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Neurotransmitter receptors in the avian brain. I. Dopamine receptors
Brain Research. 439 (1988) 354-359 Elscvicr
354 BRE 22692
Neurotransmitter receptors in the avian brain. I. Dopamine receptors M.M. Dietl and J.M. Palacios Preclinical Research, Sandoz Ltd., Basle (Switzerland)
(Accepted 29 September 1987) Key words." Dopamine receptor; D~ receptor; D 2 receptor; In vitro; autoradiography; pigeon brain; Basal ganglia
The distribution of dopamine Dj and D, receptors was investigated in the pigeon brain using in vitro receptor autoradiography. D~ receptors were labeled in slide-mounted tissue sections with [3H]SCH 23390. The agonist [3H]CV 205-502 and the antagonist [3H]spiroperidol were used to label D 2 receptors. The highest densities of both D~ and D 2 receptors were concentrated in the paleostriatum augmentatum, lobus paraolfactorius and olfactory tubercle. D~ receptors were in addition enriched in the entire pigeon telencephalon with exception of the ectostriatum. In contrast, no significant densities of D 2 receptors were observed in the pallium. D_,but not D~ receptors were present in the tectum and nucleus pretectalis. The cerebellum presented D~ but not D 2 receptors. These results demonstrate that in the avian brain, like in the mammalian one, both dopamine D~ and D, receptors are concentrated in "striatal" areas, while the telencephalon is rich in D~ but not D2 receptors. The neurotransmitter, dopamine (DA), plays important roles in the regulation of the activity of the mammalian brain particularly of the basal ganglia 1° Dopamine, on the other hand, appears to be present in the nervous system of primitive animals and has been extensively investigated in the brain of the lower vertebrates including birds and reptiles tl In the mammalian brain the physiological effects of D A are mediated through the interaction of the amine with two types of receptors, one linked in a positive manner to an adenylate cyclase, the Di type, and a second one negatively linked or unlinked to this enzyme, the D, type ~3. Radiolabeled ligands for these two types of receptors are available and have been used in their biochemical characterization 5"23 as well as in the autoradiographic investigation of their distribution Is. The autoradiographic studies have revealed that the two subtypes of the D A receptor are enriched in the basal ganglia, particularly in the nucleus caudatus-putamen. D~ and D 2 receptors are also localized in other brain areas where they present different distributions: for example, the D~ receptor being more enriched in the substantia nigra (SN) pars reticulata and the D 2 receptor in the SN pars compacta
and ventral tegmental area 3"6. Using receptor autoradiography we have detected significant differences among the distributions of these two types of receptors in the brain of several mammals (Camps et al., in preparation). In order to gain more information on the evolutionary aspects of the differential distribution of D A D~ and D2 receptors we have examined the distribution of these receptors in the brain of lowc,- vertebrates. We focused particularly on the bird ;: ain because the distribution of dopaminergic innervation in this species is known ~'9"14"1~and because recent studies on the chemoarchitecture of the basal ganglia of these anil;~als h~,.s revealed similarities between the location a~~d connection of their basal ganglia and that of the mammalian (see ref. 20). The brains of 12 adult male pigeons (Columba livia, 450-550 g bo~iv wt., provided by S. A b d e l ' A l , Basel, Switzerland) and 9 adult r.~:,te rats (Wistar, 200-250 g body w,t., from Sandoz Ltd.. Basel, Switzerland) were remt~-~ed immediately after decapitation and rapidly frozen. 10 Itm sections were cut with a cryostat and mounted onto gelatin-coated slides and stored in a freezer (-20 °C) until used. For the labeling of D~ receptors the tissue sections
Correspomlence: J.M. Palacios, Preclinical Research, Sandoz Ltd., CI-t-4002 Basle, Switzerland.
