Some observations on the binding patterns of α-bungarotoxin in the central nervous system of the rat

Brain Research, 157 (1978) 213 232 (9 Elsevier/North-Holland Biomedical Press

213

Research Reports

SOME OBSERVATIONS ON T H E B I N D I N G PATTERNS OF a-BUNGAROTOXIN IN T H E C E N T R A L NERVOUS SYSTEM OF THE RAT

STEPHEN HUNT~ and JAKOB SCHMIDT* Department of Psychiatry and Behavioral Science and Department of Biochemisto,*, State UniversiO, ()f New York at Stony Brook, Stony Brook, New York 11794 (U.S.A.)

(Accepted March 16th, 1978)

SUMMARY Patterns of a-bungarotoxin (aBuTX) binding within the brain of the rat have been studied following one of two procedures: (1) the intraventricular injection of l~5llabeled toxin followed by a survival period of 1-8 days before aldehyde perfusion, or (2) the incubation of fresh cryostat sections of brain tissue in dilute solutions of radioactive toxin. Appropriate controls with nicotine, curare, atropine and native aBuTX established the specific nature of the binding. The principal observations were that toxin binding sites are predominantly associated with central areas of the brain in direct receipt of sensory inputs (the main and accessory olfactory bulbs, superior colliculus, ventral lateral geniculate nucleus, cochlear nuclei, the substantia gelatinosa of the spinal cord and spinal trigeminal nucleus, the principal sensory nucleus of the trigeminal, and the dorsal column nuclei) and with limbic areas of the brain (hippocampus, amygdala, olfactory tubercle, medial mammillary nucleus, and the dorsal tegmental nucleus of Gudden). Toxin was not found to bind to cranial motor nuclei with the exception of the dorsal motor nucleus of the vagus and the nucleus ambiguus. The discrete distribution of clusters of silver grains within the granule layer of cerebellar folia I, IX, and X is described as well as the heavy labeling of the inferior and accessory olivary nuclei. In many areas of the brain silver grains were found to overlie cell bodies. It is suggested that this may reflect the presence of both membrane-bound toxin and internalized ligand following initial binding to a membrane receptor site. Present address: M RC Neurochemical Pharmacology Unit, Department of Pharmacology, Hills Road, Cambridge CB2 2QD, Great Britain. * To whom to address correspondence.

214 An attempt was made to correlate the localization of toxin binding sites with the terminal distributions of previously described cholinergic pathways. There appears to be a reasonably good agreement between the distribution of toxin receptors and proposed sites of cholinergic transmission within the hippocampus, interpeduncular nucleus and cerebellum. In most other cases however, lack of data precluded such correlations. The anatomical relationship of aBuTX binding activity and neurotransmitters other than acetylcholine is also considered.

INTRODUCTION The mapping of cholinergic pathways within the central nervou~ system has traditionally relied upon the histochemical localization of the degradative enzyme acetylcholinesterase (ACHE) within the brain ')6.z9.3.5.44.Sn.~v.Ss,ag.It has become apparent however that AChE may not be a specific marker for the cholinergic synapse, and attention has turned to the immunocytochem ical localization of choline acetvltransferase (CAT) 21,z,~ or to the use of ligands which selectively label either muscarinic :~3,'~'2or nicotinic acetylcholine receptors (AChR) ~4.34.4'~.a°.~°-~4. The nicotinic antagonist ~zbungarotoxin (aBuTX) has been shown to bind selectively and irreversibly to nicotinic AChR in vertebrate skeletal muscle and produce failure of synaptic transmission at the neuromuscular junction 11, and at an analogous preparation, the neuroeffector junction in electric tissuelL Evidence has accumulated to suggest that the toxin also interacts with specific binding sites in the autonomic t9,20 and central nervous system av,'~z. These neuronal toxin receptors resemble peripheral AChR with respect to biochemical properties 37 and drug affinities55. but differ from them in that the toxin does not interfere with synaptic transmission 16,a°. Nevertheless, specific binding to synaptic membranes has been observed in the central nervous system 24-34.64. In the present report patterns of aBuTX within the rat brain are described and their correlation with established and putative cholinergic pathways discussed. A preliminary report 5° and electron microscopic data ~4 have been published. MATERIALS AND METHODS aBuTX was isolated from the venom of Bungarus multicinctus (Miami serpentarium) and labeled with tZ~l as previously described 37. The specific activity of the toxin was approximately 106 Ci/mole (depending on the age of the preparation) and the concentration 5 × 10 s M.

Intraventrieular injections Rats (Sprague-Dawley) were anesthetized with Ketamine (0.2 ml/100 gm) supplemented with metofane, and placed in a stereotaxic holder. A mounted Hamilton syringe was used to inject 50-100 #1 of toxin (in 3 m M sodium phosphate p H 7.4; ca. 80 m M sodium chloride; 2 mg bovine serum albumin per ml; 0.02 ~ sodium azide) over a period of 10 min into the lateral ventricle z4. Animals were allowed to survive one ( n = 10), three (n--1) or eight ( n = 1) days, before vascular perfusion with a mixture of

215 4~/o paraformaldehyde, 0.5 ~ glutaraldehyde, 0 . 5 4 ~ dextrose, and 0.05 ~ calcium chloride in 0.1 M sodium phosphate, pH 7.263. Tissue was then embedded in paraffin wax, cut at 10 #m, and sections (1 in 20) processed by conventional light microscopic autoradiographic techniques.

