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β-Adrenergic receptors in the vasopressin-containing neurons in the paraventricular and supraptic nucleus of the rat
174
Brain Research, 499 (1989) 174-178
Elsevier
BRES 23753
I -Adrenergic receptors in the vasopressin-containing neurons in the paraventricular and supraptic nucleus of the rat T. T a k a n o I , Y. K u b o t a 1, A . W a n a k a ~, S. U s u d a ~. M . T a n a k a ~, C . C . M a l b o n 2 a n d M. T o h y a m a 1 ~Department of Anatomy II, Osaka University Medical School, Osaka (Japan) and 2Department of Pharmacology, School of Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY I1794-8651 (U.S.A.)
(Accepted 20 June 1989) Key words: fl-Adrenergic receptor; Vasopressin neuron; Paraventricular nucleus; Supraoptic nucleus; Rat; Immunocytochemistry
Immunocytochemical staining of alternate consecutive sections revealed that neurons with vasopressin-like immunoreactivity in the paraventricular and supraoptic nuclei had fl-adrenergic receptor-like immunoreactivity in the rats.
It is well known that paraventricular (PV) and supraoptic (SO) nuclei, which contain clusters of vasopressin (VAS) neurons (see ref. 16 for a review), are sites receiving a dense innervation of noradrenaline (NA) fibers 3'6. In addition, a number of studies have been carried out on the effect of VAS release by NA (see refs. 5, 8, 15, 21 for reviews), though the results of these studies have been contradictory. There is no doubt that the NA effects on the VAS neurons are mediated by adrenergic receptors ( A d R ) 2'5'9'14'21 . Leibowitz has suggested the presence of both types of AdR in the PV and they have shown that PV is a primary site mediating adrenergic stimulation of feeding and drinking 1°. Electrophysiological studies have suggested the presence of both types of AdR on the VAS neurons of the SO5'~4'21: the excitatory effect of a - A d R agonists and weak inhibitory effect of fl-AdR agonists on phasic activity of VAS neurons. However, until now, no direct evidence demonstrating the presence of AdR on the VAS neurons was shown. Therefore, we examined this possibility, using immunohistochemical staining of alternate consecutive sections with anti VAS antiserum and anti fl-AdR antiserum.
The preparation and characterization of the antiserum against fl-AdR has been described elsewhere j1'2°. In brief, the antiserum was produced in a rabbit immunized with affinity-purified fl2-AdR from guinea pig lung. The titer of the antiserum was tested with the use of solid-phase enzyme-linked immunosolvent (ELISA) and immunoprecipitation, This antiserum was proven to cross-react to the extent of 60-84% with purified fll-AdR of rat adipose cells. On the basis of the distribution determined with immunocytochemistry using this antiserum to compare the findings by autoradiography 7'12'13'1s'19'22, it is considered that there is little cross-reactivity with cq-AdR, dopamine receptors, muscarinic cholinergic receptors, or the photoreceptor rhodopsin. The findings from the inhibition test of ligand binding and displacement experiments with an excess of clonidine suggested that the antiserum did not cross-react with a2-AdR. An absorption test was also performed to check the specificity. Absorption of the antiserum with 5/~g of purified fl2-AdR resulted in a marked reduction of the immunostaining. Synthetic arginine VAS bound to bovine thyroglobulin with carbodiimide was used as an antigen for anti VAS antiserum (Miles). The
Correspondence: M. Tohyama, Department of Anatomy II, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka,
530, Japan.