1111116-899388/$113.511© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
355 were incubated with 1 nM of [sH]SCH 23390 (63.2 Ci/mmol; New England Nuclear, Dreieich, F.R.G.) in 50 mM Tris-HCl, 120 mM NaCI, 5 mM KCI, 2 mM CaCi 2, 1 mM MgCI2 (pH 7.4) for 45 min at room temperature 6. To determine the non-specific binding adjacent sections were incubated in the same medium in the presence of 10-6 M cis-flupentixoi. After incubation sections were washed for 5 min in fresh cold buffer and dried under a stream of cold air. For the labeling of D2 receptors the tissue sections were incubated either with the agonist [3H]CV 205-502 (107 Ci/mmol; synthesized and labeled by Dr. R. Voges, Biopharmaceutical Dept., Sandoz Ltd., Basle, Switzerland) 3 or with the antagonist [3H]spiroperidol (22 Ci/mmol; New England Nuclear, Dreieich, F.R.G.) 17. When using the agonist, sections were preincubated in 170 mM Tris-HCl buffer (pH 7.5) for 30 min and then incub::ted for 90 min with 1 nM [3H]CV 205-502 in the same buffer at room temperature. To determine the non-specific binding adjacent sections were incubated in the same solution in the presence of 1 aM of (+)-butaclamol. After two washes in ice-cold buffer of 1 min each and dipping in icecold distilled water, slides were dried under a stream of cold air. When using the antagonist tissue sections were incubated for 30 min with 0.4 nM [3H]spiroperidol in 170 mM Tris-HCi buffer (pH 7.5) containing 120 mM NaCI, 5 mM KCI, 2 nM CaCI.,, 1 nM MgC! at room tempcrature. Then incuoation was followed by two washes of 5 min each in the same buffer at 4 °C. dipped in cold distilled water and dried as mentioned. To block [SH]spiroperidol binding sites other than D2 binding sites, 1 uM ketanserin or haloperidoi was added to the incubation medium. Blanks for these D, binding sites were obtained using 1 !~M of the dopamine D 2 antagonist sulpiride. Autoradiograms were generated by apposing the labeled tissue sections and appropriate radioactive standards to a tritium-sensitive film (LKB, Sweden). Films were analyzed using a computerized imageanalysis system (MCID, Imaging Research, Ontario, Canada). For anatomical localization of the labeled regions the atlas of the pigeon of Karten and Hodos ~2 was employed. Both D t and D 2 receptor binding sites were found in the pigeon brain. The binding of [3H]SCH 23390 was completely blocked by cis-flupentixol, but not affected by spiperone. On the other hand, the binding
of [3H]CV 205-502 and that of [SH]spiroperidol were blocked by butaclamoi but were not affected by ke~anserin or SCH 23390. Dopamine D~ and D2 receptors presented a highly heterogeneous distribution in the pigeon brain (Fig. 1). D! receptors were concentrated in the pigeon forebrain, where the highest densities of DI binding sites were seen in the neocortex, the hyperstriatum ventrale and accessorius, and in the dorsal part of the archistriatum. The densities of receptors in these regions as estimated by computer-assisted microdensitometry was about 250 fmol/mg protein. Also rich in binding sites for [3H]SCH 23390 were the lobus paraolfactorius, the paleostriatum augmentatum, the nucleus accumbens, the neostriatum, and the nucleus superficialis parvicellularis; in these areas the density of D~ receptors was between 150 and 200 fmol/mg protein. In contrast, areas such as the paleostriatum primitivum, the layers of the tectum opticum, and the cerebellum presented much lower receptor densities in the range of 50 fmol/mg protein. No specific binding was detected in the ectostriatum. When compared with homologous regions of the rat brain incubated and measured in para!!el, in ~he pigeon brain the density was of about 10-fold lower. The rat caudate-putamen, for example, presented densities in the range of 2500-3000 fmol/mg protein, the globus pallidus of about 1500 fmol/mg protein, and the layers of the superior colliculus of about 300 fmol/mg protein. D2 receptors as labeled with the high affinity agonist. [sH]CV 205-502, were also highly heterogeneously distributed in the pigeon brain (Fig. 2). The highest densities of D, binding sites were seen in the basal ganglia, particularly in the paleostriatum augmentatum, the iobus paraolfactorius, the olfactory tubercle and the nucleus accumbens. In these areas the densities of D 2 receptors were in the range of 200-250 fmol/mg protein. In contrast, the density !a the paleostriatum primitivum was only of about 10 fmol/mg protein. Again, when compared with regions of equivalent ,,ize in the rat brain, the densities of D_, receptors were much lower in the pigeon brain although only by half. Densities of [3H]CV 205-502 binding sites in the caudate-putamen of the rat brain as examined in the same experiences as the pigeon brains were of about 500-600 fmoi/mg protein. In the globus pallidus of the ra.t the density of D 2 receptors
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Fig. 1. Photomicrographs from autoradiograms illustratipg the distribution of doamine D I receptors in the pigeon brain as labeled by [~H]SCH 23390. A,B,C are coronal sections, D is a sagittal and E a horizontal section; the hyperstriatum ventrale (HV), the cerebellum (Cb) and the layers of the rectum opticum (St) show an important labeling of D I receptors, but highest densities are observed in the paleostriatum augmentatum (PA) and the Iobus paraolfactoriu~ (I+PO) as well as in the nucleus superficialis parvicellularis (SPC). No binding is present in the paleostriatum primitivum (PP). Other abbreviations in Figs. I and 2: AHP, area parahippocampalis; Cx, neocortex; DLA, nucleus dorsolateralis anterior thalami pars lateralis; E, ectostriatum; Hy, hypothalamus; PT, nucleus pretectalis; SL, nucleus septalis laterals; TO, tuberculum olfactorium. Bar = 1 mm.
