Purification of specific antibody against aspartate and immunocytochemical localization of aspartergic neurons in the rat brain

Neuroscience Vol. 21, No. 3, pp. 755-765,

0306-4522/87$3.00+ 0.00 Pergamon Journals Ltd 0 1987 IBRO

1987

Printed in Great Britain

PURIFICATION OF SPECIFIC ANTIBODY AGAINST ASPARTATE AND IMMUNOCYTOCHEMICAL LOCALIZATION OF ASPARTERGIC NEURONS IN THE RAT BRAIN E. AOKI,* R. SEMBA, K. KATO~ and S. KASHIWAMATA Departments of Perinatology and tBiochemistry, Institute for Developmental Research, Aichi Prefecture Colony, Kasugai, Aichi 480-03, Japan

Abstract-The distribution of L-aspartate known as a putative excitatory neurotransmitter

in the central nervous system was investigated immunocytochemically in the rat brain. Anti-aspartate antiserum was raised in rabbits using L-aspartate covalently conjugated to rabbit serum albumin with glutaraldehyde as the immunogen and was found to be cross-reactive with an L-glutamate conjugate. Monospecific anti+aspartate antibody was successfully purified using affinity gels coupled with several amino acids including L-aspartate and L-glutamate and with the L-glutamate conjugate. Putative aspartergic neurons were generally immunoreactive to the purified antibody, but epithelia of the choroid plexus were also stained. These results show that the antibody is a useful tool for the immunocytochemical demonstration of possible aspartergic neurons in the central nervous system, although the immunochemical expression of L-aspartate not used as a neurotransmitter must be taken into consideration.

(rabbit) were obtained from Miles-Yeda, Israel, and glutaraldehyde (GAL) was from TAAB Laboratories U.K. Epoxy-activated Sepharose 6B and cyanogen bromideactivated Sepharose 4B gels were purchased from Pharmacia Fine Chemicals, Sweden. Pre-coated silica gel plates for high-performance thin-layer chromatography and polystyrene balls (3.18 mm diameter) were from Merck, F.R.G., and Plastic Ball Company, Chicago, respectively. Nihon white rabbits were suonlied from Chubu Kagaku, Japan. All other chemicals were of the purest grade commercially available.

L-Aspartate has been suggested to be an excitatory neurotransmitter in CNS of vertebrates.3v15*‘6 Electrophysiological studies,10.24*29 autoradiography of [3H]aspartate uptake9s’2,19 and enzyme histochemical” and morphological organization of aspartergic neurons remain obscure due to a lack of direct visual-

tative aspartergic neurons. However, precise location and morphological organization of aspartergic neurons remain obscure due to a lack of direct visualization of aspartate in the tissue. In 1983, StormMathisen et a1.16 successfully produced anti-GABA and anti-glutamate antisera, and suggested that an anti-aspartate antiserum for the histological demonstration of aspartergic neurons. In the present report, we investigated the distribution of possible aspartergic neurons in the rat brain using monospecific anti-L-aspartate antibody. EXPERIMENTAL

Preparation and puriJcation of anti-aspartate antiserum

PROCEDURES

Materials

L-Aspartate, glycine, p-alanine and rabbit serum albumin of Seikagaku Kogyo, and (RSA) were products y-aminobutyric acid (GABA), L-glutamate, taurine and 4-methylumbelliferone were from Nakarai Chemicals, Japan. 4-Methylumbelliferyl-)?-D-galactoside was from Sigma Chemical Co., St. Louis. P-o-Galactosidase [B-D-galactoside galactohydrolase, EC 3.2.1.231 from Escherichiu coli was from Boehringer-Mannheim. Anti-rabbit IgG goat serum and peroxidase-antiperoxidase complex *To whom reprint requests should be sent: Eiko Aoki, Department of Perinatology, Institute for Developmental Research, Aichi Prefecture Colony, Kasugai, Aichi 480-03, Japan. Abbreviations: GABA, y-aminobutyric acid; GAL, glutaraldehyde; RSA, rabbit serum albumin. 755

