JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 155-19 (1990)

Cholinergic Synapses in the Central Nervous System: Studies of the lmmunocytochemical Localization of Choline Acetylt ransferase CAROLYN R. HOUSER Neurology Service, Veterans Administration Medical Center, West Los Angeles, Wadsworth Division, Los Angeles, California 90073, and Department of Anatomy and Brain Research Institute, University of California, Los Angeles, California 90024

KEY WORDS

Acetylcholine, Cholinergic fibers, Electron microscopy, Ultrastructure

ABSTRACT

Cholinergic synapses can be identified in immunocytochemical preparations by the use of monoclonal antibodies and specific antisera to choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine (ACh) and a specific marker for cholinergic neurons. Electron microscopic studies demonstrate that the fibers and varicosities observed in light microscopic preparations of many brain regions are small-diameter unmyelinated axons and vesicle-containing boutons. The labeled boutons generally contain clear vesicles and one or more mitochondria1 profiles. Many of these boutons form synaptic contacts, and the synapses are frequently of the symmetric type, displaying thin postsynaptic densities and relatively short contact zones. However, ChAT-labeled synapses with asymmetric junctions are also observed, and their frequency varies among different brain regions. Unlabeled dendritic shafts are the most common postsynaptic elements in virtually all regions examined although other neuronal elements, including dendritic spines and neuronal somata, also receive some cholinergic innervation. ChAT-labeled boutons form synaptic contacts with several different types of unlabeled neurons within the same brain region. Such findings are consistent with a generally diffuse pattern of cholinergic innervation in many parts of the central nervous system. Despite many similarities in the characteristics of ChATlabeled synapses, there appears to be some heterogeneity in the cholinergic innervation within as well as among brain regions. Differences are observed in the sizes of ChAT-immunoreactive boutons, the types of synaptic contacts, and the predominant postsynaptic elements. Thus, the cholinergic system presents interesting challenges for future studies of the morphological organization and related function of cholinergic synapses.

INTRODUCTION

1983a,b; Cuello and Sofroniew, 1984; Wainer et al., 198413). Virtually all parts of the nervous system reCholinergic neurons can be identified specifically by ceive some cholinergic innervation, although the denthe presence of choline acetyltransferase (ChAT), the sity of the cholinergic fibers varies greatly among the synthesizing enzyme for acetylcholine (Ach), and for regions (Figs. 1, 5a, 6a, 8a). Presently, demany years biochemical studies have utilized this en- different tailed studies of cholinergic synapses are limited to a zyme a s a n indicator of the regional distribution of cho- relatively small number of brain regions, but several linergic elements (Hoover et al., 1978; Kuhar, 1976; Palkovits et al., 1974; McCaman and McCaman, 1976). general patterns of organization are emerging. These common patterns will be discussed first, and then findHowever, only recently has the development of mono- ings unique to particular brain regions will follow. clonal antibodies and specific antisera to ChAT (Bruce et al., 1985; Crawford et al., 1982; Eckenstein et al., Immunocytochemical methods 1981; Eckenstein and Thoenen, 1982; Levey et al., Several different monoclonal antibodies (Crawford et 1983) made it possible to identify cholinergic neurons al., 1982; Eckenstein and Thoenen, 1982; Levey et al., in immunocytochemical preparations of the central 1983) a s well a s specific antisera to ChAT (Eckenstein nervous system (CNS). Since ChAT is present through- et al., 1981; Eckenstein and Thoenen, 1982) have been out many parts of the neurons, i t has been possible to used in the immunocytochemical studies of cholinergic visualize not only the cell bodies and proximal den- synapses. The tissue has generally been processed with drites of these neurons but also their axons and termi- unlabeled antibody peroxidase-antiperoxidase or avinal fields and, for the first time, to study the characteristics and organization of cholinergic synapses. Although the cell bodies of cholinergic neurons are often concentrated within specific brain regions, their Received April 22, 1987; accepted in revised form March 10, 1988. axons and terminal fields are more widely distributed The literature covered in this review extended through March 1987. throughout much of the CNS (Armstrong et al., 1983; Address reprint requests to Carolyn R. Houser, Ph.D., UCLA-Brain Research Fibiger, 1982; Houser et al., 1983; Mesulam et al., Institute, BRI 73-364 CHS, Los Angeles, CA 90024.

1990 WILEY-LISS, INC.

CHOLINERGIC SYNAPSES IN THE CNS

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Fig. 1. Light micrographs of ChAT-immunoreactive fibers and boutons in three regions of the nervous system. A In the cerebral cortex, fine fibers with numerous varicosities (arrows) are distributed in a loose network, as illustrated in this photomicrograph of layer I11 of the somatosensory region. B: In the dentate gyrus, immunoreactive fibers with periodic varicosities (arrows) are present in all layers, but the highest concentrations are evident in the region of the molecular

layer (M) just above the granule cell layer (G). C: In the reticular nucleus of the thalamus, numerous labeled fibers with periodic swellings (arrows) are present within neuropil regions. The regions devoid of ChAT-immunoreactive fibers correspond to bundles of unstained thalamocortical and corticothalamic fibers that traverse the nucleus. Note that in all three regions the varicosities are located at irregular intervals and differ in size along the same fiber. Scale lines, 20 pm.

din-biotin methods, similar to those used by the author and colleagues (Houser et al., 1985; Phelps et al., 1985). These methods were utilized in the preparation of most of the illustrated specimens and will be described briefly. Monoclonal antibodies (3F12 and 1E6) were obtained from mice that had been immunized with a partially purified preparation of ChAT from r a t brain (Crawford et al., 1982), and the specificity of the antibodies and immunocytochemical methods was established in a previous study of several well-characterized cholinergic systems (Houser et al., 1983). Tissue was obtained from Sprague-Dawley rats that had been deeply anesthetized and perfused through the heart with a fixative solution of 4% paraformaldehyde and 0.1-0.2% glutaraldehyde in 0.12 M phosphate buffer. For light microscopic studies, blocks of tissue from selected brain regions were cryoprotected, and 40 pm-thick sections were cut on a cryostat and collected in Tris-buffered saline solution (TBS; 0.1 M Tris buffer, pH 7.4, contain-

ing 0.85% NaC1). For electron microscopy, blocks of nonfrozen tissue were sectioned on a vibratome. The sections were then incubated in the following series of reagents: normal goat serum (diluted 1:30) for 1 hour; anti-ChAT or control antibody solution (1.5 pg IgGiml for 3F12 and 12 pg IgGiml for 1E6) for 2 hours a t room temperature and a n additional 18 hours a t 4°C; speciesspecific goat anti-mouse (GAM) IgG serum (1:60; American Qualex) for 1 hour (Houser et al., 198413); and mouse monoclonal PAP complex (1:60; Sternberger-Meyer Immunocytochemicals, Inc.) for 1 hour. The GAM and PAP steps were repeated in order to intensify the staining of fibers and terminals (Houser et al., 1983).The sections were rinsed in TBS after each incubation except the first. Following the series of incubations, the sections were reacted with 0.06% 3,3’diaminobenzidine.HC1 and 0.006% H,Oz. After rinsing, sections for light microscopy were treated for 30 seconds with 0.1% buffered osmium tetroxide, rinsed, and then mounted on slides, dehydrated, and cover-

