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Background: Elevated inflammatory cytokines in the periphery have been identified as active contributors to neuroinflammation and sympathetic overactivity in heart failure (HF). Yet, the exact mechanisms by which these cytokines breach the blood-brain barrier (BBB) to exert their effects on the brain remain elusive. Interleukin 17A has been linked to BBB disruption in various neurologic disorders, and its levels were significantly augmented in circulation and the brain in HF. The present study aimed to determine whether the BBB integrity was compromised within the hypothalamic paraventricular nucleus (PVN), and if so, whether interleukin 17A contributes to BBB disruption in myocardial infarction-induced HF., Methods and Results: Male Sprague-Dawley rats underwent coronary artery ligation to induce HF or sham surgery. Some HF rats received bilateral PVN microinjections of an interleukin 17 receptor A small interfering RNA or a scrambled small interfering RNA adeno-associated virus. Four weeks after coronary artery ligation, the permeability of the BBB was evaluated by intracarotid injection of fluorescent dyes (fluorescein isothiocyanate-dextran 10 kDa+rhodamine-dextran 70 kDa). Compared with sham-operated rats, HF rats exhibited an elevated extravasation of fluorescein isothiocyanate-dextran 10 kDa within the PVN but not in the brain cortex. The plasma interleukin 17A levels were positively correlated with fluorescein isothiocyanate 10 kDa extravasation in the PVN. The expression of caveolin-1, a transcytosis marker, was augmented, whereas the expression of tight junction proteins was diminished in HF rats. Interleukin 17 receptor A was identified within the endothelium of PVN microvessels. Treatment with interleukin 17 receptor A small interfering RNA led to a significant attenuation of fluorescein isothiocyanate 10 kDa extravasation in the PVN and reversed expression of caveolin-1 and tight junction-associated proteins in the PVN., Conclusions: Collectively, these data indicate that BBB permeability within the PVN is enhanced in HF and is likely attributable to increased interleukin 17A/interleukin 17 receptor A signaling in the BBB endothelium, by promoting caveolar transcytosis and degradation of tight junction complexes.

Sex differences in the sensitivity to hypertension and inflammatory processes are well characterized but insufficiently understood. In male mice, tumor necrosis factor alpha (TNFα) in the hypothalamic paraventricular nucleus (PVN) contributes to hypertension following slow-pressor angiotensin II (AngII) infusion. However, the role of PVN TNFα in the response to AngII in female mice is unknown. Using a combination of in situ hybridization, high-resolution electron microscopic immunohistochemistry, spatial-temporal gene silencing, and dihydroethidium microfluorography we investigated the influence of AngII on both blood pressure and PVN TNFα signaling in female mice. We found that chronic (14-day) infusion of AngII in female mice did not impact blood pressure, TNFα levels, the expression of the TNFα type 1 receptor (TNFR1), or the subcellular distribution of TNFR1 in the PVN. However, it was shown that blockade of estrogen receptor β (ERβ), a major hypothalamic estrogen receptor, was accompanied by both elevated PVN TNFα and hypertension following AngII. Further, AngII hypertension following ERβ blockade was attenuated by inhibiting PVN TNFα signaling by local TNFR1 silencing. It was also shown that ERβ blockade in isolated PVN-spinal cord projection neurons (i.e. sympathoexcitatory) heightened TNFα-induced production of NADPH oxidase (NOX2)-mediated reactive oxygen species, molecules that may play a key role in mediating the effect of TNFα in hypertension. These results indicate that ERβ contributes to the reduced sensitivity of female mice to hypothalamic inflammatory cytokine signaling and hypertension in response to AngII., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

The hypothalamic paraventricular nucleus (PVN) is a specific center in the brain that regulates gastric mucosal injury following gastric ischemia-reperfusion (GI-R) injury. This study aimed to investigate whether autophagy-lysosome dysfunction in the PVN tissues of GI-R rats is involved in the gastric injury, and the underlying molecular mechanisms. The rat model of GI-R was established by clamping the celiac artery for 30 min and reperfusion for different hours (1, 3, and 6 h). The gastric injury was evaluated by hematoxylin and eosin staining of the stomach and the gastric mucosal index. The autophagy-lysosome dysfunction in the PVN was evaluated by the protein levels of LC3 II and Beclin-1 (markers for autophagosome activity) and the activity of acid phosphatase (a representative lysosomal enzyme). Immunohistochemical staining of ionized calcium-binding adaptor molecule 1 in the PVN was performed to evaluate microglial activation. Reactive oxygen species (ROS) content and phosphorylated γ-aminobutyric acid B receptor (p-GABA B R) expression in the PVN were also examined. The results revealed that, in GI-R rats, the shorter the reperfusion duration, the more severe the gastric mucosal damage. The autophagy-lysosome dysfunction exhibited by GI-R rats further enhanced microglial activation, ROS production, p-GABA B R expression, and gastric injury. In addition, activating microglial cells increased ROS production, p-GABA B R expression, and gastric injury in GI-R rats, while inhibiting microglial activation resulted in the opposite results. Taken together, autophagy-lysosome dysfunction induced by GI-R aggravated the gastric injury by inducing microglia activation., (© 2021 Wiley Periodicals LLC.)

