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Background Prostaglandin E2 (PGE2) binds to four receptor subtypes (EP1, EP2, EP3 and EP4) and plays an important role in response to stress. However, the identity of the receptor(s) responsible for PGE2 regulation of neuronal activity and signaling through activation of the hypothalamic-pituitary-adrenal (HPA) axis under immobilization stress is unknown. Purpose The present study aimed to investigate the role of the hypothalamic PGE2 receptors in the activation of the HPA axis and neuronal activity in a rat model of stress. Methods Stress was induced by immobilization of the animals, after which the stress-induced profile of PGE2 receptor signaling in the rat hypothalamus was determined by real-time polymerase chain reaction and immunohistochemistry. The effect of a selective EP3 receptor antagonist on corticosterone concentrations and c-Fos immunoreactivity was measured. Results Expression of EP2 and EP3 receptor genes, but not EP1 and EP4, was increased following immobilization stress. The EP3 receptor was localized to the paraventricular nucleus (PVN) of the hypothalamus, and the integrated density of the EP3 receptor was increased after immobilization stress. Rats given L-798,106, a selective antagonist of the EP3 receptor, showed significant attenuation of stress-increased serum corticosterone levels. EP3 antagonist also significantly suppressed the increase in the gene expression of c-Fos and the number of c-Fos-immunoreactive cells in the PVN of the hypothalamus following immobilization stress. Conclusions These results suggest that immobilization stress may result in increased activation of the HPA axis and neuronal activity through regulating the function of the EP3 receptor.

Vasoactive intestinal peptide (VIP), a neuromodulator present in the hypothalamus, plays an important role in the regulation of food intake. Paraventricular nucleus of the hypothalamus (PVN) is involved in ingestive responses and regulates the nitric oxide (NO) pathway. The main objectives of this study were to investigate metabolic changes established after different doses and times of VIP microinjection on the PVN, and the effect of VIP microinjection on the PVN on food intake and the role of NO in this control. In anesthetized rats, increased blood plasma glucose and insulin levels were observed following the doses of 40 and 80 ng/g of body weight. At the dose of 40 ng/g, VIP promoted hyperglycemia and hyperinsulinemia 5, 10, and 30 min after microinjection, and increased free fatty acids and total lipids plasma levels after 5 min, and triglycerides after 10 min. In awake animals, once again, VIP administration increased plasmatic levels of glucose, free fatty acids, corticosterone, and insulin 10 min after the microinjection. Moreover, VIP promoted hypophagia in the morning and night periods, and L-arginine (L-Arg) and monosodium glutamate (MSG) or a combination of both attenuated VIP-induced reduction on food intake. In addition, nitrate concentration in the PVN was decreased after VIP microinjection. Our data show that the PVN participates in the anorexigenic and metabolic effects of VIP, and that VIP-induced hypophagia is likely mediated by reduction of NO.

The paraventricular hypothalamic nucleus (PVN) is a complex structure with both neuroendocrine and autonomic functions including cardiovascular control. The PVN contains angiotensin II (AngII) immunoreactive cells, fibers, as well as AT1 and AT2 receptors of AngII. We microinjected AngII into the PVN of normotensive anesthetized rats and simultaneously recorded blood pressure, heart rate (HR) and single-unit responses. The roles of AT1 and AT2 receptors in these responses were also evaluated. Microinjection of AngII into the PVN produced a short excitatory single-unit response and two types of pressor responses: short duration with a decrease in HR and long with an increase in HR. Microinjection of losartan, an AT1 antagonist, into the PVN produced two response types, attenuation and augmentation of the pressor and firing rate responses to AngII. Microinjection of PD123319, an AT2 antagonist, into the PVN greatly attenuated pressor and single-unit response to AngII, indicating that the pressor response was mediated through AT2 receptors too. In conclusion, microinjection of AngII into the PVN stimulates neurons resulting in an increase in firing rate and consequently produces a short or long pressor response. These responses were mediated through AT1 and AT2 receptors; however, AT1 receptor may produce inhibition too. The results suggest that AngII of the PVN may be a neurotransmitter playing a role in arterial pressure regulation.

The aim of present study is to clarify the role of apelin in regulating energy homeostasis in brown adipose tissue (BAT). We examined the central effects of apelin-13 on the brain c-fos like immunoreactivity (c-FLI), BAT temperature and the activity of the sympathetic nerve activity innervating BAT in rats. In the hypothalamus, central infusion into the third cerebral ventricle (i3vt) of apelin-13 caused induction of c-FLI in the paraventricular nucleus (PVN) compared with the controls (PBS-treated) group. In addition, microinjection of apelin-13 into the PVN produced significant increases in BAT temperature. Furthermore, microinjection of apelin-13 treatment increased BAT sympathetic nerve activity compared with controls. We conclude that apelin-13 microinjection into PVN increases sympathetic nerve activity innervating BAT.

arginine-vasopressin in the parvocellular neurons of the hypothalamic paraventricular nucleus is known to play an important role in the control of the hypothalamo-pituitary-adrenal axis. In the present study, we verify plasma corticosterone levels, the distribution of glucocorticoid receptor- and arginine-vasopressin-positive neurons, and the co-localization of both glucocorticoid receptors and arginine-vasopressin in neurons in the anterior and medial parvocellular subdivisions of the paraventricular nucleus after manipulations of the hypothalamus-pituitary-adrenal axis. Normal, sham surgery, and adrenalectomized male rats were subjected to intraperitoneal injections of saline or dexamethasone to measure plasma corticosterone levels by a radioimmunoassay. We also examined arginine-vasopressin and glucocorticoid receptor immunofluorescence in sections from the paraventricular nucleus. Our results showed that the immunoreactivity of arginine-vasopressin neurons increased in the anterior parvocellular subdivision and decreased in the medial parvocellular subdivision from adrenalectomized rats treated with dexamethasone. On the other hand, we showed that the immunoreactivity of glucocorticoid receptors increased in the anterior and medial parvocellular subdivisions of these same animals. However, the immunoreactivity of glucocorticoid receptors is higher in the medial parvocellular than anterior parvocellular subdivision. The co-localization of arginine-vasopressin and glucocorticoid receptors was found only in the medial parvocellular subdivision. These findings indicate that glucocorticoids have direct actions on arginine-vasopressin-positive neurons in the medial parvocellular but not anterior parvocellular subdivision. There is a differentiated pattern of arginine-vasopressin-positive neuron expression between the anterior and medial parvocellular subdivisions.

