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α1-Adrenergic receptor activation releases vasopressin and oxytocin from perfused rat hypothalamic explants
Neuroscience Letters, 65 (1986) 219-223
219
Elsevier Scientific Publishers Ireland Ltd.
NSL 03844
~I-ADRENERGIC RECEPTOR ACTIVATION RELEASES VASOPRESSIN AND OXYTOCIN FROM PERFUSED RAT HYPOTHALAMIC EXPLANTS
J.C.R. R A N D L E ~, M. M A Z U R E K 2, D. K N E I F E L I, J. D U F R E S N E l and L.P. R E N A U D l,*
INeurosciences Unit, Montreal General Hospital and McGill University, 1650 Cedar Avenue, Montreal, Que. H3G 1A4 (Canada) and 2Department of Neurology, Massachusetts General Hospital, Harvard. University, Boston, MA 02114 (U.S.A.) (Received December 11 th, 1985; Accepted December 20th, 1985)
Key words: norepinephrine - ~-adrenoreceptor - vasopressin - oxytocin - hormone release - in vitro study - rat
Norepinephrine and the ~-agonist phenylephrine in concentrations of 10 -5 to 10 -3 M prompted the release of radioimmunoassayable vasopressin (up to 150 pg/min) and oxytocin (up to 20 pg/min) from intraarterially perfused explants of rat basal forebrain. Drug effects were markedly reduced or abolished in the presence of the non-specific ~-antagonists phentolamine and phenoxybenzamine, and the specific ~ antagonist prazosin. In concert with recent in vivo and in vitro electrophysiological observations, these data imply that endogenous noradrenergic pathways to magnocellular neurosecretory cells are excitatory, mediated through activation o f their ~j-receptors, thereby enhancing the release of both vasopressin and oxytocin in the neurohypophysis.
The magnocellular neurosecretory neurons of the hypothalamic supraoptic (SON) and paraventricular nuclei receive a dense catecholamine innervation from the A1 noradrenergic neurons of the ventrolateral medulla [17]. In the light microscope, this input appears to be preferentially distributed around the vasopressinergic rather than the oxytocinergic neuronal population [11]. Despite a long-standing impression that norepinephrine (NE) is involved in the release of vasopressin in the neurohypophysis, there is still uncertainty as to its actual role. On the one hand, experience with microiontophoresis [3, 4], ventrolateral medullary lesions [5] and hormone release from hypothalamic explants maintained in vitro [2] support a suppressant influence. In contrast, observations derived from intracerebral NE [13] and 6-hydroxydopamine injections [10, 12], and both in vivo [7, 8] and in vitro [15, 19] electrophysiological studies argue for a stimulatory role on vasopressin release. With respect to oxytocin secretion, both facilitatory [18] and depressant [14] influences of NE have been observed. We now report that in acutely prepared and intra-arterially perfused hypothalamic explants [6], NE prompts the release of both vasopressin and oxytocin through an ~:adrenergic mechanism.
*Author for correspondence and reprint requests. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
220 Experiments utilized male Sprague-Dawley rats (150-300 g). Following decapitation, the brain and intact pituitary were removed and an 8 × 8 x 3 mm segment of the basal forebrain was pinned to a Sylgard base in a humidified, oxygenated (95% 02-5% CO2) and temperature-controlled (33°C) environment. Within 5-7 min of decapitation, a micropipette, through which oxygenated artificial medium was flowing at 1-2 ml/min, was inserted by micromanipulation into each carotid artery stump and advanced into the anterior cerebral artery. Medium contained (in mM): NaC1 126, KCI 3.0, KH2PO4 1.2, MgSO4 1.3, CaCI2 2.0, NaHCO3 25.5, o-glucose 9.9, as well as phenol red (10 rag/l) and bovine serum albumin (1.0 g/l). Medium tonicity was confirmed at 295 + 3 mOsm/l by cryo-osmometry. Additions to the medium included norepinephrine bitartrate, phentolamine mesylate and the hydrochloride salts of phenylephrine, phenoxybenzamine and prazosin. Drugs added at regular intervals from separate infusion lines reached the preparation in known concentrations, established on the basis of drug dilution curves. After removal of the adenohypophysis, a separate micropipette connected to a peristaltic pump was positioned near the exposed neurointermediate lobe to collect the medium which flowed freely into this area after exiting from the cut ends of the cerebral arteries. Samples were collected in 2-min intervals. Vasopressin and oxytocin were measured by radioimmunoassay. Arginine vasopressin-like (AVP-LI) and oxytocin-like immunoreactivity (OXY-LI) contained in experimental samples were identical to the synthetic peptides in competition experiments and high-performance liquid chromatography. As illustrated in Fig. 1, both vasopressin and oxytocin were detected in the perfusate immediately