HYPOTHALAMIC COCAINE- AND AMPHETAMINE-REGULATED TRANSCRIPT AND CORTICOTROPHIN RELEASING FACTOR NEURONS ARE STIMULATED BY EXTRACELLULAR VOLUME AND OSMOTIC CHANGES

Several studies suggest that hypothalamic cocaine- and amphetamine-regulated transcript (CART) may interact with the hypothalamic-pituitary-adrenal (HPA) axis in the control of neuroendocrine function and may also participate in cardiovascular regulation. Therefore, this study aimed to evaluate, in experimental models of isotonic (I-EVE) and hypertonic (H-EVE) extracellular volume expansion and water deprivation (WD), the activation of CART- and corticotrophin releasing factor (CRF)-immunoreactive neurons, as well as the relative expression of CART and CRF mRNAs in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Both H-EVE (0.30M NaCl, 2mL/100g of body weight, in 1 minute) and 24 hours of WD significantly increased plasma sodium concentrations, producing, respectively, either an increase or a decrease in extracellular volume. I-EVE (0.15M NaCl, 2mL/100g of body weight, in 1 minute) evoked a significant increase in the circulating volume accompanied by unaltered plasma concentrations of sodium. CART-expressing neurons of both magnocellular and parvocellular hypothalamic divisions were activated to produce Fos in response to H-EVE but not in response to I-EVE. Furthermore, increased expression of CART mRNA was found in the PVN of H-EVE but not I-EVE rats. These data show for the first time that EVE not only activates hypothalamic CRF neurons but also increases CRF mRNA expression in the PVN. In contrast, WD increases the number of CART-immunoreactive neurons activated to produce Fos in the PVN and SON but does not change the number of neurons double labeled for Fos and CRF or expression of CRF mRNA in the PVN. These findings provided new insights into the participation of CART in diverse processes within the PVN and SON, including its possible involvement in activation of the HPA axis and cardiovascular regulation in response to changes in extracellular volume and osmolality. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Prostaglandin mediates endotoxaemia-induced hypophagia by activation of pro-opiomelanocortin and corticotrophin-releasing factor neurons in rats

Corticotrophin-releasing factor (CRF) and alpha-melanocyte-stimulating hormone (alpha-MSH), both of which are synthesized by hypothalamic neurons, play an essential role in the control of energy homeostasis. Neuroendocrine and behavioural responses induced by lipopolyssacharide (LPS) have been shown to involve prostaglandin-mediated pathways. This study investigated the effects of prostaglandin on CRF and alpha-MSH neuronal activities in LPS-induced anorexia. Male Wistar rats were pretreated with indomethacin (10 mg kg(-1); i.p.) or vehicle; 15 min later they received LPS (500 mu g kg(-1); i.p.) or saline injection. Food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the paraventricular and arcuate nuclei, respectively, were evaluated. In comparison with saline treatment, LPS administration induced lower food intake and increased plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF and Fos-alpha-MSH double-labelled neurons in vehicle-pretreated rats. In contrast, indomethacin treatment partly reversed the hypophagic effect, blunted the hormonal increase and blocked the Fos-CRF and Fos-alpha-MSH hypothalamic double labelling increase in response to the LPS stimulus. These data demonstrate that the activation of pro-opiomelanocortin and CRF hypothalamic neurons following LPS administration is at least partly mediated by the prostaglandin pathway and is likely to be involved in the modulation of feeding behaviour during endotoxaemia.

Downregulation of Melanocortin-4 Receptor during Refeeding and its Modulation by Adrenalectomy in Rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.

