The GABAA receptor complex in the chicken brain: immunocytochemical distribution of α1- and γ2-subunits and autoradiographic distribution of BZ1 and BZ2 binding sites

Journal of Chemical Neuroanatomy 25 (2003) 1 /18 www.elsevier.com/locate/jchemneu

The GABAA receptor complex in the chicken brain: immunocytochemical distribution of a1- and g2-subunits and autoradiographic distribution of BZ1 and BZ2 binding sites Maria Isabel Aller a, Miguel Angel Paniagua a, Simon Pollard b, F. Anne Stephenson b, Arsenio Fernandez-Lopez a,* b

a Facultad de Biologı´a, Departamento de Biologı´a Celular y Anatomı´a, Universidad de Leo´n, Campus de Vegazana s/n, 24071 Leo´n, Spain Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, UK

Received 7 March 2001; received in revised form 3 August 2002; accepted 3 August 2002

Abstract Two antibodies, raised against the rat GABAA receptor a1- and g2-subunits, were used for an immunocytochemical study of the distribution of these proteins in the chicken brain. The immunoreactive bands obtained by Western blotting and the similar labelling distribution found in the rat and chicken brain support the suitability of these antibodies for the labelling of GABAA receptors in birds. We found abundant a1 and g2 immunoreactivity throughout the chicken brain, mainly in the paleostriata and lobus paraolfactorius, dorsal thalamus and some nuclei of the brainstem. The a1-subunit was more abundant in the telencephalon, thalamus and cerebellum, while the presence of the g2-subunit was stronger in the optic tectum and brainstem. We also report the autoradiographic distribution of the BZ1 and BZ2 benzodiazepine receptor subtypes in the chicken brain using [3H]flunitrazepam. Benzodiazepine binding was unevenly distributed throughout the chicken brain, and the anatomical distribution of the BZ1 and BZ2 subtypes was similar to that described in mammals. The highest binding values were found in the olfactory bulb, paleostriatum primitivum, optic tectum, nucleus mesencephalicus lateralis pars dorsalis and nucleus isthmi pars parvocellularis, the BZ2 subtype being predominant in the paleostriatum primitivum and optic tectum. A general agreement in the distribution of BZ1 and a1 immunoreactivity was observed in structures such as the olfactory bulb, paleostriata, lobus parolfactorius and dorsal thalamus, although some discrepancies were observed in areas such as the optic tectum or nucleus isthmi pars parvocellularis, with high BZ1 binding and low or no a1 immunolabelling. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Polyclonal antibodies; [3H]flunitrazepam; CL218872

Abbreviations: A, archistriatum; Aa1, antibody against the a1-subunit; Ag2, antibody against the g2-subunit; Bas, nucleus basalis; BZ, benzodiazepines; BZ1, benzodiazepine receptor type I; BZ2, benzodiazepine receptor type II; Cb, cerebellum; DTh, dorsal thalamic nuclei; E, ectostriatum; EM, nucleus ectomamillaris; GL, glomerular layer; GLv, nucleus geniculatus lateralis pars ventralis; Gra, granular layer of cerebellum; GRe, external granular layer; HA, hyperstriatum accessorium; Hp, hippocampus; Hy, hypothalamus; ICo, nucleus intercollicularis; Imc, nucleus isthmi, pars magnocellularis; IO, nucleus isthmi opticum; Ipc, nucleus isthmi, pars parvocellularis; LM, nucleus lentiformis mesencephali; LPO, lobus parolfactorius; M, mitral cell layer; MLd, nucleus mesencephalicus lateralis, pars dorsalis; Mol, molecular layer of cerebellum; N, neostriatum; PA, paleostriatum augmentatum; PJ, purkinje cells; PLEe, external plexiform layer; PP, paleostriatum primitivum; PT, nucleus pretectalis; ROT, nucleus rotundus; SAC, stratum album centrale; SGC, stratum girseum centrale; SGF, stratum griseum and fibrosum superficiale; Slu, nucleus semilunaris; SN, substantia nigra; SpL, nucleus spiriformis lateralis; T, nucleus triangularis; TeO, optic tectum. * Corresponding author. Tel.:
/34-987-291-485; fax:
/34-987-291-487. E-mail address: [email protected] (A. Fernandez-Lopez). 0891-0618/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 1 - 0 6 1 8 ( 0 2 ) 0 0 0 7 1 - 6

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1. Introduction The g-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. The main binding site for this neurotransmitter in the brain is linked to a chloride channel and it is called GABAA. In mammals, the native GABAA receptor is probably a hetero-pentameric protein assembled from several different classes of GABAA receptor subunits, each of them presenting different isoforms: a(1-6), b(14), g(1-3), r(1-3), d, o, p and u. Some of these subunits also exist as alternative spliced forms (i.e. g2S and g2L, Barnard et al., 1998; Bonnert et al., 1999). Besides GABA, a variety of substances bind to different specific binding sites of the receptor modulating its activity (Olsen and Tobin, 1990; Mo¨hler et al., 1992). Benzodiazepines (BZ) bind to the channel asubunits or to the a /g-subunit interface, modulating the action of GABA on the GABAA binding sites and inducing agonist, antagonist or inverse agonist effects (Rabow et al., 1995). Two different types of BZ binding sites, BZ1 and BZ2, have been described based on the pharmacological profiles observed for native receptors (Lu¨ddens and Seeburg, 1989). Subsequent studies of the pharmacological profiles of BZ on recombinant receptors with defined subunit combinations support the idea that BZ1 are homogeneous receptors while BZ2 receptors constitute a heterogeneous class of receptors (Pritchett et al., 1989a; Pritchett and Seeburg, 1990). Studies of Xenopus oocytes expressing the a1bxg2 receptor combination (where bx is any b-subunit) have uncovered a pharmacology resembling that of the BZ1 site (high affinity for triazolopyridazines and b-carbolines) (Lu¨ddens and Wisden, 1991). Although the g2subunit is essential for conveying classical BZ sites to recombinant receptors (Pritchett et al., 1989b), the asubunit variants largely determine the receptor pharmacological profile (Pritchett et al., 1989a; Pritchett and Seeburg, 1990). BZ1 and BZ2 receptors are formed upon coexpression of the a1bxg2 and a2 or a3bxg2 isoforms, respectively (Pritchett et al., 1989a,b; Stephenson, 1995). The close association between BZ and GABAA binding sites led to the development of specific ligands that have been used to determine the distributions of GABAA and BZ binding sites in the nervous system (Mo¨hler and Okada, 1977; Niddam et al., 1987; reviewed by Fernandez-Lopez et al., 1997). GABA/BZ receptors have been shown to be abundant and heterogeneously distributed in the central nervous system (CNS) of diverse mammalian species. Physiological, biochemical, pharmacological, ligand binding, immunocytochemical and in situ hybridisation studies have revealed that GABAA/BZ receptors are also abundant in the avian brain (Glencorse et al., 1991; Veenman et al., 1994).

