Tonic noradrenergic input to neurons in the dorsal raphe nucleus mediates food intake in male mice.

The dorsal raphe nucleus (DRN) is essential for the control of food intake. Efferent projections from the DRN extend to several forebrain regions that are involved in the control of food intake. However, the neurotransmitters released in the DRN related to the control of food intake are not known. We have previously demonstrated that a tonic α1 action on DRN neurons contributes to satiety in the fed rats. In this study we investigated the participation of norepinephrine (NE) signaling in the DRN in the satiety response. Intra-DRN administration of NE causes an increase in the 2-hour food intake of sated mice, an effect that was blocked by previous administration of yohimbine, an α2 antagonist. Similarly, Intra-DRN administration of clonidine, an α2 agonist, increases food intake in sated mice. This result indicates that in the satiated mice exogenous NE acts on α2 receptors to increase food intake. Furthermore, administration of phenylephrine, an α1 agonist, decreases food intake in fasted mice and prazosin, an α1 antagonist, increases food intake in the sated mice. Taken together these results indicate that, in a satiated condition, a tonic α1 adrenergic action on the DRN neurons inhibits food intake and that exogenous NE administered to the DRN acts on α2 adrenergic receptors to increase food intake. These data reinforce the intricate neuronal functioning of the DRN and its effects on feeding.

ENDOCANNABINOID SYSTEM IN THE PARAVENTRICULAR NUCLEUS OF THE HYPOTHALAMUS MODULATES AUTONOMIC AND CARDIOVASCULAR CHANGES BUT NOT VASOPRESSIN RESPONSE IN A RAT HEMORRHAGIC SHOCK MODEL.

ABSTRACT: We evaluated the participation of the endocannabinoid system in the paraventricular nucleus of the hypothalamus (PVN) on the cardiovascular, autonomic, and plasma vasopressin (AVP) responses evoked by hemorrhagic shock in rats. For this, the PVN was bilaterally treated with either vehicle, the selective cannabinoid receptor type 1 antagonist AM251, the selective fatty acid amide hydrolase amide enzyme inhibitor URB597, the selective monoacylglycerol-lipase enzyme inhibitor JZL184, or the selective transient receptor potential vanilloid type 1 antagonist capsazepine. We evaluated changes on arterial pressure, heart rate, tail skin temperature (ST), and plasma AVP responses induced by bleeding, which started 10 min after PVN treatment. We observed that bilateral microinjection of AM251 into the PVN reduced the hypotension during the hemorrhage and prevented the return of blood pressure to baseline values in the posthemorrhagic period. Inhibition of local 2-arachidonoylglycerol metabolism by PVN treatment with JZL184 induced similar effects in relation to those observed in AM251-treated animals. Inhibition of local anandamide metabolism via PVN treatment with URB597 decreased the depressor effect and ST drop induced by the hemorrhagic stimulus. Bilateral microinjection of capsazepine mitigated the fall in blood pressure and ST. None of the PVN treatments altered the increased plasma concentration of AVP and tachycardia induced by hemorrhage. Taken together, present results suggest that endocannabinoid neurotransmission within the PVN plays a prominent role in cardiovascular and autonomic, but not neuroendocrine, responses evoked by hemorrhage.

Fasting increases circulating angiotensin levels and brain Agtr1a expression in male rats.

In addition to being recognised for involvement in cardiovascular control and hydromineral balance, the renin-angiotensin system (RAS) has also been associated with the neuroendocrine control of energy balance. One of the main brain sites for angiotensin II (ANG II)/type 1 receptor (AT1 R) signalling is the subfornical organ (SFO), a circumventricular organ related to the control of autonomic functions, motivated behaviours and energy metabolism. Thus, we hypothesised that circulating ANG II may act on the SFO AT1 R receptors to integrate metabolic and hydromineral balance. We evaluated whether food deprivation can modulate systemic RAS activity and Agrt1a brain expression, and if ANG II/AT1 R signalling influences the hypothalamic expression of mRNAs encoding neuropeptides and food and water ingestion in fed and fasted Wistar rats. We found a significant increase in both ANG I and ANG II plasma levels after 24 and 48 h of fasting. Expression of Agrt1a mRNA in the SFO and paraventricular nucleus (PVN) also increased after food deprivation for 48 h. Treatment of fasted rats with low doses of losartan in drinking water attenuated the decrease in glycemia and meal-associated water intake without changing the expression in PVN or arcuate nucleus of mRNAs encoding selected neuropeptides related to energy homeostasis control. These findings point to a possible role of peripheral ANG II/SFO-AT1 R signalling in the control of refeeding-induced thirst. On the other hand, intracerebroventricular losartan treatment decreased food and water intake over dark time in fed but not in fasted rats.

