarkedly
in the third week before increasing again to adult values of about 40% in both regions. A similar biphasic pattern
was also described in the superficial layers of the superior colliculus (involved in visual functions); whereas in its
deep layers and in the ventrolateral thalamic nucleus (areas involved in motor functions), the proportion of
5‐HTvaricosities engaged in synaptic contact showed a continuous increase frombirth to adulthood, to become
8.3 Serotonin Neurons
The growth of 5‐HT innervations was examined in numerous subcortical regions of postnatal rat brain: lateral
septum (Dinopoulos et al., 1993), lateral ventricles (Dinopoulos andDori, 1995), lateral geniculate (Dinopoulos
et al., 1995), basal forebrain (Dinopoulos et al., 1997), superior colliculus, and ventrolateral thalamic nucleus
(Dori et al., 1998). Acquisition of such data from the developing cortex (Dori et al., 1996)was complicated by the
fact that, within the first weeks after birth, thalamocortical neurons transiently express 5‐HT plasmamembrane
transporter and vesicular monoamine transporter (Lebrand et al., 1996) and are thus immunoreactive for 5‐HT
and indistinguishable from bona fide 5‐HT neurons. A constant feature of the 5‐HT innervations in the
postnatal period, albeit parenchymal or intraventricular, was their progressive change from a few, thick and
smooth unmyelinated axonal fibers at birth to a ramified and relatively dense network of fine varicose axons,
infiltrating the whole region and reaching its adult pattern of distribution and density within the first three
symmetrical axodendritic synapses, and the second developing later and affecting neuronal somata
through asymmetrical axosomatic synapses.
In the striatum, the same authors described the DA innervation of caudate‐putamen and nucleus
accumbens as exhibiting a similar type of synaptic connectivity throughout development, but evolving
from symmetrical synapses mostly located on dendritic shafts at an early stage to a later stage where
symmetrical axospinous synapses also became a prominent feature (Antonopoulos et al., 2002). Interest-
ingly, this study also indicated that after initial rises to values >90% and 80% in the islands and matrix of
caudate‐putamen, respectively, and almost 100% and 75% in the shell and core of the nucleus accumbens,
the proportion of DA varicosities making synapses declined after P7 in the caudate‐putamen and after P14
in the nucleus accumbens to respective values of 63% and 35% in the islands and matrix of caudate‐
putamen, and 71% and 47% in the shell and core of nucleus accumbens.
8.2 Noradrenaline Neurons
The noradrenergic innervations of the septum, motor and visual cortex, and dorsal lateral geniculate
nucleus were described in developing rat brain, by means of light and electron microscopic immunocyto-
chemistry with DBH antibodies (Latsari et al., 2002, 2004; Antonopoulos et al., 2004). In all four regions,
few, relatively thick NA fibers were present at birth, which arborized gradually into an adult pattern of
thinner varicose fibers by the second postnatal week, and reached the adult density of innervation one week
later. Irrespective of the postnatal age examined, only a minority of these NA varicosities displayed synaptic
specializations (10%–15%), which were usually symmetrical and found on dendritic branches. It was
concluded from these data that, in these brain regions at least, transmission by diffusion is the major
mode of NA action in the developing as well as adult brain.
a fully synaptic innervation making mostly symmetrical synapses onto dendritic shafts.
(Mechawar and Descarries, 2001). At birth, a few ChAT‐immunostained fibers capped with growth cones
Structural organization of monoamine and acetylcholine neuron systems in the rat CNS 13
are already seen throughout the cortical plate and marginal zone. By P4, faintly immunoreactive inter-
neurons are first detected. Rapid ingrowth and proliferation ensues resulting in an adult‐like distribution of
a highly elaborate network of fine varicose ACh axons by P8. In the parietal area, adult densities
of innervation are reached by P16, while development continues until the end of the first month in the
frontal area and even later in the occipital area.
