尾仲 達史

Behavioral experiments have shown that the N-methyl D-aspartate (NMDA) subclass of glutamate receptor plays an important role in acquisition of emotional memory. Exposure of a rat to conditioned fear stimuli suppresses vasopressin (VP) release and augments oxytocin (OT) or prolactin (PRL) release from the pituitary. Present experiments aimed at investigating the effect of intraperitonially administered MK-801, an antagonist of NMDA receptor on the emotional memory associated with the suppressive VP and the augmentative OT or PRL responses to conditioned fear stimuli in male rats. MK-801 injected 30 min before training impaired the VP, OT and PRL responses to the testing fear stimuli. The antagonist injected after training, however, did not block the responses. MK-801 administered before testing impaired the previously acquired VP, OT and PRL responses to conditioned fear stimuli. In the experiments with non-associatively applied fear stimuli, MK-801 did not block the VP, OT or PRL response. In the experiments with novel environmental stimuli: MK-801 did not impair VP, OT or PRL responses. The results suggest that an activation of NMDA receptors are required to acquire and recall but not to consolidate or retain the emotional memory associated with VP, OT and PRL responses to conditioned fear stimuli. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.

During prolonged exposure to morphine, oxytocin neurons of the rat supraoptic nucleus develop dependence, shown by hyperexcitation following morphine withdrawal. The present study investigated the role of afferent projections to the supraoptic nucleus in this withdrawal excitation. Rats were made morphine-dependent by continuous intracerebroventricular infusion of morphine at increasing doses (up to 50 mu g/h). On the sixth day, rats were anaesthetized with pentobarbitone and morphine withdrawal was precipitated by intraperitoneal injection of naloxone (5 mg/kg). Fos-immunoreactivity in the supraoptic nucleus, and also in the median preoptic nucleus, organum vasculosum of the lamina terminalis and subfornical organ, which project to the supraoptic nucleus, increased following morphine withdrawal. However, retrograde tracing from the supraoptic nucleus showed that, of the neurons in these regions which project to the supraoptic nucleus, only 0.4-7.1% expressed Fos in response to morphine withdrawal. Following morphine withdrawal, Fos-immunoreactivity was present in 39.2% and 19.8% of the tyrosine hydroxylase-immunoreactive neurons of the A1/C1 and A2/C2 cell groups. Of the cells in these regions identified as projecting to the supraoptic nucleus, 11.3% in the region of the A2 cell group and 12.7% in the region of the Al cell group expressed Fos after morphine withdrawal. In a second study, monoamine release was measured in the supraoptic nucleus of urethane-anaesthetized morphine-dependent and -naive rats. Retrodialysis of naloxone (10(-5) M) into the supraoptic nucleus induced a small increase in plasma oxytocin concentration in morphine-dependent rats (13.5+/-4.8 pg/ml increase) but not in naive rats (1.2+/-5.9 pg/ml decrease), with no significant change in monoamine release in either morphine-dependent or -naive rats. Intravenous injection of naloxone (5 mg/kg) 1 h later produced a further significant increase in plasma oxytocin concentration in morphine-dependent rats concomitant with a significant increase in noradrenaline release from the supraoptic nucleus. Thus, morphine-withdrawal excitation of supraoptic oxytocin neurons occurs concurrently with a modestly increased activity of their input from the brainstem, and very little activation in other known inputs. (C) 1997 IBRO. Published by Elsevier Science Ltd.

Noxious stimuli facilitate oxytocin release from the neurohypophysis. Oxytocin-secreting hypothalamic magnocellular neurosecretory neurons receive excitatory synaptic inputs from noradrenergic neurons in the medulla oblongata. The medulla oblongata includes the A2 noradrenergic and the A1 noradrenergic cells. Here we investigated whether medullary noradrenergic neurons mediate oxytocin release after noxious stimuli in male rats. 5-Amino-2,4-dihydroxy-alpha-methylphenylethylamine, a neurotoxin selective for noradrenergic fibers, was injected into the lateral cerebral ventricle or the medulla. Seven days after the injection, the hypothalamic content of noradrenaline was decreased. In the rats injected with the neurotoxin, the release of oxytocin but not vasopressin after footshocks was impaired. Surgical ablation by suction of the caudal dorsomedial medulla including the A2 cell region did not significantly affect oxytocin release after footshocks, though the surgery abolished oxytocin release after i.v. injection of cholecystokinin octapeptide. In the rats whose A2 cell region had been ablated, an i.c.v. injected ctl adrenoreceptor antagonist, benoxathian, blocked oxytocin release after footshocks. These results demonstrate that brainstem noradrenergic neurons mediate oxytocin release following noxious stimuli in the rat and suggest that responsible noradrenergic neurons are the Al cells in the caudal ventrolateral medulla.

