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Mapping of benzodiazepine-like immunoreactivity in the rat brain as revealed by a monoclonal antibody to benzodiazepines
Journal of Chemical Neuroanatomy, Vol 4:111-121 (1991)
Mapping of Benzodiazepine-like Immunoreactivity in the Rat Brain as Revealed by a Monoclonal Antibody to Benzodiazepines Marina P. S~nchez*, Monika M. Dietlt, Angd L. De Blas, and dos~ M. Palacios Preclinical Research, Sandoz Pharma Ltd, CH-4002 Basle, Switzerland ABSTRACT A monoclonal antibody against benzodiazepines (21-7F9) was used to study the distribution of benzodiazepine-like immunoreactivity in the rat brain. Immunodensitometry in combination with image analysis were used for quantification. The results showed a ubiquitous distribution of benzodiazepine-like immunoreactivity throughout the brain. Very high levels of benzodiazepine-like immunoreactivity were found in the Purkinje cell layer of the cerebellum, in the primary olfactory cortex, in the stratum pyramidale of the hippocampus and in the mitral cell layer of the olfactory bulb. High densities of benzodiazepine-like immunoreactivity were found in the granule cell layer of the cerebellum, the pyramidal cell layer of the olfactory tubercle, the granule layer of the dentate gyrus, the arcuate nucleus of the hypothalamus, the mammillary bodies, the interstitial nucleus of Cajal and superficial grey layer of superior colliculus. The substantia nigra pars compacta, the islands of Calleja and layers II, III, V and VI of the cerebral cortex had moderate levels of benzodiazepine-like immunoreactivity. Lower densities were found in the internal granular layer and the external plexiform layer of the olfactory bulb, in the molecular layer of the dentate gyrus, in layers I and IV of the cerebral cortex, in the nucleus caudate-putamen and most of the thalamic nuclei. The lowest density ofimmunoreactivity was found in the globus pallidus, and the strata radiatum, oriens and lacunosum-moleculare of the hippocampus. The distribution of endogenous benzodiazepine-like immunoreactivity was compared with the distribution of the GABA/benzodiazepine receptor by using both immunocytochemistry and receptor autoradiography. Our studies have shown a clear mismatch between the localization of the benzodiazepine-like immunoreactivity and the GABA/benzodiazepine receptors. KEY WORDS:
Endogenous benzodiazepines Benzodiazepine receptor GABA^ receptor Immunocytochemistry
INTRODUCTION Benzodiazepines (BZ) are anxiolytic, antiepileptic, hypnotic and muscle-relaxant drugs (Haefely, 1983; Martin, 1987). Their effects result from binding to specific neuronal BZ receptors (BZR) (Mrhler and Okada, 1977; Squires and Braestrup, 1977; Tallman et al., 1980), potentiating the inhibitory effect of the neurotransmitter gamma-aminobutyric acid (GABA) (Haefely and Pole, 1986; M5hler et al., 1986; Olsen et al., 1986). This interaction between BZ and GABA is due to the coexistence of binding sites for BZ, GABA and *Present address: Instituto Cajal, Doctor Arce 37, E-28002 Madrid Spain. tPresent address: Collegede France, U. 114, 11 Place Marcelin Berthelot, F-75005Paris, France. ~:Permanent address: Division of Molecular Biologyand Biochemistry, Schoolof BasicLifeSciences,Universityof MissouriKansas City, KansasCity, MO 64110-2499,USA. Correspondence to: Marina P. S~nchez,Instituto Cajal, CSlC, DoctorAree 37, E-28002Madrid, Spain. 0891-0618/91/020111-11 $05.50 © 1991 by John Wiley and Sons Ltd
barbiturates, as well as of a chloride channel within a membrane protein complex named the GABA A receptor (GABAR) (Stephenson, 1987). The Cl channel opens as a result of the binding of GABA to the complex. Both BZ and barbiturates modulate the opening of the chloride channel. The BZ increase the opening frequency of the channel while the barbiturates increase the opening time (Barker and Owen, 1986; Bormann, 1988; Mathers, 1987). Radioligand autoradiography (Palacios et al., 1980, 1981; Richards et al., 1986; Unnerstall et al., 1981; Young and Kuhar, 1979) and immunocytochemistry with monoclonal antibodies to the receptor complex (De Bias et aL, 1988; Richards et al., 1987; Vitorica et al., 1988) have been used to determine the localization in the brain of the GABA receptor/BZR/Cl channel complex. The presence of a BZ binding site in the GABAR complex raises the possibility of the existence of endogenous substances acting at this site. Benzodiazepines, BZ-like molecules and polypeptides
112 M.P. S~nchez et al. with BZ-like activity have been isolated from brain and adrenals of several animals species as well as from vegetables (De Bias et al., 1985; De Bias and Sangameswaran, 1986; Guidotti et al., 1983; Medina et al., 1988; Wildmann et al., 1987, 1988; Alho et al., 1989). In addition, BZ-like immunoreactivity (BZIi) has been described in rat and human brain using the anti-BZ monoclonal antibody (mAb) 21-7F9 (De Bias and Sotelo, 1987; De Bias et al., 1987; Sangameswaran and De Bias, 1985; Sangameswaran et al., 1986). The information available until now on the regional distribution of this BZIi is, however, limited. The purpose of the present work is to provide a detailed mapping of the distribution of BZIi using this same mAb in the rat brain. Densitometry in combination with a computerized image-analysis system has been used to quantify the relative amount of BZli in different areas of the brain. MATERIALS AND METHODS
Immunocytochemistry The production, characterization and specificities of the anti-BZ mAb 21-7F9 and the anti-GABAR mAb 62-3G1 (to the 57 000 Mr subunit) have been described elsewhere (De Bias et al., 1985, 1988, Vitorica et al., 1988). The subeellular distribution of the immunoreactivity with high resolution light and electron microscopy has also been described elsewhere (De Bias and Sotelo, 1987; De Bias et al., 1987; Sangameswaran and De Bias, 1985). The immunoreactivity was specifically blocked with antigens (De Bias et al., 1987; Sangameswaran and De Bias, 1985; Sangameswaran et al., 1986; De Bias et al., 1988). Adult male Wistar rats (200-300 g body weight) were sacrificed by decapitation and the brains were quickly removed and frozen on dry ice. Sections (10 lam thick) were cut with a cryostat microtome at - 20°C, thaw-mounted onto gelatin-coated slides and stored at -20°C. The immunocytochemical protocol is a modification of the one described by De Bias (1984). Briefly, the mounted tissue sections were placed in a fixative containing 4 per cent paraformaldehyde, 0.08 M-Lys, 0.01 M-Na-periodate in 0.05 M-phosphate buffer (PB), pH 7.4 for 2 h at room temperature. After fixation and washing, the slides were incubated in 0.1 M-PB, pH 7.4, containing 1 per cent bovine serum albumin and 0.3 per cent Triton X-100, for 30 min at room temperature and then incubated with either the anti-BZ mAb 21-7F9 (diluted 1:100) or the anti-GABAR mAb 62-3G1 (diluted 1:100) in the same incubation buffer, overnight at 4°C. The following steps were all performed at room temperature, and between incubations the slides were washed twice for 15 min each, in 0.1 M-PBwith 1 per cent bovine serum albumin. The sections were incubated with a biotinylated sheep anti-mouse IgG antibody (Amersham; diluted 1:
100) in incubation buffer for 1 h, followed by incubation in streptavidin-biotin-peroxidase complex (Amersham; diluted 1:100) in the same buffer, for 45 min. The peroxidase reaction was developed in 0.05 per cent diaminobenzidine, 0.03 per cent nickel ammonium sulfate, 0.03 per cent cobalt chloride, and 0.01 per cent H202 in 0.1 M-PB (pH 7.4). The relative density of BZIi was determined with the help of a computerized image analysis system (MCID Imaging Res. Inc. Ontario, Canada). Anatomical regions were identified by using the stereotaxic rat brain atlas of Paxinos and Watson (1982).
