Neuroscience Letters, 127 (1991) 125 128 © 199 l Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100285J
125
NSL 07804
Immunohistochemical localization of the 0 1, 2 and 3 subunit of the GABAA receptor in the rat brain Fritz Zimprich I, Jiirgen Zezula 2, Werner Sieghart 2 and Hans Lassmann 1.3 Research Unitfor Experimental Neuropathology, Austrian Academy of Sciences, Vienna (Austria), -'Department of Biochemical Psychiatry, Psychiatrische Universitiitsklinik, Vienna (Austria) and 3NeurologicalInstitute, University of Vienna, Vienna (Austria) (Received 25 February 1991; Accepted 18 March 1991)
Key words: GABAA receptor; ~-Subunit; Heterogeneity; Immunohistochemistry; Thalamic reticular nucleus; Hippocampus; Striatum; Globus pallidus The immunohistochemical distribution of the st, ~2 and ~3 subunit of the ),-aminobutyric acid-A (GABAA) receptor was investigated in the rat brain using affinity-purified antibodies against unique parts of the amino acid sequence of the respective subunits. The distribution of the 3 subunits differed markedly from each other indicating heterogeneity of the GABAA-receptor composition in different brain regions and at various receptive compartments (dendrites or somata) of neuronal cells.
7-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. It acts both via a GABA-gated chloride channel (GABAA receptor) and the G-protein coupled GABAB receptor. Several important classes of drugs like benzodiazepines, barbiturates, convulsant compounds and some steroids have been shown to interact with the GABAA receptor [5]. This receptor has been purified to apparent homogeneity by benzodiazepine affinity chromatography and seems to consist of several different protein subunits [16]. Molecular cloning studies not only have identified their amino acid sequence but also have demonstrated the existence of a significant heterogeneity of individual subunits. Thus, so far the existence of 6 ~, 3 fl, 2 7 and 1 ~ subunit of the GABAA-receptor has been demonstrated [11]. Studies on the expression in Xenopus oocytes and mammalian cells of these subunits revealed that GABA-gated chloride ion channels modulated by benzodiazepines and fl-carbolines can only be produced by the simultaneous presence of ~, fl and 7 subunit cDNAs in the cells [8]. This indicates that at least 3 different subunits are necessary to reconstitute GABAAbenzodiazepine receptors with correct pharmacology. In addition, it has been demonstrated that the subunit composition of GABAA receptors determines benzodiazepine binding properties. Thus, receptors containing the Correspondence: H. Lassmann, Research Unit for Experimental Neuropathology, Austrian Academy of Sciences, c.o. Neurological Institute, Schwarzspanierstr. 17, A-1090 Vienna, Austria.
~ subunit exhibit properties of the type I GABAA receptor whereas receptors containing the ~2 or ~3 subunits exhibit properties of the type II GABAA benzodiazepine receptors [7]. Recently polyclonal antibodies directed against specific parts of the amino acid sequence of the 0q, ct2 and ~3 subunits were raised (aq: 1-9, ~2: 416-424, 0t3: 459-467) [1]. These antibodies, each of which selectively recognized one single protein with apparent molecular weights 51 kDa, 53 kDa or 59 kDa in purified GABAA receptor preparations or in brain homogenates were used in the present study to investigate the regional distribution of the respective ~ subunits. Adult Sprague-Dawley rats (Versuchstierzucht Himberg, Austria) were perfused with Ringer solution. Brains were immediately removed and coronally cut tissue blocks rapidly frozen. Nine-/tm-thick cryocut sections were fixed in aceton for 5 min. For immunostaining, sections were blocked with 10% fetal calf serum (FCS) prior to administration of afffinity-purified [1] primary polyclonal antibodies (3/tg/ml for 1 h). Alkaline phosphatase or peroxidase coupled goat anti rabbit Fab2 fragments (Jackson Immuno Research) diluted 1: 500 and absorbed with 3% rat serum were applied in the next step. Reaction product was visualized with Fast blue BB or a diaminobenzidine reagent (Sigma, U.S.A.). All antibodies were diluted in 0.3 M Tris buffered saline (pH 7.2) containing 10% FCS. Immunoreactivity in all areas could be blocked by preabsorbing the primary antibodies for 2 h at 37°C with 16/~g/ml of peptide used