This work describes for the first time the immunocytochemical distribution of a1 and g2 GABAA receptor subunits and the autoradiographic distribution of BZ1 and BZ2 receptors in the chicken brain.

2. Methods All the animals used in this work were treated in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). 2.1. Autoradiography Five male chickens aged 30 days were killed by decapitation and their brains were rapidly removed, frozen and stored in liquid nitrogen until use. Horizontal and coronal sections, 10 mm thick, obtained with a cryostat were mounted onto gelatin-coated slides and stored at /20 8C until used. BZ receptors were labelled with [3H]flunitrazepam (82 Ci/mmol, New England Nuclear, UK) at a concentration of 2 nM, in 0.17 M Tris /HCl, pH 7.4, at 4 8C for 40 min, followed by washing in the same buffer for 2 min and drying at 4 8C, as previously described (Negro et al., 1995). Characterisation of BZ2 binding sites was carried out directly by co-incubating [3H]flunitrazepam in the presence of 1 mM CL218872, which specifically binds to BZ1 binding sites, while BZ1 binding sites were calculated as the difference between [3H]flunitrazepam-specific binding and [3H]flunitrazepam binding to BZ2 binding sites. Non-specific binding was determined in the presence of 1 mM clonazepam. Autoradiographs were generated by exposing all the tissue sections labelled with the radioligand to tritium-sensitive film (Hyperfilm, Amersham-Pharmacia, UK) for 15 days, together with the appropriate radioactive standards (Amersham-Pharmacia, UK). The autoradiographs thus generated were analysed and quantified using a computer-assisted image analysis system VIDAS (Carl Zeiss, Germany). Densitometric readings were converted into values of radioligand bound to tissue and expressed as fmol radioligand binding sites/mg tissue. 2.2. Western blotting Polyclonal antibodies were raised in rabbits against the following synthetic peptide sequences unique to the respective rat subunits (a1: aa 413/429 and g2: aa 1/15 Cys) coupled to keyhole limpet hemocyanin (for amino acid sequences, see Stephenson and Duggan, 1989; Duggan and Stephenson, 1989; Bateson et al., 1991; Stephenson et al., 1990; Glencorse et al., 1990). Antibodies were affinity-purified using the respective peptide-affinity columns (Duggan and Stephenson, 1990). Membranes from adult chicken brain were prepared as

M.I. Aller et al. / Journal of Chemical Neuroanatomy 25 (2003) 1 /18 Table 1 Different patterns of antibody labelling found throughout the chicken brain for the antibodies Aa1 (column 1) and Ag2 (column 2)

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Table 1 (Continued )

Pattern 1: individual cell somata labelling (); pattern 2: neuronal somata and dendrite labelling (w); pattern 3: cell somata labelling together with a diffusely stained neuropil ( ); pattern 4: neuronal somata and dendrite staining over a fibrous neuropil ( ); pattern 5: diffuse staining of the neuropil in which no cell somata can be recognised individually (k). The number of symbols indicates the relative intensity of subunit immunoreactivity: 4, very intense; 3, intense; 2, moderate; 1, weak; the lack of staining is indicated by a dash. Columns 3, 4 and 5 show the distribution of the autoradiographic values of 2 nM [3H]flunitrazepam binding (BZ), 2 nM [3H]flunitrazepam binding in the presence of 1 mM CL218872 (BZ2) and differences between BZ and BZ2 (BZ1), respectively, expressed in fmol/mg tissue (mean9/SEM).

described by Mertens et al. (1993) and stored at /20 8C until used. Aliquots of the washed membranes (20 mg protein/lane) were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions in 10% polyacrylamide slab gels. Western blotting was accomplished as described previously (Stephenson et al., 1986; Duggan and Stephenson, 1989) using the antibodies against the a1- and g2subunits. Immunoreactivity was detected using the ECL luminescence method (Amersham-Pharmacia, UK). 2.3. Immunocytochemical studies Five male chickens of 30 days of age were deeply anaesthetised with 100 mg/kg intravenous sodium ketamine and then perfused with 250 ml of avian saline (0.75% NaCl) followed by 300 ml of the PLM fixative at 4 8C (4% paraformaldehyde; 0.1 M L-lysine; 0.01 M sodium m -periodate in 0.1 M sodium phosphate buffer, pH 7.2), after which brains were rapidly removed and

then left in the same fixative for 6 h at 4 8C. Then, brains were kept overnight in 30% (w/v) sucrose in 0.1 M sodium phosphate buffer, pH 7.2, at 4 8C. Following this, encephalons were rapidly frozen and coronal sections were cut at a thickness of 50 mm with a cryostat. Sections were kept in cryoprotectant solution (30% (w/v) sucrose, 1% (w/v) polyvinylpyrrolidone, 30% (v/v) ethylene glycol in 0.1 M sodium phosphate buffer, pH 7.2) at /20 8C until used. Free-floating sections were incubated overnight at 4 8C in 5 mg/ml primary affinitypurified antibodies in Tris-buffered saline (pH 7.4) containing 2% (v/v) normal goat serum and 0.3% (v/v) Triton X-100. Biotinylated goat anti-rabbit antibodies (Sigma) were used as secondary antibodies. For detection, the avidin /biotin complex (Vectastain Elite† ABC kit, Vector Laboratories) and nickel-enhanced diaminobenzidine were used. Controls were performed omitting the primary antibodies and they did not label the sections in the DAB incubation times used to detect the immunoreactivity with our antibodies, thus indicat-

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Fig. 1. Autoradiographs from chicken brain coronal sections using 2 nM [3H]flunitrazepam (A, C, E, G) and 2 nM [3H]flunitrazepam in the presence of 1 mM CL218872 (B, D, F, H). The main structures of each section have been labelled using abbreviations from the list in the text (bar/2 mm).

ing that endogenous biotin expression does not interfere with the immunocytochemical procedure using these antibodies. Since the ABC DAB procedure is subjected to considerable experimental variance, we used several animals and processed three samples from each (three complete series of coronal sections of the whole brain kept in the same vial) in parallel, maintaining the same incubation times at each step of the immunocytochemical staining. The traditional subjective quantitative system (from none to four symbols) employed to indicate the grade of immunoreactivity was used, and a large number of sections from different animals were stained with the same antibody to minimise such variability.

