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Home , Fascia dentata, Nucleus accumbens, Nucleus basalis

Neuroscience Vol. 88, No. 1, pp. 241–255, 1999 Copyright  1998 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved Pergamon PII: S0306-4522(98)00202-4 0306–4522/99 $19.00+0.00

CHOLINERGIC OF THE BASALIS OF MEYNERT RECEIVE , CATECHOLAMINERGIC AND GABAERGIC SYNAPSES: AN ELECTRON MICROSCOPIC INVESTIGATION IN THE MONKEY

J. F. SMILEY* and M.-M. MESULAM The Cognitive Neurology and Alzheimer’s Disease Center, Northwestern University Medical School, Chicago, IL 60611, U.S.A.

Abstract––An electron microscopic analysis of the in the macaque monkey was carried out following the immunohistochemical labeling of choline acetyltransferase, either by itself or in conjunction with glutamate decarboxylase or tyrosine hydroxylase. Cholinergic varicosities were frequently encountered, and formed large, usually asymmetric, synapses on both choline acetyltransferase- immunopositive and -immunonegative of nucleus basalis neurons. Catecholaminergic (tyrosine hydroxylase-immunoreactive) axon varicosities formed synapses which in most cases were classified as asymmetric, and glutamate decarboxylase-immunoreactive (GABAergic) formed clearly symmetric synapses, each on to choline acetyltransferase-immunopositive or -immunonegative dendrites. These findings indicate that cholinergic cells in the nucleus basalis of the monkey, also known as Ch4 neurons, receive numerous synaptic inputs from cholinergic, catecholaminergic and GABAergic axons.  1998 IBRO. Published by Elsevier Science Ltd.

Key words: , , GABA, , primate.