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C.R. HOUSER

boutons in several brain regions form symmetric synapses that display thin postsynaptic densities (Fig. 2). These findings were not anticipated since symmetric synapses have generally been associated with inhibitory synaptic effects (Colonnier, 1981; Peters et al., 1976; Triller and Korn, 1982), and Ach has been shown to have excitatory actions in many brain regions (Kelly and Rogawski, 1985). However, physiological and Cholinergic axons and boutons-general light pharmacological evidence suggests that ACh is not microscopic observations limited to a single type of excitatory action but may Several similarities in the organization of choliner- have several different effects in the CNS; and these gic fibers and terminals have been suggested by light effects, in turn, may be related to different types of microscopic observations. In many regions of the CNS, receptors (Cole and Nicoll, 1983; Kelly and Rogawski, ChAT-containing axons can be characterized as very 1985; McCormick and Prince, 1 9 8 6 ~ ;Siggins and thin fibers with periodic varicosities that vary in size Gruol, 1986). In light of such findings, it would not be and are distributed irregularly along the axons (Fig. 1). surprising to observe ChAT-immunoreactive synapses These fibers often exhibit a diffuse pattern of organi- of several different morphological types. However, the zation, and light microscopic observations provide little initial immunocytochemical studies in several brain indication of the identity of the postsynaptic elements regions (discussed in subsequent sections) suggest since the fibers do not generally surround the somata more similarities than differences in the characterisor recognizable processes of the neurons (Fig. 1).This tics of the ChAT-labeled synapses. Furthermore, in reloosely organized pattern of ChAT-immunoreactive fi- gions where both immunocytochemical and physiologbers is common to many brain regions, but clearly con- ical data are available, the findings do not appear to trasts with that observed in some motor nuclei of the support the simple equating of symmetric and asymbrainstem and spinal cord where a population of rela- metric synaptic junctions with inhibitory and excitatively large boutons are closely associated with neu- tory effects, respectively. ChAT-labeled boutons in forebrain regions have ronal cell bodies and proximal dendrites, many of which are also labeled for ChAT (Barber et al., 1984; been described by most investigators a s small in size, Connaughton et al., 1986; Houser et al., 1983). It is although there is a moderate range of bouton sizes, likely that other patterns of cholinergic fiber organiza- consistent with the different sizes of varicosities and tion will be identified a s detailed examination of axon puncta observed in light microscopic preparations (Fig. terminal fields continues. 1).In studies of several brain regions, including the amygdala, cortex, and striatum, the reported diameCholinergic synapses-general electron ters of the terminals have ranged from 0.4 to 1.4 pm microscopic observations with a mean diameter of 0.8 pm (Carlsen and Heimer, Many of the electron microscopic studies have been 1986; de Lima and Singer, 1986; Houser et al., 1985; focused on forebrain regions with apparently diffuse Phelps et al., 1985; Phelps and Vaughn, 1986). In a few patterns of cholinergic innervation. The findings have studies of similar brain regions, the described size of confirmed that the fibers and varicosities observed the labeled terminals has been somewhat smaller, with with the light microscope correlate respectively with a range of 0.4-0.7 pm (Clarke, 1985; Wainer et al., small diameter unmyelinated axons and vesicle-con- 1984a). Several studies have suggested that the avertaining boutons (Fig. 2). It has been convincingly dem- age size of ChAT-labeled boutons may be smaller than onstrated in a number of studies that some of the ter- that of some other classes of boutons. For example, in minals and en passant boutons form synaptic contacts. the monkey neostriatum, the cross-sectional area of the However, in a single thin section, only a small percent- labeled boutons is significantly smaller than that of age of the labeled boutons may form synaptic contacts, unlabeled boutons forming asymmetric synapses with and estimates of the frequency with which boutons dendritic spines (DiFiglia, 1987). Additionally, the form synapses have ranged from “rarely” to “often.” It lengths of the synaptic conduct zones are relatively is unclear whether these estimates reflect differences short, and DiFiglia (1987) found that the synaptic juncin the subjective evaluations of the investigators or are tions of ChAT-containing boutons within the monkey due to actual differences in the frequency with which neostriatum are significantly shorter (median = ChAT-labeled boutons form synaptic contacts in differ- 0.18pm) than those of unlabeled boutons forming ent brain regions. In a study of the rat striate cortex, asymmetric synapses with dendritic spines (median = Parnavelas et al. (1986) determined by serial section 0.26pm). This is, no doubt, one of the primary reasons analysis that 22 of 24 randomly selected ChAT-positive why the synaptic contacts of many labeled boutons are varicosities formed synaptic contacts, whereas in sin- not evident in a single thin section (de Lima and gle thin sections less than 10% of the labeled boutons Singer, 1986; Wainer et al., 1984a). formed definitive synapses. Thus, in one region of the There has been general agreement that clear, agrancerebral cortex, it appears that most of the boutons ular vesicles are the predominant type within the form synaptic contacts, and similar studies are needed ChAT-immunoreactive boutons. However, descriptions to determine if this is also true in other brain regions. of the relative density, size, and shape of these vesicles One of the unexpected findings of the ultrastructural have varied. The concentration of vesicles has been destudies has been that the majority of ChAT-containing scribed as sparsely distributed in the cat striate cortex

slipped. Specimens for electron microscopy were treated with 2% osmium tetroxide for 1 hour, en bloc stained with uranyl acetate, dehydrated, and flat embedded in Epon-Araldite resin. Ultrathin sections were mounted on Formvar-coated slot grids, stained with lead citrate, and examined with a n electron microscope.

