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Neurotransmitter receptors in the avian brain. III. GABA-benzodiazepine receptors
Brain Research, 439 (1988) 366-371 Elsevier
366 BRE 22694
Neurotransmitter receptors in the avian brain. III. GABA-benzodiazepine receptors M . M . D i e t l 1, R . C o r t 6 s 2 a n d J . M . P a l a c i o s 1 t Preclinical Research, Sandoz Ltd., Basle (Switzerland) and 2Institute of Pathology, University of Basle (Swiizerland)
(Accepted 29 September 1987) Key words: y-Aminobutyricacid/benzodiazepine receptor; Pigeon brain; C1218872; y-Aminobutyricacid; Basal ganglia; Cerebellum
GABA/benzodiazepine (BZ) receptors were localized in the pigeon brain by in vitro receptor autoradiography using [3]fiunitrazepam as ligand. Highest densities of binding sites were observed in the optic rectum, in the nucleus pretectalis, the nucleus intercoilicularis and the substantia gelatinosa of the spinal cord. Intermediate densities were found in the forebrain, particularly the paleostriatum primitivum, the nucleus rotundus and the cerebellum. Low densities were detected in the midbrain and brainstem. The paleostriatum primitivum and cerebellum were enriched in receptors of the BZ type I, as indicated by their high affinity for compound CL 218872. The addition of GABA resulted in an enhanced binding of the ligand in all brain regions. All these results indicate that GABA/BZ receptors in the pigeon brain present similar pharmaco!ogica!pr,~per!iesand comparable anatomical distributior' to those in mammalian brain.
The amino acid y-aminobutyric acid ( G A B A ) is the most abundant inhibitory neurotransmitter in the vertebrate brain. GABAergic mechanisms have been identified in the brain of lower vertebrates 2°. Biochemical electrophysiologicai and anatomical studies indicate a relationship between benzodiazepines (BZ) and GABAergic transmission. Thus, the physiological actions of G A B A are mediated through several types of G A B A receptors; one of these types contains the recognition site for the anxiolytic drugs BE 19. Binding studies have demonstrated that BZ receptors appear late in the evolutionary scale. BZ binding is absent in the nervous tissue of invertebrates and lower vertebrate species such as the hagfish, appearing first in the higher bony fishes ~3. Some structural differences in the BZ receptor subunits have been reported between fishes and amphibians 7. On the other hand, GABA-stimulated BZ binding has always been found to be associated with the presence of BZ receptors ~°. However, these studies have been carried out in membrane preparations and provide little morphological evidence on the evolution of these receptors in the different brain areas. Neuro-
transmitter receptors can be used as markers for differeat neuronal populations and can be a tool for the study of the phylogeny of chemical transmission systems in the brain 2. In order to obtain more information on GABAergic mechanisms in lower vertebrates, we have examined the distribution of G A B A / B Z receptors in the pigeon brain and compared it with that seen in the mammalian brain. The brains of 18 pigeons (Columba livia, 450-550 g body wt, obtained from S. Abdel'Al, Basel, Switzerland) and 9 rats (Wistar, 200-250 g body wt, from Sandoz Ltd., Basel, Switzerland) were removed immediately after decapitation and rapidly frozen. 10 ~m sections were cut with a microtome-cryostat and mounted onto gelatin-coated slides and stored in a freezer (-20 °C) until used. G A B A / B Z receptors were labeled with [3H]flunitrazepam ([3H]Flu) as previously reported 23.25. Briefly, slide-mounted tissue sections were preincubated first for 40 min and then for 2 min at 4 °C in Tris-HCl buffer (170 mM, pH 7.4) in order to remove endogenous GABA. incubation was carried out in the same buffer containing 2 nM [3H]Fiu (92.3/~Ci/mmol; New
Correspondence: J.M. Palacios, Pr~dinical Research, Sandoz Ltd., CH-4002 Basle, Switzerland.
0006-8993/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)
367 England Nuclear, Dreieich, F.R.G.) for 40 min at 0 °C. Adjacent sections were incubated in the same medium added by 300 nM of the triazolopyridazine compound CL 218872 or 0.1 mM of G A B A . Blanks were generated by adding 2/~M diazepam to the incubation medium. After a brief dipping and a wash of 2 min in ice-cold buffer, a dipping in ice-cold distilled water followed. Finally, the slide-mounted tissue sections were dried under a stream of cold air.
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Autoradiograms were generated by apposing the labeled tissue sections and appropriate tritium standards to a [3H]Ultrofilm (LKB, Sweden). Films were analyzed using a computerized image-analysis system (MCID, Imaging Research, Ontario, Canada). To identify brain areas and nuclei, 20 !~m adjacent sections to those used for autoradiography were stained either with Cresyl violet or for acetylcholinesterase. For the pigeon brain the atlas of Karten and
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Fig. 1. Photomicrographs from autoradiograms illustrating the distribution of [3HIFlu binding in coronal sections of the pigeon brain See text for a detailed description. B~r = 3 him.
