0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd 1BRO

Neuroscience Vol. 50, No. 2, pp. 355 370, 1992 Printed in Great Britain

THE REGIONAL, CELLULAR A N D SUBCELLULAR LOCALIZATION OF GABAA/BENZODIAZEPINE RECEPTORS IN THE SUBSTANTIA NIGRA OF THE RAT L. F. B. NICHOLSON,* R. L. M. FAULL,*q" H. J. WALDVOGEL* a n d M. DRAGUNOW + *Department of Anatomy and ~cDepartment of Pharmacology, School of Medicine, University of Auckland, Private Bag, Auckland, New Zealand Abstract--The regional, cellular and subcellular distribution of GABAA/benzodiazepine receptors was investigated by light and electron microscopy in the rat substantia nigra. The regional distribution and density of GABAA/benzodiazepine receptor subtypes (Type I and II) was studied using quantitative receptor autoradiography following in vitro labelling of cryostat sections with tritiated ligands. This was followed by a detailed study of the cellular and subcellular distribution and localization of GABAA/benzodiazepine receptors by light and electron microscopy using immunohistochemical techniques with a monoclonal antibody (bd-17) to the [~2.~subunits of the GABAA/benzodiazepine receptor complex. Finally, in situ hybridization histochemistry using 35S-labelled oligonucleotide probes was used to demonstrate the cellular distribution of mRNA for the ~ and ~2 GABAA receptor subunits in the substantia nigra. The results of the autoradiographic and immunohistochemical studies showed a close correspondence in the regional distribution of GABAA/benzodiazepine receptors in the substantia nigra. A moderate-tohigh density of receptors was present throughout the full extent of the substantia nigra pars reticulata with a very low density of receptors in the substantia nigra pars compacta. Quantitative autoradiographic studies showed that: (i) the pars reticulata contained mainly central Type I receptors; (ii) the highest density of receptors was present in the caudal pars reticulata (200 + 38 fmol/mg) with successively lower densities of receptors in the middle (176 + 31 fmol/mg) and rostral (150 + 26 fmol/mg) levels of the pars reticulata; and (iii) the density of receptors in the pars reticulata was reduced by 34% following 6-hydroxydopamine-induced degeneration of dopaminergic pars compacts neurons. At the cellular level, GABAA/benzodiazepine receptor immunoreactivity was localized in a punctate fashion on dendrites and neuronal cell bodies in the pars reticulata. At the subcellular level, GABAA/benzodiazepine receptor immunoreactivity was associated with the pre- and postsynaptic membranes of axodendritic synaptic complexes along the length of small-to-large sized smooth dendrites in the pars reticulata. Two types of immunoreactive axodendritic synaptic complexes were identified: most (about 80%) immunopositive synapses showed equal staining of the pre- and postsynaptic membranes and were associated with small (< 1.01tm) axon terminals containing few mitochondria and small, round-to-pleomorphic vesicles in synaptic contact with small, peripheral dendrites; less frequently (about 20%) immunopositive synapses showed a marked immunoreactive thickening of the postsynaptic membrane and were associated with large (>1.0/~m) axon terminals containing numerous mitochondria and mainly pleomorphic vesicles in synaptic contact with large mainstem dendrites. Finally, the in situ hybridization studies showed that cells in the pars reticulata, but not in the pars compacta, expressed mRNA for the :q (but not for the :~,) subunit of the GABA A receptor complex. These results provide a detailed morphological characterization of the regional, cellular and subcellular distribution of GABAA/benzodiazepine receptors in the rat substantia nigra. These findings provide an anatomical basis for GABA-mediated synaptic inhibition in the substantia nigra.

