Neuroscience Letters, 144 (1992) 53-56 © 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$05.00
53
NSL 08918
Mapping of GABAA receptor
and subunit-like immunoreactivity in rat brain
C h r i s t o p h e r L. T h o m p s o n a, G e e r t B o d e w i t z b, F. A n n e S t e p h e n s o n a a n d J o n a t h a n D. T u r n e r b aDepartment of Pharmaceutical Chemistry, School of Pharmacy. London ( UK), bResearch Laboratories of Sehering AG, Berlin ( FRG)
(Received 3 April 1992; Revisedversion received 15 May 1992;Accepted 10 June 1992) Key words. GABAAreceptor; ct5 Subunit; ~6 Subunit; Immunohistochemistry;Rat CNS
The distribution of the ~5 and cz6subunits of the GABAAreceptor has been mapped in rat brain using affinity-purifiedantibodiesgeneratedagainst peptide sequencesunique to the respectivepolypeptides.~5 Subunit-likeimmunoreactivitywas of low density but was distributed across several cell groups including cortical interneurones, hippocampal CA3 pyramidal neurones, the anterior thalamic reticular nucleus and cerebellar Purkinje neurones. ~6 Subunit-like immunoreactivitywas observed in high density in cerebellar granule cells. These patterns are compatible with in situ hybridisation studies and provide a further anatomical substrate for GABAAreceptor heterogeneityin the CNS.
The inhibitory GABAA receptors of mammalian brain are hetero-oligomeric membrane glycoproteins, in which five subunits are thought to assemble to form individual ion channel complexes. Five distinct classes, some containing several isoforms (~1--~6, fll-fl3, y l - y 3 , 6 and p l - p 2 ) encoded by separate genes have been identified by molecular cloning (reviewed in refs. 9 and 13). Recombinant receptors assembled from these subunits possess heterogeneous pharmacological and biophysical properties (e.g. ref. 5). The combinations that assemble in vivo are not known but in situ hybridisation studies have demonstrated overlapping but also distinct distributions of the mRNAs for the different subunits and isoforms [4, 14]. To facilitate biochemical isolation and identification of possible native receptor subtypes and for the characterisation of their function, it is important also to obtain information about the relative distributions of the respective protein products. We have raised antibodies specific for peptide sequences unique to the rat and bovine ~ subunits of the GABAA receptor [2]. Here, we report the distribution of the ~5 and ~6 subunits mapped in rat brain using affinity-purified antibodies. Polyclonal antibodies were raised in rabbits to the peptide sequences Q M P T S S V Q D E T N D N I T C (rat c~5(1-16)
Correspondence: F.A. Stephenson, Department of Pharmaceutical Chemistry, School of Pharmacy, 29/39 Brunswick Square, London WC1N lAX, UK. Fax: (44) 71 2781939.
Cys [3]) and K L E D E G N F Y S K N I S R C (bovine ~6(1-I 5) Cys [6]) coupled to keyhole limpet haemocyanin and affinity-purified on the respective peptide affinity columns as described previously [2]. Conventional immunohistochemical procedures were employed. Briefly, cryostat sections, 2 0 4 0 / ~ m thick, obtained from paraformaldehyde-fixed adult rat brain were incubated free floating in primary antibody ( 0 . 0 2 5 4 / ~ g protein/ml) for 24 h at 4°C. Tissues were then processed according to the peroxidase-antiperoxidase technique (Vectastain) and the immune reaction visualised using diaminobenzidine as substrate. Specificity controls were carried out by adsorption of primary antibodies in working dilutions with their respective peptides (1-2/.tg/ml, 24 h at 4°C) prior to application to the tissue. The immunoreactivity pattern obtained with the ~5 subunit antibody showed light to moderate staining distributed throughout the CNS. This staining pattern was abolished by preadsorption of the antibody with the peptide sequence or with the peptide-KLH conjugate used as antigen. The intensity of the immune reaction, even in those areas where it was highest, was much lower than that obtained using any of the ~1, ~2 or ~3 subunit-specific antibodies (ref. 15 and results in preparation). This apparently lower abundance of ~5 subunit immunoreactivity is consistent with imunoprecipitation studies, where it has been reported that anti-~5 subunit antibodies only precipitated significant amounts of flumazenil binding sites from hippocampal extracts. Although even here this was only 10 20% of the total [8], it does indicate
54
- 5
,
k
a
b
c
-
d
Fig. 1. Distribution of cz5 subunit-like staining in rat CNS. a: horizontal section shows overall weak staining. At higher power (b-f) heavier localised staining is evident in olfactory bulb glomeruli (G in b): in pyramidal neurones in frontal cortex (c); in interneurones in parietal cortex (d); in CA3 pyramids in hippocampus (e); in primary afferent neurones in the mesencephalic trigeminal nucleus (f) and in Purkinje neurones in the cerebellum (g). Diffuse staining of processes is evident in olfactory bulb granular layer (GC in b), in layer III in parietal cortex (d) and layer V VI in entorhinal cortex (arrow's in e). Bars in b g = 100/Jm.