175 antiserum has been raised in New Zealand White rabbits by repeated injections of the conjugates emulsified in Freund's Complete adjuvant. Crossreactivity with oxytocin was tested in the Brattleboro rat, a strain that lacks the ability to synthesize VAS and demonstrated no cross-reactivity with oxytocin. Five male albino rats weighing about 100 g each were used. The animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused intracardially with 50 ml of ice-cold saline followed by 500 ml of Zamboni's fixative 23. The brains were removed, immersed in the same fixative overnight at 4 °C, and then rinsed for 48 h in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Serial frozen sections, 5/~m thick, through the PV and SO
were prepared using a cryostat. Alternate sections were processed by the indirect immunofluorescence method developed by Coons 4 to determine the VAS-like immunoreactivity (IR) or fl-AdR-IR. The dilutions of the first antiserum with phosphatebuffered saline (PBS) were as follows: VAS 1:1000; and fl-AdR 1:50. Fluorescein isothiocyanate (FITC)conjugated goat anti rabbit IgG (Miles) was used as the second antiserum. The second antiserum was diluted to 1:1000 with PBS. Fig. 1A and C show a pair of the consecutive sections through the posterior magnocellular portion of the PV, stained for vasopressin (Fig. 1A) and fl-AdR (Fig. 1C). A cluster of cells with VAS-IR and fl-AdR-IR can be seen in the posterior magnocellu-
Fig. 1. A,C: light micrographs of serial sections through the posterior magnocellular (M) and medial parovcellular part (P) of the paraventricular nucleus, incubated with anti-VAS antiserum (A) and anti-fl-AdRantiserum (C). In the M, a cluster of cells with VAS-IR and fl-AdR-IR were seen. The frames are enlarged in B and D. The cells which are numbered in B and D are identical. Most of the cells in the M contained both VAS-IRand fl-AdR-IR. A-D, frontal sections. Bar for A,C = 150 pm. Bar for B,D = 10 pm.
176 lar portion. In addition, fl-AdR-IR cells were diffusely scattered in other hypothalamic areas (Fig. 1C). The frames in Fig. 1A and C show VAS-IR and fl-AdR-IR cells in the posterior magnocellular part and are enlarged in Fig. 1B and D. Most of the neurons in the posterior magnocellular part were large and contained both VAS-IR and fl-AdR-IR. Fig. 2A and C are another pair of the serial sections through the posterior magnocellular part of the PV. The frames are located in the medial parvocellular portion and are enlarged in Fig. 2B and D, respectively. Cells containing VAS-IR were scattered in the medial palvocellular portion. Two types of cells with VAS-IR were distinguished: one was large and the other was small in size. Most of the two types of VAS-IR cells contained fl-AdR-IR. Fig. 3A and C are a pair of serial sections through the SO. Fig. 3B and D are higher magnifications of
the boxes in Fig. 3A and C. Most of the cells with fl-AdR contained VAS-IR. However, a significant number of cells with VAS-IR which were devoid of fl-AdR-IR was also found (about 30-40%) (Fig. 3). PV and SO contain numerous NA fibers most of which originate from A 1 NA neurons 16. There has been much disagreement among the studies concerning the effect of NA on VAS release (see ref. 15 for a review). Bridges and Thorn have reported the excitatory effect of NA on the release of VAS and have suggested that this effect is mediated by a - A d R 2. Conversely, there exist reports which show the inhibitory effect of central NA on VAS secretion 8. Armstrong et al. have confirmed this effect and suggested that a - A d R mediates this effect 1. Electrophysiological studies using slices through the SO have revealed the excitatory effect of a - A d R agonists and the weak inhibitory effect of fl-AdR
Fig. 2. A,C: light micrographs of serial sections through P and M of the PV, stained with anti VAS antiserum (A) and anti-fl-AdR antiserum (C). In the P, cells with VAS-IR or fl-AdR-IR were scattered. B and D are higher magnifications of the frames of A and C, respectively. Both large (arrowheads) and small (arrow) cells with VAS-IR contained fl-AdR-IR. Frontal sections. Bar for A,C = 150/~m. Bar for B,D --- 10 am.