The m a j o r inhibitory n e u r o t r a n s m i t t e r in the brain is G A B A which exerts m a n y of its effects t h r o u g h a GABAA/benzodiazepine receptor complex. 1°'25'39 Over recent years, in vitro a u t o r a d i o g r a p h i c receptor binding studies using tritiated benzodiazepine receptor ligands have s h o w n the regional distribution of G A B A A/benzodiazepine receptors in the m a m m a l i a n brain (for review, see Ref. 35). Also, with the recent

tTo whom correspondence should be addressed. Abbreviations: CL218,872, 3-methyl-6-[3-trifluoromethylphenyl]-l,2,4-triazolol-[4,3-b]pyridazine; 6-OHDA, 6hydroxydopamine; PBS, phosphate-buffered saline; SN, substantia nigra; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata. 355

d e v e l o p m e n t of m o n o c l o n a l antibodies to the G A B A A / b e n z o d i a z e p i n e r e c e p t o r c o m p l e x , 1(i.3~,50 light- a n d electron-microscopic i m m u n o h i s t o c h e m i cal studies have been u n d e r t a k e n to d e m o n s t r a t e the cellular a n d subcellular localization o f G A B A A / benzodiazepine receptors in the central nervous system.4,35,36.~9.43,44,51

In the m a m m a l i a n brain the substantia nigra (SN) contains especially high c o n c e n t r a t i o n s of G A B A : 29 in particular, the substantia nigra pars reticulata (SNr) receives m a j o r G A B A e r g i c projections from the striatum 2s and the globus pallidus, 4~ and provides G A B A e r g i c projections to the t h a l a m u s 5'46 and superior colliculus. 5't4'49 Also, receptor autoradiographic and i m m u n o h i s t o c h e m i c a l studies have

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s h o w n t h a t the SN contains high c o n c e n t r a t i o n s of G A B A ^ / b e n z o d i a z e p i n e receptors (for review, see Ref. 35). In the present study we have u n d e r t a k e n a detailed light- a n d electron-microscopic investigation o f the regional, cellular a n d subcellular distribution o f G A B A A/benzodiazepine receptors in the SN o f the rat. In these studies we have used tritiated ligands a n d q u a n t i t a t i v e receptor a u t o r a d i o g r a p h y to show the regional distribution of receptors, a GABA:,subunit specific m o n o c l o n a l a n t i b o d y a n d i m m u n o histochemistry to show the cellular a n d subcellular localization of receptors, and subunit-specific oligonucleotide probes a n d in situ hybridization to identify the cells expressing m R N A for G A B A A / benzodiazepine receptors. EXPERIMENTAL PROCEDURES

Tissue collection A total of 40 male albino Wistar rats weighing 250-300 g were used in this study on the distribution of GABAA/ benzodiazepine receptors in the SN: 25 for the autoradiographic demonstration of receptors, three for the immunohistochemioal localization of benzodiazepine receptors, and 12 for the localization of mRNA for GABA A receptor subunits using in situ hybridization. Autoradiographic localization qf GABA a/benzodiazepine receptors Rats were killed by decapitation and the brains removed and immediately frozen on dry-ice. The brains were sectioned at 16/zm on a cryostat in the coronal or sagittal plane and the sections thaw-mounted onto gelatine/chrome alum-coated slides. The slide-mounted sections were processed for the autoradiographic localization of the GABA A/benzodiazepine receptors as previously described. 8'51 Briefly, GABAA/benzodiazepine receptors were labelled by incubating the sections in 50 mM Tris-HC1 (pH 7.4) containing either 1 nM [3H]flunitrazepam (84 Ci/mmol, Amersham) or the benzodiazepine receptor antagonist [3H]Ro15-1788 (82.8Ci/mmol, New England Nuclear). Adjacent sections were incubated with [3H]flunitrazepam in the presence of CL218,872 (a ligand with high affinity for the Type I and low affinity for the Type II benzodiazepine receptors). The sections were washed (2 × 1 min in Tris-HCl buffer with a final dip in ice-cold distilled water) and dried under a stream of cold air. All procedures were carried out at 4°C. Nonspecific binding was determined by incubation of slides in the presence of 1/~M clonazepam. Once dry, the slides were brought to room temperature, taped into X-ray cassettes and apposed with 3H-sensitive Hyperfilm (Amersham) and exposed in the dark at 4°C for six to 12 weeks. The films were developed in D19, washed, fixed and dried. They were printed using standard photographic procedures to yield autoradiograms in which the autoradiographic labelled receptors appear as white dots on a dark background. After processing for autoradiography, selected sections were Nissl-stained to show the distribution of cell bodies. In order to investigate the proportion of receptors on dopaminergic neurons in the SN, seven rats were anaesthetized with sodium pentobarbitone (50 mg/kg, i.p.) and unilateral injections of 2/~1 of 6-hydroxydopamine (6OHDA; Sigma; 12#g of 6-OHDA in 2/zl of 0.9% NaCI + 0 . 2 % ascorbic acid) 6 were made into the right substantia nigra pars compacta (SNc) using stereotaxic procedures (coordinates: - 5.8 mm posterior, 1.7 mm lateral to the midline and 7 mm ventral to the pial surface). After