following preparation of the explant, gradually declining over the initial 30-50 min from rates of 10-50 pg/min to levels approaching the lower limits of detection of the assay (1-2 pg/min). NE infused into the perfusion medium so as to achieve concentrations of 10 -5 to 10 -3 M induced rapid, readily reversible and dose-related increases in the release of both vasopressin and oxytocin (Fig. 1). Values differed quantitatively but not qualitatively between experiments. In experiments ( n = 12) where the response to the first 3-min infusion of l f f 5 M NE was measured, vasopressin release reached 45.1 + 13.3 pg/min (mean+S.E.M.) compared with 6.7+2.5 pg/ min for oxytocin. The addition of 3 × 10 -5 to 10 -4 M NE achieved a vasopressin release rate of 100-150 pg/min compared with 15-20 pg/min for oxytocin. In each instance, a 3- to 20-fold greater amount of vasopressin was released. Repeated drug trials in any individual experiment (n = 3, with up to 5 trials per experiment) continued to evoke release of both hormones although values for vasopressin (but not for oxytocin) decreased with each succeeding drug challenge (Fig. 1). Receptor specificity of the response was assessed in two ways. First, the ~,-adrenergic agonist phenylephrine (10 -5 M) was observed to evoke release of both vasopressin (up to 40.7 + 19.6 pg/min) and oxytocin (up to 25.1 + 22.3 pg/min) in 3 experiments. Second, in response to repeated trials with NE, the release of both hormones was reduced following administration of phentolamine (10 -~ M), phenoxybenzamine (10-5 M) and the selective ~-antagonist prazosin (10 7 M; see Fig. 2). These observations confirm that NE evokes release of both vasopressin and oxytocin at concentrations identical to those that facilitate the firing of supraoptic neuro-
221
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NE ( 6 x 1 0 °5 M) Fig. 1. NE releases neurohypophysial hormones. Radioimmunoassay data reveal that both vasopressin (closed circles) and oxytocin (open circles) are initially released into the medium from perfused hypothalamic explants, declining to near undetectable baseline values over the first hour. Repeated 3-min infusions of NE (final concentrations listed) promote a reversible and repeatable rise in the release of both hormones. A decline in the amount of vasopressin release with successive trials was observed in each of 3 similar experiments.
secretory n e u r o n s in this p r e p a r a t i o n [15] as well as in vivo [8]. Moreover, the electrophysiological d a t a reveal that activation o f ~q-noradrenergic receptors o n S O N n e u r o n s n o t only increases their firing rate b u t also induces b u r s t i n g p a t t e r n s o f activity [15]. Interestingly, experiments using the isolated n e u r o h y p o p h y s i s [9] indicate that such p a t t e r n i n g is c o n d u c t i v e to efficient h o r m o n e release.
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100
120
140
160
180
200
PRAZOSIN ( 1 0 -7 M)
Fig. 2. cq-Adrenergic antagonists block hormone release. Profiles of hormone release reveal that the addition of the specific ~radrenergic antagonist prazosin markedly reduces the ability of NE to release both vasopressin and oxytocin. Complete reversal of the antagonist effect of prazozin (and, in separate experiment, phenoxybenzamine) was not attained over the time-course of these experiments presumably owing to its high affinity for a-receptors and hydrophobic nature. However, 70% recovery could be achieved after 10 -~M phentolamine.
The present data contradict the findings of an earlier in vitro release study using cultured hypothalamo-neurohypophysial explants [2]. Reasons for this difference are not readily apparent but may involve different methods of tissue preparation and maintenance, time of drug delivery and sampling after preparation and undefined changes in adrenergic receptor sensitivity. Relevant to this argument is a recent report that superfusion of phenylephrine in a similar explant preparation both enhances supraoptic neuronal firing and simultaneously promotes vasopressin release [1]. These combined observations support the hypothesis that the role of the ascending noradrenergic input from the ventrolateral medulla to hypothalamic magnocellular neurosecretory neurons is to augment hormone release from their posterior pituitary axon terminals through ~radrenergic receptor activation and the induction of bursting activity patterns. These studies were supported by the Quebec Heart Foundation and Canadian Medical Research Council. We thank Gwen Peard for typing the manuscript. 1 Armstrong, W.E., Gallagher, M.J. and Sladek, C.D., Noradrenergic stimulation of supraoptic neuronal activity and vasopressin release in vitro: mediation by an ~rreceptor, Brain Res., in press.