The nucleus basalis of Meynert (nbM) refers to this disease is also thought to influence the processing a collection of magnocellular and isodendritic of the amyloid-â precursor protein and the depo- neurons located in the . The nbM sition of amyloid plaques.89 Current pharmacological is a phylogenetically progressive structure which therapies for Alzheimer’s disease are based on the use reaches its highest development in primates and of cholinesterase inhibitors to reverse the loss of cetacea.43 It contains cholinergic as well as acetylcholine in the . The synaptic non-cholinergic neurons. The non-cholinergic organization of cortical cholinergic innervation is components of the nbM include GABAergic, therefore of considerable relevance to the neurobiol- NADPH-diaphorase-positive and peptidergic ogy of cognition and the treatment of Alzheimer’s neurons.11,41,44,45,103,119,125,130 The cholinergic con- disease. tingent of the nbM is also known as the Ch4 The neurophysiology of cortical cholinergic path- complex, and provides the vast majority of choliner- ways has been investigated extensively. Neurons in gic innervation for the cerebral cortex and the the nbM of the monkey are particularly sensitive to .80 novel and behaviorally relevant stimuli.96,121 Electri- The cholinergic innervation of the cerebral cortex cal or pharmacological stimulation of the nbM plays a central role in the neural modulation of induces a desynchronization of the cortical electro- attention, arousal and memory.27,77,85 A severe encephalogram, mediated in part by the direct cholin- depletion of this pathway is one of the earliest and ergic excitation of cortical neurons.13,83,94,112 Either most conspicuous neuropathological features of electrical stimulation of the nbM or iontophoresis of Alzheimer’s disease, and provides one of the several acetylcholine into areas of the primary sensory cortex anatomical substrates for the , especially the enhances the neural responses to sensory stimu- memory loss.3,40 The cortical cholinergic depletion in lation.28,75,82,84,86,95,99,104 In addition, behavioral and physiological studies indicate that cholinergic *To whom correspondence should be addressed. neurotransmission in the cerebral cortex facilitates Abbreviations: ABC, avidin–biotin–peroxidase complex; attention and memory,7,85 and enhances the for- Ch4, cholinergic neurons of the nucleus basalis of mation of long-term potentiation.50,51,113 Stimu- Meynert; ChAT, choline acetyltransferase; GAD, gluta- mate decarboxylase; NADPH, reduced nicotinamide ad- lation of the nbM also causes increased blood flow in enine dinucleotide phosphate; nbM, nucleus basalis of the cerebral cortex, a response which is likely to be Meynert; TH, tyrosine hydroxylase . mediated by cholinergic receptors on cerebral blood 241 242 J. F. Smiley and M.-M. Mesulam vessels, and perhaps also by indirect pathways that dopaminergic) axons,38,127,131 and from several types trigger the release of various substances, including of neuropeptidergic axons.9,71,130 nitric oxide.1,5,29,35,42,106,114 The pharmacological synaptology of the nbM in The neural inputs into the nbM play a crucial role the primate is much more poorly understood. We in shaping its physiological properties. In the mon- reported the presence of dopaminergic (TH-positive), key, the nbM projects to all cortical areas, but serotoninergic and noradrenergic axons in the nbM receives feedback projections from a very limited set of the monkey and human brains.107 However, the of limbic and paralimbic cortical areas.78 Additional ultrastructural synaptology of these projections was inputs to the nbM of the monkey come from the not investigated. The organization of cortical cholin- amygdala, –ventral globus pal- ergic innervation shows major inter-species differ- lidus, and from brainstem nuclei, ences: cholinergic neurons are found in the cerebral including the –ventral tegmental cortex of the rat but not of the primate, the Ch4 area, the , the pedunculopontine nucleus, neurons express calbindin-D28k in the primate but the laterodorsal tegmental nucleus and the nucleus not in the rat, and a substantial number of Ch4 .78,98,125 Similar projections have also neurons in the rat but not in the primate express been shown in the rat (for reviews, see Refs 125 NADPH-diaphorase.41,79 In view of these species and 130). In keeping with these sources of input, the differences, we decided to investigate the pharmaco- nbM in the monkey and its analog in the rat have logical synaptology of the nbM in the monkey to see been shown to contain putatively dopaminergic how the details compared to those that have been axons immunoreactive for tyrosine hydroxylase reported in the rat. (TH), -immunoreactive axons and nor- adrenergic axons immunoreactive for dopamine-â- EXPERIMENTAL PROCEDURES hydroxylase.107,111,127,131 In the rat, light microscopic investigations have also shown the nbM to receive Two adult female Macaca nemestrina monkeys were used histaminergic projections from the hypothalamus,53 for these experiments. The monkeys were received from the GABAergic projections from the amygdala92 Regional Primate Research Center at the University of Washington. They were sedated with Ketamine (5–10 mg/ (and probably also from the nucleus accum- kg, i.m.) and deeply anesthetized with sodium pentobarbital 9,46,71,129 bens ), and inputs from the (100 mg/kg, i.v.). They were then transcardially perfused cerebral cortex, amygdala, hypothalamus, over a period of 20 min, first with 350 ml of 0.9% saline with and .16,124,129 Several 2.5% heparin in 0.1 M phosphate buffer (pH 7.4) and then with 3 l of 4% paraformaldehyde in 0.1 M phosphate buffer have also been demonstrated within axons of the 130 (pH 7.4). After perfusion, the region of the cholinergic basal nbM. forebrain of the left hemisphere was removed and coronally In slice preparations of the guinea-pig nbM, sectioned on a Vibratome. Sections were then transferred to identified cholinergic Ch4 cells responded to cholin- plastic vials containing 30% sucrose in 0.1 M phosphate ergic, dopaminergic, noradrenergic, serotoninergic, buffer (pH 7.4). The vials were frozen by immersion 31,61–64 in liquid nitrogen and stored frozen until processing for glutamatergic and histaminergic agonists. immunocytochemistry. Additional studies in dissociated cholinergic cell Immunolabeling of choline acetyltransferase (ChAT) was preparations of the rat nbM demonstrated that done with a rabbit polyclonal antibody, applied at a con- centration of 1:1500, which was a gift from Lou Hersch and these cells also responded to substance P and 39 30,87 which was characterized previously. The sheep polyclonal . anti-glutamate decarboxylase (GAD) antibody, used at a The detailed pharmacological synaptology of concentration of 1:2000, was a gift of Enrico Mugnaini and inputs to the nbM has been studied primarily in was characterized previously.91 The antibody to TH was a the rat with the aid of the electron microscope. In mouse monoclonal antibody from Chemicon (Temecula, one set of experiments, anterograde tracer injec- CA), which was used at a concentration of 1:2000. All immunocytochemistry was done with the avidin–biotin– tions were combined with an electron micro- peroxidase complex (ABC) method,52 employing the scopic analysis of the immunostained nbM. Such Vectastain Elite ABC kit (Vector Laboratories, Burlingame, studies showed that putatively glutamatergic CA). The solution for antibody incubations contained 1% projections from the cerebral cortex synapsed on to bovine serum albumin, 0.1% and 0.3% normal serum, in 0.1 M phosphate buffer (pH 7.4). GABAergic cells, but not on to cholinergic cells of 124 For single labeling, sections were incubated in primary the nbM, whereas projections from the amygdala, antibody for two days at 4)C. After several rinses in hypothalamus and nucleus accumbens synapsed phosphate buffer, the tissue was treated with the bioti- directly on to the cholinergic neurons of the nylated secondary antibody for 2–4 h, rinsed several times nbM.128,129,133 and treated with ABC for 1 h. The peroxidase was visual- ized either with 0.04% diaminobenzidine plus 0.01% hydro- Additional experiments in the rat showed that gen peroxide or with the VIP-red substrate (Chemicon, GABAergic axons synapse on to cholinergic nbM Temecula, CA), which was shown previously to be suitable neurons, and that cholinergic axons synapse on to for single- and double-labeling electron microscopy.108,134 both GABAergic and cholinergic neurons in the Substituting VIP-red for diaminobenzidine did not alter the 54,73,132 distribution of labeling seen with the different antibodies nbM. Electron microscopic studies also dem- used. Control sections, incubated in a non-specific immu- onstrated synapses on to cholinergic nbM cells from noglobulin G in place of the primary antibody, lacked noradrenergic and TH-positive (presumptively labeled cells and processes. Synapses in the monkey nucleus basalis of Meynert 243

