Brain Research, 489 (1989) 21-30 21 Elsevier

BRE 14532

Autoradiographic visualization of a calcium channel antagonist, [125I] o-conotoxin GVIA, binding site in the brains of normal and

cerebellar mutant mice (pcd and weaver)

Nobuaki Maeda, Kentaro Wada, Michisuke Yuzaki and Katsuhiko Mikoshiba Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, Osaka (Japan)

(Accepted 15 November 1988)

Key words: w-Conotoxin; Calcium channel; Autoradiography; Cerebeilar mutant mouse

An in vitro autoradiographic technique has been used to localize [12sI]to-conotoxin GVIA binding sites in the brains of normal and cerebellar mutant mice. In the brains of normal mice, the highest densities of binding sites were observed at glomeruli of the olfactory bulb, cerebral cortex, caudate nucleus-putamen, hippocampus, and the nucleus of the solitary tract. Moderate densities of the silver grains occurred on the granular layer of the olfactory bulb, the molecular layer of the dentate gyrus, the molecular layer of the cerebellum, and the cochlear nucleus. No specific binding appeared in the white matter or the deep nucleus of the cerebellum, the corpus callosum, the internal capsule and the external plexiform layer of the olfactory bulb. Autoradiographic studies of the cerebella of Purkinje cell degeneration (pcd) mice showed that the distribution of binding sites on the molecular layer of the cerebellum are not affected by the degeneration of Purkinje cells. However, only background levels of the silver grains occurred on the cerebella of agranular weaver mutant mice, suggesting that the receptors for to-conotoxin GVIA in the cerebellum are predominantly distributed on the parellel fibers of granule cells.

INTRODUCTION

Voltage-sensit ive calcium channels play impor tant

roles in neurons. They are involved in neurotrans-

mi t te r release, Ca2÷-based action potent ials and

long- term poten t ia t ion 6'15. Nowycky et al. classified

voltage-sensi t ive calcium channels into three sub-

groups, L-, N-, and T-types 17. 1 ,4-Dihydropyridine

compounds (DHPs) are potent blockers of L-type

calcium channel and are used for the autoradio-

graphic localizat ion of this type of calcium channel in the bra in 3'5'7"19. Recent ly ~o-conotoxin G V I A (to-

CgTx) was purif ied from the venom of a marine

snail, Conus geographus and it has been repor ted

that it is an irreversible blocker of N-type calcium channel 4's'14,ls. to-CgTx also blocks the L-type cal-

cium channel , but this action is part ial and

reversible s. Several studies have indicated that to-

CgTx blocks neuro t ransmi t te r release, but DHPs do

not 9A°'15'21'22. This suggests that DHP-sensi t ive and

to-CgTx-sensitive calcium channels play different

roles, or that they are located in different regions in

neurons. In this study, we used [125I]to-CgTx to

localize autoradiographical ly the to-conotoxin recep-

tor in the mouse brain. A n d we tr ied to de te rmine

whether to-CgTx receptors are located on the pre-

synaptic or the postsynapt ic membrane in the cere-

bel lum by analyzing the brains of cerebel lar mutant

mice.

MATERIALS AND METHODS

Autoradiography and immunohistochemistry Adul t male C57BL/6 mice were decapi ta ted and

their brains were rapidly frozen in liquid ni trogen.

Twelve-~m-thick sections were cut on a cryostat and

thaw-mounted on gela t in-coated slides. The sections

were ai r -dr ied and then s tored at - 4 0 °C for about 15

Correspondence: N. Maeda, Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka 565, Japan.