3. Results 3.1. Autoradiographic studies The autoradiographic localisation of [3H]flunitrazepam binding sites as well as the 1 mM CL218872inhibited [3H]flunitrazepam binding sites throughout the chicken brain is summarised in Table 1. We arbitrarily designated areas with a very high density of binding sites as those with a density greater than 75% of that found in the most enriched area. Areas of high, moderate, low and very low density were considered to be those having,

respectively, 50/75, 35 /50, 10 /35 and B/10% of the density of binding sites with respect to the most enriched area. 3.1.1. Distribution of [3H]flunitrazepam binding sites [3H]flunitrazepam labelling, i.e. total BZ binding sites, were very abundant throughout the chicken brain, as shown in Table 1 and Figs. 1/3. In the telencephalon and the mesencephalon, the labelling was more regionally varied and abundant than in the diencephalon. The highest labelling density was seen in the external layers of the olfactory bulb and inner layers of the stratum griseum and fibrosum superficiale of the optic tectum (TeO) (SGF). Structures with a very high density of binding sites were observed in the paleostriatum primitivum (PP), the outer layers of SGF and the stratum griseum centrale (SGC) of the TeO, the peripheral area of the nucleus mesencephalicus lateralis, pars dorsalis (MLd), and the nucleus isthmi, pars parvocellularis (Ipc). A high density of labelling was observed in the hyperstriata, the neostriatum (N), the nucleus lateralis anterior thalami, the nucleus ovoidalis, the nucleus pretectalis (PT), the nucleus lentiformis mesencephali (LM), the central area of the MLd, and the nucleus ectomamillaris (EM). A moderate degree of labelling was observed in structures such as the paleostriatum augmentatum (PA), the hippocampus, the archistriatum, the nucleus medialis anterior thalami, the nucleus geniculatus lateralis, pars ventralis (GLv), the nucleus

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3.2. Immunological studies

Fig. 2. Autoradiographs from chicken brain coronal sections using 2 nM [3H]flunitrazepam (I, K) and 2 nM [3H]flunitrazepam in the presence of 1 mM CL218872 (J, L). The main structures of each section have been labelled using abbreviations from the list in the text (bar/2 mm).

triangularis (T), the stratum album centrale of TeO (SAC), the nucleus spiriformis lateralis (SpL), the nucleus intercollicularis (ICo), the nucleus tegmentalis (substantia nigra) (SN), the nucleus semilunaris (Slu) and the molecular and granular layers of the cerebellum (Mol, Gra). The remaining structures showed a low or very low density of binding sites.

3.1.2. Distribution of BZ2 binding sites The binding values obtained after incubation with [3H]flunitrazepam inhibited with 1 mM CL218872 are summarised in Table 1 (see Figs. 1 /3). The highest radioligand binding under these conditions was observed in the Ipc. Very high-density values were observed in the external layer of the olfactory bulb. Structures such as the external and internal layers of SGF, the peripheral area of the MLd and the EM showed a high density. A moderate density of binding sites was observed in the hyperstriata, the N, the nucleus basalis (Bas), the nucleus anterior thalami, the GLv, the LM, intermediate layers of the SGF, SAC and the central area of the MLd. The remaining structures showed a low or very low density of binding sites.

In mammals, the specificities of anti-GABAA receptor subunit-specific antibodies have been based on their characterisation by Western blotting, immunoprecipitation and immunocytochemistry (Duggan and Stephenson, 1990; Stephenson et al., 1990; Duggan et al., 1991). The results of Western blotting are shown in Fig. 4. The Western blotting bands obtained in chicken with the antibodies Aa1 and Ag2 are in keeping with the expected molecular weights for these subunits. The immunocytochemical results using Aa1 and Ag2 as primary antibodies for the distribution of a1 and g2 immunoreactivities in chicken brain are summarised in Table 1, in which the different patterns of antibody labelling previously used in the literature (Fritschy and Mo¨hler, 1995; Aller et al., 2000) found throughout the chicken brain are shown: pattern 1 */individual labelling of cell somata, sometimes including the proximal dendrites, with no background staining (Fig. 10B); pattern 2*/neuronal somata and whole dendrite labelling, where no neuropil staining can be observed (Fig. 9A); pattern 3 */cell somata labelling, sometimes including the proximal dendrites, over a background of a diffusely stained neuropil (Fig. 10D, F); pattern 4*/ neuronal somata and dendrite staining over a fibrous neuropil, sometimes including a diffusely stained neuropil (Fig. 9F); pattern 5 */diffusely stained neuropil in which no cell somata can be recognised individually (Fig. 9G). The different intensities observed with the antibodies Aa1 and Ag2 are shown in Figs. 6 and 7. 3.2.1. Regional distribution of subunits 3.2.1.1. Telencephalon. In the olfactory bulb, a strong labelling corresponding to pattern 5 (defined by a diffusely stained neuropil) was observed for both the a1- and g2-subunits in the glomerular layer of olfactory bulb (GL). The same pattern, but with only a moderate degree of staining, was found for the a1-subunit in the external granular (GRe) and external plexiform (PLEe) layers of the olfactory bulb. In the mitral cell layer of the olfactory bulb (M), a few individually labelled perikarya and proximal dendrites were visible with Aa1 over a diffusely stained neuropil, defining the pattern 3 of labelling. No staining of the a1-subunit was observed in the internal layers (Fig. 8A). Ag2 showed a moderate pattern 3 labelling in the GRe and the internal granular layer of the olfactory bulb and a weak pattern 5 for the PLEe and the internal plexiform layer of the olfactory bulb. An intense pattern 3 labelling displaying the larger cell somata was observed in the M when Ag2 were used (Fig. 8B). In the visual Wulst, medial Wulst and DVR (dorsal ventricular ridge), the a1-subunit showed a moderate-toweak staining with pattern 3. The g2-subunit in these

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Fig. 3. Autoradiographs from chicken brain horizontal sections using 2 nM [3H]flunitrazepam (A) and 2 nM [3H]flunitrazepam in the presence of 1 mM CL218872 (B). The main structures of each section have been labelled using abbreviations from the list in the text (bar/2 mm).

labelling (Fig. 9B) and the Bas a moderate degree of somata labelling with no background (pattern 1) when Ag2 were used. The basal ganglia showed intense-to-very intense labelling when Aa1 and Ag2 were used. In the case of Aa1, the lobus paraolfactorius (LPO) and the PP showed a pattern 4 labelling (Fig. 9C and E), while the PA showed a pattern 2 labelling similar to E. Using Ag2, a pattern 3 with moderate-to-intense staining was observed in the PA and LPO (Fig. 9D), and a pattern 4 with intense-to-very intense labelling was observed in the PP (Fig. 9F).