126 for i m m u n i z a t i o n . As expected, no i m m u n o s t a i n i n g was o b s e r v e d in sections f r o m liver o r k i d n e y tissues. The d i s t r i b u t i o n o f the i m m u n o r e a c t i v i t y for the 3 p o l y c l o n a l a n t i b o d i e s in the e x a m i n e d b r a i n regions is listed in T a b l e I a n d examples are shown in Fig. 1. W h e r e a s the i m m u n o r e a c t i v i t y o f the a n t i p e p t i d e ~1 was widely d i s t r i b u t e d a n d strong, staining with a n t i - p e p t i d e ~2 a n d 0~3 a n t i b o d i e s was m u c h m o r e restricted (Fig. 1a c). This is in a g r e e m e n t with in situ h y b r i d i z a t i o n studies
for ~1, ~2 a n d ~3 m R N A s [18-20] a n d the d i s t r i b u t i o n a n d relative a b u n d a n c e o f type I c o m p a r e d with type II G A B A A b e n z o d i a z e p i n e receptors [12, 13]. All 3 a n t i b o d i e s exhibited either a diffuse, n e u r o p i l i m m u n o r e a c t i v i t y o r a m o r e discrete staining at the surface o f n e u r o n a l cells a n d cell processes as s h o w n here for al (Fig. 2). In a d d i t i o n , reactivity was f o u n d within cell b o d i e s i n d i c a t i n g t u r n o v e r o f the r e c e p t o r [14]. It was interesting to note that the same a n t i b o d y c o u l d p r o d u c e
Fig. 1. Comparison of the immunoreactivity for ~ (left column), c~2(middle column) and ~3 subunit (right column) of the GABAA-receptor. a-c: frontal section at diencephalic level, serial sections, x 6, Note strong expression of c~3subunit in reticular nucleus in thalamus (arrow) (c). d-f: hippocampal formation, serial sections, × 14. Intense immunoreactivity for ~2 (e) in dentate gyrus and positive band below the granular layer (arrow). In this localizations hilar neurons are stained for ~1. g-i: globus pallidus (gp) and striatum (st), serial sections, x 113. Note the intense neuropil staining for ~l (g), only nerve cell staining for ~2 (h) and no reactivity for ~3 (i) in the globus pallidus, j-l: cerebellar cortex, x 130. There are many cells in the granular layer positive for ~1 (J), fewer cells for ~ (1), but only single cells for ~2 (k).
127
a predominant neuropil staining in one area (e.g. antipeptide ~2 in striatum), but reacted exclusively with nerve cell profiles in other nuclei (e.g. in globus pallidus or ventral and lateral thalamic nuclei) (Fig. l h). Comparable observations made by other authors using antibodies against the 0tl or fl subunits were ultrastructurally related to staining of axodendritic or axosomatic terminals [4,9]. Furthermore, we also found that within one given region (e.g. globus pallidus or ventral and lateral thalamic nuclei) one antibody (anti-peptide ~1) displayed a strong reactivity in the neuropil, the other one (antipeptide ~2) recognized only nerve cells, whereas the third TABLE I DISTRIBUTION OF IMMUNOREACTIVITY FOR THE ctt, oh OR oh SUBUNIT OF THE GABAA RECEPTOR IN VARIOUS BRAIN REGIONS. Neuropil staining (n. pil): - negative, + weak, + + strong, + + + very strong staining. Neuronal cell body staining (n.c.): - no cells, (+)single cells, + few cells, + + many cells stained. 1 Especially in layer III, V and VI; 2especially in layer III and V; 3especially in layer VI; 4more staining in CA2/3 sector than in CA1; 5 stratum lucidum (mossy fibers) negative for all 3 antibodies; ~band of diffuse immunoreactivity on the border between granule cells and hilus; 7 number of positive cells might be underestimated due to strong neuropil staining. Region
cq
~t2
cq
n.pil
n.c.
n.pil
n.c.
n.pil
n.c.