For double labeling, the first immunolabeling was done synapses per section through the cell body. Immuno- with diaminobenzidine as described above. The second reactive dendrites, in contrast, received a high density immunolabeling was then done, repeating the incubations in the second primary antibody, secondary antibody and of synapses, and these had both symmetric and ABC, but substituting the VIP-red peroxidase substrate in asymmetric synaptic densities. Although immuno- place of diaminobenzidine. In control experiments, done at reactive dendrites were not completely ensheathed in the same time as double labeling for TH and ChAT, the TH presynaptic terminals, they were typically seen to or the ChAT primary antibodies were replaced with non- have multiple synapses within 1–2 µm of dendritic specific immunoglobulin Gs of the same species. These sections lacked specific labeling for the omitted antibody length. Axons labeled for ChAT were also encoun- when evaluated both by light and electron microscopy. tered, and these included vesicle-filled varicosities After immunolabeling, tissue for electron microscopy was (Fig. 1A–D) and large unmyelinated and myelinated osmicated, dehydrated, embedded in plastic, blocked, sec- axons (Fig. 2A). Myelinated ChAT axons were com- tioned and counterstained as described previously.105 All analysis was done on series of serial sections, which were paratively common, being encountered many times in placed on consecutive grids, each of which contained a a single thin section. ribbon of 10–20 serial sections. Electron microscopy was Vesicle-filled ChAT-positive axon varicosities were done with a Jeol 100CX microscope equipped with a Z-axis relatively common. For example, when scanning a tilting grid holder. single thin section, which was small enough to be Some of the electron microscopic analysis of TH and ChAT synapses was done as a semi-quantitative survey of one of 15 serial sections on a one-hole grid, at least labeled axon varicosities. Sections were systematically two ChAT varicosities were typically encountered. scanned for varicosities which were optimally labeled, i.e. in Sampling multiple blocks from two different mon- which the label was not so dense that it obscured the keys indicated that this was a conservative estimate synaptic features. When such a varicosity was encountered, it was evaluated in serial sections to determine if it formed a of the density of these varicosities. ChAT varicosities synapse. The synapse was then followed through its entire were identified by their abundant synaptic vesicles extent in serial sections, and photographed at the section and by the presence of presynaptic specializations where it reached its largest diameter. The diameter of (Fig. 1A–D). Following 20 of these varicosities the synapse was measured from this micrograph, as was the through complete serial sections revealed that they diameter of the presynaptic axon and the postsynaptic process. The shortest diameter of all profiles was measured, always formed synaptic specializations, and these to avoid measuring the length of obliquely cut processes. were usually clearly asymmetric (18 of 20). In a The accuracy of the conclusions from these semi- few cases, the asymmetric ChAT synapses had un- quantitative samples of axons was confirmed by qualitative ambiguous subsynaptic densities (Fig. 1C). ChAT evaluations of multiple blocks from both monkeys with each of the double-labeling experiments and with single- axons occasionally formed symmetric synapses, labeling experiments. These observations were made in 85 which were similar in diameter to asymmetric ChAT grids obtained from 17 blocks of tissue, containing more synapses, but had comparatively slight membrane than 1000 thin sections. densifications (Fig. 1D). The diameter of ChAT varicosities was 0.61&0.23 µm, and their synapses RESULTS had a mean diameter of 0.33&0.13 µm (values= mean S.D., n=20). In double-labeling preparations, ChAT immuno- & The postsynaptic dendrites at ChAT synapses were reactivity was visualized with VIP-red, and GAD or sometimes ChAT immunoreactive (Fig. 1A, B). For TH immunoreactivity was visualized with diamino- example, in the above semi-quantitative analysis of benzidine. Before fixation in osmium for electron 20 ChAT axon varicosities, five ChAT synapses were microscopy, these labels were easily distinguished by on to ChAT dendrites and 15 were on to unlabeled color. After osmication, the VIP-red label was faded dendrites. Combining these and other observations of but still sufficiently visible to determine the location ChAT synapses, the diameter of postsynaptic ChAT of the cholinergic cells. The extent of the cholinergic dendrites (1.13 0.14, n=10) was similar to that of nbM was identified by the presence of ChAT cells, & unlabeled dendrites (0.92 0.45, n=17). and samples for electron microscopy were taken from & We also encountered occasional symmetric densi- the anterior and intermediate parts of Ch4 described 80 ties between ChAT dendrites. These were not associ- previously. Using electron microscopy, the VIP-red ated with synaptic vesicles, and had the appearance label was seen as a punctate reaction product, of puncta adherens93 which was easily distinguished from the floccular (Fig. 1E). diaminobenzidine label. Tyrosine hydroxylase Choline acetyltransferase Using light microscopy, TH immunoreactivity was Using light microscopy, the ChAT cells were seen in a rich bed of fine beaded axons throughout intensely labeled in their cell bodies and processes, the extent of the nbM, and also in many thick smooth and were seen to be enmeshed in a dense plexus of axons streaming dorsally and anteriorly toward the ChAT processes. Using electron microscopy, ChAT and other forebrain structures. These thick immunoreactivity was seen in cell bodies, dendrites axons were presumed to be dopaminergic axons (see and axons. Cell bodies received comparatively few Discussion). Rare TH-immunoreactive cell bodies synaptic inputs, usually having only one to three were also seen within the nbM. 244 J. F. Smiley and M.-M. Mesulam