0006-8993/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)

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,a,

B

Fig. 1. Autoradiograph of [125I]to-CgTx binding sites in a sagittal section of mouse brain. Sagittal serial sections were cut from mouse brain. A section was incubated in 0.5 nM [125I]~o-CgTx (A) and the adjacent section was incubated as in A but in the presence of 1 pM unlabelled to-CgTx (B). Only a low level of silver grains are distributed in B, indicating that most of the silver grains in A are due to the specific binding of [lzsI]to-CgTx.

h. The sections on slides were incubated in the

solution containing 0.5 nM [t25I]to-conotoxin G V I A

(1000 Ci/mmol, Amersham), 0.1% bovine serum

albumin, 150 mM choline chloride and 10 mM

HEPES-Tris, pH 7.4 in the presence or absence of 1

p M cold co-conotoxin G V I A for 30 min at room

temperature. The slides were then washed 4 times

with phosphate-buffered saline (PBS) and the sec-

tions were fixed with 2% paraformaldehyde/0.2%

glutaraldehyde/PBS for 10 min. The slides were

washed 3 times with PBS and once with distilled

water. The dried slides were dipped in Kodak

photographic emulsion NTB2 and stored for 5 days

at 4 °C in the dark. After this exposure, the slides

were processed in Kodak D19 developer at 20 °C for

4 min and then fixed for 5 min.

23

Fig. 2. Autoradiograph of [125I]w-CgTx binding sites in a coronal section of olfactory bulb. GL, glomerular layer; EPL, external plexiform layer; IPL, internal plexiform layer; IGR, internal granular layer. Note the highest density of grains at the glomernli.

To analyze the cerebella of neurological mutant mice, 12-pm-thick sections were cut from the brains of male mutants and the normal littermates, and were mounted on the same slides. The sections were treated with [125I]w-conotoxin GVIA as described above and exposed to Amersham Hyperfilm-3H at 4 °C for 8 h. The autoradiographs were analyzed with computerized image analyzer (RAS system, Amersham).

Immunohistochemistry was performed as de- scribed elsewhere 13.

Animals The Purkinje cell degeneration (pcd) mutation

was maintained on partially inbred C57BL/6 mice originating from the Jackson Lab. (Bar Harbor, Maine). Homozygous pcd/pcd were obtained by intercrossing the heterozygous mice. B6CBA/RI (wv/+) originally from the Jackson Laboratory and heterozygous for weaver (wv) mutation were inter- crossed to produce homozygous wv/wv mice.

RESULTS

Autoradiographic localization of to-conotoxin GVIA receptor in normal mouse brain

Abe et al. indicated that two kinds of specific binding sites for to-CgTx with the dissociation con- stants of 10 pm and 0.5 nM are present in the synaptic plasma membranes of rat brain 1. We also examined the binding sites using the synaptosome fraction of mouse brain and obtained essentially the same results. Therefore, we used 0.5 nM [125I]to" CgTx for autoradiographic visualization of high- and low-affinity binding sites.

Sagittal brain sections revealed high concentra- tions of receptor-associated grains in the glomerular layer of the olfactory bulb, the cerebral cortex, the hippocampus, the caudate nucleus-putamen, and the nucleus of the solitary tract (Fig. 1).

In the olfactory bulb, the glomeruli had the highest density of autoradiographic grains (Fig. 2). Cresyl violet staining of the samples indicated that the grains were confined to the glomeruli and were not distributed on the cell bodies of periglomerular cells (data not shown). The granular layer (IGR) showed a moderate density of autoradiographic grains and the external plexiform layer (EPL) had the background level of grains (Fig. 2).

Within the cerebral cortex, grains were distrib- uted evenly throughout all the layers (Fig. 3). Within the hippocampus, there was a clearly layered distri-

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CI~

B

PV NT VT CI~

~-" FX AL

Fig. 3. Autoradiograph of [~25I]to-CgTx-binding site in coronal sections of the midbrain. CA, anterior commissure; CC, corpus callosum; CPU, caudate nucleus-putamen; FX, fornix; LS, lateral septal nucleus; HPC, hippoearapus; DG, dentate gyms; FI, fimbria of the hippocampus; PV, paraventricular nucleus of the thalamus; MT, medial nucleus of the thalamus; VT, ventral nucleus of the thalamus; LT, lateral nucleus of the thalamus; RT, reticular nucleus of the thalamus; IP, internal capsule; VH, ventromedial nucleus of the hypothalamus; AL, lateral amygdaloid nucleus.

bution of the silver grains. The stratum radiatum (RD) and stratum oriens (OR) had the greatest density of the grains and between them the pyrami-

dal cell layer (PY) had a lower density. The stratum moleculare and stratum lacunosum (MO) of the hippocampus had a lower density of the grains than

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Fig. 4. Autoradiograph of [125I]to-CgTx binding sites in a sagittal section of hippocampus. HPC, hippocampus; OR, stratum oriens; PY, pyramidal cell layer; RD, stratum radiatum; MO, stratum moleculare-lacunosum of the hippocampus; DN, dentate gyrus; ML, molecular layer; GR, granular layer of the dentate gyms.

