Distribution of an α-bungarotoxin-binding cholinergic nicotinic receptor in rat brain

Brain Research, 148 (1978) 105-119 © Elsevier/North-HollandBiomedicalPress

105

DISTRIBUTION OF AN a-BUNGAROTOXIN-BINDING CHOLINERGIC NICOTINIC RECEPTOR IN RAT BRAIN

MENACHEM SEGAL, YADIN DUDAI and ABRAHAM AMSTERDAM Departments of Isotopes, Neurobiology and Hormone Research, The Weizmann Institute of Science, Rehovot (Israel)

(Accepted September 29th, 1977)

SUMMARY Cholinergic nicotinic receptors in rat brain were demonstrated by the use of the potent nicotinic antagonist [125I]a-bungarotoxin ([125I]a-Btx). Biochemical studies on binding of [125I]a-Btx to rat hippocampal homogenates revealed saturable binding sites which are protected by nicotine, D-tubocurarine and acetylcholine but not by atropine or oxotremorine. The hippocampus and hypothalamus displayed relatively high [125I]a-Btx specific binding whereas the cerebellum was devoid of specific binding. Other regions displayed intermediate binding levels. Analysis of the regional distribution of [125I]a-Btx binding by autoradiography of frontal brain sections revealed high labeling in the hippocampus, hypothalamic supraoptic, suprachiasmatic and periventricular nuclei, ventral lateral geniculate and the mesencephalic dorsal tegmental nucleus. It is suggested that the limbic forebrain and midbrain structures as well as sensory nuclei are the main nicotinic cholinoceptive structures in the brain.

INTRODUCTION The presence of cholinergic nicotinic receptors in mammalian brain has been suggested by a number of investigators. Until recently, this assertion was based on the responses of neurons to the iontophoretic application of nicotinic ligandsl,11, ~6. The potent nicotinic antagonist a-bungarotoxin (a-Btx) has been extensively used to characterize peripheral nicotinic receptors2,4,5, s. Specific binding of [125I]a-Btx to several brain regions was recently reported6,15,16,21,2L Preliminary reports of autoradiographic studies have demonstrated a-Btx binding sites in the hippocampus and several hypothalamic structures, especially the supraoptic and suprachiasmatic nuclei 20, 23. In the following we report the results of a systematic autoradiographic and biochemical analysis of the regional distribution of [l~5I]a-Btx binding sites in rat brain.

106 Our results indicate the existence of high concentrations of nicotinic receptors in limbic forebrain and midbrain structures and in several sensory nuclei. METHODS Adult (200-300 g) male Wistar rats were used. a-Btx was purified from crude Bungarus multicintus venom 5 and was iodinated with lz5I by the ICI method to an initial specific activity of 100-200 Ci/mmole, according to Vogel et al. 2s. A preparation which was iodinated by the chloramine-T method z was also used and yielded similar results. For biochemical studies, brain regions were dissected in the cold (4 °C) and homogenized (100 mg/ml) in 0.32 M sucrose in a glass-Teflon homogenizer. Alternately, the brain was frozen in dry ice, sectioned with a microtome into 500/~m-thick sections and the sections mounted on microscope slides. Samples of various identified structures were punched using a specially prepared 17-gauge needle under a dissecting microscope. The samples thus obtained (0.5-1.0 mg tissue) were homogenized in 0.32 M sucrose as above. Binding of [125I]a-Btx was assayed as follows; Aliquots of homogenate (containing up to about 0.2 mg protein) were incubated at 25 °C in 0.12 M NaCI, 2 mg/ml BSA, 0.05 M Tris.C1, pH 7.4 (buffer 1), in total vol. 0.05-0.1 ml. Reaction was started by addition of [125I]a-Btx (final concentration 15 nM, unless otherwise indicated) and terminated after 60 min by diluting with 2 ml buffer 1, followed immediately by vacuum filtration through a Millipore EGWP filter as described by Vogel and Nirenberg 27. The filter was then washed 3 times with 2 ml portions of buffer 1 and counted. Specific binding of [l~5I]a-Btx was defined as total binding minus the binding occurring in the presence of 0.1 m M nicotine. Each determination was carried out in a duplicate with and without nicotine. Protein was determined according to Lowry et al. 14. For autoradiographic studies, the brain was mounted on a cryostat chuck and sectioned (12 #m) at --15 °C. Frontal sections throughout the brain were collected in 0.5 mm intervals and mounted on glass slides. Incubation with [l~5I]a-Btx was carried out at 24 °C in a vapour-tight chamber. Incubation medium contained 8 or 15 nM [125I]a-Btx in 0.12 M NaCI, 4 mg/ml BSA, 0.05 M Tris pH 7.4. Reaction was started by covering each section with 0.15-0.2 ml of incubation medium and was terminated 40-60 min later by removing the medium. Non-specific binding was determined in adjacent sections which were preincubated for 20 min with 1 m M nicotine and reacted as above in the presence of 1 m M nicotine. Sections were dipped twice (5 min each) in coplinjars containing 40 ml of buffer 1, and subsequently by two changes (5 min each) in phosphate buffered saline. The sections were fixed in 2 ~ gluteraldehyde in 0.1 M sodium cacodylate, pH 7.4, washed ( × 3) in distilled water and air dried. Slides were coated with Ilford K5 emulsion, diluted 1 : 1 with distilled water and incubated at 4 °C for 21-28 days in a light-proof slide box containing a desiccant. The autoradiograms were developed for 4 min with Kodak D19 at 20 °C and were fixed with Kodak Fixer. Some sections were counterstained with hematoxylin eosin. The sections were examined with dark and bright field microscopy (Zeiss Photomicroscope III).