-t-+4-
4-+~
+
4-4-2
++
++a
str. o r i e n s
+ +
-
+/+
str. p y r a m i d a l e
_
+4
_
str. r a d i a t u m ~
+ +
-
4-/++4_
str. l a c u n o s u m
+ +
-
++
4-++
Cerebral cortex Hippocampus
+4_
+/+
+
+4 _ +
--
+
--
4-
(anti-peptide ~3) did not bind at all (compare Fig. lg-i). These results support the concept of GABAA receptor heterogeneity also at the level of subcellular distribution (somata or dendrites). In the cerebral cortex all 3 antibodies showed neuropil and soma staining of different intensity. Anti-peptide atl reacted strongest in layers III, V and VI and anti-peptide ~t3 especially in layer VI (Table I). In the hippocampal formation immunoreactivity of the neuropil was strong in most layers for anti-~q, in contrast to the anti-~2 antibody which showed intense staining in the molecular layer of the dentate gyrus only. Antibodies against ~3 yielded a much weaker staining signal throughout the hippocampal formation, but, interestingly the staining pattern in the CA1 sector was complementary to that observed for ~2 (compare Fig. ld-f). The granule and pyramidal cell layer appeared to be spared by all 3 antibodies. Only a few nerve cells were distinctly positive for ~l and ~t2 subunits especially in the CA2/3 sector in this layer. With anti-~2 a conspicuous band of fine, diffuse immunoreactivity was noted between the granular cell layer and the hilus of the dentate fascia (arrow in Fig. le). In this localization GABAergic synapses were described on the axon initial segments of mossy fibers [15], where the action potential is generated. In addition to axodendritic synapses in the molecular layer, these synapses are believed to mediate inhibition to one of the main excitatory hippocampal pathways very efficiently. Comparing the staining patterns of the 3 antibodies in the thalamic nuclei, the most striking observation was the intense neuropil-labelling in the reticular nucleus for the ct3 subunit (arrow in Fig. lc), whereas reactivity was
moleculare
Dentate gyrus molecular layer
+ +
-
granular layer
-
(+)
h i l u s fast. dent.
+ +
+
Medial habenula
-
-
Lateral habenula
+
+ +
(+)
--
+~
(+)
--
+
--
+
Thalamus reticular
-
-
_
(+)
+++
_7
v e n t r a l & lateral n u c l e i
+ +
(+)7
--
+4-
+
--
midline nuclei
+ +
-
+
--
++
--
Z o n a incerta
+ +
(+)
--
(+)
+
(+)
Hypothalamus
4- +
(4-)
+
+
4-
--
Striatum
+ +
-
++
(+)
++
--
Globus pallidus
+ + +
(+)7
--
-t-4-
--
--
Amygdala
+ +
(+)
+
4-
++
--
S e p t a l n u c l . (incl. n. stria t e r m . )
+ +
--
++
++
--
Nucl. olfaetorii
+ +
(+)
4-
++
4-
+
-
(+)
-
++
4-
--
4-
--
Cerebellar cortex granular layer
-
4- +
P u r k i n j e cells
-
(+)
molecular layer
+ +
Fig. 2. Pattern of immunoreactivity. Anti-peptide ~q in CA1 sector of hippocampus, x 260. Single nerve cell in stratum pyramidale and dedritic profiles in adjacent layers distinctly stained. Diffuse neuropil immunoreactivity in str. oriens (so) and radiatum (sr).
128 absent for ~1 or only weak a r o u n d very few cells as for the ~2 subunit. This was different to the a b u n d a n t staining of other thalamic nuclei for ~1 a n d ~2. A n a t o m i c a l l y in a strategic position the reticular nucleus receives axonal collaterals from thalamocortical a n d corticothalamical fibers, while its efferents project back to the t h a l a m u s a n d use G A B A as a n e u r o t r a n s m i t t e r . F u n c t i o n a l l y the reticular thalamic nucleus plays a role in gating sensory i n f o r m a t i o n a n d selective a t t e n t i o n [17]. I n a recent report n e u r o n s of the reticular nucleus were shown to have the lowest threshold for d e g e n e r a t i o n in a model of transient ischemia due to raised i n t r a c r a n i a l pressure [10]. Since excitotoxic m e c h a n i s m s are t h o u g h t to be involved in n e u r o n a l d e g e n e r a t i o n it is t e m p t i n g to speculate that the vulnerability of the reticular nucleus could be reduced by c o m p o u n d s specifically activating ~3 s u b u n i t c o n t a i n i n g G A B A A receptors. The present study is the first to c o m p a r e the i m m u n o reactivity o f antibodies specific for 3 different ~ s u b u n i t s of the G A B A A receptor. So far only antibodies directed against ~1 a n d fl s u b u n i t s have been used to study the receptor d i s t r i b u t i o n i m m u n o h i s t o c h e m i c a l l y [3, 4, 6, 9, 14]. In general our results o n the d i s t r i b u t i o n of the cq s u b u n i t agree with previous reports o n h u m a n or bovine brain. O u r d a t a are also consistent with in situ hybridization studies o n 5~1, 0~2 a n d ~3 m R N A localization in bovine b r a i n [18-20]. In conclusion, our study indicates a heterogenous n e u r o a n a t o m i c a l a n d subcellular distribution of G A B A A receptors with different molecular compositions, which seems to be a basis for the complicated a n d subtle regulation of i n h i b i t o r y m e c h a n i s m s in the central n e r v o u s system. This study was supported by the Science Research F u n d Austria. W e t h a n k Drs. M. Berger a n d K. Vass for critically reading the manuscript. 1 Fuchs, K., Adamiker, D. and Sieghart, W., Identification of ~2- and