GABAergic mediation of stress-induced secretion of corticosterone and oxytocin, but not prolactin, by the hypothalamic paraventricular nucleus

The hypothalamus-pituitary-adrenal axis (HPA) participates in mediating the response to stressful stimuli. Within the HPA, neurons in the medial parvocellular region of paraventricular nucleus (PVN) of the hypothalamus integrate excitatory and inhibitory signals triggering secretion of corticotropin-releasing hormone (CRH), the main secretagogue of adrenocorticotropic hormone (ACTH). Stressful situations alter CRH secretion as well as other hormones, including prolactin and oxytocin. Most inputs to the PVN are of local origin, half of which are GABAergic neurons, and both GABA-A and GABA-B receptors are present in the PVN. The objective of the present study was to investigate the role of GABA-A and GABA-B receptors in the PVN`s control of stress-induced corticosterone, oxytocin and prolactin secretion. Rats Were microinjected with saline or different doses (0.5, 5 and 50 pmol) of GABA-A (bicuculine) or GABA-B (phaclofen) antagonists in the PVN. Ten minutes later, they were subjected to a stressor (ether inhalation) and blood samples were collected 30 min before and 10, 30, 60, 90 and 120 min after the stressful stimulus to measure hormone levels by radioimmunoassay. Our results indicate that GABA acts in the PVN to inhibit stress-induced corticosterone secretion via both its receptor subtypes, especially GABA-B. In contrast, GABA in the PVN stimulates oxytocin secretion through GABA-B receptors and does not alter prolactin secretion. (C) 2008 Elsevier Inc. All rights reserved.

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-alpha-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POW mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 mu g/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-alpha-MSH immunoreactivity and POW mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POW neurons in the hypothalamus and an increased mRNA expression of these neuropeptides. (C) 2008 Elsevier Inc. All rights reserved.

Hypothalamic supraoptic but not paraventricular nucleus is involved in cardiovascular responses to carbachol microinjected into the bed nucleus of stria terminalis of unanesthetized rats

Microinjection of the cholinergic agonist carbachol into the bed nucleus of the stria terminalis (BST) has been reported to cause pressor response in unanesthetized rats, which was shown to be mediated by an acute release of vasopressin into the systemic circulation and followed by baroreflex-mediated bradycardia. In the present study, we tested the possible involvement of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei in the pressor response evoked by carbachol microinjection into the BST of unanesthetized rats. For this, cardiovascular responses following carbachol (1 nmol/100 nL) microinjection into the BST were studied before and after PVN or SON pretreatment, either ipsilateral or contralateral in relation to BST microinjection site, with the nonselective neurotransmission blocker cobalt chloride (CoCl(2), 1 mM/100 nL). Carbachol microinjection into the BST evoked pressor response. Moreover, BST treatment with carbachol significantly increased plasma vasopressin levels, thus confirming previous evidences that carbachol microinjection into the BST evokes pressor response due to vasopressin release into the circulation. SON pretreatment with CoCl(2), either ipsilateral or contralateral in relation to BST microinjection site, inhibited the pressor response to carbachol microinjection into the BST. However, CoCl(2) microinjection into the ipsilateral or contralateral PVN did not affect carbachol-evoked pressor response. In conclusion, our results suggest that pressor response to carbachol microinjection into the BST is mediated by SON magnocellular neurons, without significant involvement of those in the PVN. The results also indicate that responses to carbachol microinjection into the BST are mediated by a neural pathway that depends on the activation of both ipsilateral and contralateral SON. (C) 2011 Elsevier B.V. All rights reserved.

The prednisolone suppression test in depression: Dose-response and changes with antidepressant treatment