that cz5 subunit immunoreactivity colocates with functional GABAA receptor subtypes. Most prominent c~5 subunit staining was associated with hippocampal CA3 neuronal perikarya (Fig. la,e), with perikarya and processes within the anterior reticular nucleus of the thalamus (Fig. l a) and with the glomeruli of the olfactory bulb (Fig. lb). Throughout the cerebral cortex, a small population of neurones with the distribution and morphological characteristics of interneurones exhibited significant perikaryal immunoreactive staining (Fig. ld) while in frontal cortex, some pyramidal neurones and their processes were also labelled (Fig. lc). In the cerebellum, Purkinje and Golgi neurones (Fig. lg) and neurones in the deep nuclei were modestly stained while lower levels of staining were apparent in numerous additional cell groups (see Table I). A very pronounced staining was also observed in the cell bodies of the primary afferent neurones of the mesencephalic tract of the trigeminal nerve (see Fig. lf). Viewed at high magnification the immune reaction product was distributed un-
evenly, consistent with localisation on both the cell surface and within the cytoplasm. Apparent cytoplasmic labelling, as described at the optical microscope level, is a feature of other GABAA receptor subunits [1, 10, 15]. Additionally, association of GABAA receptor ~ and ,B subunit immunoreactivity was found associated with the Golgi apparatus and the endoplasmic reticulum in an ultrastructural study where it was suggested that this was due to receptor biosynthesis and assembly [12]. In most cases, dendritic arbourisations were poorly immunoreactive. Different functional properties between receptor subtypes may require differential subcellular localisations. In addition to the presence of reaction product on neuronal perikarya, diffuse, punctate staining was also evident in layer III-IV in parietal cortex and in layers II-III and V V! of entorhinal cortex and was observed also in selected subcortical nuclei (e.g. Fig. ld,e). Comparison of the immunostaining pattern with that generated by in situ hybridisation [7] reveals overall simi-
55
TABLE I DISTRIBUTION OF GABAA RECEPTOR ~5 AND ct6 SUBUNITLIKE IMMUNOREACTIVITY IN RAT CNS -, negligible staining; (+), light diffuse staining or scattered profiles; + to +++, increasing density of staining or of stained profiles. DG, dentate gyrus; ML, molecular layer; PC, Purkinje cells; GL, granule cell layer; EPC, external plexiform layer.
b
c
~
d
Fig. 2. Distribution of ct6 subunit-like staining in rat CNS. a: sagittal section (dark field) shows pronounced staining in the granular layer of the cerebellum. Dense staining of granule cells (b) is almost abolished by preadsorption with the peptide used as antigen (c). In (d) no staining of Purkinje cells (large arrows) is evident although light, apparently specific staining of cell nuclei in molecular layer (small arrows) and elsewhere in the CNS can be seen. Bars in b~t -- 100 pm.
