Estrogen reduces serotonin-1A receptor-mediated oxytocin release and Galpha(i/o/z) proteins in the hypothalamus of ovariectomized rats

The present study examined the effect of estradiol on hypothalamic serotonin-1A (5-HT(1A)) receptor signaling in female rats. We first examined the time-course effects of a single injection of the 5-HT(1A) receptor agonist (+/-)8-OH-DPAT (5, 15 or 30 min prior to decapitation), and dose response of (+)8-OH-DPAT (50, 100, 200 or 500 microg/kg, s.c.) on plasma hormones in ovariectomized rats that received a daily injection of beta-estradiol 3-benzoate (10 microg/day, s.c.) or vehicle (sesame oil) for 2 days. In vehicle- and estrogen-treated rats, the peak response of hormones occurred at 15 min after injection and the time-course of oxytocin and adrenocorticotropic hormone (ACTH) responses to an injection of 8-OH-DPAT were comparable. However, only the oxytocin response was reduced by estrogen treatment. A second experiment compared the ACTH and oxytocin responses with doses of 50 or 200 microg/kg, s.c. of (+)8-OH-DPAT vs. (+/-)8-OH-DPAT in ovariectomized rats that were treated with oil or beta-estradiol 3-benzoate (10 microg/day, s.c.) for 2 days. (+)8-OH-DPAT and (+/-)8-OH-DPAT produced a similar magnitude of increase in plasma levels of ACTH and oxytocin. Treatment with beta-estradiol 3-benzoate produced a significant and comparable reduction in the oxytocin response to the highest dose (200 microg/kg, s.c.) of both (+)8-OH-DPAT and (+/-)8-OH-DPAT but did not alter the ACTH response to either (+)8-OH-DPAT or (+/-)8-OH-DPAT. In the dose-response experiment, a dose of 50 microg/kg of (+)8-OH-DPAT produced a maximal increase in plasma levels of ACTH, while the maximal oxytocin response was achieved with a dose of 200 microg/kg, s.c. Treatment with beta-estradiol 3-benzoate reduced the maximal oxytocin response to (+)8-OH-DPAT (by 29%) but did not alter the ACTH response to any doses of (+)8-OH-DPAT. To examine potential mechanisms mediating the effects of estrogen on 5-HT(1A) receptor signaling, we measured the levels of Galpha(i), Galpha(o) and Galpha(z) proteins, which couple 5-HT(1A) receptors to their effector enzymes, in two subregions of the hypothalamus. The levels of Galpha(z) protein were reduced in the mediobasal hypothalamus (containing the ventromedial and arcuate nuclei), which mainly expresses estrogen receptor-alpha, but not in the paraventricular hypothalamus, which mainly expresses estrogen receptor-beta. Estradiol reduced the levels of Galpha(i2) and Galpha(i3 )proteins in both hypothalamic regions but did not affect Galpha(i1) levels in either area. Combined, the data suggest that racemic and stereoselective 8-OH-DPAT have similar neuroendocrine effects and that both estrogen receptor-alpha and estrogen receptor-beta mediate the reduction in levels of Galpha(i2,3) proteins.     

Ovariectomy-induced increases in hypothalamic serotonin-1A receptor function in rats are prevented by estradiol

The present study investigated the effects of long-term estradiol withdrawal (ovariectomy) on hypothalamic serotonin-1A (5-HT(1A)) receptor signaling. Changes in neuroendocrine responses to the 5-HT(1A) agonist 8-OH-DPAT and levels of G(z) protein in the hypothalamus were used to examine 5-HT(1A) receptor signaling. Five days following ovariectomy, rats received daily injections of either 2 microg of beta-estradiol 3-benzoate or vehicle (subcutaneously) for 2, 4 or 14 days. Twenty-four hours after the last injection, and 15 min prior to sacrifice, rats were injected with (+/-)8-OH-DPAT (50 micro;g/kg, s.c.) or saline. Estradiol treatment did not alter basal corticotropin (ACTH) or oxytocin levels. Injection of (+/-)8-OH-DPAT produced significant increases in plasma ACTH and oxytocin levels. In the vehicle-treated rats, hormone responses to 8-OH-DPAT were enhanced in rats that received injections for 14 days compared with rats that received injections for either 2 or 4 days. Estradiol treatment for 4 or 14 days blunted this enhanced ACTH response to 8-OH-DPAT, whereas the oxytocin response to 8-OH-DPAT was only blunted after 14 daily injections of beta-estradiol 3-benzoate. The treatment with beta-estradiol 3-benzoate (2 microg/rat) did not reduce membrane-associated G(z) protein levels in the paraventricular nucleus of the hypothalamus. Hence, the inhibitory influence of a low dose of beta-estradiol 3-benzoate on 5-HT(1A) receptor signaling in the hypothalamus is not accompanied by a change in the levels of G(z) protein in the paraventricular hypothalamic nucleus. Results from the present study indicate a supersensitivity of 5-HT(1A) receptors after withdrawal of estradiol and suggest that estradiol suppresses 5-HT(1A) receptor signaling.     