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Fig. 2. Photomicrographs from autoradiograms illustrating the distribution of dopamine D., receptors in the pigeon brain as labeled bv [3H]CV 205-502. A,B,C are coronal, D sagittal, and E horizontal sections. Highest densities are observed it, the paleostriatum augmentatum (PA), the Iobus paraolfactorius (LPO), and olfactory, tubercle (TO). Some binding is present in the lateral part of the nucleus dorsolateralis anterior thalami (DLA). the nucleus pretectalis (PT), and the layers of the tectum opticum (St). No binding is observed in the paleostriatum primitivum (PP). Bar = 1 mm.
358 was about 250 fmol/mg protein. In the pigeon brain other areas showed densities of lower than 50 fmol/mg protein. "lhus, the nucleus dorsolateralis anterior of the thalamus was highest with 50 fmol/mg protein, the nucleus superficialis parvicellularis had 30 fmol/mg protein, the layers of the optic tectum and the nucleus pretectalis showed about 15 fmoi/mg protein and regions like the neocortex, the striatal areas and the cerebellum showed lower than 10 fmol/mg protein. [sH]Spiperone labeling resulted in a similar distribution of sites (results not shown). No or very low densities of either D 1 or D 2 receptors were observed in the midbrain and brainstem. Regarding the nucleus tegmenti pedunculo-pontinus pars compacta (TPc) we did not observe significant densities of either D~ or D2 receptors in this area equivalent to the substantia nigra pars compacta of the mammalian brain 1"2"8"9"2~. This is probably not surprising because of the lower densities of dopamine receptors in the pigeon brain as compared to the rat brain. As the substantia nigra in the rat does not contain very high densities of D 2 receptors and only intermediate densities of D l receptors, it is possible that the presence of dopamine receptors in this area of the pigeon brain escape to the sensitivity of our technique. Similar results were observed in the chick brain (not shown). Thus, one of the main findings of this study is that the highest densities of dopamine receptors in the avian brain are localized in parts of the paleostriatal complex of the pigeon brain, the paleostriatum augmentatum and the Iobus paraoifactorius but not the paleostriatum primitivum. Embryological, cytoarchitectonical and histochemical criteria indicate that the paleostriatum complex of birds and reptiles can be compared to the striatal complex of the mammals, i.e. the paleostriatum augmentatum and the lobus paraolfactorius corresponding to the nucleus caudate-putamen and the paleostriatum primitivum corresponding to the giobus pallidus of the mammals (see ref. 20 for review). Interestingly, as in mammals, in the bird brain dopamine receptors appear to be concentrated in the striatum rather than in the globus pallidus. Furthermore, this appears as a general feature of the amniote brain, i.e. modern birds, reptiles and mammals7. Dopamine D~ but not dopamine D2 receptors are present in hyperstriatae areas. Comparative anatomical studies have indicated that,
in spite of clear differences in the development of the avian and reptilian pallidum and the mammalian isocortex, there are organizational similarities between portions of the avian telencephalon and the mammalian isocortex ~s. In this sense it is interesting that, while the mammalian cortex contains significant densities of the D t receptors and low or very low densities of the D2 receptors, similarly the telencephalon of the pigeon brain contains Dt but not D 2 receptors. Our autoradiographic results are in good agreement with previous biochemical and histochemical results on the presence of dopamine and dopamine receptors in the avian brain 4"8"9"1zA4"19. Coveili et al. 4 have examined the presence of dopamine receptors using binding techniques in different invertebrate and vertebrate species and found that the avian brain contains dopamine D 2 receptors. On the other hand, the distribution of dopamine in the avian brain has