Gonadotrophin and prolactin secretion in castrated male sheep following subcutaneous or intracranial treatment with testicular hormones

Interactions between testosterone, estradiol, and inhibin in the control of gonadotrophin secretion in males are poorly understood. Castrated rams were treated with steroid-free bovine follicular fluid (bFF), testosterone, or estradiol and for 7 d (2 x 2 x 2 factorial design). Given independently, none of the exogenous hormones affected follicle-stimulating hormone (FSH) concentrations, but the combination of one or both steroids with bFF reduced FSH secretion. Testosterone and estradiol reduced luteinizing hormone (LH) pulse frequency (there was no synergism), and bFF had no effect. Plasma prolactin concentrations were not affected by any treatment. To locate the central sites of steroid action, castrated rams were bilaterally implanted in the preoptic area (POA), ventromedial nucleus (VMH), or arcuate nucleus (ARC). These implants did not affect FSH or prolactin concentrations, or LH pulse amplitude. The frequency of the LH pulses was not affected by testosterone in any site. Estradiol located in the ARC, but not the POA or VMH, decreased LH pulse frequency. In summary, FSH secretion is controlled by synergistic interactions between inhibin and estradiol or testosterone, whereas GnRH/LH pulse frequency is controlled by testicular steroids. Estradiol acts partly, at least, in the ARC, but the central site of action, testosterone remains unknown.

Hypothalamic dopamine D1 receptors are involved in the stimulation of prolactin secretion by high environmental temperature in the female sheep

Recent evidence suggests that dopamine, acting via its D1 receptors, may function as a neurotransmitter in intrahypothalamic pathways involved in the stimulation of prolactin secretion. Functional dopamine D1 receptors are present in the ventromedial hypothalamic nucleus (VMH) and we hypothesized that they might be part of a prolactin-stimulatory pathway activated by stress. We tested this hypothesis in a series of experiments on sheep involving two different forms of stressors, audiovisual (barking dog) and high environmental temperature. We attempted to block the stimulation of prolactin secretion by infusion into the VMH of an antagonist specific for the D1 receptor. Ovariectomised, oestradiol-implanted merino ewes were surgically implanted with bilateral guide tubes directed at the VMH. After a 180 min pretreatment period, the ewes either were or were not exposed to a stressor (30 min of barking dog or 120 min at 35 degrees C, 65% relative humidity). D1 receptor antagonist, SCH23390 or vehicle (0.9% saline) was infused into the VMH (1.7 mu l/h, 120 nmol/h) for 60 min prior to and during the stressor period. Blood was sampled every 15 min via jugular cannulae and the plasma was assayed for prolactin, cortisol and growth hormone (GH). Both stressors significantly increased prolactin concentrations over control levels. SCH23390 infusion significantly attenuated the prolactin response to high environmental temperature, but had no effect on the prolactin response to audiovisual stress. Cortisol concentrations were significantly increased by audiovisual stress only and were not affected by SCH23390, GH concentrations were not changed by either stressor or infusion. Drug infusion alone did not affect the concentration of the hormones. The data suggest that the VMH D1 receptors are involved in a prolactin stimulatory pathway in response to high environmental temperature. The inability of the D1 antagonist to affect the response to the barking dog indicates that this pathway is stress-specific, implying that there is more than one mechanism or pathway involved in the prolactin response to different stressors.

Facilitation of 5-HT(2A/2C)-mediated neurotransmission in the ventromedial hypothalamic nucleus decreases anxiety in the elevated T-maze

Previous evidence has shown that facilitation of GABA/benzodiazepine-mediated neurotransmission in the ventromedial hypothalamus (VMH) inhibits both escape and inhibitory avoidance responses generated in the elevated T-maze test of anxiety (ETM). These defensive behaviors have been associated with panic and generalized anxiety, respectively. Aside from GABA/benzodiazepine receptors, the VMH also contains a significant number of serotonin (5-HT) receptors, including 1A, 2A and 2C subtypes. The purpose of the present study was to investigate the effect of the activation of 5-HT(1A) and 5-HT(2A/2C) receptors in the VMH on defensive behavioral responses in rats submitted to the ETM. For that, male Wistar rats were treated intra-VMH with the 5-HT(1A) agonist 8-OH-DPAT, with the 5-HT(2A/2C) agonist DOI, with the 5-HT(2C) selective agonist MK-212, or with the 5-HT(2A/2C) antagonist ketanserin and 10 min after were submitted to the ETM. Results showed that both DOI and MK-212 significantly decreased avoidance measurements, an anxiolytic-like effect, without altering escape. 8-OH-DPAT and ketanserin were without effect, although the last drug attenuated the effects of DOI. None of the drugs altered locomotor activity in an open field. These results suggest that 5-HT(2A/2C) receptors of the VMH are involved in the regulation of inhibitory avoidance and might be of relevance to the physiopathology of generalized anxiety. (C) 2010 Elsevier B.V. All rights reserved.