Depressed patients have reduced glucocorticoid receptor (GR) function, as demonstrated by resistance to the suppressive effects of the synthetic glucocorticoid hormone, and GR agonist, dexamethasone. We have developed a suppressive test with prednisolone, a synthetic glucocorticoid that is similar to cortisol in its pharmacodynamics and pharmacokinetics, and binds to both the GR and the mineralocorticoid receptor (MR). We have found that depressed patients suppress normally to prednisolone, unless they are particularly non-responsive to treatment. In the present study, we evaluated 28 inpatients with treatment-resistant depression (TRD), and compared salivary cortisol secretion (at 0900 h, 1200 h and 1700 h) after placebo or after prednisolone (5 mg), before and after an inpatient treatment admission. Half of the patients (n = 14) reached treatment response. When comparing the assessment between admission and discharge, cortisol output after placebo fell (-26% of area under the curve; p = 0.024) while the output after prednisolone did not change. Moreover, there was no change in the response to prednisolone (percentage suppression) between admission at discharge, and this was not influenced by treatment response. Finally, we could confirm and extend our previously published data with prednisolone (5 mg), showing that depressed patients (n = 12) and controls (n = 12) suppressed equally to both 5 and 10 mg doses of prednisolone. This study suggests that the response to prednisolone is similar in depressed patients and controls at different doses of prednisolone, and does not change with symptomatic improvement. This is in contrast with findings, from us and others, using other measures of hypothalamic-pituitary-adrenal axis function, such as basal cortisol levels or the response to dexamethasone. Thus, we propose that the prednisolone suppression test may offer specific biological and clinical information, related to its action at both the GR and the MR. (C) 2010 Elsevier Ltd. All rights reserved.

Neurobiology of panic disorder: From animal models to brain neuroimaging

Evidence from animal models of anxiety has led to the hypothesis that serotonin enhances inhibitory avoidance (related to anxiety) in the forebrain, but inhibits one-way escape (panic) in the midbrain periaqueductal gray (PAG). Stressing the difference between these emotions, neuroendocrinological results indicate that the hypothalamic-pituitary-adrenal axis is activated by anticipatory anxiety, but not by panic attack nor by electrical stimulation of the rat PAG. Functional neuroimaging has shown activation of the insula and upper brain stem (including PAG), as well as deactivation of the anterior cingulated cortex (ACC) during experimental panic attacks. Voxel-based morphometric analysis of brain magnetic resonance images has shown a grey matter volume increase in the insula and upper brain stem, and a decrease in the ACC of panic patients at rest, as compared to healthy controls. The insula and the ACC detect interoceptive stimuli, which are overestimated by panic patients. It is suggested that these brain areas and the PAG are involved in the pathophysiology of panic disorder. (C) 2008 Elsevier Ltd. All rights reserved.

Exposure to maternal smoking during fetal life affects food preferences in adulthood independent of the effects of intrauterine growth restriction

Experimental animal studies have shown that nicotine exposure during gestation alters the expression of fetal hypothalamic neuropeptides involved in the control of appetite. We aimed to determine whether the exposure to maternal smoking during gestation in humans is associated with an altered feeding behavior of the adult offspring. A longitudinal prospective cohort study was conducted including all births from Ribeirao Preto (Sao Paulo, Brazil) between 1978 and 1979. At 24 years of age, a representative random sample was re-evaluated and divided into groups exposed (n = 424) or not (n = 1586) to maternal smoking during gestation. Feeding behavior was analyzed using a food frequency questionnaire. Covariance analysis was used for continuous data and the chi(2) test for categorical data. Results were adjusted for birth weight ratio, body mass index, gender, physical activity and smoking, as well as maternal and subjects` schooling. Individuals exposed to maternal smoking during gestation ate more carbohydrates than proteins (as per the carbohydrate-to-protein ratio) than non-exposed individuals. There were no differences in the consumption of the macronutrients themselves. We propose that this adverse fetal life event programs the individual`s physiology and metabolism persistently, leading to an altered feeding behavior that could contribute to the development of chronic diseases in the long term.

Involvement of hypothalamic paraventricular nucleus non-N-methyl-d-aspartate receptors in the pressor response to noradrenaline microinjected into the bed nucleus of the stria terminalis of unanesthetized rats