been examined histochemically8"9'14a9 and found to correlate well with the presence of dopamine D2 receptors, in the sense that the paleostriatum augmentatum, the lobus paraolfactorius and the olfactory tubercle are richly innervated with dopaminergic terminals which appear to emanate from an area the TPc which has been proposed as homologous to the mammalian substantia nigra 1"2"8"9"-~. In a similar way to that observed in the mammalian brain, the hyperstriatal areas are poorly innervated by dopamine terminals'~'~4"~9; however, they contain significant densities of D~ receptors. These results strengthen again the above-mentioned similarities between the distribution of dopamine D~ and D 2 receptors in the avian and in the mammalian brain. Other regions like the layers of the optic tectum and the cerebellum represent further examples to support the similarity of the avian and the mammalian brain as we found comparable densities of D 1 and D 2 receptors in the rat superior colliculus and cerebellum 3"6. Our results provide a morphological basis for the neuropharmacological effects of dopaminergic agents in avian behavior and biochemistry. Several studies have shown that the paleostriatum is the site of action for the effects of dopaminergic agonists and antagonists in stereotyped and motor behavior in bii-ds. In addition, paleostriatal lesions decrease these effects while lesions of the neostriatum and hyperstriatum did not influence these actions m'2~'22. These studies, however, have been limited until now
359 to dopamine D 2 agents; we are not aware of any study involving selective D~ agents. In summary, our autoradiographic results have illustrated the distribution of dopamine receptors in the avian brain. The main conclusion of this study is that evolution appears to be conservative with regard
to the presence and distribution of dopaminergic postsynaptic markers in the basal ganglia as well as other regions. These studies provide anatomical support for further investigations on the functional role of doparni,,,,e receptors in the avian brain.
1 Brauth, S.E., Perguson, J.L. and Kitt, C.A., Prosencephalic pathways related to the paleostriatum of the pigeon (Columba livia), Brain Research, 147 (1978) 205-221. 2 Brauth, S.E., Reiner, A., Kitt, C.A. and Karten, H.J., The substance P-containing striatotegmental path in reptiles: an immunohistochemical study, J. Comp. Neurol., 219 (1983) 305-327. 3 Charuchinda, C., Supavilai, P., Karobath, M. and Palacios, J.M., Dopamine D_, receptors in the rat brain: autoradiographic visualization using a high affinity selective agonist ligand, J. Neurosci., in press. 4 CoveUi, V., Memo, M., Spano, P.F. and Trabucchi, M., Characteriz~,tion of dopamine receptors in various species of invertebrates and vertebrates, Neuroscience, 6 (1981) 2077-2079. 5 Creese, I., Sibley, D.R., Hamblin, M.W. and Left, S.E., The classification of dopamine receptors: relationship to radioligand binding, Annu. Rev. Neurosci., 6 (1983) 43-7i. 6 Dawson, T.I~, Gehlert, D.R.. McCabe, R.T., Barnett, A. and Wamsley, J.K., D I dopamine receptors in the rat brain: a quan~ita~.ive autoradiographic analysis, J. Neurosci., 6 (1986) 2352-2365. 7 Dietl, M. and Palacios, J.M., Receptor autoradiography as a tool for the study of the phylogeny of the basal ganglia, J. Ree. Res., in press. 8 Dube, L. and Parent, A., The monoamine-containing neurons in avian brain. I. A study of the brainstem of the chicken (Gallus domesticu~) by means of fluorescence and acetylcholinesterase histochemistry, J. Comp. Neurol., 196 (1981) 695-708. 9 Fuxe, K. and Ljunggren, L., Cellular localization of the monoamines in the upper brainstem of the pigeon, J. Comp. Neurol., 148 (1965) 61-90. 10 Hornykiewicz, O., Dopamine and its physiological significance in brain function. In The Structure and Function of Nervous Tissue, Vol. 6, Academic, New York, 1972, pp. 367-415. 11 Juorio, A.V. and Vogt, M., Monoamines and their metabolites in the avian brain, J. Physiol. tLond.L 189 (1967) 489-518.