Receptor autoradiogralflty The details of this procedure have been previously reported (McCabe and Wamsley, 1986; Palacios et al., 1980). [aH]Flunitrazepam was used to identify the BZ binding sites of the GABAR/BZR complex, as described elsewhere (Unnerstall et al., 1981; Young and Kuhar, 1979; Zezula et al., 1988). [3H]Muscimol was used for studying the highaffinity GABA receptors (Palacios et al., 1981; Penney et al., 1981; Snodgrass, 1978). [35S]t-Butylbicyclophosphorothionate (TBPS) was used to label the chloride channel of the GABAR/BZR complex (Gee et al., 1983; Squires et al., 1983; Supavilai and Karobath, 1984). [3H]SR 95531 was used for studying the antagonist binding site of the GABA A receptor (Heaulme et al., 1987; McCabe et al., 1988). RESULTS
Distribution of BZli The distribution of BZli in rat brain coronal sections is illustrated in Fig. 1 (for relative optical densities see Table 1). Telencephalic structures
The localization of BZIi in the olfactory bulb is shown in Fig. 1A. The mitral cell layer contained the highest BZIi in this structure. The glomeruli contained a moderate concentration of immunostaining while the internal granular and external plexiform layers showed low immunoreactivity. The olfactory tubercle (Fig. 1B) displayed high BZIi in the pyramidal cell layer but low density in the other layers (polymorphic and plexiform). The islands of Calleja showed moderate immunostaining, and the primary olfactory cortex had a very high density (Fig. 1B). Low levels of BZIi were measured in the ventral pallidum (Fig. 1C), nucleus accumbens and medial septal nucleus (Fig. 1B), while the lateral septal nucleus contained a moderate immunostaining. The bed nucleus of the stria terminalis (Fig. 1C) showed moderate immunostaining. The frontal cortex displayed moderate BZIi in layers II, III, V and VI while layers I and IV showed low immunostaining. The caudate-putamen contained a low BZli
Benzodiazepine-like Immunoreactivity in the Rat throughout its extent while the globus pallidus showed a very low immunostaining (Fig, 1C). Moderate densities were observed over the claustrum (Fig. 1B) and the amygdaloid nuclei (Fig. 1D). Very high immunostaining was found over the stratum pyramidale of the hippocampus (Fig. ID), while the other strata (lacunosum-moleculare, radiatum and oriens) had very low levels of BZIi. The granule cell layer of the dentate gyrus displayed high BZIi, in contrast to the molecular layer, which had low immunoreactivity (Fig. 1D). Diencephalic structures
Fig. 1D shows the thalamus containing intermediate to low levels of BZli. Nuclei with moderate densities of immunostaining were the paraventricular and rhomboid, while the nuclei with low BZIi were the reticular, laterodorsal, ventroposterior, mediodorsal, ventrolateral, posterior nuclear group, gelatinosus and intermediodorsal. The medial geniculate nucleus (Fig. IF), the lateral habenular nucleus (Fig. 1D) and the zona incerta (Fig. 1E) showed moderate densities of BZli. The hypothalamic nuclei (ventromedialis, dorsomedialis, lateralis and dorsalis) showed in general moderate to low densities of BZIi (Fig. 1D), but high levels of immunostaining could be found in the arcuate hypothalamic nucleus and over the mammillary bodies (Fig. 1E). Mesencephalic structures
High BZIi was found over the superficial grey layer of the superior colliculus while the other layers (optic nerve layer, intermediate grey layer and intermediate white layer) had moderate concentrations (Fig. I F). The interstitial nucleus of Cajal and the nucleus of Darkschewitseh displayed high BZIi (Fig. IF). The red nucleus and the substantia nigra pars compacta had moderate levels of immunostaining, but the substantia nigra pars reticulata showed lower immunoreactivity (Fig. 1G). The interpeduncular nucleus and the central grey matter showed moderate immunoreactivity (Fig. 1G). Cerebellum
The Purkinje cell layer of the cerebellum contained the highest concentration of BZ-like immunostaining observed in this study (Fig. 1H). A high concentration of immunostaining was also found in the granule cell layer, while the molecular layer showed a lower density. Distribution of GABAR/BZR complex in the cerebellum Fig. 2 shows the distribution of the GABAR/BZR complex in the cerebellum as revealed by different GABAR/BZR/CI channel binding probes. The highest density of [3H]flunitrazepam, binding to the BZR sites of the receptor complex (Fig. 2A), was found in the molecular layer, while the granular
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layer and the Purkinje cell layer showed much lower binding. The other ligands for the GABAR, [3H]muscimol (Fig. 2B), [3H]SR 95531 (Fig. 2D), as well as for the CI channel, [35S]TBPS (Fig. 2C) preferentially bound to the granular layer of the cerebellum. A similar distribution was revealed by the anti-GABAR mAb 62-3G1 (Fig. 2E). Comparison of cellular localization of BZH with that of Nissl staining Fig. 3 shows the cellular localization of BZIi in the cerebellum. The BZli was found mainly in the Purkinje cells and in most cells of the molecular layer. BZli was also present in some cells of the granular layer, but it was not present in the white matter. Fig. 4 shows the cellular distribution of BZIi and toluidine blue staining in the deeper layers of the somatosensory cortex. While toluidine blue stained all the cells in layer VI and white matter (Fig. 4B), BZIi was not found in all cells (Fig. 4A). DISCUSSION The main finding of the present work was the widespread distribution of cells containing BZIi in the rat brain. However, when the distribution of BZIi was compared with that of a Nissl staining, it could be observed that not all the cells were immunostained. Thus, for instance, in the cerebral cortex a large number of cells showed BZIi, but the number of Nissl-stained cells was higher. Moreover, the cell nucleoli were not immunoreactive, while they were always stained with toluidine blue. Benzodiazepine-like immunoreactivity has been detected, using the mAb 21-7F9, in rat and human brains, including brains that were kept stored in paraffin since 1940, that is, 15 years before the chemical synthesis and marketing of BZ by the pharmaceutical industry (De Bias and Sotelo, 1987; De Bias et al., 1987; Sangameswaran and De Bias, 1985; Sangameswaran et al., 1986). These results suggest a natural origin of these substances. In addition, N-desmethyldiazepam, diazepam and other BZ have been isolated from rat and cow brains, adrenals and other rat organs as well as from cow milk and vegetables (Guidotti et al., 1983; Medina et al., 1988; Wildmann et al., 1987, 1988; Alho et al., 1989). It has been proposed that the brain BZ might have their origin in a natural component of the diet (Medina et al., 1988; Sangameswaran et al., 1986; Wildmann et al., 1987, 1988). Nevertheless, the biological and/or clinical relevance of these findings needs to be assessed. For this purpose we have mapped more extensively the distribution of BZIi in the rat brain and compared it with the distribution of the GABAR/BZR complex in some areas of the brain. We have found higher densities of BZli in the Purkinje cell layer of the cerebellum, in the stratum
114 M.P. Shnchez et al.
I III
A
Fig. 1 a-d.