Microinjection of noradrenaline into the bed nucleus of the stria terminalis (BST) has been reported to cause a pressor response in unanesthetized rats, which was shown to be mediated by acute vasopressin release into the systemic circulation. In the present study we verified the involvement of magnocellular neurons of the hypothalamic paraventricular (PVN) or supraoptic (SON) nuclei and the local neurotransmitter involved in the pressor response to noradrenaline microinjection into the BST. The PVN pretreatment with the non-selective neurotransmission blocker CoCl(2) (1 nmol/100 nL) inhibited the noradrenaline-evoked pressor response. However, responses were not affected by SON treatment with CoCl(2). Further experiments were carried out to test if glutamatergic neurotransmission in the PVN mediates the pressor response evoked by noradrenaline microinjection into the BST. Pretreatment of the PVN with the selective N-methyl-d-aspartate (NMDA) receptor antagonist LY235959 (2 nmol/100 nL) did not affect the noradrenaline-evoked pressor response. However, PVN pretreatment with the selective non-NMDA receptor antagonist NBQX (2 nmol/100 nL) significantly reduced the pressor response to noradrenaline microinjection into the BST. In conclusion, our results suggest that pressor responses to noradrenaline microinjection into the BST are mediated by PVN magnocellular neurons without involvement of SON neurons. They also suggest that a glutamatergic neurotransmission through non-NMDA glutamate receptors in the PVN mediates the response.

A study of adrenocortical tumors in children and adolescents by a comparative genomic hybridization technique

Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1-p15.3 and 20p11.2-p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1-q24.3 and the more frequent losses being 3q21-q26.2, 20q12-qter, and 22q11.2-q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21-p31.2, 2p12-p21 and loss of 20p11.2-p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.

CHARACTERIZATION OF Kiss1 NEURONS USING TRANSGENIC MOUSE MODELS

Humans and mice with loss-of-function mutations of the genes encoding kisspeptins (Kiss1) or kisspeptin receptor (Kiss1r) are infertile due to hypogonadotropic hypogonadism. Within the hypothalamus, Kiss1 mRNA is expressed in the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (Arc). In order to better study the different populations of kisspeptin cells we generated Kiss1-Cre transgenic mice. We obtained one line with Cre activity specifically within Kiss1 neurons (line J2-4), as assessed by generating mice with Cre-dependent expression of green fluorescent protein or beta-galactosidase. Also, we demonstrated Kiss1 expression in the cerebral cortex and confirmed previous data showing Kiss1 mRNA in the medial nucleus of amygdala and anterodorsal preoptic nucleus. Kiss1 neurons were more concentrated towards the caudal levels of the Arc and higher leptin-responsivity was observed in the most caudal population of Arc Kiss1 neurons. No evidence for direct action of leptin in AVPV Kiss1 neurons was observed. Me lanocortin fibers innervated subsets of Kiss1 neurons of the preoptic area and Arc, and both populations expressed melanocortin receptors type 4 (MC4R). Specifically in the preoptic area, 18-28% of Kiss1 neurons expressed MC4R. In the Arc, 90% of Kiss1 neurons were glutamatergic, 50% of which also were GABAergic. In the AVPV, 20% of Kiss1 neurons were glutamatergic whereas 75% were GABAergic. The differences observed between the Kiss1 neurons in the preoptic area and the Arc likely represent neuronal evidence for their differential roles in metabolism and reproduction. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Co-Expression of Leptin and Oestrogen Receptors in the Preoptic-Hypothalamic Area

The interaction between the reproductive axis and energy balance suggests that leptin acts as a possible mediator. This hormone acts in the regulation of metabolism, feeding behaviour and reproduction. Animals homozygous for the gene `ob` (ob/ob) are obese and infertile, and these effects are reversed after systemic administration of leptin. Thus, the present study aimed to determine: (i) whether cells that express leptin also express oestrogen receptors of type-alpha (ER-alpha) or -beta (ER-beta) in the medial preoptic area (MPOA) and in the arcuate (ARC), dorsomedial (DMH) and ventromedial hypothalamic nucleus and (ii) whether there is change in the gene and protein expression of leptin in these brain areas in ovariectomised (OVX) animals when oestrogen-primed. Wistar female rats with normal oestrous cycles or ovariectomised oestrogen-primed or vehicle (oil)-primed were utilised. To determine whether there was a co-expression, immunofluorescence was utilised for double staining. Confocal microscopy was used to confirm the co-expression. The technique of real-time polymerase chain reaction and western blotting were employed to analyse gene and protein expression, respectively. The results obtained showed co-expression of leptin and ER-alpha in the MPOA and in the DMH, as well as leptin and ER-beta in the MPOA, DMH and ARC. However, we did not detect leptin in the MPOA, ARC and DMH using western blotting and there was no statistical difference in leptin gene expression in the MPOA, DMH, ARC, pituitary or adipose tissue between OVX rats treated with oestrogen or vehicle. In conclusion, the results obtained in the present study confirm that the brain is also a source of leptin and reveal co-expression of oestrogen receptors and leptin in the same cells from areas related to reproductive function and feeding behaviour. Although these data corroborate the previous evidence obtained concerning the interaction between the action of brain leptin and reproductive function, the physiological relevance of this interaction remains uncertain and additional studies are necessary to elucidate the exact role of central leptin.