Mechanisms of serotonin receptor agonist-induced activation of the hypothalamic-pituitary-adrenal axis in the rat

A substantial body of experimental evidence indicates that serotonin (5-HT) and several synthetic 5-HT receptor agonists activate the hypothalamic-pituitary-adrenal (HPA) axis. To explore the mechanism(s) by which 5-HT or 5-HT agonists enhance the activity of the HPA axis in vitro, we examined the stimulatory effects of the 5-HT1a agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5-HT1c/5-HT1b agonist m-chlorophenylpiperazine (m-CPP), and the 5-HT2/5-HT1c agonist 1-(2,5-dimethoxy-4-iodophenyl)2-amino-propane (DOI) on plasma ACTH and corticosterone secretion in the rat. To test whether 8-OH-DPAT, m-CPP, or DOI increase plasma ACTH levels by stimulating the release of endogenous CRH, catheterized conscious male Sprague-Dawley rats were pretreated with hyperimmune CRH rabbit serum (TS-6) or normal rabbit serum and subsequently challenged with a maximally stimulatory dose of the above 5-HT agonists. Pretreatment with TS-6 completely suppressed the ACTH response to m-CPP and significantly blunted the responses to 8-OH-DPAT or DOI. To examine whether the remaining ACTH response to 8-OH-DPAT or DOI was also mediated by a pituitary site of action, we administered each of these agents to pituitary stalk-transected or sham-operated rats. The ACTH responses to 8-OH-DPAT and DOI in stalk-transected rats were preserved, although significantly blunted, compared to those in sham-operated rats. This suggested that both of these 5-HT agonists may also act at the pituitary level to stimulate ACTH release in vivo. Although the ACTH responses to 8-OH-DPAT, m-CPP, and DOI were blunted after both TS-6 pretreatment and pituitary stalk transection, corticosterone responses were only slightly affected, suggesting that some of these compounds may cause corticosterone release in the rat through another mechanism. To evaluate this hypothesis, ACTH and corticosterone responses to 8-OH-DPAT, m-CPP, and DOI were examined in rats whose HPA axis had been suppressed by a single high dose injection of dexamethasone. The corticosterone responses to 8-OH-DPAT and DOI were blunted compared to those of saline-pretreated rats, but were inappropriately high compared to the ACTH responses observed in these rats. On the other hand, both ACTH and corticosterone responses to m-CPP were completely abolished by dexamethasone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stressor-responsive central nesfatin-1 activates corticotropin-releasing hormone, noradrenaline and serotonin neurons and evokes hypothalamic-pituitary-adrenal axis

A recently discovered satiety molecule, nesfatin-1, is localized in neurons of the hypothalamus and brain stem and colocalized with stress-related substances, corticotropin-releasing hormone (CRH), oxytocin, proopiomelanocortin, noradrenaline (NA) and 5-hydroxytryptamine (5-HT). Intracerebroventricular (icv) administration of nesfatin-1 produces fear-related behaviors and potentiates stressor-induced increases in plasma adrenocorticotropic hormone (ACTH) and corticosterone levels in rats. These findings suggest a link between nesfatin-1 and stress. In the present study, we aimed to further clarify the neuronal network by which nesfatin-1 could induce stress responses in rats. Restraint stress induced c-Fos expressions in nesfatin-1-immunoreactive neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus, and in the nucleus of solitary tract (NTS), locus coeruleus (LC) and dorsal raphe nucleus (DR) in the brain stem, without altering plasma nesfatin-1 levels. Icv nesfatin-1 induced c-Fos expressions in the PVN, SON, NTS, LC, DR and median raphe nucleus, including PVN-CRH, NTS-NA, LC-NA and DR-5-HT neurons. Nesfatin-1 increased cytosolic Ca2+ concentration in the CRH-immunoreactive neurons isolated from PVN. Icv nesfatin-1 increased plasma ACTH and corticosterone levels. These results indicate that the central nesfatin-1 system is stimulated by stress and activates CRH, NA and 5-HT neurons and hypothalamic-pituitary-adrenal axis, evoking both central and peripheral stress responses.     