a survival period of 13 weeks the animals were kdled and processed for the autoradiographic localization of GABA A/benzodiazepine receptors as described above. After processing for autoradiography selected sections were also Nissl-stained with Cresyl Violet to determine the extent of cell loss in the pars compacta. Quantification ofautoradiograms. The optical densities of the autoradiographic labelling in the SN were determined directly from the autoradiograms using a spot densitometer described in our previous studies on benzodiazepine receptors in the human spinal cord. 7 Briefly, a microprojector was used to project an image of the autoradiogram (magnification × 12) onto a photo-sensitive diode such that the optical density of 100-gm diameter spots could be measured. The control labelling in the presence of 1/~M clonazepam was used to zero calibrate the photo-sensitive diode. Readings from the various regions of the SN were made by moving the autoradiogram in the microprojector. In each case, over 100 optical densitometric readings were taken from sections selected from six different levels of the SN. These optical density values were then converted to nCi tritium bound/mg protein using a standard curve derived from the optical density reading of the autoradiographic standards in which log nCi/mg was plotted against log optical density. These values were then converted to femtomoles of tritiated-ligand bound/mg tissue using the known specific activity of the labelled ligand. 4v All figures on the density of GABAA/benzodiazepine receptors labelled will therefore be given as femtomoles [3H]ligand per mg tissue which for convenience will be abbreviated to fmol/mg.

Immunohistochemical localization q/' GABA A/benzodiazepine receptors The rats were deeply anaesthetized with sodium pentobarbitone (50mg/kg, i.p.) and perfused intracardially with physiological saline followed by 500ml of fixative (4% paraformaldebyde, 0.1% glutaraldehyde in 0.t M phosphate buffer at pH 7.4). The brains were immediately removed, postfixed for 2-12h in the perfusate and the midbrain sectioned in the frontal plane on a Vibratome at 60-/zm sections. The sections were collected in phosphatebuffered saline (PBS) and processed for immunohistochemistry using a monoclonal antibody, bd-17, against the fl2,3 subunits of the GABAA/benzodiazepine receptor complex. The preparation and characterization of this antibody has been previously described) °'t2'3~3~39The localization of the antibody was visualized using standard immunohistochemical methods 45 as detailed below. Sections for light microscopy and electron microscopy were treated in the same way except that the sections for light microscopy had 0.1% Triton-X added to their washes while those for electron microscopy were first treated with 50% ethanol for 20 min to aid penetration of the antibody, All sections were then incubated for I h in 20% goat serum followed by incubation in the primary antibody (a hybridoma solution of bd-17) 39 for three days at room temperature (continuous agitation). The sections were then incubated in secondary antibody (biotinylated goat antimouse IgG, Sigma) at 1:400 overnight, then in ExtrAvidin" peroxidase conjugate (Sigma) at 1:I000 for 4 h. Following each of these steps the tissue was washed (3 x 10 rain) in PBS. The sections were then reacted for 15 min with 0.05% 3,3-diaminobenzidine tetrahydrochloride (Sigma) and 0.01% H202 (pH 7.4) in 0.1 M phosphate buffer, pH 7.4, to visualize the reaction product. Light-microscopic sections were washed in PBS, mounted on chrome alum slides, rinsed in distilled water and dehydrated through a graded alcohol series to xylene and coverslipped with DePeX (Serva). The reacted sections for electron microscopy were treated as follows: postfixed for 1 h in 1% osmium tetroxide in 0.1 M phosphate buffer, washed in buffer, dehydrated in a graded series of ethanols to 100% ethanol, washed in propylene oxide, impregnated with 812 resin (TAAB) overnight and