Fig. 1. Synapses in the monkey nucleus basalis of Meynert 245

Fig. 2. Both ChAT and TH immunoreactivities were commonly found in myelinated axons in Ch4. (A) Example of ChAT immunoreactivity in a myelinated axon (arrow). (B) Example of TH immunoreactivity in a myelinated axon (arrow). Scale bar=0.5 µm and applies to both micrographs.

Electron microscopy of TH immunoreactivity and their synapses had a mean diameter of revealed numerous axons in the nbM, including 0.32&0.14 µm (values=mean&S.D., n=25). frequent large smooth axons which were unmyeli- Synapses by TH axons on to ChAT dendrites were nated or myelinated (Fig. 2B), and also vesicle-filled common. For example, in the above survey of TH varicosities (Fig. 3). A semi-quantitative analysis of varicosities, three of 18 TH synapses were on to 21 TH varicosities followed in complete serial sec- ChAT-immunoreactive dendrites (Fig. 3A–C), and tions revealed that most (18 of 21) formed clearly 15 were on to immunonegative dendrites. All post- identifiable synaptic specializations. These synapses synaptic processes were dendritic shafts, and the were usually classified as asymmetric, having some- diameter of postsynaptic ChAT dendrites (1.12& what more density on the postsynaptic membrane 0.55, n=10) was similar to that of unlabeled dendrites compared to the presynaptic membrane (Fig. 3A–C). (0.86&0.42, n=21). However, the postsynaptic density was usually not as pronounced as that of synapses formed by ChAT Glutamate decarboxylase axons in this region. Occasionally, TH synapses were seen to have distinct subsynaptic densities (Fig. 3D). Using light microscopy, GAD immunoreactivity The diameter of TH varicosities was 0.66&0.19 µm, was seen in a high density of apparent axon terminals