~3-subunitsof the GABAA-benzodiazepinereceptor complex purified from the brains of young rats, FEBS Lett., 261 (1990) 52-54. 2 Hironaka, T., Morita, Y., Hagihira, S., Tateno, E., Kita, H. and Tohyama, M., Localization of GABA^-receptor ~1 subunit mRNA-containing neurons in the lower brainstem of the rat, Mol. Brain Res., 7 (1990) 335-345. 3 Houser, C.R., Olsen, R,W., Richards, J.G. and M6hler, H., Immunohistochemical localization of benzodiazepine/GABAAreceptors in the human hippocampal formation, J. Neurosci., 8 (1988) 13701383. 4 Juiz, J.M., Helfert, R.H., Wenthold, R.J., De Blas, A.L and Altschuler, R.A., Immunocytochemical localization of the GABAA/ benzodiazepine receptor in the guinea pig cochlear nucleus, evi-
dence for receptor localization heterogeneity, Brain Res., 504 (1989) 173-179. 5 Olson, R.W., and Venter, J.C., Benzodiazepine/GABA receptors and chloride channels: structural and functional properties. In R.W. Olsen and J.C. Venter (Eds.), Receptor Biochemistry and Methodology, Vol. 5, Alan R. Liss, New York, 1986. 6 Pinard, R., Richards, J.G. and Lanoir, J., Subcellular localization of GABAA/benzodiazepinereceptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus, Neurosci. Lett., 120 (1990) 212 216. 7 Pritchett, D.B., Liiddens, H. and Seeburg, P.H., Type I and type II GABAA-benzodiazepinereceptors produced in transfected cells, Science, 245 (1989) 1389-1392. 8 Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenmann, H., Schofield, P.R. and Seeburg, P.H., Importance of a novel GABAA-receptorsubunit for benzodiazepinepharmacology, Nature, 338 (1989) 582-585. 9 Richards, J.G., Schoch, P., H~iring,P., Takacs, B. and M6hler, H., ResolvingGABAA/benzodiazepinereceptors: cellular and subcellular localization in the CNS with monoclonal antibodies, J. Neurosci., 7 (1987) 186f~1886. 10 Ross, D.T. and Duhaime, A.C., Degeneration of neurons in the thalamic reticular nucleus following transient ischemia due to raised intracranial pressure: excitotoxic degeneration mediated via non-NMDA receptors?, Brain Res., 501 (1989) 129 143. 11 Shivers, B.D., Killisch, I., Sprengel, R., Sontheimer, H., K6hler, M., Schofield, P.R. and Seeburg, P.H., Two novel GABAA-receptor subunits exist in distinct neuronal subpopulations, Neuron, 3 (1989) 327- 337. 12 Sieghart, W. and Drexler, G., Irreversible binding of [3H] flunitrazepam to different proteins in various brain regions, J. Neurochem., 41 (1983) 47 -55. 13 Sieghart, W., Multiplicity of GABAA-benzodiazepinereceptors, Trends Pharmacol. Sci., 10 (1989) 407~111. 14 Somogyi, P., Takagi, H., Richards, J.G. and M6hler H., Subcellular localization of benzodiazepine/GABAAreceptors in the cerebellum of rat, cat and monkey using monoclonal antibodies, J. Neurosci,, 9 (1989) 2197-2209. 15 Soriano, E. and Frotscher, M., A GABAergicaxo-axonic cell in the fascia dentata controls the main excitatory hippocampal pathway, Brain Res., 503 (1989) 170 174, 16 Stephenson, F.A., Understanding the GABAA-receptor: a chemically gated ion channel, Biochem. J., 249 (1988) 21 32. 17 Steriade, M., Domich, L. and Oaksen, G., Reticularis thalamic neurons revisited: activity changes during shifts in states of vigilance, J. Neurosci., 6 (1986) 68-81. 18 Wisden, W., McNaughton, LA., Darlison, M.G., Hunt, S.P. and Barnard, E.A., Differential distribution of GABAA receptor mRNAs in bovine cerebellum - - localization of cq mRNA in Bergmann glia layer, Neurosci. Lett., 106 (1989) 7 12. 19 Wisden, W., Morris, B.J., Darlison, M.G., Hunt, S.P. and Barnard, E.A., Distinct GABAA receptor ~ subunit mRNAs show differential patterns of expression in bovine brain, Neuron, 1 (1988) 937 947. 20 Wisden, W., Morris, B.J., Dartison, M.G., Hunt, S.P. and Barnard, E.A., Localization of GABAA receptor ~-subunit mRNAs in relation to receptor subtypes, Mol. Brain Res., 5 (1989) 305 310.