Fig. 5. Autoradiograph of [l~I]o~-CgTx binding sites in a coronal section of cerebellum and medulla. MC, molecular layer of the cerebellum; GC, granular layer of the cerebellum; ICN, intermediate deep nucleus of the cerebellum; LCN, lateral deep nucleus of the cerebellum; SN, nucleus of the solitary tract; CN, cochlear nucleus.

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A B

D

Fig. 6. Autoradiographic study of the cerebella of control and pcd mutant mice. A-D: analysis of 50-day-old mice. A coronal section of the cerebellum of a 50-day-old pcd/pcd mouse was immunohistochemically stained with the monoclonal antibody against Purkinje cell-characteristic P40o protein (A). A section from a normal littermate was stained as in A (B). This monoclonal antibody stains the dendrites, the soma and the axons of Purkinje cells, and so the molecular layer, the Purkinje cell layer, and the axon terminals of Purkinje ceils at the deep nucleus are stained in the cerebellum. All the Purkinje cells were stained in the cerebellum of the normal littermate, but most of the Purkinje cells had degenerated and are not stained by the antibody in the cerebellum of the mutant. In spite of the degeneration of the Purkinje cells, the results of the autoradiography with [~2sI]to-CgTx show no difference between the mutant (C) and the normal littermate (D). E,F: analysis of the 28-day-old pcd mutant. A coronal section from a 28-day-old pcd/pcd mutant mouse was immunohistochemically stained as above (F), Most of the Purkinje ceils which still survive at this age are in the vermis and the nodulus, both of which were stained by the antibody, but most Purkinje cells in the other parts of the cerebellum had already degenerated. However, there is no difference in the density of the silver grains between the molecular layers of vermis, nodulus and the other parts (E, see also Table I).

the ne ighbor ing s t ra tum rad ia tum (Fig. 4). The

m o l e c u l a r layer of the den ta t e gyrus (ML) showed

the same densi ty of the grains as the s t ra tum

molecu l a r e of h i p p o c a m p u s (Fig. 4).

Wi th in the tha lamus , the pa raven t r i cu la r nuc leus

(PV) , the media l nucleus ( M T ) and the ven t ra l

27

i !i i ii 2 :

Fig. 7. Autoradiographic study of the cerebellum of a weaver mutant mouse. Sagittal sections of the brains of a 30-day-old wv/wv mouse (B) and a normal littermate (A) were analyzed by autoradiography with [125I]~o-CgTx. The cerebellum (CR) of the wv/wv mouse displayed almost no specific binding (B). PC, posterior colliculus.

nucleus (VT) had moderate grain density, and the

other parts had lower densities (Fig. 3).

The molecular layer of the Cerebellum (MC) had a moderate level of receptor binding and the

granular layer (GC) had a lower level. The Purkinje cell layer, the white matter and the deep nucleus of

TABLE I

Binding activity of [125I#o-CgTx to the molecular layer of cerebellum

O.D. values (mean + S.D.) of the randomly selected points on the molecular layers of the cerebella were read from the autoradiographs of normal and pcd mouse brains. Number of sites examined between brackets. The O.D. values of the molecular layers of the 28-day-old pcd/pcd mouse at the points indicated by • and "A" in Fig. 6 are also shown.

O.D. values

28-day-old 50-day-old

Normal mice 0.18 + 0.02 (12) 0.20 + 0.03 (13) pcd/pcdmice 0.17 + 0.02 (19) 0.21 + 0.02 (12) • 0.16 (2) - ~t 0.16(1) -

cerebellum had the background level of the grains

(Fig. 5). Within the medulla, the nucleus of the solitary

tract (SN) had the highest grain density and the

cochlear nucleus (CN) had the moderate density,

The other parts showed the lower or background level (Fig. 5).