Bisphenol A (BPA), an organic synthetic compound found in some plastics and epoxy resins, is classified as an endocrine disrupting chemical. Exposure to BPA is especially dangerous if it occurs during specific "critical periods" of life, when organisms are more sensitive to hormonal changes (i.e., intrauterine, perinatal, juvenile or puberty periods). In this study, we focused on the effects of chronic exposure to BPA in adult female mice starting during pregnancy. Three months old C57BL/6J females were orally exposed to BPA or to vehicle (corn oil). The treatment (4 µg/kg body weight/day) started the day 0 of pregnancy and continued throughout pregnancy, lactation, and lasted for a total of 20 weeks. BPA-treated dams did not show differences in body weight or food intake, but they showed an altered estrous cycle compared to the controls. In order to evidence alterations in social and sociosexual behaviors, we performed the Three-Chamber test for sociability, and analyzed two hypothalamic circuits (well-known targets of endocrine disruption) particularly involved in the control of social behavior: the vasopressin and the oxytocin systems. The test revealed some alterations in the displaying of social behavior: BPA-treated dams have higher locomotor activity compared to the control dams, probably a signal of high level of anxiety. In addition, BPA-treated dams spent more time interacting with no-tester females than with no-tester males. In brain sections, we observed a decrease of vasopressin immunoreactivity (only in the paraventricular and suprachiasmatic nuclei) of BPA-treated females, while we did not find any alteration of the oxytocin system. In parallel, we have also observed, in the same hypothalamic nuclei, a significant reduction of the membrane estrogen receptor GPER1 expression.

Oxytocin is a well-known neurohypophysial hormone that plays an important role in behavioral anxiety and nociception. Two major forms of long-term potentiation, presynaptic LTP (pre-LTP) and postsynaptic LTP (post-LTP), have been characterized in the anterior cingulate cortex (ACC). Both pre-LTP and post-LTP contribute to chronic-pain-related anxiety and behavioral sensitization. The roles of oxytocin in the ACC have not been studied. Here, we find that microinjections of oxytocin into the ACC attenuate nociceptive responses and anxiety-like behavioral responses in animals with neuropathic pain. Application of oxytocin selectively blocks the maintenance of pre-LTP but not post-LTP. In addition, oxytocin enhances inhibitory transmission and excites ACC interneurons. Similar results are obtained by using selective optical stimulation of oxytocin-containing projecting terminals in the ACC in animals with neuropathic pain. Our results demonstrate that oxytocin acts on central synapses and reduces chronic-pain-induced anxiety by reducing pre-LTP., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus are the canonical controllers of the endocrine response to stress. Here we propose a new role for these cells as a gate for state transitions that allow the organism to engage in stress-related behaviors. Specifically, we review evidence indicating that activation of these cells at critical times allows organisms to move to a state that is permissive for motor action. This is evident when the organism is under duress (defensive behavior), when the organism has successfully vanquished a threat (coping behavior), and when an organism initiates approach to a conspecific (social behavior). The motor behavior that follows from the activation of CRH neurons is not necessarily under the control of these cells but is determined by higher order circuits that discriminate more refined features of environmental context to execute the appropriate behavior., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Multiple neurological problems have been reported in coronavirus disease-2019 (COVID-19) patients because severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) likely spreads to the central nervous system (CNS) via olfactory nerves or through the subarachnoid space along olfactory nerves into the brain's cerebrospinal fluid and then into the brain's interstitial space. We hypothesize that SARS-CoV-2 enters the subfornical organ (SFO) through the above routes and the circulating blood since circumventricular organs (CVOs) such as the SFO lack the blood-brain barrier, and infection of the SFO causes dysfunction of the hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus (SON), leading to hydroelectrolytic disorder. SARS-CoV-2 can readily enter SFO-PVN-SON neurons because these neurons express angiotensin-converting enzyme-2 receptors and proteolytic viral activators, which likely leads to neurodegeneration or neuroinflammation in these regions. Considering the pivotal role of SFO-PVN-SON circuitry in modulating hydroelectrolyte balance, SARS-CoV-2 infection in these regions could disrupt the neuroendocrine control of hydromineral homeostasis. This review proposes mechanisms by which SARS-CoV-2 infection of the SFO-PVN-SON pathway leads to hydroelectrolytic disorder in COVID-19 patients., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Scope: Gut dysbiosis and dysregulation of the gut-brain-axis contributes to the pathogenesis of hypertension. Vitamin C (VC) is a common dietary supplement that shows the ability to lower the elevated blood pressure in hypertensive animals. Thus, the hypothesis that the gut microbiota is involved in the anti-hypertensive effect of VC is proposed., Methods and Results: The changes of the gut microbiota and pathology in a spontaneously hypertensive rat (SHR) model after daily oral intake of VC in dosage of 200 or 1000 mg kg -1 are examined. After 4 weeks, the elevated blood pressure of SHRs in both VC-treated groups is attenuated. Sequencing of the gut microbiota shows improvement in its diversity and abundance. Bioinformatic analysis suggests restored metabolism and biosynthesis-related functions of the gut, which are confirmed by the improvement of gut pathology and integrity. Analysis of the hypothalamus paraventricular nucleus (PVN), the central pivot of blood pressure regulation, also shows reduced inflammatory responses and oxidative stress., Conclusions: The reduced blood pressure, enriched gut microbiota, improved gut pathology and integrity, and reduced inflammatory responses and oxidative stress in the PVN together suggest that the anti-hypertensive effects of VC involve reshaping of gut microbiota composition and function., (© 2021 Wiley-VCH GmbH.)