Thyroid hormone induces restrictive cardiomyopathy in ß1-adrenoceptor knockout mice.

The purpose of this study was to characterize the role of ß1-AR signaling and its cross-talk between cardiac renin-angiotensin system and thyroid-hormone-induced cardiac hypertrophy. T3 was administered at 0.5 mg·kg-1·day-1 for 10 days in ß1-KOT3 and WTT3 groups, while control groups received vehicle alone. Echocardiography and myocardial histology was performed; cardiac and serum ANGI/ANGII and ANP and cardiac levels of p-PKA, p-ERK1/2, p-p38-MAPK, p-AKT, p-4EBP1, and ACE were measured. WTT3 showed decreased IVSTd and increased LVEDD versus WTsal (p < 0.05). ß1-KOT3 exhibited lower LVEDD and higher relative IVSTd versus ß1-KOsal, the lowest levels of ejection fraction, and the highest levels of cardiomyocyte diameter (p < 0.05). Cardiac ANP levels decreased in WTT3 versus ß1-KOT3 (p < 0.05). Cardiac ACE expression was increased in T3-treated groups (p < 0.05). Phosphorylated-p38 MAPK levels were higher in WTT3 versus WTsal or ß1-KOT3, p-4EBP1 was elevated in ß1-KO animals, and p-ERK1/2 was up-regulated in ß1-KOT3. These findings suggest that ß1-AR signaling is crucial for TiCH.

Protective Effects of Kefir Against Unpredictable Chronic Stress Alterations in Mice Central Nervous System, Heart, and Kidney.

Kefir is a probiotic mixture with anxiolytic and antioxidant properties. Chronic stress can lead to anxiety disorders and increase oxidative damage in organs such as the heart and kidney. In this study, we examined whether kefir ameliorates the anxiety-like behavior of mice submitted to chronic unpredictable stress (CUS) by modulating brain-derived neurotrophic factor (BDNF) and corticosterone levels and whether kefir modifies the oxidative parameters in the heart and kidney of mice. Male Swiss mice received kefir (0.3 mL/100 g/day) or milk for 30 days (gavage). On the 10th day, the mice were submitted to CUS. Behavioral analysis was performed using the elevated plus maze and forced swimming tests. BDNF levels were analyzed in brain tissues. Heart and kidney superoxide dismutase (SOD), catalase, glutathione (GSH), thiobarbituric acid reactive substances (TBARS), 3-nitrotyrosine, metalloproteinase-2 (MMP-2), and plasma corticosterone were evaluated. Kefir reverted the CUS-induced decrease in the time spent in the open arms, the increase in grooming frequency, and decrease in the head dipping frequency, but not the reduced immobility time. CUS decreased the cerebellum BDNF levels and increased corticosterone levels, which were restored by Kefir. Neither catalase and SOD activities nor GSH, TBARS, 3-nitrotyrosine, and MMP-2 were modified by CUS in the heart. In the kidney, CUS increased 3-nitrotyrosine and MMP-2. Kefir increased the antioxidant defense in the heart and kidney of control and CUS mice. These results suggest that kefir ameliorated CUS-induced anxiety-like behavior by modulating brain BDNF and corticosterone levels. Kefir also increased the antioxidant defense of mice heart and kidney.

Paricalcitol Improves the Angiopoietin/Tie-2 and VEGF/VEGFR2 Signaling Pathways in Adriamycin-Induced Nephropathy.