Cryostat sectioned material Five rats were anesthetized with ether, decapitated, and the brain rapidly removed. Whole brain or pieces were frozen onto cryostat chucks sitting in dry ice, placed in the cryostat and allowed to equilibrate within the cryostat chamber. Sections in frontal, horizontal or parasagittal planes were taken at 8 to 10/~m, and collected on subbed slides. After drying at room temperature for at least 15 minutes sections were immersed in one of the following solutions: (a) 30 min 5 )< 10-1° M or 5 × 10-9 M [t251]~tBuTX; (b) 30 rain 10-3 M D-tubocurarine (or nicotine); followed by 30 min 10-3 M Dtubocurarine (or nicotine) plus 5 x 10 10 M or 5 × 10 9 M [lzSI]aBuTX; (c) 30 rain 10--3 M atropine; followed by 30 rain 10 3 M atropine plus 5 x 10 9 M [t2~I]aBuTX. All solutions were made up in 100 mM sodium phosphate, pH 7.4. The tissue sections were then washed for 30 min, fixed, dried, and dipped in Kodak NTB2 emulsion as previously described ~°. Exposure time was from 3 to 14 days. In vivo controls were not possible with curare since the amount required for blocking the binding sites exceeds the lethal dosage. However, injection of native toxin in an excess of 50-500 fold 30 rain prior to injection of labeled toxin was sufficient to abolish all binding of the radiotoxin. In describing the results only a very crude attempt at quantification has been made, with the use of such terms as 'moderately heavy', 'intense' and 'light'. It was felt that the best approach to a quantitative estimate of the numbers of binding sites within the brain was by in vitro assay of discrete brain areas. This has been accomplished 42, .~4 and there is a good correspondence between the biochemical and autoradiographic approaches. RES ULTS lntraventricular injection of [12~I]aBuTX resulted in the labeling of a large number of structures throughout the brain (Table I). Comparison with cryostat sections indicated that while this labeling was specific, it was in some cases incomplete. This was particularly true of areas distant from the injection site, notably the outer layers of the lateral portions of the cerebral cortex, the amygdaloid complex, and the central areas of the brain stem. It was therefore necessary to compare material prepared by intraventricular injection with that prepared from fresh cryostat sections and not subject to diffusion barriers. In the majority of areas examined, intraventricular injection was adequate for a complete labeling of structures. It was also vastly superior in terms of background levels of silver grains and in the potential for further studies with the electron microscope which demand mixed aldehyde perfusion for adequate histo-

216 TABLE I Distribution o f aBuTX binding sites within the rat brain Intensity of labeling rated light (-F), moderate ( F t ) or heavy (q g +). The type of A R G labeling pattern is referred to as either diffuse (d) or structured, Structured patterns are further subdivided into (i) laminar (1) silver grain distributions (cf. Fig. 2B); (ii) clusters (c) of silver grains (cf. Fig. 5) and did profiles (p) of neurons studded with silver grains (cf. Fig. 1). Area Rhinencephalon Olfactory bulb Accessory olfactory bulb Ant. olfactory n. Olfactory tubercle

Comments

lntensiO~ and type

Glomeruli and EPL Glomeruli and EPL

(d, I ) ~ (d} (d) (p)

Deep to polymorphic layer

Cerebral Cortex

Limbic Areas Hippocampus

Amygdala Septum n. Diagonal Band

Layers I, V and VI of all cortical areas. Some perikaryal labeling in 11 and III.

(l,p)

Stratum oriens Stratum lacunosum hilus of dentate gyrus All nuclei except n. centralis including n. endopiriformis Medial nucleus

(p) (p) (d) td) (d) ~d~ (d)

Basal Ganglia Globus pallidus

{p)

Thalamus n. Medialis pars medialis n. Subparafascicularis n. Rhomboideus n. Parata enialis n. Geniculatus pars ventratis

(d) : (d) (d) t (d) i

Subthalamus Zona Incerta Subthalamic n. Fields of Forel

J (d)

(d) q

, (d)

, (d)

Epithalamus Lat. habenular n. Hypothalamus Supraoptic n. Periventricular n. Arcuate n. Post. hypothalamic n. Medial mammillary n. Preoptic area Mesencephalon Pretectum Superior colliculus Inferior colliculus

-i (d) F

+

i (d) 4 (p)

I" -i (d)

Lateral and ventral portions

q -4-

Superficial layers Deeper nuclear areas

-~ (p) (d)

g (d) ÷g -~ (d) F 4- + (d?

217 Parabigeminal n. Peripeduncular n. n. lateral lemniscus Principal sensory n. of V Raphe n. Periaqueductal grey Reticular formation Interpeduncular n. Superior olivary complex Cerebellum Hindbrain and Medulla Parabrachial n. Cochlear n. Vestibular nuclei Dorsal tegmental n. of Gudden Inferior olivary complex Reticular formation: Lateral reticular n. Spinal n. of V Stratum griseum externum Dorsal motor n. X n. Ambiguus n. Solitary tract Dorsal column n. Spinal Cord Lateral cervical n. Dorsal horn Ventral horn

+ +

+ +

Groups of binding sites found within Folia I, IX, X

Dorsal and ventral n.

Principal and accessory divisions Lateral Medial

-I+ + -~. + + + + +

+ (p) 4 (p) + (p) + (p) + (p) ~ (d) ~ (d) + (d) ~ (p)

+ +-~ (c)

-~ + + + + + + + + + + + + + + +

(d) (p) (d) (d)

(p) (d) (p)

+ + (p) Marginal (1) and Layer I11

÷ + t +

+ 7+ +

-+ (d) + (p) + (d) +- (d)

(d) + + (p)

Laminae I and II Small cells only

1- + + (p) t + ¢1) + + (p)

logical preservation. Prior injection of non-radioactive toxin displaced all radioactive toxin. Incubation of cryostat sections with D-tubocurarine or nicotine, but not atropine, resulted in labeling at background levels only. No binding was evident 8 days after intraventricular injection of toxin while at three days the binding pattern was markedly attenuated.