Fig. 1. ChAT-immunoreactive axons and dendrites in Ch4. (A, B) Two examples of ChAT axons forming asymmetric synapses (arrows) on to ChAT dendrites. ChAT immunoreactivity was visualized in both the presynaptic axons and the postsynaptic dendrites by the punctate VIP-red reaction product (arrowheads in A). (C) A ChAT axon is seen forming an asymmetric synapse (arrow) on to an unlabeled . In addition to the asymmetric membrane densification, subsynaptic dense bodies (arrowheads) are seen on the postsynaptic side of the membrane. Subsynaptic dense bodies were occasionally found at ChAT synapses on to unlabeled and ChAT-labeled dendrites. (D) A ChAT axon is seen forming a symmetric synapse on to an unlabeled dendrite. This synapse was identified in nine serial sections, and had the most pronounced membrane densification in this section. (E) An apparent puncta adherens (arrowheads) is formed between two ChAT dendrites. The puncta adherens displayed a symmetrical membrane densifi- cation, and was not associated with synaptic vesicles. The ChAT-immunoreactive profiles were identified as dendrites because they received synapses from axon varicosities (open arrows). Scale bar in E=0.5 µm and applies to all micrographs. 246 J. F. Smiley and M.-M. Mesulam

Fig. 3. Synapses in the monkey nucleus basalis of Meynert 247 and small processes throughout the nbM. Cell bodies We also found that most cholinergic synapses are and proximal dendrites were only very lightly labeled. asymmetric, although an occasional symmetric syn- With electron microscopy, immunolabeling for apse was encountered. This is also different from GAD was most intense in vesicle-filled axon varicosi- findings in the rat, where nearly 70% of the choliner- ties, although an occasional lightly labeled dendrite gic synapses were described as symmetric.8,73 In other was also encountered. When GAD varicosities were respects, the ultrastructural appearance of ChAT- seen to form synapses, these were always of the immunoreactive cells in the monkey nbM was similar symmetric type. Double labeling with GAD and to that of the rat. Cholinergic cells had very few ChAT demonstrated GAD axon varicosities forming synapses on their cell bodies, but a relatively high synapses on to ChAT dendrites (Fig. 4). Synapses on density of synapses on their dendrites, and large to unlabeled profiles were also seen, but due to the myelinated cholinergic axons were commonly variability of staining intensity, a quantification encountered.8,55,73,132 of GABAergic inputs to ChAT dendrites versus Cholinergic axons in the nbM are likely to arise unlabeled dendrites was not attempted. either from local axon collaterals of neurons in the basal forebrain, or from cholinergic neurons of the brainstem pedunculopontine tegmental (Ch5) and DISCUSSION lateral dorsal tegmental (Ch6) nuclei. Double- The combination of electron microscopy with labeling studies in the rat, using retrograde tracing immunocytochemistry and axonally transported and acetylcholinesterase histochemistry or ChAT tracers has yielded a wealth of information on the immunocytochemistry, demonstrated that a small per- sources and identity of the neural inputs that reach centage of cholinergic cells in the pedunculopontine the nbM (Table 1). As shown in the table, much of and laterodorsal tegmental nuclei (the Ch5 and Ch6 this work has been done in the rat. The experiments cell groups) project into the basal forebrain.49,56,81,102 reported here add new information related to the Tract-tracing studies suggest that a similar projection pharmacological synaptology of the nbM in the is likely to be present in the monkey.98 In the rat, monkey. stimulation of the region of the pedunculopontine Our results show that cholinergic Ch4 neurons as tegmental nucleus results in a stimulation of Ch4, as well as unlabeled neurons of the nbM in the monkey demonstrated by the release of acetylcholine in the receive a substantial direct synaptic input from cerebral cortex.94 However, concurrent pharmacologi- cholinergic, catecholaminergic and GABAergic cal manipulations suggested that this stimulation of axons. These results are consistent with the findings Ch4 is not due to cholinergic projections from the in the rat, but also suggest that the monkey nbM may brainstem, but rather to glutamatergic projections.94 have a slightly different organization. For example, It remains to be shown whether cholinergic projec- the frequency of cholinergic synapses on to Ch4 tions from the brainstem make any direct synapses on neurons appeared somewhat higher than what had to cholinergic cells of the nbM. been reported in the rat. In the region of the nbM Cholinergic agonists elicit both excitatory and in the rat, ChAT-immunoreactive axon varicosi- inhibitory responses in the nbM. In rats, in situ ties were described as scarce, and appeared to hybridization and immunocytochemistry have dem- form synapses on to ChAT-positive cells only onstrated the expression of m1-, m2- and m3- rarely.8,68,73,132 In one study, only one ChAT vari- muscarinic receptors and of nicotinic receptors in the cosity per four random thin sections was reported.73 nbM.12,26,67,68,97,116,117 In vivo extracellular record- In comparison, we found at least two ChAT vari- ings in the rat showed that muscarinic and nicotinic cosities in a single thin section. Although it is possible agonists have excitatory effects on cortically project- that cholinergic terminals are more numerous in the ing cells of the nbM.65 Intracellular recordings from monkey nbM than that of the rat, it should be identified cholinergic cells in slice preparations of the considered that these estimates of synapse density are guinea-pig nbM, however, showed mainly inhibitory approximate, and may be influenced by technical effects of muscarinic agonists and excitatory effects of factors such as the intensity of immunolabeling. nicotinic agonists.64