Emotional stimuli suppress vasopressin secretion and potentiate oxytocin and prolactin secretion by the pituitary in the rat. We studied effects of central norepinephrine depletion on these hormonal responses to novel environmental or fear stimuli. Male Wistar rats were injected intracerebroventricularly with 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine, a selective neurotoxin to noradrenergic fibers. The neurotoxin treatment reduced the hypothalamic content of norepinephrine by 71% but did not significantly affect the dopamine content. Novel environmental stimuli suppressed vasopressin secretion and augmented secretion of oxytocin and prolactin in the vehicle-injected rats. The neurotoxin did not block the neuroendocrine responses. Intermittently applied electric footshocks also induced the similar neuroendocrine responses in the vehicle-injected rats. The neurotoxin significantly reduced the neuroendocrine responses. The drug, however, did not significantly alter vasopressin release after continuously applied footshocks. Environmental stimuli previously paired with footshocks (conditioned fear stimuli) suppressed vasopressin secretion and augmented secretion of oxytocin and prolactin in the vehicle-injected animals. Motor activity was suppressed during the conditioned fear stimuli. The neurotoxin impaired the neuroendocrine and behavioral responses whether the drug was injected before or after the conditioning. These data demonstrate the distinction between the neural mechanisms underlying the neuroendocrine responses to fear and to novel stimuli, suggesting that noradrenergic neurons are selectively involved in the hypothalamo-hypophysial responses to fear stimuli.

Effects of a benzodiazepine, chlordiazepoxide (CDP), on neuroendocrine responses to emotional stimuli were studied in male rats. In the experiments with conditioned fear stimuli, rats received a pure tone paired with footshocks in the training session and were tested on the following day with the same environmental stimuli. CDP injected i.p. 30 min before training impaired the suppressive vasopressin and the augmentative oxytocin or adrenocorticotrophic hormone (ACTH) responses to the conditioned fear stimuli. However, the drug, administered 30 min after the training, did not significantly alter the hormonal responses to conditioned fear stimuli. CDP administered 30 min before testing also abolished the hormonal responses to conditioned fear stimuli. The stimuli which were identical to those used for training or unconditioned fear stimuli (intermittent footshocks) also produced the vasopressin, oxytocin and ACTH responses, and CDP prevented these hormonal responses. The drug, however, did not prevent the hormonal responses to novel environmental stimuli. Novel stimuli are known to produce a state of anxiety. Thus the present experiments demonstrate that CDP discriminates between the neural circuits mediating fear- and anxiety-producing stimuli in the rat.

Noxious as well as hypertonic stimuli potentiate vasopressin and oxytocin secretion in rats. Neurohypophysial vasopressin- and oxytocin-secreting neurons receive inhibitory synaptic inputs mediated by gamma-aminobutyric acid (GABA). Benzodiazepines modulate GABA-A receptor activity in a facilitatory fashion. it is thus possible that benzodiazepines suppress vasopressin and oxytocin release after noxious stimuli. To test this hypothesis, we investigated whether chlordiazepoxide impairs the enhanced release of vasopressin and oxytocin after noxious or hypertonic stimuli in male rats. Chlordiazepoxide (5-20 mg/kg, i.p.) blocked dose-dependently the vasopressin and oxytocin responses to footshocks. Chlordiazepoxide, however, did not impair the hormonal responses to hypertonic stimulus. The results demonstrate that chlordiazepoxide selectively prevents vasopressin and oxytocin release after noxious stimuli and therefore suggest that the sites of chlordiazepoxide actions are not on the vasopressin or oxytocin neurons in rats.

Activation of abdominal vagal afferents by peripheral injection of cholecystokinin octapeptide induces oxytocin release into the circulation. To test the hypothesis that cholecystokinin increases oxytocin release via activation of noradrenergic afferents from the brainstem, we injected rats with 5-amino-2,4-dihydroxy-alpha-methylphenylethylamine, a selective neurotoxin to noradrenergic fibres, into a lateral cerebral ventricle. The neurotoxin treatment reduced the noradrenaline content in the hypothalamus by 75% and reduced the oxytocin secretion in response to cholecystokinin by over 90%. In separate experiments, the neurotoxin was injected unilaterally in the vicinity of the supraoptic nucleus to test whether direct noradrenergic afferents to the supraoptic nucleus are involved in the response to cholecystokinin. The injection reduced the immunoreactivity for dopamine beta-hydroxylase in the supraoptic nucleus and significantly decreased the number of the supraoptic neurons expressing Fos-like protein after cholecystokinin but not after hypertonic saline. In further experiments, rhodamine-conjugated latex microspheres were injected into the supraoptic nucleus to retrogradely label afferent neurons, and the brains were processed with double-immunohistochemistry for tyrosine hydroxylase and Fos-like protein. In the C2/A2 but not the C1/A1 region of the brainstem, cholecystokinin increased the expression of Fos-like protein in the population of retrogradely-labelled catecholaminergic cells. In the C2/A2 region, the majority of retrogradely labelled cells expressing Fos-like protein after cholecystokinin were catecholaminergic. We conclude that noradrenergic afferents from the A2 but not from the A1 region of the brainstem to the hypothalamus mediate, at feast in part, oxytocin release following cholecystokinin.

