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Presence of catecholaminergic axon-terminals containing β-adrenergic receptor in the periventricular zone of the rat hypothalamus
Brain Research, 479 (1989) 190-193 Elsevier
190 BRE 23335
Presence of catecholaminergic axon-terminals containing i]-adrenergic receptor in the periventricular zone of the rat hypothalamus Akio Wanaka 1, Craig C. Malbon 3, Masayasu Matsumoto 2, Takenobu Kamada 2, and Masaya Tohyama ~ t Department of Anatomy 11, ZFirstDepartment of Internal Medicine, Osaka University School of Medicine, Nakanoshima, Kizaku, Osaka (Japan) and 3Departmentof Pharmacological Sciences, State University of New York at Stony Brook, NY (U. S.A.) (Accepted 11 October 1988) Key words: Immunoelectron microscopy;fl-Adrenergic receptor; Hypothalamus; Presynaptic autoreceptor
The present study, using a light microscopic double-immunofluorescence method, has revealed the presence of fibers containing both tyrosine hydroxylase- and fl-adrenergic receptor-like immunoreactivities in the rat hypothalamic periventricular zone. Subsequent immunoelectron microscopic analysisdemonstrated that these belong to axon terminals. These findings suggest that presynaptic fl-adrenergic receptor is present in catecholaminergic terminals.
In our previous investigation 16, aatiserum against purified fl-adrenergic receptor (fl2-AdR) was employed to examine the localization of fl-AdR in the rat brain. It was found that, in addition to immunoreactive somata, there were a number of immunoreactive fibers with punctate profiles, and a few which were varicose in appearance like axonal fibers especially in the periventricular zone of the rat hypothalamus 16. Other investigators have shown by pharmacological means that the fl-AdR is localized in the pres~laptic catecholaminergic fibers (autorecep~..-..X ,,,1 / 1.5. "r'k^_^~:__^ • ~,~,~tL,~~, we tried to elucidate immunocytochemically whether or not the varicose fibers labeled by the anti-fl-AdR antiserum are catecholaminergic axons in the periventricular zone of the rat hypothalamus. Tyrosine hydroxylase (TH) was used as a marker for catecholaminergic systems. Preparation and characterization of the antiserum has been described elsewhere 6.16. Briefly, the antiserum was raised in rabbit immunized with affinity-pudried fle-AdR. The titer of the antiserum was
checked by solid-phase enzyme-linked immunosolvent assay (ELISA) and immunoprecipitation 6. This antiserum was proved to crossreact to the extent of 60-84% with purified f l r A d R of rat adipose cell 6. However. on the basis of the distributions determined by autoradiography 3'7-9,14,15a7, it is considered that there is little crossreaction with a r A d R , dopamine receptor, muscadnic cholinergic receptor, and rhodepsin receptor 16. We also performed absorption control test with 5/~g of purified fle-AdR to check the immunostaining. This test showed a marked reduction of the immunoreaction, in addition, on the basis of inhibition of ligand binding, displacement experiments with an excess of clonidine revealed that this antiserum did not crossreact with a2-AdR 16. Immunoelectron microscopic procedures used here were similar to those described by Somogyi and Takagi ~2. Sprague-Dawley rats were perfused transcardially with modified Zamboni's fixative 12, following which their brains were dissected out and postfixed overnight. These were then immersed in a solu-
Correspondence: A Wanaka, Department of Anatomy II, Osaka UniversitySchool of Medicine, Nakanoshima 4-3-57, Kitaku, Osaka 530, Japan. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
/ tion of 30% sucrose in 0.1 M phosphate buffer (pH 7.4). Vibratome sections (10 /~m) were cut and reacted with anti-/~2-AdR antiserum (diluted 50 times in phosphate-buffered saline). After rinsing with
191
phosphate-buffered saline, sections were reacted ~vith goat anti-rabbit IgG antiserum and then with peroxidase-antiperoxidase (PAP) cemplex. Antigens were visualized by means of diaminobenzidine
Fig. 1. a and b: fluorescence photomicrographs of a double-labeled section of the hypothalamic periventricular zone (a, TH-like immunorcactivity, b,/3-AdR-like immunoreactivity). Note that the running patterns of both types of immunoreactivefiber (arrowheads) are very similar. Bar = 50 #m. c: electron photomicrograph offl-AdR-like immunoreactivefiber (presented in Fig. lb). Note that immunoreaction products are mainly associated with synapticvesicles and sometimes with cytoplasmicmembrane (arrows). × 41,600.