125
NSL 07804
Immunohistochemical localization of the 0 1, 2 and 3 subunit of the GABAA receptor in the rat brain Fritz Zimprich I, Jiirgen Zezula 2, Werner Sieghart 2 and Hans Lassmann 1.3 Research Unitfor Experimental Neuropathology, Austrian Academy of Sciences, Vienna (Austria), -'Department of Biochemical Psychiatry, Psychiatrische Universitiitsklinik, Vienna (Austria) and 3NeurologicalInstitute, University of Vienna, Vienna (Austria) (Received 25 February 1991; Accepted 18 March 1991)
Key words: GABAA receptor; ~-Subunit; Heterogeneity; Immunohistochemistry; Thalamic reticular nucleus; Hippocampus; Striatum; Globus pallidus The immunohistochemical distribution of the st, ~2 and ~3 subunit of the ),-aminobutyric acid-A (GABAA) receptor was investigated in the rat brain using affinity-purified antibodies against unique parts of the amino acid sequence of the respective subunits. The distribution of the 3 subunits differed markedly from each other indicating heterogeneity of the GABAA-receptor composition in different brain regions and at various receptive compartments (dendrites or somata) of neuronal cells.
7-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. It acts both via a GABA-gated chloride channel (GABAA receptor) and the G-protein coupled GABAB receptor. Several important classes of drugs like benzodiazepines, barbiturates, convulsant compounds and some steroids have been shown to interact with the GABAA receptor [5]. This receptor has been purified to apparent homogeneity by benzodiazepine affinity chromatography and seems to consist of several different protein subunits [16]. Molecular cloning studies not only have identified their amino acid sequence but also have demonstrated the existence of a significant heterogeneity of individual subunits. Thus, so far the existence of 6 ~, 3 fl, 2 7 and 1 ~ subunit of the GABAA-receptor has been demonstrated [11]. Studies on the expression in Xenopus oocytes and mammalian cells of these subunits revealed that GABA-gated chloride ion channels modulated by benzodiazepines and fl-carbolines can only be produced by the simultaneous presence of ~, fl and 7 subunit cDNAs in the cells [8]. This indicates that at least 3 different subunits are necessary to reconstitute GABAAbenzodiazepine receptors with correct pharmacology. In addition, it has been demonstrated that the subunit composition of GABAA receptors determines benzodiazepine binding properties. Thus, receptors containing the Correspondence: H. Lassmann, Research Unit for Experimental Neuropathology, Austrian Academy of Sciences, c.o. Neurological Institute, Schwarzspanierstr. 17, A-1090 Vienna, Austria.
~ subunit exhibit properties of the type I GABAA receptor whereas receptors containing the ~2 or ~3 subunits exhibit properties of the type II GABAA benzodiazepine receptors [7]. Recently polyclonal antibodies directed against specific parts of the amino acid sequence of the 0q, ct2 and ~3 subunits were raised (aq: 1-9, ~2: 416-424, 0t3: 459-467) [1]. These antibodies, each of which selectively recognized one single protein with apparent molecular weights 51 kDa, 53 kDa or 59 kDa in purified GABAA receptor preparations or in brain homogenates were used in the present study to investigate the regional distribution of the respective ~ subunits. Adult Sprague-Dawley rats (Versuchstierzucht Himberg, Austria) were perfused with Ringer solution. Brains were immediately removed and coronally cut tissue blocks rapidly frozen. Nine-/tm-thick cryocut sections were fixed in aceton for 5 min. For immunostaining, sections were blocked with 10% fetal calf serum (FCS) prior to administration of afffinity-purified [1] primary polyclonal antibodies (3/tg/ml for 1 h). Alkaline phosphatase or peroxidase coupled goat anti rabbit Fab2 fragments (Jackson Immuno Research) diluted 1: 500 and absorbed with 3% rat serum were applied in the next step. Reaction product was visualized with Fast blue BB or a diaminobenzidine reagent (Sigma, U.S.A.). All antibodies were diluted in 0.3 M Tris buffered saline (pH 7.2) containing 10% FCS. Immunoreactivity in all areas could be blocked by preabsorbing the primary antibodies for 2 h at 37°C with 16/~g/ml of peptide used
126 for i m m u n i z a t i o n . As expected, no i m m u n o s t a i n i n g was o b s e r v e d in sections f r o m liver o r k i d n e y tissues. The d i s t r i b u t i o n o f the i m m u n o r e a c t i v i t y for the 3 p o l y c l o n a l a n t i b o d i e s in the e x a m i n e d b r a i n regions is listed in T a b l e I a n d examples are shown in Fig. 1. W h e r e a s the i m m u n o r e a c t i v i t y o f the a n t i p e p t i d e ~1 was widely d i s t r i b u t e d a n d strong, staining with a n t i - p e p t i d e ~2 a n d 0~3 a n t i b o d i e s was m u c h m o r e restricted (Fig. 1a c). This is in a g r e e m e n t with in situ h y b r i d i z a t i o n studies