177
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i
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Fig. 3. A,C: light micrographs of serial sections through the supraoptic nucleus (SO), stained with anti VAS antiserum (A) and anti-fl-AdR antiserum (C). Cells with fl-AdR-IR were less numerous than those with VAS-IR, B,D: enlargements of the frames in A and C. Arrowheads indicate the cells which contained both VAS-IR and fl-AdR-IR. Frontal sections. Bar for A,C = 150/~m. Bar for B,D = 15/~m.
agonists on the phasic activity of V A S cells 14'21. The p r e s e n t study shows the presence of cells with f l - A d R - I R on the V A S neurons in the PV and SO, indicating that the N A effect on V A S neurons via f l - A d R exists and is monosynaptically regulated by an ascending N A system from A 1 N A neurons. H o w e v e r , the p r o p o r t i o n of the cells with V A S - I R which contain differences in f l - A d R - I R were n o t e d b e t w e e n the PV and SO: most of the V A S cells in the PV contained f l - A d R , while about 60% of V A S cells in the SO contained f l - A d R . This m a y be attributed to the difference in character between the V A S neurons in the PV and SO. H o w e v e r , p r o b l e m s of
This study was s u p p o r t e d in part by grants from the Ministry of E d u c a t i o n of J a p a n and from M o c h i d a F o u n d a t i o n . C . C . M . is s u p p o r t e d by N I H Grants DK25410 and D K 30111.
1 Armstrong, W.E., Sladek, C.D. and Sladek Jr., J.R., Characterization of noradrenergic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system, Endocrinology, 111 (1982) 273-279. 2 Bridges, T.E. and Thon, N.A., The effect of autonomic blocking agents on vasopressin release in vivo by osmore-
ceptor stimulation, J. Endocrinol., 48 (1970) 265-276. 3 Carlsson, A., Faick, B. and Hillarp, N.-A., Cellular localization of brain monoamines, Acta Physiol. Scand., 56, Suppl. 196 (1962) 1-27. 4 Coons, A.H., Fluorescent antibody method. In J.E Danielli (Ed.), General Cytochemical Methods, Academic,
the titer should also be considered. T h e presence of a - A d R on the V A S neurons has also b e e n suggested 2,9,14,21. T h e possibility exists that a - A d R and f l - A d R are localized in the same neurons in some brain regions 17. The complex nature of the effects of N A on the V A S neurons dictates further analysis by the i m m u n o c h e m i c a l approach.
178 New York, 1958, pp. 399-422. 5 Day, T.A., Randle, J.C.R. and Renaud, L.P., Opposing alpha-adrenergic and beta-adrenergic mechanisms mediate dose-dependent actions of noradrenaline on supraoptic vasopressin neurons in vivo, Brain Research, 358 (1985) 171-179. 6 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine nerve terminals in the central nervous system, Acta Physiol. Scand., 64, Suppl. 247 (1965) 37-85. 7 Joness, L.S., Gauger, L.L. and Davis, J.N., Anatomy of brain alpha-l-adrenergic receptors: in vitro autoradiography with [125I]-HEAT, J. Comp. Neurol., 231 (1985) 190-208. 8 Kimura, T., Share, L., Wang, B.C. and Crofton, J.T., The role of central adrenoreceptors in the control of vasopressin release and blood pressure, Endocrinology, 108 (1981) 1829-1836. 9 Kuhn, E.R., Cholinergic and adrenergic release mechanisms for vasopressin in the male rat: a study with injections of neurotransmitters and blocking agents into the third ventricle, Neuroendocrinology, 16 (1974) 255-264. 10 Leibowitz, S.E, Paraventricular nucleus: a primary site mediating adrenergic stimulation of feeding and drinking, Pharmacol. Biochem. Behav., 8 (1978) 163-175. 11 Moxham, C.P., George, S.T., Graziano, M.P., Brandwein, H.J. and Malbon, C.C., Mammalian betas- and beta 2adrenergic receptors. Immunological and structural comparisons, J. Biol. Chem., 261 (1986) 14562-14570. 12 Palacios, J.M. and Wamsley, J.K., Catecholamine receptors. In A. BjOrklund, T. H6kfelt and M.J. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, Vol. 3, Elsevier, Amsterdam, 1984, pp. 325-351. 13 Rainbow, T.C., Parson, B. and Wolfe, B.B., Quantitative autoradiography of betas- and beta2-adrenergic receptors in rat brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1585-1589. 14 Sakai, K.K., Marks, B.H., George, J.M. and Koestler, A., The isolated organ-cultured supraoptic nucleus as a neu-