Dopaminergic neurons of the hypothalamic tuberoinfundibular dopaminergic (TIDA) system exert a tonic inhibitory control on prolactin (PRL) secretion whereas estrogen, known to inhibit TIDA neuron function, has been postulated to be toxic to TIDA neurons when it is chronically high. In order to determine whether estrogen in high doses can cause permanent damage to TIDA function, we submitted young female rats to continue high doses of estrogen administered, either centrally (intrahypothalamic estrogen implants) or peripherally (subcutaneous estrogen implants or weekly intramuscular (i.m.) injections for 7 weeks), subsequently withdrawing the steroid and observing the evolution of lactotrophes, serum PRL and TIDA neurons. Serum PRL was measured by radioimmunoassay whereas tyrosine hydroxylase positive (TH+) neurons and PRL cells were morphometrically assessed in sections of fixed hypothalami and pituitaries, respectively. After 30 days, hypothalamic estrogen implants induced a significant increase in serum PRL, whereas TH+ neurons were not detectable in the arcuate-periventricular hypothalamic (ARC) region of estrogen-implanted rats. Removal of implants on day 30 restored TH expression in the ARC and brought serum PRL back to basal levels 30 days after estrogen withdrawal. Subcutaneous or i.m. administration of estrogen for 7 weeks induced a marked hyperprolactinemia. However, 30 weeks after estrogen withdrawal, TH neuron numbers in the ARC were back to normal and serum PRL returned to basal levels. After peripheral but not central estrogen withdrawal, pituitary weight and lactotrophic cell numbers remained slightly increased. Our data suggest that estrogen even at high doses, does not cause permanent damage to TIDA neurons. Fil: Morel, Gustavo Ramón. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Caron, Ruben Walter. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Console de Avegliano, Gloria Miriam. Universidad Nacional de La Plata; Argentina Fil: Soaje, Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Sosa, Yolanda Elena. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Rodríguez, Silvia Susana. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina Fil: Jahn, Graciela Alma. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Goya, Rodolfo Gustavo. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina

The mechanisms by which the CB1 receptor antagonist rimonabant exerts its appetite-suppressing and energy-dissipating effects are still incompletely resolved. To shed further light on the central pathways influenced by CB1 receptor modulation we examined the expression of the immediate early gene c-fos in male Sprague-Dawley rats at 60, 120 and 240 min after intraperitoneal administration of the CB1R antagonist rimonabant (10 mg/kg) and the CB1R agonist WIN55,212-2 (3 mg/kg). Perfusion-fixed brains were processed for immunohistochemistry and the localization of c-Fos immunoreactive neuronal profiles was assessed qualitatively throughout the brain. Nine areas, including specific hypothalamic and brainstem nuclei known to be involved in appetite regulation, were selected for quantitative analyses. Whereas WIN55,212-2 induced c-Fos immunoreactivity in a time-specific manner in the striatum, the central nucleus of amygdala, the hypothalamic paraventricular nucleus and the arcuate nucleus, no significant increases in c-Fos positive nuclei were found in any forebrain areas following rimonabant administration. In contrast, rimonabant and WIN55,212-2 were both found to significantly increase c-Fos immunoreactivity in the brainstem lateral parabrachial nucleus, the nucleus of the solitary tract and the area postrema. To characterize the phenotype of activated neurons in the nucleus of the solitary tract, a triple immunohistochemical staining technique was used to simultaneously label c-Fos protein and tyrosine hydroxylase (TH), GLP-1 or CART. Interestingly, rimonabant was found to significantly increase c-Fos protein expression in TH-positive neurons. Collectively, these results suggest that brainstem areas including ascending catetholaminergic A2/C2 neurons could play a role in rimonabant-induced inhibition of food intake.

We previously demonstrated that while diabetic animals receiving long-term insulin treatment exhibited some impairment in their corticosterone response to hypoglycemia, the stress response to hypoglycemia was completely absent when these animals were subjected to recurrent hypoglycemia. In the current study, we examined potential mechanisms that may contribute to defects in the adrenocortical response to hypoglycemia in long-term insulin-treated diabetic animals exposed to antecedent hypoglycemia. Whereas insulin-treated diabetic animals exhibited a significant rise in corticotrophin-releasing hormone (CRH) mRNA levels during hypoglycemia, exposure to antecedent hypoglycemia completely abolished this response. Moreover, expression of hippocampal mineralocorticoid receptors (MR) mRNA, which normally act to suppress hypothalamo-pituitary-adrenal activity, decreased in the normal control and insulin-treated diabetic groups in response to hypoglycemia, whereas MR mRNA levels remained at baseline in animals subjected to antecedent hypoglycemia. Interestingly, hippocampal glucocorticoid receptor (GR) mRNA levels decreased in all three treatment groups following the hypoglycemic clamp. While GR mRNA levels in the paraventricular nucleus were lower in normal controls following hypoglycemia, this trend just failed to reach statistical significance in the two diabetic groups. These data suggest that (1) recurrent hypoglycemia, much like uncontrolled diabetes, has a pronounced effect on hippocampal mineralocorticoid receptor mRNA expression that may prevent it, and presumably also the stress axis, from responding properly to a subsequent bout of hypoglycemia, and (2) while long-term insulin treatment was sufficient to restore some of these responses in diabetic animals, tighter glycemic control may be necessary to see full restoration of the stress response.

Functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei was investigated in response to acute treatment with Zolpidem (a GABA(A) receptor agonist with selectivity for alpha(1) subunits) utilizing dual Fos/OXY immunohistochemistry. Zolpidem was administered intraperitoneally in dose 10 mg/kg of BW and 60 min later the animals were sacrificed by transcardial perfusion with fixative. The Fos/OXY co-labelings were analyzed on 40 microm thick serial coronal sections using computerized light microscopy. Zolpidem elicited a concordant Fos/OXY staining in all four PVN sub-areas investigated, including the anterior (15.71+/-2.35%), middle (14.52+/-2.53%), dorsal (13.34+/-2.61%), and periventricular (18.21+/-4.75%) ones, however, had no significant stimulatory effect on OXY cells in the SON. In response to Zolpidem, statistically significant activations were also seen in certain groups of accessory structures including the circular nucleus (13.99+/-3.43%), small clusters of accessory neurons (10.55+/-1.94%), and the lateral hypothalamic perivascular nucleus (9.42+/-2.74%). Between the naive and vehicle controls, the dual Fos/OXY labelings did not elicit any significant differences. Our data provide insight into the topographic patterns of brain activity within the clusters of magnocellular OXY cells in the hypothalamus associated with stimulation of GABA(A) benzodiazepine receptors and for the first time illustrate the triggering contemporaneousness within the cells of the principal and accessory magnocellular nuclei in response to Zolpidem treatment. The present study provides a comparative background that may help in the further understanding of a possible extend of Zolpidem effect on the brain.

Growth hormone-releasing hormone (GHRH) and somatostatin are the two main hypothalamic neurohormones, which stimulate or inhibit directly hypophysial growth hormone (GH) release. Majority of the GHRH neurons projecting to the median eminence is situated in the arcuate nucleus and the somatostatin neurons in the anterior periventricular nucleus. Data suggest that the excitatory amino acid glutamate may play an important role in the control of hypothalamic neuroendocrine neurons and processes including the control of GH. There is a dense plexus of glutamatergic fibres in the hypothalamic arcuate and anterior periventricular nucleus. The aim of the present studies was to examine the relationship of these fibres to the GHRH neurons in the arcuate nucleus and to somatostatin neurons in the anterior periventricular nucleus. Double-labelling immuno-electron microscopy was used. Glutamatergic structures were identified by the presence of vesicular glutamate transporter 2 (VGluT2) (a selective marker of glutamatergic elements) immunoreactivity. A significant number of VGluT2-immunoreactive boutons was observed to make asymmetric type of synapses with GHRH-immunostained nerve cells in the arcuate and with somatostatin neurons in the anterior periventricular nucleus. A subpopulation of somatostatin-immunoreactive neurons displayed also VGluT2 immunoreactivity. Our findings provide direct neuromorphological evidence for the view that the action of glutamate on GH release is exerted, at least partly, directly on GHRH and somatostatin neurons releasing these neurohormones into the hypophysial portal blood.