Fig. 3. Double labeling with TH and ChAT demonstrated TH synapses on to ChAT dendrites and on to unlabeled dendrites. TH was visualized with the diaminobenzidine reaction product and ChAT with the VIP-red reaction product (arrowheads in A). (A, B) Two serial sections show a TH axon forming two synapses (arrows), one to an unlabeled dendrite on the left and one to a ChAT dendrite on the right. In A, the synapse on the right reaches its largest size, whereas in B the synapse on the left reaches its largest extent. Most TH synapses were asymmetric, but they usually had less pronounced postsynaptic densities than synapses of ChAT axons (compare these synapses to those in Fig. 1A and B). (C) A TH axon forming a synapse (arrow) on to a ChAT dendrite. As in A and B, the postsynaptic membrane has a moderate amount of membrane densification. (D) Occasional TH synapses with subsynaptic dense bodies were also seen. In this example, the TH synapse is asymmetric and has subsynaptic dense bodies, and is on to an unlabeled dendrite. Scale bar in C=0.5 µm and applies to all micrographs. 248 J. F. Smiley and M.-M. Mesulam

Fig. 4. (A, B) Double labeling with GAD and ChAT demonstrated GAD synapses (arrows) on to ChAT dendrites. GAD was visualized with the diaminobenzidine reaction product and ChAT with the VIP-red reaction product, which is seen as punctate particles in the postsynaptic dendrites (arrowheads in A). Synapses from GAD axons were consistently symmetric. Scale bar=0.5 µm and applies to both micrographs.

The present study demonstrates that most cholin- pus and thalamus, there is evidence that symmetric ergic synapses in the nbM of the monkey are asym- and asymmetric synapses are found on different metric and large. This contrasts with most areas of postsynaptic cells.22,33,101 Notably, reconstruction of the CNS of rats, monkeys and humans, where cholin- individual cholinergic axons in the thalamus of cats ergic synapses are predominantly symmetric and showed that they formed symmetric synapses mainly small (for references see Ref. 108). In the hippocam- on to and asymmetric synapses mainly Table 1. in the nucleus basalis of Meynert and their sources

Identity of postsynaptic nbM neurons deter- Type of substance shown in mined by electron microscopy ([r] in rat, [m] the nbM by light microscopy in monkey) Source of neurotransmitter 249 References Meynert of basalis nucleus monkey the in Synapses

