Strychnine-sensitive glycine receptors in rat caudatoputamen are expressed by cholinergic interneurons

Neuroscience Vol. 96, No. 1, pp. 33–39, 2000 33 Copyright q 2000 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

Strychnine-sensitive glycine receptors in rat caudatoputamen

Pergamon PII: S0306-4522(99)00535-7 www.elsevier.com/locate/neuroscience

STRYCHNINE-SENSITIVE GLYCINE RECEPTORS IN RAT CAUDATOPUTAMEN ARE EXPRESSED BY CHOLINERGIC INTERNEURONS M. DARSTEIN,* G. B. LANDWEHRMEYER,* C. KLING,† C.-M. BECKER† and T. J. FEUERSTEIN*‡ *Sektion Klinische Neuropharmakologie der Neurologischen Universita¨tsklinik, Neurozentrum, Breisacherstrasse 64, D-79106, Freiburg, Germany †Institut fu¨r Biochemie, Universita¨t Erlangen-Nu¨rnberg, Fahrstrasse 17, D-91054 Erlangen, Germany

Abstract—Strychnine-sensitive glycine receptors are ligand-gated anion channels widely expressed in spinal cord and brainstem. Recent functional studies demonstrating glycine-induced release of [ 3H]acetylcholine in rat caudatoputamen suggested the existence of excitatory glycine receptors in that region. Since the expression of glycine receptors in the caudatoputamen had not been reported earlier, we studied the glycine receptor-like immunoreactivity in this structure using a monoclonal antibody (mAb4a) recognizing an epitope common to all of the ligand-binding a-subunit variants of the glycine receptor. [Becker et al. (1993) Brain Res. 11, 327–333; Nicola et al. (1992) Neurosci. Lett. 138, 173–178]. Immunohistochemistry with mAb4a disclosed a specific staining of sparsely distributed large neurons in rat caudatoputamen, displaying an immunoreactive signal of lower intensity than that observed in motoneurons in spinal cord. Fluorescent dual labelling demonstrated that glycine receptor-like immunoreactivity co-localizes with choline acetyltransferase-like immunoreactivity in rat caudatoputamen. All neurons with glycine receptor-like immunoreactivity in the caudatoputamen studied were immunoreactive with choline acetyltransferase, and represented a subpopulation of cholinergic neurons (approximately 90% of the somata with choline acetyltransferase-like immunoreactivity). These results suggest that strychnine-sensitive glycine receptors are present on cholinergic interneurons in rat caudatoputamen, supporting the hypothesis that glycine receptors inducing striatal release of [ 3H]acetylcholine may be localized to cholinergic neurons. q 2000 IBRO. Published by Elsevier Science Ltd. Key words: cholinergic interneurons, glycine receptor, immunoreactivity, rat caudatoputamen, strychnine-sensitive.

Glycine is the major inhibitory neurotransmitter in spinal cord and brainstem of adult mammals. 13 In the anterior horn of the spinal cord, glycine mediates recurrent inhibition of a-motoneurons via postsynaptic glycine receptors (GlyR). 7,13 The presence of GlyR on these cholinergic neurons has been demonstrated in immunohistochemical studies. 16,30 The inhibitory actions of glycine are selectively blocked by the alkaloid strychnine acting as competitive antagonist. 12,32 Activation of inhibitory strychnine-sensitive GlyR leads to an increased chloride conductance through a ligand-gated ion channel resulting in a hyperpolarization of the postsynaptic membrane. 8,13 Besides its inhibitory role in spinal cord and brainstem and its well-characterized modulatory action at N-methyl-daspartate receptors, glycine has been found to induce the release of several neurotransmitters in rat brain via a strychnine-sensitive mechanism. 14,29,31 As previously described, glycine induces the release of tritiated acetylcholine in rat caudatoputamen (CP). 14,29 Since glycine-induced [ 3H]acetylcholine ([ 3H]Ach) release was not observed in the presence of tetrodotoxin, 14 it was suggested that this effect is mediated by strychnine-sensitive GlyR located on the somatodendritic region of cholinergic interneurons. Strychnine competitively inhibited glycine-evoked [ 3H]ACh release with pA2-values in

the range of those of glycine receptors in spinal cord (pA2values: 6.86, CI95 ˆ [6.61, 7.08] in rat CP 14 vs 7.08, CI95 ˆ [6.67, 7.49] in rat spinal cord 26). Moreover, the glycine-evoked release of [ 3H]ACh in rat CP showed a steep concentration–response curve resembling glycineinduced inhibition of neuronal activity in the spinal cord. 7,17 The strychnine binding site of the GlyR protein complex is located on a-subunit variants (a1–a4) which are constitutive subunits of all GlyR isoforms. 1,7 The strychnine sensitivity of glycine-induced [ 3H]ACh release is indicative of a-subunits being present in rat CP. Here, we show that GlyR a-subunits are expressed in CP, as detected by immunomethods employing monoclonal antibody (mAb4a) which recognizes an epitope common to all of the GlyR a-subunit variants known. 3,4,6,21,24,28 EXPERIMENTAL PROCEDURES