12 Karten, H.J. and Hodos, W., A Stereotaxic Atlas of the Brain of Pigeon (Columba livia), Johns Hopkins Press, Baltimore, 1967. 13 Kebabian, J.W. and Calne, D.B., Multiple receptors for dopamine, Nature (Lond. ~, 227 (1979) 93-96. 14 Kiss. J.Z. and P6czely, P., Distribution of tyrosine-hydro×ylase (TH) immunoreactive neurons in the diencephaIon of the pigeon ( Columba livia domestica), J. Comp. Neurol., 257 (1987) 333-346. 15 Nauta, W.J.H. and Karten. H.J., A general profile of the 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. i0 Nistico'. G. and Stcphenson. J.D., Dopamine~-gic mechanisms and stereotyped behavior in birds, Pharmacol. Res. Commun., 11 (1979) 555-570. 17 Palacios, J.M., Niehoff. D.L. and Kuhar, M.J., [3H]Spiperone binding sites in brain" autcradiographic localization of multiple receptors, Brain Research, 213 (1981) 277-289. 18 Palacios, J.M. and Pazos, A., Visualization of dopamine receptors: a progress review. In I. Creese and C. Frazer, (Eds.), Receptor Biochenlistrv and Methodology, I/ol. 9, Structure and Function of Dopamine Receptors, Alan R. Liss, New York, 1987. in press. 19 Reiner, A., Karten, H.J. aod Solina, A.R.. Substance P: localization within paleostriatal-tegmental pathways in the pigeon, Neuroseience, 9 (1983) 61-85. 20 Reiner, A., Brauth, S.E. and Karten, H.J., Evolution of the amniote basal ganglia, Trends Neurosci., 7 (1984) 32O-325. 21 Rieke, G.K.. The TPc, the avian substantia nigra: pharmacology and behavior, Physiol. Behav., 28 (1982) 755-763. 22 Sandberg, P.R. and Mark, R.F.. The effect of striatal lesions in the chick on haloperidol-potentiated tonic immobility, Neuropharmacology, 22 (1983) 253-257. 23 Seeman, P., Brain dopamine receptors. Pharmacol. Rev., 32 (1981) 229-313.
354 BRE 22692
Neurotransmitter receptors in the avian brain. I. Dopamine receptors M.M. Dietl and J.M. Palacios Preclinical Research, Sandoz Ltd., Basle (Switzerland)
(Accepted 29 September 1987) Key words." Dopamine receptor; D~ receptor; D 2 receptor; In vitro; autoradiography; pigeon brain; Basal ganglia
The distribution of dopamine Dj and D, receptors was investigated in the pigeon brain using in vitro receptor autoradiography. D~ receptors were labeled in slide-mounted tissue sections with [3H]SCH 23390. The agonist [3H]CV 205-502 and the antagonist [3H]spiroperidol were used to label D 2 receptors. The highest densities of both D~ and D 2 receptors were concentrated in the paleostriatum augmentatum, lobus paraolfactorius and olfactory tubercle. D~ receptors were in addition enriched in the entire pigeon telencephalon with exception of the ectostriatum. In contrast, no significant densities of D 2 receptors were observed in the pallium. D_,but not D~ receptors were present in the tectum and nucleus pretectalis. The cerebellum presented D~ but not D 2 receptors. These results demonstrate that in the avian brain, like in the mammalian one, both dopamine D~ and D, receptors are concentrated in "striatal" areas, while the telencephalon is rich in D~ but not D2 receptors. The neurotransmitter, dopamine (DA), plays important roles in the regulation of the activity of the mammalian brain particularly of the basal ganglia 1° Dopamine, on the other hand, appears to be present in the nervous system of primitive animals and has been extensively investigated in the brain of the lower vertebrates including birds and reptiles tl In the mammalian brain the physiological effects of D A are mediated through the interaction of the amine with two types of receptors, one linked in a positive manner to an adenylate cyclase, the Di type, and a second one negatively linked or unlinked to this enzyme, the D, type ~3. Radiolabeled ligands for these two types of receptors are available and have been used in their biochemical characterization 5"23 as well as in the autoradiographic investigation of their distribution Is. The autoradiographic studies have revealed that the two subtypes of the D A receptor are enriched in the basal ganglia, particularly in the nucleus caudatus-putamen. D~ and D 2 receptors are also localized in other brain areas where they present different distributions: for example, the D~ receptor being more enriched in the substantia nigra (SN) pars reticulata and the D 2 receptor in the SN pars compacta