Patterns of Toxin Binding Autoradiograms produced by either the cryostat or intraventricular perfusion methods demonstrated a number of distinct types of binding pattern within the brain. These patterns can be classified as either diffuse or structured. Diffuse labeling patterns as seen over the superior colliculus (Fig. 2B) and ventral geniculate nucleus (Fig. 3B) consisted of silver grains distributed homogeneously over neuropil and cell bodies of the structure in question. In contrast, structured patterns of silver grains were of several types: silver grains accumulating in either bursts or clusters as in the cerebellar cortex (Fig. 5), laminar patterns, as in the cerebral cortex (Fig. 2B), or specifically overlying and outlining underlying perikarya and dendrites, for example within the cochlear nuclei (Fig. 4) and lateral cervical nucleus (Fig. 2A). In cryostat sections it was found that the appearance of structured patterns of labeling, particularly those overlying single neurons, was to an extent dependent upon the concentration of ligand

218

8

tit ~

Fig. 1. Bright-field photomicrograph of cell bodies (c)and their associated processes labeled 24 h after intraventricular (IVt) injection of [1251]aBuTX. A: fusiform cell found within the pyramidal cell layer of area CAI of the hippocampuS, d dendrite. B: labeled Perikarya within cortical layer 11 of the cingulate cortex. 1, the molecular layer of the cortex. C: small labeled cell bodies (Renshaw cells?) amongst unlabeled cell bodies of motor neurons (M) in the ventral horn of the spinal cord (level CI) Scale bar, 30 ~ m in all cases.

219

Fig. 2. Dark-field photomicrograph o f frontal section through A : spinal cord at level CI 24 hrs after IVt injection o f [I~'sI]aBuTX. nG, nucleus gracilis; LCn, lateral cervical nucleus; I, marginal layer; I I and l l l, substantia gelatinosa; VH, ventral horn. B: the superior colliculus (SC) and retrosplenial cortex (layers I and VI labeled) after the same treatment as in A. Scale bar 0.5 mm for both.

22l

Fig. 4. Light- (A) and dark-field (B) photomicrographs of the same frontal section through the cochlear nuclei, a, b, c - corresponding cells in the two micrographs 24 h after IVt injection of labeled toxin. Scale bar, 200 l~rn; nChd, dorsal cochlear n. ; nChv, ventralcochlear n. used and on the duration of the incubation with iodinated toxin. At a concentration of 10 -10 M, lOW background levels were obtained and structured patterns of labeling were seen in much the same fashion as those observed in material produced following intraventricular injection. Increasing the molarity of [12aI]aBuTX to 10-8 M resulted in a dramatic rise in background levels of silver grains and an increase in the number of labeled 'neuronal' profiles seen throughout the neuraxis. It is therefore quite probable that the most favorable labeling conditions (i.e. low molarity incubation or intraventricular injection) resulted in the marking of only a small percentage of available toxin receptors. It was apparent that within certain areas of the central nervous system 'neuronal profiles' of silver grains were rarely observed regardless of incubation conditions. These areas included the olfactory bulb, the superior colliculus and ventral geniculate body, the cerebellum and the dorsal horn of the spinal cord. Primary sensoo' areas

Binding of [1251]aBuTX was found at all levels from the olfactory bulb to the spinal cord but from an analysis of these patterns some general conclusions could be reached. With the exception of the dorsal motor nucleus of the vagus (Fig. 6A) and n.

222

Fig. 5. A: dark-field photomicrograph of a parasagittal section through the cerebellunl showing the distribution of silver grain clusters (cl) within folia IX and X; Scale bar, 400/~m. B: bright-field phOtomicrograph of three silver grain clusters (cl) within cerebellar folium X. gin, glomcrulus; Scale bar, 20/*m. Both observed after IVt injection of [i m~l]aBuTX,

223

Fig. 6. Dark-field photomicrographs of heavily labeled areas of the brain stem. A : dorsal motor nucleus of the vagus (DMN X). Frontal section prepared from a fresh cryostat section, nS nucleus of the solitary tract; AP, area postrema; nXII, hypoglossal nucleus. B : the inferior (01) and medial accessory (01 m) olivary nuclei, following 1Vt injection of [a2~l]~tBuTX 24 h survival. Frontal section. C: dorsal tegmental nucleus of Gudden (DTG) prepared from a fresh cryostat section. V, ventricle. Frontal section. Scale bar for all micrographs, 200 ~m.

224 ambiguus no cranial motor nuclei were labeled, in contrast, areas of the brain in direct receipt of sensory afferents generally demonstrated distinct heavy patterns of toxin binding although again certain exceptions were observed. The glomeruli of the main olfactory bulb and the inner portion of the external plexiform layer (Fig. 3A) were heavily labeled while the periglomerular and granule cell bodies were devoid of binding sites. The same layers of the accessory olfactory bulb were also intensely labeled. Similarly heavy binding was seen within areas of optic nerve termination, the ventral geniculate body (Fig. 3B), and the superficial layers of the superior colliculus (Fig. 2B) but not within the dorsal lateral geniculate nucleus (Fig. 3B). The cochlear nuclei, particularly the ventral nucleus, were labeled but in a less diffuse fashion than the previously discussed structures. It was c o m m o n to find bursts of silver grains overlying the neuropil and certain large perikarya (Fig. 4). The medial and lateral vestibular nuclei were lightly and diffusely labele& Silver grains could be seen to cover the soma and dendrites of celts of the dorsal column nuclei. The principal sensory nucleus of the trigeminal and the marginal layer and layer 1II of the substantia gelatinosa of both the dorsal horn of the spinal cord (Fig. 2B) and the spirtat trigeminal nucleus were heavily labeled. The nucleus of the solitary tract was lightly covered in silver grains (Fig. 6A). The cerebellum Folia I, IX. and X of the cerebellum were heavily labeled but in a punctate fashion {Fig. 5A). Bursts of silver grains were observed throughout the granular layer of these fotia and were generally found to overlie areas of neuropil rather than celt bodies (Fig. 5B). Occasional bursts were seen within other folia but these were rare. The vermal regions of folia I. IX. and X were the most heavily labeled areas. The deep cerebellar nuclei were not labeled. Cerebral cortex and limbic system Layers I. V, and VI of the neocortex bind [I~5I]uBuTX. Heavily labeled cell bodies within the superficial layers of the cortex (1-IV) could also be seen particularly in the cingulate cortex (Fig. I B and 2A). Limbic structures of the telencephalon and a number of hypothalamic nuclei were covered in fairly dense accumulations of silver grains. The hippocampus was labeled in a laminar fashion, with silver grains lying over perikarya and the neuropil of the stratum oriens, stratum lacunosum and the hilus of the dentate gyrus. Occasionally labeled fusiform cell bodies were found within the pyramidal cell layer but pyramidal cell bodies were never associated with silver grains (Fig. 1A) s°. The majority of amygdaloid nuclei (and the endopiriform nucleus) with the exception of the central nucleus* were moderately heavily labeled. Hypothalamic structures that were covered in heavy accumulations of silver grains included the