larity but also some differences. Thus, the immune reaction p r o d u c t was most prominent in h i p p o c a m p a l pyramidal neurones and cortex, especially frontal cortex, agreeing with the distribution o f in situ hybridisation signal. However, a prominent in situ signal is observed in olfactory granule cells which demonstrated little perikaryal immunolabelling. This could be explained by either a lack o f translation or by translation at sites distant f r o m the cell soma, the latter possibly consistent with the presumptive dendritic labelling in the granular layer (Fig. ld). Conversely, a significant, specific c~5 subunit-like i m m u n o r e a c t i o n was obtained in cerebellar Purkinje and Golgi neuronal perikarya where ~5 subunit m R N A has not been detected [7]. This discrepancy could arise if the m R N A were unstable or if protein turnover were very slow. A n o t h e r possibility is that the ~5 peptide sequence is contained in another u n k n o w n protein in these cerebellar neurones. The immunoreactivity pattern obtained with the ~6 subunit a n t i b o d y showed very dense staining associated
Brain region
~5
ct6
Cortex
(+)
-
Hippocampus DG CA1 CA3 Interneurones
(+) + ++
Striatum
(+)
Pallidum
(+)
Septal nuclei
(+)
Thalamus
(+)
Hypothalamus
(+)
Substantia nigra
(+)
Colliculus sup.
(+)
Colliculus inf.
(+)
Cerebellum ML PC GL
(+) ++ (+)
Brainstem
(+)
Olfactory bulb Glomeruli EPL Mitrals GL
+++
+++ (+) ++
with m o s t granule cells in the cerebellum (Fig. 2a~:l). In the molecular layer in some preparations (e.g. Fig. 2d) the a n t i b o d y recognised processes radiating perpendicular to the pial surface. These processes m a y be derived from Bergmann glial cells or represent bundles o f immunoreactive, ascending, granule cell axons. In addition, particularly in very well- fixed tissue, less prominent staining o f nuclei o f neurones elsewhere in the C N S was also evident. All staining was abolished by adsorption o f
56 the a n t i b o d y either with peptide or conjugate, a n d it was equally susceptible to p r i m a r y a n t i b o d y dilution. The d i s t r i b u t i o n o f ~t6 subunit-like i m m u n o r e a c t i v i t y reflects the d i s t r i b u t i o n o f c~6 m R N A in that it shows a high c o n c e n t r a t i o n in cerebellar granule cells [6]. Furthermore, it closely resembles the d i s t r i b u t i o n in rat brain o f benzodiazepine-insensitive b i n d i n g of the imidazodiazepine R o 15-4513 [11], which has been shown to b i n d to ct6 s u b u n i t - c o n t a i n i n g r e c o m b i n a n t receptors in transfected cells [6]. In summary, we have used anti-peptide antibodies to m a p the d i s t r i b u t i o n of G A B A A receptor ~5 a n d ~6 subunits in rat brain. They d e m o n s t r a t e highly distinctive patterns, largely consistent with the cellular localisation of their respective m R N A s a n d with the b i n d i n g of selective ligands. A l t h o u g h they are of limited a b u n d a n c e , their distinct localisation suggests approaches for the further biochemical a n d functional characterisation of native receptors which c o n t a i n them. M o r e o v e r it renders them suitable targets for novel drug development. This work was supported in part by the Medical Research Council (UK). We t h a n k Dr. M.J. D u g g a n a n d S. Pollard for a n t i b o d y p r o d u c t i o n and preparation. 1 Benke, D., Mertens, S. and Mohler, H., Ubiquitous presence of GABA,x receptors containing cd-subunit in rat brain demonstrated by immunoprecipitation and immunohistochemistry, Mol. Neuropharmacol.. 1 (1991)103 110. 2 Duggan, M.J. and Stephenson, F.A., Biochemicalevidence for the existence of y-aminobutyrate.x receptor iso-oligomers, J. Biol. Chem., 265{1990) 3831 3835. 3 Khrestchatisky, M., MacLennan, J., Chiang, M.-Y., Xu, W., Jackson, M.B., Brecha, N., Sternini, C., Olsen, R.W. and Tobin. A.J., A novel 0t subunit in rat brain GABAA receptors, Neuron. 3 (1989) 745 753.