223 2 Armstrong, W.E., Sladek, C.D. and Sladek, J.R., Jr., Characterization of noradrenergic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system, Endocrinology, 111 (1982) 273-279. 3 Arnauld, E., Cirino, M., Layton, B.S. and Renaud, L.P., Contrasting actions of amino acids, acetylcholine, noradrenaline and leucine-enkephalin on the excitability of supraoptic vasopressin-secreting neurons, Neuroendocrinology, 36 (1983) 187-196. 4 Barker, J.L., Crayton, J.W. and Nicoll, R.A., Noradrenaline and acetylcholine response of supraoptic neurosecretory cells, J. Physiol., (London), 218 ( 1971) 19-32. 5 Blessing, W.W., Sved, A.F. and Reis, D.J., Destruction of noradrenergic neurons in rabbit brainstem elevates plasma vasopressin, causing hypertension, Science, 217 (1982) 661-663. 6 Bourque, C.W. and Renaud, L.P., A perfused in-vitro preparation of hypothalamus for electrophysiological studies on neurosecretory neurons, J. Neurosci. Meth., 7 (1983) 203-214. 7 Day, T.A., Ferguson, A.V. and Renaud, L., Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells, J. Physiol. (London), 355 (1984) 237249. 8 Day, T.A., Randle, J.C.R. and Renaud, L.P., Opposing ~t- and fl-adrenergic mechanisms mediate dosedependent actions of noradrenaline on supraoptic vasopressin neurones in vivo, Brain Res., 358 (1985) 171-179. 9 Dutton, A. and Dyball, R.E.J., Phasic firing enhances vasopressin release from the neurohypophysis, J. Physiol. (London), 290 (1979)433-440. 10 Lightman, S.L., Todd, K. and Everitt, B.J., Ascending noradrenergic projections from the brainstem: evidence for a major role in the regulation of blood pressure and vasopressin secretion, Exp. Brain Res., 55 (1984) 145-151. 11 McNeill, T.H. and Sladek, J.R., Jr., Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry. II. Correlative distribution of catecholamine varicosities and magnocellular neurosecretory neurons in the rat supraoptic and paraventricular nuclei, J. Comp. Neurol., 193 (1980) 1023-1033. 12 Milton, A.S. and Paterson, A.T., Intracranial injections of 6-hydroxydopamine (6-OHDA) in cats: effects on the release of antidiuretic hormone, Brain Res., 61 (1973) 423-427. 13 Milton, A.S. and Paterson, A.T., A microinjection study on the control of antidiuretic hormone release by the supraoptic nucleus of the hypothalamus in the cat, J. Physiol. (London), 241 (1974) 607-628. 14 Moos, F. and Richard, P., The inhibitory role of ct-adrenergic receptors in oxytocin release during suckling, Brain Res., 169 (1979) 595-599. 15 Randle, J.C.R., Bourque, C.W. and Renaud, L.P., at-adrenergic activation of rat hypothalamic supraoptic neurons maintained in vitro, Brain Res., 307 (1984) 374-378. 16 Renaud, L.P., Day, T.A., Randle, J.C.R. and Bourque, C.W., In-vivo and in-vitro electrophysiological evidence that central noradrenergic pathways enhance the activity of hypothalamic vasopressinergic neurosecretory cells. In R.W. Schrier (Ed.), Vasopressin, Raven Press, New York, 1985, pp. 385-393. 17 Sawchenko, P.E. and Swanson, L.W., The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev., 4 (1982) 275-325. 18 Tribollet, E., Clarke, G., Dreifuss, J.J. and Lincoln, D.W., The role of central adrenergic receptors in the reflex release of oxytocin, Brain Res., 142 (1978) 69-84. 19 Wakerley, J.B., Noble, R. and Clarke, G., In vitro studies of the control of phasic discharges in neurosecretory cells of the supraoptic nucleus. Prog. Brain Res., 60 (1983) 53-59.
219
Elsevier Scientific Publishers Ireland Ltd.