Acetylcholine ChAT-positive [r, m], GABA-positive [r], Possibly from Ch4 cells in the nbM, or from 8,49,56,68,73,81,98,100,102 , this report unlabeled [r, m] extrinsic sources in the basal forebrain or brainstem GABA ChAT-positive, unlabeled [r, m] Amygdala (probably also from cells in the 9,46,54,71,92,129,132 , this report nbM and striatum) Glutamate GABA-positive, ChAT-negative [r] Cerebral cortex 124 Glutamate Unknown Amygdala, hypothalamus, thalamus, lateral 16,23,124,128 septum, brainstem TH ChAT-positive, unlabeled [r, m] Presumably mostly from the substantia 36,38,56,72,98,102,107,115,127,131 , this report nigra– Norepinephrine ChAT-positive [r], unlabeled [r, m] Nucleus locus coeruleus 10,36,56,57,58,98,107,115,127,131 Serotonin Unlabeled [m] Raphe nuclei 4,56,61,66,98,107,111,115 Unknown Hypothalamus 2,53 ChAT-positive, unlabeled [r] Probably cells in the nbM and striatum 18,34,47,48,71,103,119,123 Substance P ChAT-positive, unlabeled [r] Probably striatum and brainstem 6,9,14,34,47,48,103,118,123 Somatostatin ChAT-positive, unlabeled [r] Probably cells in the nbM 14,20,103,119,123,125,126,130 Y ChAT-positive, unlabeled [r] Probably cells in the nbM 109,119,125,126,130 Other neuropeptides: VIP, neurotensin, Unknown Some are probably from cells in the nbM 14,19,20,48,60,70,76,90,103,110,119,123,130 galanin, pro-opiomelanocortin, , calcitonin gene-related peptide, luteinizing hormone-releasing hormone, cholecysto- kinin, á-melanocyte-stimulating hormone, , bombesin, vasopressin 250 J. F. Smiley and M.-M. Mesulam on to relay cells.22 The different morphological types myelinated as they approach their synaptic targets in of cholinergic synapses in the nbM may thus mediate the forebrain. different postsynaptic effects, possibly using different Our finding that GAD-immunoreactive terminals cholinergic receptors. synapse on to cholinergic cells in the nbM demon- Our results also show that synapses from TH strates a GABAergic inhibitory input to these cells. axons are abundant in the nbM, and that a sub- These GABAergic terminals are likely to arise both stantial fraction of these synapses are on to cho- from local neurons in the nbM and from afferent linergic dendrites. Although dopaminergic as well sources such as the amygdala and nucleus as noradrenergic axons contain TH, the latter accumbens.92,129,132 In the rat nbM, GABAergic cells usually label poorly with antibodies to TH.37,69 are intermingled with cholinergic cells, and are about Another possible source of axonal TH is from TH- twice as numerous as cholinergic cells.11,45 While positive interneurons in the basal forebrain,44 but some of these cells are large, with efferent projections these cells were rare in our preparation. The most to the cerebral cortex,15,32,59 others are small and likely source of TH-immunoreactive axons in our might be local interneurons.45 In the monkey nbM, preparations therefore appears to be the dopaminer- a similar population of GABAergic cells is also gic cells of the substantia nigra–ventral tegmental present.120 Application of GABA antagonists to the area.107 nbM of rats causes a dramatic increase in the release Tracing studies in the rat and monkey showed that of acetylcholine in the cerebral cortex.17,21,122 Our neurons in the dopaminergic nuclei of the ventral findings suggest that GABAergic substances are also tegmental area and substantia nigra project on to the likely to influence the activity of Ch4 and other nbM cholinergic nuclei of the basal forebrain.38,56,72,98,102 neurons in primates. In the rat, TH-positive terminals and anterogradely labeled axons from the region of dopaminergic nuclei 38,127,131 formed synapses on to cholinergic cells. CONCLUSIONS

Physiological studies showed that agonists of the D1 subtype elicited excitatory re- Our results demonstrate that cholinergic cells in sponses from most cells of the rat nbM, whereas D2 the primate nbM receive direct synaptic input from dopamine agonists were usually inhibitory.74,88 These cholinergic, catecholaminergic and GABAergic findings are consistent with the observation that axons. The results are largely consistent with pre- systemically applied D1 agonists cause increased vious electron microscopic findings in the nbM of the acetylcholine release in the cerebral cortex, whereas rat, with the exception of a somewhat higher 24,25 D2 agonists cause a slight decrease. density of predominantly large and asymmetrical Myelinated axons were often seen to contain cholinergic synapses. The TH and perhaps some TH or ChAT immunoreactivity in the monkey of the ChAT axons synapsing on to Ch4 cells in nbM. Similar myelinated axons labeled with ChAT the nbM may represent a pathway by which the were noted previously in the nbM of rats.8,73,132 brainstem reticular activating system could regu- This finding contrasts with descriptions of cate- late the cholinergic projections from the nbM to cholaminergic and cholinergic axons in the striatum the cerebral cortex. These findings provide new and cerebral cortex, which are rarely or never details concerning the pharmacological synaptology myelinated. This difference indicates that some of the nbM in the monkey and may become cholinergic and catecholaminergic axons arising from relevant to the development of pharmacological the brainstem and basal forebrain are myelinated manipulations for enhancing cortical cholinergic near the sites of their origin, but become un- neurotransmission.

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(Accepted 1 April 1998)