pyramidale of the hippocampus, in the mitral cell layer of the olfactory bulb and in the primary olfactory cortex. In contrast, lower levels of BZIi were localized in the globus pallidus and in the strata radiatum, oriens and lacunosum-moleeulare of the hippocampus. It has been shown that BZIi is mostly found in neurons (perikarya and dendritic processes) both in the soluble cytoplasm and also in the cytoplasmic face of the cell membranes (De Bias and Sotelo,
1987; De Bias et al., 1987). In the same study it was reported that both GABAergic and non-GABAergic neurons showed BZli. In addition, their axon terminals remained mostly unlabelled, suggesting that BZ might not be co-released with GABA (De Bias and Sotelo, 1987). Radioligand autoradiography (McCabe and Wamsley, 1986; McCabe et al., 1988; Palacios et al., 1980; Richards et al., 1986; Sieghart and Karobath, 1980; Young and Kuhar, 1979; Young et al., 1981;
Benzodiazepine-like Immunoreactivity in the Rat
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Fig. 1 e-h. Fig. 1. Distribution of BZIi in coronal sections at different levels of the rat brain. Dark regions correspond to areas enriched in BZli. See Table 1 for relative optical density. (A) The olfactory bulb; (B) olfactory tubercle; (C) caudate-putamen and globus pallidus; (D) medial thalamus, hypothalamus and hippocampal formation; (E) posterior thalamus and zona incerta; (F) mammillary bodies, interstitial nucleus of Cajal and nucleus of Darksehewitsch; (G) substantia nigra, superior colliculus, central grey and medial geniculate body; (H) cerebellum. Bar = 2 mm.
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Table 1. Relative optical densities of benzodiazepine-like immunoreactivity in the rat brain Olfactory bulb Glomerular layer External plexiform layer Internal granular layer Mitral cell layer
680 465 475 965
Olfactory tubercle Polymorphic layer Pyramidal layer Plexiform layer Islands of Calleja Primary olfactory cortex
490 740 475 650 1000
Basal ganglia Ventral pallidum Caudate-putamen Globus pallidus Claustrum Nucleus aceumbens
445 465 300 535 485
Frontal cortex Lamina I Lamina II Lamina III Lamina IV Lamina V Lamina VI
460 615 640 485 590 520
Hippocampus Stratum oriens Stratum pyramidale Stratum lacunosum-moleculare Stratum radiatum
380 975 360 335
Dentate gyrus Molecular layer Granule cell layer
505 765
Septum Lateralis Medialis
560 415
Bed nucleus stria terminalis
605
Hypothalamus Ventromedial nucleus Dorsomedial nucleus Lateral nucleus Dorsal nucleus Arcuate nucleus
505 540 420 525 855
Thalamus Reticular nucleus Laterdorsal nucleus Ventroposterior nucleus Ventrolateral nucleus Mediodorsal nucleus Paraventficular nucleus Rhomboideus nucleus Posteromedial nucleus Intermediodorsal nucleus Gelatinosus nucleus Medial genieulate nucleus Lateral habenula
470 495 440 470 430 535 585 455 490 470 615 520
Amygdatoid nuclei
580
Mammillary bodies
740
Zona incerta
520
Red nucleus
545
Interpeduncular nucleus
675
Periaqueduetal grey matter
670
Interstitial nucleus of Cajal
760
Nucleus of Darkschewitsch
905
Substantia nigra Pars reticulata Pars compacta
480 580
Superior colliculus Superficial grey layer Optic nerve layer Intermediate grey layer Intermediate white layer
740 575 585 605
Cerebellum Granule cell layer Purkinje cell layer Molecular layer
810 1125 420
Data are the mean values of relative optical densities ( x 10 000) of at least two animals for each brain region. In all cases, SEMs were equal to or less than 10%.
Zezula et al., 1988) and specific m A b s (De Bias et al., 1988; Richards et al., 1987; Vitorica et al., 1988) have been used to study the distribution o f the GABAR/BZR/CI channel complex in the brain. A l t h o u g h there is co-localization o f G A B A and its biosynthetic enzyme glutamic acid decarboxylase, with the G A B A R / B Z R complex in some brain areas, there are also m a n y mismatches (De Bias et al., 1988; H e r k e n h a m , 1987; K u h a r , 1985; Mariani et al., 1987; Mugnaini and Oertel, 1985; Seguela et al., 1984). The localization mismatch between transmitter (or transmitter-synthesizing enzyme) and receptor is a c o m m o n finding reported t h r o u g h o u t the
literature (Herkenham, 1987; Kuhar, 1985). A lack o f correlation between the distribution o f BZIi and that o f the k n o w n G A B A A receptors also appears to be the case. The cerebellum contains several classes o f G A B A A receptors (Fig. 2). This is supported by the differential distribution o f ligand binding sites with different binding capacities (e.g., see M c C a b e et al., 1988; Olsen et al., 1990; Unnerstall et al., 1981), and by recent 'in situ' hybridization studies showing differential cellular expression o f the m R N A coding for the subunits o f the G A B A A receptor in bovine and rat cerebellum (e.g. Siegel, 1988; Wisden et al., 1988; Shivers et al., 1989).
Benzodiazepine-like Immunoreactivity in the Rat
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Fig. 2. Distribution of GABAR/BZR/CI- channel complex in rat cerebellum as revealed by radioligand autoradiography (A-D) and immunocytochemistry (E). (A) [3H]Flunitrazepam binding to BZR. (B) [3H]Muscimol binding to bigh-affmity GABA^ receptor. (C) [35S]TBPSbindingto the chloride channel. (D) [3H]SR95531 bindingto the antagonist high-affinity bindingconformation of the GABAR. (E) GABA^ receptor-like immunoreactivity, with the mAb 62-3G 1 to the receptor complex (De Bias et al., 1988). Bar = 2 ram.
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Fig. 3. Cellular localization of BZIi in the cerebellum. BZIi is present in most cells of the Purkinje and molecular layers. In the granular layer some cells are immunoreactive while the white matter is immunoreactivity-free. Bar = 100 ~m.
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Fig. 4. Comparison of cellular localization of BZli and that of toluidine blue staining in the deeper layers of the somatosensory cortex. (A) BZli distribution in layer VI (Via and VIb) and white matter. (B) Toluidine blue staining in layer VI and white matter. Bar = 100 Hm.