NSL 03844
~I-ADRENERGIC RECEPTOR ACTIVATION RELEASES VASOPRESSIN AND OXYTOCIN FROM PERFUSED RAT HYPOTHALAMIC EXPLANTS
J.C.R. R A N D L E ~, M. M A Z U R E K 2, D. K N E I F E L I, J. D U F R E S N E l and L.P. R E N A U D l,*
INeurosciences Unit, Montreal General Hospital and McGill University, 1650 Cedar Avenue, Montreal, Que. H3G 1A4 (Canada) and 2Department of Neurology, Massachusetts General Hospital, Harvard. University, Boston, MA 02114 (U.S.A.) (Received December 11 th, 1985; Accepted December 20th, 1985)
Key words: norepinephrine - ~-adrenoreceptor - vasopressin - oxytocin - hormone release - in vitro study - rat
Norepinephrine and the ~-agonist phenylephrine in concentrations of 10 -5 to 10 -3 M prompted the release of radioimmunoassayable vasopressin (up to 150 pg/min) and oxytocin (up to 20 pg/min) from intraarterially perfused explants of rat basal forebrain. Drug effects were markedly reduced or abolished in the presence of the non-specific ~-antagonists phentolamine and phenoxybenzamine, and the specific ~ antagonist prazosin. In concert with recent in vivo and in vitro electrophysiological observations, these data imply that endogenous noradrenergic pathways to magnocellular neurosecretory cells are excitatory, mediated through activation o f their ~j-receptors, thereby enhancing the release of both vasopressin and oxytocin in the neurohypophysis.
The magnocellular neurosecretory neurons of the hypothalamic supraoptic (SON) and paraventricular nuclei receive a dense catecholamine innervation from the A1 noradrenergic neurons of the ventrolateral medulla [17]. In the light microscope, this input appears to be preferentially distributed around the vasopressinergic rather than the oxytocinergic neuronal population [11]. Despite a long-standing impression that norepinephrine (NE) is involved in the release of vasopressin in the neurohypophysis, there is still uncertainty as to its actual role. On the one hand, experience with microiontophoresis [3, 4], ventrolateral medullary lesions [5] and hormone release from hypothalamic explants maintained in vitro [2] support a suppressant influence. In contrast, observations derived from intracerebral NE [13] and 6-hydroxydopamine injections [10, 12], and both in vivo [7, 8] and in vitro [15, 19] electrophysiological studies argue for a stimulatory role on vasopressin release. With respect to oxytocin secretion, both facilitatory [18] and depressant [14] influences of NE have been observed. We now report that in acutely prepared and intra-arterially perfused hypothalamic explants [6], NE prompts the release of both vasopressin and oxytocin through an ~:adrenergic mechanism.
*Author for correspondence and reprint requests. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
220 Experiments utilized male Sprague-Dawley rats (150-300 g). Following decapitation, the brain and intact pituitary were removed and an 8 × 8 x 3 mm segment of the basal forebrain was pinned to a Sylgard base in a humidified, oxygenated (95% 02-5% CO2) and temperature-controlled (33°C) environment. Within 5-7 min of decapitation, a micropipette, through which oxygenated artificial medium was flowing at 1-2 ml/min, was inserted by micromanipulation into each carotid artery stump and advanced into the anterior cerebral artery. Medium contained (in mM): NaC1 126, KCI 3.0, KH2PO4 1.2, MgSO4 1.3, CaCI2 2.0, NaHCO3 25.5, o-glucose 9.9, as well as phenol red (10 rag/l) and bovine serum albumin (1.0 g/l). Medium tonicity was confirmed at 295 + 3 mOsm/l by cryo-osmometry. Additions to the medium included norepinephrine bitartrate, phentolamine mesylate and the hydrochloride salts of phenylephrine, phenoxybenzamine and prazosin. Drugs added at regular intervals from separate infusion lines reached the preparation in known concentrations, established on the basis of drug dilution curves. After removal of the adenohypophysis, a separate micropipette connected to a peristaltic pump was positioned near the exposed neurointermediate lobe to collect the medium which flowed freely into this area after exiting from the cut ends of the cerebral arteries. Samples were collected in 2-min intervals. Vasopressin and oxytocin were measured by radioimmunoassay. Arginine vasopressin-like (AVP-LI) and oxytocin-like immunoreactivity (OXY-LI) contained in experimental samples were identical to the synthetic peptides in competition experiments and high-performance liquid chromatography. As illustrated in Fig. 1, both vasopressin and oxytocin were detected in the perfusate immediately following preparation of the explant, gradually declining over the initial 30-50 min from rates of 10-50 pg/min to levels approaching