for ~1, ~2 a n d ~3 m R N A s [18-20] a n d the d i s t r i b u t i o n a n d relative a b u n d a n c e o f type I c o m p a r e d with type II G A B A A b e n z o d i a z e p i n e receptors [12, 13]. All 3 a n t i b o d i e s exhibited either a diffuse, n e u r o p i l i m m u n o r e a c t i v i t y o r a m o r e discrete staining at the surface o f n e u r o n a l cells a n d cell processes as s h o w n here for al (Fig. 2). In a d d i t i o n , reactivity was f o u n d within cell b o d i e s i n d i c a t i n g t u r n o v e r o f the r e c e p t o r [14]. It was interesting to note that the same a n t i b o d y c o u l d p r o d u c e
Fig. 1. Comparison of the immunoreactivity for ~ (left column), c~2(middle column) and ~3 subunit (right column) of the GABAA-receptor. a-c: frontal section at diencephalic level, serial sections, x 6, Note strong expression of c~3subunit in reticular nucleus in thalamus (arrow) (c). d-f: hippocampal formation, serial sections, × 14. Intense immunoreactivity for ~2 (e) in dentate gyrus and positive band below the granular layer (arrow). In this localizations hilar neurons are stained for ~1. g-i: globus pallidus (gp) and striatum (st), serial sections, x 113. Note the intense neuropil staining for ~l (g), only nerve cell staining for ~2 (h) and no reactivity for ~3 (i) in the globus pallidus, j-l: cerebellar cortex, x 130. There are many cells in the granular layer positive for ~1 (J), fewer cells for ~ (1), but only single cells for ~2 (k).
127
a predominant neuropil staining in one area (e.g. antipeptide ~2 in striatum), but reacted exclusively with nerve cell profiles in other nuclei (e.g. in globus pallidus or ventral and lateral thalamic nuclei) (Fig. l h). Comparable observations made by other authors using antibodies against the 0tl or fl subunits were ultrastructurally related to staining of axodendritic or axosomatic terminals [4,9]. Furthermore, we also found that within one given region (e.g. globus pallidus or ventral and lateral thalamic nuclei) one antibody (anti-peptide ~1) displayed a strong reactivity in the neuropil, the other one (antipeptide ~2) recognized only nerve cells, whereas the third TABLE I DISTRIBUTION OF IMMUNOREACTIVITY FOR THE ctt, oh OR oh SUBUNIT OF THE GABAA RECEPTOR IN VARIOUS BRAIN REGIONS. Neuropil staining (n. pil): - negative, + weak, + + strong, + + + very strong staining. Neuronal cell body staining (n.c.): - no cells, (+)single cells, + few cells, + + many cells stained. 1 Especially in layer III, V and VI; 2especially in layer III and V; 3especially in layer VI; 4more staining in CA2/3 sector than in CA1; 5 stratum lucidum (mossy fibers) negative for all 3 antibodies; ~band of diffuse immunoreactivity on the border between granule cells and hilus; 7 number of positive cells might be underestimated due to strong neuropil staining. Region
cq
~t2
cq
n.pil
n.c.
n.pil
n.c.
n.pil
n.c.
-t-+4-
4-+~
+
4-4-2
++
++a
str. o r i e n s
+ +
-
+/+
str. p y r a m i d a l e
_
+4
_
str. r a d i a t u m ~
+ +
-
4-/++4_
str. l a c u n o s u m
+ +
-
++
4-++
Cerebral cortex Hippocampus
+4_
+/+
+
+4 _ +
--
+
--
4-
(anti-peptide ~3) did not bind at all (compare Fig. lg-i). These results support the concept of GABAA receptor heterogeneity also at the level of subcellular distribution (somata or dendrites). In the cerebral cortex all 3 antibodies showed neuropil and soma staining of different intensity. Anti-peptide atl reacted strongest in layers III, V and VI and anti-peptide ~t3 especially in layer VI (Table I). In the hippocampal formation immunoreactivity of the neuropil was strong in most layers for anti-~q, in contrast to the anti-~2 antibody which showed intense staining in the molecular layer of the dentate gyrus only. Antibodies against ~3 yielded a much weaker staining signal throughout the hippocampal formation, but, interestingly the staining pattern in the CA1 sector was complementary to that observed for ~2 (compare Fig. ld-f). The granule and pyramidal cell layer appeared to be spared by all 3 antibodies. Only a few nerve cells were distinctly positive for ~l and ~t2 subunits especially in the CA2/3 sector in this layer. With anti-~2 a conspicuous band of fine, diffuse immunoreactivity was noted between the granular cell layer and the hilus of the dentate fascia (arrow in Fig. le). In this localization GABAergic synapses were described on the axon initial segments of mossy fibers [15], where the action potential is generated. In addition to axodendritic synapses in the molecular layer, these synapses are believed to mediate inhibition to one of the main excitatory hippocampal pathways very efficiently. Comparing the staining patterns of the 3 antibodies in the thalamic nuclei, the most striking observation was the intense neuropil-labelling in the reticular nucleus for the ct3 subunit (arrow in Fig. lc), whereas reactivity was
moleculare
Dentate gyrus molecular layer
+ +
-
granular layer
-
(+)
h i l u s fast. dent.