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GABAA/benzodiazepine receptors in the substantia nigra then flat-embedded in fresh resin according to the method of Aides and Boone.t Blocks of tissue from the SNr were dissected out and glued onto blank blocks. Thin sections were cut on an ultramicrotome (LKB Ultratome IV), collected on single slot formvar coated grids, counterstained with Reynolds lead citrate and viewed in a Hitachi H7000 transmission electron microscope. Control sections for nonspecific labelling were also processed as above except that the primary antibody was substituted with 1% goat serum. As a further control for immunohistochemical processing artefacts, two rat brains were processed for electron microscopy as outlined above except that all immunohistochemical procedures were omitted. In situ hybridization histochernistry The distribution of GABA A receptor mRNAs for the ~l and ~2 subunits was investigated using in situ hybridization histochemistry and 35S-labelled oligodeoxyribonucleotide probes complementary to least conserved sequences in the cytoplasmic loop between the putative membrane-spanning domains M3 and M4. The oligonucleotide probes were kindly provided by Dr J. G. Richards (Hoffman La Roche, Basel); they were prepared on a DNA synthesizer by Med. prob A.S., Oslo. The a~-probe (54mer) was complementary to nucleotides 1144~1197 and the e2-probe (50mer) was complementary to nucleotides 1137 1186.32 Sense cq and ct2 35-base oligonucleotides, exactly complementary to the :q and ~2 antisense probes, were used as controls. The probes were 35S-labelled as described by Persohn et al. 32 The in situ hybridization method is detailed below. Rats were killed by decapitation; the brains removed and immediately frozen on dry-ice. Brains were then sectioned at 12 ~m in the coronal plane on a cryostat and mounted on slides previously coated with 2% 3-aminopropylthriethoxysilane solution in acetate. 34 Sections were stored at - 20 'C

Sections were processed for the localization of mRNA for the respective subunits of the GABAa receptor as described by Persohn et al? 2 Briefly, sections were brought to room temperature 1 h before hybridization then incubated in 50/~1 of hybridization buffer containing the 35S-labelled probe (3 x l0 s c.p.rn.). Sections were covered with Fujifilm" coverslips and incubated in a moist chamber at 3T'C for 24 h. Sections were then washed, dehydrated in ethanol and exposed (for up to 10 days) to sheet film (Hyperfilm, B-Max ", Amersham). The film was developed in Kodak PL12 then transfered to Kodak rapid fix. Following development, selected sections were counterstained with Cresyl Violet. RESULTS The principal aims of this study were to investigate the light-microscopic and electron-microscopic localization of GABAA/benzodiazepine receptors in the SN of the rat midbrain. First, the distribution of GABAA/benzodiazepine receptors was studied using autoradiography following in vitro labelling of cryostat sections with tritiated ligands. This was followed by a detailed study using light and electron microscopy to reveal the cellular and subcellular localization of the GABAA/benzodiazepine receptor using immunohistochemical techniques with a monoclonal antibody to the ~2.3 subunits of the receptor complex. Finally, in situ hybridization histocbemistry was used to identify the presence of the subunits of the GABAA/benzodiazepine receptor in the SN of the rat.