the lower limits of detection of the assay (1-2 pg/min). NE infused into the perfusion medium so as to achieve concentrations of 10 -5 to 10 -3 M induced rapid, readily reversible and dose-related increases in the release of both vasopressin and oxytocin (Fig. 1). Values differed quantitatively but not qualitatively between experiments. In experiments ( n = 12) where the response to the first 3-min infusion of l f f 5 M NE was measured, vasopressin release reached 45.1 + 13.3 pg/min (mean+S.E.M.) compared with 6.7+2.5 pg/ min for oxytocin. The addition of 3 × 10 -5 to 10 -4 M NE achieved a vasopressin release rate of 100-150 pg/min compared with 15-20 pg/min for oxytocin. In each instance, a 3- to 20-fold greater amount of vasopressin was released. Repeated drug trials in any individual experiment (n = 3, with up to 5 trials per experiment) continued to evoke release of both hormones although values for vasopressin (but not for oxytocin) decreased with each succeeding drug challenge (Fig. 1). Receptor specificity of the response was assessed in two ways. First, the ~,-adrenergic agonist phenylephrine (10 -5 M) was observed to evoke release of both vasopressin (up to 40.7 + 19.6 pg/min) and oxytocin (up to 25.1 + 22.3 pg/min) in 3 experiments. Second, in response to repeated trials with NE, the release of both hormones was reduced following administration of phentolamine (10 -~ M), phenoxybenzamine (10-5 M) and the selective ~-antagonist prazosin (10 7 M; see Fig. 2). These observations confirm that NE evokes release of both vasopressin and oxytocin at concentrations identical to those that facilitate the firing of supraoptic neuro-
221
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NE ( 6 x 1 0 °5 M) Fig. 1. NE releases neurohypophysial hormones. Radioimmunoassay data reveal that both vasopressin (closed circles) and oxytocin (open circles) are initially released into the medium from perfused hypothalamic explants, declining to near undetectable baseline values over the first hour. Repeated 3-min infusions of NE (final concentrations listed) promote a reversible and repeatable rise in the release of both hormones. A decline in the amount of vasopressin release with successive trials was observed in each of 3 similar experiments.
secretory n e u r o n s in this p r e p a r a t i o n [15] as well as in vivo [8]. Moreover, the electrophysiological d a t a reveal that activation o f ~q-noradrenergic receptors o n S O N n e u r o n s n o t only increases their firing rate b u t also induces b u r s t i n g p a t t e r n s o f activity [15]. Interestingly, experiments using the isolated n e u r o h y p o p h y s i s [9] indicate that such p a t t e r n i n g is c o n d u c t i v e to efficient h o r m o n e release.
222 60
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100
120
140
160
180
200
PRAZOSIN ( 1 0 -7 M)
Fig. 2. cq-Adrenergic antagonists block hormone release. Profiles of hormone release reveal that the addition of the specific ~radrenergic antagonist prazosin markedly reduces the ability of NE to release both vasopressin and oxytocin. Complete reversal of the antagonist effect of prazozin (and, in separate experiment, phenoxybenzamine) was not attained over the time-course of these experiments presumably owing to its high affinity for a-receptors and hydrophobic nature. However, 70% recovery could be achieved after 10 -~M phentolamine.
The present data contradict the findings of an earlier in vitro release study using cultured hypothalamo-neurohypophysial explants [2]. Reasons for this difference are not readily apparent but may involve different methods of tissue preparation and maintenance, time of drug delivery and sampling after preparation and undefined changes in adrenergic receptor sensitivity. Relevant to this argument is a recent report that superfusion of phenylephrine in a similar explant preparation both enhances supraoptic neuronal firing and simultaneously promotes vasopressin release [1]. These combined observations support the hypothesis that the role of the ascending noradrenergic input from the ventrolateral medulla to hypothalamic magnocellular neurosecretory neurons is to augment hormone release from their posterior pituitary axon terminals through ~radrenergic receptor activation and the induction of bursting activity patterns. These studies were supported by the Quebec Heart Foundation and Canadian Medical Research Council. We thank Gwen Peard for typing the manuscript. 1 Armstrong, W.E., Gallagher, M.J. and Sladek, C.D., Noradrenergic stimulation of supraoptic neuronal activity and vasopressin release in vitro: mediation by an ~rreceptor, Brain Res., in press.