4 Laurie, D.J., Seeburg, P.H. and Wisden, W., The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum, J. Neurosci., 12 (1992) 1063 1076. 5 Luddens, H. and Wisden, W., Function and pharmacology of multiple GABAA receptor subunits, Trend Pharmacol. Sci.. 12 (1991) 49-51. 6 Luddens, H., Pritchen, D.B., Kohler, M., Killisch, 1., Keinanen, K., Monyer, H., Sprengel, R. and Seeburg, RH., Cerebellar GABAa receptor selective for a behavioural alcohol antagonist, Nature, 346 (1990) 648-651. 7 Malherbe, R, Sigel, E., Baur, R., Pershon, E., Richards, J.G. and Mohler, H., Functional expression and sites ofgene transcription of a novel ~-subunit of the GABAAreceptor of rat brain, FEBS Lett., 260 (1990) 261 265. 8 McKernan, R.M., Quirk, K., Prince, R., Cox, P.A., Gillard, N.R, Ragan, C.I. and Whiting, P., GABAA receptor subtypes immunopurified from rat brain with ~ subunit-specificantibodies have unique pharmacological properties, Neuron, 7 (1991) 667 676. 90lsen, R.W. and Tobin, A.J., Molecular biology of GABAA receptors, FASEB J., 4 (1990) 1469 1480. 10 Richards, J.G., Schoch, P., Haring, R, Takacs, B. and Mohler. H., ResolvingGABAA/benzodiazepinereceptors: cellular and subcellular localization in the CNS with monoclonal antibodies, J. Neurosci., 7 (1987) 1866,1886. 11 Sieghart, W., Eichinger, A., Richards, J.G. and Mohler, H., Photoaffinity labelling of benzodiazepine receptor proteins with the partial inverse agonist [~H]Ro 15-4513: A biochemical and autoradiographic study, J. Neurochem., 48 (1987) 46-52. 12 Somogyi, P., Takagi, H., Richards, J.G. and Mohler, H., Subcellular localisation of benzodiazepine/GABAAreceptors in the cerebellum of rat, cat and monkey using monoclonal antibodies. J. Neurosci., 9 (1989) 2197 2209. 13 Stephenson, F.A., The GABAA receptors: structure and function, Curr. Asp. Neurosci.. 3 (1991) 177 194. 14 Wisden, W., Laurie, D.J., Monyer, H., and Seeburg, P.H., The distribution of 13 GABAAreceptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon, J. Neurosci., 12 (1992) 1040 1062. 15 Zimprich, F., Zezula, J., Sieghart, W. and Lassmann, H., lmmunohistochemical localisation of the ~1, ~2 and c~3 subunits of the GABAA receptor in the rat brain, FEBS Lett., 127 (1991) 125 128.
53
NSL 08918
Mapping of GABAA receptor
and subunit-like immunoreactivity in rat brain
C h r i s t o p h e r L. T h o m p s o n a, G e e r t B o d e w i t z b, F. A n n e S t e p h e n s o n a a n d J o n a t h a n D. T u r n e r b aDepartment of Pharmaceutical Chemistry, School of Pharmacy. London ( UK), bResearch Laboratories of Sehering AG, Berlin ( FRG)
(Received 3 April 1992; Revisedversion received 15 May 1992;Accepted 10 June 1992) Key words. GABAAreceptor; ct5 Subunit; ~6 Subunit; Immunohistochemistry;Rat CNS
The distribution of the ~5 and cz6subunits of the GABAAreceptor has been mapped in rat brain using affinity-purifiedantibodiesgeneratedagainst peptide sequencesunique to the respectivepolypeptides.~5 Subunit-likeimmunoreactivitywas of low density but was distributed across several cell groups including cortical interneurones, hippocampal CA3 pyramidal neurones, the anterior thalamic reticular nucleus and cerebellar Purkinje neurones. ~6 Subunit-like immunoreactivitywas observed in high density in cerebellar granule cells. These patterns are compatible with in situ hybridisation studies and provide a further anatomical substrate for GABAAreceptor heterogeneityin the CNS.
The inhibitory GABAA receptors of mammalian brain are hetero-oligomeric membrane glycoproteins, in which five subunits are thought to assemble to form individual ion channel complexes. Five distinct classes, some containing several isoforms (~1--~6, fll-fl3, y l - y 3 , 6 and p l - p 2 ) encoded by separate genes have been identified by molecular cloning (reviewed in refs. 9 and 13). Recombinant receptors assembled from these subunits possess heterogeneous pharmacological and biophysical properties (e.g. ref. 5). The combinations that assemble in vivo are not known but in situ hybridisation studies have demonstrated overlapping but also distinct distributions of the mRNAs for the different subunits and isoforms [4, 14]. To facilitate biochemical isolation and identification of possible native receptor subtypes and for the characterisation of their function, it is important also to obtain information about the relative distributions of the respective protein products. We have raised antibodies specific for peptide sequences unique to the rat and bovine ~ subunits of the GABAA receptor [2]. Here, we report the distribution of the ~5 and ~6 subunits mapped in rat brain using affinity-purified antibodies. Polyclonal antibodies were raised in rabbits to the peptide sequences Q M P T S S V Q D E T N D N I T C (rat c~5(1-16)
Correspondence: F.A. Stephenson, Department of Pharmaceutical Chemistry, School of Pharmacy, 29/39 Brunswick Square, London WC1N lAX, UK. Fax: (44) 71 2781939.