Autoradiographic localization diazepine receptors

of

G A B A A/benzo-

The localization of GABAA/benzodiazepine receptors in the rat SN was investigated using [3H]flunitrazepam (a benzodiazepine receptor agonist with a high affinity for Type I and II receptors) and [3H]RoI5-1788 (a central benzodiazepine receptor antagonist with a high affinity for Type I and II receptors). The details on the anatomy and density of receptors in the two major subdivisions of the SN, SNr and SNc, are shown in coronal sections of the SN in Fig. 1, and in parasagittal sections in Fig. 2. The autoradiograms in both figures show a very similar overall distribution of GABAA/ benzodiazepine receptors in the SN. The autoradiograms in Fig. 1 show a moderate-to-high density of receptors throughout the full extent of the SNr; by contrast the SNc shows a very low density of receptors. Comparison of the density of receptors throughout the rostrocaudal extent of the SN, shown in Fig. la, c and e, clearly demonstrates that the intensity of receptor labelling is greatest in the caudal region of the SNr (Fig. le) when compared with the middle (Fig. Ic) and rostral (Fig. la) regions. Quantitative analysis of the autoradiograms from coronal sections of the SN incubated with [3H]flunitrazepam confirms this differential regional distribution in the density of receptors in the SNr. The highest densities of GABAA/benzodiazepine receptors were found in the caudal regions of the SNr. Here the density of receptors in the caudal SNr (200 + 38 fmol/mg; Fig. le) was 19% higher than that at mid-levels of the SNr (176 ___31 fmol/mg; Fig. lc), and over 30% greater than the density of receptors in the rostral SNr (150 + 26 fmol/mg; Fig. la). These quantitative studies reveal that the overall density of receptors throughout the SNr averaged 179 + 38 fmol/mg. This regional difference in the density of GABAA/benzodiazepine receptors in the SN is especially obvious in parasagittal sections of the brain (Fig. 2). The autoradiograms in Fig. 2a, b clearly show a higher density of receptors in the caudal region of the SNr (see insets, Fig. 2a, b). Quantitative comparative analyses of the density of ligand binding in parasagittal sections of the SNr incubated with [3H]RolS-1788 (Fig. 2a) and [3H]flunitrazepam (Fig. 2b) demonstrated that the density of receptors in the caudal SNr ([3H]RoI5-1788 = 259 +_ 43 fmol/mg; [3H]flunitrazepam = 267 + 51 fmol/mg) was substantially higher than the density of receptors in the rostral SNr ([3H]RoI5-1788 = 163 +_ 21 fmol/mg; [3H]flunitrazepam = 161 + 31 fmol/mg). In order to determine the proportion of Type I and Type II GABAA/benzodiazepine receptors in the SNr, the density of labelling in nigral sections incubated with [3H]flunitrazepam alone (a ligand with a high affinity for both Type I and II receptors; Fig. 2b) was compared with the density of labelling in adja-

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cent sections incubated with [3H]flunitrazepam in the presence of CL218,872 (a discriminating ligand with a preferential high affinity for Type I receptors; ~5~7 Fig. 2c). A visual comparison of the autoradiograms (Fig. 2b, c) suggests that the SNr contains mainly Type I GABAA/benzodiazepine receptors. These visual impressions are confirmed and extended by detailed quantitative comparative analyses of the autoradiograms. These studies show that the density of binding in parasagittal sections of the SNr incubated with [3H]flunitrazepam alone averages 217 _+ 68 fmol/mg (Fig. 2b), whereas adjacent sections incubated with [3H]flunitrazepam in the presence of CL218,872 (Fig. 2c) show a density of binding of 16 _+ 2.5 fmol/mg. In order to investigate the proportion of GABAA/benzodiazepine receptors which were localized on the cell bodies and dendrites of dopaminergic neurons in the SN, quantitative autoradiographic studies were undertaken on the SN in animals which had received unilateral ventral midbrain injections of 6-OHDA. On the lesioned side in these cases, Nisslstained sections of the midbrain showed a virtual total loss of neurons in the SNc (Fig. 3a) and the autoradiograms demonstrated an almost complete loss of receptors in the pars compacta (Fig. 3b). In the SNc on the normal side there was a moderate-tolow density of receptors (133 _+ 30 fmol/mg; Fig. 3b) whereas on the lesioned side the density of receptor labelling in the SNc was extremely low (11 7 fmol/mg; Fig. 3b). A loss of receptors was also evident in the SNr on the lesioned side. Quantitative studies showed that in comparison to the density of receptors in the SNr on the normal side (294 + 55 fmol/mg), the density of receptors in the SNr on the lesioned side (195 ___27 fmol/mg) was reduced by 34% and this reduction was due entirely to a loss of Type I GABAA/benzodiazepine receptors. Immunohistochemical localization o f G A B A A / benzo diazepine receptors