Fig. 4. (A) Western blot of chicken telencephalon (lane 1) and cerebellum homogenates (lane 2) exposed to affinity-purified Aa1. (B) Western blot of chicken optic tectum (lane 1) and cerebellum homogenates (lane 2) exposed to affinity-purified Ag2. Molecular weights (kDa) of the labelled bands are shown.

areas showed the same pattern but more somata were labelled. The ectostriatum (E) and the Bas displayed the same pattern, with a moderate-to-intense staining of somata and dendrites (pattern 2), for the a1-subunit (Fig. 9A), while the E showed a moderate pattern 3

3.2.1.2. Diencephalon. Using Ag2, the pattern 3 of staining was observed in the nucleus habenularis, while no labelling was observed with the Aa1. In the thalamic nuclei, the use of Aa1 revealed a pattern 5 with very intense labelling (Fig. 9G), while Ag2 displayed a pattern 3, with a moderate intensity of labelling (Fig. 9H). The a1-subunit was revealed in the nucleus rotundus (ROT) and T as a pattern 3 with moderate staining (Fig. 10A) while the g2-subunit was observed in a pattern 1 with moderate-to-intense staining (Fig. 10B). No Aa1 labelling could be observed in the hypothalamus but a pattern 3 with moderate-to intense labelling was observed using Ag2.

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Fig. 5

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3.2.1.3. Brainstem. The PT showed a very intense pattern 5 of staining with Aa1, while a moderate labelling corresponding to pattern 3 was observed for Ag2. The nucleus subpretectalis showed a weakly stained pattern 2 for the a1-subunit and a pattern 4 with moderate staining for the g2-subunit. All layers of the TeO, except the stratum opticum, appeared with a weak, diffusely stained neuropil for Aa1. From layers 2 /12, the use of this antibody disclosed a few weakly stained fibres and also labelled neuronal somata and their proximal dendrites, mainly in layers 8 and 10, where a pattern 4 was observed. This pattern 4 was also observed in layer 13 (SGC). Ag2 revealed a moderate pattern 3 staining in layers 2, 4, 6, 8 and 10 as well as in layers 5 and 9 but these latter with scarce somata. An intense pattern 4 labelling of the g2subunit was observed in layer 13. Throughout the tectum, scarce radial fibres, some of them from tectal neurons, were labelled with Ag2. The Ag2 labelling in the LM showed pattern 3 with weak-to-moderate staining while no Aa1 labelling was detected. A very weak pattern 1 labelling for the a1subunit and a moderate pattern 4 labelling for the g2subunit were observed in the nucleus spiriformis medialis. The SpL showed pattern 4 for both subunits but moderate staining for Aa1 and intense labelling for Ag2. Two different areas in the MLd were immunoreactive for Aa1: a peripheral area, with an intense pattern 5 labelling, and a central area, with intense somata labelling and a moderate staining of a diffuse neuropil (pattern 3) (Fig. 10C). This pattern 3, with moderate-tointense staining of somata and a diffuse neuropil, was observed with the Ag2. No different areas were detected with this antibody (Fig. 10D). The ICo showed a similar weak pattern 4 labelling for a1- and g2-subunits. The substantia grisea centralis, the red nucleus, the SN and the EM showed a pattern 4 labelling for the a1subunit, with intense staining in the SN, and moderate staining in the remaining structures. A similar pattern 4, with intense-to-very intense g2-subunit staining, was observed for these structures. The Ipc displayed pattern 3 with a weak a1-subunit labelling and a moderate g2-subunit labelling and the nucleus isthmi pars magno cellularis (Imc) showed pattern 4 with a moderate labelling for the a1- and g2subunit (Fig. 10E and F). Aa1 and Ag2 intensely labelled the nucleus nervi oculomotorii with a pattern 4 and the Edinger-Westphal nucleus with pattern 3 (Fig. 10 G and H). The nucleus nervi trochlearis showed a moderate pattern 4 staining with Aa1 and an intense pattern 4 staining with the Ag2. The nucleus isthmi opticum (IO) and Slu showed

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moderate and intense pattern 3 staining, respectively, when labelled with Ag2. A week pattern 3 staining was observed with Aa1 in the Slu and no staining was observed in the IO. The nucleus pontis lateralis and the nucleus reticularis pontis displayed an intense-to-very intense pattern 4 labelling for both the a1- and g2subunit (Fig. 11 A and B) as did the nucleus cerebellaris. In the Mol, a moderate-to-intense labelling of radial fibres and some cell somata roughly corresponding to pattern 4, although in a special arrange was found for Aa1 (Fig. 11C) and a moderate pattern 3 labelling for Ag2 (Fig. 11D). Purkinje cells (PJ) somata and proximal dendrites (pattern 2) were observed with Ag2 but no labelling was obtained with Aa1. The Gra showed moderate-to-intense pattern 3 for both Aa1 and Ag2 (Fig. 11 C and D).