368 emp!oyed and the atlas of Paxinos and Watson 16for the rat brain. GABA/BZ receptors as labeled by [3H]FIu were widely and heterogeneously distributed throughout the pigeon brain (Figs. 1, 2). In general, white matter tracts were unlabeled by this ligand. Non-specific binding was not different from the film background. [3H]FIu binding was highest (in the range of 3250-2500 fmol/mg protein) in the tectum opticum, particularly in the stratum griseum et fibrosum superficiale (Fig. 1E; a) and in the nucleus intercodicularis (Fig. 1F; ICo). Several areas presented also high densities of [3H]FIu binding (in the range of 2500-1500 faaol/mg protein) such as the paleostriaturn primitivum (Fig. 1A; PP), the nucleus ectomammiUaris (Fig. 1D; EM), the nucleus pretectalis (Fig. 1D; PT), the internal laminae of the tectum opticum (Fig. 1E; b) and the nucleus semilumaris (Fig. IF; Slu). Intermediate densities of binding (in the range of 1500-1000 fmol/mg protein) were observed in the hippocampus (Fig. 1A; Hp), the hyperstriatum ventrale (Fig. 1A; HV), the paleostriatum augmentatum (Fig. 1A, B; PA), the nucleus lateralis anterior thalami (Fig. 1B; LA), the ventral part of the lateral geniculus (Fig. 1B, C; GLv), the nucleus subhabenularis pars medialis (Fig. 1C; SHM), the nucleus lentiformis mesencephali pars magnocellularis (Fig. 1C; LMmc), the nucleus rotundus (Fig. 1C; Rt), the hypothalamus (Fig. !D; Hy), the nucleus tegmenti pedunculopontinus pars compacta (Fig. IE; TPc), the mesencephalic central grey (Fig. IF" CG) and the substantia gelatinosa of both the trigeminai nucleus and the spinal cord (Fig. 11, J. K; SG). Moderate densities of binding (of 1000-500 fmol/mg protein) were present in the area parahippocampalis (Fig. 1A; APH), some telencephalic areas such as the ectostriatum (Fig. 1A; E) and the archistriatum dorsale (Fig. 1B; Ad). Moderate densities were also seen in the nucleus nervi trochlearis (Fig. IF; nlV), the nucleus olivaris superior (Fig. 1G; OS), the nucleus ti-actus solitarius and the nucleus of the vagus (Fig. 1H; S and nX), the nucleus proprius as well as the central grey of the spinal cord (Fig. 1J, K). Moderate binding densities were also observed in the cerebellum. Here GABA/BZ receptors were present in both the molecular and the granule cell layer, with lower densities in the Purkinje cell layer. The mean cerebellar density was of about 800 fmol/mg protein. H o d o s 9 was
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Fig. 2. Photomicrographs from autoradiograms of horizontal sections of the pigeon brain illustrating the distribution of [~H]Flu binding (A) and [3HIFIu binding in the presence of 300 nM CI 218872 (B) or 10-4 M GABA (C). For abbreviations see text. Bar = 5 mm.
Regional differences existed in the distribution of GABA/BZ receptors in the pigeon cerebellum, thus the anterior and posterior lobes showed slightly higher densities than the intermediate lobe. Low densities of [3H]FIu binding (below 500 fmol/mg protein) were observed in the lateral septum (Fig. 1A; SL), the stratum album centrale of the tectum opticum (Fig. 1E; c), in several regions of the midbrain and brainstem (Fig. 1D-I) and the ventral horn of the spinal cord (Fig. 1J, K). Some characteristics of [3H]FIu binding to mounted tissue sections were examined (Figs. 2, 3, 4). The triazolopyridazine CL 2188722~ added to the
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Fig. 3. Displacement of [3l-llFlu binding by the subtype selective compound CL 218872 in several areas of the pigeon brain. T.he bars represent the remaining binding activity in % of total binding. PA, paleostriatum augmentatum; Tec, tectum opticum: Ad, archistriatum dorsale: Hp, hippocampus; TPc, nucleus tegmenti pedunculopontinus, pars compacta; GLv, ventral part of the :ateral geniculus; SLu. nucleus semilunaris; PT, nucleus pretectalis; Cb. cerebellum; EM. nucleus ectomammillaris; PP, paleostril,tum primitivum.
incubation medium at a concentration of 300 nM resuited in a differential regional blockade of [3H]Flu binding (Fig. 2B and 3). The most sensitive areas were (Fig. 3) the paleostriatum primitivum (PP), the nucleus ectomammillaris (EM), the cerebellum (Cb) and the nucleus pretectalis (PT), while the paleostriatum augmentatum (PA), the tectum opticum, the archistriatum dorsale (Ad) and the hippocampus presented only a very low sensitivity to CL 218872. On the other hand, GABA stimulation of [3H]Flu binding by addition of 100/lM GABA to the incubation medium resulted in an increased [3H]Fiu binding throughout the pigeon brain (Figs. 2C and 4). The increase was proportional to the density of [3H]FIu binding (Fig. 4). Thus, in agreement with previous biochemical studies6"7"1°-13we find that the avian brain contains GABA/BZ receptors. Many similarities with the mammalian brain GABA/BZ receptors are observed regarding their pharmacological characteristics and
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Fig. 4. GABA stimulation of BZ receptor binding in the pigeon brain. The addition of GABA ( 10-4 M) to the incubation medium homogeneousl) increased specific [3H]Flu binding by about 20% throughout the whole brain. The graph illustrates the correlation of this increase in selected brain structures which were measured in at least 3 pigeon brains. Correlation coefficient = 0.92.