107 RESULTS

(,4) Biochemical studies At the first stage of our study basic properties of [125I]a-Btx binding sites were determined. The hippocampus was selected as a representative region because of its relatively high binding levels (see below). Under the conditions employed, binding observed with a given concentration of labeled toxin was linearly proportional to the amount of homogenate present. Specific binding of toxin was saturable (Fig. 1). After incubation for 1 h, maximum specific binding was obtained with toxin concentrations higher than 10 nM. Non-specific binding at that concentration was 30-40 ~o of total and was not abolished even by preincubation with 1 m M nicotine or with 0.1 F M non-labeled a-Btx. Of the cholinergic ligands studied the nicotinic ligands nicotine and D-tubocurarine were most potent in protecting against [125I]a-Btx binding, both displaying EDs0 values (i.e., concentration of drug displacing 50 ~o of specific toxin binding) of 7 × l0 -6 M (Fig. 2). Acetylcholine was also effective in protecting[125I]a-Btx binding sites (EDs0 ---- 6 × l0 -5 M). The potent muscarinic ligands atropine and oxotremorine were not effective at concentrations as high as 1 mM. Thus the affinity spectrum of the [125I]a-Btx binding sites is of a nicotinic nature, in accordance with previous reports for rat cerebral cortex and whole brain extracts6, 21,22. Concentration of specific [125I]a-Btx binding sites was determined for homogenates of various brain regions, dissected in toto. Results are presented in Fig. 3. The highest binding was found in the hypothalamus and the hippocampus. High

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Fig. 2. The effect of cholinergic ligands on specific [z25I]a-Btx binding to hippocampal homogenate. Aliquots (containing about 0.2 mg protein) were preincubated for 25 min at 25 °C in buffer 1 containing the appropriate concentration of ligand. Reaction was started by addition of [12SI]a-Btx (10 nM) and terminated after 60 min as described under Methods. (3 (3, D-tubocurarine; • ~ , nicotine; ID I), acetylcholine, in the presence of 10-5 M diethylfluorophosphate, which completely inhibits acetylcholinesterase; [] [], atropine; • • , oxotremorine.

binding levels were also found in the tectum. The tegmentum, pons, neocortex and the cingulate cortex displayed intermediate binding, followed by the medulla oblongata, septal area, thalamus and caudate. The cerebellum was essentially devoid of specific [l~5I]a-Btx binding.

( B) Autoradiographic analysis Sections from 3 brains were systematically scanned for specific [125I]a-Btx labeling. Comparison between adjacent sections which were or were not preincubated 5025 e~

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Fig. 3. Specific [125I]a-Btx binding to homogenates of various parts of rat brain, dissected in toto. Each value represents the meat + S.E.M. of 3--4 determinations, performed on different rats. Each assay was carried out in a duplicate with 10-4 Mnicotine and a duplicate with no nicotine, to determine specificity. Homogenization and assay procedures are described under Methods. Hth, hypothalamus; Hpc, hippocampus; Tec, tectum; Teg, tegmentum; Pon, ports; Cx, cortex; Cing, cingulate cortex; Med, medulla; Sep, septum; Th, thalamus; Cd, caudate; Cb, cerebellum.