Male Wistar rats (Charles River, Sulzfeld, Germany) weighing about 300 g were used for the experiments. All efforts were made to minimize animal suffering and to reduce the number of animals used, according to the obtained licence of the local ethical committee on animal care. Preparation of membrane fractions and immunodetection CP, spinal cord and liver were dissected, immediately frozen in liquid nitrogen and stored at 2708C. Tissue of indicated areas was homogenized in 20 volumes of ice-cold 10 mM KPi, pH 7.4, containing 5 mM EGTA, 5 mM EDTA, and a cocktail of the protease inhibitors 5 mM o-phenanthroline, 30 mg/ml pefabloc-C, 10 mg/ml pepstatin, 10 mg/ml leupeptin, 1 mM benzamidine, 100 mg/ml bacitracin and 1 mM PMSF. After centrifugation at 35,000 g for 20 min and repeated washing, membranes were suspended in 25 mM KPi, pH 7.4, containing 200 mM KCl and protease inhibitors, and immediately frozen in liquid nitrogen. 3,18

‡To whom correspondence should be addressed. Abbreviations: ABC, avidin–biotin–peroxidase complex; ACh, acetylcholine; ChAT, choline acetyltransferase; CI95, 95% confidence interval; CP, caudatoputamen; Cy2, carbocyanin; Cy3, indocarbocyanin; DAB, diaminobenzidine tetrahydrochloride; EDTA, ethylenediaminetetra-acetate; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; GlyR, glycine receptor; GlyRN, neonatal glycine receptor isoform; IR, like immunoreactivity; mAb, monoclonal antibody; NGS, normal goat serum; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride. 33

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dehydrated in graded ethanols, embedded in Eukitt w, inspected and photographed using light microscopy. Double-label immunohistochemistry Free-floating sections of brain and spinal cord from three different rats were washed three times in PBS and blocked in a solution of 5% normal goat serum (NGS) and 0.3% Triton X-100 in PBS for 1 h at room temperature. Afterwards, sections were washed three times in PBS. For immunohistochemical double labelling they were incubated in a solution containing mouse mAb4a (dilution 1:50 PBS containing 5% NGS) and a rat mAb recognizing protein of the enzyme choline acetyltransferase (mAbChAT, concentration 2.5 mg/ml in PBS containing 5% NGS) at 48C for 48 h on a shaking platform. After rinsing in PBS, the immunhistochemical staining was visualized by incubation with a goat anti-mouse IgG conjugated to indocarbocyanin (Cy3, dilution 1:400) and a goat anti-rat IgG conjugated carbocyanin (Cy2, dilution 1:200) for 3 h at room temperature. Control experiments were performed omitting either mAbChAT, mAb4a or both primary antibodies. Sections were then mounted on gelatin-coated slides, coverslipped with a mixture of 0.2% p-phenylenediamine dihydrochloride and 10% Tris buffer (pH 8) in glycerol and scanned with a Leica confocal laser microscope. Statistical analysis

Fig. 1. Western blot analysis of liver (L), spinal cord (SC) and caudatoputamen (CP) from adult rats. GlyR a-subunit antigen was visualized using the monoclonal antibody mAb4a. Note that film exposure to chemiluminescence substrate was 1 min for spinal cord and 10 min for caudatoputamen and liver membrane fractions, since in the latter region immunoreactivity was much lower than in the spinal cord. Positions of size markers (mol. wt × 1000) were as indicated.

For western blot analysis, crude membranes (50 mg of protein per lane) were subjected to electrophoresis on 10% sodium dodecyl sulphate (SDS)–polyacrylamide gels and blotted on to nitrocellulose. Immunodetection of GlyR proteins was performed using the monoclonal antibody mAb4a (diluted 1:100). The monoclonal antibody mAb4a used in this study had been obtained by immunization with affinity-purified GlyR and was purified as described previously. 24 Binding of mAb4a was detected by goat anti-mouse IgG antibody coupled to horseradish peroxidase (diluted 1:8000) using BM Chemiluminescence as a substrate.