regional distribution ='23"25. In the pigeon brain the BZ receptors also seem to be associated to a GABA receptor because they are, as in the mammalian brain, modulated by the addition of GABA to the inccbation medium 22. When compared with that in the rat brain, GABA/BZ receptor distribution in the pigeon brain appears quite similar. Thus, GABA/BZ receptors are observed in the mammalian basal ganglia where the globus pallidus showed high concentrations while the caudate-putamen presented moderate to low densities of receptors; the same was observed in the homologous areas of the pigeon brain, high densities of GABA/BZ receptors in the paleostriatum primitivum and moderate to low densities in the paleostriatum augmentatum 12.14.I5.zs. In addition, the socalled "external striatum" of the pigeon telencephalon (ectostriatum, hyperstriatum, neostriatum, and archistriatum) which is enriched in GABA/BZ receptors, may be homologous, at least in part to the mammalian neocortex 12"14. which is also enriched in GABA/BZ receptors. As in the mammalian brain, parts of the olfactory system such as for example the olfactory bulb, showed high densities of GABA/BZ receptors. Intermediate densities of GABA/BZ re-
370 tors, as it has been shown that not all G A B A A receptors are associated with BZ receptors. On the other hand, another subtype of G A B A receptor, the socalled GABAB receptors had been demonstrated in the rat brain and presents a different regional distribution i. The distribution of these other types of G A B A receptors in the avian brain is unknown at the present time although the presence of high affinity muscimol binding to G A B A A receptors has been demonstrated biochemically in the chick brain 10.17.2..
ceptvrs were observed in the rat as well as in the pigeon hippocampus. Intermediate to moderate binding densities are present in both :he rat and pigeon thalamus and hypothalamus. Also comparable densities of GABA/BZ receptors to those in the mammalian visual pathway are found in the pigeon visual system. Thus, we observe very high densities of GABA/BZ receptors in the optic tectum, a region homologous to the mammalian superior colliculus, and the lateral geniculate body (GLv), as well as intermediate to high binding densities in the nucleus dorsolateralis anterior thalami (DLA), the nucleus rotundus (Rt), the hyperstriati accessorium and dorsale and in the ectostriatum, as already mentioned, areas probably homologous to the mammalian visual cortex 12'~4. Finally, also in the midbrain and brainstem are comparable concentrations found in both species. Thus, for example, the nucleus tegmentopeduncularis pars compacta (TPc) of the pigeon is the area corresponding to the substantia nigra of the rat and presented in both species intermediate densities. The same was observed in the substantia gelatinosa. In conclusion, as for other receptor systems, such as the dopaminergic, muscarinic-cholinergic and others 2-4. the location of GABA/BZ receptors appears to be preserved during evolution in homologous areas. The preservation of the G A B A / B Z system agrees well with the studies on GABAergic pathways in the pigeon brain. Like in the mammalian striatonigral system, G A B A seems to be a neurotransmitter in the corresponding striatotegmental system of the avian brain 5. Another system where a role for GABA as a neurotransmitte: is documented is the visual pathway. High densities of G A B A had been found in the optic tectum with corresponding similar high densities of GABA and G A D in the mammalian superior colliculus 8. However, G A B A / B Z receptors represent only a subtype of G A B A A recep-
The similarities in the pharmacological characteristics and the anatomical distribution suggest that benzodiazepines could exert pharmacological effects in the pigeon brain which should be comparable to those reported in mammals. Although these effects have not been examined in detail, a recent report 24 indicates that benzodiazepine administration can modify behavior in pigeons. Interestingly, the effects of the benzodiazepine antagonist Ro 15-1788 in pigeons appear to differ from those seen in mammals.
1 Bowery, N.G., Hudson, A.L. and Price, G.W., GABA A and GABAa receptors site distribution in the rat central nervous system, Neuroscience, 20 (1987) 365-383. 2 Dietl, M.M. and Palacios, J.M., Receptor autoradiography as a tool for the study of the phylogeny of the basal ganglia, J. Rec. Res., in press. 3 Dietl, M.M. and Palacios, J.M., Neurotransmitter receptors in the avian brain. I. Dopamine receptors, Brain Research, 439 (1988) 354-359. 4 Dietl, M.M., Cort6s, R. and Palacios, J.M., Neurotrans-
mitter receptors in the av'~anbrain. II. Muscariniccholinergic receptors. Brain Research, 439 (1988) 360-365. 5 Hall. K.. Brauth. S. and Kitt. C.A., Retrograde transport ,f I3H]GABA in the st6atotegmental system of the pigeon, ~,ain Research, 3 i0 (1984) 157-163. 6 ~rebebrand. J.. Friedl. W.. Unverzagt. B. and Propping. P.. Benzodiazepine receptor subunits in avian brain. J. Neurochem., 47 (1986) 790-793. 7 Hebebrand. J.. Friedl, W.. Breidenbach, B. and Propping, P.. Phylogenetic comparison of the photoaffinity-labeled
These two subtypes of the G A B A / B Z receptors have been differentiated on the basis of the differential affinities of compounds such as the triazolopy-. ridazine CL 218872 (ref. 23). In the mammalian brain these two subtypes present marked differences in their regional distribution 23-26, Our results in the pigeon brain indicate that these differences are already present in the avian brain. Thus, CL 218872 preferentially inhibited the binding of [3H]Fiu to the cerebellum and the paleostriatum primitivum, while the binding of i'H]Flu to the hippocampus or the optic tectum was less affected. These results agree very well with the preferential enrichment of BZ Type I sites (CL 218872-sensitive) in the mammalian cerebellum and globus pailidus and of Type II BZ sites in the mammalian hippocampus and superior colliculus26.