CHOLINERGIC SYNAPSES IN THE CNS

Fig. 2. Electron micrographs illustrating characteristic features of many ChAT-immunoreactive synapses. A: This portion of a ChATlabeled fiber consists of two boutons interconnected by a thin intervening segment. The upper bouton forms a symmetric synapse (arrow) with a small dendrite (D). Both boutons contain numerous vesicles and a mitochondria1 profile. B: A labeled terminal forms a

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symmetric synaptic contact (arrow) with a small dendrite (D). The thin postsynaptic density of the labeled synapse contrasts with the much thicker postsynaptic density of the asymmetric synapse (arrowhead) formed by an unlabeled terminal in the upper part of the field. Both micrographs are from the amygdala. Scale lines, 0.25 km.

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C.R. HOUSER TABLE 1. Postsynaptic targets of ChAT-immunoreactiue boutons % of labeled boutons on each

Region Cerebral cortex-frontoparietal (rat) Cerebral cortex-striate (cat) Dentate gyrus (rat1 Ventral striatum-substriatal grey (rat) Neostriatum (monkevl ~~

Investigators

No. of synapses in quantitative analysis

Clarke and Dunnett, 1986 de Lima and Singer, 1986 Clarke, 1985 Phelps and Vaughn, 1986 DiFiglia, 1987

170 72 79 113 170

type of postsynaptic element Dendritic shafts 65.0 84.7 64.6 61.0 37.0

Dendritic spines

Cell bodies

20.0 9.7 21.5 13.0 53.0

2.0 5.6 5.1 26.0 9.0

Other elements 13.0' -

8.g2 -

LO?

~

'Ambiguous dendritic shafts or spines. 'Ambiguous small dendrites or spines (6.3%)and non- identified elements (2.6%I "Axon initial segments.

(de Lima and Singer, 1986) and highly concentrated in the monkey neostriatum (DiFiglia, 1987). Likewise, the sizes and shapes of the vesicles have been variously described as large, round (Clarke, 1985; Wainer et al., 1984a) and pleomorphic of varying sizes (Carlsen and Heimer, 1986; Houser e t al., 1985; Phelps et al., 1985). Since the vesicles within peroxidase-labeled boutons are often very difficult to visualize, it is not surprising that there are variations in their descriptions. Further clarifications of the shapes and sizes of the vesicles may be obtained by immunolabeling with small, electrondense markers such as ferritin or colloidal-gold that should permit clearer visualization of the contents of the boutons. However, different fixative and immunocytochemical solutions could also influence the vesicle shapes (Peters et al., 1976) and may need to be considered in such studies. Despite their small size, the synaptic terminals frequently contain one or more mitochondrial profiles (Fig. 2; de Lima and Singer, 1986; DiFiglia, 1987; Wainer et al., 1984a). In the monkey neostriatum, significantly more mitochondrial profiles have been observed in ChAT-containing boutons forming symmetric synapses than in nearby unlabeled terminals that form asymmetric synapses with dendritic spines (DiFiglia, 1987). A greater number of mitochondrial profiles has also been observed in glutamic acid decarboxylase (GAD)-containing terminals, which generally form symmetric synapses, than in unlabeled terminals forming asymmetric synapses within the cerebral cortex (Houser et al., 1984a; Ribak et al., 1982). Thus, the presence of several mitochondrial profiles, occupying a relatively large area of the bouton, appears to be a common feature of several types of terminals that form symmetric synapses. Cholinergic synapses have been observed on many parts oE the postsynaptic neurons, but the most common postsynaptic elements in many brain regions are dendritic shafts, followed by dendritic spines and neuronal somata. The data from several quantitative studies of the postsynaptic elements of cholinergic synapses are summarized in Table 1.However, some interesting variations in this general scheme have been found in specific brain regions, and these will be considered in the following sections. The review will focus on the types of neurons that receive cholinergic input and the location of the synapses on these neurons.

Regional localization of cholinergic synapses Cerebral cortex. Cholinergic innervation appears to be a basic feature of cortical organization. Cholinergic fibers and boutons have been found within all cytoarchitectural areas of the cerebral cortex, and, in the regions that have been studied a t the ultrastructural level, ChAT-immunoreactive synapses have been observed in all cortical layers (Clarke and Dunnett, 1986; de Lima and Singer, 1986; Houser et al., 1985; Parnavelas et al., 1986). The morphological characteristics of these synapses appear to be quite similar among different cortical areas as well as among species. As in many other brain regions, the majority of ChAT-labeled synapses are of the symmetric type and are located predominantly on dendritic processes (Fig. 3; Table 1).While labeled terminals form synaptic contacts with dendrites of virtually all sizes, small to mediumsized dendritic shafts are the most common postsynaptic elements (de Lima and Singer, 1986; Houser et al., 1985). A very small percentage of ChAT-labeled terminals form asymmetric synaptic contacts, and such synapses are also located on dendritic shafts (Houser et al., 1985; Parnavelas et al., 1986). Identification of the types of neurons that receive a cholinergic innervation is critical for understanding the functional role of this system within the cortex, and several studies have demonstrated that both pyramidal and nonpyramidal neurons receive a cholinergic innervation (Clarke and Dunnett, 1986; de Lima and Singer, 1986; Houser et al., 1985).ChAT-immunoreactive synapses have been observed on several regions of pyramidal neurons, including apical (Fig. 3a) and basilar dendrites (Clarke and Dunnett, 1986; de Lima and Singer, 1986; Houser et al., 1985).While it is presumed that many of the small dendritic shafts and spines that receive cholinergic innervation also originate from pyramidal neurons, this has not been demonstrated directly by double labeling methods. The cell bodies of pyramidal neurons in the cat striate cortex also receive synaptic contacts from ChAT-containing boutons, but such synapses appear to be relatively rare (de Lima and Singer, 1986). Cholinergic synapses on the cell bodies of unlabeled nonpyramidal neurons may be somewhat more common and have been observed in all regions of the cerebral cortex examined (Clarke and Dunnett, 1986; de Lima and Singer, 1986; Houser et al.,

CHOLINERGIC SYNAPSES IN THE CNS

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Fig. 3. ChAT-labeled boutons in the cerebral cortex. A: A labeled bouton in layer I1 of the motor cortex forms a symmetric synapse (arrow) with a distal apical dendrite (D) of a pyramidal neuron. An unlabeled terminal forms an asymmetric synapse (arrowhead) with

the same process. B-D: ChAT-labeled terminals of various sizes form symmetric synapses (arrows) with medium-sized and small dendrites (D) as well as a probable dendritic spine (S).Scale lines, 0.25 pm. (Panel A from Houser et al., 1985.)

1985; Parnavelas et al., 1986). These nonpyramidal neurons exhibit many of the morphological features of y-aminobutyric acid (GABA) neurons (Houser et al., 1985), but cholinergic synapses with GABA neurons have not yet been demonstrated directly.