Estradiol modulates stress reactions in female rats. Several studies showed anxiolytic effects of estradiol in behavioral tests, but the underlying mechanisms are still unclear. The aim of the current study was to explore how estradiol-treated rats respond to acute and chronic stress compared to ovariectomized rats. Ovariectomized rats received vehicle or 17 beta-estradiol injections (10 mu g/250 g) once every 4 days, which induced alternating high and low plasma 17 beta-estradiol levels. Stress was presented by daily exposure to an adverse environment in which the animals received five footshocks for either 3 or 22 days. Under control conditions no differences were observed, but as soon as stress was applied, reactions of ovariectomized and estradiol-treated rats diverged. Both acute and chronic stress increased the c-Fos protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Cyclic estradiol treatment reduced this stress-induced activation of the PVN, an effect that seems to be dependent on the plasma estradiol levels. No differences in stress-induced corticosterone responses were revealed between the treatment groups. An increase in the number of ER beta-expressing cells in the PVN of ovariectomized and estradiol-treated rats during chronic stress implied increased ER beta-mediated mechanisms during these conditions. The dampening effect of estradiol on the excessive stress-induced activity in the PVN may be beneficial for the animal in its response to chronic recurrent stress by reducing the output of the PVN. (c) 2005 Elsevier Inc. All rights reserved.

Interleukin-1beta (IL-1beta), a cytokine with pronounced central effects such as fever, anorexia, analgesia, etc., is also known to activate the hypothalamo-pituitary-adrenal (HPA) axis. Corticotropin releasing hormone (CRH) neurons located in the hypothalamus are important for HPA activation. The cell bodies of CRH neurons are located in the paraventricular nucleus (PVN) and their terminals are present in the median eminence (ME). Although the catecholamines, norepinephrine (NE) and dopamine (DA) are believed to be crucial factors in the stimulation of CRH neurons, it is not clear if they affect the cell bodies or terminals of these neurons to cause HPA activation. This study was done to determine if IL-1beta affects NE and DA release at the level of CRH cell bodies or their terminals. Adult male Sprague-Dawley rats were implanted with two push-pull cannulae, one in the PVN and another in the ME, and were subjected to push-pull perfusion. They were treated either with 0, 1 or 5 microg of IL-1beta. Perfusates were collected for 2 h after treatment and analyzed for NE concentrations using HPLC-EC. NE levels in the control and low dose groups did not change significantly during the entire period of observation both in the PVN and ME. In contrast, treatment with 5 microg of IL-1beta produced a marked increase in NE release in the PVN at 20 and 40 min post-treatment. NE release in the ME increased from 10 to 140 min post-treatment. There were no significant changes in the release of DA from both these areas. These results indicate that IL-1beta increases NE levels both in the PVN and in the ME and this could be a possible mechanism by which it stimulates the HPA axis.

The anteroventral region of the third ventricle (AV3V) is critical in mediating osmotic sensitivity. AV3V lesions increase plasma osmolality and block osmotic-induced vasopressin (VP) and oxytocin (OT) secretion. The aim was to evaluate the effects of AV3V lesions on neurosecretion under control/water replete conditions and after 48 h dehydration. The focus was on central peptidergic changes with measurement of OT and VP content in the hypothalamic paraventricular (PVN) and supraoptic (OT) regions and the posterior pituitary. AV3V-lesioned rats exhibited an elevated plasma osmolality and higher OT content in SON and PVN. There was an increase in VP content in PVN, but no change in SON. As predicted, the plasma peptide response to dehydration was absent in lesioned animals. However, dehydration produced depletion in posterior pituitary VP in lesioned animals with no change in OT. No changes in nuclear VP and OT levels were seen after dehydration. These results demonstrate that AV3V lesions alter the VP and OT neurosecretory system, seen as a blockade of osmotic-induced release and an increase in basal nuclear peptide content. The data indicate that interruption of the osmotic sensory system affects the central neurosecretory axis, resulting in a backup in content and likely changes in synthesis and processing.

Atrial natriuretic peptide (ANP), first discovered in the heart, has been also detected in various brain regions involved in the control of cardiovascular function and water and sodium balance. The anteroventral region of the third ventricle (AV3V) and the subfornical organ (SFO) have ANP-immunoreactive projections towards the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Extracellular fluid (ECF) hyperosmolality stimulates the secretion of oxytocin (OT) which induces ANP release by the atrium. On the other hand, passive immunoneutralization of ANP reduces OT secretion in response to ECF hypertonicity. Previous studies have shown the co-localization of ANP and OT in PVN and SON neurons and in the periventricular region, as well as the presence of ANPergic and oxytocinergic neurons in the median eminence. The aim of the present study was to investigate the OT and ANP content in the SON and PVN of the hypothalamus and in the posterior pituitary (PP) after an osmotic stimulus that induces OT secretion. The results showed that intracerebroventricular microinjection of normal rabbit serum (NRS) or of ANP antiserum followed or not by an intraperitoneal injection of isotonic saline did not alter OT secretion or OT content in the PVN, SON, and PP; passive ANP immunoneutralization reduced the basal content of ANP in the PVN, SON, and PP of animals in a situation of isotonicity; the ANP antiserum inhibited the increase of OT secretion and content of OT and ANP in the PVN, SON and PP induced by the osmotic stimulus. Thus, the increase in plasma OT and oxytocinergic neurons of the hypothalamus-posterior pituitary system in response to hypertonicity depends on the action of endogenous ANP, i.e., ECF hypertonicity must activate ANPergic neurons which directly or indirectly stimulate OT release.

The present study was designed to examine whether both induction and adaptation of brain Fos expression during acute stress depend on the intensity and duration of stressors. For this purpose, different durations of two types of acute stress, mild (restraint) and severe (immobilization) stress, were employed. Stress-induced Fos expression was analyzed quantitatively by immunohistochemistry. Adaptation of Fos expression to the acute stressors was not apparent in the hypothalamic paraventricular nucleus (PVN) or locus coeruleus (LC) but was observed in the amygdala, hippocampus, and cerebral cortex. A higher level of Fos expression was seen in the PVN, LC, and amygdala, following severe stress than was seen following mild stress. In the hippocampus, the dentate gyrus showed reduced Fos expression in response to stressors, although both mild and severe acute stress increased Fos expression in other regions of the hippocampus. The cingulate cortex showed increased Fos expression during mild stress, whereas long-duration severe stress reduced Fos expression. In the somatosensory cortex, both stressors increased Fos expression. These results indicate that the PVN and LC are relatively resistant to adaptation to acute stress compared to other brain regions. In addition, the PVN, LC, and amygdala may play important roles in the perception of the severity of stress.

The present study was designed to examine the effects of prenatal stress on the morphological development of sexually dimorphic structures in the anterior hypothalamus in male rats and to determine if there is a relationship between morphologic development of the brain and copulatory behavior in individual animals. Dams in the stress group were subjected to treatments of heat-light restraint during the third trimester of gestation (day 14 to parturition) three times daily for 45-min periods. At 90 days of age, prenatally stressed and control male offspring were tested during the dark cycle for spontaneous male sexual behavior. Volumes of the sexually dimorphic nucleus of the preoptic area (SDN-POA) and the anteroventral periventricular nucleus (AVPV) were measured. Comparisons were made between copulatory behavior and hypothalamic nuclear volumes. SDN-POA volumes were significantly reduced (feminized; males have a larger SDN-POA than females) in prenatally stressed males that did not copulate, whereas, SDN-POA volumes in prenatally stressed males that copulated were not altered. The few control males that did not copulate (sexually non-active) also had significantly reduced SDN-POA volumes compared to the control males that did copulate (sexually active). The volume of the AVPV was significantly increased (feminized; males have a smaller AVPV than females) in prenatally stressed males that were sexually non-active compared to AVPV volumes in sexually active males. The results obtained in this study provide a strong positive relationship between sexual behavior and the morphology of the two sexually dimorphic structures measured.