For quantitative estimation of the co-localization of GlyR and choline acetyltransferase (ChAT) in the rat CP three rats were studied. Double labelling was performed using three to six sections of each rat brain. The number of GlyR-positive neurons and of ChAT-positive neurons per visual field of the microscope in the right and left CP from each brain section were counted. The ratio GlyR/ChAT ˆ (number of GlyR-positive neurons)/(number of ChAT-positive neurons) per visual field was calculated. The ratio GlyR/ChAT was given as means with respective 95% confidence intervals (CI95). Materials BM Chemiluminescence and mAbChAT (Boehringer Mannheim Biochemica, Germany), Cy2 goat anti-rat IgG (Rockland, Gilbertsville, PA, U.S.A.), Cy3 goat anti-mouse IgG and horseradish peroxidase goat anti-mouse IgG (Jackson Immuno Research, Dianova-Gesellschaft, Hamburg, Germany), Vectastain w Elite ABC Kit and Peroxidase Substrate Kit (Serva, Heidelberg, Germany), Eukitt w (O. Kindler GmbH and Co., Freiburg, Germany), Pentobarbital (Nembutal w, Sanofi Ceva, Hannover, Germany), protease inhibitors (pefabloc-C, o-phenanthroline, pepstatin, leupeptin, benzamidine, bacitracin, PMSF; Sigma, Deisenhofen, Germany) and p-phenylene diamine dihydrochloride (Sigma, Deisenhofen, Germany) were used. RESULTS

Tissue preparation for immunohistochemical methods Three adult rats were anaesthetized with pentobarbital (100 mg/kg body weight, i.p.) and intracardially perfused with 0.9% NaCl solution and 4% paraformaldehyde in PBS (phosphate-buffered saline, pH 7.4). The brains and spinal cords were rapidly removed, postfixed for 4 h in the same solution of 4% paraformaldehyde in PBS at 48C and cryoprotected in 30% sucrose for several days. Forty micrometer sections were cut on a Leica freezing sledge and stored in 30% glycerol in PBS at 48C until use. Single-label immunohistochemistry Free-floating sections of brain and spinal cord were washed three times in PBS and pretreated with 40% methanol and 1% H2O2 for 10 min to inhibit endogenous peroxidases. Sections were blocked by 5% horse serum and 0.3% Triton X-100 in PBS for 1 h at room temperature. Subsequently, the sections were rinsed in PBS and incubated with the mouse GlyR antibody mAb4a at 48C overnight (dilution 1:100 in PBS containing 5% horse serum). Afterwards, the sections were washed in PBS and incubated with a biotinylated anti-mouse antibody (dilution 1:400; Vector) at room temperature for 30 min. Immunohistochemical staining was visualized using the avidin– biotin–peroxidase complex (ABC; Vector) and 3,3 0 -diaminobenzidine tetrahydrochloride (DAB). Control experiments were performed omitting mAb4a. The sections were then mounted on gelatin-coated slides,

Western blot analysis To verify the presence of GlyR protein in CP tissue, GlyR polypeptide expression was analysed using the mAb4a which recognizes all GlyR a-subunit variants known. In western blot analysis of CP and spinal cord membranes, an immunoreactive band of 48,000–49,000 mol. wt became apparent corresponding to the molecular weight of a-polypeptides (Fig. 1). When rat liver membranes were used as an antigen control, specific immunoreactivity was not detectable. Compared with the mAb4a reactivity obtained with spinal cord membranes, immunosignals from CP were significantly weaker. Single-label immunohistochemistry Following incubation with mAb4a, specific immunohistochemical staining was found in tissue sections of both CP and spinal cord. In the anterior horn of the spinal cord, somata and dendrites of first and higher order of medium-sized and large neurons displayed glycine receptor-like immunoreactivity

Strychnine-sensitive glycine receptors in rat caudatoputamen

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Fig. 2. mAb4a-IR of different regions in rat brain. (A) Immunohistochemical staining of medium-sized and large neurons in the anterior horn of the spinal cord. (B) Negative control of the spinal cord omitting the mAB4a antibody. Scattered neurons in the striatum disclosing intense labelling (C) with mAb4a and (D) control without mAB4a.