Physiological studies have indicated that both pyramidal and nonpyramidal neurons respond to acetylcholine (Lamour et al., 1982; McCormick and Prince, 1985), and such findings are consistent with the morphological observations. However, McCormick and

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Prince (1985, 198613)have suggested that the effects of ACh are different on the two types of neurons. Several studies have demonstrated that ACh produces a slow depolarization of pyramidal neurons that results in facilitatory effects on these neurons (KrnjeviC, 1981; McCormick and Prince, 1985; Sillito and Kemp, 1983). In contrast, McCormick and Prince (1985) have shown that ACh produces a rapid excitation of nonpyramidal neurons that are presumed to be GABA neurons, and these neurons, in turn, inhibit pyramidal cells. However, despite the apparently different effects of ACh on the two types of neurons in the cerebral cortex, no morphological differences in the cholinergic synapses on pyramidal and nonpyramidal neurons have yet been identified. Furthermore, the synapses on both types of neurons are of the symmetric type, even though ACh may have depolarizing, excitatory effects on both types of neurons (McCormick and Prince, 1985). Such observations suggest that the routine association of symmetric synapses with inhibitory effects requires reassessment (see also the chapter by Clements et al., this volume). Only the groundwork for understanding the morphological organization of the cholinergic innervation of the cerebral cortex has been established, and many important details remain to be determined. For example, intrinsic cholinergic neurons have been observed in the cerebral cortex of some species, including the rat (Clarke and Dunnett, 1986; Eckenstein and Thoenen, 1983; Eckenstein and Baughnman, 1984; Houser et al., 1983,1985; Levey et al., 1984; Parnavelas et al., 1986) and cat (Stichel e t al., 1987). These neurons provide a small, intrinsic source of cholinergic innervation to the cortex in addition to the well recognized extrinsic source in the basal forebrain (Johnston et al., 1979, 1981; Mesulam et al., 1983a,b).However, the axon terminals of these two types of cholinergic neurons have not been distinguished morphologically. In addition, laminar variations in the density of cholinergic innervation in some cortical regions have been described (Brady and Vaughn, 1988; de Lima and Singer, 1986; Houser et al., 1985; Lysakowski et al., 1986; Parnavelas et al., 1986; Stichel and Singer, 19871, but the ultrastructural correlates of these laminar patterns have not been determined. Detailed knowledge of the normal distribution of cholinergic synapses in the cerebral cortex is critical not only for understanding the functional role of ACh in this region but also for analyzing the distribution of cholinergic elements in experimentally altered tissue. In studies designed to determine if transplanted cholinergic neurons establish synaptic contacts in the rat cerebral cortex, Clarke and Dunnett (1986) observed numerous ChAT-immunoreactive synapses in experimental animals that had previously been deprived of much of the normal cholinergic innervation of the cortex. In addition, a n abnormal distribution of these synapses was observed in the deep cortical layers. Whereas only a small percentage (2%) of ChAT-containing boutons established axosomatic synapses in the normal animals, a much larger percentage (19%) formed axosomatic synapses in the experimental animals. Such findings provide ultrastructural evidence for the

growth potential of cholinergic neurons and suggest that the development and plasticity of cholinergic synapses will be a n exciting field for future studies. Hippocampal formation. ChAT-containing fibers and boutons have been observed in all layers of the hippocampal formation and display a more distinct pattern than in many regions of the cerebral cortex. Such ChAT-containing elements are particularly prominent in the supragranular region of the stratum moleculare (Fig. l b ) and the infrapyramidal region of the stratum oriens (Clarke, 1985; Frotscher and Leranth, 1985; Houser et al., 1985; Matthews et al., 1987). Since the proximal ascending dendrites of granule cells and basilar dendrites of pyramidal neurons are located in these regions respectively, i t is not surprising that dendritic shafts are the most common postsynaptic elements of the cholinergic synapses in the hippocampal formation (Table 1; Clarke, 1985; Frotscher and Leranth, 1985, 1986). Furthermore, by serial section analysis and the combination of immunocytochemistry with Golgi-impregnation of the postsynaptic neurons, Frotscher and Leranth (1985, 1986) determined that several cell types in the hippocampal formation receive a cholinergic innervation, and these include pyramidal, nonpyramidal and granule cells. ChAT-positive synaptic contacts of both the symmetric and asymmetric types have been described in the hippocampal formation, and it appears that different types of synaptic contacts are preferentially established with specific postsynaptic targets. For example, symmetric immunoreactive synapses are located on the cell bodies, dendritic shafts and the stalks of large complex spines of identified granule cells, whereas asymmetric ChAT-positive synapses are established with the heads of small spines on these same neurons in the dentate gyrus (Frotscher and Leranth, 1985). Similarly, within the CA1 and CA3 fields of the hippocampus, ChAT-labeled synapses of the asymmetric type are confined primarily to small dendritic spines while labeled synapses of the symmetric type are found on the cell bodies and dendritic shafts (Frotscher and Leranth, 1985). ChAT-immunoreactive synapses have also been observed on the cell bodies and proximal dendrites of nonpyramidal neurons in the hilus. In a n interesting series of experiments, Leranth and Frotscher (1987) demonstrated that some of these cholinoceptive hilar neurons are GAD- or somatostatin-immunoreactive by labeling the cholinergic terminals with peroxidase and the GAD- or somatostatin-containing postsynaptic neurons with ferritin (Fig. 4).Then, in a triple-labeling study, the investigators demonstrated that some of the GAD- or somatostatin-containing neurons that are postsynaptic to cholinergic boutons also project t o the contralateral hippocampus. In several recent studies, the ultrastructural features of ChAT-containing elements have been analyzed following the transplantation of fetal septa1 neurons to the hippocampal formation. These studies demonstrated that transplanted ChAT-containing neurons form numerous synaptic contacts with neurons in the dentate gyrus and hippocampus. However, the age of the host animals and the specific experimental conditions appeared to influence the postsynaptic targets of

CHOLINERGIC SYNAPSES IN THE CNS

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Fig. 4. Electron micrographs of the hilar region of the hippocampal formation immunolabeled for both ChAT and somatostatin. ChAT-immunoreactive terminals, labeled with peroxidase, form symmetric synaptic contacts (white arrows) with somatostatin-containing

dendrites (D), labeled with avidinated ferritin (black arrows). A large mossy fiber terminal (MI is unlabeled. Scale lines, 0.25 pm. (Adapted from Leranth and Frotscher, 1987.)