Analysis of the location of w-conotoxin GVIA recep- tors by autoradiography of pcd and weaver mice cerebella

From the above autoradiographic studies of nor-

mal brain, it can not be determined on what kinds of

cells the receptors are distributed, and whether the

receptors are present on the presynaptic site or on the postsynaptic membrane. We studied these points

for the cerebellum by using cerebellar mutant mice. In the normal cerebellum, moderate concentrations

of receptor-associated silver grains occurred on the molecular layer. These grains may be attributed to the Purkinje cell dendrites, or the parallel fibers of granule cells, or the interneurons.

Purkinje cell degeneration mutant mice (pod) lose

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almost all of the Purkinje cells and the major loss occurs between postnatal days 22 and 28 t6. But the

granule cells are only moderately affected by post- natal day 50. Fig. 6A,B shows the immunohisto- chemistry of the cerebella from 50-day-old normal and pcd mutant mice. They were stained by the ABC method with the monoclonal antibody against Pur- kinje cell-characteristic P4oo protein. In the normal mouse cerebella, all of the Purkinje cells were stained (Fig. 6B). In contrast, in the cerebella of the pcd mutants of the same age, almost all of the Purkinje cells degenerated and no stained cells appeared (Fig. 6A). But the densities of the a~-CgTx receptors at the molecular layers of the normal and mutant cerebella were almost the same (Fig. 6C,D and Table I). At postnatal day 28, the Purkinje cells persisted at the vermis and nodulus but most of these cells were lost in the other part of the cerebellum (Fig. 6F). There was almost no difference in the densities of the autoradiographic grains between the molecular layers of the severely affected regions and the regions where most of the Purkinje cells per- sisted (Fig. 6E,F and Table I). These results sug- gested that most of the autoradiographic grains in the molecular layer of the cerebellum are not present on the dendrites of Purkinje cells but are on the parallel fibers or the interneurons.

Next, we analyzed the cerebella of weaver (wv) mutant mice. Weaver mutant mice are characterized by the degeneration of granule cells in the cerebel- lum. Purkinje cells and interneurons persist in the cerebella of these mutants, although the dendritic arborization of Purkinje cells is poor and disoriented 2°,23. Fig. 7 shows the results of autora- diography of weaver mutant mice. The cerebella of these mice showed only the background level of autoradiographic grains (Fig. 7B), but there was a moderate density of grains on the molecular layer of the cerebella of normal littermates (Fig. 7A). These results suggested that most of the autoradiographic grains on the molecular layer of the cerebellum can be explained by the receptors on the parallel fibers

of granule cells.

DISCUSSION

The present results show a heterogeneous local- ization of ~o-CgTx-binding sites in the mouse brain.

Such a distribution in the brain is quite different from that of DHP receptors. For example, in the olfactory bulb, the highest density of the DHP receptor was located at the external plexiform layer 3'7, but the density of ~o-CgTx receptors is extremely low in that region. The greatest density of ~o-CgTx receptors in the olfactory bulb was at the glomeruli and a moderate density of DHP receptors was also located in that region 3'7. In the glomeruli, the olfactory fibers form synapses on the dendrites of the mitral cells, the tufted cells and of the periglo- merular cells. There also are dendro-dendritic syn- aptic connections between dendrites of mitral cells and periglomerular cells. The external plexiform layer is abundant in the dendro-dendritic synapses between the dendrites of mitral cells and granule cells. It has been suggested that DHP receptors in this layer are involved in the dendro-dendritic interactions between these cells 7. The precise local- ization of the e)-CgTx and DHP receptors in the glomeruli is unknown, but ~o-CgTx-sensitive calcium channels are considered to be involved in the neurotransmitter-release at presynaptic terminals, and so the ~o-CgTx receptors may be on the presynaptic terminal of olfactory fibers and the DHP receptors may function also in the dendro-dendritic interactions between mitral cells and periglomerular cells.