Involvement of endogeneous glutamate in the stimulatory effect of norepinephrine and serotonin on the hypothalamo-pituitary-adrenocortical axis

The effects of ionotropic glutamate receptor antagonists on the pituitary adrenal responses following injections of norepinephrine (NE) and serotonin (5-HT) receptor agonists into the hypothalamic paraventricular nucleus (PVN) or electrical stimulation of central NE and 5-HT pathways were studied in anesthetized male rats. PVN injections of an alpha(1)-adrenergic receptor agonist or a serotonergic 5-HT(1A) receptor agonist markedly increased both adrenocorticotropin (ACTH) and corticosterone (CS) serum levels. These responses were significantly inhibited by separate pre-injection of the selective non-NMDA and NMDA glutamate receptor subtype antagonists into the PVN in a dose-dependent manner. Electrical stimulation of either the ventral noradrenergic bundle or the dorsal raphe nucleus markedly increased serum ACTH and CS. These responses were also significantly attenuated by pre-injection of the above glutamate ionotropic receptor antagonists in a dose-dependent manner. These results suggest that glutamatergic interneurons in the PVN, acting via non-NMDA and NMDA receptors, may act as an excitatory mechanism in the NE and 5-HT control of hypothalamic ACTH secretagogues.     

Prolactin-releasing peptide releases corticotropin-releasing hormone and increases plasma adrenocorticotropin via the paraventricular nucleus of the hypothalamus

Intracerebroventricular (ICV) injection of prolactin-releasing peptide (PrRP) is known to increase plasma adrenocorticotropin (ACTH) and cause c-fos expression in the hypothalamic paraventricular nucleus (PVN). We hypothesize that this is the site at which PrRP acts to increase plasma ACTH. We have used ICV injection and direct intranuclear injection of PrRP into the PVN to investigate the sites important in the stimulation of ACTH release in vivo. To investigate the mechanism of action by which PrRP increases ACTH, we have used primary culture of pituitary cells and measured neuropeptide release from in vitro hypothalamic incubations. ICV administration of PrRP increased plasma ACTH 10 min post-injection (PrRP 5 nmol 81.0 +/- 23.5 pg/ml vs. saline 16.8 +/- 14.1 pg/ml, p < 0.05). Intra-PVN injection of PrRP increased ACTH 5 min post-injection (PrRP 1 nmol 22.9 +/- 5.0 pg/ml vs. saline 10.3 +/- 1.4 pg/ml, p < 0.05). This effect continued until 40 min post-injection (PrRP 1 nmol 9.9 +/- 1.5 pg/ml vs. saline 6.2 +/- 0.5 pg/ml, p < 0.05). In vitro PrRP (1-100 nmol/l) did not effect basal or corticotropin-releasing hormone (CRH)-stimulated ACTH release from dispersed anterior pituitary cells. PrRP increased hypothalamic release of CRH (PrRP 100 nmol/l 1.4 +/- 0.2 nmol/explant vs. the basal 1.1 +/- 0.2 nmol/explant, p < 0.05) but not arginine vasopressin. PrRP also stimulated neuropeptide Y release (PrRP 100 nmol/l 56.5 +/- 11.8 pmol/explant vs. basal 24.0 +/- 1.9 pmol/explant, p < 0.01), a neuropeptide known to stimulate the hypothalamo-pituitary-adrenal axis. Our data suggest that in vitro PrRP does not have a direct action on the corticotrope but increases plasma ACTH via the PVN and this effect involves the release of hypothalamic neuropeptides including CRH and neuropeptide Y.     