(GlyR-IR) (Fig. 2A). In the CP, in contrast, the neuropil appeared unstained. The somata and primary dendrites of scattered large striatal neurons, however, were labelled. The labelling of dendrites in striatum was restricted to dendrites of large size; no axonal staining was observed (Fig. 2C). Control incubations with omission of the primary antibody in the spinal cord and in the CP resulted in a diffuse weak coloration of the neuropil with weak staining of some vessel (Fig. 2B, D). Fluorescent dual-label immunohistochemistry Simultaneous immunostaining of tissue sections with mAb4a and mAbChAT using fluorescent antibodies disclosed a coexpression of GlyR- and ChAT-IR in neurons of the ventral horn of the spinal cord (data not shown). In the CP, large, scattered cells showed an intense labelling with mAbChAT (Fig. 3A). Most of these cells were also labelled with the GlyR-specific antibody mAb4a (Fig. 3B). GlyR-IR appeared to be restricted to neurons which were ChAT-IR. ChAT immunoreactivity, in contrast, was also found in neurons unstained with mAb4a recognizing the GlyR. Statistical analysis showed that the extent of co-localization of GlyR and ChAT in the CP was about 90% (mean GlyR/ ChAT ratio ˆ 0.89, CI95 ˆ [0.85, 0.93]) for all counted visual fields. When calculated for each of the three rats used individually, GlyR/ChAT ratios were very similar (Table 1). Omission of mAb4a (Fig. 4A, right) or mAbChAT (Fig. 4B, left)

from the double-staining procedure resulted in weak background labelling only. No fluorescence was detected when sections were incubated with Cy3-conjugated goat antimouse IgG and Cy2-conjugated goat anti-rat IgG without the primary antibody (data not shown).

DISCUSSION

Using a monclonal antibody recognizing an epitope conserved in all a-subunit variants of the strychnine-sensitive GlyR, 5,22 we observed a specific immunohistochemical staining of scattered large neurons in rat CP. Fluorescent dual labelling disclosed that cells expressing GlyR-IR corresponded to cholinergic striatal interneurons. Specificity of antibody and immunohistochemical signal In western blot analysis of mammalian CNS preparations, mAb4a detected a polypeptide of 48,000–49,000 mol. wt as expected for GlyR a-subunit variants. Epitope mapping demonstrated that mAb4a recognizes a determinant within the extracellular N-terminal domain that is conserved in all known a-subunits of GlyR. 5,28 In immunohistochemical studies, mAb4a has demonstrated GlyR-IR in human and rodent cerebral cortex and in spinal cord. 3,21 The immunohistochemical staining of spinal cord observed here agrees well with the staining pattern previously reported. Specificity of

Fig. 3. Immunofluorescence reactions for the co-localization of ChAT and the GlyR. Immunostaining with (A) mAbChAT and (B) mAb4a and (C) composition of mABChAT and mAB4a in the CP.

Strychnine-sensitive glycine receptors in rat caudatoputamen

this signal is confirmed by the absence of neuronal staining in control experiments omitting mAb4a. Striatal glycine receptor immunoreactivity: localization and putative subunit variant

cellular

The main finding of the present study is the detection of GlyR-IR in a subpopulation of striatal neurons. In human CNS, quantitative immunoanalysis disclosed similar mAb4a

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reactivities for CP and spinal cord, 21 providing evidence for the presence of strychnine-sensitive GlyR in this brain region in humans. In the present study, western blot analysis revealed that the mAb4a immunoreactivity detectable in rat CP indeed corresponds to an antigen of 48,000–49,000 mol. wt as expected for GlyR a-subunit variants. 2 The population of GlyR-IR cells in rat striatum consisted of large scattered somata reminiscent of the distribution and density of cholinergic interneurons of CP. 11 To confirm the

Fig. 4. Negative controls of the dual immunofluorescence. (A) Shows ChAT immunoreactivity only (left, no signal on the right) since mAbChAT was used together with both secondary antibodies (Cy2 against rat antibody for ChAT and Cy3 against mouse antibody for GlyR). (B) Shows GlyR immunoreactivity only (right, no signal on the left) since mAb4A was used together with both secondary antibodies (Cy2 against rat antibody for ChAT and Cy3 against mouse antibody for GlyR).

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Table 1. Extent of co-localization of glycine receptor and choline acetyltransferase in rat caudatoputamen Individuals Rat 1 Rat 2 Rat 3

Mean GlyR/ChAT-ratios, CI95 0.89, [0.83, 0.94], n ˆ 43 0.86, [0.76, 0.96], n ˆ 20 0.94, [0.86, 1.00], n ˆ 13

Results are given as mean GlyR/ChAT-ratios for the three individual rats used (n ˆ number of visual fields counted).