the ChAT-labeled terminals. In young rats with previous cholinergic denervation of the host hippocampus, alterations in the normal pattern of cholinergic innervation were observed. Anderson et al. (1986) found that the majority of ChAT-labeled terminals formed synapses with dendritic shafts and spines a s in the control animals. However, a number of immunoreactive terminals were also located adjacent to unlabeled axon terminals and possibly formed axo-axonal connections. Such relationships were not observed in the normal animals. In a similar study of young, partially deafferented animals, Clarke et al. (1986a) found that the quantitative relationships of ChAT-immunoreactive soma1 and dendritic synapses were abnormal and that the extent of these changes was even greater than that described previously in the cerebral cortex. ChAT-immunoreactive synapses on cell bodies, which compose a very small percentage of the labeled synapses in the normal animal, constituted over 60% of labeled synapses in the grafted animals. Thus, in the two experimental studies of the hippocampus, the predominant postsynaptic elements of ChAT-immunoreactive synapses were different. Experimental factors that may have contributed to the findings include differences in the time interval between the deafferentation and transplantation.

Transplanted cholinergic neurons were also capable of forming numerous ChAT-positive synapses in aged, behaviorally impaired rats that had not undergone cholinergic denervation of the hippocampus a t the time of grafting (Clarke et al., 198613).Furthermore, the new synapses showed a normal morphology and distribution within the dentate gyrus of the host. Thus, the findings of each of the studies suggest that cholinergic neurons in the septa1 grafts may be able to modulate the function of the host hippocampus by specific synaptic contacts rather than only by non-synaptic release of ACh. However, the pattern of cholinergic innervation appears to be influenced by the synaptic organization within the hippocampus at the time of the implants. Amygdala. Several studies have shown that the basolateral nucleus of the amygdala possesses one of the highest concentrations of ChAT-immunoreactive fibers and boutons in the CNS (Armstrong, 1986; Carlsen et al., 1985; Carlsen and Heimer, 1986; Hellendall et al., 1986). Since the adjacent nuclei exhibit low to moderate levels of ChAT-containing elements, the boundaries of the basolateral nucleus are clearly delineated in light microscopic preparations (Fig. 5a). Despite the distinctive, heavy staining in this region, the ultrastructural characteristics of the ChAT-containing fi-

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bers and boutons are remarkably similar to those in the brain regions just reviewed (Armstrong, 1986; Carlsen and Heimer, 1986). Indeed, there are striking similarities between the organization of the cholinergic system in the basolateral nucleus and the cerebral cortex, including a dual innervation from extrinsic and intrinsic sources (see Carlsen and Heimer, 1986 for further discussion). Due t o the high concentration of ChAT-immunoreactive elements in the basolateral nucleus, several labeled profiles are frequently present within the same thin section. These ChAT-positive processes include small-diameter axons (Fig. 5c) and synaptic terminals (Fig. 5b-d). Occasionally even branching axons can be observed (Fig. 5d), and frequent branching of labeled fibers might be expected in a brain region with such a dense innervation. The vast majority of ChAT-containing boutons in the basolatera1 nucleus form symmetric synaptic contacts with the shafts of small and medium-sized dendrites (Fig. 5 b 4 , present report; see also Carlsen and Heimer, 1986; Wainer et al., 1984a). In addition, symmetric synapses are formed with dendritic spines and, infrequently, with the somata of large, unlabeled neurons presumed to be projection neurons (Carlsen and Heimer, 1986). Finally, ChAT-containing boutons establish synaptic contacts with the somata of unlabeled nonpyramidal neurons and, occasionally, with ChAT-labeled neurons (Carlsen and Heimer, 1986). Thus, as in the cerebral cortex and hippocampus, cholinergic synapses are established with several types of neurons. Furthermore, the vast majority of ChAT-immunoreactive synapses are on small dendritic profiles, presumably of distal dendrites. Due to the high density of cholinergic innervation, the basolateral nucleus could be a particularly important region for detailed studies of the synaptic effects of ACh. As yet, there appear to be few clues as to why this brain region receives such a heavy cholinergic innervation when the majority of this innervation arises from neurons in the basal forebrain, in parallel with the more moderate cholinergic projections to the cerebral cortex and hippocampus (Carlsen et al., 1985; Emson et al., 1979; Nagai et al., 1982; Woolf and Butcher, 1982). In addition, this subdivision of the amygdala warrants attention because of the potential alterations of the cholinergic system within this nucleus in Alzheimer’s disease and some forms of epilepsy (Olney et al., 1983; Rossor et al., 1982). Striatum. The neostriatum contains a high concentration of ChAT-containing fibers and boutons, and, in contrast to the regions just discussed, the majority of these elements originate from intrinsic cholinergic neurons rather than from extrinsic sources (Takagi et al., 1984; Woolf and Butcher, 1981). Several groups of investigators have classified the cholinergic neurons of the neostriatum as large aspiny or sparsely spinous neurons since they have large cell bodies (30 pm, mean diameter) and exhibit long dendrites that possess few spines (Fig. 6a; Bolam et al., 1984; DiFiglia, 1987; Phelps et al., 1985). These cholinergic neurons constitute approximately 2% of the neuronal population within the neostriatum (Phelps et al., 1985). Thus, in order to provide the extensive cholinergic innervation