Renal endothelial cell (EC) injury and microvascular dysfunction contribute to chronic kidney disease (CKD). In recent years, increasing evidence has suggested that EC undergoes an endothelial-to-mesenchymal transition (EndoMT), which might promote fibrosis. Adriamycin (ADR) induces glomerular endothelial dysfunction, which leads to progressive proteinuria in rodents. The activation of the vitamin D receptor (VDR) plays a crucial role in endothelial function modulation, cell differentiation, and suppression of the expression of fibrotic markers by regulating the production of nitric oxide (NO) by activating the endothelial NO synthase (eNOS) in the kidneys. This study aimed to evaluate the effect of paricalcitol treatment on renal endothelial toxicity in a model of CKD induced by ADR in rats and explore mechanisms involved in EC maintenance by eNOS/NO, angiopoietins (Angs)/endothelium cell-specific receptor tyrosine kinase (Tie-2, also known as TEK) and vascular endothelial growth factor (VEGF)-VEGF receptor 2 (VEGFR2) axis. The results show that paricalcitol attenuated the renal damage ADR-induced with antiproteinuric effects, glomerular and tubular structure, and function protection. Furthermore, activation of the VDR promoted the maintenance of the function and structure of glomerular, cortical, and external medullary endothelial cells by regulating NO production. In addition, it suppressed the expression of the mesenchymal markers in renal tissue through attenuation of (transforming growth factor-beta) TGF-ß1/Smad2/3-dependent and downregulated of Ang-2/Tie-2 axis. It regulated the VEGF/VEGFR2 pathway, which was ADR-deregulated. These effects were associated with lower AT1 expression and VDR recovery to renal tissue after paricalcitol treatment. Our results showed a protective role of paricalcitol in the renal microvasculature that could be used as a target for treating the beginning of CKD.

Alterations in Kidney Structures Caused by Age Vary According to Sex and Dehydration Condition.

Aging is a complex biological process, resulting in gradual and progressive decline in structure and function in many organ systems. Our objective is to determine if structural changes produced by aging vary with sex in a stressful situation such as dehydration. The expression of Slc12a3 mRNA in the renal cortex, α-smooth muscle actin (α-SMA), and fibronectin was evaluated in male and female rats, aged 3 and 18 months, submitted and not submitted to water deprivation (WD) for 48 h, respectively. When comparing ages, 18-month-old males showed a lower expression of Slc12a3 mRNA than 3-month-old males, and control and WD 18-month-old male and female rats exhibited a higher expression of α-SMA than the respective 3-month-old rats. Fibronectin was higher in both control and WD 18-month-old males than the respective 3-month-old males. In females, only the control 18-month-old rats showed higher fibronectin than the control 3-month-old rats. When we compared sex, control and WD 3-month-old female rats had a lower expression of Slc12a3 mRNA than the respective males. The WD 18-month-old male rats presented a higher expression of fibronectin and α-SMA than the WD 18-month-old female rats. When we compared hydric conditions, the WD 18-month-old males displayed a lower relative expression of Slc12a3 mRNA and higher α-SMA expression than the control 18-month-old males. Aging, sex, and dehydration lead to alterations in kidney structure.

High-fat diet changes the behavioural and hormonal responses to water deprivation in male Wistar rats.

NEW FINDINGS: What is the central question of this study? What is the effect of an obesogenic diet on the control of hydromineral balance in rats? What is the main finding and its importance? The results showed that, when dehydrated, rats fed a high-fat diet drink less water than their control-diet-fed counterparts. Changes in aquaporin-7 and peroxisome proliferator-activated receptor α expression in the white adipose tissue might be involved. ABSTRACT: High-fat diet (HFD) increases fat accumulation, glycaemia and blood triglycerides and is used as a model to study obesity. Besides the metabolic changes, obesity likely affects water intake. We assessed the effects of HFD on behavioural and hormonal responses to water deprivation. Additionally, we measured if the adipose tissue is differentially affected by water deprivation in control and HFD-fed rats. HFD rats showed a decreased basal water intake when compared to control-fed rats. When subjected to 48 h of water deprivation, as expected, both control and HFD rats drank more water than the hydrated rats. However, the increase in water intake was lessened in HFD dehydrated rats. Similarly, the increase in haematocrit in dehydrated rats was less pronounced in HFD dehydrated rats. These results suggest that HFD diminishes drinking behaviour. White adipose tissue weight, glycaemia and plasma glycerol concentration were increased in HFD rats; however, after 48 h of water deprivation, these parameters were significantly decreased in dehydrated HFD rats, when compared to controls. The increase in adipose tissue caused by HFD may mitigate the effects of dehydration, possibly through the increased production of metabolic water caused by lipolysis in the adipocytes. Oxytocin possibly mediates the lipolytic response, since both its secretion and receptor expression are affected by dehydration in both control and HFD rats, which suggests that oxytocin signalling is maintained in these conditions. Changes in mediators of lipolysis, such as aquaporin-7 and peroxisome proliferator-activated receptor α, might contribute to the different effects observed in control and HFD rats.