1. This study aimed to establish the site at which morphine acts to inhibit oxytocin release in response to peripheral administration of cholecystokinin (CCK). 2. Conscious rats were given morphine or vehicle followed by CCK or vehicle (I.V.). Fos immunoreactivity was apparent 90 min after CCK injection in the supraoptic nucleus of vehicle- but not morphine-pretreated animals. 3. In the dorsomedial (C2/A2) and the ventrolateral (C1/A1) regions of the brainstem, about half of the cells immunoreactive for tyrosine hydroxylase (TH) expressed Fos-like protein after CCK injection. In the C2/A2 region, 20% of the Fos-positive cells also showed TH immunoreactivity, whereas in the C1/A1 region 68% did so. Morphine treatment did not significantly change the number of cells expressing Fos immunoreactivity, or the percentage of TH-positive cells expressing Fos-like protein. 4. Amine release was measured in the supraoptic nucleus of urethane-anaesthetized rats using a microdialysis probe. An I.V. injection of CCK increased the concentrations in the dialysate of noradrenaline and serotonin, but not of either adrenaline or dopamine. Pretreatment with morphine (I.V.) blocked the effects of CCK in a naloxone-reversible manner. 5. Inclusion of morphine in the dialysate also blocked the increase in noradrenaline and serotonin in response to CCK in a naloxone-reversible manner. 6. These observations indicate that morphine acts near or within the supraoptic nucleus to block CCK-evoked noradrenaline release presynaptically. This presynaptic action of morphine may be a cause of the blockade of oxytocin release after CCK.

The nucleus tractus solitarii (NTS) projects directly to the oxytocin neurones of the supraoptic nucleus (SON), and relays afferent stimuli arising from the birth canal during parturition. About 80% of these projecting neurones are noradrenergic, and these same neurones are activated following systemic administration of cholecystokinin (CCK), which also results in an increased electrical and secretory activity in oxytocin neurones. Oxytocin release in response to CCK is abolished following selective neurotoxic destruction of these noradrenergic neurones. Oxytocin release following CCK (and that during parturition) is potently inhibited by morphine, which blocks the local noradrenaline release in the supraoptic nucleus. This acute opiate action involves presynaptic inhibition of the noradrenergic terminals, and occurs without marked suppression of the activity of noradrenergic cells in the NTS. During chronic exposure to morphine the oxytocin system becomes tolerant to, and dependent upon morphine. In the course of tolerance, oxytocin cell activation in response to CCK recovers from initial inhibition. However, the pathway that mediates this response does not appear to become dependent: the oxytocin cell response to CCK is unchanged by opiate withdrawal induced by naloxone, despite a large increase in the background electrical activity of oxytocin cells provoked by withdrawal. Nevertheless, expression of withdrawal excitation by oxytocin neurones is curiously contingent upon the activity of the noradrenergic input in that prior lesioning of this input has no effect upon the subsequent withdrawal excitation of oxytocin cells. Yet under urethane anaesthesia, acute pharmacological blockade of the noradrenergic input suppresses withdrawal. We discuss how these paradoxical observations might be reconciled, and note that the difference may be related to differing levels of tonic activity in the noradrenergic input. It is possible that dependence relies upon the input when it is there, but not when it is not.

Effects of novel environmental stimuli on vasopressin and oxytocin secretion by the pituitary were studied in dehydrated male rats. As the novel environmental stimuli, rats were transferred to an experimental room, placed in a box painted black and given a pure tone auditory stimulus of 2 kHz. Exposure of rats to the novel environmental stimuli for a period of 2 min decreased plasma concentrations of vasopressin and increased plasma levels of adrenocorticotrophic hormone (ACTH) and prolactin, but did not significantly change the plasma level of oxytocin. The stimuli, however, became ineffective for producing the suppressive vasopressin response as the period of exposure was prolonged to more than 5 and up to 30 min, although the prolonged stimuli were still effective for inducing facilitatory ACTH and prolactin responses. After repeated exposures of rats to the environmental stimuli once a day for 5 or 10 days, the stimuli became disabled from producing the suppressive vasopressin response. However, the rats were still capable of responding to the novel stimuli of another kind. All these data suggest that novelty stress suppresses vasopressin secretion but does not change oxytocin secretion. In order to test the possibility that glucocorticoids expectedly secreted by the adrenals in response to the stress might have suppressed vasopressin secretion, a large amount of dexamethasone was administered to the rat before testing. Dexamethasone pretreatment depressed plasma levels of ACTH and vasopressin as reported previously and blocked the facilitatory ACTH response to the novelty stress. However, dexamethasone treatment did not affect the suppressive vasopressin response to the novelty stress. Thus, it is likely that the suppressive vasopressin response to novelty stress does not primarily depend upon endogenous glucocorticoids.