of the region, the neurons presumably form rich axonal arborizations within the striatum. Consistent with the light microscopic observations of dense staining in neuropil regions, numerous immunoreactive fibers and boutons are observed with the electron microscope (DiFiglia, 1987; Phelps et al., 1985). Interconnected boutons may form a series of contacts with different postsynaptic profiles (Fig. 6b), and several boutons may contact the same postsynaptic profile (Fig. 6c). The ChAT-immunoreactive boutons are relatively small and establish synapses of the symmetric type in both the monkey and rat (DiFiglia, 1987; Phelps et al., 1985; Wainer et al., 1984a). This morphology is similar to that of local axon collaterals of large Golgi-impregnated neurons of the striatum (Takagi et al., 19841, thus supporting the suggestion that the ChATcontaining terminals originate from this group of neurons. Several studies have shown that small to mediumsized dendritic shafts are the most common postsynaptic elements in the rat neostriatum (Phelps et al., 1985; Wainer et al., 1984a). In contrast, DiFiglia (1987) has found that dendritic spines are the primary postsynaptic target in the monkey neostriatum, and that over half of the ChAT-immunoreactive boutons form synapses with these elements (see Table 1).In addition, ChAT-containing terminals form some synaptic contacts with virtually all regions of unlabeled neurons. Ultrastructural studies in both the rat and monkey have indicated that striatal neurons of several different types receive a cholinergic innervation. One principal target appears t o be the medium-sized spiny neuron, and ChAT-immunoreactive terminals form synaptic contacts with the somata, proximal dendrites, and axon initial segments of these neurons (DiFiglia, 1987; Phelps et al., 1985; Wainer et al., 1984). The proximal locations of the cholinergic synapses suggest that they could exert substantial control over these postsynaptic neurons. Such neurons are major projection neurons of the neostriatum and have many of the characteristics of GABA neurons within this region (Oertel and Mugnaini, 1984; Somogyi and Smith, 1979). Thus, a morphological basis for cholinergicGABAergic interactions is suggested in the neostriatum as well as in the cerebral cortex and hippocampal formation. In addition, cholinergic terminals form synapses with the somata of large, unlabeled neurons in the rat (Phelps et al., 1985) and with medium-sized, aspiny neurons in the monkey (DiFiglia, 1987). The substriatal grey of the ventral striatum also contains a high concentration of ChAT-immunoreactive elements, and the synaptic organization appears to be very similar to that observed in the neostriatum (Phelps and Vaughn, 1986). Dendritic shafts are again the principal postsynaptic site, but a relatively large percentage of the ChAT-positive synapses, approximately one-fourth, are with the cell bodies of unlabeled neurons (Table 1;Fig. 7). In both the neostriatum and ventral striatum, some ChAT-immunoreactive terminals are closely associated with ChAT-containing dendrites and occasionally appear to form synaptic contacts, but the dense reaction product within both the pre- and postsynaptic elements has made unequivocal

CHOLINERGIC SYNAPSES IN THE CNS

Fig. 5. ChAT localization in the basolateral nucleus of the amygdala. A This light micrograph from a coronal section of the forebrain illustrates the very high density of ChAT-immunoreactive elements in the basolateral (BL) nucleus. The density of staining is significantly higher than that of adjacent amygdaloid nuclei as well as the neostriatum (S). Labeled cell bodies are evident in the basal nucleus (B) and neostriatum. B: A small ChAT-labeled bouton forms a synaptic contact (arrow) with a small dendritic shaft (D). C: A la-

11

beled terminal forms a symmetric synaptic contact (arrow) with a medium-sized dendrite (Dj. A small immunolabeled element in the upper part of the field (arrowhead)does not form a synaptic contact and is presumably an intervaricose segment of a transversely-sectioned axon. D: A labeled synaptic bouton forms a symmetric synapse with a small dendrite (D) and is located near the junction of two branches (arrowheads) of a labeled axon. Scale lines, A, 300 pm; B-D, 0.25 pm.

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C.R. HOUSER

Fig. 6 . ChAT-immunoreactive elements in the neostriatum. A: In this light micrograph, three labeled neurons, with cell bodies of various shapes, display dendrites that branch several times within the field (arrowheads).The neurons are surrounded by a densely-stained neurophil containing numerous fibers and terminals B: Intercon-

nected ChAT-positive boutons establish synaptic contacts (arrows) with different profiles of unlabeled dendritic shafts. C: Two ChATpositive terminals form symmetric synaptic contacts (arrows) with the same unlabeled dendritic shaft. Scale lines, A, 50 pm; B, 0.25 pm; C, 0.5 pm. (From Phelps et al., 1985.)

identification difficult (Phelps et al., 1985; Phelps and Vaughn, 1986). Thalamus. Light microscopic studies have demonstrated that the concentration of ChAT-immunoreactive fibers and boutons varies among the different thalamic nuclei (Houser et al., 1988; Levey et al., 1987; Sofroniew et al., 1984). As one example, the reticular nucleus of the thalamus receives a substantial cholinergic innervation, and the density of ChAT-immunoreactive fibers and boutons within the region differen-

tiates it from adjacent, more lightly stained, thalamic nuclei in light microscopic preparations (Fig. 8a). ChAT localization has been studied with the electron microscope in only a few thalamic nuclei, and these include the perigeniculate nucleus, the homologue of the reticular nucleus in the visual system of the cat (de Lima et al., 1985).The investigators found that ChATimmunoreactive boutons form predominantly symmetric synapses in the perigeniculate nucleus as in many other brain regions. They also demonstrated that den-

CHOLINERGIC SYNAPSES IN THE CNS

13

Fig. 7. ChAT-labeledboutons in the substriatal grey of the ventral striatum. A Several ChAT-positiveboutons (arrows) are seen around the soma (SJ of an unlabeled neuron. One of the terminals (larger arrow) forms a symmetric axosomatic synapse (arrow in B) in a serial

section. Numerous other ChAT-labeled boutons are present within the field. Scale lines, A, 2 pm; B, 0.25 pm. (From Phelps and Vaughn, 1986.1

dritic shafts are the most common postsynaptic target and that a single axon often establishes several en passant synapses with the same neuron (de Lima et al., 1985). Preliminary studies have indicated that the characteristics and distribution of ChAT-immunoreactive elements in the reticular nucleus of the rat are very similar to those described in the perigeniculate nucleus (Houser, unpublished observations). Again,

the labeled boutons primarily form symmetric synapses with dendritic elements (Fig. 8b,c), even though the source of the innervation differs from that of previously described regions. Much of the cholinergic innervation of the thalamus is from brainstem sources (Mesulam et al., 1983131, and double-labeling studies have demonstrated that ChAT-immunoreactive neurons in both the pedunculopontine and dorsolateral

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C.R. HOUSER

Fig. 8. ChAT-immunoreactive elements in the reticular nucleus of the thalamus. A In this light micrograph, the immunolabeling in the reticular nucleus (R) delineates this nucleus from the adjacent, less heavily labeled regions of the thalamus (T), and the virtually unlabeled internal capsule (IC). Labeled neurons of the basal nucleus (B) and densely stained neuropil of the striatum (S) are evident in this section. B: A small ChAT-labeled bouton forms a symmetric synapse

(arrow) with a medium-sized dendritic shaft (D). C: A large immunoreactive bouton is located adjacent to the soma (S) of an unlabeled neuron, but in this section it forms short synaptic contacts (arrows) with an adjacent decdritic shaft (D). Note several mitochondria1 profiles near the synaptic contacts. Scale lines, A, 300 pm; B, 0.25 pm; C, 0.5 pm.