109 with nicotine indicated the specificity of labeling. Specific binding pattern was essentially similar in all 3 brains examined. A differential distribution of [125I]a-Btx was found in the brain. The regional distribution of specific binding sites is summarized in Fig. 4. Representative radioautographs are shown in Figs. 5-8. The telencephalon. Relatively high grain concentrations were found mainly in limbic areas; the cingulate cortex, pyriform cortex, entorhinal cortex and the subicular regions. The labeling was concentrated in the molecular layers (Figs. 4 and 5). The hippocampus contained particularly high concentration of [l~5I]a-Btx labeling. Labeling was found in the dentate polimorphic layer, adjacent to the granular cell layer. Fewer grains were seen in the CA4 region of the dentate hilus. A narrow band was found in the outer third of stratum oriens, near the alveus, in region CA1-CA3 of the hippocampus and was continuous with a thicker band in the subiculum. Labeling was slightly higher in ventral than in dorsal hippocampus. The amygdala contained medium labeling mainly in its central and basolateral nuclei. Specific labeling was also A

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Fig. 4, Representative frontal sections drawn according to K6nig and Klippel 1° summarizing the main labeled nuclei in the brain from anterior (A) to posterior (L) regions. Based on subjective judgement of the degree of labeling, 3 categories were determined: dark gray represents high labeling (e.g. supraoptic nucleus, dentate polimorphic layer, see Figs. 5 and 6); medium gray represents medium labeling (e.g., cingulate cortex (Fig. 5), amygdala) and light gray represents above-background labeling (e.g., septal area, pyriform cortex). The neocortex which contained above-background labeling was not marked in these sections for sake of simplicity. Asterisks indicate ventricles.

Fig. 5. a: labeling of [l~I]a-Btx in the cingulate cortex. The label is concentrated in the molecular layer and is relatively absent over neuronal somata. Asterisks indicate midline, b: labelling of the outer third of stratum oriens in the hippocampus region CA1. The other layers of the hippocampus, with the exception of the pyramidal layer, also contain labelling above the level seen in the overlying corpus callosum, c: labeling in the dentate hilus. Grains are heavily concentrated in the polymorphic layer and are relatively absent in the granular layer. Magnification 63 x.

112

Fig. 6. a: intensely labeled neurons (arrowheads) and high neuropil labeling in the mediobasal hypothalamus, in the region of the ventromedial nucleus of the hypothalamus, b: the suprachiasmatic nucleus, labeled clearly above background in the region of the anterior nucleus of the hypothalamus. c: the supraoptic nucleus located above the lateral aspect of the optic chiasm (OC). d : a section adjacent to c but preincubated with nicotine. Note the blood vessel (BV) and the optic chiasm for orientation. Magnification, 63 ×.

Fig. 7. a: labeling in the ventral lateral geniculate nucleus. Note the relative absence of labeling over fiber bundles in the nucleus, b: intense labeling in the hypothalamic periventricular region in the posterior dorsal aspect of the third ventricle, c: control section, adjacent to b, preincubated with nicotine. Magnification, 63 x .