Le rôle de CRTC1 dans la prise de poids induite par les antipsychotiques

Les antipsychotiques atypiques, de deuxième génération, ont largement contribué à améliorer le traitement des patients souffrant de schizophrénie. Cependant, leur mécanisme d'action reste mal compris et leurs effets secondaires sont importants, notamment la prise de poids. CRTC1 (CREB-regulated transcription coactivator 1), aussi appelé TORC1 (transducers of regulated CREB activity 1), est un coactivateur de CREB. Il régule la transcription de Bdnf1 qui joue un rôle essentiel dans le contrôle de la balance énergétique dépendant du VMH 2, 3, 4. Nous pensons que CRTC1 est impliqué dans la prise de poids induite par certains antipsychotiques. En effet, il a été démontré que les souris Crtc1-/- devenaient hyperphagiques et obèses 5, 6, que la régulation de l'activité de CRTC1 se faisait par l'AMPK et que les antipsychotiques atypiques activaient cette kinase dans l'hypothalamus.7 L'AMPK de l'hypothalamus est liée à la régulation de la prise alimentaire, elle inverse l'action de la leptine, hormone anorexigène. Suite à ces constatations, nous proposons de suivre l'hypothèse de travail suivante : l'activation de l'AMPK par les antipsychotiques atypiques dans l'hypothalamus peut maintenir la phosphorylation de CRTC1 et le bloquer dans le cytoplasme, l'empêchant ainsi d'activer les gènes anorexigènes, comme Bdnf par exemple. En effet, la forme phosphorylée inactive de CRTC1 est séquestrée dans le cytoplasme et sa migration dans le noyau nécessite en même temps l'activation de la phosphatase calcineurine et l'inactivation des kinases de la famille de l'AMPK. Dans le travail que nous avons entamé, nous cherchons donc, par western blot et par immunohistochimie, à comprendre si les antipsychotiques atypiques inactivent CRTC1 en induisant sa phosphorylation par l'AMPK et sa rétention dans le cytoplasme.

Faster gastric emptying of a liquid meal in rats after hypothalamic dorsomedial nucleus lesion

The effects of dorsomedial hypothalamic (DMH) nucleus lesion on body weight, plasma glucose levels, and the gastric emptying of a liquid meal were investigated in male Wistar rats (170-250 g). DMH lesions were produced stereotaxically by delivering a 2.0-mA current for 20 s through nichrome electrodes (0.3-mm tip exposure). In a second set of experiments, the DMH and the ventromedial hypothalamic (VMH) nucleus were lesioned with a 1.0-mA current for 10 s (0.1-mm tip exposure). The medial hypothalamus (MH) was also lesioned separately using a nichrome electrode (0.3-mm tip exposure) with a 2.0-mA current for 20 s. Gastric emptying was measured following the orogastric infusion of a liquid test meal consisting of physiological saline (0.9% NaCl, w/v) plus phenol red dye (6 mg/dl) as a marker. Plasma glucose levels were determined after an 18-h fast before the lesion and on the 7th and 15th postoperative day. Body weight was determined before lesioning and before sacrificing the rats. The DMH-lesioned rats showed a significantly faster (P<0.05) gastric emptying (24.7% gastric retention, N = 11) than control (33.0% gastric retention, N = 8) and sham-lesioned (33.5% gastric retention, N = 12) rats, with a transient hypoglycemia on the 7th postoperative day which returned to normal by the 15th postoperative day. In all cases, weight gain was slower among lesioned rats. Additional experiments using a smaller current to induce lesions confirmed that DMH-lesioned rats had a faster gastric emptying (25.1% gastric retention, N = 7) than control (33.4% gastric retention, N = 17) and VMH-lesioned (34.6% gastric retention, N = 7) rats. MH lesions resulted in an even slower gastric emptying (43.7% gastric retention, N = 7) than in the latter two groups. We conclude that although DMH lesions reduce weight gain, they do not produce consistent changes in plasma glucose levels. These lesions also promote faster gastric emptying of an inert liquid meal, thus suggesting a role for the DMH in the regulation of gastric motility

Prolonged consumption of soy or fish-oil-enriched diets differentially affects the pattern of hypothalamic neuronal activation induced by refeeding in rats