* Silver and Billiar(~0reported that the central nucleus of the amygdala was labeled following theintra ventricular injection of tritiated aBuTX. While we were unable to find evidence of amygdatoid labeling after intraventricular application, cryostat sections revealed a pattern of toxin bindinginalt amygdaloid nuclei except the central nucleus, a result in close agreement with a recent study of CAT and AChE distribution within this complex4. We are unable to explain the discrepancy between the two studies.

225 supraoptic and arcuate nuclei 6° and to a lesser extent the posterior hypothalamic nucleus, paraventricular nucleus, and the medial portion of the medial mammillary nucleus. There was however light labeling of the whole hypothalamic and subthalamic area contrasting with the absence of grains over the majority of dorsal thalamic nuclei. This light, diffuse covering of silver grains continued rostrally and was found over all ventral telencephalic areas including the preoptic area, and the nucleus of the diagonal band. More dorsally the medial septal nucleus was lightly labeled. At most rostral and ventral levels, the olfactory tubercle and anterior olfactory nucleus were covered with large numbers of silver grains. Within the olfactory tubercle however, toxin binding sites were predominantly found within the layers deep to the polymorphic layer but columns of silver grains were seen to pass more superficially towards the pyramidal cell layer.

Thalamus, basal ganglia and mesencephalon The dorsal thalamus and basal ganglia appear to be almost devoid of toxin binding sites. There are exceptions to this however in that the globus pallidus, certain midline thalamic nuclei (n.medialis, n.subparafascicularis, n.rhomboideus, n. parataenialis) and the ventral geniculate nucleus (Fig. 3B) were covered with moderate numbers of silver grains. The parabigeminal and peripeduncular nuclei were heavily overlaid with silver grains while moderate amounts of label were found over the central grey and nuclei of the raph6. The substantia nigra was lightly labeled while large numbers of silver grains were found over the interpeduncular nucleus, nucleus of the lateral lemniscus, and deeper nuclear areas of the inferior colliculus.

The hind brain, medulla, and spinal cord The reticular formation was covered in moderately dense accumulations of silver grains which could be traced rostrally into apparent continuity with the silver grains lying over the subthalamic nucleus, zona incerta and hypothalamus. Within the hind brain there was evidence of a heavier labeling of medial reticular areas. The dorsal territories of the tegmentum contained large numbers of toxin binding sites as judged autoradiographically. Embedded within this region (the stratum griseum externum) was an area of intense labeling, in which silver grains were found to overlie the dorsal tegmental nucleus of Gudden (Fig. 6C). Particularly heavy accumulations of silver grains were found to overlie the superior, inferior, and accessory olivary nuclei. (Fig. 6B). Within the spinal cord labeled motor neuron cell bodies were rarely seen. However, small cell bodies were intensely labeled (Fig. 1C).

Fiber tracts Examination of cortical white matter or of fiber bundles throughout the brain failed to reveal any evidence of toxin binding sites on either the fibers or surrounding glial cells.

226 DISCUSSION

The significance of toxin binding To interpret the observed patterns of [r~5l]aBuTX binding sites within the brain as indicative of sites of cholinergic transmission presupposes that toxin is bound at the synapticjunction and that the presynaptic component is always a cholinergic terminal. Several lines of investigation have indeed suggested that this is the case. Working with tissue homogenates of the whole brain or of discrete brain areas it has been shown that nicotinic but not muscarinic drugs inhibit toxin binding 4e,5~ and that the toxin receptor is preferentially found within the synaptosomal fraction3, 5t~, Using iodinated 24 or horseradish peroxidase-tagged aBuTX 64 the toxin receptor has been localized at anatomically defined synaptic complexes within the hippocampus and retina. It was proposed that the toxin binds to cholinergic synapses in these areas, a suggestion which, in the case of hippocampus, is supported by morphological arguments 24. Using HRP-conjugated aBuTX it has been reported that within certain areas of the rat midbrain and hypothalamus 34 and the chick retina 64 toxin binding sites are found predominantly upon the postsynaptic membrane. Such a distinction is beyond the resolution of both electron and light autoradiographic techniques. Surgical interruption of the cholinerglc septohippocampal pathway failed to reduce the total number of either muscarinic or nicotinic binding sites within the hippocampus 2~.6~. This would indicate that at least within the hippocampus cholinergic receptors are only located postsynaptically. To show that the presynapt~c component releases acetylcholine has proved extremely difficult. The specific cholinergm enzyme CAT has recently been identified immunohistochemically a9 but the approach has not been widely used to define cholinergic pathways within the brain. Acetylcholinesterase, while found throughout the neuraxis, has been shown to be a relatively unreliable indicator of cholinergic synapses 5s,~9. Thus the enzyme is present at sites of physiologically defined cholinergic transmission, but also occurs within clearly non-cholinergic neurons such as the dopaminergic neurons of the substantia nigra 9. A further complication in the application of aBuTX as a nicotinic cholinergic ligand is the apparent failure of the toxin to block synaptic transmission within certain central sites 16,4°. On the basis of experiments with a pheochromocy toma cell line Patrick and Stallcup 47 proposed that neuronal toxin receptors may play a role in trophic interactions rather than in synaptic transmission. Interestingly, a recent experiment has provided support not only for the trophic receptor hypothesis, but also for the assumption that toxin receptors on nerve cells are in fact acetylcholine receptors. Application of aBuTX onto the optic tectum of the toad Bufo marinus was found by Freeman to produce postsynaptic blockade 18. In addition he was able to demonstrate that toxin application results in sprouting of retinal terminals and inhibits reinnervation of the rectum by the regenerating optic nerve.

