European Journal of Pharmacology, 211 (1992)427-428
4~
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 0334R
Rapid communication
GABA A antagonists reveal binding sites for [35S]TBPS in cerebellar granular cell layer E s a R. Korpi, H a r t m u t L f i d d e n s a n d P e t e r H. S e e b u r g Laboratory of Molecular Neuroendocrinology, ZMBH, University of Heidelberg, Heidelberg, F.R.G.
Received 14 January 1992, accepted 17 January 1992
Autoradiography revealed picrotoxin-sensitive [35S]TBPS ([35S]t-butylbicyclophosphorothionate) binding sites in the cerebeli granule cell layer only in the presence of GABA A receptor antagonists. However, under the same conditions, the binding w decreased in other brain regions, including the cerebellar molecular cell layer. Thus, the convulsant binding sites at GAB• receptors in the granule cell layer are highly sensitive to down-regulation by GABA, which may be due to a unique combinatic of receptor subunits. Granule cells; GABA A receptors; TBPS (t-butylbicyclophosphorothionate)
In the cerebellar cortex, several mismatches have been observed in the localization of various binding domains of the classical G A B A A receptor. The oligomeric r e c e p t o r complex should contain a GABAa-type agonist site, a benzodiazepine receptor, and a convulsant binding site, which seems to hold true for the cerebellar molecular layer. However, in the granular cell layer, neither benzodiazepine agonist nor convulsant sites are abundant, whereas G A B A A agonist sites are highly concentrated (Olsen et al., 1990). A significant proportion of the granular cell GABA A receptors have no affinity to benzodiazepine receptor full agonists (Korpi et al., 1992), which apparently explains the mismatch between benzodiazepine agonist and G A B A A agonist site locations. There is still no explanation for the lack of convulsant sites, Peris et al. (1991) reported that binding of the chloride ionophore ligand, [35S]TBPS (t-butylbicyclophosphorothionate), to cerebellar membranes is enhanced by blockade of the G A B A A agonist site by specific antagonists, bicuculline and SR 95531 (2'-(3'carboxy-2',3'-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide). We now report on receptor autoradiographic localization of this observation, which appears to explain why the convulsant site in the granular cell layer has remained undetected,
Correspondence to: E.R. Korpi, Laboratory of Molecular Neuroendocrinology, ZMBH, University of Heidelberg, Im Neuenheimer Feld 282, D-6900 Heidelberg, F.R.G. Tel. 49.6221.566 894, fax 49.6221.565 894.
Horizontal 12-~m cryostat sections were cut fro non-fixed brains of four adult Sprague-Dawley ma rats. After preincubation (30 min in 50 mM Tris-HCl ice-water bath), the sections mounted on gelatin coated slides were incubated (90 rain at 22°C) in 400 j of 50 mM Tris-HCI (pH 7.4) supplemented with 1~ mM NaCI in the presence of [35S]TBPS (6 nM, 0 m C i / 1 , NEN) with and without GABA A antagonis and G A B A (modified from Olsen et al. (1990)). Tl sections were then washed three times for 15 s ice-cold buffer, dipped into distilled H 2 0 , quickly ai dried at room temperature, and exposed to Kod~ X - O M A T AR-film for 2 - 4 days. The basal binding of [35S]TBPS was lower in tl cerebellar granular cell layer than in the molecular c~ layer (fig. 1). This distribution of binding was revers~ by bicuculline, an effect reversible by exogeno~ GABA. SR 95531 (10 ~ M ) and RU 5135 (3a-hydrox 11-oxo-16-imino-17-aza-5~-androstane; 0.l p~M) ga' results similar to those with bicuculline (data n shown). The data indicate that the convulsant bindil sites are present in high concentrations in the cereb~ lar granular cell layer, but can only be detected if tl GABA A agonist sites are blocked. Figure 1 also illustrates the finding that the antag nists (SR 95531 and R U 5135 not shown) decrea [35S]TBPS binding in other brain regions, indicatil that residual G A B A concentrations stimulate [355 TBPS binding in brain regions other than the cereb~ lar granular cell layer. Preliminary data from our lab ratory suggest that this phenomenon can indeed l reproduced in membrane preparations from mar
428 m a l i a n cells t r a n s f e c t e d w i t h c e r t a i n p l a s m i d v e c t c c o d i n g for v a r i o u s G A B A A r e c e p t o r s u b u n i t s . T h e a n o m a l o u s r e g u l a t i o n o f c o n v u l s a n t b i n d i n g sit in t h e c e r e b e l l u m is l i m i t e d to t h e g r a n u l a r cell lay~ S i n c e t h e g r a n u l a r cells h a v e a u n i q u e c o l l e c t i o n G A B A A r e c e p t o r s u b u n i t s ( S h i v e r s et al., 198 L i i d d e n s et al., 1990), t h e a p p a r e n t l y d i f f e r e n t i a l co p i i n g o f G A B A A a g o n i s t sites a n d [35S]TBPS bindil sites m a y t h u s b e d u e to a s p e c i f i c G A B A A r e c e p t subunit composition.
