Immunohistochemical localization of γ-aminobutyric acid- and aspartate-containing neurons in the rat deep cerebellar nuclei

Brain Research, 439 ( 1988) 3(12-310 Elsevier

302 BRE 15225

Immunohistochemical localization of y-aminobutyric acid- and aspartate-containing neurons in the rat deep cerebellar nuclei Kazuo Kumoi, Naoaki Saito, Takayoshi Kuno and Chikako Tanaka Department of Pharmacology, Kobe University School of Medicine, Kobe (Japan) (Accepted 14 July 1987) Key words: y-Aminobutyric acid; Aspartate; Immunohistochemistry, Deep cerebellar nucleus; Rat

The immunohistochemical localization of y-aminobutyric acid (GABA)- and aspartate-containing neurons was evidenced in the rat deep cerebellar nuclei, using purified antisera to GABA and aspartate, respectively. Most GABA-containing neurons had small cell bodies and were scattered unevenly throughout the deep cerebellar nuclei. The medial cerebellar nucleus had a few GABA-containing ceil bodies and interpositus and lateral nuclei contained many GABA-containing cell bodies. GABA-containing terminals were distributed throughout the nuclei. A large number of various-sized aspartate-containing cells were present in the deep cerebellar nuclei and most of these cells were large or medium-size. Aspartate-containing cells in the medial, interpositus and lateral nucleus were surrounded by GABA-Iike immunoreactive terminals, thereby suggesting the modulation of aspartate-containJng neurons by GABAergic fibers from Purkinje cells.

INTRODUCTION Neurons containing amino acids which function as transmitters have been identified in physiological and pharmacological studies 4. y-Aminobutyric acid (GABA) is an inhibitory neurotransmitter in various brain regions. Aspartate and glutamate are thought to be excitatory amino acids and are closely related to each other in brain functions. In the cerebellum in particular, aspartate may be a transmitter associated with the climbing fibers 22 while glutamate may be a transmitter for the cerebeUar granule cells 6. To visualize neurons contaning an amino acid, immunohistochemical techniques make use of antisera for the enzymes related to the metabolism, such as glutamic acid decarboxylase ( G A D ) for G A B A TM 14,17 aspartate aminotransferase (AAT) I and glutaminase (GLNase) 2 for aspartate and glutamate. An-

tisera for G A B A , aspartate and glutamate have been developed and are used in the demonstration of immunohistochemical distribution of these amino acids in various tissues 3"5'15"2°'21. We prepared G A B A and aspartate antisera and the antisera were purified using affinity chromatography 1°'18a9 The cytoarchitecture and connectivity of the deep cerebellar nuclei have been well demonstrated. G A B A seems to be a transmitter in afferent fibers of nuclei from Purkinje cells, determined in physiological studies 7'8. On the other hand, AATase, GLNase and 7-glutamyl-glutamate-like immunoreactivity has been noted in the efferent neurons of the nuclei ~2. The aim of the present study was to investigate the localization of the G A B A and aspartate immunoxeactivity within the deep cerebellar nuclei and to search for possible interactions between afferent and efferent neurons of the nuclei by staining consecutive

Correspondence: C. Tanaka, Department of Pharmacology, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku. Kobe 650, Japan. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

Nb sections with GABA and aspartate antisera, respectively. MATERIALS AND METHODS