(DAB) reaction. After samples were dehydrated and embedded in resin, ultrathin sections were produced for observation under an electron microscope.
Fig. 2. Electron photomicrographs showing fl-AdR-immunoreactive structures in the periventricular zone. a: axo-dendritic synapse. Immunoreaction products are localized not only at the postsynaptic density (arrowheads) but also in the dendroplasm. b: axo-somatic synapse with fl-AdR immunoreactivity (arrowheads), c: immunoreaction products are associated with endoplasmic reticulum, a, x 16,700; b, x 13,900; c, x21,600.
Some vibratome sections were used for the doublelabeling experiment n. These were incubated with a mixture of anti-fl2-AdR and monoclonal anti-TH antisera, then allowed to react with a solution containing fluorescein isothiocyanate-labeled goat anti-rabbit IgG antiserum and Texas red-labeled anti-mouse IgG antiserum. Distributions offl-AdR-like immunoreactive and TH-like immunoreactive structures were separately observed under a fluorescence microscope equipped with the appropriate mirror filter system. Subsequently, in order to visualize only the fl-AdR-like immunoreactivities for electron microscopy, the sections were processed using the rabbit PAP-DAB procedure and subjected to immunoelectron microscopy as described above. Fig. ib shows fibers with fl-AdR-like immunoreactivity in the periventricular zone of the hypothalamus. The localization of the TH-like immunoreactive structures in the same area is presented in Fig. la. Many of the latter fibers and neurons lacked fl-AdRlike immunoreactivity, while the fl-AdR-immunoreactive fibers shown in Fig. la (arrowheads) displayed concurrent TH-like immunoreactivity (Fig. lb, arrowheads). In Fig. lc, the ultrastructure of the fl-AdRdmmunoreactive fiber from Fig. lb is shown. The labeled fiber was filled with synaptic vesicles, indicating that it was an axon terminal. Immunoreactive end-products were mainly associated with these synaptic vesicles, and sometimes with either the cellular (arrows) or mitochondrial surface membrane (Fig. lc). However, throughout the analysis of the fine structures of fl-AdR-immunoreactive fibers and neurons, the above-mentioned pro~:les were not very frequent, and most of the fl-AdR-like immunoreaction occurred in ttle dendrites (Fig. 2a). In addition, immunoreactive soma were also frequently detected (Fig. 2b). Immunoreactive end-products were associated with the postsynaptic densities where non-labeled terminals terminated (Fig. 2a,b) and were also found scattered in the dendroplasm (Fig. 2a) and cytoplasm, some of which were associated with endoplasmic reticulum (Fig. 2c). Thus, the present study has clearly demonstrated that catecholaminergic axon terminals contain flAdR, strongly suggesting that this fl-AdR belongs to the presynaptic autoreceptor. The ultrastructural localization of fl-AdR-like immunoreactivity with synaptic vesicles may represent the receptors which are
193 being transferred from soma to axon term.~nals or which are recycling in the cytoplasm4. In general, in the peripheral and central nervous systems the a2-adrenergic receptor is the major subtype involved in modulating the release of catecholamines. However, there has been some evidence that/~-adrenergic receptors play a modulatory role at presynaptic sites~, s,10. Our findings lead us to believe that/~-adrenergic rece ptor-mediated modulation of catecholamine release takes place in the periventricular zone of the hypothalamus. In addition, as shown in Fig. la and b, many of the TH-like immunoreactive fibers lacked 8AdR-like immunoreactivity, implying that there are two subpopulations of these fibers in the hypotha1 Adler-Graschinsky, E. and Langer, S.Z., Possible role of a beta-adrenoceptor in the regulation of noradrenaline release by nerve stimulation through a positive feed-back mechanism, Br. J. Pharmacol., 53 (1975) 43-50. 