and ventral tegmental area 3"6. Using receptor autoradiography we have detected significant differences among the distributions of these two types of receptors in the brain of several mammals (Camps et al., in preparation). In order to gain more information on the evolutionary aspects of the differential distribution of D A D~ and D2 receptors we have examined the distribution of these receptors in the brain of lowc,- vertebrates. We focused particularly on the bird ;: ain because the distribution of dopaminergic innervation in this species is known ~'9"14"1~and because recent studies on the chemoarchitecture of the basal ganglia of these anil;~als h~,.s revealed similarities between the location a~~d connection of their basal ganglia and that of the mammalian (see ref. 20). The brains of 12 adult male pigeons (Columba livia, 450-550 g bo~iv wt., provided by S. A b d e l ' A l , Basel, Switzerland) and 9 adult r.~:,te rats (Wistar, 200-250 g body w,t., from Sandoz Ltd.. Basel, Switzerland) were remt~-~ed immediately after decapitation and rapidly frozen. 10 Itm sections were cut with a cryostat and mounted onto gelatin-coated slides and stored in a freezer (-20 °C) until used. For the labeling of D~ receptors the tissue sections
Correspomlence: J.M. Palacios, Preclinical Research, Sandoz Ltd., CI-t-4002 Basle, Switzerland.
1111116-899388/$113.511© 1988 Elsevier Science Publishers B.V. (Biomedical Division)
355 were incubated with 1 nM of [sH]SCH 23390 (63.2 Ci/mmol; New England Nuclear, Dreieich, F.R.G.) in 50 mM Tris-HCl, 120 mM NaCI, 5 mM KCI, 2 mM CaCi 2, 1 mM MgCI2 (pH 7.4) for 45 min at room temperature 6. To determine the non-specific binding adjacent sections were incubated in the same medium in the presence of 10-6 M cis-flupentixoi. After incubation sections were washed for 5 min in fresh cold buffer and dried under a stream of cold air. For the labeling of D2 receptors the tissue sections were incubated either with the agonist [3H]CV 205-502 (107 Ci/mmol; synthesized and labeled by Dr. R. Voges, Biopharmaceutical Dept., Sandoz Ltd., Basle, Switzerland) 3 or with the antagonist [3H]spiroperidol (22 Ci/mmol; New England Nuclear, Dreieich, F.R.G.) 17. When using the agonist, sections were preincubated in 170 mM Tris-HCl buffer (pH 7.5) for 30 min and then incub::ted for 90 min with 1 nM [3H]CV 205-502 in the same buffer at room temperature. To determine the non-specific binding adjacent sections were incubated in the same solution in the presence of 1 aM of (+)-butaclamol. After two washes in ice-cold buffer of 1 min each and dipping in icecold distilled water, slides were dried under a stream of cold air. When using the antagonist tissue sections were incubated for 30 min with 0.4 nM [3H]spiroperidol in 170 mM Tris-HCi buffer (pH 7.5) containing 120 mM NaCI, 5 mM KCI, 2 nM CaCI.,, 1 nM MgC! at room tempcrature. Then incuoation was followed by two washes of 5 min each in the same buffer at 4 °C. dipped in cold distilled water and dried as mentioned. To block [SH]spiroperidol binding sites other than D2 binding sites, 1 uM ketanserin or haloperidoi was added to the incubation medium. Blanks for these D, binding sites were obtained using 1 !~M of the dopamine D 2 antagonist sulpiride. Autoradiograms were generated by apposing the labeled tissue sections and appropriate radioactive standards to a tritium-sensitive film (LKB, Sweden). Films were analyzed using a computerized imageanalysis system (MCID, Imaging Research, Ontario, Canada). For anatomical localization of the labeled regions the atlas of the pigeon of Karten and Hodos ~2 was employed. Both D t and D 2 receptor binding sites were found in the pigeon brain. The binding of [3H]SCH 23390 was completely blocked by cis-flupentixol, but not affected by spiperone. On the other hand, the binding