The hippocampus displayed clear differences in grain density between constituent layers. The high- est densities of the receptors were in the stratum oriens and stratum radiatum; a moderate density of grains is located at the pyramidal cell layer and at the stratum lacunosum-moleculare. The pyramidal cells receive characteristic inputs from different afferents at each layer. For example, CA3 pyramidal neuron dendrites receive entorhinal afferents at the stratum lacunosum-moleculare, and commissural afferents and septal inputs are confined to the stratum oriens and stratum radiatum. The mossy fibers from the dentate gyrus terminate on the proximal region of the dendrites and basket cells synapse upon the cell soma. The density differences between the layers may reflect the characteristics of these afferent fibers. The dentate gyrus displayed a moderate density of ~o-CgTx receptors that is lower than that of the stratum radiatum of the hippocampus. How- ever, the molecular layer of the dentate gyrus has a

great density of DHP receptors 7. Lesion of granule cells by colchicine deleted the binding sites of DHP, suggesting that the DHP receptors are on the dendrites of granule ceUs 2. A similar experiment should reveal the site of ~o-CgTx receptors in the dentate gyrus.

In the cerebellum, the molecular layer displayed a moderate density of to-CgTx receptors, the gran- ular layer had a low density, and a background level appeared in the white matter and in the deep nucleus. This distribution is again quite different from that of DHP-receptors. The greatest density of

DHP receptors in the cerebellum was localized at the granular layer, and the molecular layer displayed lower densities 3. It is interesting to determine whether to-CgTx receptors are present on the den- drites of Purkinje cells, since they display potent calcium spikes ~2. But the analysis of the pcd and weaver mouse brains indicated that there is no, or an extremely low density of to-CgTx receptors on the

Purkinje cells. Because the density of the DHP receptors on the molecular layer of the cerebellum is quite low 3, it is not known what type of calcium channel is responsible for the calcium spikes gener- ated on the dendrites of Purkinje cells. Analysis of the mutants suggested that the to-CgTx receptors at the molecular layer of the cerebellum are present

REFERENCES

1 Abe, T., Koyano, K., Saisu, H., Nishiuchi, Y. and Sakakibara, S., Binding of to-conotoxin to receptor sites associated with voltage-sensitive calcium channel, Neuro- sci. Lett., 71 (1986) 203-208.

2 Cortes, R., Supavilai, P., Karobath, M. and Palacios, J.M., The effects of lesions in the rat hippocampus suggest the association of calcium channel blocker binding sites with specific neuronal population, Neurosci. Lett., 42 (1983) 249-254.

3 Cortes, R., Supavilai, P., Karohath, M. and Palacios, J.M., Calcium antagonist binding sites in the rat brain: quanti- tative autoradiographic mapping using the 1,4-dihydropy- ridines [3H]PN200-110 and [3H]PY108-068, J. Neural Transm., 60 (1984) 169-197.

4 Cruz, L.J. and Olivera, B.M., Calcium channel antagonist, to-conotoxin defines a new high affinity site, J. Biol. Chem., 261 (1986) 6230-6233.

5 Ferry, D.R., Goll, A., Gadow, C. and Glossmann, H., (-)-3H-desmethoxyverapamil labelling of putative calcium channels in brain: autographic distribution and allosteric coupling to 1,4-dihydropyridine and diltiazem binding sites, Naunyn-Schmiedeberg's Arch. Pharmacol., 327 (1984) 183-187.

6 Gamble, E. and Koch, C., The dynamics of free calcium

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predominantly on the parallel fibers of granule cells.

They are presumably involved in the neurotransmit-

ter-release from the parallel fibers. This is consistent with the suggestion by Takemura et al. 24 that

to-CgTx binding sites are confined to presynaptic sites. But to-CgTx-sensitive or N-type channels were detected on the neurite and on the cell bodies of cultured neurons 8"11"21, so it seems premature to say

that co-CgTx receptors are restricted to the presyn- aptic sites. Further, to-CgTx-sensitive channels may not be the only ones that are responsible for the

neurotransmitter-release in the brain, since there is no specific binding of to-CgTx at the cerebellar nucleus, where the Purkinje cell axons terminate.

In summary, the distribution of ~o-CgTx receptors in the brain is quite different from that of DHP receptors. This suggests that they play different roles in the brain. Further, it was suggested that the to-CgTx receptors in the cerebellum are present mostly on the parallel fibers.