Cys [3]) and K L E D E G N F Y S K N I S R C (bovine ~6(1-I 5) Cys [6]) coupled to keyhole limpet haemocyanin and affinity-purified on the respective peptide affinity columns as described previously [2]. Conventional immunohistochemical procedures were employed. Briefly, cryostat sections, 2 0 4 0 / ~ m thick, obtained from paraformaldehyde-fixed adult rat brain were incubated free floating in primary antibody ( 0 . 0 2 5 4 / ~ g protein/ml) for 24 h at 4°C. Tissues were then processed according to the peroxidase-antiperoxidase technique (Vectastain) and the immune reaction visualised using diaminobenzidine as substrate. Specificity controls were carried out by adsorption of primary antibodies in working dilutions with their respective peptides (1-2/.tg/ml, 24 h at 4°C) prior to application to the tissue. The immunoreactivity pattern obtained with the ~5 subunit antibody showed light to moderate staining distributed throughout the CNS. This staining pattern was abolished by preadsorption of the antibody with the peptide sequence or with the peptide-KLH conjugate used as antigen. The intensity of the immune reaction, even in those areas where it was highest, was much lower than that obtained using any of the ~1, ~2 or ~3 subunit-specific antibodies (ref. 15 and results in preparation). This apparently lower abundance of ~5 subunit immunoreactivity is consistent with imunoprecipitation studies, where it has been reported that anti-~5 subunit antibodies only precipitated significant amounts of flumazenil binding sites from hippocampal extracts. Although even here this was only 10 20% of the total [8], it does indicate
54
- 5
,
k
a
b
c
-
d
Fig. 1. Distribution of cz5 subunit-like staining in rat CNS. a: horizontal section shows overall weak staining. At higher power (b-f) heavier localised staining is evident in olfactory bulb glomeruli (G in b): in pyramidal neurones in frontal cortex (c); in interneurones in parietal cortex (d); in CA3 pyramids in hippocampus (e); in primary afferent neurones in the mesencephalic trigeminal nucleus (f) and in Purkinje neurones in the cerebellum (g). Diffuse staining of processes is evident in olfactory bulb granular layer (GC in b), in layer III in parietal cortex (d) and layer V VI in entorhinal cortex (arrow's in e). Bars in b g = 100/Jm.
that cz5 subunit immunoreactivity colocates with functional GABAA receptor subtypes. Most prominent c~5 subunit staining was associated with hippocampal CA3 neuronal perikarya (Fig. la,e), with perikarya and processes within the anterior reticular nucleus of the thalamus (Fig. l a) and with the glomeruli of the olfactory bulb (Fig. lb). Throughout the cerebral cortex, a small population of neurones with the distribution and morphological characteristics of interneurones exhibited significant perikaryal immunoreactive staining (Fig. ld) while in frontal cortex, some pyramidal neurones and their processes were also labelled (Fig. lc). In the cerebellum, Purkinje and Golgi neurones (Fig. lg) and neurones in the deep nuclei were modestly stained while lower levels of staining were apparent in numerous additional cell groups (see Table I). A very pronounced staining was also observed in the cell bodies of the primary afferent neurones of the mesencephalic tract of the trigeminal nerve (see Fig. lf). Viewed at high magnification the immune reaction product was distributed un-
evenly, consistent with localisation on both the cell surface and within the cytoplasm. Apparent cytoplasmic labelling, as described at the optical microscope level, is a feature of other GABAA receptor subunits [1, 10, 15]. Additionally, association of GABAA receptor ~ and ,B subunit immunoreactivity was found associated with the Golgi apparatus and the endoplasmic reticulum in an ultrastructural study where it was suggested that this was due to receptor biosynthesis and assembly [12]. In most cases, dendritic arbourisations were poorly immunoreactive. Different functional properties between receptor subtypes may require differential subcellular localisations. In addition to the presence of reaction product on neuronal perikarya, diffuse, punctate staining was also evident in layer III-IV in parietal cortex and in layers II-III and V V! of entorhinal cortex and was observed also in selected subcortical nuclei (e.g. Fig. ld,e). Comparison of the immunostaining pattern with that generated by in situ hybridisation [7] reveals overall simi-
55
TABLE I DISTRIBUTION OF GABAA RECEPTOR ~5 AND ct6 SUBUNITLIKE IMMUNOREACTIVITY IN RAT CNS -, negligible staining; (+), light diffuse staining or scattered profiles; + to +++, increasing density of staining or of stained profiles. DG, dentate gyrus; ML, molecular layer; PC, Purkinje cells; GL, granule cell layer; EPC, external plexiform layer.