Two parameters of this ingrowth have been quantified (Mechawar and Descarries, 2001): the elongation
(and branching) of ACh axons and their number of axon varicosities per unit length. This latter value was
shown to increase steadily in all cortical layers and areas, doubling from 2 varicosities per 10 mm of axon at
P4 to the adult ratio of 4 per 10 mm at P16. It was thus possible to calculate that, within the first two weeks
after birth, a single basalocortical neuron produced an average of 2 cm of axon and 9,000 varicosities/day,
i.e., almost 1 mm of axon and 400 varicosities/hour.
A similar study has also been carried out in CA1, CA3, and the dentate gyrus of dorsal hippocampus
(Aznavour et al., 2005). At P8, an elaborate network of varicose ChAT‐immunostained axons was already
present in all three hippocampal regions. As in neocortex, the number of axon varicosities per unit length of
ACh axon increased during the first two weeks after birth to reach the adult value of 4 per 10 mm at P16.
At this age, the laminar distribution of this network resembled that of maturity, but adult densities of axons
and axon varicosities were reached only by P32. Between P8 and P32, the mean densities in the three regions
increased from 8.4 to 14 m of axons, and 2.3 to 5.7 million varicosities/mm3 of tissue. This suggested that,
on average, the septohippocampal ACh neurons are capable of generating 7.5 cm of axon and 27,000
varicosities/day, i.e., more than 3 mm of axon and 1,120 varicosities/hour. Such growth rates are even higher
than previously estimated for nucleus basalis ACh neurons innervating the neocortex, and emphasize the
remarkable growth capacities of ACh neurons.
These studies have also examined the intrinsic and relational features of ACh varicosities in the
developing neocortex and hippocampus (Mechawar et al., 2002; Aznavour et al., 2005). In both regions,
these varicosities were of similar size throughout development and only slightly smaller than in the adult.
They were endowed with aggregated synaptic vesicles, and the frequency with which they showed a
mitochondrion increased gradually with age, from about 20% at P8 to >40% at P32. As in the adult, the
vast majority of these varicosities were asynaptic throughout the postnatal period. Since the proportion of
synaptic ACh varicosities was stable during development, it could be inferred that the number of ACh
synapses had already reached its adult value by the end of the second week, at least in the parietal cortex
(0.55 � 106/mm3 at P16, compared to 0.53 � 106 at >P60).
The postnatal growth and ultrastructural characteristics of a developing ACh innervation have also been
examined in the neostriatum, where this innervation arises almost completely from a limited number oflarge interneurons. As in cortex and hippocampus, the invasion of neostriatum by ACh axons mostly takes
place during the first two weeks after birth, but continues at a slower rate until the fourth postnatal week
(Aznavour et al., 2003), in keeping with biochemical measurements of ChATactivity (Coyle and Yamamura,
1976). As in neocortex and hippocampus, the intrinsic and relational ultrastructural features of these
rapidly growing axons were examined at three developmental time points, i.e., after the first, second, and
fourth postnatal weeks (P8, P16, and P32) (Aznavour et al., 2003). Again, a low synaptic incidence was
measured at all three ages, indicating that this structural feature is an intrinsic determinant of the
functioning of this system during development as well as in the adult.
9 Concluding Remarks: A New Image of the Neuron
The detailed comparison of the structural attributes of neuromodulatory systems, whether at the level of
their general organization, regional distribution, or fine structure, underscores some consistent features of
8.4 Acetylcholine Neurons
A recent description of the ACh innervation in the frontal, parietal, and occipital neocortex of the postnatal
rat has revealed that this ACh system develops rapidly and much earlier than previously suspected
1
the major modulatory systems. First and foremost, it requires a drastic revision of what may be called the
14 1 Structural organization of monoamine and acetylcholine neuron systems in the rat CNS
traditional (textbook) image of the neuron, mostly inherited from the study of the neuromuscular junction.