2 Aoki, C., .Ioh, T.H. and Pickel, V.M., Uitrastructural localization of beta-adrenergic receptor-like immunoreactivity in the cortex and neostriatum of rat brain, Brain Research, 437 (1988) 264-282. 3 Jones, L.S., Gauger, L.L. and Davis, J.N., Anatomy of brain alpha-l-adrenergic receptors: in vitro autoradiography with [lZ~I]-HEAT, J. Comp. Neurol., 231 (1985) 190-208. 4 Laduron, P.M., Axonal transport of neuroreceptors: possible involvement in long term memory, Neuroscience, 22 (1987) 767-779. 5 Langer, S.Z., Presynaptic regulation of monoaminergic neurons. In H.Y. Meltzer (Ed.), Psychopharmacology: The Third Generation of Progress, Raven, New York, 1987, pp. 151-157. 6 Moxham, C.P., George, S.T., Graziano, M.P., Brandwein, H.J. and Malbon, C.C., Mammalian beta l- and beta2-adrenergic receptors. Immunological and structural comparisons, J. Biol. Chem., 261 (1986) 14562-14570. 7 Palacios, J.M. and Kuhar, M.J., Beta-adrenergic receptor localization by light microscopic autoradiography, Science, 208 (1980) 1378-1380. 8 Palacios, J.M. and Wamsley, if.K, Catecholamine receptors. in A. Bj~rklund, T. H6kfelt and M..I. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, Vol. 3, Elsevier: Amsterdam, 1984, pp. 325-351. 9 Rainbow, T.C., Parsons, B. and Wolfe, B.B., Quantitative autoradiography of betas- and betae-adrenergic receptors in rat brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1585-1589. 10 Rand, M.J., Majewaki, H., McCulloch, M.W. and Story, D.F., An adrenaline-mediated positive feedback loop in
lamic periventricular zone. The localization pattern of/3-AdR in the dendrites and soma of the hypothalamic periventricular zone determined here coincides well with distributions in the amphibian cerebellum~3, rat cerebral cortex and neostriatum2, which has been shown immunocytochemically using anti-/~AdR antiserum. Although Aoki et al. have reported the presence of/~-AdR-like immunoreactivity in the glial element2, the present ~tudy failed to demonstrate this in the rat hypothalamus. This discrepancy may arise from the experimental differences between the conditions or materials (including antisera) used or in the brain regions examined.
sympathetic transmission and its possible role in hypertension. In S.Z. Langer, K. Starke and M.L. Dubocovich (Eds.), Advances in Biosciences, Voi. 18, Presynaptic Receptors, Pergamon, Oxford, 1979, pp. 263-269. 11 Slfiosaka, S. and Tohyama, M., Immunohistochemical techniques. In P.C. Emson, M.N. Rossor, M. Tohyaraa (Eds.), Progress in Brain Research, Vol. 66, Elsevier, Amsterdam, pp. 3-32. 12 Somogyi, P. and Takagi, H., A note on the use of p;.'a'ic acid-paraformaldehyde-glutaraldehyde fixative for correlated fight and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 1979-1983. 13 Strader, C.D., Pickel, V.M., Job, T.H., Strohsacker, M.W., Shorr, R., Lefkowitz, R.J. and Caron, M.G., Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain, Proc. Natl. Acad. Sci. U.S.A., 80 (1984) 1840-1844. 14 Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J., Distribution of a2-agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents, Br~,n Res. Rev., 7 (1984) 69-101. 15 Wamsley, J.K., Lewis, M.S., Young, W.M. HI and Kuhar, M.J., Autoradiographic localization of muscarinic cholinergic receptors in rat brain stem, J. Neurosci., 1 (1981) 176-191. 16 Wanaka, A., Kiyama, H., Murakami, T., Matsumoto, M., Kamada, T., Malbon, C.C. and Tohyama, M., Immunocytochemical localization of/~-adlenergic receptors in the rat brain, Brain Research, in press. 17 Young, W.S. IIl and Kuhar, M.J., Noradrenergic alph~-I and alpha-2 receptors: light microscopic autoradiographic localization, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1969-1700.