Anti-aspartate antiserum was produced essentially according to the procedure of Storm-Mathisen et al,,26 except RSA were substituted for and aspartate that GABA/glutamate and bovine serum albumin, respectively. Purity of aspartate was verified by thin-layer chromatography developed with three solvent systems, that is, 96% ethanol/distilled water (63/37, v/v); n-butanollacetic acid/distilled water (60/20/20, v/v); and 96% ethanol/34% ammonia (67/23, v/v). Aspartate (lOOpmoB and RSA (12 mg) were dissolved in 2 ml of 0.2 M sodium phosphate buffer, pH 7.4, and GAL (100 pmol) was added to initiate the reaction to produce an aspartateGAL-RSA complex. After incubation for 20 h at 20°C the reaction was terminated by adding 0.1 ml of sodium borohydride solution (4 mg/ml). The aspartateGAL-RSA conjugate was dialysed for 48 h at 4°C against 2 litres of 0.1 M sodium phosphate buffer, pH 7.4, and emulsified with an equal volume of complete Freund’s adjuvant. One millilitre of the emulsion was injected intracutaneously into the multiple sites on the back of the rabbit to raise anti-aspartate antiserum. Forty and sixty days later immunizations were repeated with the same volume as mentioned above, and the blood was sampled 10 days after the last injection. For the purification of anti-aspartate antibody, the antiserum was passed in sequence through affinity gel columns of glutamate, GABA and taurine immobilized on epoxy-activated Sepharose 6B to eliminate antibodies possibly cross-reactive to these neurotransmitter candidates. Then the anti-aspar-

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tate antibody was recovered from an affinity gel column of aspartate,” and finally was passed through a column of glutamate-GAL-RSA immobilized on cyanogen bromideactivated Sepharose 4B gel.

Enzyme immunoassay Purity and specificity of the antibody were checked by a sandwich-type enzyme immunoassay system.’ Polystyrene balls were coated with aspartate_GAL-RSA conjugate solution at 0.3 mg/ml (optical concentration) in 0.1 M sodium phosphate buffer, pH 7.0, for 16 h at 4°C. After this period, the balls were rinsed three times with the above buffer. Then, they were filled with buffer A (0.01 M sodium phosphate buffer, pH 7.0, containing 0. I M NaCl, 0.1% bovine serum albumin and 0.1% NaN,) and left overnight at 4°C to saturate the balls perfectly and to prevent the non-specific binding of antibody. Purified antibody or unpurified antiserum, which was preincubated for 16 h at 4°C with one of the conjugated amino acids or non-conjugated aspartate, was applied onto the balls coated with aspartateGALRSA. After incubation at 30°C for 5 h with shaking, the reaction medium was removed by aspiration. Each ball was washed three times with 1 ml of buffer A in a test tube (10 x 75 mm), then transferred to another test tube containing b-o-galactosidase-labelled anti-rabbit IgG in 200 ~1 of buffer A, and incubated at 4°C overnight. The ball was washed again three times with buffer A and transferred to a third test tube. The activity of j?-D-galactosidase activity bound to the polystyrene ball was assayed as follows. The ball was preincubated in 0.1 ml of buffer A at 30°C for 5 min and the enzyme reaction was started by adding 50~1 of 3 x lO-4 M 4-methylumbelliferyl-/?-o-galactoside. After incubation for 20 min at 30°C with shaking, 2.5 ml of 0.1 M glycine_NaOH buffer, pH 10.3, was added and the amount of 4-methylumbelliferone liberated was measured with a Hitachi 204 spectrofluorometer (excitation at 360nm and emission at 450 nm) using 4-methylumbelliferone used as a standard.

Immunocytochemical procedure SpragueeDawley rats were obtained from a closed colony of our laboratory. At postnatal day 30 or 40, they were perfused via the heart with a mixture of 1% GAL, 4% paraformaldehyde, 0.2% picric acid and 2% sucrose in 0.1 M sodium acetate buffer, pH 6.0.23 Each brain was sectioned in slices of about 3 mm thickness, kept in the above fixative for 45 h, rinsed twice with 50 mM Tris-HCl buffer, pH 7.6, containing 150mM NaCI, and kept overniaht in the same buffer at 4°C. Slices were embedded in described.2 Three-pmpolyethylene glycoln as previously thick sections cut with a glass knife were floated on the surface of the purified anti-aspartate antibody (0.1 pg/ml) in 50 mM Tris-HCl buffer, pH 7.6, containing 500 mM NaCl and incubated overnight. The location of aspartate was disclosed by the peroxidasc-antiperoxidase methodz5 with diaminobenzidine as chromogen. To ascertain the location of immunonegative cells, some sections were counterstained with Toluidine Blue (not shown). Control sections were prepared by using the antiserum exhaustively preabsorbed with the aspartateGAL-RSA complex which was coupled with epoxy-activitated Sepharose 6B. Sections were mounted with Malinol (Muto Pure Chemicals, Ltd, Japan). Identification of nuclei in CNS was done according to the atlas of Pellegrino ef al.*O