Benzodiazepine-like Immunoreactivity in the R a t
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ABBREVIATIONS USED IN FIGURES Acb Amyg Arc BST CG CI CPu Dk EPI Fr GI GP Gr GrDG ICj IGr InC InG InWh
aecumbens nucleus amygdaloid nuclei arcuate hypothalamic nucleus bed nucleus stria terminalis central grey matter claustrum caudate-putamen nucleus nucleus of Darkschewitsch externalplexiform layer of the olfactory bulb frontal cortex giomerular layer of the olfactory bulb globus pallidus granular layer of the cerebellum granular layer of the dentate gyrus islands of Calleja internal granular layer of the olfactory bulb interstitialnucleus of Cajal intermediate grey layer of the superior colliculus intermediate white layer of the superior colliculus
interpeduncular nucleus laterodorsal thalamic nucleus lateral hypothalamic nuclei lateral habenular nucleus lateral septal nucleus layer IV of the frontal cortex layer Via of the somatosensory cortex L VIb layer VIb of the somatosensory cortex MB mammillary bodies mediodorsal thalamic nucleus MD medial geniculate nucleus MG Mi mitral cell layer of the olfactory bulb MolDG molecular layer of the dentate gyrus Mol molecular layer of the cerebellum MS medial septal nucleus optic nerve layer of the Op superior colliculus PO primary olfactory cortex Po posterior thalamic nuclear group IPC LD LH LHb LS L IV L Via
The BZIi was f o u n d mainly in the Purkinje cell layer and in the granular layer o f the cerebellum (Fig. 1H), while the [3H]flunitrazepam binding sites were mainly localized in the molecular layer (Fig. 2A). The binding sites for [3H]muscimol, [35S]TBPS, [3H]SR 95531 and m A b 62-3G1 were mostly localized on the granular layer o f the cerebellum (Fig. 2B-E). Nevertheless, there were some regions o f the brain where the distribution patterns o f BZli and B Z R overlapped as for instance, the central (periaqueductal) grey matter, superior and inferior colliculi and mammillary bodies. The distribution o f brain BZli is consistent with the hypothesis that e n d o g e n o u s brain BZ might m o d u l a t e G A B A neurotransmission, by interacting with the G A B A A receptor complex. Nevertheless, brain BZ could also be exogenous natural products taken with the diet and that accumulate in the brain (Medina et al., 1988; S a n g a m e s w a r a n and De Bias, 1985; S a n g a m e s w a r a n et al., 1986; W i l m a n n et al., 1987, 1988). I f that was the case, the brain BZIi could be revealing either the m o d u l a t i o n o f G A B A transmission or, like other dietetic substances, the process o f biotransformation, accumulation and elimination.
ACKNOWLEDGEMENTS We thank Dr Alfonso Fairrn for his help and financial support during the preparation of the manuscript (CSIC/CAICYT grant 154/1985, Spain). We gratefully acknowledge Dr Gonzalo Alvarez-Bolado (I. Cajal, Madrid) for helpful advice, and Christian Waeber, of the Sandoz Pharma Ltd Laboratories (Basel), for help and support.
Purk Py R Rt SNC SNR SuG TuP1 TuPo TuPy VMH VP WM ZI
Purkinje cell layer of the cerebellum stratum pyramidalis of the hippocampus red nucleus reticular thalamic nucleus substantia nigra, pars compacta substantia nigra, pars reticulata superficial grey layer of the superior colliculus plexiform layer of the olfactory tubercle polimorphic layer of the olfactory tubercle pyramidal layer of the olfactory tubercle ventromedial hypothalamic nucleus ventral pallidum white matter zona incerta
This research was supported in part by grant NS 17708 from the National Institute of Neurological and Communicative Disorders and Stroke, USA, to A.L.D.B. during a sabbatical leave in the laboratory of J.M.P.
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structure and location. In Molecular Aspects of Neurobiology (eds Levi-Montalcini, R. et al.), pp. 91-96. Springer-Verlag, Berlin. Mugnaini, E. and Oertel, W. H. (1985). An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In Handbook of Chemical Neuroanatomy, Vol. 4, GABA andneuropeptides in the CNS, part I, (eds Bjfrklund, A. and H6kfelt, T.), pp. 436-608. Elsevier Sci. Publ., B.V., Amsterdam. Olsen, R. W., McCabe, R. T. and 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. Olsen, R. W., Yang, J., King, R. G., Dilber, A., Stauber, G. B. and Ransom, R. W. (1986). Barbiturate and benzodiazepine modulation of GABA receptor binding and function. Life Sci. 39, 1969-1976. Palacios, J. M., Young, W. S. III and Kuhar, M. J. (1980). GABA and benzodiazepine receptors in rat and human brain: autoradiographic localization by a novel technique. In Enzymes and Neurotransmitters in Mental Disease (eds Usdin, E., Sourkes, T. L. and Youdin, M. B. H.), pp. 573-583. John Wiley and Sons, New York. Palacios, J. M., Wamsley, J. K. and Kuhar, M. J. (1981). High affinity GABA receptors--autoradiography localization. Brain Res. 222, 285--307. Paxinos, G. and Watson, C. (1982). The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Penney, J. B. Jr., Pan, H. S., Young, A. B., Frey, K. A. and Dauth, D. W. (1981). Quantitative autoradiography of [3H] muscimol binding in rat brain. Science 214, 10361038. Richards, G., Mfhler, H. and Haefely, W. (1986). Mapping benzodiazepine receptors in the CNS by radiohistochemistry and immunohistochemistry. In Neurology and Neurobiology, Vol. 16, Neurohistochemistry, Modern Methods and Applications (eds Panula, P., Paivarinta, H. and Soinila, S.), pp. 629-677. Liss, New York. Richards, J. G., Schoch, P., Haring, P., Takacs, B. and M6hler, H. (1987). Resolving GABAA/benzodiazepine receptors: Cellular and subcellular localization in the CNS with monoclonal antibodies. J. Neurosci. 7, 18661886. Sangameswaran, L. and De Bias, A. L. (1985). Demonstration of benzodiazepine-like molecules in the mammalian brain with a monoclonal antibody to benzodiazepines. Proc. Natl. Acad. Sci. USA 82, 55605564. Sangameswaran, L., Fales, H. M., Friedrich, P. and De Bias, A. L. (1986). Purification of a benzodiazepine from bovine brain and detection ofbenzodiazepine-like immunoreactivity in human brain. Proc. Natl. Acad. Sci. USA 83, 9236--9240. Seguela, P., Geffard, M., Buijs, R. M. and Le Moal, M. (1984). Antibodies against gamma-amino butyric acid: specific studies and immunoeytochemical results. Proc. Natl. Acad. Sci. USA 81, 3888-3892. Shivers, B. D., Killisch, I., Sprengel, R., Sontheimer, H., Kfhler, M., Schofield, P. R. and Seeburg, P. H. (1989). Two novel GABAA receptor subunits exist in distinct neuronal subpopulations. Neuron 3, 327-337. Siegel, R. E. (1988). The mRNAs encoding GABAA/benzodiazepine receptor subunits are localized in different
Benzodiazepine-like Immunoreactivity in the Rat cell populations of the bovine cerebellum. Neuron 1, 579-584. Sieghart, W. and Karobath, M. (1980). Molecular heterogeneity of benzodiazepine receptors. Nature 286, 285287. Snodgrass, S. R. (1978). Use of 3H muscimol for GABA receptor studies. Nature 273, 392-394. Squires, R. F. and Braestrup, C. (1977). Benzodiazepine receptors in rat brain. Nature 266, 732-734. Squires, R. F., Casida, J. E., Richardson, M. and Saederup, E. (1983). [3~S]t-butylbicyclophosphorothionate binds with high affinity to brain-specific sites coupled to gamma-aminobutyric acid-A and ion recognition sites. Mol. Pharmacol. 23, 326-336. Stephenson, F. A. (1987). Understanding the GABAA receptor: a chemically gated ion channel. Biochem. J. 249, 21-32. Supavilai, P. and Karobath, M. (1984). [35S]t-butylbicyclophosphorothionate binding sites are constituents of the gamma-aminobutyric acid benzodiazepine receptor complex. J. Neurosci. 41, 1193-I 200. Tallman, J. F., Paul, S. M., Skolnick, P. and Gallager, D. W. (1980). Receptors for the age of anxiety: Pharmacology of the benzodiazepines. Science 207, 274-281. Unnerstall, J. R., Kuhar, M. J., Niehoff, D. L. and Palacios, J. M. (1981). Benzodiazepine receptors are coupled to a subpopulation of gamma-aminobutyric acid (GABA) receptors: evidence from a quantitative autoradiographic study. J. Pharmac. Exp. Ther. 218, 797-804.