Using immunohistochemical detection of the Fos protein as a cellular marker of neuronal activation, we examined forebrain areas that may be activated upon chemical stimulation of the laryngeal opening. Anesthetized rats were subject to multiple infusions of a chemical solution into the laryngopharynx. These animals were compared to two control groups: a surgical control group in which the animals were subject to the surgical procedure but received no stimulus infusions and a flow control group in which physiological saline replaced the chemical stimulus. Comparing the numbers of Fos-like-immunoreactive neurons in regions of the forebrain across groups revealed that infusing the chemical stimulus solution into the laryngopharyngeal opening selectively increased the number of Fos-like-immunoreactive nuclei in the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala, two autonomic-visceral related forebrain regions. Within the paraventricular nucleus of the hypothalamus, Fos-like-immunoreactive nuclei were significantly increased in the parvocellular subdivision while in the central nucleus of the amygdala, significant increases in Fos-like-immunoreactive nuclei were limited to the lateral capsular subdivision. These data suggest that in the rat laryngopharyngeal chemosensory stimulation activates forebrain regions that receive oral sensory information and are involved in visceral and autonomic functions.

The purpose of this study was to examine specificity in the effects of interleukin-1beta (IL-1beta) on monoamines in various areas of the hypothalamus. Adult male rats were injected i.p. with saline or 2.5 or 5.0 microg of IL-1beta or were pretreated with 500 microg of IL-1 receptor antagonist (IL-1ra) followed 5 min later by 5 microg of IL-1beta. The paraventricular nucleus (PVN), arcuate nucleus (AN), median eminence (ME), and medial preoptic area (MPA) were microdissected and analyzed for neurotransmitter concentrations by high-performance liquid chromatography with electrochemical detection (HPLC-EC). In the PVN, IL treatment produced significant increases in the concentrations of norepinephrine (NE), dopamine (DA), DA metabolite dihydroxyphenylacetic acid (DOPAC), serotonin (5-HT), and its metabolite 5-hydroxyindoleacetic acid (5-HIAA). IL-1 treatment increased the concentrations of NE and DA in the AN but only of NE in the ME, and it was without any effect in the MPA. Pretreatment with IL-1ra completely blocked the IL-1 effects. It is concluded that IL-1 induces highly specific changes in monoamine metabolism in the hypothalamus, and the nature of these changes depends on specific hypothalamic nuclei.

This study examined the role of the area postrema (AP) in transducing peripheral immune signals, represented by intravenous (i.v.) interleukin-1β (IL-1), into neuroendocrine responses. The AP, a circumventricular organ with a leaky blood–brain barrier, lies adjacent to the nucleus of the solitary tract (NTS) in the medulla. The AP was removed by aspiration, and 2 weeks later, AP-lesioned or sham-lesioned rats were injected i.v. with 0.5 μg/kg IL-1 or sterile saline. After 30 min, brains were removed and analyzed for c- fos mRNA levels in various structures implicated in the hypothalamic-pituitary-adrenal axis response to peripheral cytokine challenge. The sham-lesioned animals responded to IL-1 with large elevations in adrenocorticotropic hormone (ACTH) and corticosterone levels in the plasma and c- fos mRNA levels in cells of the AP, NTS, central nucleus of the amygdala, bed nucleus of the stria terminalis, hypothalamic paraventricular nucleus (PVN), and meninges. Prior AP removal abolished the IL-1-induced increases in ACTH and corticosterone in the plasma and c- fos mRNA levels in the NTS and PVN. However, AP removal had no effect on IL-1-induced increases in c- fos mRNA levels in the other areas examined. The selective AP lesion effects suggest that the AP and adjacent NTS play a pivotal role in transducing a circulating IL-1 signal into hypothalamic-pituitary-adrenal axis activation by a pathway that may be comprised of known anatomical links between the AP, NTS, and corticotropin-releasing hormone neurons of the PVN.

To clarify the role of the hypothalamic cholinergic system in the regulation of peripheral glucose metabolism, we investigated hypothalamic cholinergic activities after administration of 2-deoxyglucose (2-DG). Intravenous administration of 2-DG (500 mg/kg) caused neuroglycopenia and marked hyperglycemia; the level of plasma glucose increased to 210% of the initial levels at 20 min. For evaluation of the cholinergic activity, we employed a microwave device and subsequently analyzed the contents of acetylcholine (ACh) and choline after microdissection of the hypothalamic nuclei, ventromedial hypothalamic nucleus (VMH), lateral hypothalamus (LH), and paraventricular nucleus (PVN). In addition, we analyzed fluctuation of extracellular levels of ACh using in vivo brain microdialysis. A decrease in the ACh content, and a corresponding increase in the choline content, was observed in those hypothalamic nuclei 20 min after administration of 2-DG. In the microdialysis perfusate, on the other hand, extracellular level of ACh was increased by 2-DG administration. These data show that ACh release, which is cholinergic activity, was increased after 2-DG administration. Our results suggest the involvement and importance of the hypothalamic cholinergic system in 2-DG-induced hyperglycemia.

Normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) received aminopeptidase M (AmM) delivered into the paraventricular nucleus of the hypothalamus (PVN). Resulting changes in blood pressure were recorded in both anesthetized and alert animals. The findings indicate significant dose-determined decreases in blood pressure in members of both strains with SHR more responsive than WKY rats. The respective drops in blood pressure for members within each strain were equivalent for the anesthetized and alert conditions. Pretreatment with the specific angiotensin receptor antagonist, sarthran, [Sar1, Thr2] Angll, into the PVN greatly diminished these responses, suggesting the involvement of the brain angiotensin system. Additionally, a sympathetic nervous system blocker, hexamethonium, and the arginine vasopressin antagonist, Pmp1, O-Me-Tyr2-[Arg] vasopressin, were peripherally administered to assess the potential contributions of these systems to cardiovascular regulation by the brain angiotensin system. The use of these blockers, individually and combined, attenuated responsiveness to infusion of AmM into the PVN. We conclude that AmM can act as a hypotensive agent in both SHR and WKY rats, and that this decrease in blood pressure is at least partially mediated via the brain angiotensin system although other systems may play a role.

Peripheral administration of interleukin-1 (IL-1) has been shown to activate induction of Fos in the brain, but the mechanism is not known. Because cerebral noradrenergic systems have been implicated in Fos induction, we studied the IL-1-induced appearance of Fos in mice pretreated with 6-hydroxydopamine (6-OHDA) which depleted cerebral norepinephrine (NE) by more than 90%, but did not significantly alter dopamine. Intraperitoneally injected IL-1β increased Fos in several brain regions, but most obviously in the hypothalamic paraventricular nucleus (PVN). Pretreatment with 6-OHDA substantially reduced the IL-1-induced Fos increase in the PVN which was no longer statistically significant. When the 6-OHDA treatment was preceded by administration of desmethylimipramine which prevents NE depletion, IL-1 treatment increased Fos in the PVN, suggesting that the effect of 6-OHDA was indeed related to the depletion of NE. These results suggest that the noradrenergic innervation of the PVN is involved in the IL-1-induced induction of Fos in the PVN. By contrast with previous experiments in rats, the IL-1-induced increase in plasma corticosterone was not significantly altered by the 6-OHDA pretreatment in mice.