RESULTS

Purity and specificity of anti-aspartate

antibody

The anti-aspartate antiserum was found to be cross-reactive mainly to the glutamate-GAL-RSA complex (Fig. 1). As the cross-reactivity was not

-7

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log

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c

Fig. 1. Displacement curve of unpurified anti-aspartate antiserum established with an enzyme immunoassay system. Competitions were performed between aspartate_GALRSA coated on polystyrene balls and either each of the conjugated amino acids or non-conjugated aspartate, previously incubated with unpurified anti-aspartate antiserum for 17 h at 4°C. B/B,, is the ratio between the fluorescence intensity with competition (B) and without (I&,). C denotes the concentration of each competitor mentioned below. Each point represents the average of two experiments. 0, aspartateGAL-RSA; 0, glutamate-GAL-RSA; A, GABA-GAL-RSA; n , glycine-GAL-RSA; A, taurineGAL-RSA; 0, p-alanineGAL-RSA; jl-, non-conjugated aspartate.

satisfactorily eliminated by affinity chromatography using each of aspartate, glutamate, GABA and taurine immobilized on epoxy-activated Sepharose 6B, the antiserum was passed through a column of glutamate-GAL-RSA immobilized on cyanogen bromide-activated Sepharose 4B. The finally purified anti-aspartate antibody was no longer cross-reactive to the glutamate, GABA, taurine and glycine-GALRSA complexes as judged by the enzyme immunoassay (Fig. 2, Table 1). Immunocytochemical

observations

The anti-aspartate antibody was effective in visualizing the location of aspartate in the rat brain. Aspartate appeared to be concentrated mostly in putative aspartergic neurons. Aspartate-like immunoreactivity was not observed in glial cells. Telencephalon Most of neurons in the external and internal pyramidal cell layers of the cerebral cortex, especially those in the cingulate gyrus, were intensely stained with the anti-aspartate antibody (Fig. 3). Neurons in the pyramidal and granule cell layers of the hippocampus were labelled a little, while some strongly immunopositive neurons were found scattered in the reticular and molecular layers (Fig. 4). Most neurons in the medial habenular nucleus were highly immunoreactive (Fig. 5). The dark-coloured cell bodies appeared in close proximity to each other. Some neurons of the lateral habenular nucleus were also stained.

Immunocytochemistry

of aspartergic

neurons

in rat

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strongly immunoreactive to the anti-aspartate antibody. Fibres in the lateral lemniscus were well disclosed (Fig. 8). Pons

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Fig. 2. Displacement curve of purified anti-aspartate antibody established with an enzyme immunoassay system. Competitions were performed between aspartateGALRSA coated on polystyrene balls and either each of the conjugated amino acids, previously incubated with purified anti-aspartate antibody for 17 h at 4°C. B/B0 is the ratio between the fluorescence intensity with competition (B) and without (Et,). C denotes the concentration of each competitor mentioned below. Each point represents the average of two experiments. 0, aspartate_GAL-RSA; 0, glutamate-GAL-RSA; A, GABAGAL-RSA; n , glycine GAL-RSA; A, taurin+GAL-RSA; 0, b-alanine GAL-RSA.

Diencephalon

Staining intensity of the nuclei in the thalamus showed some regional variations. In the ventral thalamic nucleus, its ventral part was more intensely stained than the dorsal part (Fig. 6). In the lateral thalamic nucleus, fibres were weakly stained while cell bodies showed little reactivity. The reuniens and rhomboid nuclei, but not the dorsomedial nucleus, were immunopositive. Relatively intense reaction was observed in spindle-shaped cells of the zona incerta. In the hypothalamus, a few round and weakly stained cells were found. Mesencephalon

Figure 7 shows large monopolar cells and their dendrites in the oculomotor nucleus, which were

Table

1. Specificity

of anti-aspartate

antibodies

Cross-reactivity Compounds AspartateGAL-RSA Glutamate-GAL-RSA GlycineeGAL-RSA GABAGAL-RSA Taurine-GAL-RSA /I-alanine-GAL-RSA Non-conjugated aspartate

Unpurified antiserum l/8191 l/2000 l/3550 l/7080 l/8910 l/20000

ratios Purified antibody

l/1:600 1I56200 l/44700 l/35500 l/56200 undetermined

Cross-reactivity ratios were calculated from the ratio between asparate conjugate and other amino acid conjugates or non-conjugated aspartate in the same point of E/B0 (refer to the legend of Fig. 1).