Molecular neurobiological markers in the onset of sodium appetite.

Sodium appetite is a motivational state involving homeostatic behavior, seeking the ingest of salty substances after sodium loss. There is a temporal dissociation between sodium depletion (SD) and the appearance of sodium appetite. However, the responsible mechanisms for this delay remain poorly elucidated. In the present study, we measured the temporal changes at two and 24 h after SD in the gene expression of key elements within excitatory, inhibitory, and sensory areas implicated in the signaling pathways involved in the onset of sodium appetite. In SD rats, we observed that the expression of critical components within the brain control circuit of sodium appetite, including Angiotensin-type-1 receptor (Agtr1a), Oxytocin-(OXT-NP)-neurophysin-I, and serotonergic-(5HT)-type-2c receptor (Htr2c) were modulated by SD, regardless of time. However, we observed reduced phosphorylation of mitogen-activated protein kinases (MAPK) at the paraventricular nucleus (PVN) and increased oxytocin receptor (Oxtr) mRNA expression at the anteroventral of the third ventricle area (AV3V), at two hours after SD, when sodium appetite is inapparent. At twenty-four hours after SD, when sodium appetite is released, we observed a reduction in the mRNA expression of the transient receptor potential channel 1gene (Trpv1) and Oxtr in the AV3V and the dorsal raphe nucleus, respectively. The results indicate that SD exerts a coordinated timing effect, promoting the appearance of sodium appetite through changes in MAPK activity and lower Trpv1 channel and Oxtr expression that trigger sodium consumption to reestablish the hydroelectrolytic homeostasis.

Nicotine exposure through breastfeeding affects brain-derived neurotrophic factor and synaptic proteins levels in the brain of stressed adult female mice.

Nicotine has been used during pregnancy and lactation as a tobacco harm reduction strategy. However, it is unclear whether nicotine exposure during a critical development period negatively impacts stress responses in adulthood. This study investigated how nicotine, administered via breastfeeding, affects the brain-derived neurotrophic factor (BDNF), synaptic proteins levels, and anxiety-like behavior in adult female mice subjected to stress. Female Swiss mice were exposed to saline or nicotine (8 mg/kg/day) through breastfeeding between their fourth and 17th postnatal days (P) via implanted osmotic mini pumps. The unpredictable chronic mild stress (UCMS) protocol was performed during their adulthood (P65) for 10 consecutive days, followed by the elevated plus maze (EPM) test 1 day after the protocol. Animals were euthanized and their blood, collected for plasma corticosterone measurements and their brain structures, dissected for BDNF and synaptic proteins analyses. We found no significant differences in corticosterone levels between groups (Saline/Non-stress, Nicotine/Non-stress, Saline/Stress, and Nicotine/Stress). The UCMS protocol hindered weight gain. Mice exposed to nicotine through breastfeeding with or without the UCMS protocol in adulthood showed higher grooming and head dipping frequency; decreased BDNF levels in cerebellum and striatum; increased postsynaptic density protein 95 (PSD-95), synapsin I, and synaptophysin levels in cerebellum; and decreased PSD-95 and synapsin I levels in brainstem. Our results indicate that nicotine exposure through breastfeeding leads to long-lasting behavioral effects and synaptic protein changes, most of which were independent of the UCMS protocol, even after a long nicotine-free period, highlighting the importance of further studies on nicotine exposure during development.

Vasoactive intestinal peptide promotes hypophagia and metabolic changes: Role of paraventricular hypothalamic nucleus and nitric oxide.

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.

Arcuate AgRP, but not POMC neurons, modulate paraventricular CRF synthesis and release in response to fasting.

BACKGROUND: The activation of the hypothalamic-pituitary-adrenal (HPA) axis is essential for metabolic adaptation in response to fasting. However, the neurocircuitry connecting changes in the peripheral energy stores to the activity of hypothalamic paraventricular corticotrophin-releasing factor (CRFPVN) neurons, the master controller of the HPA axis activity, is not completely understood. Our main goal was to determine if hypothalamic arcuate nucleus (ARC) POMC and AgRP neurons can communicate fasting-induced changes in peripheral energy stores, associated to a fall in plasma leptin levels, to CRFPVN neurons to modulate the HPA axis activity in mice. RESULTS: We observed increased plasma corticosterone levels associate with increased CRFPVN mRNA expression and increased CRFPVN neuronal activity in 36 h fasted mice. These responses were associated with a fall in plasma leptin levels and changes in the mRNA expression of Agrp and Pomc in the ARC. Fasting-induced decrease in plasma leptin partially modulated these responses through a change in the activity of ARC neurons. The chemogenetic activation of POMCARC by DREADDs did not affect fasting-induced activation of the HPA axis. DREADDs inhibition of AgRPARC neurons reduced the content of CRFPVN and increased its accumulation in the median eminence but had no effect on corticosterone secretion induced by fasting. CONCLUSION: Our data indicate that AgRPARC neurons are part of the neurocircuitry involved in the coupling of PVNCRF activity to changes in peripheral energy stores induced by prolonged fasting.