+ +
+
Medial habenula
-
-
Lateral habenula
+
+ +
(+)
--
+~
(+)
--
+
--
+
Thalamus reticular
-
-
_
(+)
+++
_7
v e n t r a l & lateral n u c l e i
+ +
(+)7
--
+4-
+
--
midline nuclei
+ +
-
+
--
++
--
Z o n a incerta
+ +
(+)
--
(+)
+
(+)
Hypothalamus
4- +
(4-)
+
+
4-
--
Striatum
+ +
-
++
(+)
++
--
Globus pallidus
+ + +
(+)7
--
-t-4-
--
--
Amygdala
+ +
(+)
+
4-
++
--
S e p t a l n u c l . (incl. n. stria t e r m . )
+ +
--
++
++
--
Nucl. olfaetorii
+ +
(+)
4-
++
4-
+
-
(+)
-
++
4-
--
4-
--
Cerebellar cortex granular layer
-
4- +
P u r k i n j e cells
-
(+)
molecular layer
+ +
Fig. 2. Pattern of immunoreactivity. Anti-peptide ~q in CA1 sector of hippocampus, x 260. Single nerve cell in stratum pyramidale and dedritic profiles in adjacent layers distinctly stained. Diffuse neuropil immunoreactivity in str. oriens (so) and radiatum (sr).
128 absent for ~1 or only weak a r o u n d very few cells as for the ~2 subunit. This was different to the a b u n d a n t staining of other thalamic nuclei for ~1 a n d ~2. A n a t o m i c a l l y in a strategic position the reticular nucleus receives axonal collaterals from thalamocortical a n d corticothalamical fibers, while its efferents project back to the t h a l a m u s a n d use G A B A as a n e u r o t r a n s m i t t e r . F u n c t i o n a l l y the reticular thalamic nucleus plays a role in gating sensory i n f o r m a t i o n a n d selective a t t e n t i o n [17]. I n a recent report n e u r o n s of the reticular nucleus were shown to have the lowest threshold for d e g e n e r a t i o n in a model of transient ischemia due to raised i n t r a c r a n i a l pressure [10]. Since excitotoxic m e c h a n i s m s are t h o u g h t to be involved in n e u r o n a l d e g e n e r a t i o n it is t e m p t i n g to speculate that the vulnerability of the reticular nucleus could be reduced by c o m p o u n d s specifically activating ~3 s u b u n i t c o n t a i n i n g G A B A A receptors. The present study is the first to c o m p a r e the i m m u n o reactivity o f antibodies specific for 3 different ~ s u b u n i t s of the G A B A A receptor. So far only antibodies directed against ~1 a n d fl s u b u n i t s have been used to study the receptor d i s t r i b u t i o n i m m u n o h i s t o c h e m i c a l l y [3, 4, 6, 9, 14]. In general our results o n the d i s t r i b u t i o n of the cq s u b u n i t agree with previous reports o n h u m a n or bovine brain. O u r d a t a are also consistent with in situ hybridization studies o n 5~1, 0~2 a n d ~3 m R N A localization in bovine b r a i n [18-20]. In conclusion, our study indicates a heterogenous n e u r o a n a t o m i c a l a n d subcellular distribution of G A B A A receptors with different molecular compositions, which seems to be a basis for the complicated a n d subtle regulation of i n h i b i t o r y m e c h a n i s m s in the central n e r v o u s system. This study was supported by the Science Research F u n d Austria. W e t h a n k Drs. M. Berger a n d K. Vass for critically reading the manuscript. 1 Fuchs, K., Adamiker, D. and Sieghart, W., Identification of ~2- and