We used c-Fos immunoreactivity to estimate neuronal activation in hypothalamic feeding-regulatory areas of 3-month-old rats fed control or oil-enriched diets (soy or fish) since weaning. While no diet effect was observed in c-Fos immunoreactivity of 24-h fasted animals, the acute response to refeeding was modified by both hyperlipidic diets but with different patterns. Upon refeeding, control-diet rats had significantly increased c-Fos immunoreactivity only in the paraventricular hypothalamic nucleus (PVH, 142%). In soy-diet rats, refeeding with the soy diet increased c-Fos immunoreactivity in dorsomedial hypothalamic nucleus (DMH, 271%) and lateral hypothalamic area (LH, 303%). Refeeding fish-diet rats with the fish diet increased c-Fos immunoreactivity in PVH (161%), DMH (177%), VMH (81%), and ARC (127%). Compared to the fish-diet, c-Fos immunoreactivity was increased in LH by the soy-diet while it was decreased in ventromedial hypothalamic nucleus (VMH) and arcuate hypothalamic nucleus (ARC). Based on the known roles of the activated nuclei, it is suggested that, unlike the fish-diet, the soy-diet induced a potentially obesogenic profile, with high LH and low VMH/PVH activation after refeeding.

HYPOTHALAMIC-LESIONS INCREASE SALINE INGESTION INDUCED BY INJECTION OF ANGIOTENSIN-II INTO AV3V IN RATS

Water and 3% NaCl intake were increased by the injection of 4 ng angiotensin II (ANG II) into the anteroventral third ventricle (AV3V) region of rats. Pretreatment with two specific ANG II receptor antagonists, [octanoyl-Leu8]ANG II and [Leu8]ANG II, significantly reduced ANG II-induced water and saline intake. This inhibition lasted approximately 30 min, with partial recovery at 60 min. In rats with electrolytic lesion of the bilateral ventromedial nucleus of hypothalamus (VMH), the effect of ANG II on water intake was not different from that observed in sham rats, but saline ingestion increased. In summary, the present results show that the AV3V region is an important central structure for ANG II-induced saline ingestion. Lesion of the VMH increases the response to ANG II, showing an interaction between the AV3V region and the VMH in the regulation of salt ingestion.

Effect of ibotenate lesions of the ventromedial hypothalamus on the water and salt intake induced by activation of the median preoptic nucleus in sodium-depleted rats

In this study we investigated the influence of a ventromedial hypothalamus (VMH) lesion with ibotenic acid on water and sodium intake and presser responses induced by combined treatment of the median preoptic nucleus (MnPO) with angiotensin Il (ANG II) and adrenergic agonists (phenylephrine, norepinephrine, isoproterenol and clonidine). Male Holtzman rats with a stainless steel cannula implanted into the MnPO and bilateral sham (vehicle) or VMH lesions with ibotenic acid were used. The ingestion of water and sodium and mean arterial pressure (MAP) were determined in separate groups submitted to sodium depletion with the diuretic furosemide (20 mg/rat). ANG II (10 pmol) injection into the MnPO of sham-lesioned rats induced water and sodium intake and presser responses. VMH-lesion reduced ANG II-induced water intake and increased saline intake, In sham rats phenylephrine (80 nmol) into MnPO increased, whereas norepinephrine (80 nmol) and clonidine (40 nmol) reduced ANG II-induced water intake while sodium intake was reduced only by clonidine into MnPO. In VMH-lesioned rats, phenylephrine reduced, noradrenaline increased and clonidine produced no effect on ANG II-induced water intake. In lesioned rats ANG II-induced sodium intake was reduced by phenylephrine and noradrenaline, whereas clonidine produced no change. ANG II-induced presser response was reduced in VMH-lesioned rats, but the presser response combining ANG II and phenylephrine or noradrenaline in VMH-lesioned rats was bigger than sham rats. These results show that the VMH is important for the changes in water and sodium intake and cardiovascular responses induced by angiotensinergic and adrenergic activation of the MnPO. (C) 1997 Elsevier B.V. B.V.