115 found in the amygdala cortical nucleus, which appeared to be continuous with the pyriform cortex. The neocortex contained low, but above background labeling. This was scattered homogenously throughout the cortex with no apparent differences among the various cortical fields. Diencephalon. Intense labeling of [125I]a-Btx was found in the supraoptic nucleus (Fig. 6) of the hypothalamus. High grain concentration was found in the suprachiasmatic nucleus and the posterior hypothalamic periventricular region (Fig. 7). Medium levels of labeling were found in the lateral mammillary nuclei and the lateral part of the medial mammillary nuclei. Labeling was also found in the lateral and dorsomedial hypothalamus and in the medial preoptic region. Among the thalamic nuclei, a high concentration of specific [l~sI]a-Btx binding-sites was found in the ventral lateral geniculate nucleus. The lateral habenula and the subthalamic area also contained high grain concentration. Labeling was also found in the nucleus paratenealis, the internal medullary lamina of the thalamus, and nucleus reuniens. The rest of the thalamus displayed relatively low [125I]a-Btx labeling. The hindbrain. Autoradiograms of the superior and inferior colliculi displayed relatively high grain concentrations. These were apparent mainly in the superficial lamina of both structures and were present throughout the entire length of the colliculi. Among the tegmental structures which contained significant labeling were the dorsal and median raphe nuclei, nucleus tegmenti pontis and the nuclei of the lateral lemniscus. The dorsal tegmental nuclei (Fig. 8) displayed among the highest labeling densities of the whole brain. The labeling covered the entire nuclei and was essentially abolished in sections which were pretreated with nicotine. In order to compare the binding of the latter region with that of other structures, the amount of binding of [125I]a-Btx in small (1 mg tissue) punches of the dorsal tegmental nucleus was compared with similar size punches obtained from other brain regions. Specific binding in this nucleus amounted to 0.15-0.20 pmole/mg, about 6 times the values obtained for the hypothalamus in toto. Control punches of adjacent cerebellar tissue did not display any specific binding. High grain concentrations were also found in autoradiograms of the parabrachial nuclei. Only a few structures in the medulla contained above background levels of labeling. The nucleus commissuralis, adjacent to the nucleus tractus solitarius, was labeled. The cerebellum was devoid of specific labeling of [l~5I]a-Btx. DISCUSSION The present studies have demonstrated specific binding of [125I]a-Btx to structures in rat brain. Earlier suggestions6,15,16, ~o-z3 for the presence of cholinergic nicotinic

Fig. 8. Autoradiograms of [125I]a-Btxlabeling of the dorsal tegmental nuclei (dtn). a: a bright field photograph of a cresyl violet stained section in the region of the central gray matter. The dtn, marked by arrowheads for ease of comparison with b and c, is stained slightly darker than the rest of the central gray area. Magnification 25 x. b: dark field photograph of the labeled dtn. Note the low background labeling in the ventricle (v) and the above-background labeling outside the dtn. c: a control section, adjacent to b, preincubated with 10-4 M nicotine. The dtn is barely visible whereas the rest of the section has background labeling (as in the ventricle in b). Magnification for b and c 63 ×.

116 receptors in rat brain have thus been confirmed and extended to provide a more complete map of the distribution of nicotinic receptors in the brain. The results of the biochemical and autoradiographic studies appear to correlate quite well; the hippocampus and hypothalamus displayed relatively high concentrations of nicotinic receptors as revealed both by binding to homogenates and by autoradiograms, whereas the cerebellum was almost devoid of specifically bound [125I]a-Btx as demonstrated by both methods. Structures such as the caudate or thalamus, with only a few exceptions (i.e., the ventral lateral geniculate), were relatively low in specific [125I]a-Btx binding sites. Specific binding levels obtained for various brain regions homogenized in toto, are in the range previously reported for brain homogenates6,21,9z. Such binding levels represent the average for each region and could be employed to compare various gross brain areas (Fig. 3), but fine distribution of receptors within each region could be analyzed only by autoradiography (Figs. 4-8). An analysis of the differential distribution of [1251]a-Btx labeling reveals that most of the limbic forebrain structures contain nicotinic receptors. Among these are the main limbic cortex structures: the cingulate cortex, entorhinal cortex, pyriform cortex and the subiculum. The hippocampus, a major component of the limbic system, contained high concentrations of nicotinic receptors (see below), and so did several nuclei of the amygdala. Among the diencephalic structures, the most conspicuously labeled structures were the supraoptic and suprachiasmatic nuclei and, among others, the mammillary nuclei. The dorsal tegmental nucleus, a central limbic midbrain nucleus 13 contained among the highest concentrations of [125I]a-Btx labeling in the brain. The dorsal and median raphe nuclei also contained specific binding sites. Primary visual and auditory nuclei comprise another category of nicotinic cholinoceptive structures. Among these are the bed nuclei of the lateral lemniscus, the superior and inferior colliculi and the ventral component of the lateral geniculate body. Interestingly, somatosensory nuclei of the thalamus did not contain significant nicotinic labeling. The distribution of the cholinergic enzymes, choline acetyltransferase (CAT) and acetylcholinesterase (ACHE), reported elsewhere 7,9'13'1s'19, corresponds only partially with that of the nicotinic receptors. AChE staining was found in the zona incerta, lateral mammillary nucleP 9, ventral lateral geniculate, dorsal tegmental nucleus 13, hippocampus and to a lesser extent in the cerebral cortex. All of the above structures contained nicotinic receptors. However, several structures, e.g. the caudate nucleus, contain high AChE activity but low concentrations of nicotinic receptors. The correlation between CAT activity and concentration of nicotinic receptors is not as clear. Many structures rich in CAT activity, e.g. medial habenula, nucleus of the diagonal band, lateral amygdala, medial septal nucleus, caudate nucleus and motor nucleig, is, displayed low [125I]a-Btx binding. The comparison between the distribution of muscarinic receptors in the brain, as revealed by binding of [aH]quinuclidinyl benzilate ([3H]QNB) (ref. 24), and the nicotinic receptors as revealed by [125I]a-Btx binding in our study, is important for the understanding of possible functional significance of the cholinergic receptor dichotomy. Altogether muscarinic receptors appear to be more abundant than nicotinic receptors