4. Discussion The antibodies used in this study were raised against a peptide sequence of the a1-subunit which is identical in both rat and chicken (Bateson et al., 1991), and the bands obtained for the chicken in Western blotting with Aa1 (Fig. 4A) fit those described previously using this and different antibodies (Stephenson and Duggan, 1989; Duggan and Stephenson, 1989, 1990). Furthermore, the a1-subunit immunocytochemical staining found in chicken nuclei is in agreement with that found in the rat nuclei considered to be homologous in birds and mammals when different antibodies (Turner et al., 1993; Fritschy and Mo¨hler, 1995) against this subunit were used, thus providing additional support to the suitability of using this antibody in birds. The molecular weight of the bands labelled with the Ag2 antibody in Western blot from chicken brain homogenates is in agreement with the theoretical molecular weight (55 kDa) calculated from the chicken g2-subunit mRNA sequence (Glencorse et al., 1990) (Fig. 4B). The same Ag2 used here have been assayed previously in the retina of mammals using immunocytochemistry (Greferath et al., 1995), and comparison of the g2-subunit distribution in the chicken brain using this antibody with that described in the rat with different antibodies against the g2-subunit (Fritschy and Mo¨hler, 1995) also supports the suitability of the Ag2 used in this study for labelling this subunit in birds and mammals. The distribution of chicken brain [3H]flunitrazepam binding sites is in agreement with that described for the pigeon using 2 nM [3H]flunitrazepam (Dietl et al., 1988) or 5 nM [3H]flunitrazepam (Veenman et al., 1994). Although data from other species and orders are

Fig. 5. Schematics of representative transverse sections from rostral to caudal (A /J) levels of the chicken brain. The nomenclature of the brain regions described and is largely in accordance with Karten and Hodos (1966) and Kuenzel and Masson (1988).

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Fig. 6. Schematics of representative transverse sections from rostral to caudal (A /J) levels of the chicken brain. The immunolabelling density of Aa1 is shown.

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Fig. 7. Schematics of representative transverse sections from rostral to caudal (A /J) levels of the chicken brain. The immunolabelling density of Ag2 is shown.

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obtained with in situ hybridisation should be regarded with caution since differences in development exist. 4.1. Olfactory bulb The presence of both BZ1 and BZ2 binding sites in the chicken is similar to that found in mammals (Niddam et al., 1987; reviewed by Fernandez-Lopez et al., 1997). The strong a1 and g2 labelling in the chick GL is consistent with the predominance of BZ1 observed in this structure. In the rat, in situ hybridisation studies have described high levels of both a1- and g2subunit mRNAs in mitral cells (Wisden et al., 1992), corresponding to the strong immunolabelling for these subunits in the somata and dendrites of these cells (Fritschy and Mo¨hler, 1995). This supports the idea of the presence of postsynaptic a1- and g2-subunits in rat mitral cells. The coincidence in the immunocytochemistry in the rat and chicken suggests that these subunits could be postsynaptic in chicken, although in situ hybridisation data for this structure would be necessary for further support. 4.2. Visual Wulst, dorsal ventricular ridge and hippocampal complex 4.2.1. Visual Wulst Our data show that both BZ1 and BZ2 are present in these areas. Although we detected a1 and g2 in the whole of the visual Wulst by immunocytochemistry, accordingly with BZ1 presence, the data from in situ hybridisation revealed a strong mRNA expression of these subunits only in the hyperstriatum accessorium (HA) (Bateson et al., 1991; Glencorse et al., 1991). This indicates that at least some of the GABAA isoforms with BZ1 pharmacology come from HA neurons.

Fig. 8. Patterns of immunolabelling in the olfactory bulb using Aa1 (A) and Ag2 (B). Toluidine blue staining (C). The arrow shows the mitral cell immunostaining with Aa1 (bar/100 mm).

lacking, the chicken belongs to a primitive group (Galliforms) while the pigeon belongs to the Columbiforms, a considerably more evolved order (Sibley and Ahlquist, 1990), which suggests that birds present a conservative distribution of [3H]flunitrazepam binding sites. No previous data exist about a1, g2 and BZ1 and BZ2 in birds, and in situ hybridisation studies have been performed on chick (P1) (Glencorse et al., 1991; Bateson et al., 1991). Thus, comparison of our data and those

4.2.2. Hippocampal complex The parahippocampal area and the hippocampus are considered to be the avian equivalent to Ammon’s horn and the dentate gyrus of mammals, respectively (Veenman et al., 1994; Veenman and Reiner, 1994; Butler and Hodos, 1996). Our data show an agreement between the moderate g2- and a1-subunit labelling and the moderate BZ1 binding in birds. [3H]flunitrazepam binding is lower in birds than in mammals, but our data show an abundance of BZ2 binding sites in the chicken, resembling that described in mammals. However, some differences in distribution appeared. Thus, we found a similar degree of BZ2 binding throughout the hippocampal complex in birds, while in mammals the dentate gyrus has higher BZ2 densities than Ammon’s horn (Lo et al., 1983; Niddam et al., 1987; reviewed by Fernandez-Lopez et al., 1997). In the dentate gyrus of mammals, immunocytochemical and in situ hybridisation studies have described a strong mRNA expression

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Fig. 9. Patterns of immunolabelling in the ectostriatum (A, B), lobus parolfactorius (C, D), paleostriatum primitivum (E, F) and nucleus anterior thalami (G, H). Aa1 labelling (A, C, E, G) and Ag2 labelling (B, D, F, H) (bar /100 mm).

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Fig. 10. Patterns of immunolabelling in the nucleus rotundus (A, B), nucleus mesencephalicus lateralis, pars dorsalis (C, D), nucleus isthmi (E, F) and nucleus of Edinger-Westphal and nucleus nervi oculomotorii (G, H). Aa1 labelling (A, C, E, G) and Ag2 labelling (B, D, F, H) (bar /100 mm).

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Fig. 11. Patterns of immunolabelling in the nucleus reticularis pontis (A, B) and cerebellum (C, D). Aa1 labelling (A and C) and Ag2 labelling (B and D) (bar/100 mm).

and immunolabelling for the a2- and g2-subunits; low for a1 and very low or non-existent for a3 (Turner et al., 1993; Wisden et al., 1992; Fritschy and Mo¨hler, 1995). We have previously described a strong a5 immunolabelling in the chicken hippocampus (Aller et al., 2000), which suggests that the presence of BZ2 in birds could depend on the a5-subunit. In mammals, the immunolabelling of a5 is strong in the hippocampus and low in the dentate gyrus, while a2 are similar in these areas. This contrasts with the homogeneous a5 immunolabelling found in the chicken hippocampal complex, indicating differences in a5 expression between birds and mammals. Knowledge of the distribution of the a2-subunit in this area of the chicken brain seems to be important to clear up the patterns of expression in the chicken and their relationships with those seen in mammals. 4.2.3. Dorsal ventricular ridge (DVR) The Bas and E show low or non-specific [3H]flunitrazepam binding in the chicken and pigeon, in agreement with the scarce GABAA binding sites observed using [3H]muscimol as a radioligand (Stewart et al.,