371 benzodiazepine receptor subunits, J. Neurochem., 48 (1987) 1103-1108. 8 Henkc, H. and Fonnum, F., Topographical and subcellular distribution of choline acetyltransferase and glutamate decarboxylase in pigeon optic tectum, J. Neurochem., 27 (1976) 387-391. 9 Karten, H.J. and Hodos, W., A Stereotaxic Atlas of the Brain of Pigeon (Columba livia), Johns Hopkins Press, Baltimore, 1967. 10 Maggi, A., Satinover, J., Oberdorfer, M., Mann, E. and Enna, S.J., Phylogenetic characteristics of muscimol-actirated benzodiazepine receptor binding, Brain Res. Bull., 5 (1980) 167-171. 11 Mann, E. and Enna, S.J., Phylogenetic distribution of bicuculine-sensitive 7-aminobutyric acid (GABA) receptor binding, Brain Research, 184 (1980) 367-373. 12 Nauta, W.J.H. and Karten, H.J., A general profile of ~Le vertebrate brain, with side lights on the anthestry of cerebral cortex. In F.O. Schmitt (Ed.), The Neurosciences: Second Study Program, Rockefeller University Press, New York, 1970, pp. 7-26. 13 Nielsen, M., Braestrup, C. and Squires, R.F., Evidence for a late evolutionary appearance of brain-specific benzodiazepine receptors: an investigation of 18 vertebrate and 5 invertebrate species, Brain Research. 141 (1978) 342-346. 14 Northcutt, R.G., Evolution of the telencephalon in nonmammals, Annu. Rev. Neurosci., 4 (1981)301-350. 15 Parent, A. and Oliv'.'er, A., Comparative histochemicai study of the corpus striatum, J. Hirnforsch., 12 (1970) 73-81. 16 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinate~, Academic, New York, 1982. 17 Ramirez, G., Barat, A., Gomez-Barriocanal, J., Manrique, E. and Batuecas, A., Development of specific binding sites for [3H]kainic acid and [-~H]muscimol in the chick optic
tectum: mod~¢iation by earl}' cha, 2es in vis:3al input. In P. Mandei and F.V. DeFedis (kds. ~. CN5 rc.,c:ep:,,,;:~ -- From Molecular Pharmacology to Beha~ior, Raven, New York, 1983, pp. ! 87- i 97. 18 Reiner, A., Brauth, S.E. and Karten, H.J., Evolution of the amniote basal ganglia, Trends Neurosci., 7 (1984) 320 -325. 19 Richards, J.G., M6hler, H. and Haefely, W., Benzodiazepine receptors and their ligands. In C.N. Woodruff (Ed.), Mechanisms of Drug Action, Macmillan, London, 1986, pp. 131-176. 20 Roberts, E., Chase, T.N. and Tower, D.B., G A B A in Nervous System Function, Raven, New York, 1976. 21 Stewart, M.G. and Bourne, R.C., Ontogeny of [3H]muscimol binding to membranes of chick forebrain, Exp. Brain Res., 65 (1987) 707-710. 22 Unners~all, J.R., Kuhar, M.J., Niehoff, D.L. and Palacios, J.M., Benzodiazepine receptors are coupled to a subpopulation of GABA receptors: evidence from a quantitative autoradiographic study, J. Pharmacol. Exp. Ther., 211~; (1981) 787-804. 23 Unnerstall, J.R., Niehoff, D.L., Kuhar, M.J. and Palacios, J.M., Quantitative receptor autoradiography using [3H]UItrofilm: Application to multiple benzodiazepine receptors, J. Neursci. Meth., 6 (1982) 59-73. 2/1 Witkin, J.M. and Barrett, J.E., Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (Flumazepil) in pigeon, Life Sci., 37 (1985) 1587-1595. 25 Young III, W.S. and Kuhar, M.J., Radiohistochemical localization of benzodiazepine receptors in rat brain, J. Pharmacol. Exp. Ther., 212 (1980) 337-346. 26 Young III, W.S., Niehoff, D., Kuhar, M.J., Beer, B. and Lippa, A.S., Multiple benzodiazepine receptor localization by light microscopic radiohistochemistry, J. Pharmacol. Exp. Ther.. 216 (1981) 425-430.