15
16
17
18
19
20
21
22
23
ropharmacological test system, J. Pharmacol. Exp. Ther., 190 (1974) 482-491. Sladek, C.D., Regulation of vasopressin release by neurotransmitters, neuropeptides and osmotic stimuli. In B.A. Cross and G. Leng (Eds.), The Neurohypophysis: Structure, Function and Control, Progress in Brain Research, Vol. 60 (1983) 71-90. Swanson, L.W. and Sawchenko, P.E., Hypothalamic integration of paraventricular and supraoptic nuclei, Annu. Rev. Neurosci., 6 (1983) 275-323. Szabodi, E., Adrenoceptors on central neurons: microelectrophoretic studies, Neuropharmacology, 18 (1979) 831843. 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, Brain Res. Rev., 7 (1984) 69-101. Wamsley, J.K., Lewis, M.S., Young III, W.M. and Kuhar, M.J., Autoradiographic localization of muscarinic cholinergic receptors in rat brain stem, J. Neurosci., 1 (1981) 176-191. Wanaka, A., Kiyama, H., Murakami, T., Matsumoto, M., Kamada, T., Malbon, C.C. and Tohyama, M., Immunocytochemical localization of fl-adrenergic receptors in the rat brain, Brain Research, 485 (1989) 125-140. Warkerley, J.B., Noble, R. and Clarke, G., In vitro studies of the control of phasic discharge in neurosecretory cells of the supraoptic nucleus. In B.A. Cross and G. Leng (Eds.), The Neurophysis: Structure, Function and Control, Progress in Brain Research, Vol. 60, 1983, pp. 53-59. Young III, W.S. and Kuhar, M.J., Noradrenergic alpha-1 and alpha-2 receptors: light microscopic autoradiographic localization, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1969-1700. Zamboni, L. and De Martino, C., Buffered picric acid: a new rapid fixative for electron microscopy, J. Cell Biol., 35 (1967) 148A.
Brain Research, 499 (1989) 174-178
Elsevier
BRES 23753
I -Adrenergic receptors in the vasopressin-containing neurons in the paraventricular and supraptic nucleus of the rat T. T a k a n o I , Y. K u b o t a 1, A . W a n a k a ~, S. U s u d a ~. M . T a n a k a ~, C . C . M a l b o n 2 a n d M. T o h y a m a 1 ~Department of Anatomy II, Osaka University Medical School, Osaka (Japan) and 2Department of Pharmacology, School of Medicine, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY I1794-8651 (U.S.A.)
(Accepted 20 June 1989) Key words: fl-Adrenergic receptor; Vasopressin neuron; Paraventricular nucleus; Supraoptic nucleus; Rat; Immunocytochemistry
Immunocytochemical staining of alternate consecutive sections revealed that neurons with vasopressin-like immunoreactivity in the paraventricular and supraoptic nuclei had fl-adrenergic receptor-like immunoreactivity in the rats.
It is well known that paraventricular (PV) and supraoptic (SO) nuclei, which contain clusters of vasopressin (VAS) neurons (see ref. 16 for a review), are sites receiving a dense innervation of noradrenaline (NA) fibers 3'6. In addition, a number of studies have been carried out on the effect of VAS release by NA (see refs. 5, 8, 15, 21 for reviews), though the results of these studies have been contradictory. There is no doubt that the NA effects on the VAS neurons are mediated by adrenergic receptors ( A d R ) 2'5'9'14'21 . Leibowitz has suggested the presence of both types of AdR in the PV and they have shown that PV is a primary site mediating adrenergic stimulation of feeding and drinking 1°. Electrophysiological studies have suggested the presence of both types of AdR on the VAS neurons of the SO5'~4'21: the excitatory effect of a - A d R agonists and weak inhibitory effect of fl-AdR agonists on phasic activity of VAS neurons. However, until now, no direct evidence demonstrating the presence of AdR on the VAS neurons was shown. Therefore, we examined this possibility, using immunohistochemical staining of alternate consecutive sections with anti VAS antiserum and anti fl-AdR antiserum.