190 BRE 23335
Presence of catecholaminergic axon-terminals containing i]-adrenergic receptor in the periventricular zone of the rat hypothalamus Akio Wanaka 1, Craig C. Malbon 3, Masayasu Matsumoto 2, Takenobu Kamada 2, and Masaya Tohyama ~ t Department of Anatomy 11, ZFirstDepartment of Internal Medicine, Osaka University School of Medicine, Nakanoshima, Kizaku, Osaka (Japan) and 3Departmentof Pharmacological Sciences, State University of New York at Stony Brook, NY (U. S.A.) (Accepted 11 October 1988) Key words: Immunoelectron microscopy;fl-Adrenergic receptor; Hypothalamus; Presynaptic autoreceptor
The present study, using a light microscopic double-immunofluorescence method, has revealed the presence of fibers containing both tyrosine hydroxylase- and fl-adrenergic receptor-like immunoreactivities in the rat hypothalamic periventricular zone. Subsequent immunoelectron microscopic analysisdemonstrated that these belong to axon terminals. These findings suggest that presynaptic fl-adrenergic receptor is present in catecholaminergic terminals.
In our previous investigation 16, aatiserum against purified fl-adrenergic receptor (fl2-AdR) was employed to examine the localization of fl-AdR in the rat brain. It was found that, in addition to immunoreactive somata, there were a number of immunoreactive fibers with punctate profiles, and a few which were varicose in appearance like axonal fibers especially in the periventricular zone of the rat hypothalamus 16. Other investigators have shown by pharmacological means that the fl-AdR is localized in the pres~laptic catecholaminergic fibers (autorecep~..-..X ,,,1 / 1.5. "r'k^_^~:__^ • ~,~,~tL,~~, we tried to elucidate immunocytochemically whether or not the varicose fibers labeled by the anti-fl-AdR antiserum are catecholaminergic axons in the periventricular zone of the rat hypothalamus. Tyrosine hydroxylase (TH) was used as a marker for catecholaminergic systems. Preparation and characterization of the antiserum has been described elsewhere 6.16. Briefly, the antiserum was raised in rabbit immunized with affinity-pudried fle-AdR. The titer of the antiserum was
checked by solid-phase enzyme-linked immunosolvent assay (ELISA) and immunoprecipitation 6. This antiserum was proved to crossreact to the extent of 60-84% with purified f l r A d R of rat adipose cell 6. However. on the basis of the distributions determined by autoradiography 3'7-9,14,15a7, it is considered that there is little crossreaction with a r A d R , dopamine receptor, muscadnic cholinergic receptor, and rhodepsin receptor 16. We also performed absorption control test with 5/~g of purified fle-AdR to check the immunostaining. This test showed a marked reduction of the immunoreaction, in addition, on the basis of inhibition of ligand binding, displacement experiments with an excess of clonidine revealed that this antiserum did not crossreact with a2-AdR 16. Immunoelectron microscopic procedures used here were similar to those described by Somogyi and Takagi ~2. Sprague-Dawley rats were perfused transcardially with modified Zamboni's fixative 12, following which their brains were dissected out and postfixed overnight. These were then immersed in a solu-
Correspondence: A Wanaka, Department of Anatomy II, Osaka UniversitySchool of Medicine, Nakanoshima 4-3-57, Kitaku, Osaka 530, Japan. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
/ tion of 30% sucrose in 0.1 M phosphate buffer (pH 7.4). Vibratome sections (10 /~m) were cut and reacted with anti-/~2-AdR antiserum (diluted 50 times in phosphate-buffered saline). After rinsing with
191
phosphate-buffered saline, sections were reacted ~vith goat anti-rabbit IgG antiserum and then with peroxidase-antiperoxidase (PAP) cemplex. Antigens were visualized by means of diaminobenzidine
Fig. 1. a and b: fluorescence photomicrographs of a double-labeled section of the hypothalamic periventricular zone (a, TH-like immunorcactivity, b,/3-AdR-like immunoreactivity). Note that the running patterns of both types of immunoreactivefiber (arrowheads) are very similar. Bar = 50 #m. c: electron photomicrograph offl-AdR-like immunoreactivefiber (presented in Fig. lb). Note that immunoreaction products are mainly associated with synapticvesicles and sometimes with cytoplasmicmembrane (arrows). × 41,600.