Hypothalamic-pituitary-adrenal responses to 5-HT1A and 5-HT2A/C agonists are differentially altered in female and male rats prenatally exposed to ethanol

BACKGROUND: Prenatal ethanol exposure alters the development of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in HPA hyper-responsiveness to stressors in adulthood. Prenatal ethanol exposure also alters the development and activity of the serotoninergic (5-HT) system. We have previously shown that 5-HT(1A) and 5-HT(2A/C) receptor-mediated behavioral and physiological function are altered in fetal ethanol-exposed offspring. As there are extensive interactions between the HPA axis and the 5-HT system, the present study tested the hypothesis that prenatal ethanol exposure would alter 5-HT(1A) and 5-HT(2A/C) receptor-mediated HPA function. METHODS: The 5-HT(1A) agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.2 mg/kg), and the 5-HT(2A/C) agonist, (+)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI; 0.3 mg/kg), or vehicle (1 mL/kg) were administered to adult female and male offspring from prenatal ethanol-exposed (E), pair-fed control (PF), and ad libitum-fed control (C) dams. The plasma concentration of adrenocorticotropin (ACTH) and corticosterone (CORT) were determined at 0, 15, 30, 60, and 120 minutes postinjection. In addition, corticotropin releasing hormone (CRH) mRNA expression in the paraventricular nucleus of the hypothalamus, and 5-HT(1A) and 5-HT(2A/C) receptor mRNA expression in the hippocampus and prefrontal cortex, respectively, were determined by in situ hybridization. RESULTS: Ethanol-exposed females showed a blunted ACTH response to 8-OH-DPAT at 15 and 30 minutes, and conversely, an increased ACTH response to DOI at all time points postinjection, compared with PF and C females. Differences among E, PF, and C males failed to reach significance. Centrally, however, DOI resulted in a trend toward lower CRH mRNA levels in E and PF compared with C females, but higher CRH mRNA levels in E compared with control males. There were no differences among prenatal groups in 5-HT(2A) receptor expression in the prefrontal cortex following either 8-OH-DPAT or DOI treatment. However, following 8-OH-DPAT, hippocampal 5-HT(1A) receptor expression was higher in E than in PF females in CA1, with a trend toward higher expression in E than in C females in CA2, whereas following DOI, a prenatal group by subfield interaction suggests lower 5-HT(1A) mRNA levels in E and PF compared with C females in CA1 and the dentate gyrus. CONCLUSIONS: These data are the first to demonstrate that prenatal ethanol exposure has differential long-term effects on 5-HT(1A)-mediated and 5-HT(2A)-mediated neuroendocrine function in females and males, and suggest a sex-specific ethanol-induced alteration in the interaction between the HPA axis and the serotonin system.     

Effect of acute ether stress on monoamine metabolism in median eminence and discrete hypothalamic nuclei of the rat brain and on anterior pituitary hormone secretion

This study was designed to correlate the endocrine responses elicited by acute ether stress with the changes in metabolism of several monoamines in discrete nuclei of the rat brain. Concentrations of norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) and also of the specific metabolites of NE, DA, and 5-HT, 3-methoxy-4-hydroxyphenylethylene glycol, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid, respectively, were concurrently measured in microdissected nuclei using high-performance liquid chromatography with electrochemical detection. The ratio of the metabolites to their respective amines was used as an estimate of the metabolism of NE, DA, and 5-HT. Acute exposure to ether vapors induced, within 5-15 min, large increments in plasma levels of adrenocorticotropic hormone (ACTH), beta-endorphin, and prolactin (PRL), and decrements in the levels of plasma growth hormone (GH). Significant increases in NE metabolism were observed in the rostral (ANr) and caudal (ANc) divisions of the arcuate nucleus, as well as in the paraventricular (PVN) and dorsomedial nuclei, 15 min after ether stress. A significant decrease in 5-HT metabolism was observed in the PVN, supraoptic nucleus, and ANc, whereas significant increases in 5-HT metabolism were detected in the suprachiasmatic nucleus and ANr. DA metabolism selectively increased in the ANr. The present results indicate that the acute changes in ACTH, beta-endorphin, PRL, and GH release induced by ether exposure are temporally correlated with increases in NE metabolism in many hypothalamic nuclei; a selective increase in DA metabolism restricted to the ANr, and differential effects on 5-HT metabolism, probably reflecting selective activation or inhibition of different populations of 5-HT neurons.     

Diurnal corticotropin-releasing hormone mRNA variation in the hypothalamus exhibits a rhythm distinct from that of plasma corticosterone.