The distribution and localization of GABAA/ benzodiazepine receptors at the light- and electronmicroscopic levels were studied using standard immunohistochemical procedures and a monoclonal antibody (bd-17) specific for the /~2,~ subunits of the GABAA/benzodiazepine receptor complex. Details of the immunohistochemical localization of receptors in the SN are shown in Fig. 4 at the light-microscopic level and in Fig. 5 at the electronmicroscopic level.

Light microscopy. When sections which had been reacted with monoclonal antibody bd-17 were viewed at low-magnification in the light microscope (Fig. 4a), they showed a pattern of receptor immunoreactivity which was remarkably similar to the autoradiographic pattern of GABAA/benzodiazepine receptor distribution (Fig. la, c, e). The immunohistochemicat sections showed a moderate-to-high density of' receptor immunoreactivity throughout the SNr which was interrupted by the fascicles of myelinated fibres traversing the rostrocaudal length of especially the medial two thirds of the SNr, as shown in Fig. 4a. In contrast, the SNc showed a very low level of immunoreactivity. When viewed at higher magnification with the light microscope, the dense immunoreactivity in the SNr was localized on what appeared to be immunoreactive dendrites and the outer membrane of medium to large neuronal somata (small arrows Fig. 4c, e). At higher magnification, receptor immunoreactivity was distributed in a discrete punctate fashion along the outer membrane of the dendritic processes in the SNr (Fig. 4d). Sections in which the primary antibody was omitted from the immunohistochemical procedures showed no immunolabelling (Fig. 4b). Electron microscopy. Ultrastructural studies on the immunohistochemical localization of GABAA/ benzodiazepine receptors in the SN were undertaken on tissue blocks selected from the pars reticulata, the region which consistently showed the most intense immunoreactivity (Fig. 4a) and which is known to receive major GABAergic projections. 2~'4~ The electron-microscopic distribution of GABAA/ benzodiazepine receptors in the SNr is shown in a series of electromicrographs in Fig. 5 (Fig. 5b, d, f). In order to demonstrate the ultrastructural distribution of GABAA/benzodiazepine receptors in the SNr, results which were obtained from sections processed for immunohistochemistry in the presence of the primary antibody, bd-17 (Fig. 5b, d, f), were compared with the results from sections processed in the absence of the primary antibody (Fig. 5a, c, e). Comparison of these results consistently showed that receptor immunoreactivity in the SN was characterized by an increased density of staining which was only associated with the membranes of axons and dendrites (Fig. 5b, d, f). In virtually all instances, the immunoperoxidase reaction end-product was associa~ted with synaptic complexes where it was localized on both the pre- and postsynaptic membranes; in sections cut normal to the synaptic membranes there

Fig. 1. A series of darkfield-type autoradiograms of the SN showing the distribution of GABAA/benzodiazepine receptors labelled with [3H]flunitrazepam from rostral (a), middle (c) and caudal (e) levels of the SN. For orientation purposes, the sections used to generate the autoradiograms in a, c and e have been counterstained for Nissl substance and fibres and are shown in b, d and f, respectively. The boundary between the SNr and the SNc is indicated by arrows. The autoradiograms show a moderate-to-high density of receptors throughout virtually the full extent of the SNr and a low density of receptors in the SNc. CC, crus cerebri; FR, fasiculus retroflexus; ML, medial lemniscus. Scale bar = I mm.

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benzodiazepine receptors in the substantia nigra of the rat.

The regional, cellular and subcellular distribution of GABAA/benzodiazepine receptors was investigated by light and electron microscopy in the rat sub...
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