Fragile X Syndrome (FXS) is an inherited developmental disorder caused by the non-expression of the Fmr1 gene. FXS is associated with abnormal social and anxiety behavior that is more prominent among males. Given that oxytocin (OXT) regulates both social and anxiety behavior, we studied the effect of FXS in the hypothalamic paraventricular nucleus (PVN), the major central source of OXT. We observed a significant suppression of protein kinase C epsilon (PKCε) (34%) in the ventral hippocampal CA1 region of postnatal day-18 (P18) male Fmr1 knockout (KO) mice, which displayed social behavior deficits and hyper-anxiety in adulthood. These mice also displayed a 39% increase in cell surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) at P18 (measured by the surface level of the AMPAR subunit GluR2), thereby indicating excitation of the CA1 neurons. It is known that neuronal activation at CA1 is linked to an inhibition of the PVN neurons. As expected, these mice also displayed a 25% suppression of oxytocin+ (OXT+) cells in the PVN at P20. Stimulating PKCε during postnatal days 6-,14 (P6-14) mice using a selective activator, dicyclopropyl-linoleic acid (DCP-LA), corrected AMPAR externalization in CA1 and suppression of OXT+ cell number in PVN in a PKCε dependent manner. Most notably, neonatal DCP-LA treatment rescued social behavior deficits and hyper-anxiety, displayed by adult (≥P60) male but not female KO mice. Thus, neonatal stimulation of PKCε could be a strategy to correct endophenotypic anomalies during brain development and aberrant adult behavior of the FXS males to the wild-type levels., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The arcuate nucleus (ARC) of the hypothalamus comprises two antagonistic neuron populations critical for energy balance, namely, the anorexigenic pro-opiomelanocortin (POMC) and the orexigenic agouti-related peptide (AgRP) neurons that act as agonists and antagonists, respectively, for neurons expressing the type IV melanocortin receptor (MC4R) (Andermann ML and Lowell BB. Neuron 95: 757-778, 2017). MC4R activation increases energy expenditure and decreases food intake during positive energy balance states to prevent diet-induced obesity (DIO). Work from our group identified aberrant neuronal cell cycle events both as a novel biomarker and druggable target in the ARC for the treatment of DIO, demonstrating pharmacological restoration of retinoblastoma protein function in the ARC using cyclin-dependent kinase 4/6 (CDK4/6) inhibitors could treat DIO in mice by increasing lipid oxidation to selectively decrease fat mass. However, the role of CDK4/6 inhibitors on food intake was not examined. Four-week-old Mc4r-lox TB mice were continuously administered high-fat diet (60% kcal fat). At 8 wk of age, animals were administered 60 mg/kg abemaciclib orally or a saline control and monitored every 2 wk for fat mass changes by MRI. At 11 wk of age, all animals were injected bilaterally in the paraventricular hypothalamus with AAV8 serotype virus expressing a Cre-mCherry and monitored for another 5 wk. Restoration of Mc4r expression in the paraventricular hypothalamic nucleus (PVN/PVH) reduced food intake in hyperphagic obese mice when given CDK4/6 inhibitor therapy. The reduced food intake was responsible for reduced fat mass in mice treated with abemaciclib. These results indicate that targeting POMC neurons could be an effective strategy in treating diet-related obesity. NEW & NOTEWORTHY We have defined some of the necessary components to prevent high-fat diet-induced obesity at the molecular and cellular level. Within POMC neurons, the retinoblastoma protein must remain active and prevented from phosphoinactivation by cyclin-dependent kinases. The downstream neurons within the PVH must also properly express MC4R for the circuit to appropriately regulate feeding behavior.

Studies have shown that arginine-vasopressin (AVP) is an important neuropeptide regulating social behaviors. The present work aimed to detect changes in the AVP numbers and level in a valproic acid (VPA)-induced rat model of autism and the underlying mechanism of its pathogenesis. Our results indicated that infants exposed to VPA showed obviously impaired communication and repetitive behaviors with reduced number of AVP-ir cells in paraventricular nucleus (PVN) and cerebrospinal fluid (CSF). The postnatal subcutaneous injection of AVP can alleviate social preference deficits and stereotyped behaviors, accompanied with the increase of the AVP concentrations in the CSF. We concluded that AVP system was involved in etiology of VPA-induced autism-like symptoms and postnatal AVP treatment rescued the behavioral deficits,which could be a promising treatment for autism., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Exercise (Ex) has long been recognized to produce beneficial effects on hypertension (HTN). This coupled with evidence of gut dysbiosis and an impaired gut-brain axis led us to hypothesize that reshaping of gut microbiota and improvement in impaired gut-brain axis would, in part, be associated with beneficial influence of exercise. Male spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were randomized into sedentary, trained, and detrained groups. Trained rats underwent moderate-intensity exercise for 12 weeks, whereas, detrained groups underwent 8 weeks of moderate-intensity exercise followed by 4 weeks of detraining. Fecal microbiota, gut pathology, intestinal inflammation, and permeability, brain microglia and neuroinflammation were analyzed. We observed that exercise training resulted in a persistent decrease in systolic blood pressure in the SHR. This was associated with increase in microbial α diversity, altered β diversity, and enrichment of beneficial bacterial genera. Furthermore, decrease in the number of activated microglia, neuroinflammation in the hypothalamic paraventricular nucleus, improved gut pathology, inflammation, and permeability were also observed in the SHR following exercise. Interestingly, short-term detraining did not abolish these exercise-mediated improvements. Finally, fecal microbiota transplantation from exercised SHR into sedentary SHR resulted in attenuated SBP and an improved gut-brain axis. These observations support our concept that an impaired gut-brain axis is linked to HTN and exercise ameliorates this impairment to induce antihypertensive effects.