CHOLINERGIC SYNAPSES IN THE CNS

tegmental nuclei project to the reticular nucleus (Hallanger et al., 1987; Satoh and Fibiger, 1986; Sofroniew et al., 1984; Woolf and Butcher, 1986). However, neurons of the basal forebrain are a n additional source of cholinergic input to this thalamic region (Hallanger et al., 1987; Steriade et al., 1987), and future studies may identify subtle morphological differences in the cholinergic afferents from these different brain regions. In fact, variations in the size of ChAT-immunoreactive boutons are particularly striking in the reticular nucleus of the r a t (Fig. 8b,c), and the diameter of the boutons ranged from 0.3 pm to 1.6 km (Houser, unpublished findings). Although some of the size differences could be related to different sources of afferent fibers, substantial variations in the size of the varicosities can also be observed along the same fiber (Fig. 1). Physiological studies have shown that ACh has inhibitory actions in the reticular nucleus (Kelly et al., 1979; McCormick and Prince, 1986a); and, thus, this is one region where the labeled symmetric synapses could be associated with inhibitory effects. Since virtually all neurons within the reticular and perigeniculate nuclei are GABA neurons (Houser et al., 1980; Montero and Singer, 1984; Oertel et al., 1983; Yen e t al., 1985), there is strong evidence for a cholinergic innervation of GABA neurons in this region. Thus, it appears that the cholinergic portion of the ascending reticular system of the brainstem may influence thalamocortical transmission, in part, by actions on GABA neurons of the reticular nucleus, which, in turn, can modify neuronal activity in many other thalamic nuclei (Kelly et al., 1979; McCormick and Prince, 1986a). Different patterns of cholinergic innervation can be found in other types of thalamic nuclei, such a s the thalamic relay nuclei. In studies of the dorsal lateral geniculate nucleus, de Lima et al. (1985) demonstrated that ChAT-immunoreactive boutons form symmetric synaptic contacts within synaptic glomeruli as well as in the extraglomerular neuropil. Within the glomeruli, ChAT-containing terminals typically occupy a peripheral position and make synaptic contacts with classical dendrites a s well a s with profiles containing pleomorphic synaptic vesicles. These latter profiles, presumed to be presynaptic dendrites of GABA neurons, are also postsynaptic to retinal terminals and presynaptic to other dendrites. In the extraglomerular neuropil, ChAT-containing terminals form synaptic contacts predominantly with dendritic profiles, and such postsynaptic dendrites in both intra- and extraglomerular locations are known to arise from relay neurons. Thus, de Lima et al. (1985) have suggested that there are morphological substrates for cholinergic modulation of both relay cells and inhibitory interneurons within the dorsal lateral geniculate nucleus. Septa1 region. Some interesting variations in the characteristics and locations of cholinergic synapses have been observed in the septal complex (Bialowas and Frotscher, 1987). This complex can be subdivided on the basis of the morphological characteristics of the cholinergic and noncholinergic neurons (Amaral and Kurz, 1985), and it now appears that the pattern of cholinergic innervation also differs among the subre-

15

gions (Bialowas and Frotscher, 1987). The medial septal and diagonal band regions contain many cholinergic cell bodies, yet virtually no ChAT-immunoreactive boutons contact these labeled somata (Bialowas and Frotscher, 1987). Indeed, these neurons, a s well a s cholinergic neurons in other forebrain groups, appear to receive very few axosomatic synapses of any type (Armstrong, 1986; Dinopoulos e t al., 1986; Ingham et al., 1985). Although contacts are occasionally observed between ChAT-immunoreactive terminals and labeled dendrites in the medial septal nucleus, the accumulation of reaction product masked the pre- and postsynaptic specializations, making it difficult to definitively identify the contacts as synaptic junctions (Bialowas and Frotscher, 1987). Within the dorsal septal nucleus, a very different pattern of cholinergic innervation was observed (Bialowas and Frotscher, 1987). This region contains virtually no labeled cell bodies but numerous ChAT-immunoreactive boutons. These elements often form basket-like arrangements around unlabeled cell bodies and primary dendrites, and one immunoreactive fiber may give rise to several of the boutons around the same neuron. Furthermore, many of the synaptic contacts display thickened postsynaptic densities and have been classified as asymmetric synapses (Bialowas and Forscher, 1987). Thus, the cholinergic synapses in the dorsal septal nucleus differ in several respects from those described in other brain regions. Brainstem and spinal cord. Other regions of the CNS also contain cholinergic synapses with varying morphological characteristics, and two such regions are located in the brainstem. The interpeduncular nucleus contains a dense concentration of immunoreactive fibers and boutons (Cuello and Sofroniew, 1984; Eckenrode et al., 1987; Houser et al., 1983; Lenn et al., 1985), and by comparing ChAT-labeled preparations with conventionally prepared specimens, Lenn et al. (1985) were able to distinguish four types of cholinergic synapses on the basic of their morphological characteristics and location in different subnuclei. They found that three types of ChAT-labeled terminals form asymmetric contacts and contain round vesicles, whereas one type forms symmetric synapses and contains pleomorphic vesicles. However, small dendrites are the common postsynaptic elements in all of these synaptic types (Lenn e t al., 1985). The hypoglossal nucleus is a second brainstem region where cholinergic presynaptic elements have been studied with the electron microscope (Connaughton et al., 1986), and two types of cholinergic terminals have been described in this region. One type forms symmetric synapses predominantly with small immunoreactive dendrites and was hypothesized to originate from recurrent axon collaterals of the hypoglossal neurons (Connaughton et al., 1986). A second type of immunoreactive terminal is located adjacent to labeled neuronal somata and proximal dendrites. These terminals do not form classical synaptic junctions but are located near subsynaptic cisterns that follow the postsynaptic membrane and then merge with the underlying rough endoplasmic reticulum (Connaughton et al., 1986). The source of these terminals is unknown, but the investi-

16

C.R. HOUSER

gators suggested that they might originate from cholinergic neurons in the reticular formation. Within the spinal cord, several different types of ChAT-labeled terminals are also observed (Barber et al., 1984; Houser et al., 1983). In the ventral horn, the boutons are large (frequently 2 pm or greater in major diameter) and are often distributed around the somata and proximal dendrites of labeled neurons. Electron microscopic studies confirm that the ChAT-positive terminals form synaptic contacts with these elements as well as with unlabeled cell bodies and dendrites (Houser et al., 1983). The sites of synaptic contact are generally small, and some labeled terminals exhibit multiple synaptic sites within a single section. Synaptic contacts of the asymmetric type were observed, but many others displayed postsynaptic densities of intermediate thickness and could not be classified easily as symmetric o r asymmetric. In the dorsal horn of the spinal cord, the ChAT-positive terminals are much smaller (less than 1 pm in major diameter) and frequently form symmetric synaptic contacts with unlabeled dendritic processes (Barber et al., 1984). Thus, within the brainstem and spinal cord, a pattern of large ChAT-labeled terminals with axosomatic relationships may predominate in motor nuclei, whereas a more diffuse innervation pattern, similar to those of the forebrain, may be common in regions with other, non-motor functions.