The preparation and characterization of the antiserum against fl-AdR has been described elsewhere j1'2°. In brief, the antiserum was produced in a rabbit immunized with affinity-purified fl2-AdR from guinea pig lung. The titer of the antiserum was tested with the use of solid-phase enzyme-linked immunosolvent (ELISA) and immunoprecipitation, This antiserum was proven to cross-react to the extent of 60-84% with purified fll-AdR of rat adipose cells. On the basis of the distribution determined with immunocytochemistry using this antiserum to compare the findings by autoradiography 7'12'13'1s'19'22, it is considered that there is little cross-reactivity with cq-AdR, dopamine receptors, muscarinic cholinergic receptors, or the photoreceptor rhodopsin. The findings from the inhibition test of ligand binding and displacement experiments with an excess of clonidine suggested that the antiserum did not cross-react with a2-AdR. An absorption test was also performed to check the specificity. Absorption of the antiserum with 5/~g of purified fl2-AdR resulted in a marked reduction of the immunostaining. Synthetic arginine VAS bound to bovine thyroglobulin with carbodiimide was used as an antigen for anti VAS antiserum (Miles). The
Correspondence: M. Tohyama, Department of Anatomy II, Osaka University Medical School, 4-3-57 Nakanoshima, Kitaku, Osaka,
530, Japan.
175 antiserum has been raised in New Zealand White rabbits by repeated injections of the conjugates emulsified in Freund's Complete adjuvant. Crossreactivity with oxytocin was tested in the Brattleboro rat, a strain that lacks the ability to synthesize VAS and demonstrated no cross-reactivity with oxytocin. Five male albino rats weighing about 100 g each were used. The animals were anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and perfused intracardially with 50 ml of ice-cold saline followed by 500 ml of Zamboni's fixative 23. The brains were removed, immersed in the same fixative overnight at 4 °C, and then rinsed for 48 h in 0.1 M phosphate buffer (pH 7.4) containing 30% sucrose. Serial frozen sections, 5/~m thick, through the PV and SO
were prepared using a cryostat. Alternate sections were processed by the indirect immunofluorescence method developed by Coons 4 to determine the VAS-like immunoreactivity (IR) or fl-AdR-IR. The dilutions of the first antiserum with phosphatebuffered saline (PBS) were as follows: VAS 1:1000; and fl-AdR 1:50. Fluorescein isothiocyanate (FITC)conjugated goat anti rabbit IgG (Miles) was used as the second antiserum. The second antiserum was diluted to 1:1000 with PBS. Fig. 1A and C show a pair of the consecutive sections through the posterior magnocellular portion of the PV, stained for vasopressin (Fig. 1A) and fl-AdR (Fig. 1C). A cluster of cells with VAS-IR and fl-AdR-IR can be seen in the posterior magnocellu-
Fig. 1. A,C: light micrographs of serial sections through the posterior magnocellular (M) and medial parovcellular part (P) of the paraventricular nucleus, incubated with anti-VAS antiserum (A) and anti-fl-AdRantiserum (C). In the M, a cluster of cells with VAS-IR and fl-AdR-IR were seen. The frames are enlarged in B and D. The cells which are numbered in B and D are identical. Most of the cells in the M contained both VAS-IRand fl-AdR-IR. A-D, frontal sections. Bar for A,C = 150 pm. Bar for B,D = 10 pm.