Preparation and purification of antisera to GA BA and aspartate GABA and aspartate antisera were prepared according to Storm-Mathisen et al. 21 and were purified using affinity chromatography ~8,w. In brief, GABA or aspartate was conjugated to bovine serum albumin by glutaraldehyde and the products were mixed with adjuvant and injected intracutaneously into rabbits. Antisera were obtained after 5 to 7 boosters had been given. For the purification of antisera, crude GABA (or aspartate) antiserum was applied to an epoxy-activated Sepharose 4B column coupled with GABA (1.2 pmoi/mg gel) (or aspartate, 1.3 pmol/mg gel), washed with 0.1 M phosphate buffer, pH 7.4 and eluted by 0.1 M glycine buffer, pH 3.0. The eluent was neutralized by Tris and used as the purified GABA (or aspartate) antiserum. Immunohistochemical staining Adult rats of either sex, weighing 200 g, were anesthetized with pentobarbital and per_fiased through the left ventricle with 50 ml of ice-cold Krebs-Ringer ~olution, pH 7.4 followed by 4% paraformaldehyde, 0.2% picric acid and 0.5% glutaraldehyde in 300 mi of 0.1 M phosphate buffer, pH 7.4. The brains were removed and immersed in the post fixative containing 4% paraformaldehyde and 0.2% picric acid for 2 days, then were transferred to 30% sucrose in phosphate buffer and washed for 2 days before sectioning. Coronal sections were cut at 20 pm on cryostat and sections were collected in phosphate-buffered saline containing 0.3% Triton X-100 (PBS-T). Free-floating coronal sections of the rat cerebellum were incubated with 0.2% hydrogen peroxide for 20 min followed by 10% normal goat serum for 20 min to inhibit endogenous peroxidase and non-specific binding of the first antiserum, respectively. The preparations were incubated with purified G A B A or aspartate antiserum diluted to 45 or 873 ng/mi (IgG content) in PBS-T at 4 °C for 3 days, respectively, then were washed with PBS-T and incubated overnight with goat anti-rabbit IgG (Miles) diluted 1:1000 in PBS-T at 4 °C. After another wash with PBS-T, the

sections were incubated overnight with peroxidaserabbit antiperoxidase (Miles) diluted 1:5000 in PBST at 4 °C. The slices were then washed with PBS-T. and reacted with 3.3'-diaminobenzidine (Sigma. 20 mg/100 ml) and 0.005% hydrogen peroxide in 50 mM Tris-HCi buffer, pH 7.4. The sections were transferred to phosphate-buffered saline, collected on a glass slide, air-d~.ied, mounted under coverslip and studied under a bright-field microscope. For the correlation study, two thin (7 pm) consecutive sections were cut on the cryostat, mounted on a glass slide and incubated overnight with purified GABA (0.45 pg IgG/ml) and aspartate (8.73 pg IgG/ml) antiserum at 4 °C, respectively. The sections were washed with PBS-T for 10 min, incubated with fluorescein isothiocynate (~:'ITC)-conjugated goat antirabbit IgG (Miles) diluted 1:100 for 30 min. rinsed. mounted in phosphate buffered glycerol, pH 7.4 and examined under a Zeiss fluorescence microscope. RESULTS

Specificity of the antiserum The purified GABA and aspartate antisera produced selective immunostaining in the rat deep cerebellar nucleus (Figs. 1 and 2). The specificity of immunostaining has been described elsewhere ~s'~9. In brief, absorption of GABA (or aspartate) antiserum with 10 -3 M of free GABA (or aspartate) did not alter the paitern of immunostaining (Figs. 3A,4A). When the purified GABA (45 ng IgG/ml) (or aspartate (873 IgG/ml)) antiserum was absorbed with 10 -3 M of GABA (or aspartate) conjugated to bovine serum albumin used for the immunization, the G A B A (or aspartate)-like immunoreactivity disappeared (Figs. 3B,4B). Furthermore, 10 -3 M of aspartate (or GABA) conjugated to bovine serum albumin was unable to absorb away the GABA (or aspartate)-like immunoreactivity (Figs. 3C,4C). No immunoreactivity was observed when the GABA (or aspartate) antiserum was replaced with preimmunized rabbit serum (Figs. 3D,4D). Distribution of GABA- and aspartate-like immunoreactivities in the deep cerebellar nuclei The cerebellar nuclei were delineated according to the description of Korneliussen 9. GABA-like immunoreacti~,ity was found through-

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Fig. 3. Microphotographs of control experiments for GABA immunostaining. GABA-Iike immunoreactivity in the deep cerebellar nuclei is not altered when 45 ng IgG/ml of the purified GABA antiserum was absorbed with 10 -3 M of free GABA (A). lmmunoreactivity in the nuclei is nil when 45 ng/ml of the purified GABA antiserum was absorbed with 10 -3 M of GABA conjugated to bovine serum albumin (B). GABA-like immunoreactivity in the deep cerebellar nuclei cannot be absorbed away with 10 -3 M of aspartate conjugated to bovine serum albumin (C). There is no immunoreactivity when the GABA antiserum was replaced by preimmunized rabbit serum (D). Bar = 100ltm. Medial