Cholinergic pathways A number of publications have previously dealt with the distribution of acetyl-

227 cholinesterase and choline acetyltransferase within the central nervous sytem of the rat26,2s,44,45. Such distribution studies coupled with surgical interruption of fiber systems have provided a certain amount of evidence concerning the presence of three cholinergic pathways within the rat brain. These are the septohippocampal pathway 36; the habenulo-interpeduncular pathway21,32; and a less well understood cholinergic pathway probably terminating as mossy fibers within the archicerebellum (folia IX and X) 27. In all cases it has been possible to find toxin binding within discrete areas of the terminal regions of these pathways. In the case of the hippocampus, it was possible to demonstrate at the ultrastructural level that silver grains were associated with synaptic complexes which were closely similar to those identified as cholinergic in previous studies 24. While such detailed data are not available for the other two pathways, there are certain points which deserve consideration. Cholinergic receptors within the interpeduncular nucleus were not found to bind the muscarinic receptor ligand QNB 3z which presumably implies that the receptors at this site are indeed nicotinic. The pattern of cerebellar labeling illustrates dramatically the correspondence between the distribution of acetylcholine-related enzymes and aBuTX binding sites. CAT and AChE are heavily concentrated within cerebellar folia IX and X and thought to reflect the presence of a cholinergic mossy fiber input 2v. The closely overlapping distribution of these enzymes and aBuTX binding activity provides a strong argument in favor of an intimate relation between nicotinic AChR and the toxin receptor. The bursts of silver grains found within the granule layer of certain cerebellar folia clearly resemble mossy fiber terminal structures and may well reflect toxin binding to postsynaptic receptors of a cholinergic mossy fiber input. The origin of this pathway is unknown but the distribution pattern of grain bursts shows an obvious correspondence with the fields of termination of both primary and secondary vestibular inputs 4:~.

Sensory pathways The aBuTX binding pattern within certain areas in direct receipt of sensory input prompts the question as to whether these pathways utilize acetylcholine as their neurotransmitter. In the case of the olfactory and optic nerves of the rat this is almost certainly not the case. Examination of the labeled olfactory glomerulus at the electron microscope level 25 indicated that silver grains were not associated with the synaptic contacts formed by incoming olfactory nerve fibers. Biochemical studies have also indicated that section of either the olfactory 3s or optic nerve 5 does not result in a fall in the levels of CAT or AChE within the relevant brain areas. Similarly, low levels of the two cholinergic enzymes were found within the dorsal roots of the spinal cord 4s. A possibility that should be considered is that cholinergic pathways from central areas of the brain terminate within the more peripherally located areas of sensory termination. Such centrifugal pathways have been reported to terminate within the olfactory bulb s, originating within the horizontal limb of the diagonal band .51 and within the cochlear nuclei 56. That toxin binds to the receptors associated with these structures is unlikely in the case of the olfactory bulb. Centrifugal fibers terminate

228 within the periglomerular domain, not within the glomeruli where toxin binding sites were found 49.

The Renshaw celt The termination of motor neuron axon collaterals upon Renshaw cells within the ventral horn of the spinal cord is perhaps the most fully documented nicotinic cholinergic synapse within the central nervous system. Recent attempts to label these synapses with aBuTX in the cat were unsuccessful; only non-specific toxin binding was observed 17. After intraventricular injection of toxin in the rat however we were able to observe a number of small labeled cell bodies lying between, and ventral to, motor neuron perikarya (Fig. 1C). These may be Renshaw cells. The lack of success of the earlier workers is difficult to explain. Insufficient specific radioactivity of the toxin used (ca. 25 times lower than in the present study), high local background levels generated by direct injection into the structure under investigation and obscuring specific binding patterns, species differences, or a combination of these factors may be responsible.

Muscarinic and nicotinic receptors The distribution of nicotinic and muscarinic ligands 33,~2,52 within the central nervous system of the rat reveals both areas of overlap and disparity. For example both nicotinic and muscarinic ligands bind to sites within the substantia nigra; the corpus striatum on the other hand contains very high levels of muscarlnic receptors 33. but is virtually devoid of aBuTX binding activity. Within the hippocampus the point has previously been made 24 that the aBuTX receptor appears to be associated with the interneuronal population while muscarinic ligands are primarily found in association with the pyramidal cells of Ammon's horn 83. Within the cortex a degree of overlap is found in the distribution of receptor sites within the superficial but not deep cortical layers. Muscarinic binding has also been associated with receptors on cranial motor nuclei 52 and within the basal ganglia 33. but apparently not within areas in receipt of sensory input 3z. It is impossible to state from the presently available data that muscarinic and nicotinic receptors are found on the same neurons although in a large number of cases this would seem to be an unwarranted assumption at least in the rat 3t. It has been proposed that in the central nervous system the classification of acetytcholine receptors as nicotinic or muscarinic may be inadequate and that receptors of a mixed type, i.e., responding to both nicotinic and muscarinic ligands, may exist zl,62. In most cases the discrimination between two types of receptor on the same cell. and a single receptor displaying mixed binding characteristics, has been difficult. In a recent study of rat hippocampus pyramidal cells, Bird and Aghajanian 6 observed competition between nicotinic and muscarinic drugs and concluded that receptors there have mixed pharmacological properties. We did not observe toxin binding over pyramidal cells: thus, mixed-type receptors probably do not bind aBuTX. Species differences may well be an important variable in the study of receptor distribution within the central nervous system. Comparing the distribution of aBuTX binding sites within the brain of the rat and the mouse revealed a number of important

229 differences. Thus no binding sites were found within the cerebellum of the mouse, while the caudate-putamen was massively labeled (unpublished obs.)