Prolonged Activation of Brain CB2 Signaling Modulates Hypothalamic Microgliosis and Astrogliosis in High Fat Diet-Fed Mice.

Low-grade inflammation of the hypothalamus is associated with the disturbance of energy balance. The endocannabinoid system has been implicated in the development and maintenance of obesity as well as in the control of immune responses. The type 2 cannabinoid receptor (CB2) signaling has been associated with anti-inflammatory effects. Therefore, in high fat diet (HFD)-induced obese mice, we modulated CB2 signaling and investigated its effects on energy homeostasis and hypothalamic microgliosis/astrogliosis. We observed no effect on caloric intake and body weight gain in control diet-fed animals that received prolonged icv infusion of the CB2 receptor agonist HU308. Interestingly, we observed a decrease in glucose tolerance in HFD-fed animals treated with HU308. Prolonged icv infusion of HU308 increases astrogliosis in the ventromedial nucleus (VMH) of obese animals and reduced HFD-induced microgliosis in the hypothalamic arcuate (ARC) but not in the paraventricular (PVN) or VMH nuclei. These data indicate that central CB2 signaling modulates glucose homeostasis and glial reactivity in obesogenic conditions, irrespective of changes in body weight.

The AT-1 Angiotensin Receptor is Involved in the Autonomic and Neuroendocrine Responses to Acute Restraint Stress in Male Rats.

The renin-angiotensin system (RAS) is involved in cardiovascular and hydroelectrolytic control, being associated with the development of hypertension. The restraint stress (RS) model is an aversive situation, which promotes a sustained increase in blood pressure and heart rate, and stimulation of the hypothalamic-pituitary-adrenal axis. Stress leads to an increase of angiotensin-II contents both in the circulation and the central nervous system (CNS), as well as an increased expression of AT-1 receptors in CNS structures related to stress. Stressful stimuli are associated with the modulation of autonomic nervous system, as well as baroreflex; changes in this adjustment mechanism are related to cardiovascular diseases. We hypothesized that RAS is involved in the modulation of autonomic, neuroendocrine, and functional RS-caused alterations. The intravenous (i.v) pretreatment of rats with lisinopril, an angiotensin-converting-enzyme inhibitor, reduced the RS-evoked pressor response. The doses of 0.1 and 0.3 mg/kg also reduced the RS-evoked tachycardia, while in the dose of 1 mg/kg of lisinopril potentiated the tachycardic one. Additionally, i.v. pretreatment with losartan, a selective AT-1 receptor antagonist, reduced the pressor and the tachycardic responses caused by RS. Pretreatment with lisinopril 0.3 mg/kg increased the power of the low frequency (LF) band of the systolic BP spectrum after the treatment without affecting this parameter during RS. The pretreatment with losartan 1 mg/kg increased the power of the high frequency (HF) band and reduced the LF (n.u.) and the LF/HF ratio of the pulse interval spectrum in the first hour of RS. Concerning baroreflex sensitiveness (SBR), pretreatments with losartan or lisinopril did not affect the gain of the baroreflex sequences. However, the pretreatment with losartan reduced the baroreflex effectiveness index of the total sequences in the third hour of the RS. These results indicate that Ang-II, via the AT-1 receptor, plays a facilitating influence on the cardiovascular response caused by RS; facilitates sympathetic activation and reduces parasympathetic activity related to RS; facilitates the baroreflex activation during RS and favors corticosterone release under this stress model. The impairment of Ang-II synthesis, as well as the blockade of AT-1 receptors, may constitute an important pharmacological strategy to treat cardiovascular consequences caused by stress.

α-1 Adrenoceptor Activation in the Dorsal Raphe Nucleus Decreases Food Intake in Fasted Rats.