b
c
~
d
Fig. 2. Distribution of ct6 subunit-like staining in rat CNS. a: sagittal section (dark field) shows pronounced staining in the granular layer of the cerebellum. Dense staining of granule cells (b) is almost abolished by preadsorption with the peptide used as antigen (c). In (d) no staining of Purkinje cells (large arrows) is evident although light, apparently specific staining of cell nuclei in molecular layer (small arrows) and elsewhere in the CNS can be seen. Bars in b~t -- 100 pm.
larity but also some differences. Thus, the immune reaction p r o d u c t was most prominent in h i p p o c a m p a l pyramidal neurones and cortex, especially frontal cortex, agreeing with the distribution o f in situ hybridisation signal. However, a prominent in situ signal is observed in olfactory granule cells which demonstrated little perikaryal immunolabelling. This could be explained by either a lack o f translation or by translation at sites distant f r o m the cell soma, the latter possibly consistent with the presumptive dendritic labelling in the granular layer (Fig. ld). Conversely, a significant, specific c~5 subunit-like i m m u n o r e a c t i o n was obtained in cerebellar Purkinje and Golgi neuronal perikarya where ~5 subunit m R N A has not been detected [7]. This discrepancy could arise if the m R N A were unstable or if protein turnover were very slow. A n o t h e r possibility is that the ~5 peptide sequence is contained in another u n k n o w n protein in these cerebellar neurones. The immunoreactivity pattern obtained with the ~6 subunit a n t i b o d y showed very dense staining associated
Brain region
~5
ct6
Cortex
(+)
-
Hippocampus DG CA1 CA3 Interneurones
(+) + ++
Striatum
(+)
Pallidum
(+)
Septal nuclei
(+)
Thalamus
(+)
Hypothalamus
(+)
Substantia nigra
(+)
Colliculus sup.
(+)
Colliculus inf.
(+)
Cerebellum ML PC GL
(+) ++ (+)
Brainstem
(+)
Olfactory bulb Glomeruli EPL Mitrals GL
+++
+++ (+) ++
with m o s t granule cells in the cerebellum (Fig. 2a~:l). In the molecular layer in some preparations (e.g. Fig. 2d) the a n t i b o d y recognised processes radiating perpendicular to the pial surface. These processes m a y be derived from Bergmann glial cells or represent bundles o f immunoreactive, ascending, granule cell axons. In addition, particularly in very well- fixed tissue, less prominent staining o f nuclei o f neurones elsewhere in the C N S was also evident. All staining was abolished by adsorption o f
56 the a n t i b o d y either with peptide or conjugate, a n d it was equally susceptible to p r i m a r y a n t i b o d y dilution. The d i s t r i b u t i o n o f ~t6 subunit-like i m m u n o r e a c t i v i t y reflects the d i s t r i b u t i o n o f c~6 m R N A in that it shows a high c o n c e n t r a t i o n in cerebellar granule cells [6]. Furthermore, it closely resembles the d i s t r i b u t i o n in rat brain o f benzodiazepine-insensitive b i n d i n g of the imidazodiazepine R o 15-4513 [11], which has been shown to b i n d to ct6 s u b u n i t - c o n t a i n i n g r e c o m b i n a n t receptors in transfected cells [6]. In summary, we have used anti-peptide antibodies to m a p the d i s t r i b u t i o n of G A B A A receptor ~5 a n d ~6 subunits in rat brain. They d e m o n s t r a t e highly distinctive patterns, largely consistent with the cellular localisation