Thyroid hormone (T3) inhibits thyrotropin-releasing hormone (TRH) synthesis in the hypothalamic paraventricular nucleus (PVN). Although the T3 receptor (TR) β2 is known to mediate the negative regulation of the prepro-TRH gene, its molecular mechanism remains unknown. Our previous studies on the T3-dependent negative regulation of the thyrotropin β subunit (TSHβ) gene suggest that there is a tethering mechanism, whereby liganded TRβ2 interferes with the function of the transcription factor, GATA2, a critical activator of the TSHβ gene. Interestingly, the transcription factors Sim1 and Arnt2, the determinants of PVN differentiation in the hypothalamus, are reported to induce expression of TRβ2 and GATA2 in cultured neuronal cells. Here, we confirmed the expression of the GATA2 protein in the TRH neuron of the rat PVN using immunohistochemistry with an anti-GATA2 antibody. According to an experimental study from transgenic mice, a region of the rat prepro-TRH promoter from nt. -547 to nt. +84 was able to mediate its expression in the PVN. We constructed a chloramphenicol acetyltransferase (CAT) reporter gene containing this promoter sequence (rTRH(547)-CAT) and showed that GATA2 activated the promoter in monkey kidney-derived CV1 cells. Deletion and mutation analyses identified a functional GATA-responsive element (GATA-RE) between nt. -357 and nt. -352. When TRβ2 was co-expressed, T3 reduced GATA2-dependent promoter activity to approximately 30%. Unexpectedly, T3-dependent negative regulation was maintained after mutation of the reported negative T3-responsive element, site 4. T3 also inhibited the GATA2-dependent transcription enhanced by cAMP agonist, 8-bromo-cAMP. A rat thyroid medullary carcinoma cell line, CA77, is known to express the preproTRH mRNA. Using a chromatin immunoprecipitation assay with this cell line where GATA2 expression plasmid was transfected, we observed the recognition of the GATA-RE by GATA2. We also confirmed GATA2 binding using gel shift assay with the probe for the GATA-RE. In CA77 cells, the activity of rTRH(547)-CAT was potentiated by overexpression of GATA2, and it was inhibited in a T3-dependent manner. These results suggest that GATA2 transactivates the rat prepro-TRH gene and that liganded TRβ2 interferes with this activation via a tethering mechanism as in the case of the TSHβ gene., Competing Interests: The authors have declared that no competing interests exist.

BACKGROUND The neuroinflammation of paraventricular nucleus (PVN) of the hypothalamus has been implicated in the development of hypertension. The promoted invasion of peripheral immune cells into PVN may be attributed to the upregulation of chemokines, then exacerbating neuroinflammation. We studied the expressions of chemokines, activation of microglial cells, and inflammatory mediators in PVN of rats with stress-induced hypertension (SIH). MATERIAL AND METHODS SIH was induced by electrical foot shock combined with noise for 2 h twice a day, at an interval of 4 h for 14 consecutive days. At the end of the 14th day, fresh PVN tissues were collected to measure the expressions of chemokines using the RayBiotech antibody array. RESULTS We are the first to report that the expression of CXCL7 was extremely high in PVN of control rats, and was significantly lower in SIH rats. The expressions of CCL2 and CX3CL1 in PVN of SIH rats significantly exceeded those of control rats. The numbers of CX3CR1 (receptor of CX3CL1)-immunostained cells and oxycocin-42 (OX-42, marker of microglia)-positive cells increased in PVN of the SIH rats. The stress enhanced the protein expressions of proinflammatory cytokines IL-6 and IL-17 and reduced those of anti-inflammatory cytokines TGF-ß and IL-10 in PVN. CONCLUSIONS In PVN of SIH rats, chronic stress induced neuroinflammation characterized by the activated microglia and upregulated proinflammatory cytokines. Expressions of chemokines CXCL7, CX3CL1, and CCL2 were altered. The causal link of chemokines to PVN neuroinflammation and hypertension remain to be determined.