Labeling of cell bodies and dendrites Large numbers of cell bodies and dendrites were labeled throughout the neuraxis after either cryostat or intraventricular administration of labeled toxin. There are a number of possible explanations for this effect. Silver grains lying over cell bodies and dendrites could reflect (1) labeling of both axosomatic and axodendritic cbolinergic synapses, (2) intracellular movement of label secondary to binding to postsynaptic receptors upon the cell membrane or (3) direct binding to a cytoplasmic component of a damaged neuron iv. The labeling of cells was seen after both cryostat and intraventricular routes of toxin administration and, within the majority of areas examined, the distribution of labeled profiles was the same. The binding of toxin to receptors on both cell bodies and dendrites appears to be the most obvious explanation for this phenomenon. In addition, intracellular uptake may occur in some situations. For example, electron microscopic study of the hippocampus (where labeled neuronal profiles are seen) has revealed a significant number of silver grains over the cytoplasm of dendritic elements, i.e. intracellular labeling 24. Damage to neurons could occur during cryostat sectioning but probably not following intraventricular injection of toxin. Thus it appears that intracellular labeling is not a procedural artifact as suggested by Duggan et al. 17, but arises from some in vivo mechanism. Such an internalization of BuTX has previously been analyzed in muscle fibers. Extrajunctional acetylcholine receptors in denervated muscle turn over rapidly; prior to their degradation they are taken up into the cell, a process that can be followed by exposing the fiber to fluorescently labeled or radioactive aBuTX which binds to the receptor and is internalized with it2,15. Similarly, uptake of radioactivity into nerve cells could be a reflection of receptor turnover in a neuronal synapse that is metabolically more labile (adaptable, 'plastic') than a peripheral neuroeffector junction. Preliminary ultrastructural data indicate that internalized radioactivity may, on occasion, be associated with lysosomes, a phenomenon also observed in denervated muscle 1~. Presynaptic endocytosis (in the course of vesicle recycling) could also lead to internalization of label, however, as a rule, it was the postsynaptic element that contained the silver grains. Further electron microscopic studies are required to settle this question. Certain areas are notably almost devoid of perikaryal labeling under any preparative conditions (e.g. the cerebellar cortex and olfactory bulb) and this would tend to suggest that receptors can be located either upon some part of the postsynaptic dendritic tree or, in other areas of the brain, upon cell bodies where they would in fact be indicative of an axosomatic cholinergic input. Correspondence with the distribution of other neurotransmitter substances A striking aspect of the distribution of aBuTX binding sites, and therefore presumably nicotinic cholinergic receptors is the close correspondence with the reported distribution of serotoninergic axons and terminals. Notable examples of this overlap include the ventral lateral geniculate nucleus 1, the inferior olive 7, the superior

230 colliculus 1, certain portions of the cerebellar granule layer ~0 and the cerebral cortex TM. W i t h i n the h i p p o c a m p u s the p a t t e r n of t e r m i n a t i o n of s e r o t o n i n - c o n t a i n i n g raph6 n e u r o n s exactly matches that of the d i s t r i b u t i o n of toxin b i n d i n g sites found within the structure 41. W h e t h e r the relationship between cholinergic nicotinic receptors a n d serotoninergic i n p u t is m a i n t a i n e d at the single cell level remain~ to be seen.

ACKNOWLEDGEMENTS This research was supported by a g r a n t from the Council of T o b a c c o Research to J . S . S . H . was partly supported by N I H grant No. NS-12078 to H. J. Karten, to w h o m we are greatly indebted for the use of facilities. We also wish to t h a n k him as well as N. Brecha for reading the manuscript.

REFERENCES 1 Aghajanian, G. K., Haigler, H. J. and Bennett. J. L., Amine receptors in CNS. II I. 5-Hydroxytryptamine in brain. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.) Handbook c~/P.s;vchopharmacology, Vol. 5, Plenum Press, New York, 1975. pp. 63-126. 2 Axelrod, D. A., Ravdin, P., Koppel, D. E., Schlessinger, J., Webb, W. W., Elson, E. L. and Podteski T. R., Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers, Proe. nat. Acad. Sci. [ Wash.), 73 (1976)4594-4598. 3 Bartfai, T., Berg, P., Schultzberg, M. and Heilbronn, E.. Isolation of synaptic membrane fraction enriched in cholinergic receptors by controlled phospholipase A2 hydrolysis of synaptic membranes. Biochim. biophys. Acta (Amst.), 426 (1976) 186-197. 4 Ben-Ari, Y., Zigmond, R. E.. Sbute, C. C. D. and Lewis, P. R., Regional distribution of choline acetyltransferase and acetylcholinesterase within the amygdaloid complex and stria terminatis system, Brain Research, 120 (1977)435-445. 5 Bigt, V. and Schober, W., Cholinergic transmission in subcortical and cortical visual centers of rats: no evidence for the involvement of primary optic system, Exp. Brain Res., 27 (1977) 211-223. 6 Bird, S. J. and Aghajanian, G. K., The cholinergic pharmacology of hippocampal pyramidal cells: a microiontophoretic study, Neuropharmacology, 15 (1976) 273-282. 7 Bj6rklund, A., Baumgarten, H. G. and Nobin, A., Chemical lesioningof central monoamine axons by means of 5,6-dihydroxytryptamine and 5,7-dihydroxytryptamine. In E. Costa, G. L. Gessa and M. Sandler (Eds.), Serotonin-new vistas. Histochemistry and pharmacology. Advances in Biochemical Psychopharmacology, Vol. 10, Raven Press, New York. 1974, pp. 13-33. 8 Broadwell, R. D. and Jacobowitz, D. M., Olfactory relationships of the telencephalon and diencephalon in the rabbit. III. The ipsilateral centrifugal fibers to the olfactory bulbar and retrobulbar formations, J. eomp. NeuroL, 170 (1976) 321-346. 9 Butcher, L. L., Talbot, K. and Bilezikjian, L., Acetylchotinesterase neurons in dopamine-containing regions of the brain, J. Neural. Trans., 37 (1975) 127-153. 10 Chan-Palay, V., Fine structure of labeled axons in the cerebellar cortex and nuclei of rodents and primates after intraventricular infusions with tritiated serotonin, Anat. Embryol., 148 (1975) 235265. 11 Chang, C. C. and Lee, C. Y., Isolation of neurotoxins from the venom of Bungarus multicinctus and their modes of neuromuscular blocking action, Arch. int. Pharmacodyn. Ther.. 144 (1963) 241 257. 12 Changeux, J. P., Kasai, M. and Lee, C. V., Use of a snake venom toxin to characterize the cholinergic receptor protein, Proc. nat. Aead. Sci. (Wash.), 67 (1970) 1241-1247. 13 Cowan, W. M., Gottlieb, D. I., Hendriekson, A. E., Price, L. J. and Woolsey, T. A., The autoradiographic demonstration of axonal connections in the central nervous system, Brain Research, 37 (1972) 21-51. 14 Descarries, L., Beaudet, A. and Watkins, K. C., Serotonin nerve terminals in adult rat neocortex, Brain Research, 100 (1975) 563-588.