The effects of intracerebroventricularly (i.c.v.) administered histaminergic receptor antagonists on plasma levels of vasopressin, oxytocin, prolactin and adrenocorticotrophic hormone (ACTH) after fear-related emotional stress were investigated in the male rat. Pyrilamine, a histaminergic H-1-receptor antagonist did not significantly alter the suppressive vasopressin or the facilitative prolactin response to nonassociatively applied emotional stress. On the other hand, i.c.v. administered ranitidine, a histaminergic H-2-receptor antagonist, blocked these responses to stress. Pyrilamine again did not significantly change the suppressive vasopressin response to the associatively applied emotional stress. However, the drug attenuated the prolactin response slightly but significantly. Ranitidine blocked the suppressive vasopressin and the facilitative prolactin responses to the associatively applied emotional stress, but the drug did not change the facilitative oxytocin or ACTH response to the stress. Suppression of motor activity during the associatively applied emotional stress was not significantly changed by either of these antagonists. These results suggest that histaminergic H-2 receptors are selectively involved in the neural pathways which mediate the suppressive vasopressin and the facilitative prolactin responses to fear-related emotional stress.

In an attempt to test the possibility that sino-aortic baroreceptors may mediate the previously reported stress response in hypothalamic magnocellular neurosecretory cell activity in rats, effects of deafferentation of sino-aortic baroreceptors on plasma levels of vasopressin and oxytocin after fear-related emotional stress were studied in male rats 28-33 days after the surgery. An alpha-1-adrenergic receptor agonist, phenylephrine (1 mg/kg) injected i.p. under anesthesia increased arterial blood pressure in the rats that had received surgical operation of sino-aortic denervation (SAD) and in the rats of sham-operation control (SHAM). Reflex bradycardia after phenylephrine occurred in the SHAM but not in the SAD group. These results indicate that afferent signals originating from sino-aortic baroreceptors were effectively blocked by the SAD surgery. In the similarly prepared SAD group, plasma level of vasopressin was decreased and plasma level of oxytocin was increased significantly to the same extent as in the SHAM group after low-frequency shocks (0.05 Hz, 5 min) or environmental cue signals previously paired with shocks. It is therefore suggested that afferent neural signals originating from sino-aortic baroreceptors are not primarily involved in the suppressive vasopressin or the facilitatory oxytocin response to fear-related emotional stress in rats.

Effects of a dopamine D1 receptor antagonist, SCH 23390, were investigated on plasma level of vasopressin after stressful stimuli in rats. The antagonist markedly attenuated the increase in plasma level of vasopressin after electric footshocks but not after s.c. injected hypertonic saline. The antagonist, however, did not significantly change the suppressive vasopressin response to fear-related emotional stress, though the drug suppressed motor behavior of the rat during testing period. These data suggest that dopamine D1 receptors play an important role selectively in the facilitatory vasopressin response to noxious stimuli in rats.

The possibility that arterial baroreceptors may be involved in the potentiation of vasopressin or oxytocin secretion observed after noxious stimuli was tested in male rats after sino-aortic denervation (SAD). There was no significant difference in plasma level of vasopressin or oxytocin between the SAD and the corresponding sham-operation control (SHAM) groups with or without electric shocks. An i.p. injected alpha-1-adrenergic receptor antagonist, prazosin, decreased arterial blood pressure both in the SAD and in the SHAM groups. However, the increased levels of these hormones after prazosin were significantly lower in the SAD than in the SHAM groups. Reflexly evoked tachycardia after prazosin occurred in the SHAM but not in the SAD groups. These results suggest that afferent neural signals originating from arterial baroreceptors are not involved in potentiation of vasopressin and oxytocin secretion after noxious stimuli in the rat.

Interactions between emotional stress due to fear and hypovolemic stimuli on vasopressin secretion were studied in rats. Intraperitoneally injected dextran did not significantly change plasma osmolality osmolality and arterial blood pressure but increased blood hemoglobin and plasma vasopressin level. An i.v. infused physiological solution reversed these changes. Emotional stress due to fear acquired by learning suppressed plasma vasopressin level in dextran-injected rats. Emotional stress due to fear produced by low-frequency footshocks also suppressed the increased plasma vasopressin level. These results suggest that emotional stress due to fear interacts with afferent neural signals originating from cardio-vascular volume receptors in the control of vasopressin secretion.