Acknowledgements The study was supported by the Alexander von Humboldt For dation, the Deutsche Forschungsgemeinschaft (SFB317/B9), and t Finnish Academy of Sciences.
References
Fig. 1. Positive images of autoradiograms of [35S]TBPS binding to rat brain horizontal sections. (A) Basal binding showing wide distribution of binding sites. (B,D) In the presence of bicuculline (50 /xM) binding generally decreased, except in the cerebellar granular cell layer (Gr, arrow), where it increased greatly. (C,E) Reversal of the bicuculline effects by GABA (0.5 ~M) in the cerebellar granular and molecular cell layer (Mol, arrow) and in other brain regions. Cb, cerebellum; Cx, cerebral cortex; Hi, hippocampus; OB, olfactory bulb. Similar results were obtained for all rats used in the study. Picrotoxin (10 /xM) abolished virtually all the binding (data not shown).
Korpi, E.R., M. Uusi-Oukari and K. Wegelius, 1992, Substr~ specificity of diazepam-insensitive cerebellar [3H]Ro 15-45 binding sites, Eur. J. Pharmacol. 213 (in press). Liiddens, H., D.B., Pritchett, M. K6hler, I. Killisch, K. Kein~inen, Monyer, R. Sprengel and P.H. Seeburg, 1990, Cerebellar GABt receptor selective for a behavioral alcohol antagonist, Nature 3, 648. Olsen, R.W., R.T. McCabe and J.K. Wamsley, 1990, GABA A rece tor subtypes: autoradiographic comparison of GABA, benzo, azepine, and convulsant binding sites in the rat central nerve system, J. Chem. Neuroanat. 3, 59. Peris, J., A. Shawley, R. Dawson and K.H. Abendschein, 19! Regulation of 35S-TBPS binding by bicuculline is region speci in rat brain, Life Sci. 49, PL-49. Shivers, B.D., 1. Killisch, R. Sprengel, H. Sontheimer, M. K6hl P.R. Schofield and P.H. Seeburg, 1989, Two novel GAB/ receptor subunits exist in distinct neuronal populations, Neur 3, 327.
4~
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 0334R
Rapid communication
GABA A antagonists reveal binding sites for [35S]TBPS in cerebellar granular cell layer E s a R. Korpi, H a r t m u t L f i d d e n s a n d P e t e r H. S e e b u r g Laboratory of Molecular Neuroendocrinology, ZMBH, University of Heidelberg, Heidelberg, F.R.G.
Received 14 January 1992, accepted 17 January 1992
Autoradiography revealed picrotoxin-sensitive [35S]TBPS ([35S]t-butylbicyclophosphorothionate) binding sites in the cerebeli granule cell layer only in the presence of GABA A receptor antagonists. However, under the same conditions, the binding w decreased in other brain regions, including the cerebellar molecular cell layer. Thus, the convulsant binding sites at GAB• receptors in the granule cell layer are highly sensitive to down-regulation by GABA, which may be due to a unique combinatic of receptor subunits. Granule cells; GABA A receptors; TBPS (t-butylbicyclophosphorothionate)
In the cerebellar cortex, several mismatches have been observed in the localization of various binding domains of the classical G A B A A receptor. The oligomeric r e c e p t o r complex should contain a GABAa-type agonist site, a benzodiazepine receptor, and a convulsant binding site, which seems to hold true for the cerebellar molecular layer. However, in the granular cell layer, neither benzodiazepine agonist nor convulsant sites are abundant, whereas G A B A A agonist sites are highly concentrated (Olsen et al., 1990). A significant proportion of the granular cell GABA A receptors have no affinity to benzodiazepine receptor full agonists (Korpi et al., 1992), which apparently explains the mismatch between benzodiazepine agonist and G A B A A agonist site locations. There is still no explanation for the lack of convulsant sites, Peris et al. (1991) reported that binding of the chloride ionophore ligand, [35S]TBPS (t-butylbicyclophosphorothionate), to cerebellar membranes is enhanced by blockade of the G A B A A agonist site by specific antagonists, bicuculline and SR 95531 (2'-(3'carboxy-2',3'-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide). We now report on receptor autoradiographic localization of this observation, which appears to explain why the convulsant site in the granular cell layer has remained undetected,
Correspondence to: E.R. Korpi, Laboratory of Molecular Neuroendocrinology, ZMBH, University of Heidelberg, Im Neuenheimer Feld 282, D-6900 Heidelberg, F.R.G. Tel. 49.6221.566 894, fax 49.6221.565 894.