Background: We previously found that high-dose methylprednisolone increased the incidence of critical illness-related corticosteroid insufficiency (CIRCI) and mortality in rats with traumatic brain injury (TBI), whereas low-dose hydrocortisone but not methylprednisolone exerted protective effects. However, the receptor-mediated mechanism remains unclear. This study investigated the receptor-mediated mechanism of the opposite effects of different glucocorticoids on the survival of paraventricular nucleus (PVN) cells and the incidence of CIRCI after TBI., Methods: Based on controlled cortical impact (CCI) and treatments, male SD rats (n = 300) were randomly divided into the sham, CCI, CCI + GCs (methylprednisolone 1 or 30 mg/kg/day; corticosterone 1 mg/kg/day), CCI + methylprednisolone+RU486 (RU486 50 mg/kg/day), and CCI + corticosterone+spironolactone (spironolactone 50 mg/kg/day) groups. Blood samples were collected 7 days before and after CCI. Brain tissues were collected on postinjury day 7 and processed for histology and western blot analysis., Results: We examined the incidence of CIRCI, mortality, apoptosis in the PVN, the receptor-mediated mechanism, and downstream signaling pathways on postinjury day 7. We found that methylprednisolone and corticosterone exerted opposite effects on the survival of PVN cells and the incidence of CIRCI by activating different receptors. High-dose methylprednisolone increased the nuclear glucocorticoid receptor (GR) level and subsequently increased cell loss in the PVN and the incidence of CIRCI. In contrast, low-dose corticosterone but not methylprednisolone played a protective role by upregulating mineralocorticoid receptor (MR) activation. The possible downstream receptor signaling mechanism involved the differential effects of GR and MR on the activity of the Akt/CREB/BDNF pathway., Conclusion: The excessive activation of GR by high-dose methylprednisolone exacerbated apoptosis in the PVN and increased CIRCI. In contrast, refilling of MR by corticosterone protects PVN neurons and reduces the incidence of CIRCI by promoting GR/MR rebalancing after TBI.

Previous studies have shown that melatonin (Mel) can effectively ameliorate myocardial ischemia/reperfusion (MI/R) injury, but the mechanism is yet to be fully elucidated. Mel receptors are expressed in the paraventricular nucleus (PVN), which is also involved in regulating cardiac sympathetic nerve activity. The aim of this study was to examine whether Mel receptors in the PVN are involved in the protective effects of Mel against MI/R injury. The results of quantitative polymerase chain reaction, western blot, and immunofluorescence assays indicated that Mel receptor 2 (MT2) expression in the PVN was upregulated after MI/R. Intraperitoneal administration of Mel significantly improved post-MI/R cardiac function and reduced the infarct size, whereas shRNA silencing of MT2 in the PVN partially blocked this effect. Intraperitoneal administration of Mel reduced sympathetic nerve overexcitation caused by MI/R, whereas shRNA silencing of MT2 in the PVN partially diminished this effect. Furthermore, enzyme-linked immunosorbent assay and western blot results indicated that intraperitoneal administration of Mel lowered the levels of inflammatory cytokines in the PVN after MI/R injury, whereas the application of sh-MT2 in the PVN reduced this effect of Mel. Mel significantly reduced the levels of NF-κB after astrocyte oxygen and glucose deprivation/reoxygenation injury, and this effect was offset when MT2 was silenced. The above experimental results suggest that MT2 in the PVN partially mediated the protective effects of Mel against MI/R injury, and its underlying mechanisms may be related to postactivation amelioration of PVN inflammation and reduction of cardiac sympathetic nerve overexcitation.

Background: Cardiovascular diseases, including heart failure, are the most common cause of death globally. Recent studies support a high degree of comorbidity between heart failure and cognitive and mood disorders resulting in memory loss, depression, and anxiety. While neuroinflammation in the hypothalamic paraventricular nucleus contributes to autonomic and cardiovascular dysregulation in heart failure, mechanisms underlying cognitive and mood disorders in this disease remain elusive. The goal of this study was to quantitatively assess markers of neuroinflammation (glial morphology, cytokines, and A1 astrocyte markers) in the central amygdala, a critical forebrain region involved in emotion and cognition, and to determine its time course and correlation to disease severity during the progression of heart failure., Methods: We developed and implemented a comprehensive microglial/astrocyte profiler for precise three-dimensional morphometric analysis of individual microglia and astrocytes in specific brain nuclei at different time points during the progression of heart failure. To this end, we used a well-established ischemic heart failure rat model. Morphometric studies were complemented with quantification of various pro-inflammatory cytokines and A1/A2 astrocyte markers via qPCR., Results: We report structural remodeling of central amygdala microglia and astrocytes during heart failure that affected cell volume, surface area, filament length, and glial branches, resulting overall in somatic swelling and deramification, indicative of a change in glial state. These changes occurred in a time-dependent manner, correlated with the severity of heart failure, and were delayed compared to changes in the hypothalamic paraventricular nucleus. Morphometric changes correlated with elevated mRNA levels of pro-inflammatory cytokines and markers of reactive A1-type astrocytes in the paraventricular nucleus and central amygdala during heart failure., Conclusion: We provide evidence that in addition to the previously described hypothalamic neuroinflammation implicated in sympathohumoral activation during heart failure, microglia, and astrocytes within the central amygdala also undergo structural remodeling indicative of glial shifts towards pro-inflammatory phenotypes. Thus, our studies suggest that neuroinflammation in the amygdala stands as a novel pathophysiological mechanism and potential therapeutic target that could be associated with emotional and cognitive deficits commonly observed at later stages during the course of heart failure.