of their respective m R N A s a n d with the b i n d i n g of selective ligands. A l t h o u g h they are of limited a b u n d a n c e , their distinct localisation suggests approaches for the further biochemical a n d functional characterisation of native receptors which c o n t a i n them. M o r e o v e r it renders them suitable targets for novel drug development. This work was supported in part by the Medical Research Council (UK). We t h a n k Dr. M.J. D u g g a n a n d S. Pollard for a n t i b o d y p r o d u c t i o n and preparation. 1 Benke, D., Mertens, S. and Mohler, H., Ubiquitous presence of GABA,x receptors containing cd-subunit in rat brain demonstrated by immunoprecipitation and immunohistochemistry, Mol. Neuropharmacol.. 1 (1991)103 110. 2 Duggan, M.J. and Stephenson, F.A., Biochemicalevidence for the existence of y-aminobutyrate.x receptor iso-oligomers, J. Biol. Chem., 265{1990) 3831 3835. 3 Khrestchatisky, M., MacLennan, J., Chiang, M.-Y., Xu, W., Jackson, M.B., Brecha, N., Sternini, C., Olsen, R.W. and Tobin. A.J., A novel 0t subunit in rat brain GABAA receptors, Neuron. 3 (1989) 745 753.
4 Laurie, D.J., Seeburg, P.H. and Wisden, W., The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum, J. Neurosci., 12 (1992) 1063 1076. 5 Luddens, H. and Wisden, W., Function and pharmacology of multiple GABAA receptor subunits, Trend Pharmacol. Sci.. 12 (1991) 49-51. 6 Luddens, H., Pritchen, D.B., Kohler, M., Killisch, 1., Keinanen, K., Monyer, H., Sprengel, R. and Seeburg, RH., Cerebellar GABAa receptor selective for a behavioural alcohol antagonist, Nature, 346 (1990) 648-651. 7 Malherbe, R, Sigel, E., Baur, R., Pershon, E., Richards, J.G. and Mohler, H., Functional expression and sites ofgene transcription of a novel ~-subunit of the GABAAreceptor of rat brain, FEBS Lett., 260 (1990) 261 265. 8 McKernan, R.M., Quirk, K., Prince, R., Cox, P.A., Gillard, N.R, Ragan, C.I. and Whiting, P., GABAA receptor subtypes immunopurified from rat brain with ~ subunit-specificantibodies have unique pharmacological properties, Neuron, 7 (1991) 667 676. 90lsen, R.W. and Tobin, A.J., Molecular biology of GABAA receptors, FASEB J., 4 (1990) 1469 1480. 10 Richards, J.G., Schoch, P., Haring, R, Takacs, B. and Mohler. H., ResolvingGABAA/benzodiazepinereceptors: cellular and subcellular localization in the CNS with monoclonal antibodies, J. Neurosci., 7 (1987) 1866,1886. 11 Sieghart, W., Eichinger, A., Richards, J.G. and Mohler, H., Photoaffinity labelling of benzodiazepine receptor proteins with the partial inverse agonist [~H]Ro 15-4513: A biochemical and autoradiographic study, J. Neurochem., 48 (1987) 46-52. 12 Somogyi, P., Takagi, H., Richards, J.G. and Mohler, H., Subcellular localisation of benzodiazepine/GABAAreceptors in the cerebellum of rat, cat and monkey using monoclonal antibodies. J. Neurosci., 9 (1989) 2197 2209. 13 Stephenson, F.A., The GABAA receptors: structure and function, Curr. Asp. Neurosci.. 3 (1991) 177 194. 14 Wisden, W., Laurie, D.J., Monyer, H., and Seeburg, P.H., The distribution of 13 GABAAreceptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon, J. Neurosci., 12 (1992) 1040 1062. 15 Zimprich, F., Zezula, J., Sieghart, W. and Lassmann, H., lmmunohistochemical localisation of the ~1, ~2 and c~3 subunits of the GABAA receptor in the rat brain, FEBS Lett., 127 (1991) 125 128.