n ucleus

The medial cerebellar nucleus is composed of 3 subdivisions, viz., a dorsolateral protuberance, a middle part and a caudomedial part 9. Round or oval small GABA-Iike immunoreactive cell bodies were scattered in the nucleus. GABA-Iike immunoreactive terminals were observed in the nucleus with the medium density (Fig. 1). GABA-like immunoreactive terminals were seen to surround aspartate-like immunoreactive cells in the nucleus, by staining of the consecutive sections with G A B A and aspartate antiserum, respectively (Fig. 5A,B). Aspartate-like immunoreactivity was found in round or oval, multipolar large and oval small cells of the dorsolateral protuberance (Fig. 2B), round or oval, multipolar medium-sized and oval small cells of

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Though the anterior interpositus nucleus and the posterior interpositus nucleus were demonstrated in Korneliussen's study, it proved impossible to discern the border between the anterior and posterior interpositus nuclei in the coronal sections 9. Thus, we describe here the anterior and posterior interpositus nucleus, as the interpositus nucleus. Round or oval G A B A - l i k e immunoreactive cells were scattered in the interpositus nucleus and were small. The number of G A B A - l i k e immunoreactive cells was larger than the medial cerebeUar nucleus (Fig. 1). GABA-like immunoreactive terminals were observed in the nucleus, with a medium density. In

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Fig. 4. Microphotographs of control experiments for aspartate immunostaining. Aspartate-like immunoreactivity in the deep cerebellar nuclei is not altered when 873 ng lgG/mi of the purified aspartate antiserum was absorbed with 10-~ M of free aspartate (A). lmmunoreactivity in the nuclei is nil when 873 ng/ml of the purified aspartate antiserum was absorbed with 11)-3 M of aspartate conjugated to bovine serum albumin (B). Aspartate-like immunoreactivity cannot be absorbed away with 10-3 M of G A B A conjugated to bovine serum albumin (C). lmmunoreactivitv is absent when the aspartate antiserum was replaced with preimmunized rabbit serum (D). Bar = 100 !t m.

the consecutive sections, many GABA-like immunoreactive dots surrounded the aspartate-like immunoreactive cells in the nucleus and some small cells contained both G A B A - and aspartate-like immunoreactivities (Fig. 5C,D). Oval or triangular, mulfipolar large and oval small aspartate-like immunoreactive cells were found in the nucleus. Most of the large cells had aspartate-like immunoreactive dendrites (Fig. 2). Lateral nucleus Based on the cytoarchitectonics, two subnuclei were demonstrated, viz. a large-celled part and a small-celled part 9. Round or oval small cells exhibiting GABA-like

imrnunoreactivity were scattered in the nucleus. The number of GABA-like immunoreactive cells were larger than those in the interpositus nucleus. GABAlike immunoreactive terminals were observed in the nucleus, with a medium density (Fig. 1).GABA-like immunoreactive terminals surrounded the aspartatelike immunoreactive cells in the nucleus (Fig. 5E,F). Oval, various sized aspartate-like immunoreactive cells were observed in the large-celled part of the nucleus. There were few aspartate-like immunoreactive cells in the small-celled part of the nucleus (Fig.

2). Other nuclear divisions Two other nuclear divisions, a dorsomedial "hump"

308

Fig. 5. Immunofluorescence microphotographs showing GABA-like immunoreactivities (A,C,E) and aspartate-like immunoreactivities (B,D,F) of two consecutive sections of the medial nucleus (A,B), interpositus nucleus (C,D) and lateral nucleus (E,F), respectively. The purified GABA and aspartatc antisera were diluted to 0.45 l~g IgG/ml and 8.73 ~tg IgG/ml, respectively. GABA-Iike immunoreactive terminals surround aspartate-like immunoreactive cells, in consecutive sections. C,D: in the interpositus nucleus, GABA-Iike immuno'eactive small cells show simultaneously aspartate-like immunoreactivity (arrow). Bar = 20 ¢tm.