231 15 Devreotes, P. N. and Fambrough, D. M., Turnover of acetylcholine receptors in skeletal muscle, Cold Spr. Harb. Syrup. quant. Biol., 40 (1975) 237 251. 16 Duggan, A. W., Hall, J. G. and Lee, C. Y., Alpha-bungarotoxin, cobra neurotoxin, and excitation of Renshaw cells by acetylcholine, Brain Research, 107 (1976) 166-170. 17 Duggan, A. W., Hall, J. G., Headly, P. M., Hendry, I. A. and Minchin, M. C. W., Absence of binding of ct-bungarotoxin and cobra neurotoxin to central acetylcholine receptors an autoradiographic study, Neurosci. Lett., 3 (1976) 123-127. 18 Freeman, J. A., Possible regulatory function of acetylcholine receptor in maintenance of retinotectal synapses, Nature (Lond.), 269 (1977) 218-222. 19 Fumagalli, L., DeRenzis, G. and Miani, N., Acetylcholine receptors : number and distribution in intact and deafferented superior cervical ganglion of the rat, J. Neurochem., 27 (1976) 47 52. 20 Greene, L. A., Binding of a-bungarotoxin to chick sympathetic ganglia: properties of the receptor and its rate o f appearance during development, Brain Research, 111 (1976) 135-145. 2l Hattori, T., McGeer, E. G., Singh, V. K. and McGeer, P. L., Cholinergic synapses of the interpeduncular nucleus, Exp. Neurol., 55 (1977) 666-679. 22 Hiley, C. R. and Burgen, A. S. V., The distribution of muscarinic receptor sites in the nervous system of the dog, J. Neurochem., 22 (1974) 159 162. 23 H6kfelt, T., Erde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A., lmmunohistochemical evidence for separate populations of somatostatin and substance P-containing primary afferent neurons in the rat, Neuroscience, 1 (1976) 131 136. 24 Hunt, S. P. and Schmidt, J., The electron microscopic autoradiographic localization of a-bungarotoxin binditag sites within the central nervous system of the rat, Brain Research, 142 (1978) 152-159. 25 Hunt, S. P. and Schmidt, J., Are mitral cells cholinergic? Neuroscience Symposia, Vol. 3 (1978) in press. 26 Jacobowitz, D. M. and Palkovits, M.,Topographic atlas ofcatecholamine- and acetylcholinesterasecontaining neurons in the rat brain. 1. Forebrain (Telencephalon, Diencephalon), J. comp. Neurol., 157 (1974) 13 28. 27 Kasa, P. and Silver, A., The correlation between chotine acetyltransferase and acetylcholinesterase activity in different areas of the cerebellum of rat and guinea pig, J. Neurochem., 16 (1969) 389 396. 28 Kobayashi, R. M., Brownstein, M., Saavedra, J. M. and Palkovits, M., Choline acetyltransferase content in discrete regions of the rat brain stem, J. Neurochem., 24 (1975) 637 640. 29 Koelle, G. B., The histochemical localization of cholinesterases in the central nervous system of the rat, J. comp. Neurol., 100 (1954) 211-235. 30 Kouvelas, E. D. and Greene, L. A., The binding properties and regional ontogeny of receptors for a-bungarotoxin in chick brain, Brain Research, 1| 3 (1976) 111-126. 31 Krnjevi6, K., Acetylcholine receptors in vertebrate CNS. In L. L. lversen, S. D. Iversen and S. H. Snyder (Eds.) Handbook ofPsychopharmacology, Vol. 6, Biogenic Amine Receptors, Plenum Press, New York, 1975, pp. 97 126. 32 Kuhar, M. J., DeHaven, R. N., Yamamura, H. I., Rommelspacher, H. and Simon, J. R., Further evidence for cholinergic habenulo-interpeduncu[ar neurons: pharmacologic and functional characteristics, Brain Research, 97 (1975) 265-275. 33 Kuhar, M. J. and Yamamura, H. l., Localization of cholinergic muscarinic receptors in rat brain by light microscopic radioautography, Brain Research, 110 (1976) 229 243. 34 Lentz, T. L. and Chester, J., Localization of acetylcholine receptors in central synapses, J. Cell Biol., 75 (1977) 258 267. 35 Lewis, P. R. and Shute, C. C. D., The cholinergic limbic system: Projections to the hippocampal formation, medial cortex, nuclei of the ascending cholinergic reticular system and the subfornical organ arid supraoptic crest, Brain, 90 (1967) 521 540. 36 Lewis, P. R., Shute, C. C. D. and Silver, A., Confirmation from choline acetylase analyses of a massive cholinergic innervation of the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215 224. 37 Lowy, J., MacGregor, J., Rosenstone, J. and Schmidt, J., Solubilization of an a-bungarotoxinbinding component from rat brain, Bioehemistry, 15 (1976) 1522 1527. 38 Margolis, F. L., Roberts, N., Ferriero, D. and Feldman, J., Denervation in the primary olfactory pathway of mice: biochemical and morphological effects, Brain Research, 81 (1974) 469 483. 39 McGeer, P. L., McGeer, E. G., Singh, V. K. and Chase, W. H., Choline acetyltransferase localization in the central nervous system by immunohistochemistry, Brain Research, 81 (1974) 373 379. 40 Miledi, R. and Szczepaniak, A. C., Effect of Dendroaspis neurotoxins on synaptic transmission in the spinal cord of the frog, Proe. roy. Soc. B, 190 (1975) 267-274.