117 in the brain ~1,24. The basal nuclei, caudate and putamen contain the highest concentration of muscarinic receptors in the brain 24. The cerebral neocortex as well as the hippocampus contain relatively high [SH]QNB binding sites, whereas fewer binding sites were found in thalamic and hypothalamic structures. It appears that nicotinic receptors are present mainly in limbic structures whereas muscarinic receptors are more abundant in extrapyramidal structures. Several structures contain relatively high concentrations of both nicotinic and muscarinic receptors, e.g. the neocortex. A clear anatomical dissociation of both receptor types within those regions is not yet apparent. Such a dissociation could be detected in the hippocampus. The muscarinic receptor was found in high concentration in the dentate stratum moleculare12. In contrast, stratum polimorphe did not contain specific [SH]QNB labelinglL The hippocampus proper contained [3H]QNB labeling in both stratum oriens and radiatum of areas CA3-1 (ref. 12). [125I]a-Btx binding sites, on the other hand, were found mainly in stratum polimorphe of the dentate gyrus and were restricted to a narrow band in stratum oriens of region CA3-1. Since the distributions of the two receptor types do not overlap, it is of interest to correlate the receptor distributions with the distribution of the cholinergic enzymes, primarily ACHE, as well as the distribution of the septal afferents, known to contain the cholinergic input to the hippocampus. AChE levels are high in stratum polimorphe and granulare of the dentate gyrusZL and also in stratum oriens and pyramidale of area CA1. Lower AChE activity was found in other hippocampal strata 25. This distribution generally parallels that of the nicotinic receptors, with the exception of stratum granulare of the dentate which does not contain nicotinic receptors. The distribution of AChE activity does not appear to correlate as well with the muscarinic receptors distribution. The septal afferents terminate in regions rich in both nicotinic or muscarinic receptors ~7, thus it is most likely that cholinergic synapses for both nicotinic and muscarinic receptors arise from the same nucleus. The latter observation brings up the question of the physiological significance of each receptor type. Elsewhere in the brain, nicotinic and muscarinic effects are opposite, e.g. in the supraoptic nucleus nicotinic agents are excitatory and muscarinic-inhibitory1. In the hippocampus cholinergic excitation can be blocked by atropine a, but iontophoretic application of nicotinic antagonists, e.g. D-tubocurarine, is not efficient in antagonizing cholinergic excitation (Segal, unpublished observations). The question of the differential role of nicotinic and muscarinic receptors in the hippocampus remains therefore unsolved. In conclusion, although their exact physiological role is not yet known, the wide and differential distribution of nicotinic receptors in the brain suggests that these cholinergic receptors have specific roles in brain that are different from those of muscarinic receptors. ACKNOWLEDGEMENTS The skilled technical assistance of Varda Grinberger, Anat Azrad and Shoshana Nahum is gratefully acknowledged.