176 lar portion. In addition, fl-AdR-IR cells were diffusely scattered in other hypothalamic areas (Fig. 1C). The frames in Fig. 1A and C show VAS-IR and fl-AdR-IR cells in the posterior magnocellular part and are enlarged in Fig. 1B and D. Most of the neurons in the posterior magnocellular part were large and contained both VAS-IR and fl-AdR-IR. Fig. 2A and C are another pair of the serial sections through the posterior magnocellular part of the PV. The frames are located in the medial parvocellular portion and are enlarged in Fig. 2B and D, respectively. Cells containing VAS-IR were scattered in the medial palvocellular portion. Two types of cells with VAS-IR were distinguished: one was large and the other was small in size. Most of the two types of VAS-IR cells contained fl-AdR-IR. Fig. 3A and C are a pair of serial sections through the SO. Fig. 3B and D are higher magnifications of
the boxes in Fig. 3A and C. Most of the cells with fl-AdR contained VAS-IR. However, a significant number of cells with VAS-IR which were devoid of fl-AdR-IR was also found (about 30-40%) (Fig. 3). PV and SO contain numerous NA fibers most of which originate from A 1 NA neurons 16. There has been much disagreement among the studies concerning the effect of NA on VAS release (see ref. 15 for a review). Bridges and Thorn have reported the excitatory effect of NA on the release of VAS and have suggested that this effect is mediated by a - A d R 2. Conversely, there exist reports which show the inhibitory effect of central NA on VAS secretion 8. Armstrong et al. have confirmed this effect and suggested that a - A d R mediates this effect 1. Electrophysiological studies using slices through the SO have revealed the excitatory effect of a - A d R agonists and the weak inhibitory effect of fl-AdR
Fig. 2. A,C: light micrographs of serial sections through P and M of the PV, stained with anti VAS antiserum (A) and anti-fl-AdR antiserum (C). In the P, cells with VAS-IR or fl-AdR-IR were scattered. B and D are higher magnifications of the frames of A and C, respectively. Both large (arrowheads) and small (arrow) cells with VAS-IR contained fl-AdR-IR. Frontal sections. Bar for A,C = 150/~m. Bar for B,D --- 10 am.
177
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i
il ¸
Fig. 3. A,C: light micrographs of serial sections through the supraoptic nucleus (SO), stained with anti VAS antiserum (A) and anti-fl-AdR antiserum (C). Cells with fl-AdR-IR were less numerous than those with VAS-IR, B,D: enlargements of the frames in A and C. Arrowheads indicate the cells which contained both VAS-IR and fl-AdR-IR. Frontal sections. Bar for A,C = 150/~m. Bar for B,D = 15/~m.
agonists on the phasic activity of V A S cells 14'21. The p r e s e n t study shows the presence of cells with f l - A d R - I R on the V A S neurons in the PV and SO, indicating that the N A effect on V A S neurons via f l - A d R exists and is monosynaptically regulated by an ascending N A system from A 1 N A neurons. H o w e v e r , the p r o p o r t i o n of the cells with V A S - I R which contain differences in f l - A d R - I R were n o t e d b e t w e e n the PV and SO: most of the V A S cells in the PV contained f l - A d R , while about 60% of V A S cells in the SO contained f l - A d R . This m a y be attributed to the difference in character between the V A S neurons in the PV and SO. H o w e v e r , p r o b l e m s of
This study was s u p p o r t e d in part by grants from the Ministry of E d u c a t i o n of J a p a n and from M o c h i d a F o u n d a t i o n . C . C . M . is s u p p o r t e d by N I H Grants DK25410 and D K 30111.
1 Armstrong, W.E., Sladek, C.D. and Sladek Jr., J.R., Characterization of noradrenergic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system, Endocrinology, 111 (1982) 273-279. 2 Bridges, T.E. and Thon, N.A., The effect of autonomic blocking agents on vasopressin release in vivo by osmore-
ceptor stimulation, J. Endocrinol., 48 (1970) 265-276. 3 Carlsson, A., Faick, B. and Hillarp, N.-A., Cellular localization of brain monoamines, Acta Physiol. Scand., 56, Suppl. 196 (1962) 1-27. 4 Coons, A.H., Fluorescent antibody method. In J.E Danielli (Ed.), General Cytochemical Methods, Academic,
the titer should also be considered. T h e presence of a - A d R on the V A S neurons has also b e e n suggested 2,9,14,21. T h e possibility exists that a - A d R and f l - A d R are localized in the same neurons in some brain regions 17. The complex nature of the effects of N A on the V A S neurons dictates further analysis by the i m m u n o c h e m i c a l approach.