Thus, relatively dense and widespread innervations by fine, unmyelinated axons whose multiple branches
bear innumerable varicosities (terminals) appears to be the rule for most if not all modulatory neurons in
the adult and developing CNS. It may also be assumed that these axonal arborizations, endowed with
varicosities that often lack a junctional specialization, are highly plastic, not only in a functional sense
but also in their structural configuration. As initially postulated by Descarries and coworkers (1975, 1977;
Beaudet and Descarries, 1978), it is likely that such small varicosities, lying free in the neuropil, undergo
incessant movements of remodeling and translocation along their parent fibers, resulting in release of their
transmitter or modulator in different microlocations of the tissue at different moments in time. Several
lines of evidence suggest that many of the peptidergic systems might also share such properties. It is also
apparent that these structural determinants apply to both extrinsic and intrinsic neuromodulatory systems,
as defined by Katz and Frost (1996).
By definition, extrinsic neuromodulation refers to the capacity of a system to cause global changes,
affecting many functional circuits simultaneously. The best examples of extrinsic modulatory systems are
the DA, NA, 5‐HT, and almost all ACh systems. To varying degrees, these systems pervade most CNS regions
and originate from relatively small numbers of neuronal cell bodies, grouped in discrete nuclei. Thus,
through their innervation of numerous cortical and subcortical regions, the DA subsystems contribute to
various aspects of motor function, neuroendocrine control, motivation, and behavioral learning. The NA,
5‐HT, and ACh systems are even more widespread, innervating most if not all regions of the CNS. They
seem to be involved in capacities of a rather global nature and rely on the simultaneous functioning of
widely distributed numerous neuronal circuits, such as waking and sleep, arousal, attention, emotional
states, learning, memory, and ultimately consciousness. In this perspective, state‐ and context‐dependent
responsiveness of the nervous system may be viewed as resulting from the joint activity of multiple,
spatially and temporally overlapping extrinsic neuromodulatory systems, as well as that of more specialized,
function‐specific systems.
Intrinsic neuromodulation arises from neurons entirely contained within a given circuitry (i.e., inter-
neurons). The massive ACh innervation of neostriatum issued from a fraction of its relatively small
population of scattered interneurons is a good example of an intrinsic modulatory system. These tonically
active neurons appear to be critical elements in the striatal circuitry controlling motor planning, movement,
and associative learning (Graybiel et al., 1994; Bennett and Wilson, 1999). Theoretically, intrinsic neuro-
modulation produces local changes in neuronal computation within a circuit. In view of the largely
asynaptic nature of the neostriatal ACh system, its influence is certainly exerted beyond point‐to‐point
synaptic connections, and presumably reaches a variety of more‐or‐less distant cellular targets within the
neostriatal circuitry. As an intrinsic component, the level of activity of this ACh system would be directly
dependent on that of the overall circuitry, the latter being itself modulated by extrinsic inputs (e.g., DA
and 5‐HT). It is tempting to speculate that the activity of such a circuitry is reflected by fluctuations in
the ambient level of its intrinsic neuromodulators (see Descarries et al., 1997). On the other hand, the
ambient levels of extrinsic neuromodulators should more closely depend on the activity of these systems
themselves.
Much as the progressive unraveling of the numerous modulatory systems, several fairly recent data and
concepts pertaining to chemical neurotransmission per se emphasize the previously unsuspected multiplicity
of transmission modes in the CNS. The coexistence of transmitter substances within the same neurons
(Ho¨kfelt et al., 1980; Merighi, 2002) is now recognized as a common if not universal trait in the CNS, much as
the release of transmitter from dendrites, and presumably from cell bodies (Cheramy et al., 1981; He´ry et al.,
1982). Spillover beyond the synaptic clefts has been demonstrated for most of the highly if not exclusively
synaptic, amino acid transmitters (Isaacson et al., 1993; Kullmann, 2000). There is also ample evidence that
multiple metabotropic and/or ionotropic receptors exist for every neurotransmitter/modulator, and receptors
for a wide variety of transmitters, and even different receptor subtypes for the same transmitter, are expressed
by a given neuron. Moreover, whenever visualized by electron microscopic immunocytochemistry,
most neuronal receptors, whether somatodendritic and/or axonal, appear to be located on extra‐ as well
as intrasynaptic portions of the plasma membrane (e.g., Riad et al., 2000). Lastly, a growing number of
signaling cascades, accounting for short‐, medium‐, and long‐term effects, have been identified for most
a
ca
nd
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capable of processing highly diversified information a
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