1988; Aller et al., unpublished data). However, these data contrast with the strong labelling of the a1-subunit in cell somata as well as the high expression of a1 mRNA in these nuclei (Bateson et al., 1991; Glencorse et al., 1991). It has been hypothesised that some lowprobability subunit assemblies would give rise to receptor complex isoforms unable to bind [3H]flunitrazepam and other benzodiazepines (Costa, 1998), which could perhaps explain this controversy. However, the strong presence of a1 would imply a relevant presence of b-subunits. In fact, the presence of b2 has been confirmed (Aller et al., unpublished data). This would produce a moderate-to-strong [3H]muscimol binding in Bas and E, since this radioligand binds to b-subunits (Bureau and Olsen, 1989; Olsen et al., 1990), which was not the case. We believed that a possible explanation for the controversy between areas without autoradiographic labelling but with a strong immunocytochemical labelling arranged in disperse ‘‘dots’’, such as occurs in isolated cell somata, would be that these scattered dots might not be detected by autoradiography (Aller et al., 2000).

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4.3. Basal ganglia 4.3.1. Paleostriatum primitivum The predominance of the BZ1 subtype in this area is similar to that described in the mammalian homologous nuclei (globus pallidus) (Niddam et al., 1987; reviewed by Fernandez-Lopez et al., 1997), and is consistent with the strong expression of a1-subunit mRNAs in the chick (Bateson et al., 1991; Glencorse et al., 1991) and the intense a1- and g2-subunit immunostaining found in chicken, although we found higher levels of [3H]flunitrazepam binding in the chicken and pigeon than in the rat when we performed parallel autoradiographic experiments in these species. Interestingly, the results described here show that Aa1 labels the periphery of the cell somata while Ag2 labels the whole somata. The strong expression of both a1and g2-subunit mRNAs described in this area in the chick (Glencorse et al., 1991) supports the presence of both subunits in postsynaptic receptor complexes expressed in PP cell somata. A possible explanation for the intriguing differences in the location of a1- and g2subunits in these cell somata could be that complexes with the a1-subunit, which have a more restricted distribution, would appear only in synapses associated with specific afferent fibres arriving at the somata, while complexes with g2 would have a wider distribution over the somata. This would imply a regionalisation of the somata depending on the fibres that they receive. 4.3.2. Paleostriatum augmentatum and lobus paraolfactorius These areas show high levels of BZ1 and BZ2 binding, in a similar way to those described in homologous mammalian regions (caudate-putamen) (Niddam et al., 1987; reviewed by Fernandez-Lopez et al., 1997). The intense a1-subunit labelling in both the PA and LPO is consistent with the presence of BZ1. The a5-subunit is not immunolabelled in this area in the chick (Aller et al., 2000). Since the a2-subunit shows an intense labelling in the mammalian caudate-putamen (Fritschy and Mo¨hler, 1995), it is likely that a2 would be the main a-subunit involved in the BZ2 binding of this area in the chicken. The intense a1- and g2-subunit immunostaining as well as the moderate autoradiographic labelling observed in LPO contrasts with the very low mRNA expression of these subunits observed in the chick (Bateson et al., 1991; Glencorse et al., 1991). This suggests that the complexes observed in this area would be located in synapses of fibres from areas of the brain other than the LPO. 4.4. Thalamus An abundance of GABAergic fibres, but not GABAergic neurons, in the dorsal area of the thalamus has

been described in the pigeon, suggesting that GABAergic fibres come from reticular GABAergic neuronal areas located more ventrally in the thalamus (Veenman et al., 1994). This seems consistent with the low a1 mRNA expression described in the chick (Bateson et al., 1991; Glencorse et al., 1991) and high a1 immunolabelling of the neuropil that we observed in the dorsal area of the chicken thalamus. It also suggests that the dorsal area of the chicken thalamus receives extrinsic fibres with GABAA isoforms containing a1-subunits. The characteristic distribution of a1 in the periphery of cell somata in the ROT, similar to that described in the PP, suggests a regionalisation of the somata in these nuclei. 4.5. Optic tectum The TeO is considered to be one of the most conservative structures in the vertebrate brain with visual and somatosensorial inputs arranged in a somatotopic and retinotopic organisation (Frost et al., 1990; Butler and Hodos, 1996). The outer layers show the highest levels of [3H]flunitrazepam binding in the chicken, similar to the situation in the pigeon (Dietl et al., 1988; Veenman and Reiner, 1994) and mammals (Unnerstall et al., 1982; Niddam et al., 1987; Negro et al., 1995; Soria et al., 1995; reviewed by FernandezLopez et al., 1997). The strongly labelled outer and inner layers in the chicken SGF are not observed in the rat, where a very strongly labelled outer layer can only be observed in the superior colliculus. A striking amount of BZ2 appears in the outer and inner layers of the SGF in birds as well as in the outer layer of the rat. This different distribution in the outer TeO between birds and mammals probably mirrors differences in visual function and suggests an important role for BZ2 in the retino-tectal pathway. This makes the avian optic tectum a very interesting area for the study of the GABAA receptor complex. In situ hybridisation in the chick indicates a strong labelling of some mRNA subunits, such as a1 and g2 (Glencorse et al., 1991) and a strong labelling of BZ1 can be observed in the chicken, in contrast with the low immunolabelling for the a1-subunit. This controversy, as opposed to that seen in the E and Bas, is also observed in structures such as the LM and is difficult to explain, unless [3H]flunitrazepam is able to label molecules other than GABAA receptor complexes. 4.6. The nucleus mesencephalicus lateralis, pars dorsalis The immunocytochemical labelling of the subunits studied in this work, as well as the strong labelling for the g2- and a1-subunit mRNA by in situ hybridisation in the chick (Glencorse et al., 1991) is consistent with the [3H]flunitrazepam binding observed in this area. The MLd, together with the ICo of birds, are considered to