Therapeutic advances for pulmonary hypertension (PH) have been incremental because of the focus on the pulmonary vasculature in PH pathology. Here, we evaluate the concept that PH is, rather, a systemic disorder involving interplay among multiorgan systems, including brain, gut, and lungs. Therefore, the objective of this study was to evaluate the hypothesis that PH is associated with a dysfunctional brain-gut-lung axis and that global overexpression of ACE2 (angiotensin-converting enzyme 2) rebalances this axis and protects against PH. ACE2 knockin and wild-type (WT; C57BL/6) mice were subjected to chronic hypoxia (10% FIO2) or room air for 4 weeks. Cardiopulmonary hemodynamics, histology, immunohistochemistry, and fecal 16S rRNA microbial gene analyses were evaluated. Hypoxia significantly increased right ventricular systolic pressure, sympathetic activity as well as the number and activation of microglia in the paraventricular nucleus of the hypothalamus in WT mice. This was associated with a significant increase in muscularis layer thickening and decreases in both villi length and goblet cells and altered gut microbiota. Global overexpression of ACE2 prevented changes in hypoxia-induced pulmonary and gut pathophysiology and established distinct microbial communities from WT hypoxia mice. Furthermore, WT mice subjected to fecal matter transfer from ACE2 knockin mice were resistant to hypoxia-induced PH compared with their controls receiving WT fecal matter transfer. These observations demonstrate that ACE2 ameliorates these hypoxia-induced pathologies and attenuates PH. The data implicate dysfunctional brain-gut-lung communication in PH and provide novel avenues for therapeutic interventions.

Aims: This study aimed to investigate the functions of the amygdala in rat asthma model., Main Methods: Wheat germ agglutinin-horseradish peroxidase (WGA-HRP) was used for tracing from the paraventricular nucleus (PVN) to the amygdala, and nuclear lesions were performed to observe changes in respiratory function and airway inflammation., Results: This study showed that the extracellular neuronal discharged in the medial amygdala (MeA) and central amygdala (CeA), and the expression of Fos significantly increased in asthmatic rat compared to control group. The distribution of Fos- and oxytocin (OT)-positive neurons and Fos/OT dual-positive neurons evidently increased in the PVN. WGA-HRP was injected into the PVN for tracing, and Fos/HRP-dual-positive neurons were observed to be distributed in the MeA. By using kainic acid (KA) to injure the MeA and CeA in asthmatic rats, expiratory and inspiratory times (TE/TI) and airway resistance (Raw) decreased, and minute ventilation volume (MVV) and dynamic pulmonary compliance (Cdyn) increased accordingly. In the bronchoalveolar lavage fluid (BALF), the number of eosinophils and the concentration of IL-4 were lower than those of the control group, and the ratio of Th1/Th2 cells was higher than that of the control group. In the PVN, the distribution of Fos-, OT-positive cells and Fos/OT double-positive cells decreased compared with those of the control group. The activities of the MeA and CeA and of OT neurons in the PVN of the rats were correlated with the occurrence of asthma., Conclusions: Asthma attack could induce neural activities in the MeA and CeA, and OT neurons in the PVN may be involved in the process of asthma attack., (© 2020 The Authors. CNS Neuroscience & Therapeutics Published by John Wiley & Sons Ltd.)

Obstructive sleep apnea is characterized by interrupted breathing that leads to cardiovascular sequelae including chronic hypertension that can persist into the waking hours. Chronic intermittent hypoxia (CIH), which models the hypoxemia associated with sleep apnea, is sufficient to cause a sustained increase in blood pressure that involves the central nervous system. The median preoptic nucleus (MnPO) is an integrative forebrain region that contributes to blood pressure regulation and neurogenic hypertension. The MnPO projects to the paraventricular nucleus (PVN), a preautonomic region. We hypothesized that pathway-specific lesions of the projection from the MnPO to the PVN would attenuate the sustained component of chronic intermittent hypoxia-induced hypertension. Adult male Sprague-Dawley rats (250-300 g) were anesthetized with isoflurane and stereotaxically injected bilaterally in the PVN with a retrograde Cre-containing adeno-associated virus (AAV; AAV9.CMV.HI.eGFP-Cre.WPRE.SV40) and injected in the MnPO with caspase-3 (AAV5-flex-taCasp3-TEVp) or control virus (AAV5-hSyn-DIO-mCherry). Three weeks after the injections the rats were exposed to a 7-day intermittent hypoxia protocol. During chronic intermittent hypoxia, controls developed a diurnal hypertension that was blunted in rats with caspase lesions. Brain tissue processed for FosB immunohistochemistry showed decreased staining with caspase-induced lesions of MnPO and downstream autonomic-regulating nuclei. Chronic intermittent hypoxia significantly increased plasma levels of advanced oxidative protein products in controls, but this increase was blocked in caspase-lesioned rats. The results indicate that PVN-projecting MnPO neurons play a significant role in blood pressure regulation in the development of persistent chronic intermittent hypoxia hypertension. NEW & NOTEWORTHY Chronic intermittent hypoxia associated with obstructive sleep apnea increases oxidative stress and leads to chronic hypertension. Sustained hypertension may be mediated by angiotensin II-induced neural plasticity of excitatory median preoptic neurons in the forebrain that project to the paraventricular nucleus of the hypothalamus. Selective caspase lesions of these neurons interrupt the drive for sustained hypertension and cause a reduction in circulating oxidative protein products. This indicates that a functional connection between the forebrain and hypothalamus is necessary to drive diurnal hypertension associated with intermittent hypoxia. These results provide new information about central mechanisms that may contribute to neurogenic hypertension.