assumption that GlyR carrying neurons of CP are cholinergic, mAb4a was combined with the monoclonal ChAT antibody in a dual-label immunofluorescent study. In the CP, most cellular profiles labelled the ChAT antibody (90%) were also labelled with the GlyR mAb4a. Some of the cells displaying immunoreactivity for ChAT (approx. 10%), however, did not yield unequivocal GlyR-IR. In contrast, all GlyR-positive neurons were labelled with the ChAT antibody, suggesting that a small subpopulation of cholinergic striatal interneurons does not express detectable levels of strychnine-sensitive GlyRs. The detection of GlyR-IR in cholinergic interneurons affirms the assumption that strychnine-sensitive GlyR are located somatodendritically on cholinergic interneurons in CP. This is consistent with our recent study on glycineevoked release of striatal [ 3H]ACh mediated by strychninesensitive receptors. 14 Previous studies did not yield clear evidence for the existence of strychnine-sensitive GlyR in striatum. Binding of [ 3H]strychnine did not exceed background levels in the CP of rat brain 10 and was very low in the striatum of human post mortem tissue, 25 whereas the gray matter of the spinal cord revealed a high density of [ 3H]strychnine binding sites. 10,25 Investigations on rat brain using a monoclonal antibody mAb7a directed against the GlyR associated protein gephyrin disclosed only a few supratentorial neurons. 20 In situ hybridization studies showed the expression of b- and some a-subunit mRNAs in supratentorial regions. 19 In seven-day-old rats a2-subunit mRNA expression was detectable in a few large neurons in the CP 27 which showed morphological criteria of cholinergic interneurons. So far, no in situ hybridization signals have been detected in CP of adult rats using a1-subunit-specific DNA oligonucleotide probes. 19 Therefore, it appears likely that the GlyR-IR detected by mAb4a in CP of adult rats corresponds to the a2 polypeptide, a subunit characteristic of the neonatal glycine receptor isoform (GlyRN), a protein isoform initially characterized in neonatal rats. 1,3 The presence of GlyRN could offer an explanation for the apparent absence of [ 3H]strychnine binding sites in the striatum, 10,25 as this receptor isoform is characterized by low strychnine binding affinity. 1,3 This conclusion, however, contrasts with the report by Malosio and co-workers 19 who did not report a2-specific in situ hybridization signals in adult CP. The limited sensitivity and spatial resolution of the DNA oligonucleotide probes, however, may have precluded the detection of moderate levels of a2 mRNA in cholinergic

neurons, not least because cholinergic interneurons represent only a small fraction of striatal neurons. Clearly, the issue of GlyR-mRNA expression in cholinergic striatal interneurons requires further study. In the experiments presented, rat spinal motoneurons displayed an intense immunoreactivity with the monoclonal antibody mAb4a. This is in agreement with previous studies 3 and the well-known function of glycine to mediate synaptic inhibition through postsynaptic receptors located on cholinergic a-motoneurons. In rat spinal cord, a-motoneurons were found to co-localize ChAT and GlyR immunoreactivity. Striatal glycine receptor immunoreactivity: functional implications The present results demonstrate that the GlyR antigen detectable in rat CP is expressed by cholinergic neurons. This suggests that the strychnine-sensitive, glycine-evoked [ 3H]ACh release in this brain region is mediated by GlyRs. This conclusion, however, contradicts the well-established role of glycine as a mediator of inhibitory impulses. However, the loop diuretic furosemid, a blocker of chloride transport, 9 significantly diminished GlyR-mediated [ 3H]ACh release in rat CP (own unpublished results), indicating that glycineinduced excitatory actions can be attributed to chloride channels. This hypothesis is consistent with alterations in neuronal chloride potential that have recently been correlated with excitatory actions of strychnine-sensitive GlyRs in dorsal root ganglia and cortical neurons of mature rats. 16 A similar depolarizing effect through GABAA receptors has been demonstrated in neonatal cortical neurons where intracellular chloride is high. 23 Under these circumstances, activation of striatal GlyR would result in a chloride efflux from cholinergic interneurons as a consequence of high intracellular chloride concentrations. To our knowledge it is unknown whether striatal cholinergic interneurons resemble neonatal cortical neurons with respect to their intracellular chloride levels. Interestingly, neonatal cortical neurons displaying excitatory effects of glycine express GlyR a2 transcripts 15,19,27 and the GlyRN protein complex. 4 CONCLUSIONS

Striatal cholinergic interneurons in adult rats display GlyRIR. Combined with functional studies demonstrating glycineevoked release of [ 3H]ACh, we assume that striatal cholinergic interneurons may express functional strychnine-sensitive GlyRs. However, strychnine-sensitive GlyRs from rat CP and spinal cord appear different in excitatory versus inhibitory function, probably owing to a reversed direction of chloride currents.

Acknowledgements—This study was supported by grants from the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie (BMBF 01EB9413 and 01KO9702) and the Deutsche Forschungsgemeinschaft (SFB 505).

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