of [3H]CV 205-502 and that of [SH]spiroperidol were blocked by butaclamoi but were not affected by ke~anserin or SCH 23390. Dopamine D~ and D2 receptors presented a highly heterogeneous distribution in the pigeon brain (Fig. 1). D! receptors were concentrated in the pigeon forebrain, where the highest densities of DI binding sites were seen in the neocortex, the hyperstriatum ventrale and accessorius, and in the dorsal part of the archistriatum. The densities of receptors in these regions as estimated by computer-assisted microdensitometry was about 250 fmol/mg protein. Also rich in binding sites for [3H]SCH 23390 were the lobus paraolfactorius, the paleostriatum augmentatum, the nucleus accumbens, the neostriatum, and the nucleus superficialis parvicellularis; in these areas the density of D~ receptors was between 150 and 200 fmol/mg protein. In contrast, areas such as the paleostriatum primitivum, the layers of the tectum opticum, and the cerebellum presented much lower receptor densities in the range of 50 fmol/mg protein. No specific binding was detected in the ectostriatum. When compared with homologous regions of the rat brain incubated and measured in para!!el, in ~he pigeon brain the density was of about 10-fold lower. The rat caudate-putamen, for example, presented densities in the range of 2500-3000 fmol/mg protein, the globus pallidus of about 1500 fmol/mg protein, and the layers of the superior colliculus of about 300 fmol/mg protein. D2 receptors as labeled with the high affinity agonist. [sH]CV 205-502, were also highly heterogeneously distributed in the pigeon brain (Fig. 2). The highest densities of D, binding sites were seen in the basal ganglia, particularly in the paleostriatum augmentatum, the iobus paraolfactorius, the olfactory tubercle and the nucleus accumbens. In these areas the densities of D 2 receptors were in the range of 200-250 fmol/mg protein. In contrast, the density !a the paleostriatum primitivum was only of about 10 fmol/mg protein. Again, when compared with regions of equivalent ,,ize in the rat brain, the densities of D_, receptors were much lower in the pigeon brain although only by half. Densities of [3H]CV 205-502 binding sites in the caudate-putamen of the rat brain as examined in the same experiences as the pigeon brains were of about 500-600 fmoi/mg protein. In the globus pallidus of the ra.t the density of D 2 receptors
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Fig. 1. Photomicrographs from autoradiograms illustratipg the distribution of doamine D I receptors in the pigeon brain as labeled by [~H]SCH 23390. A,B,C are coronal sections, D is a sagittal and E a horizontal section; the hyperstriatum ventrale (HV), the cerebellum (Cb) and the layers of the rectum opticum (St) show an important labeling of D I receptors, but highest densities are observed in the paleostriatum augmentatum (PA) and the Iobus paraolfactoriu~ (I+PO) as well as in the nucleus superficialis parvicellularis (SPC). No binding is present in the paleostriatum primitivum (PP). Other abbreviations in Figs. I and 2: AHP, area parahippocampalis; Cx, neocortex; DLA, nucleus dorsolateralis anterior thalami pars lateralis; E, ectostriatum; Hy, hypothalamus; PT, nucleus pretectalis; SL, nucleus septalis laterals; TO, tuberculum olfactorium. Bar = 1 mm.
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Fig. 2. Photomicrographs from autoradiograms illustrating the distribution of dopamine D., receptors in the pigeon brain as labeled bv [3H]CV 205-502. A,B,C are coronal, D sagittal, and E horizontal sections. Highest densities are observed it, the paleostriatum augmentatum (PA), the Iobus paraolfactorius (LPO), and olfactory, tubercle (TO). Some binding is present in the lateral part of the nucleus dorsolateralis anterior thalami (DLA). the nucleus pretectalis (PT), and the layers of the tectum opticum (St). No binding is observed in the paleostriatum primitivum (PP). Bar = 1 mm.