Horizontal 12-~m cryostat sections were cut fro non-fixed brains of four adult Sprague-Dawley ma rats. After preincubation (30 min in 50 mM Tris-HCl ice-water bath), the sections mounted on gelatin coated slides were incubated (90 rain at 22°C) in 400 j of 50 mM Tris-HCI (pH 7.4) supplemented with 1~ mM NaCI in the presence of [35S]TBPS (6 nM, 0 m C i / 1 , NEN) with and without GABA A antagonis and G A B A (modified from Olsen et al. (1990)). Tl sections were then washed three times for 15 s ice-cold buffer, dipped into distilled H 2 0 , quickly ai dried at room temperature, and exposed to Kod~ X - O M A T AR-film for 2 - 4 days. The basal binding of [35S]TBPS was lower in tl cerebellar granular cell layer than in the molecular c~ layer (fig. 1). This distribution of binding was revers~ by bicuculline, an effect reversible by exogeno~ GABA. SR 95531 (10 ~ M ) and RU 5135 (3a-hydrox 11-oxo-16-imino-17-aza-5~-androstane; 0.l p~M) ga' results similar to those with bicuculline (data n shown). The data indicate that the convulsant bindil sites are present in high concentrations in the cereb~ lar granular cell layer, but can only be detected if tl GABA A agonist sites are blocked. Figure 1 also illustrates the finding that the antag nists (SR 95531 and R U 5135 not shown) decrea [35S]TBPS binding in other brain regions, indicatil that residual G A B A concentrations stimulate [355 TBPS binding in brain regions other than the cereb~ lar granular cell layer. Preliminary data from our lab ratory suggest that this phenomenon can indeed l reproduced in membrane preparations from mar
428 m a l i a n cells t r a n s f e c t e d w i t h c e r t a i n p l a s m i d v e c t c c o d i n g for v a r i o u s G A B A A r e c e p t o r s u b u n i t s . T h e a n o m a l o u s r e g u l a t i o n o f c o n v u l s a n t b i n d i n g sit in t h e c e r e b e l l u m is l i m i t e d to t h e g r a n u l a r cell lay~ S i n c e t h e g r a n u l a r cells h a v e a u n i q u e c o l l e c t i o n G A B A A r e c e p t o r s u b u n i t s ( S h i v e r s et al., 198 L i i d d e n s et al., 1990), t h e a p p a r e n t l y d i f f e r e n t i a l co p i i n g o f G A B A A a g o n i s t sites a n d [35S]TBPS bindil sites m a y t h u s b e d u e to a s p e c i f i c G A B A A r e c e p t subunit composition.
Acknowledgements The study was supported by the Alexander von Humboldt For dation, the Deutsche Forschungsgemeinschaft (SFB317/B9), and t Finnish Academy of Sciences.
References
Fig. 1. Positive images of autoradiograms of [35S]TBPS binding to rat brain horizontal sections. (A) Basal binding showing wide distribution of binding sites. (B,D) In the presence of bicuculline (50 /xM) binding generally decreased, except in the cerebellar granular cell layer (Gr, arrow), where it increased greatly. (C,E) Reversal of the bicuculline effects by GABA (0.5 ~M) in the cerebellar granular and molecular cell layer (Mol, arrow) and in other brain regions. Cb, cerebellum; Cx, cerebral cortex; Hi, hippocampus; OB, olfactory bulb. Similar results were obtained for all rats used in the study. Picrotoxin (10 /xM) abolished virtually all the binding (data not shown).
Korpi, E.R., M. Uusi-Oukari and K. Wegelius, 1992, Substr~ specificity of diazepam-insensitive cerebellar [3H]Ro 15-45 binding sites, Eur. J. Pharmacol. 213 (in press). Liiddens, H., D.B., Pritchett, M. K6hler, I. Killisch, K. Kein~inen, Monyer, R. Sprengel and P.H. Seeburg, 1990, Cerebellar GABt receptor selective for a behavioral alcohol antagonist, Nature 3, 648. Olsen, R.W., R.T. McCabe and J.K. Wamsley, 1990, GABA A rece tor subtypes: autoradiographic comparison of GABA, benzo, azepine, and convulsant binding sites in the rat central nerve system, J. Chem. Neuroanat. 3, 59. Peris, J., A. Shawley, R. Dawson and K.H. Abendschein, 19! Regulation of 35S-TBPS binding by bicuculline is region speci in rat brain, Life Sci. 49, PL-49. Shivers, B.D., 1. Killisch, R. Sprengel, H. Sontheimer, M. K6hl P.R. Schofield and P.H. Seeburg, 1989, Two novel GAB/ receptor subunits exist in distinct neuronal populations, Neur 3, 327.