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Vitorica, J., Park, D., Chin, G. and De Bias, A. L. (1988). Monoclonal antibodies and conventional antisera to the GABAAreceptor/benzodiazepine receptor/Cl" channel complex. J. Neurosci. 8, 615-622. Wildmann, J., M6hler, H., Vetler, W., Ranalder, V., Schmidt, K. and Maurer, R. (1987). Diazepam and Ndesmethyldiazepam are found in rat brain and adrenal and may be of plant origin. J. Neural Transm. 70, 383-388. Wildmann, J., Vetter, W., Ranalder, U. B., Schmidt, K., Maurer, R. and M6hler, H. (1988). Occurrence of pharmacologically active benzodiazepines in trace amounts in wheat and potato. Biochem. Pharmacol. 37, 35493559. Wisden, W., Morris, B. I., Darlison, M. G., Hunt, S. P. and Barnard, E. A. (1988). Distinct GABAA receptor alpha-subunit mRNAs show differential patterns of expression in bovine brain. Neuron 1, 937-947. Young, W. S. III and Kuhar, M. J. (1979). Autoradiographic localization of benzodiazepine receptors in the brains of humans and animals. Nature 280, 393-395. Young, W. S. III, Niehoff, D., Kuhar, M. J., Beer, B. and Lippe, A. S. (1981). Multiple benzodiazepine receptor localization by light microscopic radiohistochemistry. J. Pharmac. Exp. Ther. 216, 425-430. Zezula, J., Cort6s, R., Probst, A. and Palacios, J. M. (1988). Benzodiazepine receptor sites in the human brain: autoradiographic mapping. Neuroscience 25, 771-795.
Accepted 9 June 1990
Mapping of Benzodiazepine-like Immunoreactivity in the Rat Brain as Revealed by a Monoclonal Antibody to Benzodiazepines Marina P. S~nchez*, Monika M. Dietlt, Angd L. De Blas, and dos~ M. Palacios Preclinical Research, Sandoz Pharma Ltd, CH-4002 Basle, Switzerland ABSTRACT A monoclonal antibody against benzodiazepines (21-7F9) was used to study the distribution of benzodiazepine-like immunoreactivity in the rat brain. Immunodensitometry in combination with image analysis were used for quantification. The results showed a ubiquitous distribution of benzodiazepine-like immunoreactivity throughout the brain. Very high levels of benzodiazepine-like immunoreactivity were found in the Purkinje cell layer of the cerebellum, in the primary olfactory cortex, in the stratum pyramidale of the hippocampus and in the mitral cell layer of the olfactory bulb. High densities of benzodiazepine-like immunoreactivity were found in the granule cell layer of the cerebellum, the pyramidal cell layer of the olfactory tubercle, the granule layer of the dentate gyrus, the arcuate nucleus of the hypothalamus, the mammillary bodies, the interstitial nucleus of Cajal and superficial grey layer of superior colliculus. The substantia nigra pars compacta, the islands of Calleja and layers II, III, V and VI of the cerebral cortex had moderate levels of benzodiazepine-like immunoreactivity. Lower densities were found in the internal granular layer and the external plexiform layer of the olfactory bulb, in the molecular layer of the dentate gyrus, in layers I and IV of the cerebral cortex, in the nucleus caudate-putamen and most of the thalamic nuclei. The lowest density ofimmunoreactivity was found in the globus pallidus, and the strata radiatum, oriens and lacunosum-moleculare of the hippocampus. The distribution of endogenous benzodiazepine-like immunoreactivity was compared with the distribution of the GABA/benzodiazepine receptor by using both immunocytochemistry and receptor autoradiography. Our studies have shown a clear mismatch between the localization of the benzodiazepine-like immunoreactivity and the GABA/benzodiazepine receptors. KEY WORDS:
Endogenous benzodiazepines Benzodiazepine receptor GABA^ receptor Immunocytochemistry
INTRODUCTION Benzodiazepines (BZ) are anxiolytic, antiepileptic, hypnotic and muscle-relaxant drugs (Haefely, 1983; Martin, 1987). Their effects result from binding to specific neuronal BZ receptors (BZR) (Mrhler and Okada, 1977; Squires and Braestrup, 1977; Tallman et al., 1980), potentiating the inhibitory effect of the neurotransmitter gamma-aminobutyric acid (GABA) (Haefely and Pole, 1986; M5hler et al., 1986; Olsen et al., 1986). This interaction between BZ and GABA is due to the coexistence of binding sites for BZ, GABA and *Present address: Instituto Cajal, Doctor Arce 37, E-28002 Madrid Spain. tPresent address: Collegede France, U. 114, 11 Place Marcelin Berthelot, F-75005Paris, France. ~:Permanent address: Division of Molecular Biologyand Biochemistry, Schoolof BasicLifeSciences,Universityof MissouriKansas City, KansasCity, MO 64110-2499,USA. Correspondence to: Marina P. S~nchez,Instituto Cajal, CSlC, DoctorAree 37, E-28002Madrid, Spain. 0891-0618/91/020111-11 $05.50 © 1991 by John Wiley and Sons Ltd
barbiturates, as well as of a chloride channel within a membrane protein complex named the GABA A receptor (GABAR) (Stephenson, 1987). The Cl channel opens as a result of the binding of GABA to the complex. Both BZ and barbiturates modulate the opening of the chloride channel. The BZ increase the opening frequency of the channel while the barbiturates increase the opening time (Barker and Owen, 1986; Bormann, 1988; Mathers, 1987). Radioligand autoradiography (Palacios et al., 1980, 1981; Richards et al., 1986; Unnerstall et al., 1981; Young and Kuhar, 1979) and immunocytochemistry with monoclonal antibodies to the receptor complex (De Bias et aL, 1988; Richards et al., 1987; Vitorica et al., 1988) have been used to determine the localization in the brain of the GABA receptor/BZR/Cl channel complex. The presence of a BZ binding site in the GABAR complex raises the possibility of the existence of endogenous substances acting at this site. Benzodiazepines, BZ-like molecules and polypeptides
112 M.P. S~nchez et al. with BZ-like activity have been isolated from brain and adrenals of several animals species as well as from vegetables (De Bias et al., 1985; De Bias and Sangameswaran, 1986; Guidotti et al., 1983; Medina et al., 1988; Wildmann et al., 1987, 1988; Alho et al., 1989). In addition, BZ-like immunoreactivity (BZIi) has been described in rat and human brain using the anti-BZ monoclonal antibody (mAb) 21-7F9 (De Bias and Sotelo, 1987; De Bias et al., 1987; Sangameswaran and De Bias, 1985; Sangameswaran et al., 1986). The information available until now on the regional distribution of this BZIi is, however, limited. The purpose of the present work is to provide a detailed mapping of the distribution of BZIi using this same mAb in the rat brain. Densitometry in combination with a computerized image-analysis system has been used to quantify the relative amount of BZli in different areas of the brain. MATERIALS AND METHODS