The development of the hypothalamic vasopressin (VP) and oxytocin (OT) systems has been studied in rats from the 16th embryonic day (E16) until the 11th postnatal day (P11). The VP and OT mRNA-producing neurons were identified on cryostat sections by in situ hybridization using oligonucleotide probes labeled by [35S], [3H] or digoxigenin. Moreover, VP and OT gene expressions were evaluated either at E21 or at P11 following chronic depletion of catecholamines (CA). For this purpose, pregnant rats were daily injected with alpha-methyl-m(p)-tyrosine from gestational day 13 to 20 while neonates were daily injected with alpha-methyl-m(p)-tyrosine and neurotoxin 6-hydroxydopamine from postnatal day 2 to 10. No VP mRNA- or OT mRNA-expressing cells were observed in the hypothalamus of intact fetuses at E16, while 2 days later rather numerous VP and OT neurons occupied the anterior hypothalamus. One major bilateral group of VP and OT neurons was located in the supraoptic nucleus (SON). Less numerous labeled cells were found in the developing paraventricular nucleus (PVN). Some VP and OT neurons were also spread along the ventrolateral surface of the hypothalamus from the level of the median eminence, caudally, to the level of the optic nerves, rostrally. From E18 until birth, the OT neurons were localized in the dorsal portion of the SON, while its ventral portion was occupied by the VP neurons. The VP mRNA- and OT mRNA-expressing cells seemed to increase both in size and in number over the perinatal period. Frequent relatively long neuronal processes contained VP and OT mRNAs in fetuses and in newborns. When performed during the second half of the fetal life, the chronic depletion of CA did not cause any change in the VP and OT mRNA concentrations in the SON and PVN of fetuses. By contrast, similar treatment of neonates resulted in a significant increase of both mRNA levels in the SON. These data suggest that at least in the SON VP and OT gene expression might be under the inhibitory control of CA during the neonatal period.

Siberian hamsters were maintained in a short-day photoperiod (8 h light/day) at 15 degrees C; body temperature (Tb) was telemetrically monitored at 10-min intervals over the course of 4 months. Animals manifesting repeated torpor bouts (Tb30 degrees C) were subjected to lesions of the paraventricular nucleus (PVN) or a sham operation. In the 8 weeks after surgery, 67% of the animals with complete bilateral ablation of the PVN failed to express torpor; circadian Tb rhythms, as determined by periodogram analysis, were normal in all operated and sham-operated animals. Body mass did not change in PVN-ablated animals that continued to express torpor; in contrast, rapid and sustained increases in body mass were manifested by all hamsters that terminated expression of torpor. Expression of torpor is attenuated in the absence of the PVN; it is suggested that the PVN influences torpor indirectly by regulating body mass or the availability of metabolic fuels.

Neuropeptide Y (NPY) produces a vigorous feeding response in several species when it is injected into hypothalamic structures involved in eating behavior. The purpose of this study was to determine whether a unique carboxy terminal fragment of NPY would alter the pattern of eating induced in the rat either by NPY injected into the hypothalamus or by a 24-h period of food deprivation. In this case, two l -tyrosine residues and one t.-threonine residue of the NPY27–36 fragment were transformed to their D-conformation to produce [ d -Tyr27,36, d -Thr32]-NPY (27–36), i.e., D-NPY27–36. Guide cannulae for microinjection were implanted stereotaxically just dorsal to the paraventricular nucleus (PVN) or ventromedial hypothalamus (VMH) of 24 adult male Sprague-Dawley rats. Following postoperative recovery, a microinjection of artificial CSF or 1.1 jig or 3.3 μg of a peptide was made directly into the PVN or VMH as follows; native NPY; D-NPY27–36; or [L-Tyr27,36 L-Thr32]-NPY (27–36), i.e., L-NPY27–36. Food intakes were measured at intervals of 0.25, 0.5, 1.1, 2.0, 4.0, and 24 h. When D-NPY27–36 was microinjected at NPY reactive sites in the PVN or VMH of the rat 15 min before a similar microinjection of NPY, the intense eating response induced by the peptide was reduced significantly. Not only was the effect dose dependent, but D-NPY27–36 also augmented the latency to feed. A mixture of the two doses of NPY and DNPY27–36 injected at the same hypothalamic loci did not attenuate the intake of food but tended to enhance the feeding response in the rats. After the rats were deprived of food for 24 h, D-NPY27–36 microinjected in the same hypothalamic sites similarly caused a dose-dependent suppression of normal feeding behavior. However, the CSF control vehicle and L-NPY27–36 microinjected in the PVN or VMH were without effect on the pattern of eating. Further, D-NPY27–38 injected in the same hypothalamic sites affected neither body temperature nor water intakes of the rats significantly. These results demonstrate that the D substitution of this C-fragment of the NPY molecule, i.e., D-NPY27–36, serves to inhibit feeding evoked in the rat by hypothalamic NPY as well as the natural eating response to food deprivation. Thus, the D-NPY27–36 molecule may act as an antagonist at one or more subtypes of the NPY receptor in the brain of the rat.

The effect of the central administration of nitroglycerin, a potent organic nitrate vasodilator, on penile erection and yawning was studied in male rats. When given intracerebroventricularly (ICV), nitroglycerin (33-99 micrograms) induced the above responses dose-dependently. The minimal effective dose was 33 micrograms, which was active in 60% of the rats. Nitroglycerin (1.65-6.6 micrograms) induced penile erection and yawning also when injected in the paraventricular nucleus of the hypothalamus. Nitroglycerin responses were prevented by methylene blue (200-400 micrograms ICV), by d(CH2)5Tyr(Me)2-Orn8-vasotocin (0.5-1 micrograms ICV) but not hemoglobin (100-200 micrograms ICV). In contrast methylene blue (10-20 micrograms), d(CH2)5Tyr(Me)2-Orn8-vasotocin (0.05-0.1 microgram) and hemoglobin (10-20 micrograms) were ineffective when injected in the paraventricular nucleus. Systemic haloperidol (0.5-1 mg/kg IP) was also ineffective. The results suggest that nitroglycerin induces penile erection and yawning by activating brain oxytocinergic transmission through the formation of nitric oxide in the paraventricular nucleus of the hypothalamus.

In female rats, stimulation of the uterine cervix during mating induces two daily surges of prolactin. Inhibition of hypothalamic dopamine release and stimulation of oxytocin neurons in the paraventricular nucleus (PVN) are required for prolactin secretion. We aim to better understand how stimulation of the uterine cervix is translated into two daily prolactin surges. We hypothesize that noradrenergic neurons in the A1, A2, and locus coeruleus (LC) are responsible for conveying the peripheral stimulus to the PVN. In order to determine whether projections from these neurons to the PVN are activated by cervical stimulation (CS), we injected a retrograde tracer, Fluoro-Gold (FG), into the PVN of ovariectomized rats. Fourteen days after injection, animals were submitted to artificial CS or handling and perfused with a fixative solution. Brains were removed and sectioned from the A1, A2, and LC for c-Fos, tyrosine hydroxylase (TH), and FG triple-labeling using immunohistochemistry. CS increased the percentage of TH/FG+ double-labeled neurons expressing c-Fos in the A1 and LC. CS also increased the percentage of TH+ neurons expressing c-Fos within the A1 and A2, independent of their projections to the PVN. Our data reinforce the significant contributions of the A1 and A2 to carry sensory information during mating, and provide evidence of a functional pathway in which CS activates A1 and LC neurons projecting to the PVN, which is potentially involved in the translation of CS into two daily prolactin surges.