Pons showed a characteristic staining pattern with the anti-aspartate antibody. The antibody labelled intensely neurons in the facial (VII) (Fig. 9a) and raphe and medial lemniscal (Fig. 9b) nuclei. Large multipolar neurons in these nuclei were found scattered around fibres. In the cochlear nucleus some round cells were immunoreactive and the other cells showed no or very weak staining (Fig. 10). Immunopositive neurons in the vestibular nuclei were large and multipolar. Cerebellum

Basket cells and their axon terminals were intensely stained and stellate cells were weakly stained (Fig. 1la). A few Purkinje cells were also strongly immunoreactive while the rest were not labelled or only slightly. Reactions were observed in the perikarya of granule cells. Golgi cells were not reactive. In the deep cerebellar nuclei, immunopositive cells appeared to be larger in size than immunonegative cells (Fig. 11b). Figure 1lc shows a magnified area arrowed in Fig. llb. Medulla oblongata

In the olivary nucleus, fibres and round cells were well immunostained, while a few immunonegative neurons were also observed (Fig. 12). Choroid plexus

Epithelia of the choroid plexus were found immunoreactive to the anti-aspartate antibody as shown in Fig. 5. DISCUSSION

For a direct visualization of aspartate by an immunocytochemical method, it is critical to get a specific anti-aspartate antibody. The present study showed the successful use of affinity columns for the preparation of such an antibody. Though Saito and Tanaka” recommended GABA immobilized on epoxy-activated Sepharose 6B for the purification of anti-GABA antibody, aspartate immobilized on the gel was not sufficient to remove antibodies crossreactive to other amino acid-GAL-RSA conjugates, especially to the glutamate conjugate. This may be probably due to recognition by cross-reactive antibodies of the glutamate-GAL part but not of the glutamate-Sepharose complex. A passage of the antiaspartate antibody through cyanogen bromideactivated Sepharose 4B coupled with glutamateGAL-RSA could successfully eliminate the crossreactivity to the glutamate conjugate. [3H]Aspartate uptake has been shown in all layers of the cerebral neocortex except layer IV.18 In the

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Fig. 3. Photographs showing the distribution of aspartate-like immunoreactivity in the cingulate a frus. (a) Pyramidal cells of layers III and V (arrow) were strongly labelled and granule cells of layers IV and VI were weakly labelled x 100. (b) Magnification of an area arrowed in (a). Pyramidal cells (arrowh and their dendrites (double arrow). x 800.

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759

Fig. 4. Photograph of anti-aspartate antibody-labelled neurons in the hippocampus dentate gyrus. A few aspartate-positive neurons are found scattered between the pyramidal (P) and granule (G) cell layers. x loo. present study on the dist~bution of aspartate-like immunoreactivity, the staining of internal and external py~i~l neurons and of their dendrites extending through several cortical layers could be followed. It was also demonstrated that some neurons in layers IV and VI were weakly stained (Fig. 3). In the hippocampus our results were almost similar to those of Ottersen and Storm-Mathisen. A pharmacological study has suggested that neurons in the habenular nuclei are the major source of the cholinergic

afferents to the inte~uncular nucleus8 Houser et aL6 evidenced immun~yt~hemically the presence of choline a~tyltransfera~-positive somata in the ventral part of the medial habenuiar nucleus, but not in the lateral nucleus. Virtually all of the somata in the former region seemed to contain choline acetyltransferase.6 In our study, aspartate-like immunoreactivity was found in almost all neurons of the medial habenular nucleus (Fig. 5). Consequently, many neurons in the nucleus may contain the two

Fig. 5. Photograph showing the location of aspartate-like immunoreactivity in the habenular region. Most of neurons in the medial habenular nucleus (H) are strongly stained. x LOO.Note that epithelia of the choroid plexus (arrow) are also coloured.

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al.

Fig. 6. Photograph showing the localization of aspartate-like immunoreactivity in the ventral part (VE) of the ventral thalamic nucleus. MD, dorsomedial nuclei of the thalamus; RH, rhomboid nucleus; RT, reticular nucleus of the thalamus. VD, dorsal part of the ventral thalamic nuclei. x 30.

transmitter candidates, aspartate and acetylcholine, simultaneously. Ottersen et al.” revealed thalamocortical projections of aspartergic fibers by retrograde labelling. ” In agrement with these reports, aspartate-like immunoreactivity was detected in many of the thalamic nuclei, although there were some regional variations in the staining intensity. On the other hand, the hypothalamus was found low in aspartate-like immunoreactivity, coinciding with low or intermediate uptake of D-[%I]aspartate.”