178 New York, 1958, pp. 399-422. 5 Day, T.A., Randle, J.C.R. and Renaud, L.P., Opposing alpha-adrenergic and beta-adrenergic mechanisms mediate dose-dependent actions of noradrenaline on supraoptic vasopressin neurons in vivo, Brain Research, 358 (1985) 171-179. 6 Fuxe, K., Evidence for the existence of monoamine neurons in the central nervous system. IV. The distribution of monoamine nerve terminals in the central nervous system, Acta Physiol. Scand., 64, Suppl. 247 (1965) 37-85. 7 Joness, L.S., Gauger, L.L. and Davis, J.N., Anatomy of brain alpha-l-adrenergic receptors: in vitro autoradiography with [125I]-HEAT, J. Comp. Neurol., 231 (1985) 190-208. 8 Kimura, T., Share, L., Wang, B.C. and Crofton, J.T., The role of central adrenoreceptors in the control of vasopressin release and blood pressure, Endocrinology, 108 (1981) 1829-1836. 9 Kuhn, E.R., Cholinergic and adrenergic release mechanisms for vasopressin in the male rat: a study with injections of neurotransmitters and blocking agents into the third ventricle, Neuroendocrinology, 16 (1974) 255-264. 10 Leibowitz, S.E, Paraventricular nucleus: a primary site mediating adrenergic stimulation of feeding and drinking, Pharmacol. Biochem. Behav., 8 (1978) 163-175. 11 Moxham, C.P., George, S.T., Graziano, M.P., Brandwein, H.J. and Malbon, C.C., Mammalian betas- and beta 2adrenergic receptors. Immunological and structural comparisons, J. Biol. Chem., 261 (1986) 14562-14570. 12 Palacios, J.M. and Wamsley, J.K., Catecholamine receptors. In A. BjOrklund, T. H6kfelt and M.J. Kuhar (Eds.), Handbook of Chemical Neuroanatomy, Vol. 3, Elsevier, Amsterdam, 1984, pp. 325-351. 13 Rainbow, T.C., Parson, B. and Wolfe, B.B., Quantitative autoradiography of betas- and beta2-adrenergic receptors in rat brain, Proc. Natl. Acad. Sci. U.S.A., 81 (1984) 1585-1589. 14 Sakai, K.K., Marks, B.H., George, J.M. and Koestler, A., The isolated organ-cultured supraoptic nucleus as a neu-
15
16
17
18
19
20
21
22
23
ropharmacological test system, J. Pharmacol. Exp. Ther., 190 (1974) 482-491. Sladek, C.D., Regulation of vasopressin release by neurotransmitters, neuropeptides and osmotic stimuli. In B.A. Cross and G. Leng (Eds.), The Neurohypophysis: Structure, Function and Control, Progress in Brain Research, Vol. 60 (1983) 71-90. Swanson, L.W. and Sawchenko, P.E., Hypothalamic integration of paraventricular and supraoptic nuclei, Annu. Rev. Neurosci., 6 (1983) 275-323. Szabodi, E., Adrenoceptors on central neurons: microelectrophoretic studies, Neuropharmacology, 18 (1979) 831843. 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, Brain Res. Rev., 7 (1984) 69-101. Wamsley, J.K., Lewis, M.S., Young III, W.M. and Kuhar, M.J., Autoradiographic localization of muscarinic cholinergic receptors in rat brain stem, J. Neurosci., 1 (1981) 176-191. Wanaka, A., Kiyama, H., Murakami, T., Matsumoto, M., Kamada, T., Malbon, C.C. and Tohyama, M., Immunocytochemical localization of fl-adrenergic receptors in the rat brain, Brain Research, 485 (1989) 125-140. Warkerley, J.B., Noble, R. and Clarke, G., In vitro studies of the control of phasic discharge in neurosecretory cells of the supraoptic nucleus. In B.A. Cross and G. Leng (Eds.), The Neurophysis: Structure, Function and Control, Progress in Brain Research, Vol. 60, 1983, pp. 53-59. Young III, W.S. and Kuhar, M.J., Noradrenergic alpha-1 and alpha-2 receptors: light microscopic autoradiographic localization, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 1969-1700. Zamboni, L. and De Martino, C., Buffered picric acid: a new rapid fixative for electron microscopy, J. Cell Biol., 35 (1967) 148A.