366 BRE 22694
Neurotransmitter receptors in the avian brain. III. GABA-benzodiazepine receptors M . M . D i e t l 1, R . C o r t 6 s 2 a n d J . M . P a l a c i o s 1 t Preclinical Research, Sandoz Ltd., Basle (Switzerland) and 2Institute of Pathology, University of Basle (Swiizerland)
(Accepted 29 September 1987) Key words: y-Aminobutyricacid/benzodiazepine receptor; Pigeon brain; C1218872; y-Aminobutyricacid; Basal ganglia; Cerebellum
GABA/benzodiazepine (BZ) receptors were localized in the pigeon brain by in vitro receptor autoradiography using [3]fiunitrazepam as ligand. Highest densities of binding sites were observed in the optic rectum, in the nucleus pretectalis, the nucleus intercoilicularis and the substantia gelatinosa of the spinal cord. Intermediate densities were found in the forebrain, particularly the paleostriatum primitivum, the nucleus rotundus and the cerebellum. Low densities were detected in the midbrain and brainstem. The paleostriatum primitivum and cerebellum were enriched in receptors of the BZ type I, as indicated by their high affinity for compound CL 218872. The addition of GABA resulted in an enhanced binding of the ligand in all brain regions. All these results indicate that GABA/BZ receptors in the pigeon brain present similar pharmaco!ogica!pr,~per!iesand comparable anatomical distributior' to those in mammalian brain.
The amino acid y-aminobutyric acid ( G A B A ) is the most abundant inhibitory neurotransmitter in the vertebrate brain. GABAergic mechanisms have been identified in the brain of lower vertebrates 2°. Biochemical electrophysiologicai and anatomical studies indicate a relationship between benzodiazepines (BZ) and GABAergic transmission. Thus, the physiological actions of G A B A are mediated through several types of G A B A receptors; one of these types contains the recognition site for the anxiolytic drugs BE 19. Binding studies have demonstrated that BZ receptors appear late in the evolutionary scale. BZ binding is absent in the nervous tissue of invertebrates and lower vertebrate species such as the hagfish, appearing first in the higher bony fishes ~3. Some structural differences in the BZ receptor subunits have been reported between fishes and amphibians 7. On the other hand, GABA-stimulated BZ binding has always been found to be associated with the presence of BZ receptors ~°. However, these studies have been carried out in membrane preparations and provide little morphological evidence on the evolution of these receptors in the different brain areas. Neuro-
transmitter receptors can be used as markers for differeat neuronal populations and can be a tool for the study of the phylogeny of chemical transmission systems in the brain 2. In order to obtain more information on GABAergic mechanisms in lower vertebrates, we have examined the distribution of G A B A / B Z receptors in the pigeon brain and compared it with that seen in the mammalian brain. The brains of 18 pigeons (Columba livia, 450-550 g body wt, obtained from S. Abdel'Al, Basel, Switzerland) and 9 rats (Wistar, 200-250 g body wt, from Sandoz Ltd., Basel, Switzerland) were removed immediately after decapitation and rapidly frozen. 10 ~m sections were cut with a microtome-cryostat and mounted onto gelatin-coated slides and stored in a freezer (-20 °C) until used. G A B A / B Z receptors were labeled with [3H]flunitrazepam ([3H]Flu) as previously reported 23.25. Briefly, slide-mounted tissue sections were preincubated first for 40 min and then for 2 min at 4 °C in Tris-HCl buffer (170 mM, pH 7.4) in order to remove endogenous GABA. incubation was carried out in the same buffer containing 2 nM [3H]Fiu (92.3/~Ci/mmol; New
Correspondence: J.M. Palacios, Pr~dinical Research, Sandoz Ltd., CH-4002 Basle, Switzerland.
0006-8993/88/$03.50 (~ 1988 Elsevier Science Publishers B.V. (Biomedical Division)
367 England Nuclear, Dreieich, F.R.G.) for 40 min at 0 °C. Adjacent sections were incubated in the same medium added by 300 nM of the triazolopyridazine compound CL 218872 or 0.1 mM of G A B A . Blanks were generated by adding 2/~M diazepam to the incubation medium. After a brief dipping and a wash of 2 min in ice-cold buffer, a dipping in ice-cold distilled water followed. Finally, the slide-mounted tissue sections were dried under a stream of cold air.
HV t
A
Autoradiograms were generated by apposing the labeled tissue sections and appropriate tritium standards to a [3H]Ultrofilm (LKB, Sweden). Films were analyzed using a computerized image-analysis system (MCID, Imaging Research, Ontario, Canada). To identify brain areas and nuclei, 20 !~m adjacent sections to those used for autoradiography were stained either with Cresyl violet or for acetylcholinesterase. For the pigeon brain the atlas of Karten and
i
PP
X C
61 .•,.~ •
H
,,k
P J
•
Fig. 1. Photomicrographs from autoradiograms illustrating the distribution of [3HIFlu binding in coronal sections of the pigeon brain See text for a detailed description. B~r = 3 him.