ACKNOWLEDGEMENT

This study was supported by grants from the Japanese Ministry of Education, Science, and Cul- ture.

in dendritic spines in response to repetitive synaptic input, Science, 236 (1987) 1311-1315.

7 Gould, R., Murphy, K.M. and Snyder, S.H., Autoradio- graphic localization of calcium channel antagonist recep- tors in rat brain with [3H]nitrendipine, Brain Research, 330 (1985) 217-223.

8 Kasai, H., Aosaki, T. and Fukuda, J., Presynaptic Ca- antagonist ~o-conotoxin irreversibly blocks N-type Ca- channels in chick sensory neurons, Neurosci. Res., 4 (1987) 228-235.

9 Kerr, L.M. and Yoshikarni, D., A venom peptide with a novel presynaptic blocking action, Nature (Lond.), 308 (1984) 282-284.

10 Koyano, K., Abe, T., Nishiguchi, Y. and Sakakibara, S., Effects of synthetic to-conotoxin on synaptic transmission, Eur. J. Pharmacol., 135 (1987) 337-343.

11 Lipscombe, D., Madison, D.V., Poenie, M., Reuter, H., Tsien, R.Y. and Tsien, R.W., Spatial distribution of calcium channels and Cytosolic calcium transients in growth cones and cell bodies of sympathetic neurons, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 2398-2402.

12 Llinas, R. and Sugimori, M., Electrophysiological proper- ties of in vitro Purkinje cell dendrites in mammalian cerebellar slices, J. Physiol. (Lond.), 305 (1980) 197-213.

13 Maeda, N., Niinobe, M., Nakahira, K. and Mikoshiba, K., Purification and characterization of P4oo protein, a glyco-

30

protein characteristic of Purkinje cells, from mouse cere- bellum, J. Neurochem., 51 (1988) 1724-1730.

14 McCleskey, E.W., Fox, A.P., Feldman, D.H., Cruz, L.J., Olivera, B.M. and Tsien, R.W., w-Conotoxin: direct and persistent blockade of specific types of calcium channels in neurons but not muscle, Proc..Natl. Acad. Sci. U.S.A., 84 (1987) 4327-4331.

15 Miller, R.J., Multiple calcium channels and neuronal function, Science, 235 (1987) 46-52.

16 Mullen, R.J., Eicher, E.M. and Sidman, R.L., Purkinje cell degeneration, a new neurological mutation in the mouse, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 208-212.

17 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Three types of neuronal calcium channel with different calcium agonist sensitivity, Nature (Lond.), 316 (1985) 440-443.

18 Olivera, B.M., Gray, W.R., Zeikos, R., Mclntosh, J.M., Varga, J., Rivier, J., Santos, V. and Cruz, L.J., Peptide neurotoxins from fish hunting cone snails, Science, 230 (1985) 1338-1343.

19 Quirion, R., Autoradiographic localization of a calcium channel antagonist, [3H]nitrendipine binding site in rat

brain, Neurosci. Lett., 36 (1983) 267-271. 20 Rakic, P. and Sidman, R.L., Organization of cerebellar

cortex secondary to deficit of granule cells in weaver mutant mice, J. Comp. Neurol., 152 (1973) 133-162.

21 Reynolds, I.J., Wayner, J.A., Snyder, S.H., Thayer, S.A., Olivera, B.M. and Miller, R.J., Brain voltage-sensitive calcium channel subtypes differentiated by a)-conotoxin fraction GVIA, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 8804-8807.

22 Sano, K., Enomoto, K. and Maeno, T., Effects of synthetic w-conotoxin, a new type Ca ~+ antagonist, on frog and mouse neuromuscular transmission, Eur. J. Pharmacol., 141 (1987) 235-241.

23 Sotelo, C., Anatomical, physiological and biochemical studies of the cerebellum from mutant mice II. Morpho- logical study of cerebellar cortical neurons and circuits in the weaver mouse, Brain Research, 94 (1975) 19-44.

24 Takemura, M., Kiyama, H., Fukui, H., Tohyama, M. and Wada, H., Autoradiographic visualization in rat brain of receptors for to-conotoxin GVIA, a newly discovered calcium antagonist, Brain Research, 451 (1988) 386--389.