Energy imbalance due to excess of calories is considered to be a major player in the current worldwide obesity pandemic and could be accompanied by systemic and central inflammation and mitochondrial dysfunctions. This hypothesis was tested by comparing the wild-derived diet-induced obesity- (DIO-) resistant mouse strain WSB/EiJ to the obesity-prone C57BL/6J strain. We analysed circulating and hypothalamic markers of inflammatory status and hypothalamic mitochondrial activity in both strains exposed to high-fat diet (HFD). We further analysed the regulations of hypothalamic genes involved in inflammation and mitochondrial pathways by high throughput microfluidic qPCR on RNA extracted from laser micro-dissected arcuate (ARC) and paraventricular (PVN) hypothalamic nuclei. HFD induced increased body weight gain, circulating levels of leptin, cholesterol, HDL and LDL in C57BL/6J whereas WSB/EiJ mice displayed a lower inflammatory status, both peripherally (lower levels of circulating cytokines) and centrally (less activated microglia in the hypothalamus) as well as more reactive mitochondria in the hypothalamus. The gene expression data analysis allowed identifying strain-specific hypothalamic metabolic pathways involved in the respective responses to HFD. Our results point to the involvement of hypothalamic inflammatory and mitochondrial pathways as key factors in the control of energy homeostasis and the resistance to DIO.

New Findings: • What is the central question of this study? We hypothesized that central inflammatory processes that involve activation of microglia and astrocytes contribute to the development of Gαi 2 protein-dependent, salt-sensitive hypertension. • What is the main finding and its importance? The main finding is that PVN-specific inflammatory processes, driven by microglial activation, appear to be linked to the development of Gαi 2 protein-dependent, salt-sensitive hypertension in Sprague-Dawley rats. This finding might reveal new mechanistic targets in the treatment of hypertension., Abstract: The central mechanisms underlying salt-sensitive hypertension, a significant public health issue, remain to be established. Researchers in our laboratory have reported that hypothalamic paraventricular nucleus (PVN) Gαi 2 proteins mediate the sympathoinhibitory and normotensive responses to high sodium intake in salt-resistant rats. Given the recent evidence of central inflammation in animal models of hypertension, we hypothesized that PVN inflammation contributes to Gαi 2 protein-dependent, salt-sensitive hypertension. Male Sprague-Dawley rats received chronic intracerebroventricular infusions of a targeted Gαi 2 or control scrambled oligodeoxynucleotide (ODN) and were maintained for 7 days on a normal-salt (NS; 0.6% NaCl) or high-salt (HS; 4% NaCl) diet; in subgroups on HS, intracerebroventricular minocycline (microglial inhibitor) was co-infused with ODNs. Radiotelemetry was used in subgroups of rats to measure mean arterial pressure (MAP) chronically. In a separate group of rats, plasma noradrenaline, plasma renin activity, urinary angiotensinogen and mRNA levels of the PVN pro-inflammatory cytokines TNFα, IL-1β and IL-6 and the anti-inflammatory cytokine IL-10 were assessed. In additional groups, immunohistochemistry was performed for markers of PVN and subfornical organ microglial activation and cytokine levels and PVN astrocyte activation. High salt intake evoked salt-sensitive hypertension, increased plasma noradrenaline, PVN pro-inflammatory cytokine mRNA upregulation, anti-inflammatory cytokine mRNA downregulation and PVN-specific microglial activation in rats receiving a targeted Gαi 2 but not scrambled ODN. Minocycline co-infusion significantly attenuated the increase in MAP and abolished the increase in plasma noradrenaline and inflammation in Gαi 2 ODN-infused animals on HS. Our data suggest that central Gαi 2 protein prevents microglial-mediated PVN inflammation and the development of salt-sensitive hypertension., (© 2019 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Brain renin angiotensin system within the paraventricular nucleus plays a critical role in balancing excitatory and inhibitory inputs to modulate sympathetic output and blood pressure regulation. We previously identified ACE2 and ADAM17 as a compensatory enzyme and a sheddase, respectively, involved in brain renin angiotensin system regulation. Here, we investigated the opposing contribution of ACE2 and ADAM17 to hypothalamic presympathetic activity and ultimately neurogenic hypertension. New mouse models were generated where ACE2 and ADAM17 were selectively knocked down from all neurons (AC-N) or Sim1 neurons (SAT), respectively. Neuronal ACE2 deletion revealed a reduction of inhibitory inputs to AC-N presympathetic neurons relevant to blood pressure regulation. Primary neuron cultures confirmed ACE2 expression on GABAergic neurons synapsing onto excitatory neurons within the hypothalamus but not on glutamatergic neurons. ADAM17 expression was shown to colocalize with angiotensin-II type 1 receptors on Sim1 neurons, and the pressor relevance of this neuronal population was demonstrated by photoactivation. Selective knockdown of ADAM17 was associated with a reduction of FosB gene expression, increased vagal tone, and prevented the acute pressor response to centrally administered angiotensin-II. Chronically, SAT mice exhibited a blunted blood pressure elevation and preserved ACE2 activity during development of salt-sensitive hypertension. Bicuculline injection in those models confirmed the supporting role of ACE2 on GABAergic tone to the paraventricular nucleus. Together, our study demonstrates the contrasting impact of ACE2 and ADAM17 on neuronal excitability of presympathetic neurons within the paraventricular nucleus and the consequences of this mutual regulation in the context of neurogenic hypertension.