(DAB) reaction. After samples were dehydrated and embedded in resin, ultrathin sections were produced for observation under an electron microscope.
Fig. 2. Electron photomicrographs showing fl-AdR-immunoreactive structures in the periventricular zone. a: axo-dendritic synapse. Immunoreaction products are localized not only at the postsynaptic density (arrowheads) but also in the dendroplasm. b: axo-somatic synapse with fl-AdR immunoreactivity (arrowheads), c: immunoreaction products are associated with endoplasmic reticulum, a, x 16,700; b, x 13,900; c, x21,600.
Some vibratome sections were used for the doublelabeling experiment n. These were incubated with a mixture of anti-fl2-AdR and monoclonal anti-TH antisera, then allowed to react with a solution containing fluorescein isothiocyanate-labeled goat anti-rabbit IgG antiserum and Texas red-labeled anti-mouse IgG antiserum. Distributions offl-AdR-like immunoreactive and TH-like immunoreactive structures were separately observed under a fluorescence microscope equipped with the appropriate mirror filter system. Subsequently, in order to visualize only the fl-AdR-like immunoreactivities for electron microscopy, the sections were processed using the rabbit PAP-DAB procedure and subjected to immunoelectron microscopy as described above. Fig. ib shows fibers with fl-AdR-like immunoreactivity in the periventricular zone of the hypothalamus. The localization of the TH-like immunoreactive structures in the same area is presented in Fig. la. Many of the latter fibers and neurons lacked fl-AdRlike immunoreactivity, while the fl-AdR-immunoreactive fibers shown in Fig. la (arrowheads) displayed concurrent TH-like immunoreactivity (Fig. lb, arrowheads). In Fig. lc, the ultrastructure of the fl-AdRdmmunoreactive fiber from Fig. lb is shown. The labeled fiber was filled with synaptic vesicles, indicating that it was an axon terminal. Immunoreactive end-products were mainly associated with these synaptic vesicles, and sometimes with either the cellular (arrows) or mitochondrial surface membrane (Fig. lc). However, throughout the analysis of the fine structures of fl-AdR-immunoreactive fibers and neurons, the above-mentioned pro~:les were not very frequent, and most of the fl-AdR-like immunoreaction occurred in ttle dendrites (Fig. 2a). In addition, immunoreactive soma were also frequently detected (Fig. 2b). Immunoreactive end-products were associated with the postsynaptic densities where non-labeled terminals terminated (Fig. 2a,b) and were also found scattered in the dendroplasm (Fig. 2a) and cytoplasm, some of which were associated with endoplasmic reticulum (Fig. 2c). Thus, the present study has clearly demonstrated that catecholaminergic axon terminals contain flAdR, strongly suggesting that this fl-AdR belongs to the presynaptic autoreceptor. The ultrastructural localization of fl-AdR-like immunoreactivity with synaptic vesicles may represent the receptors which are
193 being transferred from soma to axon term.~nals or which are recycling in the cytoplasm4. In general, in the peripheral and central nervous systems the a2-adrenergic receptor is the major subtype involved in modulating the release of catecholamines. However, there has been some evidence that/~-adrenergic receptors play a modulatory role at presynaptic sites~, s,10. Our findings lead us to believe that/~-adrenergic rece ptor-mediated modulation of catecholamine release takes place in the periventricular zone of the hypothalamus. In addition, as shown in Fig. la and b, many of the TH-like immunoreactive fibers lacked 8AdR-like immunoreactivity, implying that there are two subpopulations of these fibers in the hypotha1 Adler-Graschinsky, E. and Langer, S.Z., Possible role of a beta-adrenoceptor in the regulation of noradrenaline release by nerve stimulation through a positive feed-back mechanism, Br. J. Pharmacol., 53 (1975) 43-50. 