Ventromedial hypothalamus lesions increase the dipsogenic responses and reduce the pressor responses to median preoptic area activation

In this study, we investigated the participation of adrenergic receptors of the median preoptic area (MnPO) and the participation of ventromedial hypothalamus (VMH) in angiotensin II- (ANG II)-induced water intake and presser responses. Male rats with sham or electrolytic VMH lesions and a stainless steel cannula implanted into the MnPO were used. Noradrenaline, clonidine (an alpha(2)-adrenergic receptor agonist), or phenylephrine (an alpha(1)-adrenergic receptor agonist) injected into the MnPO of sham-lesioned rats reduced water ingestion induced by ANG II injected into the same area. In VMH-lesioned rats ANG II-induced water intake increased with a previous injection of noradrenaline, phenylephrine, or isoproterenol. The presser response induced by ANG II injected into the MnPO was reduced in VMH-lesioned rats, whereas the presser response induced by clonidine was abolished. Previous treatment with noradrenaline and phenylephrine into the MnPO of sham-lesioned rats produced a presser response, and a hypotensive response was obtained with the previous administration of noradrenaline, phenylephrine or isoproterenol into the MnPO of VMH-lesioned rats. These results show that VMH is essential for the dipsogenic and presser responses induced by adrenergic and angiotensinergic activation of the MnPO in rats. (C) 1997 Elsevier B.V.

AV3V LESION REDUCES THE PRESSOR, DIPSOGENIC, AND NATRIURETIC RESPONSES TO VENTROMEDIAL HYPOTHALAMUS ACTIVATION

In the present study, we investigated the effect of anteroventral third ventricle (AV3V) lesion on pressor, tachycardic, dipsogenic, natriuretic, and kaliuretic responses induced by the injection of the cholinergic agonist carbachol into the ventromedial hypothalamic nucleus (VMH) of rats. Male rats with sham or AV3V lesion and a stainless steel cannula implanted into the VMH were used. Carbachol (2 nmol) injected into the VMH of sham rats produced pressor (32 +/- 4 mmHg). tachycardic (83 +/- 14 bpm), dipsogenic (8.2 +/- 1.1 ml/h). natriuretic (320 +/- 46-mu-Eq/120 min), and kaliuretic (155 +/- 20-mu-Eq/120 min) responses. In AV3V-lesioned rats (2 and 15 days), the pressor (4 +/- 2 and 15 +/- 2 mmHg. respectively), dipsogenic (0.3 +/-0.2 and 1.4 +/- 0.7 ml/h), natriuretic (17 +/- 7 and 99 +/- 21-mu-Eq/120 min), and kaliuretic (76 +/- 14 and 79 +/- 7-mu-Eq/120 min) responses induced by carbachol injection into the VMH were reduced. The tachycardia was also abolished (27 +/- 15 and -23 +/-29 bpm, respectively). These results show that the AV3V region is essential for the pressor, tachycardic, dipsogenic, natriuretic. and kaliuretic responses induced hy cholinergic activation of the VMH in rats.

AN ELECTROPHYSIOLOGICAL ANALYSIS OF THE AMYGDALOID AND SUBICULAR PROJECTIONS TO THE VENTROMEDIAL NUCLEUS OF THE HYPOTHALAMUS IN THE RABBIT

Electrophysiological experiments were performed on 96 male New Zealand white rabbits, anesthetized with urethane. Glass electrodes, filled with 2M NaCl, were used for microstimulation of three fiber pathways projecting from "limbic" centers to the ventromedial nucleus of the hypothalamus (VMH). Unitary and field potential recordings were made in the VMH after stimulation.^ Stimulation of the lateral portion of the fimbria, which carries fibers from the ventral subiculum of the hippocampal formation, evokes predominantly an inhibition of neurons medially in the VMH, and excitation of neurons located laterally.^ Stimulation of the dorsal portion of the stria terminalis, which carries fibers from the cortical nucleus of the amygdala, also produces predominantly an inhibition of cells medially and excitation laterally.^ Stimulation of the ventral component of the stria terminalis, which carries fibers from the medial nucleus of the amygdala, evokes excitation of cell medially, with little or no response seen laterally.^ Cells recorded medially in the VMH received convergent inputs from each of the three fiber systems: inhibition from fimbria and dorsal stria stimulation, excitation from ventral stria stimulation.^ The excitatory unitary responses recorded medially to ventral stria stimulation and laterally to fimbria and dorsal stria stimulation were subjected to a series of threshold stimulus intensities. From these tests it was determined that each of these three projections terminates monosynaptically on VMH neurons.^ The evidence for convergence upon single VMH neurons of projections from the amygdala and the hippocampal formation suggests this area of the brain to be important for integration of information from these two limbic centers. The VMH has been implied in a number of behavioral states: eating, reproduction, defense and aggression; it has further been linked to control of the anterior pituitary. These data provide a functional circuit through which the amygdaloid complex and the hippocampal formation can channel information from higher cortical centers into a hypothalamic area capable of coordinating behavioral and hormonal responses. ^