Neural connections linking the four magnocellular nuclei i.e.,. the paired supraoptic (SON) and paraventricular (PVN) nuclei, may contribute to the simultaneous and parallel changes in firing patterns of oxytocinergic neurons during reflex milk ejection. To investigate these neural connections in the absence of suckling, intranuclear release of oxytocin (OT) was stimulated by microdialysis of hypertonic CSF containing 1 M NaCl (HS-CSF) into the right SON area and glucose metabolism of both SONs and PVNs and the neural lobe of virgin and lactating (10–12 day) rats was mapped by the autoradiographic [14C]deoxyglucose (DG) method. OT in the microdialysates and in plasma, obtained before and after 80–90 min of dialysis with CSF or HS-CSF, was quantified by RIA. In both virgin and lactating rats, microdialysis of HS-CSF unilaterally into the SON area significantly (p < 0.05) increased release of OT in the nucleus and into plasma, which was associated with enhanced (p < 0.05) metabolic activity in the ipsilateral and contralateral SON and the neural lobe but not in either PVN. Compared with virgins, lactating rats were less active, had lower (p < 0.05) glucose utilization in the hypothalamo-neurohypophysial system, and less (p < 0.05) OT in plasma during microdialysis of HS-CSF into the SON area. The osmotic stimulus did not activate neural structures (suprachiasmatic and medial amygdaloid nuclei) near the SON in either hemisphere. Thus, neural connections or, less likely, transport of OT via the subarachnoid space, may function to recruit activation of cells in the contralateral SON following hypertonic stimulation of cells in the other SON.

In the preceding article, a dorsochiasmatic area (DCh) was described that projects to both paraventricular (PVN) and supraoptic (SON) nuclei. The main afferents of the DCh, revealed by local injections of retrograde tracers, are the hypothalamic PVN and SON, lateral septal nuclei (LSV and SHy), bed nuclei of the stria terminalis (BST), anteroventral third ventricle region, particularly the median preoptic nucleus (MnPO), the subfornical organ, medial preoptic areas, arcuate hypothalamic nucleus, ventromedial hypothalamic nuclei, paraventricular thalamic nucleus, and, more caudally, several structures of the posterior hypothalamus and mesencephalon. The relations between DCh and BST, LSV, SHy, or MnPO appeared reciprocal. In view of their reciprocal relationships with the hypothalamo-neurohypophyseal system and some of their related extrahypothalamic structures, the DCh might be involved in the regulation of the vasopressin (AVP) and/or oxytocin (OT) systems, or in reproductive behavior.

To elucidate hypothalamic involvement in blood calcium homeostasis, the effects of unilateral electrical stimulation (0.1 mA 0.5 ms, 30 Hz, 60 min) of the lateral hypothalamic area (LHA), the paraventricular nucleus (PVN), and the ventromedial nucleus of the hypothalamus (VMH) on the blood concentration of ionized calcium were examined in the anesthetized rats. LHA stimulation induced a sustained decrease (0.05–0.07 mM fall) in the blood calcium level during the period of 60 to 150 min (end of the measurements) after stimulation. In contrast, PVN stimulation elicited a transient hypocalcemia (0.07 mM decrease) 60 min after stimulation. The hypocalcemie effects of LHA and PVN stimulation were eliminated by vagotomy of the gastric branches and the thyroid/parathyroid branches, respectively. VMH stimulation, using the same parameters, did not induce any significant change in blood calcium. The results suggest that the LHA and the PVN have a hypocalcemie function that is mediated, at least in part, by the vagus nerve innervating the stomach and the thyroid/parathyroid glands, respectively.

The aim of the present study was to assess the effects of intravenous (i.v.) cytidine-5'-diphosphate (CDP)-choline administration on the activation of oxytocin and vasopressin neurons in the supraoptic (SON) and paraventricular nuclei (PVN), using the immunohistochemical identification of c-Fos expression as a marker of neuronal activation and to correlate this with the plasma hormone levels. Rats were catheterized under sevofluorane anesthesia and experiments were conducted 24 h later. Blood samples were withdrawn from arterial catheter at 2, 5, 10, 20, 40 and 60 min after CDP-choline (0.5, 1.0 and 2.0 g/kg; i.v.) or saline (1.0 ml/kg; i.v.) for the measurement of plasma oxytocin and vasopressin levels by radioimmunoassay. Animals were sacrificed 90 min after CDP-choline administration for dual immunohistochemistry which was performed on paraformaldehyde-fixed vibratome sections. Dual immunohistochemistry for c-Fos and oxytocin or vasopressin revealed that CDP-choline activates these neurons in a dose-dependent manner. Light microscopic analyses showed that, about 41%, 75% or 87% of the oxytocin neurons and about 18%, 46% or 82% of the vasopressin neurons in SON express c-Fos, thus activated, by the dosages of 0.5, 1.0 or 2.0 g/kg CDP-choline, respectively. Increases in c-Fos expression were about 29%, 62% or 81% for the oxytocin neurons and about 38%, 70% or 78% for the vasopressin neurons in PVN with the dosages of 0.5, 1.0 or 2.0 g/kg CDP-choline, respectively. When compared to the control groups (8% and 7% oxytocin or 2% and 5% vasopressin neuronal activation in SON or PVN, respectively), these increases were found to be statistically significant (p < 0.05). In the PVN most of the magnocellular neurons were activated while less number of parvocellular neurons expressed c-Fos in response to CDP-choline challenge. In correlation with c-Fos data, CDP-choline increased plasma oxytocin and vasopressin levels both dose- and time-dependently. Results of the present study suggested that peripheral administration of CDP-choline is able to increase plasma oxytocin and vasopressin levels while activating the respective neurons.

Losartan is a novel nonpeptidergic antagonist of angiotensin (ANG) II subtype 1 (AT1) receptors, which effectively lowers blood pressure in high-renin hypertensive rat and blocks the pressor response to systemic ANG II. It is well known that high densities of ANG II receptors exist in the hypothalamic paraventricular nucleus (PVN). In addition, activation of putative angiotensinergic afferents to the PVN originating in subfornical organ (SFO) elevates blood pressure and facilitates the activity of PVN neurons. We report here that systemic administration of losartan (3 mg/kg) significantly attenuates the pressor response to electrical stimulation of SFO. The excitatory responses of PVN neurons to SFO stimulation or local pressure microinjection of ANG II were also significantly inhibited in 58.8% and 88.9% of PVN cells, respectively, by intravenous administration of losartan. These pharmacological effects were rapid and reversible, and were accompanied by little change of basal arterial blood pressure or spontaneous neuronal activity. These observations suggest that systemic losartan crosses the blood-brain barrier (BBB) and acts at AT1 receptors within the PVN.