Immunocytochemistry The production, characterization and specificities of the anti-BZ mAb 21-7F9 and the anti-GABAR mAb 62-3G1 (to the 57 000 Mr subunit) have been described elsewhere (De Bias et al., 1985, 1988, Vitorica et al., 1988). The subeellular distribution of the immunoreactivity with high resolution light and electron microscopy has also been described elsewhere (De Bias and Sotelo, 1987; De Bias et al., 1987; Sangameswaran and De Bias, 1985). The immunoreactivity was specifically blocked with antigens (De Bias et al., 1987; Sangameswaran and De Bias, 1985; Sangameswaran et al., 1986; De Bias et al., 1988). Adult male Wistar rats (200-300 g body weight) were sacrificed by decapitation and the brains were quickly removed and frozen on dry ice. Sections (10 lam thick) were cut with a cryostat microtome at - 20°C, thaw-mounted onto gelatin-coated slides and stored at -20°C. The immunocytochemical protocol is a modification of the one described by De Bias (1984). Briefly, the mounted tissue sections were placed in a fixative containing 4 per cent paraformaldehyde, 0.08 M-Lys, 0.01 M-Na-periodate in 0.05 M-phosphate buffer (PB), pH 7.4 for 2 h at room temperature. After fixation and washing, the slides were incubated in 0.1 M-PB, pH 7.4, containing 1 per cent bovine serum albumin and 0.3 per cent Triton X-100, for 30 min at room temperature and then incubated with either the anti-BZ mAb 21-7F9 (diluted 1:100) or the anti-GABAR mAb 62-3G1 (diluted 1:100) in the same incubation buffer, overnight at 4°C. The following steps were all performed at room temperature, and between incubations the slides were washed twice for 15 min each, in 0.1 M-PBwith 1 per cent bovine serum albumin. The sections were incubated with a biotinylated sheep anti-mouse IgG antibody (Amersham; diluted 1:
100) in incubation buffer for 1 h, followed by incubation in streptavidin-biotin-peroxidase complex (Amersham; diluted 1:100) in the same buffer, for 45 min. The peroxidase reaction was developed in 0.05 per cent diaminobenzidine, 0.03 per cent nickel ammonium sulfate, 0.03 per cent cobalt chloride, and 0.01 per cent H202 in 0.1 M-PB (pH 7.4). The relative density of BZIi was determined with the help of a computerized image analysis system (MCID Imaging Res. Inc. Ontario, Canada). Anatomical regions were identified by using the stereotaxic rat brain atlas of Paxinos and Watson (1982).
Receptor autoradiogralflty The details of this procedure have been previously reported (McCabe and Wamsley, 1986; Palacios et al., 1980). [aH]Flunitrazepam was used to identify the BZ binding sites of the GABAR/BZR complex, as described elsewhere (Unnerstall et al., 1981; Young and Kuhar, 1979; Zezula et al., 1988). [3H]Muscimol was used for studying the highaffinity GABA receptors (Palacios et al., 1981; Penney et al., 1981; Snodgrass, 1978). [35S]t-Butylbicyclophosphorothionate (TBPS) was used to label the chloride channel of the GABAR/BZR complex (Gee et al., 1983; Squires et al., 1983; Supavilai and Karobath, 1984). [3H]SR 95531 was used for studying the antagonist binding site of the GABA A receptor (Heaulme et al., 1987; McCabe et al., 1988). RESULTS
Distribution of BZli The distribution of BZli in rat brain coronal sections is illustrated in Fig. 1 (for relative optical densities see Table 1). Telencephalic structures
The localization of BZIi in the olfactory bulb is shown in Fig. 1A. The mitral cell layer contained the highest BZIi in this structure. The glomeruli contained a moderate concentration of immunostaining while the internal granular and external plexiform layers showed low immunoreactivity. The olfactory tubercle (Fig. 1B) displayed high BZIi in the pyramidal cell layer but low density in the other layers (polymorphic and plexiform). The islands of Calleja showed moderate immunostaining, and the primary olfactory cortex had a very high density (Fig. 1B). Low levels of BZIi were measured in the ventral pallidum (Fig. 1C), nucleus accumbens and medial septal nucleus (Fig. 1B), while the lateral septal nucleus contained a moderate immunostaining. The bed nucleus of the stria terminalis (Fig. 1C) showed moderate immunostaining. The frontal cortex displayed moderate BZIi in layers II, III, V and VI while layers I and IV showed low immunostaining. The caudate-putamen contained a low BZli
Benzodiazepine-like Immunoreactivity in the Rat throughout its extent while the globus pallidus showed a very low immunostaining (Fig, 1C). Moderate densities were observed over the claustrum (Fig. 1B) and the amygdaloid nuclei (Fig. 1D). Very high immunostaining was found over the stratum pyramidale of the hippocampus (Fig. ID), while the other strata (lacunosum-moleculare, radiatum and oriens) had very low levels of BZIi. The granule cell layer of the dentate gyrus displayed high BZIi, in contrast to the molecular layer, which had low immunoreactivity (Fig. 1D). Diencephalic structures
Fig. 1D shows the thalamus containing intermediate to low levels of BZli. Nuclei with moderate densities of immunostaining were the paraventricular and rhomboid, while the nuclei with low BZIi were the reticular, laterodorsal, ventroposterior, mediodorsal, ventrolateral, posterior nuclear group, gelatinosus and intermediodorsal. The medial geniculate nucleus (Fig. IF), the lateral habenular nucleus (Fig. 1D) and the zona incerta (Fig. 1E) showed moderate densities of BZli. The hypothalamic nuclei (ventromedialis, dorsomedialis, lateralis and dorsalis) showed in general moderate to low densities of BZIi (Fig. 1D), but high levels of immunostaining could be found in the arcuate hypothalamic nucleus and over the mammillary bodies (Fig. 1E). Mesencephalic structures
High BZIi was found over the superficial grey layer of the superior colliculus while the other layers (optic nerve layer, intermediate grey layer and intermediate white layer) had moderate concentrations (Fig. I F). The interstitial nucleus of Cajal and the nucleus of Darkschewitseh displayed high BZIi (Fig. IF). The red nucleus and the substantia nigra pars compacta had moderate levels of immunostaining, but the substantia nigra pars reticulata showed lower immunoreactivity (Fig. 1G). The interpeduncular nucleus and the central grey matter showed moderate immunoreactivity (Fig. 1G). Cerebellum