Dual Effects of Hyperprolactinemia on Carrageenan-Induced Inflammatory Paw Edema in Rats

Objectives: The effects of short-term 5-day and long-term 30-day hyperprolactinemia induced by domperidone (1.7 mg/kg/day, s.c.) or ectopic pituitary graft on the acute inflammatory response induced by carrageenan were evaluated in male rats. Both models of hyperprolactinemia effectively increased serum prolactin (PRL) levels. Methods: The volume in milliliters of inflammatory edema was measured by plethysnnography 1, 2, 3, 4, 6, 8 and 24 h after carrageenan injection. The areas under the inflammatory time-response curves were compared. Additionally, the effects of hyperprolactinemia on body weight and serum corticosterone levels were evaluated. Results: In both domperidone-treated and pituitary graft-implanted animals, short-term 5-day hyperprolactinemia increased the inflammatory response, while long-term 30-day hyperprolactinemia had anti-inflammatory effects. Body weight was not affected by either short- or long-term hyperprolactinennia. Conclusion: These results show that PRL has biphasic effects on the carrageenan-induced inflammatory response. Copyright (C) 2011 S. Karger AG, Basel

Long-Term Maternal Separation Differentially Alters Serum Corticosterone Levels and Blood Neutrophil Activity in A/J and C57BL/6 Mouse Offspring

Objectives: In this work, we searched for maternal separation effects on serum corticosterone levels and blood neutrophil activity in adult male A/J and C57BL/6 mouse offspring. Methods: 40 male A/J mice and 40 male C57BL/6 mice were divided within each strain into two groups. Mice in the maternal separation group were separated from their mothers (1 h/day) on postnatal days 0-13. Mice in the control group were left undisturbed. On postnatal day 45, blood was drawn from all mice and used to assess neutrophil activity by flow cytometry and serum corticosterone levels by radioimmunoassay. Results: The results showed that each mouse strain responded differently to maternal separation, but in both cases, serum corticosterone levels were affected. In both strains, adult mice that experienced maternal separation showed lower serum corticosterone levels than control mice. In relation to control mice kept together with their mothers, the levels of serum corticosterone were 72.7 and 36.36% lower in A/J and C57BL/6 mice submitted to maternal separation, respectively. The current findings showed that maternal separation increased neutrophil activity in mice after reaching adulthood. The observed effects, although in the same direction, differed between A/J and C57BL/6 mice. Maternal separation increased both the percentage and intensity of phagocytosis in C57BL/6 mice, but had no effects on A/J mice. Furthermore, maternal separation increased basal and propidium iodide-labeled Staphylococcus aureus-induced oxidative burst in A/J mice but did not affect oxidative burst in C57BL/6 mice. Finally, phorbol myristate acetate-induced oxidative burst increased in both strains. Conclusion: These results indicate that early maternal separation increases innate immunity, most likely by modifying hypothalamus-pituitary-adrenal axis activity. This suggests that maternal separation is a good model for stress which produces long-term neuroimmune changes whatever the animal species and strain used. Copyright (C) 2011 S. Karger AG, Basel