358 was about 250 fmol/mg protein. In the pigeon brain other areas showed densities of lower than 50 fmol/mg protein. "lhus, the nucleus dorsolateralis anterior of the thalamus was highest with 50 fmol/mg protein, the nucleus superficialis parvicellularis had 30 fmol/mg protein, the layers of the optic tectum and the nucleus pretectalis showed about 15 fmoi/mg protein and regions like the neocortex, the striatal areas and the cerebellum showed lower than 10 fmol/mg protein. [sH]Spiperone labeling resulted in a similar distribution of sites (results not shown). No or very low densities of either D 1 or D 2 receptors were observed in the midbrain and brainstem. Regarding the nucleus tegmenti pedunculo-pontinus pars compacta (TPc) we did not observe significant densities of either D~ or D2 receptors in this area equivalent to the substantia nigra pars compacta of the mammalian brain 1"2"8"9"2~. This is probably not surprising because of the lower densities of dopamine receptors in the pigeon brain as compared to the rat brain. As the substantia nigra in the rat does not contain very high densities of D 2 receptors and only intermediate densities of D l receptors, it is possible that the presence of dopamine receptors in this area of the pigeon brain escape to the sensitivity of our technique. Similar results were observed in the chick brain (not shown). Thus, one of the main findings of this study is that the highest densities of dopamine receptors in the avian brain are localized in parts of the paleostriatal complex of the pigeon brain, the paleostriatum augmentatum and the Iobus paraoifactorius but not the paleostriatum primitivum. Embryological, cytoarchitectonical and histochemical criteria indicate that the paleostriatum complex of birds and reptiles can be compared to the striatal complex of the mammals, i.e. the paleostriatum augmentatum and the lobus paraolfactorius corresponding to the nucleus caudate-putamen and the paleostriatum primitivum corresponding to the giobus pallidus of the mammals (see ref. 20 for review). Interestingly, as in mammals, in the bird brain dopamine receptors appear to be concentrated in the striatum rather than in the globus pallidus. Furthermore, this appears as a general feature of the amniote brain, i.e. modern birds, reptiles and mammals7. Dopamine D~ but not dopamine D2 receptors are present in hyperstriatae areas. Comparative anatomical studies have indicated that,
in spite of clear differences in the development of the avian and reptilian pallidum and the mammalian isocortex, there are organizational similarities between portions of the avian telencephalon and the mammalian isocortex ~s. In this sense it is interesting that, while the mammalian cortex contains significant densities of the D t receptors and low or very low densities of the D2 receptors, similarly the telencephalon of the pigeon brain contains Dt but not D 2 receptors. Our autoradiographic results are in good agreement with previous biochemical and histochemical results on the presence of dopamine and dopamine receptors in the avian brain 4"8"9"1zA4"19. Coveili et al. 4 have examined the presence of dopamine receptors using binding techniques in different invertebrate and vertebrate species and found that the avian brain contains dopamine D 2 receptors. On the other hand, the distribution of dopamine in the avian brain has been examined histochemically8"9'14a9 and found to correlate well with the presence of dopamine D2 receptors, in the sense that the paleostriatum augmentatum, the lobus paraolfactorius and the olfactory tubercle are richly innervated with dopaminergic terminals which appear to emanate from an area the TPc which has been proposed as homologous to the mammalian substantia nigra 1"2"8"9"-~. In a similar way to that observed in the mammalian brain, the hyperstriatal areas are poorly innervated by dopamine terminals'~'~4"~9; however, they contain significant densities of D~ receptors. These results strengthen again the above-mentioned similarities between the distribution of dopamine D~ and D 2 receptors in the avian and in the mammalian brain. Other regions like the layers of the optic tectum and the cerebellum represent further examples to support the similarity of the avian and the mammalian brain as we found comparable densities of D 1 and D 2 receptors in the rat superior colliculus and cerebellum 3"6. Our results provide a morphological basis for the neuropharmacological effects of dopaminergic agents in avian behavior and biochemistry. Several studies have shown that the paleostriatum is the site of action for the effects of dopaminergic agonists and antagonists in stereotyped and motor behavior in bii-ds. In addition, paleostriatal lesions decrease these effects while lesions of the neostriatum and hyperstriatum did not influence these actions m'2~'22. These studies, however, have been limited until now
359 to dopamine D 2 agents; we are not aware of any study involving selective D~ agents. In summary, our autoradiographic results have illustrated the distribution of dopamine receptors in the avian brain. The main conclusion of this study is that evolution appears to be conservative with regard
to the presence and distribution of dopaminergic postsynaptic markers in the basal ganglia as well as other regions. These studies provide anatomical support for further investigations on the functional role of doparni,,,,e receptors in the avian brain.
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