~3-subunitsof the GABAA-benzodiazepinereceptor complex purified from the brains of young rats, FEBS Lett., 261 (1990) 52-54. 2 Hironaka, T., Morita, Y., Hagihira, S., Tateno, E., Kita, H. and Tohyama, M., Localization of GABA^-receptor ~1 subunit mRNA-containing neurons in the lower brainstem of the rat, Mol. Brain Res., 7 (1990) 335-345. 3 Houser, C.R., Olsen, R,W., Richards, J.G. and M6hler, H., Immunohistochemical localization of benzodiazepine/GABAAreceptors in the human hippocampal formation, J. Neurosci., 8 (1988) 13701383. 4 Juiz, J.M., Helfert, R.H., Wenthold, R.J., De Blas, A.L and Altschuler, R.A., Immunocytochemical localization of the GABAA/ benzodiazepine receptor in the guinea pig cochlear nucleus, evi-
dence for receptor localization heterogeneity, Brain Res., 504 (1989) 173-179. 5 Olson, R.W., and Venter, J.C., Benzodiazepine/GABA receptors and chloride channels: structural and functional properties. In R.W. Olsen and J.C. Venter (Eds.), Receptor Biochemistry and Methodology, Vol. 5, Alan R. Liss, New York, 1986. 6 Pinard, R., Richards, J.G. and Lanoir, J., Subcellular localization of GABAA/benzodiazepinereceptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus, Neurosci. Lett., 120 (1990) 212 216. 7 Pritchett, D.B., Liiddens, H. and Seeburg, P.H., Type I and type II GABAA-benzodiazepinereceptors produced in transfected cells, Science, 245 (1989) 1389-1392. 8 Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenmann, H., Schofield, P.R. and Seeburg, P.H., Importance of a novel GABAA-receptorsubunit for benzodiazepinepharmacology, Nature, 338 (1989) 582-585. 9 Richards, J.G., Schoch, P., H~iring,P., Takacs, B. and M6hler, H., ResolvingGABAA/benzodiazepinereceptors: cellular and subcellular localization in the CNS with monoclonal antibodies, J. Neurosci., 7 (1987) 186f~1886. 10 Ross, D.T. and Duhaime, A.C., Degeneration of neurons in the thalamic reticular nucleus following transient ischemia due to raised intracranial pressure: excitotoxic degeneration mediated via non-NMDA receptors?, Brain Res., 501 (1989) 129 143. 11 Shivers, B.D., Killisch, I., Sprengel, R., Sontheimer, H., K6hler, M., Schofield, P.R. and Seeburg, P.H., Two novel GABAA-receptor subunits exist in distinct neuronal subpopulations, Neuron, 3 (1989) 327- 337. 12 Sieghart, W. and Drexler, G., Irreversible binding of [3H] flunitrazepam to different proteins in various brain regions, J. Neurochem., 41 (1983) 47 -55. 13 Sieghart, W., Multiplicity of GABAA-benzodiazepinereceptors, Trends Pharmacol. Sci., 10 (1989) 407~111. 14 Somogyi, P., Takagi, H., Richards, J.G. and M6hler H., Subcellular localization of benzodiazepine/GABAAreceptors in the cerebellum of rat, cat and monkey using monoclonal antibodies, J. Neurosci,, 9 (1989) 2197-2209. 15 Soriano, E. and Frotscher, M., A GABAergicaxo-axonic cell in the fascia dentata controls the main excitatory hippocampal pathway, Brain Res., 503 (1989) 170 174, 16 Stephenson, F.A., Understanding the GABAA-receptor: a chemically gated ion channel, Biochem. J., 249 (1988) 21 32. 17 Steriade, M., Domich, L. and Oaksen, G., Reticularis thalamic neurons revisited: activity changes during shifts in states of vigilance, J. Neurosci., 6 (1986) 68-81. 18 Wisden, W., McNaughton, LA., Darlison, M.G., Hunt, S.P. and Barnard, E.A., Differential distribution of GABAA receptor mRNAs in bovine cerebellum - - localization of cq mRNA in Bergmann glia layer, Neurosci. Lett., 106 (1989) 7 12. 19 Wisden, W., Morris, B.J., Darlison, M.G., Hunt, S.P. and Barnard, E.A., Distinct GABAA receptor ~ subunit mRNAs show differential patterns of expression in bovine brain, Neuron, 1 (1988) 937 947. 20 Wisden, W., Morris, B.J., Dartison, M.G., Hunt, S.P. and Barnard, E.A., Localization of GABAA receptor ~-subunit mRNAs in relation to receptor subtypes, Mol. Brain Res., 5 (1989) 305 310.