The hypothalamopituitary-adrenal axis exhibits a diurnal rhythm as witnessed by the daily excursion of corticosterone in plasma. The rhythm appears to be mediated largely by the stimulation of CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus. In the present study, we investigated the effects of circadian influence on CRH mRNA levels in the paraventricular hypothalamus. Animals were sacrificed through a 24-hour period to establish a detailed time course of CRH mRNA fluctuations. Levels of both type I and type II corticosterone receptor mRNAs were also measured in this area to see whether changes correlate with that of CRH mRNA. Plasma levels of ACTH were quantified as an index for CRH peptide secretion. The results indicate that changes in ACTH closely paralleled alterations in corticosterone levels with an increasing trend starting at 1 PM, suggesting that the diurnal secretory drive commences around this time. The CRH mRNA rhythm as determined by RNase protection assays appeared to change in an anticipatory fashion to these endocrine fluctuations, increasing during the light phase and reaching maximal levels just prior to dark (5-6 PM). An abrupt decrease of 30% in the CRH mRNA content was detected in the hypothalamus within 2 h after dark (8 PM) and coincided with the peak of plasma corticosterone levels. However, other periodic variations in the CRH mRNA content were not accompanied by changes in plasma corticosterone. Neither types of corticosterone receptor mRNAs showed any diurnal change suggesting that the expression of steroid receptors in the hypothalamus is not regulated by circadian influences. We conclude that CRH mRNA levels fluctuate diurnally but are inversely related to corticosterone levels only in the early evening.     

Central type 2 corticotropin-releasing hormone receptor mediates hypothalamic-pituitary-adrenocortical axis activation in the rat

In an attempt to clarify the role of the type 2 corticotropin-releasing hormone (CRH) receptor (CRHR-2) in the brain in activation of the hypothalamic-pituitary-adrenocortical axis, we conducted experiments using male Wistar rats. First, an injection of urocortin-2 (7.5 microg) into the lateral ventricle resulted in transient increases in CRH heteronuclear RNA (hnRNA) in parvocellular paraventricular nucleus (PVN) and in plasma adrenocorticotropic hormone (ACTH), whereas sustained increases in arginine vasopressin (AVP) hnRNA and c-fos mRNA in the parvocellular PVN were observed as compared with vehicle treatment. Pretreatment with the selective CRHR-2 antagonist antisauvagine-30 (20 microg) into the lateral ventricle 15 min prior to agonist injection attenuated the stimulatory effects of urocortin-2 on the above-mentioned hypothalamic-pituitary-adrenal axis variables. These effects were similar or rather more potent than those induced by pretreatment with 50 microg of alpha-helical CRH. Second, we found longer-lasting increases in CRH and AVP hnRNA and c-fos mRNA in parvocellular PVN and in plasma ACTH following central administration of urocortin-3 (7.5 microg) than following urocortin-2. Pretreatment with antisauvagine-30 antagonized the effects of urocortin-3 on the above-mentioned variables. Finally, central administration of antisauvagine-30 as well as alpha-helical CRH profoundly attenuated restraint-stress-induced increases in AVP hnRNA. However, alpha-helical CRH, but not antisauvagine-30, attenuated restraint-stress-induced increases in CRH hnRNA in the PVN. Both antagonists transiently attenuated stress responses of c-fos mRNA in PVN and plasma ACTH. These results indicate that there is a CRHR-2-mediated mechanism in the brain that stimulates CRH- and AVP-producing neurons in the PVN which results in the promotion of plasma ACTH secretion.     

Correlation between the stress-induced transient increase in corticotropin-releasing hormone content of the median eminence of the hypothalamus and adrenocorticotropic hormone secretion

Intravenous angiotensin II and ether stress were found to produce a rapid, transient increase in the corticotropin-releasing hormone (CRH) content of the median eminence as measured by a radioimmunoassay employing an antibody against rat CRH(1-41). This confirms previous reports of transient increases in CRH measured by bioassay. The increase did not occur in the paraventricular region or in other parts of the brain. It occurred along with an increase in plasma adrenocorticotropic hormone (ACTH) when a second ether stress was administered 1 h after the first, and it also occurred when rats that had been adrenalectomized for 5 days were exposed to ether. The increases in CHR and the ACTH responses to ether were reduced or abolished by dexamethasone and pentobarbital. Four days after semicircular knife cuts in the posterior hypothalamus, resting CRH in the median eminence was increased but there was no further rise after ether stress. Plasma ACTH was normal at rest after the cuts, but the increase produced by ether was reduced. The ACTH responses to angiotensin II and immobilization were also reduced. Because the posterior knife cuts reduced hypothalamic catecholamine content, the effects of reducing hypothalamic norepinephrine and epinephrine by administration of the dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDC) were tested. Five hours after DDC, plasma ACTH was elevated but there was no further increase with ether stress. The median eminence CRH content was normal but failed to increase after exposure to ether.(ABSTRACT TRUNCATED AT 250 WORDS)     

The stimulatory effect of 5HT and the role of the paraventricular nucleus on PMS induced ovulation in the immature rat.