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be homologous to the mammalian inferior colliculus (Butler and Hodos, 1996). The two different areas of [3H]flunitrazepam binding described here in the chicken and also indicated for the rat inferior colliculus (Fernandez-Lopez et al., 1997) suggest the existence of two different and evolutionarily conserved regions in both birds and mammals. The stronger a1 immunolabelling seen in the peripheral area in the chicken could correspond to the external and dorsal cortex of the inferior colliculus, described as showing a strong labelling of the a1-subunit in the rat (Fritschy and Mo¨hler, 1995), suggesting conservative differences in native receptors between these two areas. 4.7. Nucleus isthmi The most striking aspects of the nucleus isthmi appear in the Ipc, which displays very high [3H]flunitrazepam binding levels in which BZ2 sites are strongly represented. Although the presence of g2 immunolabelling explains the high level of BZ binding, discrepancies appear between the immunocytochemical, BZ1 and BZ2 labelling results. Thus, the low immunolabelling of the a1-subunit does not agree with the BZ1 binding. On the other hand, we have no information about a-subunits other than a5 (Aller et al., 2000), although the immunolabelling of this subunit is not in agreement with the high levels of BZ2. Thus, this area shows discrepancies between the high levels of autoradiographic labelling and the low immunolabelling, in a similar way to that described for the TeO and LM. The other part of this nucleus, the Imc, would present similar characteristics to those described for the PP and ROT. 4.8. Cerebellum The findings of the autoradiographic studies carried out in the chicken cerebellum are similar to those described in the pigeon (Dietl et al., 1988; Veenman et al., 1994) and mammals (Niddam et al., 1987; Fernandez-Lopez et al., 1997). The immunolabelling of a1 and g2 in the cerebellum is consistent with the predominance of BZ1 binding. However, some discrepancies appear in the chicken PJ with respect to the data described in mammals. Thus, a strong expression of a1-subunit mRNA has been described in both the rat (Wisden et al., 1992) and chick (Glencorse et al., 1991) and a moderate degree of a1 immunolabelling in the rat PJ (Turner et al., 1993; Fritschy and Mo¨hler, 1995). We were unable to find a1 immunolabelling in chicken PJ somata, while our parallel study in rat cerebellum did demonstrate this a1 immunostaining. These data support differences in the cellular distribution of this subunit between the rat and chicken in a similar way to the distribution described for the a5-subunit (Aller et al., 2000).

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Acknowledgements This work has been supported by FEDER-MEC 1FD97-0004 and FIS 99/0604.

References Aller, M.I., Paniagua, M.A., Gimenes, C.C., Araujo, F., Vitorica, J., Ferna´ndez-Lo´pez, A., 2000. Distribution of the g-aminobutyric acid A receptor complex alpha5 subunit in chick brain: an immunocytochemical and autoradiographic study. Neurosci. Lett. 291, 49 /53. Barnard, E.A., Skolnick, P., Olsen, R.W., Mohler, H., Sieghart, W., Biggio, G., Braestrup, C., Bateson, A., Langer, S.Z., 1998. International Union of Pharmacology. XV. Subtypes of gammaaminobutyric acid A receptors: classification on the basis of subunit structure and receptor function. Pharmacol. Rev. 50, 291 /313. Bateson, A.N., Harvey, R.J., Wisden, W., Glencorse, T.A., Hicks, A.A., Hunt, S.P., Barnard, E.A., Darlison, M.G., 1991. The chicken GABAA receptor a1 subunit: cDNA sequence and localization of the corresponding mRNA. Brain Res. Mol. Brain Res. 9, 333 /339. Bonnert, T.P., McKernan, R.M., Farrar, S., Le Bourdelles, B., Heavens, R.P., Smith, D.W., Hewson, L., Rigby, M.R., Sirinathsinghji, D.J., Brown, N., Wafford, K.A., Whiting, P.J., 1999. Theta, a novel gamma-aminobutyric acid type A receptor subunit. Proc. Natl. Acad. Sci. USA 96, 9891 /9896. Bureau, M., Olsen, R.W., 1989. Isolation of multiple pharmacologically distinct GABA and benzodiazepine binding subunits of the GABAA receptor. Abstr. Soc. Neurosci. 15, 642 /649. Butler, A.B., Hodos, W., 1996. Comparative vertebrate neuroanatomy. In: Butler, A.B., Hodos, W. (Eds.), Evolution and Adaptation. Wiley-Liss, New York. Costa, E., 1998. From GABAA receptor diversity emerges a unified vision of GABAergic inhibition. Annu. Rev. Pharmacol. Toxicol. 38, 321 /350. Dietl, M.M., Cortes, R., Palacios, J.M., 1988. Neurotransmitter receptors in the avian brain. III. GABA-benzodiazepine receptors. Brain Res. 439, 366 /371. Duggan, M.J., Stephenson, F.A., 1989. Bovine gamma-aminobutyric acid A receptor sequence-specific antibodies: identification of two epitopes which are recognized in both native and denatured gamma-aminobutyric acid A receptors. J. Neurochem. 53, 132 / 139. Duggan, M., Stephenson, F.A., 1990. Biochemical evidence for the existence of g-aminobutyric acidA, receptors iso-oligomers. J. Biol. Chem. 265, 3.831 /3.835. Duggan, M.J., Pollard, S., Stephenson, F.A., 1991. Immunoaffinity purification of GABAA receptor alpha-subunit iso-oligomers: demonstration of receptor populations containing alpha 1 alpha 2, alpha 1 alpha 3, and alpha 2 alpha 3 subunit pairs. Biol. Chem. 266, 24778 /24784. Fernandez-Lopez, A., Chinchetru, M.A., Calvo, P., 1997. The autoradiographic perspective of central benzodiazepine receptors: a short review. Gen. Pharm. 29, 173 /180. Fritschy, J.M., Mo¨hler, H., 1995. GABAA-receptor heterogeneity in the adult rat brain: differential, regional and cellular distribution of seven major subunits. J. Comp. Neurol. 359, 154 /194. Frost, B.J., Wise, L.Z., Morgan, B., Bird, D., 1990. Retinotopic representation of the bifoveate eye of the kestrel (Falco sparverius) on the optic tectum. Visual Neurosci. 5, 231 /239.