118 This work was supported by grants from the United States - Israel Binational Science F o u n d a t i o n , Jerusalem (to M.S., Y.D. a n d A.A.). M.S., i n c u m b e n t of the W o r m s e r Career D e v e l o p m e n t Chair; A.A., i n c u m b e n t of the Gestetner Career D e v e l o p m e n t Chair. LIST OF ABBREVIATIONS A, nucleus accumbens; ABL, nucleus amygdaloideus basalis pars lateralis; ACO, nucleus amygdaloideus cortical is; AL, alveus of hippocampus; AQ, aqueduct; BV, blood vessel; C, cingulate cortex; CA, nucleus commissurae anterioris; CAIR, capsula interna, pars retrolenticularis; CB, cerebellum; CC, corpus callosum; CG, central gray matter; CI, colliculus inferior; CP, nucleus caudatus putamen ; CPF, cortex piriformis; DCGL, nucleus dorsalis corporis geniculati lateralis; DR, nucleus dorsMis raphes; DTN, nucleus dorsalis tegmenti; EL, cortex entorhinalis, pars lateralis; EM, cortex entorhinalis, pars medialis ; FH, fimbria hippocampi; FMP, fasciculus medialis prosencephali; FOR, formatio reticularis; G, dentate granular layer; GD, gyrus dentatus; GP, globus pallidus; HA, nucleus anterior hypothalami; HDV, nucleus dorsomedialis hypothalami, HI, hippocapmus; HL, nucleus lateralis hypothalami; HPV, nucleus periventricularis hypothalami ; LAMI, lamina medullaris interna thalami; M, stratum molecularis; MH, medial habenula; MR, nucleus medianus raphes; O, stratum oriens hippocampi; OC, optic chiasm; P, stratum pyramidale; PN, nucleus parabrachialis; PNM, nucleus parabrachialis, pars medialis; PO, nucleus pontis: POM, nucleus preopticus medialis; PT, nucleus paratenialis; RE, nucleus reuniens; S, subiculum; SC, nucleus suprachiasmaticus; SCS, colliculi superioris; SL, nucleus septi lateralis; SO, nucleus supraopticus; ST, nucleus interstitialis striae terminalis; SUT, nucleus subthalamicus; TD, nucleus tractus diagonalis; TL, nucleus lateralis thalami; TP, nucleus tegmenti pontis; TV, nucleus ventralis thalami; TVD, nucleus ventralis thalami pars dorsomedialis; V, ventricle; VCGL, nucleus ventralis corporis geniculati lateralis; VCLL, nucleus ventralis caudalis lemnisci lateralis; VRLL, nucleus ventralis rostralis lemnisci lateralis; ZI, zona incerta; Vm, nucleus trigeminus motoria; Vs, nucleus trigeminus sensoria; Vmes, nucleus trigeminus mesencephalis. REFERENCES 1 Barker, J. C., Crayton, J. W. and Nicoll, R. A., Noradrenaline and acety|choline responses of supraoptic neurosecretory cells, J. Physiol. (Lond.), 218 (1971) 19-32. 2 Berg, D. K., Kelly, R. B., Sargent, P. B., Williamson, P. and Hall, Z. W., Binding of a-bungarotoxin to acetylcholine receptors in mammalian muscle, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 147-151. 3 Biscoe, T. J. and Straughn, D. W., Microelectrophoretic studies of neurons in the cat hippocampus, J. Physiol. (Lond.), 183 (1966) 341-359. 4 Changeux, J. P., The cholinergic receptor protein from fish electric organ. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook of Psyebopharmacology, Vol. 6, Plenum Press, N.Y., 1976, pp. 235-301. 5 Changeux, J. P., Kasai, M. and Lee, C. Y., Use of a snake venom toxin to characterize the cholinergic receptor protein, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 1241-1247. 6 Eterovic, V. A. and Bennett, E. L., Nicotinic cholinergic receptor in brain detected by binding of a-[aH]bungarotoxin, Biochim. biophys. Acta (Amst.), 362 (1974) 346-355. 7 Jacobowitz, D. M.and Palkovits, M.,Topographicatlasofcatecholamineandacetylcholinesterasecontaining neurons in the rat brain 1. Forebrain, J. comp. Neurok, 157 (1974) 13-28. 8 Karlin, A., The acetylcholine receptor: progress report, Life Sci., 14 (1974) 1385-1415. 9 Kobayashi, R. M., Brownstein, M., Saavedra, J. M. and Palkovits, M., Choline acetyltransferase content in discrete regions of the rat brainstem, J. Neurochem., 24 0975) 637-640. 10 K6nig, J. F. R. and Klippel, R. A., The Rat Brain. A Stereotaxic Atlas, Williams and Wilkins, New York, 1963. 11 Krnjevic, K., Acetylcholine receptors in vertebrate CNS. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook ofPsycbopharmacology, VoL 6, Plenum Press, N.Y., 1976. 12 Kuhar, M. J. and Yamamura, H. I., Localization of cholinergic muscarinic receptors in rat brain by light microscopic radioautography, Brain Research, 110 (1976) 229-243.

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