The Purkinje cell layer of the cerebellum contained the highest concentration of BZ-like immunostaining observed in this study (Fig. 1H). A high concentration of immunostaining was also found in the granule cell layer, while the molecular layer showed a lower density. Distribution of GABAR/BZR complex in the cerebellum Fig. 2 shows the distribution of the GABAR/BZR complex in the cerebellum as revealed by different GABAR/BZR/CI channel binding probes. The highest density of [3H]flunitrazepam, binding to the BZR sites of the receptor complex (Fig. 2A), was found in the molecular layer, while the granular
113
layer and the Purkinje cell layer showed much lower binding. The other ligands for the GABAR, [3H]muscimol (Fig. 2B), [3H]SR 95531 (Fig. 2D), as well as for the CI channel, [35S]TBPS (Fig. 2C) preferentially bound to the granular layer of the cerebellum. A similar distribution was revealed by the anti-GABAR mAb 62-3G1 (Fig. 2E). Comparison of cellular localization of BZH with that of Nissl staining Fig. 3 shows the cellular localization of BZIi in the cerebellum. The BZli was found mainly in the Purkinje cells and in most cells of the molecular layer. BZli was also present in some cells of the granular layer, but it was not present in the white matter. Fig. 4 shows the cellular distribution of BZIi and toluidine blue staining in the deeper layers of the somatosensory cortex. While toluidine blue stained all the cells in layer VI and white matter (Fig. 4B), BZIi was not found in all cells (Fig. 4A). DISCUSSION The main finding of the present work was the widespread distribution of cells containing BZIi in the rat brain. However, when the distribution of BZIi was compared with that of a Nissl staining, it could be observed that not all the cells were immunostained. Thus, for instance, in the cerebral cortex a large number of cells showed BZIi, but the number of Nissl-stained cells was higher. Moreover, the cell nucleoli were not immunoreactive, while they were always stained with toluidine blue. Benzodiazepine-like immunoreactivity has been detected, using the mAb 21-7F9, in rat and human brains, including brains that were kept stored in paraffin since 1940, that is, 15 years before the chemical synthesis and marketing of BZ by the pharmaceutical industry (De Bias and Sotelo, 1987; De Bias et al., 1987; Sangameswaran and De Bias, 1985; Sangameswaran et al., 1986). These results suggest a natural origin of these substances. In addition, N-desmethyldiazepam, diazepam and other BZ have been isolated from rat and cow brains, adrenals and other rat organs as well as from cow milk and vegetables (Guidotti et al., 1983; Medina et al., 1988; Wildmann et al., 1987, 1988; Alho et al., 1989). It has been proposed that the brain BZ might have their origin in a natural component of the diet (Medina et al., 1988; Sangameswaran et al., 1986; Wildmann et al., 1987, 1988). Nevertheless, the biological and/or clinical relevance of these findings needs to be assessed. For this purpose we have mapped more extensively the distribution of BZIi in the rat brain and compared it with the distribution of the GABAR/BZR complex in some areas of the brain. We have found higher densities of BZli in the Purkinje cell layer of the cerebellum, in the stratum
114 M.P. Shnchez et al.
I III
A
Fig. 1 a-d.
pyramidale of the hippocampus, in the mitral cell layer of the olfactory bulb and in the primary olfactory cortex. In contrast, lower levels of BZIi were localized in the globus pallidus and in the strata radiatum, oriens and lacunosum-moleeulare of the hippocampus. It has been shown that BZIi is mostly found in neurons (perikarya and dendritic processes) both in the soluble cytoplasm and also in the cytoplasmic face of the cell membranes (De Bias and Sotelo,
1987; De Bias et al., 1987). In the same study it was reported that both GABAergic and non-GABAergic neurons showed BZli. In addition, their axon terminals remained mostly unlabelled, suggesting that BZ might not be co-released with GABA (De Bias and Sotelo, 1987). Radioligand autoradiography (McCabe and Wamsley, 1986; McCabe et al., 1988; Palacios et al., 1980; Richards et al., 1986; Sieghart and Karobath, 1980; Young and Kuhar, 1979; Young et al., 1981;
Benzodiazepine-like Immunoreactivity in the Rat
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115
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Fig. 1 e-h. Fig. 1. Distribution of BZIi in coronal sections at different levels of the rat brain. Dark regions correspond to areas enriched in BZli. See Table 1 for relative optical density. (A) The olfactory bulb; (B) olfactory tubercle; (C) caudate-putamen and globus pallidus; (D) medial thalamus, hypothalamus and hippocampal formation; (E) posterior thalamus and zona incerta; (F) mammillary bodies, interstitial nucleus of Cajal and nucleus of Darksehewitsch; (G) substantia nigra, superior colliculus, central grey and medial geniculate body; (H) cerebellum. Bar = 2 mm.
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M . P . Sfinchez et al.
Table 1. Relative optical densities of benzodiazepine-like immunoreactivity in the rat brain Olfactory bulb Glomerular layer External plexiform layer Internal granular layer Mitral cell layer
680 465 475 965
Olfactory tubercle Polymorphic layer Pyramidal layer Plexiform layer Islands of Calleja Primary olfactory cortex
490 740 475 650 1000
Basal ganglia Ventral pallidum Caudate-putamen Globus pallidus Claustrum Nucleus aceumbens
445 465 300 535 485
Frontal cortex Lamina I Lamina II Lamina III Lamina IV Lamina V Lamina VI
460 615 640 485 590 520
Hippocampus Stratum oriens Stratum pyramidale Stratum lacunosum-moleculare Stratum radiatum
380 975 360 335
Dentate gyrus Molecular layer Granule cell layer
505 765
Septum Lateralis Medialis
560 415
Bed nucleus stria terminalis
605
Hypothalamus Ventromedial nucleus Dorsomedial nucleus Lateral nucleus Dorsal nucleus Arcuate nucleus
505 540 420 525 855
Thalamus Reticular nucleus Laterdorsal nucleus Ventroposterior nucleus Ventrolateral nucleus Mediodorsal nucleus Paraventficular nucleus Rhomboideus nucleus Posteromedial nucleus Intermediodorsal nucleus Gelatinosus nucleus Medial genieulate nucleus Lateral habenula
470 495 440 470 430 535 585 455 490 470 615 520
Amygdatoid nuclei
580
Mammillary bodies
740
Zona incerta
520
Red nucleus
545
Interpeduncular nucleus
675
Periaqueduetal grey matter
670
Interstitial nucleus of Cajal
760
Nucleus of Darkschewitsch
905
Substantia nigra Pars reticulata Pars compacta
480 580
Superior colliculus Superficial grey layer Optic nerve layer Intermediate grey layer Intermediate white layer
740 575 585 605
Cerebellum Granule cell layer Purkinje cell layer Molecular layer
810 1125 420
Data are the mean values of relative optical densities ( x 10 000) of at least two animals for each brain region. In all cases, SEMs were equal to or less than 10%.