Systemic administration of CCK-8 increased plasma oxytocin (OXT) level in rats anesthetized with a mixture of urethane and alpha-chloralose. Extracellular recordings were made from magnocellular neurosecretory neurons in the paraventricular nucleus (PVN) of the hypothalamus in anesthetized rats to examine the effects of CCK-8 on the firing of PVN neurons. Thirteen out of 16 nonphasic neurons (putative OXT-secreting neurons) were excited by intravenous and/or intraperitoneal administration of CCK-8. By contrast, 8 out of 10 phasic cells, vasopressin(AVP)-secreting cells, were inhibited by systemic administration of CCK-8. Four out of five nonneurosecretory neurons in the PVN were excited by the administration of CCK-8. Moreover, microiontophoretically applied phentolamine blocked the excitatory responses induced by CCK-8 in nonphasic neurons. We measured extracellular noradrenaline (NA) level in the PVN, using in vivo microdialysis. Intravenous administration of CCK-8 induced NA release in the PVN. These results suggest that CCK-8 activates the excitatory afferent pathway to putative OXT-secreting neurons in the PVN which may, at least in part, be involved in the central noradrenergic projection.

The organization of angiotensin (Ang)-(l-7) immunoreactivity with respect to neurons containing the nitric oxide synthase, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), in the hypothalamo-neurohypophysial (HNS) system was examined. The majority of neurons in this system were single labeled with Ang-(1–7) or NADPH-d and were found localized in two separate populations of neurons within the HNS. A small number of double-labeled neurons were observed in the supraoptic (SO) and paraventricular (PV) nuclei. Double-labeled accessory neurosecretory neurons were also found distributed between the SO and PV. Although a well-defined function for nitric oxide in the magnocellular hypothalamic system has not been determined, the codistribution and limited coexistence of Ang-(1–7), a heptapeptide involved in antidiuresis, and NADPH-d in the HNS suggests a potential role for these substances in mechanisms modulating fluid homeostasis.

The distribution of corticotropin-releasing hormone-immunoreactive (CRH-IR) neurons and fibers was observed in golden hamsters. CRH-IR neurons and fibers were observed within the hypothalamus, thalamus, amygdala, cortex, midbrain, and hindbrain. The largest numbers of CRH-IR neurons were seen within the magno- and parvocellular divisions of the paraventricular nucleus of the hypothalamus and within the septum, bed nucleus of the stria terminalis, preoptic area continuum. The highest density of immunoreactive fibers was observed in the external zone of the median eminence. In addition, many immunoreactive fibers were observed within the bed nucleus of the stria terminalis and the preoptic area. The distribution obtained in hamsters was compared with previously reported distributions from rats, and both were generally similar.

Descending projections from the hypothalamic paraventricular nucleus (PVN) to the spinal cord mediate the effects of photoperiod on the reproductive system of hamsters. To elucidate the course of these PVN efferent fibers, injections of horseradish peroxidase conjugated to either cholera toxin or wheat germ agglutinin were made into the T1-C7 region of the spinal cord of hamsters. Four sets of descending tracts were identified in males and females. Two sets of fibers originated from the medial PVN and exited the nucleus dorsally and ventrally, respectively. Most of the descending fibers, however, organized themselves into two tracts that exited the PVN laterally. In earlier experiments, destruction of these lateral pathways prevented photoperiodic responses in hamsters of both sexes. Photoperiodism Paraventricular nucleus Seasonal cycles Spinal cord

A histochemical study of the distribution of NADPH diaphorase activity in the hypothalamus of normal rats was carried out. Our study demonstrates the presence of NADPH-diaphorase activity in the circularis and anterior and posterior fornicals nuclei for the first time. Additionally, we confirm the presence of NADPH-diaphorase-stained neurons in the paraventricular (both magno- and parvicellular neurons) and supraoptic nuclei, as well as a population of isolated positive neurons (not included in any hypothalamic nuclei) located among the different nuclei. Because NADPH diaphorase has recently been shown to be a nitric oxide synthase. our study reveals a wide presence of this enzymatic activity in the hypothalamus of the rat.

The effect of the intracerebroventricular (ICV) administration of pertussis toxin on penile erection and yawning induced by apomorphine and oxytocin was studied in male rats. Pertussis toxin (2 micrograms ICV) prevented the above behavioral responses to apomorphine (80 micrograms/kg SC) and oxytocin (30 ng ICV) on day 3 and 4, but not on day 0 and 1 after treatment. Oxytocin and apomorphine responses were restored on day 6. Similar results were obtained by microinjecting pertussis toxin (0.5 microgram) in the paraventricular nucleus of the hypothalamus, the most sensitive brain area for the induction of penile erection and yawning by oxytocin and apomorphine. The results suggest that G proteins are involved in the expression of above responses to apomorphine and oxytocin.

The mechanisms controlling central and systemic oxytocin (OT) release were examined using in vivo microdialysis of the paraventricular (PVN) region. Dialysate and plasma samples were collected from conscious male rats and stimuli were administered via the dialysate fluid. Characterization studies showed that microdialysis was a viable technique for the study of peptide secretion in the conscious animal. OT was consistently detected in the PVN dialysate and a partially purified extract crossreacted in parallel fashion with the synthetic peptide. In vitro studies showed that peptide recovery was positively correlated with the pore size of the dialysis membrane and that there was an inverse relationship between flow rate and recovery. Hypertonic saline administered centrally caused an increase in dialysate and plasma oxytocin while the intravenous injection affected only plasma oxytocin. The excitatory amino acid, glutamate (0.05-0.5 M), caused an increase in plasma, but not dialysate oxytocin, while depolarization with potassium chloride (0.05-0.15 M) had no significant effects. Histological examination showed that the dialysis probe was located in the rostral, lateral PVN. Our results show that in vivo microdialysis provides a method for the delivery of drugs into specific brain regions as well as a useful technique for the evaluation of in vivo neuropeptide release.

The distribution of calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat was studied using the avidin-biotin-immunoperoxidase method and highly specific monoclonal antibodies. Incubation with anticalbindin D-28K-antiserum revealed immunoreactive neurons in the following nuclei: supraoptic, paraventricular (both in the magnocellular and parvicellular regions), circularis, fornicals and medial forebrain bundle. Incubation with parvalbumin antiserum displayed immunoreactive neurons only in the circularis nucleus. Additionally, it was possible to observe scattered calbindin and parvalbumin immunoreactive neurons (which do not form part of the nuclei considered) located in the hypothalamic area between the supraoptic and the paraventricular nuclei, especially for the calbindin D-28K antiserum.

5 páginas The elevated plus-maze is an animal model used to study anxiety. In a second session, rats show a reduction in the exploratory behavior even when the two sessions are separated by intervals as large as 7 days. The aim of the present study was to investigate whether the reduction in the exploratory behavior is maintained after intervals larger than 7 days. Additionally, we aimed at investigating eventual correlations between behaviors in the plus-maze and activation of limbic structures as measured by Fos protein expression after the second session. Rats were tested for 5 min in the elevated plus-maze and re-tested 3, 9 or 33 days later. Other groups were tested only once. The rat brains were processed for immunohistochemical detection of Fos protein. The results show a decrease in the open arms exploration in the second trial with intervals of 3, 9 and 33 days. The expression of Fos protein in the piriform cortex, septal nucleus and paraventricular hypothalamic nucleus in the groups tested with intervals of 9 and 33 days were statistically different from the other groups. The alterations observed in exploratory behavior in the second session in the plus-maze did not correlate with Fos expression. In conclusion, although the specific test conditions were sufficient to evoke behavioral alterations in exploration in the elevated plus-maze, they were enough to induce significant Fos protein expression in piriform cortex, septal nucleus and thalamic and hypothalamic paraventricular nuclei but not in other areas such as dorsomedial nucleus of the hypothalamus and amygdala nuclei, known to be also active participants in circuits controlling fear and anxiety

The present studies have examined the hypothesis that food intake and sympathetic activity are inversely related. Following lateral hypothalamic lesions, food intake decreases and sympathetic activity rises. When pair-gained animals are used, the sympathetic activity of the LH-lesioned animals is above that of ad lib fed controls, and the sympathetic activity of the pair-gained animals is below that of the controls. With VMH-lesions, food intake rises and sympathetic activity falls. Several drugs, including 2-deoxy-D-glucose, fenfluramine, corticotropin releasing hormone, and neuropeptide Y, all show this reciprocal relationship. Three exceptions to this rule, the rat with a PVN lesion, cold exposure and animals eating a highly palatable diet, are discussed. Under the usual circumstances, the sympathetic activity appears to play a tonic role in regulating the onset of a meal, and the acute changes in sympathetic activity after eating may play a role in the onset of satiety.