223 2 Armstrong, W.E., Sladek, C.D. and Sladek, J.R., Jr., Characterization of noradrenergic control of vasopressin release by the organ-cultured rat hypothalamo-neurohypophyseal system, Endocrinology, 111 (1982) 273-279. 3 Arnauld, E., Cirino, M., Layton, B.S. and Renaud, L.P., Contrasting actions of amino acids, acetylcholine, noradrenaline and leucine-enkephalin on the excitability of supraoptic vasopressin-secreting neurons, Neuroendocrinology, 36 (1983) 187-196. 4 Barker, J.L., Crayton, J.W. and Nicoll, R.A., Noradrenaline and acetylcholine response of supraoptic neurosecretory cells, J. Physiol., (London), 218 ( 1971) 19-32. 5 Blessing, W.W., Sved, A.F. and Reis, D.J., Destruction of noradrenergic neurons in rabbit brainstem elevates plasma vasopressin, causing hypertension, Science, 217 (1982) 661-663. 6 Bourque, C.W. and Renaud, L.P., A perfused in-vitro preparation of hypothalamus for electrophysiological studies on neurosecretory neurons, J. Neurosci. Meth., 7 (1983) 203-214. 7 Day, T.A., Ferguson, A.V. and Renaud, L., Facilitatory influence of noradrenergic afferents on the excitability of rat paraventricular nucleus neurosecretory cells, J. Physiol. (London), 355 (1984) 237249. 8 Day, T.A., Randle, J.C.R. and Renaud, L.P., Opposing ~t- and fl-adrenergic mechanisms mediate dosedependent actions of noradrenaline on supraoptic vasopressin neurones in vivo, Brain Res., 358 (1985) 171-179. 9 Dutton, A. and Dyball, R.E.J., Phasic firing enhances vasopressin release from the neurohypophysis, J. Physiol. (London), 290 (1979)433-440. 10 Lightman, S.L., Todd, K. and Everitt, B.J., Ascending noradrenergic projections from the brainstem: evidence for a major role in the regulation of blood pressure and vasopressin secretion, Exp. Brain Res., 55 (1984) 145-151. 11 McNeill, T.H. and Sladek, J.R., Jr., Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry. II. Correlative distribution of catecholamine varicosities and magnocellular neurosecretory neurons in the rat supraoptic and paraventricular nuclei, J. Comp. Neurol., 193 (1980) 1023-1033. 12 Milton, A.S. and Paterson, A.T., Intracranial injections of 6-hydroxydopamine (6-OHDA) in cats: effects on the release of antidiuretic hormone, Brain Res., 61 (1973) 423-427. 13 Milton, A.S. and Paterson, A.T., A microinjection study on the control of antidiuretic hormone release by the supraoptic nucleus of the hypothalamus in the cat, J. Physiol. (London), 241 (1974) 607-628. 14 Moos, F. and Richard, P., The inhibitory role of ct-adrenergic receptors in oxytocin release during suckling, Brain Res., 169 (1979) 595-599. 15 Randle, J.C.R., Bourque, C.W. and Renaud, L.P., at-adrenergic activation of rat hypothalamic supraoptic neurons maintained in vitro, Brain Res., 307 (1984) 374-378. 16 Renaud, L.P., Day, T.A., Randle, J.C.R. and Bourque, C.W., In-vivo and in-vitro electrophysiological evidence that central noradrenergic pathways enhance the activity of hypothalamic vasopressinergic neurosecretory cells. In R.W. Schrier (Ed.), Vasopressin, Raven Press, New York, 1985, pp. 385-393. 17 Sawchenko, P.E. and Swanson, L.W., The organization of noradrenergic pathways from the brainstem to the paraventricular and supraoptic nuclei in the rat, Brain Res. Rev., 4 (1982) 275-325. 18 Tribollet, E., Clarke, G., Dreifuss, J.J. and Lincoln, D.W., The role of central adrenergic receptors in the reflex release of oxytocin, Brain Res., 142 (1978) 69-84. 19 Wakerley, J.B., Noble, R. and Clarke, G., In vitro studies of the control of phasic discharges in neurosecretory cells of the supraoptic nucleus. Prog. Brain Res., 60 (1983) 53-59.