2 Aoki, C., .Ioh, T.H. and Pickel, V.M., Uitrastructural localization of beta-adrenergic receptor-like immunoreactivity in the cortex and neostriatum of rat brain, Brain Research, 437 (1988) 264-282. 3 Jones, L.S., Gauger, L.L. and Davis, J.N., Anatomy of brain alpha-l-adrenergic receptors: in vitro autoradiography with [lZ~I]-HEAT, J. Comp. Neurol., 231 (1985) 190-208. 4 Laduron, P.M., Axonal transport of neuroreceptors: possible involvement in long term memory, Neuroscience, 22 (1987) 767-779. 5 Langer, S.Z., Presynaptic regulation of monoaminergic neurons. In H.Y. Meltzer (Ed.), Psychopharmacology: The Third Generation of Progress, Raven, New York, 1987, pp. 151-157. 6 Moxham, C.P., George, S.T., Graziano, M.P., Brandwein, H.J. and Malbon, C.C., Mammalian beta l- and beta2-adrenergic receptors. Immunological and structural comparisons, J. Biol. Chem., 261 (1986) 14562-14570. 7 Palacios, J.M. and Kuhar, M.J., Beta-adrenergic receptor localization by light microscopic autoradiography, Science, 208 (1980) 1378-1380. 8 Palacios, J.M. and Wamsley, if.K, Catecholamine receptors. in A. Bj~rklund, T. H6kfelt and M..I. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, Vol. 3, Elsevier: Amsterdam, 1984, pp. 325-351. 9 Rainbow, T.C., Parsons, B. and Wolfe, B.B., Quantitative autoradiography of betas- and betae-adrenergic receptors in rat brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1585-1589. 10 Rand, M.J., Majewaki, H., McCulloch, M.W. and Story, D.F., An adrenaline-mediated positive feedback loop in
lamic periventricular zone. The localization pattern of/3-AdR in the dendrites and soma of the hypothalamic periventricular zone determined here coincides well with distributions in the amphibian cerebellum~3, rat cerebral cortex and neostriatum2, which has been shown immunocytochemically using anti-/~AdR antiserum. Although Aoki et al. have reported the presence of/~-AdR-like immunoreactivity in the glial element2, the present ~tudy failed to demonstrate this in the rat hypothalamus. This discrepancy may arise from the experimental differences between the conditions or materials (including antisera) used or in the brain regions examined.
sympathetic transmission and its possible role in hypertension. In S.Z. Langer, K. Starke and M.L. Dubocovich (Eds.), Advances in Biosciences, Voi. 18, Presynaptic Receptors, Pergamon, Oxford, 1979, pp. 263-269. 11 Slfiosaka, S. and Tohyama, M., Immunohistochemical techniques. In P.C. Emson, M.N. Rossor, M. Tohyaraa (Eds.), Progress in Brain Research, Vol. 66, Elsevier, Amsterdam, pp. 3-32. 12 Somogyi, P. and Takagi, H., A note on the use of p;.'a'ic acid-paraformaldehyde-glutaraldehyde fixative for correlated fight and electron microscopic immunocytochemistry, Neuroscience, 7 (1982) 1979-1983. 13 Strader, C.D., Pickel, V.M., Job, T.H., Strohsacker, M.W., Shorr, R., Lefkowitz, R.J. and Caron, M.G., Antibodies to the beta-adrenergic receptor: attenuation of catecholamine-sensitive adenylate cyclase and demonstration of postsynaptic receptor localization in brain, Proc. Natl. Acad. Sci. U.S.A., 80 (1984) 1840-1844. 14 Unnerstall, J.R., Kopajtic, T.A. and Kuhar, M.J., Distribution of a2-agonist binding sites in the rat and human central nervous system: analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents, Br~,n Res. Rev., 7 (1984) 69-101. 15 Wamsley, J.K., Lewis, M.S., Young, W.M. HI and Kuhar, M.J., Autoradiographic localization of muscarinic cholinergic receptors in rat brain stem, J. Neurosci., 1 (1981) 176-191. 16 Wanaka, A., Kiyama, H., Murakami, T., Matsumoto, M., Kamada, T., Malbon, C.C. and Tohyama, M., Immunocytochemical localization of/~-adlenergic receptors in the rat brain, Brain Research, in press. 17 Young, W.S. IIl and Kuhar, M.J., Noradrenergic alph~-I and alpha-2 receptors: light microscopic autoradiographic localization, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1969-1700.