Stimulation of the ventral noradrenergic ascending bundle (VNAB) at low frequencies (0.5/5 Hz) excited the majority (37/46, 80%) of single paraventricular nucleus (PVN) tuberoinfundibular neurones, with high frequency (50 Hz) trains of stimuli reversing the direction of the response to inhibition for 7/16 (44%) of these excited cells. Iontophoretic application of noradrenaline, or the alpha 1-adrenoceptor agonist 1-phenylephrine, increased the spontaneous electrical activity of most of the cells tested (94% and 72%), whilst application of the alpha 1-antagonist, ergotamine reduced the spontaneous activity of 44% of the cells tested and prevented the excitation following VNAB stimulation for 84% of the cells examined. Application of the beta-adrenoceptor antagonist, propranolol, increased the spontaneous activity of 77% of cells and prevented the inhibitory PVN neuronal responses following high frequency VNAB stimulation of 94% of the cells, often reversing the response to excitation similar to that observed following low frequency VNAB stimulation. The alpha 2-adrenoceptor antagonist, tolazoline, was found to evoke mixed responses from the cells examined but a trend towards a suppression of spontaneous activity and potentiation of VNAB stimulation-evoked responses was observed. The alpha 2-adrenoceptor agonist, clonidine, elicited an initial excitation from the majority of cells tested, with most of the cells then exhibiting an inhibition, either with or without continued application. Excitatory responses following stimulation of the sciatic nerve were recorded from the majority of cells (82.5%) and ergotamine was able to suppress this response for all four cells so tested.(ABSTRACT TRUNCATED AT 250 WORDS)

The timing of the proestrous surge of follicle-stimulating hormone (FSH) was examined in female hamsters with hypothalamic knife cuts that prevented reproductive responses to photoperiod. All animals received either a horizontal knife cut aimed between the suprachiasmatic nuclei (SCN) and the paraventricular nuclei (PVN), or sham surgery, and were housed in long (16 h of light/24 h) or short (6 h of light/24 h) photoperiods. Following exposure to either photo-period for 11-12 weeks, a subset of the animals was fitted with an indwelling jugular cannula. Blood samples were taken hourly over a 24-h period and plasma levels of FSH were determined by RIA. Knife cuts placed ventral to or through the ventral portions of the PVN prevented short day-induced anestrus. On the day of proestrus, peak elevations of FSH in cycling animals with knife cuts in both photoperiods, as well as in sham-operated females in long days, occurred 4-5 h before lights out. In contrast, sham-operated anestrous females in short days showed peak elevations of FSH approximately 3-4 h after lights out. The present results support the view that neural connections between the SCN and the PVN mediate the effects of short days on reproductive physiology, including changes in the timing of the FSH surge.

In the rat, medullary afferents to the hypothalamic magnocellular nuclei mediate the baroreceptor reflexes of vasopressinergic neurons and the cholecystokinin- or gastric distention-induced excitation of oxytocinergic neurons. One strategy that reflexes such as these may use to coordinate the activity of magnocellular neuroendocrine neurons is collateral branching of input. Previous work has shown that the distributions of medullary neurons projecting to the paraventricular and the supraoptic nuclei overlap and that their axons branch. Thus, we hypothesized that single neurons in the ventral lateral medulla and/or the nucleus tractus solitarius would project to both the paraventricular and supraoptic nuclei via collateral branches of their axons. Medullary afferent neurons were retrogradely labeled after injection into the paraventricular and the supraoptic nucleus on one side of the brain with two different fluorescent tracers: Fluoro-Gold or rhodamine-labeled latex microspheres. The topographic distribution of labeled cells in the medulla containing either a single fluorescent tracer or both tracers were plotted. Of these labeled neurons, a small percentage (7%) contained both dyes, suggesting that they send collateral branches to both of the magnocellular neuroendocrine nuclei injected. Single labeled cells were both ipsi- and contralateral to the injected side (53% ipsilateral), but most double-labeled cells were ipsilateral (84%). In rats, areas that project to both the paraventricular and the supraoptic nuclei may act upon both nuclei together. Thus, afferent inputs, in conjunction with the known inter- and intracellular changes that take place within the magnocellular nuclei, may be involved with the coordinated responses throughout magnocellular neuroendocrine system during medullary reflexes, i.e., the baroreceptor-mediated reflexes or the gastric distention reflexes.

To clarify some of the actions of Neurotropin (NSP), intra- and extracellular measurements were made of 140 paraventricular (PVN) and 48 ventromedial nucleus (VMH) neurons in rat hypothalamic tissue slices in vitro while perfusing NSP, an extract from the inflamed skin of rabbits inoculated with vaccinia virus, through the recording chamber. NSP mostly decreased activity in PVN neurons (inhibition, 46; excitation, 27; excitation-inhibition, 5; no response, 62), and mostly increased it in VMH neurons (excitation, 12; inhibition, 3; no response, 33). Inhibition of PVN neurons by NSP was due to hyperpolarization with no change in membrane conductance. Since ouabain antagonized the NSP-induced inhibition of PVN neurons, the effect was probably due to activation of the sodium pump. Activity of some NSP-responsive PVN neurons was increased by increase of extracellular osmolarity, and activity of some NSP-responsive VMH neurons was increased by glucose application. The results suggest central modulation of autonomic or neuroendocrinological function by distinct NSP influence on PVN and VMH neural activity.

Extracellular recordings were made from 44 spontaneously active neurosecretory and 43 unidentified neurons in the paraventricular nucleus (PVN) of the hypothalamus in urethane-anesthetized rats. Physiologically hypertonic (+30 mOsm/kg, NaCl or mannitol) and hypotonic ( − 30 mOsm/kg) solutions were applied by pressure through multibarrel micropipettes to the immediate vicinity of neurons. Of 44 neurosecretory neurons tested, 31 (70%) were unaffected by locally applied osmotic stimulation, and the remaining 13 were excited. Of these 31 osmotically unresponsive neurosecretory neurons, 21 were further tested with very strong hypertonic (+260 mOsm/kg, NaCl) solution, and only 7 were affected. Of 43 unidentified neurons, 28 (65%) were unaffected by osmotic stimulation, and 12 were excited. Accordingly, only parts (30%) of both the neurosecretory and unidentified neurons in the PVN were found to be osmosensitive. We concluded that some neurons in the rat PVN are osmosensitive and osmosensitivity is not specific to neurosecretory neurons in the PVN.