The dorsal raphe (DR) nucleus is involved in a myriad of physiological functions, such as the control of sleep-wake cycle, motivation, pain, energy balance, and food intake. We have previously demonstrated that in ad libitum fed rats the intra-DR administration of phenylephrine, an α-1 receptor agonist, does not affect food intake, whereas clonidine, an α-2 receptor agonist, potently stimulates food intake. These results indicated that in fed rats an increased adrenergic tonus blocked food intake, since the activation of α-2 auto-receptors, which decreases pre-synaptic release of adrenaline/noradrenaline, affected food intake. Thus, in this study we assessed whether the response to adrenergic stimuli would differ after overnight fasting, a situation of low adrenergic activity in the DR. Intra-DR administration of adrenaline and noradrenaline blocked food intake evoked by overnight fasting. Similarly, phenylephrine administration decreased hunger-induced food intake. These changes in food intake were accompanied by changes in other behaviors, such as increased immobility time and feeding duration. On the other hand, intra-DR administration of clonidine did not affect food-intake or associated behaviors. These results further support the hypothesis that in fed animals, increased adrenergic tonus in DR neurons inhibiting feeding, while in fasted rats the adrenergic tonus decreases and favors food intake. These data indicate a possible mechanism through which adrenergic input to the DRN contributes to neurobiology of feeding.

Effects of gonadotropin inducible ovarian transcription factor 1 in the paraventricular nucleus on fluid intake after dehydration of ovariectomized female rats.

NEW FINDINGS: What is the central question of this study? Giot1, the gene for gonadotropin inducible ovarian transcription factor 1 (GIOT1), is upregulated in osmotically challenged rats: does Giot1 gene expression in the paraventricular nucleus have a role in controlling fluid intake following dehydration and what is the role of ovarian hormones in the modulation of GIOT1 actions? What is the main finding and its importance? GIOT1 acts to regulate water and salt intake as well as hormone secretion after dehydration. The identification of genes that participate in the hormone and behavioural responses involved with hydromineral homeostasis is essential for future exploration of novel drug targets for the treatment of metabolic disease. ABSTRACT: In order to maintain body fluid balance after dehydration, hypothalamic neurons of the paraventricular nucleus (PVN) are activated to promote secretion of vasopressin (AVP) and oxytocin (OXT) from the neurohypophysis, and to modulate the behavioural allostatic responses of thirst and salt appetite. Gonadotropin inducible transcription factor (GIOT1) is a Krüppel-type zinc finger protein induced by gonadotropins and oestradiol (E2). This transcription factor is expressed in the hypothalamus, specifically in the PVN where expression of Giot1 mRNA increases following hydromineral challenges such as water deprivation or salt loading, although its physiological role is not clear. We hypothesize that GIOT1 has a central role in the integrated homeostatic and allostatic responses to disturbances in hydromineral balance, especially in the presence of female gonadal hormones. Female rats with intact ovaries or ovariectomized rats were subjected to specific microinjection of a lentiviral vector mediating Giot1 knockdown in the PVN. Three weeks after injection, rats were subjected to 48 h water deprivation, and thereafter water and salt intake were evaluated. Giot1 knockdown in PVN reduced water and saline intake as well as AVP and OXT secretion. Furthermore, Giot1 knockdown had profound effects on gene expression in the PVN, reducing the abundance of transcripts encoded by the Avp, Oxt, Nr4a1 and Crh genes. In conclusion, the present study shows for the first time that GIOT1 in the PVN regulates both transcription and fluid intake, although any connection to ovarian hormones remains to be established.

Vasopressinergic Activity of the Suprachiasmatic Nucleus and mRNA Expression of Clock Genes in the Hypothalamus-Pituitary-Gonadal Axis in Female Aging.