Zezula et al., 1988) and specific m A b s (De Bias et al., 1988; Richards et al., 1987; Vitorica et al., 1988) have been used to study the distribution o f the GABAR/BZR/CI channel complex in the brain. A l t h o u g h there is co-localization o f G A B A and its biosynthetic enzyme glutamic acid decarboxylase, with the G A B A R / B Z R complex in some brain areas, there are also m a n y mismatches (De Bias et al., 1988; H e r k e n h a m , 1987; K u h a r , 1985; Mariani et al., 1987; Mugnaini and Oertel, 1985; Seguela et al., 1984). The localization mismatch between transmitter (or transmitter-synthesizing enzyme) and receptor is a c o m m o n finding reported t h r o u g h o u t the
literature (Herkenham, 1987; Kuhar, 1985). A lack o f correlation between the distribution o f BZIi and that o f the k n o w n G A B A A receptors also appears to be the case. The cerebellum contains several classes o f G A B A A receptors (Fig. 2). This is supported by the differential distribution o f ligand binding sites with different binding capacities (e.g., see M c C a b e et al., 1988; Olsen et al., 1990; Unnerstall et al., 1981), and by recent 'in situ' hybridization studies showing differential cellular expression o f the m R N A coding for the subunits o f the G A B A A receptor in bovine and rat cerebellum (e.g. Siegel, 1988; Wisden et al., 1988; Shivers et al., 1989).
Benzodiazepine-like Immunoreactivity in the Rat
117
GF
A
|
B
)
Fig. 2. Distribution of GABAR/BZR/CI- channel complex in rat cerebellum as revealed by radioligand autoradiography (A-D) and immunocytochemistry (E). (A) [3H]Flunitrazepam binding to BZR. (B) [3H]Muscimol binding to bigh-affmity GABA^ receptor. (C) [35S]TBPSbindingto the chloride channel. (D) [3H]SR95531 bindingto the antagonist high-affinity bindingconformation of the GABAR. (E) GABA^ receptor-like immunoreactivity, with the mAb 62-3G 1 to the receptor complex (De Bias et al., 1988). Bar = 2 ram.
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Fig. 3. Cellular localization of BZIi in the cerebellum. BZIi is present in most cells of the Purkinje and molecular layers. In the granular layer some cells are immunoreactive while the white matter is immunoreactivity-free. Bar = 100 ~m.
,
.
j,
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8
Fig. 4. Comparison of cellular localization of BZli and that of toluidine blue staining in the deeper layers of the somatosensory cortex. (A) BZli distribution in layer VI (Via and VIb) and white matter. (B) Toluidine blue staining in layer VI and white matter. Bar = 100 Hm.
Benzodiazepine-like Immunoreactivity in the R a t
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ABBREVIATIONS USED IN FIGURES Acb Amyg Arc BST CG CI CPu Dk EPI Fr GI GP Gr GrDG ICj IGr InC InG InWh
aecumbens nucleus amygdaloid nuclei arcuate hypothalamic nucleus bed nucleus stria terminalis central grey matter claustrum caudate-putamen nucleus nucleus of Darkschewitsch externalplexiform layer of the olfactory bulb frontal cortex giomerular layer of the olfactory bulb globus pallidus granular layer of the cerebellum granular layer of the dentate gyrus islands of Calleja internal granular layer of the olfactory bulb interstitialnucleus of Cajal intermediate grey layer of the superior colliculus intermediate white layer of the superior colliculus
interpeduncular nucleus laterodorsal thalamic nucleus lateral hypothalamic nuclei lateral habenular nucleus lateral septal nucleus layer IV of the frontal cortex layer Via of the somatosensory cortex L VIb layer VIb of the somatosensory cortex MB mammillary bodies mediodorsal thalamic nucleus MD medial geniculate nucleus MG Mi mitral cell layer of the olfactory bulb MolDG molecular layer of the dentate gyrus Mol molecular layer of the cerebellum MS medial septal nucleus optic nerve layer of the Op superior colliculus PO primary olfactory cortex Po posterior thalamic nuclear group IPC LD LH LHb LS L IV L Via
The BZIi was f o u n d mainly in the Purkinje cell layer and in the granular layer o f the cerebellum (Fig. 1H), while the [3H]flunitrazepam binding sites were mainly localized in the molecular layer (Fig. 2A). The binding sites for [3H]muscimol, [35S]TBPS, [3H]SR 95531 and m A b 62-3G1 were mostly localized on the granular layer o f the cerebellum (Fig. 2B-E). Nevertheless, there were some regions o f the brain where the distribution patterns o f BZli and B Z R overlapped as for instance, the central (periaqueductal) grey matter, superior and inferior colliculi and mammillary bodies. The distribution o f brain BZli is consistent with the hypothesis that e n d o g e n o u s brain BZ might m o d u l a t e G A B A neurotransmission, by interacting with the G A B A A receptor complex. Nevertheless, brain BZ could also be exogenous natural products taken with the diet and that accumulate in the brain (Medina et al., 1988; S a n g a m e s w a r a n and De Bias, 1985; S a n g a m e s w a r a n et al., 1986; W i l m a n n et al., 1987, 1988). I f that was the case, the brain BZIi could be revealing either the m o d u l a t i o n o f G A B A transmission or, like other dietetic substances, the process o f biotransformation, accumulation and elimination.
ACKNOWLEDGEMENTS We thank Dr Alfonso Fairrn for his help and financial support during the preparation of the manuscript (CSIC/CAICYT grant 154/1985, Spain). We gratefully acknowledge Dr Gonzalo Alvarez-Bolado (I. Cajal, Madrid) for helpful advice, and Christian Waeber, of the Sandoz Pharma Ltd Laboratories (Basel), for help and support.
Purk Py R Rt SNC SNR SuG TuP1 TuPo TuPy VMH VP WM ZI
Purkinje cell layer of the cerebellum stratum pyramidalis of the hippocampus red nucleus reticular thalamic nucleus substantia nigra, pars compacta substantia nigra, pars reticulata superficial grey layer of the superior colliculus plexiform layer of the olfactory tubercle polimorphic layer of the olfactory tubercle pyramidal layer of the olfactory tubercle ventromedial hypothalamic nucleus ventral pallidum white matter zona incerta
This research was supported in part by grant NS 17708 from the National Institute of Neurological and Communicative Disorders and Stroke, USA, to A.L.D.B. during a sabbatical leave in the laboratory of J.M.P.
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Accepted 9 June 1990