232 41 Moore, R. Y. and Halaris, A. E., Hippocampal innervation by serotonin neurons of the midbrain raphe in the rat, J. comp. Neurol., 164 (1975) 171--184. 42 Morley, B. J., Lorden, J. F., Brown, G. B., Kemp, G. E. and Bradley, R. J., Regional distribution of nicotinic acetylcholine receptor in rat brat n, Brain Research, 134 (1977) t 6 I- 166. 43 Palay, S. L. and Chan-Palay, V., Cerebellar cortex, Cytology and Organization, Springer, Berlin, (1974) 348 pp. 44 Palkovits, M. and Jacobowitz, D. M. ,Topographic atlas ofcatecholamine and acetylcholinesterasecontaining neurons in the rat brain. II. Hindbrain (Mesencephalon, Rhombencephalon), J. comp. Neurol., 157 (1974) 14-28. 45 Palkovits, M.,Saavedra, R. M., Kobayashi, R. M. and Brownstein, M. B., Choline acetyltransferase content of limbic nuclei of the rat, Brain Research, 79 (1974) 443450. 46 Parent, A. and Butcher, L. L., Organization and morphologies of acetylcholinesterase-containing neurons in the thalamus and hypothalamus of the rat, J. comp. Neurol., 170 (1976) 205-~226. 47 Patrick, J. and Stallcup, W. B., Immunological distinction between acetylcholine receptor and the a-bungarotoxin-binding component on sympathetic neurons, Proc. nat. Acad. Set. : Wash,), 74 (1977) 4689-4692. 48 Phillis, J. W., The Pharmacology oJSynapses, Pergamon Press, Oxford, 1970. 49 Pinching, A. J. and Powell, T. P. S., The termination of centrifugal fibers in the glomerular layer of the olfactory bulb, J. Cell Set., 10 (1972) 621-635. 50 Polz-Tejera, G., Schmidt, J. and Karten, H. J., Autoradiographic localization of ¢~-bungarotoxinbinding sites in the central nervous system, Nature (Lond.), 258 (1975) 349-351. 51 Price, J.L. andPowell, T. P.S.,Anexperimentalstudyoftheoriginandthecourseofthecentrifugai fibers to the olfactory bulb in the rat, J. Anat. (Lond.), 107 (1970) 215-237. 52 Rotter, A., Birdsall, N. J. M., Burgen, A. S. V., Field, P. M. and Raisman, G., Axotomy causes loss of muscarinic receptors and loss of synaptic contacts in the hypoglossal nucleus, Nature (Lond.), 266 (1977) 734-735. 53 Salvaterra, P. M., Mahler, H. R. and Moore, W. J., Subcellular and regional distribution of levilabeled a-bungarotoxin binding in rat brain and its relationship to acetylchotinesterase and choline acetyltransferase, J. biol. Chem., 250 (1975) 6459-6475. 54 Scheehter, N., Handy, I. C., Pezzementi, L. and Schmidt, J., Distribution of binding sites tbr Czbungarotoxin in the central nervous system and in peripheral organs of the rat, Toxieon, 16 (I 978) 245-251. 55 Schmidt, J., Drug binding properties of an a-bungarotoxin-binding component from rat brain, Molec. Pharmacol., 13 (1977) 283--290. 56 Shute, C. C. D. and Lewis, P. R., Cholinesterase-containing pathways of the hindbrain. Afferent cerebellum and centrifugal cochlear fibers, Nature (Lond.), 205 (1965) 242--246, 57 Shute, C. C. D. and Lewis, P. R., The ascending cholinergic reticular system : Neocorticai, olfactory and subcortical projections, Brahe, 90 (1967) 497-520. 58 Silver, A., Cholinesterases of the central nervous system with special reference to the cerebellum, Int. Rev. NeurobioL, 10 (1967) 57--109. 59 Silver, A., The Biology of Cholinesterases, Frontiers in Biology, Vol. 36, North-Holland, Amsterdam, 1974, 596 pp. 60 Silver, J. and Billiar, R. B., An autoradiographic analysis of [aH]a-bungarotoxin distribution in the rat brain after intraventricular injection, J. Cell Biol., 71 (1976) 956-963. 61 Storm-Mathisen, J., Localization of transmitter candidates in the brain. The hippocampal formation as a model, Progr. Neurobiol., 8 (1977) 119-181. 62 Tebecis, A. K., Cholinergic and noncholinergic transmission in the medial geniculate nucleus of the cat, J. Physiol. (Lond.), 226 (1972) 153 172. 63 Vaughn, J. E. and Peters, A" ,Aldehyde fixati°n ° f nerve fibers, J" Anat" ( Lond. ) , 100(1966)687-705. 64 Vogel, Z., Maloney, G. J., Ling, A. and Daniels, M. P., Identification of synaptic acetylcholine receptor sites in retina with peroxidase-labeled a-bungarotoxin, Proc. nat. Acad. Sci. (Wash:), 74 (1977) 3268-3272.

Note added in proof. Since this report went to press, Segal et al. published a study on a-bungarotoxin binding sites in rat brain (Brain Research, 148 (1978) 105-120)~ Their results, though less extensive, are comparable to those reported here.