368 emp!oyed and the atlas of Paxinos and Watson 16for the rat brain. GABA/BZ receptors as labeled by [3H]FIu were widely and heterogeneously distributed throughout the pigeon brain (Figs. 1, 2). In general, white matter tracts were unlabeled by this ligand. Non-specific binding was not different from the film background. [3H]FIu binding was highest (in the range of 3250-2500 fmol/mg protein) in the tectum opticum, particularly in the stratum griseum et fibrosum superficiale (Fig. 1E; a) and in the nucleus intercodicularis (Fig. 1F; ICo). Several areas presented also high densities of [3H]FIu binding (in the range of 2500-1500 faaol/mg protein) such as the paleostriaturn primitivum (Fig. 1A; PP), the nucleus ectomammiUaris (Fig. 1D; EM), the nucleus pretectalis (Fig. 1D; PT), the internal laminae of the tectum opticum (Fig. 1E; b) and the nucleus semilumaris (Fig. IF; Slu). Intermediate densities of binding (in the range of 1500-1000 fmol/mg protein) were observed in the hippocampus (Fig. 1A; Hp), the hyperstriatum ventrale (Fig. 1A; HV), the paleostriatum augmentatum (Fig. 1A, B; PA), the nucleus lateralis anterior thalami (Fig. 1B; LA), the ventral part of the lateral geniculus (Fig. 1B, C; GLv), the nucleus subhabenularis pars medialis (Fig. 1C; SHM), the nucleus lentiformis mesencephali pars magnocellularis (Fig. 1C; LMmc), the nucleus rotundus (Fig. 1C; Rt), the hypothalamus (Fig. !D; Hy), the nucleus tegmenti pedunculopontinus pars compacta (Fig. IE; TPc), the mesencephalic central grey (Fig. IF" CG) and the substantia gelatinosa of both the trigeminai nucleus and the spinal cord (Fig. 11, J. K; SG). Moderate densities of binding (of 1000-500 fmol/mg protein) were present in the area parahippocampalis (Fig. 1A; APH), some telencephalic areas such as the ectostriatum (Fig. 1A; E) and the archistriatum dorsale (Fig. 1B; Ad). Moderate densities were also seen in the nucleus nervi trochlearis (Fig. IF; nlV), the nucleus olivaris superior (Fig. 1G; OS), the nucleus ti-actus solitarius and the nucleus of the vagus (Fig. 1H; S and nX), the nucleus proprius as well as the central grey of the spinal cord (Fig. 1J, K). Moderate binding densities were also observed in the cerebellum. Here GABA/BZ receptors were present in both the molecular and the granule cell layer, with lower densities in the Purkinje cell layer. The mean cerebellar density was of about 800 fmol/mg protein. H o d o s 9 was
8
A ,
i ......
,,_._...__
Fig. 2. Photomicrographs from autoradiograms of horizontal sections of the pigeon brain illustrating the distribution of [~H]Flu binding (A) and [3HIFIu binding in the presence of 300 nM CI 218872 (B) or 10-4 M GABA (C). For abbreviations see text. Bar = 5 mm.
Regional differences existed in the distribution of GABA/BZ receptors in the pigeon cerebellum, thus the anterior and posterior lobes showed slightly higher densities than the intermediate lobe. Low densities of [3H]FIu binding (below 500 fmol/mg protein) were observed in the lateral septum (Fig. 1A; SL), the stratum album centrale of the tectum opticum (Fig. 1E; c), in several regions of the midbrain and brainstem (Fig. 1D-I) and the ventral horn of the spinal cord (Fig. 1J, K). Some characteristics of [3H]FIu binding to mounted tissue sections were examined (Figs. 2, 3, 4). The triazolopyridazine CL 2188722~ added to the
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PA Tec Ad Hp TPcGLvSLu PT Cb EM PP
Fig. 3. Displacement of [3l-llFlu binding by the subtype selective compound CL 218872 in several areas of the pigeon brain. T.he bars represent the remaining binding activity in % of total binding. PA, paleostriatum augmentatum; Tec, tectum opticum: Ad, archistriatum dorsale: Hp, hippocampus; TPc, nucleus tegmenti pedunculopontinus, pars compacta; GLv, ventral part of the :ateral geniculus; SLu. nucleus semilunaris; PT, nucleus pretectalis; Cb. cerebellum; EM. nucleus ectomammillaris; PP, paleostril,tum primitivum.
incubation medium at a concentration of 300 nM resuited in a differential regional blockade of [3H]Flu binding (Fig. 2B and 3). The most sensitive areas were (Fig. 3) the paleostriatum primitivum (PP), the nucleus ectomammillaris (EM), the cerebellum (Cb) and the nucleus pretectalis (PT), while the paleostriatum augmentatum (PA), the tectum opticum, the archistriatum dorsale (Ad) and the hippocampus presented only a very low sensitivity to CL 218872. On the other hand, GABA stimulation of [3H]Flu binding by addition of 100/lM GABA to the incubation medium resulted in an increased [3H]Fiu binding throughout the pigeon brain (Figs. 2C and 4). The increase was proportional to the density of [3H]FIu binding (Fig. 4). Thus, in agreement with previous biochemical studies6"7"1°-13we find that the avian brain contains GABA/BZ receptors. Many similarities with the mammalian brain GABA/BZ receptors are observed regarding their pharmacological characteristics and
3H-FLU
Fig. 4. GABA stimulation of BZ receptor binding in the pigeon brain. The addition of GABA ( 10-4 M) to the incubation medium homogeneousl) increased specific [3H]Flu binding by about 20% throughout the whole brain. The graph illustrates the correlation of this increase in selected brain structures which were measured in at least 3 pigeon brains. Correlation coefficient = 0.92.