Immature rats can be induced to ovulate with pregnant mare serum (PMS) as long as they weigh over 60 g. In the rats weighing less than 60 g 5-hydroxytryptamine (5HT)-60 micrograms/rat intraventricularly or 2 micrograms/rat in the paraventricular nucleus (PVN)-stimulated ovulation. Injections into several other hypothalamic nuclei were ineffective. In rats weighing greater than 60 g, lesions of the PVN or injection of 10 micrograms/rat p-chlorophenylalanine into the PVN inhibited ovulation. The hypothalamic levels of 5HT and 5-hydroxy indole acetic acid (5HIAA) were generally lower in the afternoon and evening in the less than 60 g when compared with the greater than 60 g rats. It is possible that increased 5HT activity is required to stimulate ovulation and that its site of action is the PVN.     

Activity of vasoactive intestinal peptide and serotonin in the paraventricular nucleus reflects the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion.

PRL secretion in the female rat is regulated by an endogenous stimulatory rhythm (ESR) of prolactin-releasing factors of hypothalamic origin which has a bimodal periodicity with distinct nocturnal (N) and diurnal (D) phases. The N phase reaches peak magnitude by 0300 h and the D phase reaches peak magnitude by 1700 h. This rhythm was first unmasked in ovariectomized rats by correctly timed injection of a dopamine antagonist. OT, vasoactive intestinal peptide (VIP), and serotonin (5-HT) are differentially involved in generating the ESR. Pharmacological studies suggest that OT is the neurohormone and VIP and 5-HT are neuromodulators which act to stimulate OT release. Recently, we reported that activity of OTergic neurons in the paraventricular nucleus (PVN) and OT concentrations in the anterior pituitary mirror the periodicity of the ESR. The present experiments were conducted to determine if VIP and 5-HT activity in the hypothalamus also mirrors the periodicity of the ESR. Push-pull cannulae were surgically implanted in the PVN of ovariectomized female rats. Following recovery, push-pull perfusion was conducted from either 0600-1400 h, 1400-2200 h, or 2200-0600 h. VIP was measured in perfusates by RIA. There was no difference in VIP pulse frequency between rats perfused during the three periods studied. However, animals perfused from 2200-0600 h had significantly greater pulse amplitude as compared to rats at either 0600-1400 h or 1400-2200 h. Activity of 5-HTergic neurons in the hypothalamus was studied by estimating the turnover of 5-HT 10 min following the injection of pargyline. Hypothalamic nuclei were dissected using Palkovits' punch technique and 5-HT concentration assayed by HPLC in conjunction with electrochemical detection. Turnover of 5-HT was estimated by calculating the slope of the accumulation of 5-HT over 10 min at differing times of day using least squares regression analysis. There was a distinct diurnal rhythm of 5-HT accumulation in the PVN. Rats killed at 1700 h had significantly greater slopes of 5-HT accumulation in the PVN than rats killed at either 0300 or 1200 h. Similarly, there was a diurnal rhythm of 5-HT turnover in the suprachiasmatic nucleus. Rats sampled at either 1200 or 1700 h had significantly greater slopes of 5-HT accumulation in the suprachiasmatic nucleus than rats sampled at 0300 h. There was no diurnal rhythm of 5-HT turnover evident in either the median eminence or the supraoptic nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Centrally administered murine-leptin stimulates the hypothalamus-pituitary- adrenal axis through arginine-vasopressin