Fig. 7. lmmunocytochemical

localization

There is substantial evidence which suggests a possible role of aspartate as a neurotransmitter in the auditory system. Altschuler et al.’ have shown that the immunoreactivity of aspartate aminotransferase, an enzyme associated with the metabolism of aspartate and glutamate, is concentrated in axons and axon terminals of the auditory nerve and around spherical cells in the anteroventral cochlear nucleus. Biochemical and pharmacological data support the role of glutamate and aspartate as neurotransmitters

of aspartate-positive

neurons

in the oculomotor

nucleus.

x 100.

Immunocytochemistry

of aspartergic neurons in rat

Fig. 8. Photograph of the lateral lemniscus. Fibres were well stained. x 100.

Fig. 9. Photographs of the facial [(a) x 3501 and medial lemniscal [(b) x 3501 nuclei. Large immunoreactive neurons were found scattered around the well-stained fibres.

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Fig. 10. Photograph of the cochlear nucleus. x 100.

for the auditory nerve.* In our study, aspartate-like immunoreactivity was also observed in neurons of the cochlear and vestibular nuclei. Recently the enzyme histochemical and immunocytochemical localization of aspartate aminotransferase in the cerebellum have been extensively studied. A strong histochemical reaction of the enzyme was shown in the nerve fibres of basket cells around Purkinje cells and also on the plasma and nuclear membranes of granule cells, while a very weak positive reaction in the perikarya of Purkinje neurons.” Immunocytochemical studies of the enzyme revealed intense labellings on granule, stellate and basket cell bodies and basket cell terminals, while Pnrkinje cell bodies were unlabelled or lightly labelled?’ In the deep cerebellar nuclei, Monaghan et aLI described that aspartate aminotransferase-like immunor~cti~ty was observed in neurons of the lateral, interpositus and medial nuclei. The localization of aspartate-like immunoreactivity in the present study was almost similar to and confirmed the above observations with the anti-aspartate aminotransferase antibody.

The inferior olivary nucleus is thought to be the origin of climbing fibres, which make synapses on Purkinje cell dendrites and may use aspartate as a transmitter.22.2*. Evidence for the latter is also given by the decrease of cerebellar aspartate content after destruction of olivary neurons with 3-acetylpyridine. 15*2*In our immunocytochemical study, the inferior olivary nucleus was strongly stained with the anti-aspartate antibody, supporting the hypothesis that aspartate was a neurotransmitter of olivary neurons. Nevertheless, some immunonegative neurons were also found in the nucleus (Fig. 12). CONCLUSIONS

Our observations on the distribution of aspartate in rat brain generally agreed with previous reports based on other methods, whilst providing a direct and more detailed view. Thus, immunostaining using this anti-aspartate antibody will be useful for demonstrating the precise distribution of endogenous aspartate and candidates for aspartergic neurons. However, as shown in Fig. 5, epithelial cells of the choroid plexus were also stained. Though the antibody has been

Fig. 1I. Photo~aphs showing the localization of aspartate-like immuno~ctivity in the cerebellum. (a) cerebellar cortex. x 400. (b) deep cerebellar nuclei. x 40. (c) Magnification of an area arrowed in (b). x 400. Immunopositive cells in the cerebellar nuclei appear to be larger in size than immunonegative cells. Arrowhead, Basket cell axon terminals; B, Basket cells; G, granule cells; P, Purkinje cells; S, stellate cell.

Fig. II

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Fig. 12. Photograph showing the distribution of aspartate-like lmmunoreactivity in the olivary nucleus. A few aspartate-negative neurons are also observed. x 100.

evidenced to recognize aspartate immobilized on proteins (Fig. 1, Table l), it is not certain that all of the aspartate detected immunocytochemicaly may he used as a neurotransmitter. Thus the positive staining on the choroid plexus may suggest the presence of highly concentrated aspartate in these epithelial cells, where no aspartate will serve as a transmitter.

Note. It is noted that just prior to submission of the present paper, a brief report of a similar study using antibodies

without

purification

has appeared.”

Acknowledgements-A part of this research was supported by Grant No. 85-14 and 8612 from the National Centre for Nervous, Mental and Muscular Disorders (NCNMMD) of the Ministry of Health and Welfare, Japan.

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