Functional relationships Present knowledge of the morphology of cholinergic synapses can now be considered in relation to current views of the physiological actions of ACh. Several lines of evidence suggest that ACh may have neuromodulatory actions in many regions of the nervous system (Kelly and Rogawski, 1985; Krnjevid, 1981; Siggins and Groul, 1986; Brown et al., 1986), and some of the morphological observations could be viewed a s consistent with such a role. First, the widespread distribution of cholinergic fibers and terminals within the nervous system and their apparent origin from a limited number of cholinergic cell groups would be consonant with neuromodulatory actions of these neurons. In addition, the cholinergic fibers appear to innervate many different types of neurons a s might be expected of modulatory elements. While the cholinergic synapses are located on most parts of the postsynaptic neurons, they are found predominantly on dendritic elements, where terminals with known excitatory influences are also distributed. For example, cholinergic synapses on dendritic spines are generally accompanied by unlabeled synapses of the asymmetric type (DiFiglia, 1987; Houser et al., 1985; Phelps and Vaughn, 19861, and ACh could presumably modulate the effects of other neurotransmitters a t such sites. However, beyond the general suggestion that ACh may act as a neuromodulator, physiological studies have indicated that the actions of ACh within the CNS are quite diverse and that multiple types of cholinergic receptors may exist (Cole and Nicoll, 1983; Kelly et al., 1979; Rovira et al., 1983; Watson et al., 1986). For example, both rapid and slow forms of excitation as well as rapid inhibitory actions of ACh have been demon-

strated in forebrain regions (McCormick and Prince, 1986a,b). Presently, the morphological observations do not appear to demonstrate the same degree of diversity. Rather, in most regions of the CNS, similarities in the appearance of cholinergic synapses exist, and these include the generally small size of the terminals, their periodic distribution along the fibers, the formation of symmetric synaptic contacts, and the selection of small dendritic profiles as the major postsynaptic element. Although such features are common to cholinergic terminals in the cerebral cortex, reticular nucleus of the thalamus, and the striatum, ACh has been reported to have quite different physiological effects in these brain regions (McCormick and Prince, 1986c; Dodt and Misgeld, 1986). Despite the similarities of many cholinergic synapses, some heterogeneity has been observed. While symmetric synapses predominate in most regions, labeled asymmetric synapses are commonly observed in both the lateral septa1 and interpeduncular nuclei. Additionally, moderate numbers of ChAT-positive asymmetric synapses have been described in the hippocampal formation, and occasional synapses of this type have been observed in several regions of the cerebral cortex and the striatum. The functional implications of the different types of synaptic contacts are presently unknown. Physiological studies have also demonstrated that intrinsic neurons, presumed to be GABA neurons, respond to ACh (McCormick and Prince, 19851, and the findings of several of the immunocytochemical studies have suggested that cholinergic synapses are present on GABA neurons. Such synaptic connections have been demonstrated conclusively in the hippocampus and basal forebrain by double-labeling methods (Leranth and Frotscher, 1987; Zaborszky et al., 1986). There is also strong evidence for such synaptic connections in the reticular and perigeniculate nuclei of the thalamus (de Lima et al., 1985; present observations) where a substantial cholinergic innervation is found within a nucleus that contains predominantly, if not exclusively, GABA neurons. In addition, there are suggestions of cholinergic connections with GABA neurons in the cerebral cortex (Houser et al., 1985) and striatum (Phelps et al., 1985; DiFiglia, 1987) where neurons with many of the morphological characteristics of GABA neurons receive synaptic contacts from ChAT-immunoreactive terminals. The present morphological observations in conjunction with physiological findings suggest that studies of cholinergic synapses can be expanded in several interesting directions. Efforts to relate the physiology and morphology of cholinergic synapses appear to be particularly important for our understanding of this system. Some of the complexity of the synaptic actions of ACh may be related to different subtypes of postsynaptic cholinergic receptors (Kelly and Rogawski, 1985; McCormick and Prince, 1986c; Palacios et al., 1986; Watson et al., 19861, and yet the morphology of many cholinergic synapses in different brain regions appears to be quite similar. However, a s specific antibodies to receptors become available, immunocytochemical methods should be useful in distinguishing between

CHOLINERGIC SYNAPSES IN THE CNS

different types of postsynaptic receptors just a s they have been in identifying chemically-unique types of presynaptic terminals. Subdivisions of cholinergic synapses could then be identified in morphological preparations and possibly associated with the symmetric and asymmetric contacts that are observed in several brain regions or with subpopulations of the common symmetric synapses. Further chemical and morphological identification of the postsynaptic neurons will also be important for a thorough understanding of cholinergic function. Although small dendritic profiles receive the majority of cholinergic synapses in many brain regions, the identity of the postsynaptic neurons giving rise to these dendrites is generally unknown. Furthermore, while ChAT-immunoreactive synapses on several different morphological types of neurons have been observed, there is, as yet, no detailed comparison of the quantity and distribution of cholinergic synapses on these neurons. It is possible that different types of neurons may receive characteristic patterns of cholinergic innervation. Finally, it will be important to determine the relationships of other chemically identified synapses to those of cholinergic synapses. The continued development of multiple labeling methods and their use in detailed ultrastructural studies will no doubt provide important new insights into the organization of cholinergic synapses within the CNS. Such descriptions could aid in the interpretation of physiological findings suggesting that ACh may modulate the neuronal response to other neurotransmitters (KrnjeviC, 1981; Brown et al., 1986) and that other neuroactive substances may influence the efyects of ACh (Mancillas et aI., 1986). It is such apparent complexities in the physiological actions of ACh within the CNS a s well a s knowledge of the importance of the cholinergic system for normal brain function that make i t a n intriguing system for further study.

ACKNOWLEDGMENTS I thank Dr. Patricia Phelps for many helpful discussions and review of the manuscript; Dr. James Vaughn, Dr. Paul Salvaterra and colleagues for their contributions to much of the basic work discussed in this paper; Drs. J. Carlsen, M. Frotscher, C. Leranth, and W. Singer for providing recent manuscripts for inclusion in this review; and Janet Miyashiro and Mariko Lee for their excellent technical assistance. This work was supported by VA Medical Research Funds, a research grant from the Epilepsy Foundation of America, and NIH grant NS21908.

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