regional distribution ='23"25. In the pigeon brain the BZ receptors also seem to be associated to a GABA receptor because they are, as in the mammalian brain, modulated by the addition of GABA to the inccbation medium 22. When compared with that in the rat brain, GABA/BZ receptor distribution in the pigeon brain appears quite similar. Thus, GABA/BZ receptors are observed in the mammalian basal ganglia where the globus pallidus showed high concentrations while the caudate-putamen presented moderate to low densities of receptors; the same was observed in the homologous areas of the pigeon brain, high densities of GABA/BZ receptors in the paleostriatum primitivum and moderate to low densities in the paleostriatum augmentatum 12.14.I5.zs. In addition, the socalled "external striatum" of the pigeon telencephalon (ectostriatum, hyperstriatum, neostriatum, and archistriatum) which is enriched in GABA/BZ receptors, may be homologous, at least in part to the mammalian neocortex 12"14. which is also enriched in GABA/BZ receptors. As in the mammalian brain, parts of the olfactory system such as for example the olfactory bulb, showed high densities of GABA/BZ receptors. Intermediate densities of GABA/BZ re-
370 tors, as it has been shown that not all G A B A A receptors are associated with BZ receptors. On the other hand, another subtype of G A B A receptor, the socalled GABAB receptors had been demonstrated in the rat brain and presents a different regional distribution i. The distribution of these other types of G A B A receptors in the avian brain is unknown at the present time although the presence of high affinity muscimol binding to G A B A A receptors has been demonstrated biochemically in the chick brain 10.17.2..
ceptvrs were observed in the rat as well as in the pigeon hippocampus. Intermediate to moderate binding densities are present in both :he rat and pigeon thalamus and hypothalamus. Also comparable densities of GABA/BZ receptors to those in the mammalian visual pathway are found in the pigeon visual system. Thus, we observe very high densities of GABA/BZ receptors in the optic tectum, a region homologous to the mammalian superior colliculus, and the lateral geniculate body (GLv), as well as intermediate to high binding densities in the nucleus dorsolateralis anterior thalami (DLA), the nucleus rotundus (Rt), the hyperstriati accessorium and dorsale and in the ectostriatum, as already mentioned, areas probably homologous to the mammalian visual cortex 12'~4. Finally, also in the midbrain and brainstem are comparable concentrations found in both species. Thus, for example, the nucleus tegmentopeduncularis pars compacta (TPc) of the pigeon is the area corresponding to the substantia nigra of the rat and presented in both species intermediate densities. The same was observed in the substantia gelatinosa. In conclusion, as for other receptor systems, such as the dopaminergic, muscarinic-cholinergic and others 2-4. the location of GABA/BZ receptors appears to be preserved during evolution in homologous areas. The preservation of the G A B A / B Z system agrees well with the studies on GABAergic pathways in the pigeon brain. Like in the mammalian striatonigral system, G A B A seems to be a neurotransmitter in the corresponding striatotegmental system of the avian brain 5. Another system where a role for GABA as a neurotransmitte: is documented is the visual pathway. High densities of G A B A had been found in the optic tectum with corresponding similar high densities of GABA and G A D in the mammalian superior colliculus 8. However, G A B A / B Z receptors represent only a subtype of G A B A A recep-
The similarities in the pharmacological characteristics and the anatomical distribution suggest that benzodiazepines could exert pharmacological effects in the pigeon brain which should be comparable to those reported in mammals. Although these effects have not been examined in detail, a recent report 24 indicates that benzodiazepine administration can modify behavior in pigeons. Interestingly, the effects of the benzodiazepine antagonist Ro 15-1788 in pigeons appear to differ from those seen in mammals.
1 Bowery, N.G., Hudson, A.L. and Price, G.W., GABA A and GABAa receptors site distribution in the rat central nervous system, Neuroscience, 20 (1987) 365-383. 2 Dietl, M.M. and Palacios, J.M., Receptor autoradiography as a tool for the study of the phylogeny of the basal ganglia, J. Rec. Res., in press. 3 Dietl, M.M. and Palacios, J.M., Neurotransmitter receptors in the avian brain. I. Dopamine receptors, Brain Research, 439 (1988) 354-359. 4 Dietl, M.M., Cort6s, R. and Palacios, J.M., Neurotrans-
mitter receptors in the av'~anbrain. II. Muscariniccholinergic receptors. Brain Research, 439 (1988) 360-365. 5 Hall. K.. Brauth. S. and Kitt. C.A., Retrograde transport ,f I3H]GABA in the st6atotegmental system of the pigeon, ~,ain Research, 3 i0 (1984) 157-163. 6 ~rebebrand. J.. Friedl. W.. Unverzagt. B. and Propping. P.. Benzodiazepine receptor subunits in avian brain. J. Neurochem., 47 (1986) 790-793. 7 Hebebrand. J.. Friedl, W.. Breidenbach, B. and Propping, P.. Phylogenetic comparison of the photoaffinity-labeled
These two subtypes of the G A B A / B Z receptors have been differentiated on the basis of the differential affinities of compounds such as the triazolopy-. ridazine CL 218872 (ref. 23). In the mammalian brain these two subtypes present marked differences in their regional distribution 23-26, Our results in the pigeon brain indicate that these differences are already present in the avian brain. Thus, CL 218872 preferentially inhibited the binding of [3H]Fiu to the cerebellum and the paleostriatum primitivum, while the binding of i'H]Flu to the hippocampus or the optic tectum was less affected. These results agree very well with the preferential enrichment of BZ Type I sites (CL 218872-sensitive) in the mammalian cerebellum and globus pailidus and of Type II BZ sites in the mammalian hippocampus and superior colliculus26.