309 region and dorsomedial crest region, have been documented 9. In the dorsolateral 'hump' region, GABA-Iike immunoreactivity was observed in scattered small cell bodies and terminals with a medium density. Aspartate-like immunoreactivity was found in oval various sized cell bodies (Figs. 1 and 2). In the dorsomedial crest region, GABA-like immunoreactivity was found in small cells and ~erminais with a medium density. Aspartate-like immunoreactivity was observed in triangular or spindle-shaped medium-sized and small cell bodies (Fig ~. 1 and 2). DISCUSSION The present immunohistochemical study revealed the precise localization of GABA- and aspartatecontaining neurons in the deep cerebeilar nuclei of the rat. G A B A and aspartate are differentially distributed in this region. GABA-like immunoreactivity was found in small neurons scattered within the deep cerebellar nuclei and aspartate-like immunoreactivity was observed especially in large and mediumsized cells. lmmunohistochemical studies of G A D , a synthesizing enzyme of G A B A , in the rat deep cerebellar nuclei demonstrated small GAD-positive cells in the medial, interpositus and lateral nuclei ~4. The distribution of GABA-Iike immunoreactive cells is in good agre ;ment with these results. Immunoreactivity in perfusion-fixed sections reflects the t~lal amount of a particular amino acid in the cell, which probably consists of a 'metabolic pool" and a 'transmitter pool '15. In a correlation study, GABA- and aspartate-like immunoreactivities were demonstrated in the same small cells in adjacent sections of the interpositus nucleus (Fig. 5C,D). As G A B A and aspartate antiserum did not react with other amino acids in immunochemical studies 18"~9, our findings may indicate a 'metabolic pool' of asoartate as G A B A precursor a~ well as the coexistence of G A B A and aspartate in a 'transmitter pool'. As the metabolic pathways of G A B A and aspartate are closely interconnected 16, the possible role of aspartate in G A B A metabolism has to be given attention. These data are in contrast with the finding that the distribution of ,:,ells negative for glutamate-like immunoreactivity matched that of neurons strongly

stained for GABA-Iike immunoreactivity 15. Most GABA-containing neurons in the deep cerebellar complex were small. Some of these cells may correspond to the inhibitory short neurons. The cerebellar nuclei were small. Some of these cells may axon produce inh~.bitory postsynaptic potentials (IPSP), monosynaptically, in their target neurons of the deep cerebellar nuclei 7. This inhibitory response could be blocked by picrotoxin and the G A B A antagonist, bicuculline s. Our irnmunohistochemical studies demonstrated the presence of nerve terminals containing GABA in the deep cerebellar nuclei. This evidence indicates that the cerebellar corticofugal pathway terminating in the deep cerebellar nuclei is GABAergic. In the present study, some aspartate containing neurons of the nuclei were surrounded by GABA-containing terminals (Fig. 5). This would suggest that GABAergic fibers originating from Purkinje cells can influence aspartate-containing neurons of the deep cerebellar efferent pathway. According tO Monaghan et al.~2 a number of neurons in ~he deep cerebellar nuclei were AATase-, GLNase- and ),-glutamyl-glutamate-like immunoreactive and the AATase-like immunoreactive neurons were most abundant. They considered the possibility that there were at least 3 populations of cells present in the deep cerebellar nuclei, containing glutamate, aspartate and G A B A as a neurotransmitter, respectively. The present study revealed high aspartate-like immunoreactivity in neurons of deep cerebellar nuclei. Measurement of the concentration of amino acids in different regions of the cerebellum revealed that the content of aspartate in the deep cerebeilar nuclei was larger than that in the white matter, while glutamate concentration in the nuclei was the same as that ip white matter ~3. These results are in good agreement with our findings on aspartate staining. Small cells contained both G A B A and aspartate. while large and medium-sized aspartate-like immunoreactive cells were not GABA-like immunoreactive. These findings suggest that aspartate-like immunoreactivity in some small neurons would show a 'metabolic pool' of aspartate as a G A B A precursor and the immunoreactivity in large and medium-sized cells would indicate a 'transmitter pool" of aspartate or a 'metabolic pool'. As one criterion for proof of the neurotransmitter status of amine acids is demonstration of a selective

310 localization in neurons, we suggest that aspartate may be an excitatory neurotransmitter of some efferent fibers in the deep cerebellar nuclei. Pharmacological and physiological studies on the role of aspartate in these neurons are underway.

ACKNOWLEDGEMENTS This work was funded by grants from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare, Japan. We thank M. Ohara of Kyushu University for critical comments on the manuscript.

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