18

M.I. Aller et al. / Journal of Chemical Neuroanatomy 25 (2003) 1 /18

Glencorse, T.A., Bateson, A.N., Darlison, M.G., 1990. Sequence of the chicken GABAA receptor gamma 2-subunit cDNA. Nucl. Acids Res. 11 (18), 7157. Glencorse, T.A., Bateson, A.N., Hunt, S.P., Darlison, M.G., 1991. Distribution of the GABAA receptor alpha 1- and gamma 2subunit mRNAs in chick brain. Neurosci. Lett. 133, 45 /48. Greferath, U., Grunert, U., Fritschy, J.M., Stephenson, F.A., Mohler, H., Wassle, H., 1995. GABAA receptor subunits have differential distributions in the rat retine: in situ hybridisation and immunohistochemistry. J. Comp. Neurol. 353, 553 /571. Karten, H.J., Hodos, W., 1966. A Stereotaxic Atlas of the Brain of the Pigeon (Columba livia ). The Johns Hopkins Press, Baltimore, MD. Kuenzel, W.J., Masson, M., 1988. A Stereotaxic Atlas of the Brain of the Chick (Gallus domesticus ). The Johns Hopkins Press, London. Lo, M.S., Niehoff, D.L., Kuhar, M.J., Snyder, S.H., 1983. Autoradiographic differentiation of multiple benzodiazepine receptors by detergent solubilization and pharmacologic specificity. Neurosci. Lett. 39, 37 /44. Lu¨ddens, H., Seeburg, P.H., 1989. Type I and type II GABAA/ benzodiazepine receptors. Soc. Neurosci. Abstr. 15, 641. Lu¨ddens, H., Wisden, W., 1991. Function and pharmacology of multiple GABAA receptors subunits. Trends Pharmacol. Sci. 12, 49 /51. Mertens, S., Benke, D., Mohler, H., 1993. GABAA receptor populations with novel subunit combinations and drug binding profiles identified in brain by alpha 5- and delta-subunit-specific immunopurification. J. Biol. Chem. 268, 5965 /5973. Mo¨hler, H., Okada, T., 1977. Benzodiazepine receptor: demonstration on the central nervous system. Science 198, 849 /851. Mo¨hler, H., Benke, D., Mertens, S., Fritschy, J.M., 1992. GABAA receptor subtypes differing in a subtype subunit composition display unique pharmacological properties. Adv. Biochem. Psychopharmacol. 47, 41 /53. Negro, M., Fernandez-Lopez, A., Calvo, P., 1995. Autoradiographical study of types 1 and 2 of benzodiazepine receptors in rat brain after chronic ethanol treatment and its withdrawal. Neuropharmacology 34, 1.177 /1.182. Niddam, R., Dubois, A., Scatton, R., Arbilla, S., Langer, Z., 1987. Autoradiographic localization of [3H]-zolpidem binding sites in the rat CNS: comparison with the distribution of [3H]-flunitrazepam binding sites. J. Neurochem. 49, 890 /899. Olsen, R.W., Tobin, A.J., 1990. Molecular biology of GABAA receptors. FASEB J. 4, 1.469 /1.480. Olsen, R.W., McCabe, R.T., Wamsley, J.K., 1990. GABAA receptor subtypes: autoradiographic comparison of GABA, benzodiazepine and convulsant binding sites in the rat central nervous system. J. Chem. Neuroanat. 3, 59 /76. Pritchett, D.B., Seeburg, P.H., 1990. g-Aminobutyric acidA receptor a5-subunit creates a novel type II benzodiazepine receptor pharmacology. J. Neurochem. 54, 1.802 /1.804.

Pritchett, D.B., Luddens, H., Seeburg, P.H., 1989a. Type I and type II GABAA/benzodiazepine receptors produced in transfected cells. Science 245, 1.389 /1.392. Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenman, H., Schofield, P.R., Seeburg, P.H., 1989b. Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology. Nature 52, 338 /585. Rabow, L.E., Russek, S.J., Farb, D.H., 1995. From ion currents to genomic analysis: recent advances in GABAA receptor research. Synapse 21, 189 /274. Sibley, C.G., Ahlquist, J.E., 1990. Phylogeny and Classification of Birds: A Study in Molecular Evolution. Yale University Press. Soria, C., Revilla, V., Candelas, M.A., Calvo, P., Fernandez-Lopez, A., 1995. An autoradiographical saturation kinetic study of the different benzodiazepine binding sites in rat brain using [3H]flunitrazepam as a radioligand. Biochem. Pharmacol. 50, 1.619 /1.635. Stephenson, F.A., 1995. The GABAA receptors. J. Biochem. 310, 1 /9. Stephenson, F.A., Duggan, M.J., 1989. Mapping the benzodiazepine photoaffinity-labelling site with sequence-specific gamma-aminobutyric acid A-receptor antibodies. Biochem. J. 264 (1), 199 /206. Stephenson, F.A., Casalotti, S.O., Mamalaki, C., Barnard, E.A., 1986. Antibodies recognising the GABAA/benzodiazepine receptor including its regulatory sites. J. Neurochem. 46, 854 /861. Stephenson, F.A., Duggan, M.J., Pollard, S., 1990. The gamma 2 subunit is an integral component of the gamma-aminobutyric acid A receptor but the alpha 1 polypeptide is the principal site of the agonist benzodiazepine photoaffinity labeling reaction. J. Biol. Chem. 265, 21160 /21165. Stewart, M.G., Bourne, R.C., Chmielowska, J., Kalman, M., Csillag, A., Stanford, D., 1988. Quantitative autoradiographic analysis of the distribution of [3H]muscimol binding to GABA receptors in chick brain. Brain Res. 26 (456), 387 /391. Turner, J.D., Bodewitz, G., Thompson, C.L., Stephenson, F.A., 1993. Immunohistochemical mapping of gamma-aminobutyric acid typeA receptor alpha subunits in rat central nervous system. Psychopharmacol. Ser. 11, 29 /49. Unnerstall, J., Niehoff, D., Kuhar, M., Palacios, J., 1982. Quantitative receptor autoradiography using [3H] ultrafilm: application to multiple benzodiazepine receptors. J. Neurosci. Meth. 6, 59 /73. Veenman, C.L., Reiner, A., 1994. The distribution of GABA-containing perikarya, fibers, and terminals in the forebrain and midbrain of pigeons, with particular reference to the basal ganglia and its projection targets. J. Comp. Neurol. 339, 209 /250. Veenman, C.L., Albin, R.L., Richfield, E.K., Reiner, A., 1994. Distributions of GABAA, GABAB, and benzodiazepine receptors in the forebrain and midbrain of pigeons. J. Comp. Neurol. 344, 161 /189. Wisden, W., Laurie, D.J., Monyer, H., Seeburg, P.H., 1992. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J. Neurosci. 12, 1.040 /1.062.