Starvation induces a decrease in circulating leptin levels and activation of the hypothalamus-pituitary-adrenal (HPA) axis. Leptin inhibits the HPA axis in unfed rodents or genetically leptin-deficient ob/ob mice, whereas it stimulates corticotropin-releasing hormone (CRH) gene expression in the paraventricular nucleus (PVN). However, the interactions between leptin, CRH and the HPA axis are poorly understood and are likely to be complex. We recently demonstrated that central leptin administration caused increases in plasma arginine-vasopressin (AVP) and AVP gene expression of the PVN in nonstressful rats. AVP stimulates the release of adrenocorticotropic hormone (ACTH), but it also potentiates the action of CRH on ACTH release. In this study, we investigated the effects of leptin on plasma ACTH and corticosterone levels, CRH mRNA of the PVN and proopiomelanocortin (POMC) mRNA of the pituitary in nonstrained rats. Intracerebroventricularly administered leptin caused increases in plasma ACTH and corticosterone levels in dose-dependent manners. In Northern blot analyses, the leptin injection induced significant increases in the expression of CRH mRNA in the PVN and POMC mRNA in the pituitary. The increased plasma ACTH and corticosterone levels by leptin were attenuated with intracerebroventricular pretreatment of a V(1a) receptor antagonist (OPC-21268) or a V(1a)/V(1b) receptor antagonist (dP[Tyr(Me)(2)]AVP), but not with that of a V(2) receptor antagonist (OPC-31260). The leptin-induced CRH mRNA expression in the PVN and POMC mRNA expression in the pituitary were also reduced by the pretreatment with OPC-21268 and dP[Tyr(Me)(2)]AVP. These results suggest that intracerebroventricular leptin administration activates the HPA axis by AVP receptor activation through V(1a) receptors in the PVN which in turn activates CRH neurons to drive ACTH and corticosterone secretion in concert with AVP in nonstrained rats.     

The amygdala regulates the pituitary-adrenocortical response and release of hypothalamic serotonin following electrical stimulation of the dorsal raphe nucleus in the rat

The amygdala is known to modulate the function of the hypothalamo-pituitary-adrenocortical (HPA) axis, but the mechanism of this effect is still not clear. In the present study we examined the specific role of the serotonin (5-HT) system in mediating the effect of the amygdala on the activity of the HPA axis. Bilateral lesions of the amygdala in rats reduced the adrenocorticotropin (ACTH) and corticosterone responses to electrical stimulation of the dorsal raphe nucleus, where the cell bodies of serotonergic neurons are located. Amygdala lesions had no effect on the ACTH and corticosterone responses to administration of a 5-HT(1A) receptor agonist directly into the paraventricular nucleus (PVN) of the hypothalamus, indicating that there was no impairment in the activity of postsynaptic 5-HT(1A) receptors in the hypothalamus. In vivo microdialysis showed that amygdala lesions markedly attenuated the effect of electrical stimulation of the dorsal raphe to increase extracellular secretion of 5-HT in the PVN. This is the first demonstration that the amygdala has a facilitatory effect on the function of dorsal raphe 5-HT neurons which project to the PVN, and suggests a mechanism by which the amygdala may modulate the function of the HPA axis.     

Hypothalamo-pituitary-adrenal axis sensitization after chronic salt loading

Hypothalamic parvocellular vasopressin (VP) and corticotropin-releasing hormone (CRH) in the paraventricular nucleus (PVN) are major secretagogues of corticotropin (ACTH), and central plasticity including their alteration is closely related to hypothalamic-pituitary-adrenal (HPA) axis modulation. Chronic hyperosmotic stress caused by 2% salt loading has been known to alter VP and CRH expression. We recently reported that rehydration, a recovery stage from salt loading, induced a prolonged increase in parvocellular VP mRNA expression and suggested that rehydration can modulate HPA axis function without obvious external stress. In the present study, we examined hypothalamic VP and CRH mRNA expression and their responsiveness to acute immobilization stress in control, salt-loaded and rehydrated animals, in order to clarify the precise mechanism of HPA axis regulation during rehydration. The results were further compared with plasma corticosterone and ACTH levels. Plasma corticosterone decreased during salt loading, whereas it increased during rehydration at 1 week. Basal ACTH concentration increased in 1-week-rehydrated animals, with enhanced responsiveness to the acute immobilization stress. In the hypothalamic parvocellular PVN, basal CRH mRNA levels also decreased during salt loading and increased during rehydration. Basal VP mRNA was up-regulated during both salt loading and rehydration. VP mRNA responded to additional acute stress during salt loading and rehydration, but CRH mRNA did not. These results indicate that the HPA axis activity of parvocellular neurons is still altered at 1 week of rehydration and that VP plays a dominant role in regulating ACTH release in response to acute stress. This rehydration stage may thus be a good model for analysis of post-stress sensitization of the HPA axis.     