371 benzodiazepine receptor subunits, J. Neurochem., 48 (1987) 1103-1108. 8 Henkc, H. and Fonnum, F., Topographical and subcellular distribution of choline acetyltransferase and glutamate decarboxylase in pigeon optic tectum, J. Neurochem., 27 (1976) 387-391. 9 Karten, H.J. and Hodos, W., A Stereotaxic Atlas of the Brain of Pigeon (Columba livia), Johns Hopkins Press, Baltimore, 1967. 10 Maggi, A., Satinover, J., Oberdorfer, M., Mann, E. and Enna, S.J., Phylogenetic characteristics of muscimol-actirated benzodiazepine receptor binding, Brain Res. Bull., 5 (1980) 167-171. 11 Mann, E. and Enna, S.J., Phylogenetic distribution of bicuculine-sensitive 7-aminobutyric acid (GABA) receptor binding, Brain Research, 184 (1980) 367-373. 12 Nauta, W.J.H. and Karten, H.J., A general profile of ~Le vertebrate brain, with side lights on the anthestry of cerebral cortex. In F.O. Schmitt (Ed.), The Neurosciences: Second Study Program, Rockefeller University Press, New York, 1970, pp. 7-26. 13 Nielsen, M., Braestrup, C. and Squires, R.F., Evidence for a late evolutionary appearance of brain-specific benzodiazepine receptors: an investigation of 18 vertebrate and 5 invertebrate species, Brain Research. 141 (1978) 342-346. 14 Northcutt, R.G., Evolution of the telencephalon in nonmammals, Annu. Rev. Neurosci., 4 (1981)301-350. 15 Parent, A. and Oliv'.'er, A., Comparative histochemicai study of the corpus striatum, J. Hirnforsch., 12 (1970) 73-81. 16 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinate~, Academic, New York, 1982. 17 Ramirez, G., Barat, A., Gomez-Barriocanal, J., Manrique, E. and Batuecas, A., Development of specific binding sites for [3H]kainic acid and [-~H]muscimol in the chick optic
tectum: mod~¢iation by earl}' cha, 2es in vis:3al input. In P. Mandei and F.V. DeFedis (kds. ~. CN5 rc.,c:ep:,,,;:~ -- From Molecular Pharmacology to Beha~ior, Raven, New York, 1983, pp. ! 87- i 97. 18 Reiner, A., Brauth, S.E. and Karten, H.J., Evolution of the amniote basal ganglia, Trends Neurosci., 7 (1984) 320 -325. 19 Richards, J.G., M6hler, H. and Haefely, W., Benzodiazepine receptors and their ligands. In C.N. Woodruff (Ed.), Mechanisms of Drug Action, Macmillan, London, 1986, pp. 131-176. 20 Roberts, E., Chase, T.N. and Tower, D.B., G A B A in Nervous System Function, Raven, New York, 1976. 21 Stewart, M.G. and Bourne, R.C., Ontogeny of [3H]muscimol binding to membranes of chick forebrain, Exp. Brain Res., 65 (1987) 707-710. 22 Unners~all, J.R., Kuhar, M.J., Niehoff, D.L. and Palacios, J.M., Benzodiazepine receptors are coupled to a subpopulation of GABA receptors: evidence from a quantitative autoradiographic study, J. Pharmacol. Exp. Ther., 211~; (1981) 787-804. 23 Unnerstall, J.R., Niehoff, D.L., Kuhar, M.J. and Palacios, J.M., Quantitative receptor autoradiography using [3H]UItrofilm: Application to multiple benzodiazepine receptors, J. Neursci. Meth., 6 (1982) 59-73. 2/1 Witkin, J.M. and Barrett, J.E., Behavioral effects and benzodiazepine antagonist activity of Ro 15-1788 (Flumazepil) in pigeon, Life Sci., 37 (1985) 1587-1595. 25 Young III, W.S. and Kuhar, M.J., Radiohistochemical localization of benzodiazepine receptors in rat brain, J. Pharmacol. Exp. Ther., 212 (1980) 337-346. 26 Young III, W.S., Niehoff, D., Kuhar, M.J., Beer, B. and Lippa, A.S., Multiple benzodiazepine receptor localization by light microscopic radiohistochemistry, J. Pharmacol. Exp. Ther.. 216 (1981) 425-430.