The important involvement of the suprachiasmatic nucleus (SCN) and the activity of vasopressinergic neurons in maintaining the rhythmicity of the female reproductive system depends on the mRNA transcription-translation feedback loops. Therefore, circadian clock function, like most physiological processes, is involved in the events that determine reproductive aging. This study describes the change of mRNA expression of clock genes, Per2, Bmal1, and Rev-erbα, in the hypothalamus-pituitary-gonadal axis (HPG) of female rats with regular cycle (RC) and irregular cycle (IC), and the vasopressinergic neurons activity in the SCN and kisspeptin neurons in the arcuate nucleus (ARC) of these animals. Results for gonadotropins and the cFos/AVP-ir neurons in the SCN of IC were higher, but kisspeptin-ir was minor. Change in the temporal synchrony of the clock system in the HPG axis, during the period prior to the cessation of ovulatory cycles, was identified. The analysis of mRNA for Per2, Bmal1, and Rev-erbα in the reproductive axis of adult female rodents shows that the regularity of the estrous cycle is guaranteed by alternation in the amount of expression of Bmal1 and Per2, and Rev-erbα and Bmal1 between light and dark phases, which ceases to occur and contributes to determining reproductive senescence. These results showed that the desynchronization between the central and peripheral circadian clocks contributes to the irregularity of reproductive events. We suggest that the feedback loops of clock genes on the HPG axis modulate the spontaneous transition from regular to irregular cycle and to acyclicity in female rodents.

Restricted feeding modulates peripheral clocks and nutrient sensing pathways in rats.

OBJECTIVE: Feeding restriction in rats alters the oscillators in suprachiasmatic, paraventricular, and arcuate nuclei, hypothalamic areas involved in food intake. In the present study, using the same animals and experimental protocol, we aimed to analyze if food restriction could reset clock genes (Clock, Bmal1) and genes involved in lipid metabolism (Pgc1a, Pparg, Ucp2) through nutrient-sensing pathways (Sirt1, Ampk, Nampt) in peripheral tissues. METHODS: Rats were grouped according to food access: Control group (CG, food ad libitum), Restricted night-fed (RF-n, food access during 2 h at night), Restricted day-fed (RF-d, food access during 2 h in the daytime), and Day-fed (DF, food access during 12 h in the daytime). After 21 days, rats were decapitated at ZT3 (0900-1000 h), ZT11 (1700-1800 h), or ZT17 (2300-2400 h). Blood, liver, brown (BAT) and peri-epididymal (PAT) adipose tissues were collected. Plasma corticosterone and gene expression were evaluated by radioimmunoassay and qPCR, respectively. RESULTS: In the liver, the expression pattern of Clock and Bmal1 shifted when food access was dissociated from rat nocturnal activity; this phenomenon was attenuated in adipose tissues. Daytime feeding also inverted the profile of energy-sensing and lipid metabolism-related genes in the liver, whereas calorie restriction induced a pre-feeding increased expression of these genes. In adipose tissues, Sirt1 expression was modified by daytime feeding and calorie restriction, with concomitant expression of Pgc1a, Pparg, and Ucp2 but not Ampk and Nampt. CONCLUSION: Feeding restriction reset clock genes and genes involved in lipid metabolism through nutrient-sensing-related genes in rat liver, brown, and peri-epididymal adipose tissues.

N-Methyl-D-aspartate Glutamate Receptor Modulates Cardiovascular and Neuroendocrine Responses Evoked by Hemorrhagic Shock in Rats.

Here, we report the participation of N-methyl-D-aspartate (NMDA) glutamate receptor in the mediation of cardiovascular and circulating vasopressin responses evoked by a hemorrhagic stimulus. In addition, once NMDA receptor activation is a prominent mechanism involved in nitric oxide (NO) synthesis in the brain, we investigated whether control of hemorrhagic shock by NMDA glutamate receptor was followed by changes in NO synthesis in brain supramedullary structures involved in cardiovascular and neuroendocrine control. Thus, we observed that intraperitoneal administration of the selective NMDA glutamate receptor antagonist dizocilpine maleate (MK801, 0.3 mg/kg) delayed and reduced the magnitude of hemorrhage-induced hypotension. Besides, hemorrhage induced a tachycardia response in the posthemorrhage period (i.e., recovery period) in control animals, and systemic treatment with MK801 caused a bradycardia response during hemorrhagic shock. Hemorrhagic stimulus increased plasma vasopressin levels during the recovery period and NMDA receptor antagonism increased concentration of this hormone during both the hemorrhage and postbleeding periods in relation to control animals. Moreover, hemorrhagic shock caused a decrease in NOx levels in the paraventricular nucleus of the hypothalamus (PVN), amygdala, bed nucleus of the stria terminalis (BNST), and ventral periaqueductal gray matter (vPAG). Nevertheless, treatment with MK801 did not affect these effects. Taken together, these results indicate that the NMDA glutamate receptor is involved in the hemorrhagic shock by inhibiting circulating vasopressin release. Our data also suggest a role of the NMDA receptor in tachycardia, but not in the decreased NO synthesis in the brain evoked by hemorrhage.