Central dysregulation of the hypothalamic-pituitary-adrenal axis in neuron-specific proopiomelanocortin-deficient mice

Proopiomelanocortin (POMC) is synthesized predominantly in pituitary corticotrophs, melanotrophs, and arcuate hypothalamic neurons. Corticotroph-derived ACTH mediates basal and stress-induced glucocorticoid secretion, but it is uncertain whether POMC peptides produced in the brain also regulate the hypothalamic-pituitary-adrenal axis. To address this question, we generated neuron-specific POMC-deficient mice by transgenic (Tg) replacement of pituitary POMC in a global Pomc(-/-) background. Selective restoration of pituitary POMC prevented the adrenal insufficiency and neonatal mortality characteristic of Pomc(-/-) mice. However, adult Pomc(-/-)Tg/+ mice expressing the pituitary-specific transgene exhibited adrenal cortical hypertrophy, elevated basal plasma corticosterone, elevated basal but attenuated stress-induced ACTH secretion, and inappropriately elevated CRH expression in the hypothalamic paraventricular nucleus. In addition, Pomc(-/-)Tg/+, Pomc(+/-)Tg/+, and Pomc(+/-) mice, which all displayed varying degrees of elevated CRH, frequently developed melanotroph adenomas after 1 yr of age, whereas Pomc(-/-) mice, with maximal CRH expression and glucocorticoid disinhibition, developed corticotroph and melanotroph adenomas. These results indicate that neuronal POMC peptides are necessary to regulate CRH within physiological limits and that a chronic reduction or absence of hypothalamic POMC leads to trophic stimulation of pituitary cells directly or indirectly through elevated CRH levels.     

Decreased type 2 corticotropin-releasing hormone receptor mRNA expression in the ventromedial hypothalamus during repeated immobilization stress.

Chronic or repeated stress results in reduction of food intake and body weight in rats. Stress-induced anorexia has been attributed to increased corticotropin-releasing hormone (CRH) function in the central nervous system. To explore possible roles of other neuropeptides and peripheral hormones involved in food intake and energy utilization during continuing stress, we examined the impact of repeated immobilization stress on expression of mRNAs coding for CRH, neuropeptide Y (NPY), galanin and pro-opiomelanocortin (POMC) mRNAs in such hypothalamic nuclei as the paraventricular nucleus (PVN), arcuate nucleus (ARC) and dorsomedial hypothalamus (DMH), as well as plasma insulin and leptin concentrations. Changes in type 2 CRH receptor (CRHR-2) mRNA in the ventromedial hypothalamus (VMH), a possible target of anorectic CRH effect, were also examined. Rats were immobilized for 2 h daily for 6 days and sacrificed 24 h after the last immobilization. Immobilized rats had lower food intake and body weight and higher levels of PVN CRH mRNA than controls. Repeated immobiliza tion also lowered plasma insulin and leptin concentrations and VMH CRHR-2 mRNA levels. These results provide additional evidence linking VMH CRHR-2 mRNA levels to plasma leptin concentration. ARC NPY and DMH galanin mRNAs increased following repeated immobilization, while ARC POMC mRNA decreased. DMH NPY mRNA and ARC galanin mRNA were unaltered by immobilization. Since NPY and galanin are considered orexigenic, while the POMC-melanocortin-4 receptor system is apparently anorexigenic, the changes in neuropeptide mRNAs and VMH CRHR-2 mRNA may play counterregulatory roles against anorectic CRH effects.     

Age- and stress-induced changes in corticotropin-releasing hormone mRNA expression in the paraventricular nucleus of the hypothalamus

In reaction to acute stress, prepubertal (25-28 days of age) animals demonstrate a prolonged adrenocorticotropic hormone (ACTH) and corticosterone response compared to adults (>65 days of age), while after chronic stress, prepubertal animals show a higher peak ACTH and corticosterone response, but a faster return to baseline compared to adults. Differential activation of corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) of prepubertal and adult animals have been suggested to mediate these changes in stress responsiveness. The purpose of the present set of experiments was to further elucidate possible differences in PVN structure and function in prepubertal (28 days of age) and adult (77 days of age) male rats. The results indicate that PVN volume and somal size and cell number are similar in the parvocellular and magnocellular subdivision of the PVN before and after pubertal development. Furthermore, after a peripheral injection of the retrograde tracer Fluoro-Gold (FG), prepubertal and adult males demonstrate similar numbers of anterior pituitary projecting neurosecretory neurons in the parvocellular region of the PVN. Finally, using in situ hybridization we show that in response to acute stress, CRH mRNA in the PVN was affected by both age and stress such that prepubertal males have greater CRH expression than adults and both prepubertal and adult males show significant stress-induced increases in CRH mRNA. Interestingly, in response to repeated restraint, neither age nor stress significantly influence CRH expression. Together, these data indicate that both age and experience with stress interact to modulate CRH expression in the PVN.