Accumulating evidence has demonstrated that there is a growing trend of menopausal women suffering from depression. However, the pathogenesis of menopausal depression still remains unclear. Hence, this paper aims to reveal the pathological mechanisms involved in postmenopausal depression by using a novel peri- to postmenopausal depression model induced by a two-step ovariectomy plus chronic mild stress (CMS). The results of metabolic chambers and serum hormone/cytokine determination revealed that peri/postmenopausal depressive mice exhibited endocrine and metabolic disorders. Electrophysiological recordings indicated that the hippocampal synaptic transmission was compromised. Compared to the sham group, the microRNA-99a (miR-99a) level decreased significantly in the hypothalamus, and its target FK506-binding protein 51 (FKBP51) enormously increased; in contrast, the nuclear translocation of the progesterone receptor (PR) decreased in hypothalamic paraventricular nucleus (PVN) in the peri/postmenopausal depression mouse model. Additionally, synaptic proteins, including postsynaptic density protein 95 (PSD-95) and synaptophysin (SYN), showed a similar decrease in the hypothalamus. Accordingly, the present work suggests that miR-99a may be involved in the regulation of hypothalamic synaptic plasticity and that it might be a potential therapeutic target for peri/postmenopausal depression.

Chronic intermittent hypoxia (CIH) is known to induce hypertension, but the mechanism is not well understood. We hypothesized that sensory plasticity of the carotid body (CB) and oxidative stress in the paraventricular nucleus (PVN) are involved in CIH-induced hypertension. In this study, rats were exposed to CIH for 28 days (intermittent hypoxia of 21% O 2 for 60 s and 5% O 2 for 30 s, cyclically repeated for 8 hr/day) and then randomly grouped for intracerebroventricular injection of 5-HT2 receptor antagonist ritanserin, Rho-associated protein kinase (ROCK) inhibitor Y-27632, and NADPH oxidase (NOX) inhibitor diphenyleneiodonium (DPI), respectively. We found that CIH increased blood pressure (BP), elevated carotid sinus nerve (CSN) and renal sympathetic nerve (RSN) activities, oxidative stress, and cell apoptosis in PVN. NOX-derived reactive oxygen species (ROS) production and cell apoptosis decreased when CIH-induced activation of 5-HT/5-HT2AR/PKC signaling was inhibited by ritanserin. In addition, RhoA expression was downregulated when oxidative stress was attenuated by DPI, while Y-27632 decreased the expression of endothelin-1, which is overexpressed in the vascular wall during hypertension. Moreover, treatment with ritanserin, DPI or Y-27632 attenuated the sensory plasticity and sympathetic hyperactivity as well as CIH-induced elevation of BP. In conclusion, CIH-induced activation of 5-HT/5-HT2AR/PKC signaling contributes to NOX-derived oxidative stress in PVN, which may cause sensory plasticity of CB, RSN hyperactivity, and elevated BP., (© 2019 Wiley Periodicals, Inc.)

Objective: Sustained hypertension is a major cause of heart failure in aging hypertensive patients. Salusin β, a novel bioactive peptide of 20 amino acids, has been reported to participate in various cardiovascular diseases, including hypertension. We therefore hypothesized that central knockdown of salusin β might be effective for hypertension-induced heart failure treatment., Methods and Results: Eighteen-month-old male aged spontaneously hypertensive rats (SHR) with heart failure and WKY rats were microinjected with either a specific adenoviral vector encoding salusin β shRNA (Ad-Sal-shRNA) or a scramble shRNA (Ad-Scr-shRNA) in the hypothalamic paraventricular nucleus (PVN) for 4 weeks. Radiotelemetry and echocardiography were used for measuring blood pressure and cardiac function, respectively. Blood samples and heart were harvested for evaluating plasma norepinephrine, tyrosine hydroxylase, and cardiac morphology, respectively. The mesenteric arteries were separated for measurement of vascular responses. The PVN was analyzed for salusin β, proinflammatory cytokines (PICs), mitogen-activated protein kinase (MAPK), NF-κB, and reactive oxygen species (ROS) levels. Compared with normotensive rats, aging SHR with heart failure had dramatically increased salusin β expression. Silencing salusin β with Ad-Sal-shRNA attenuated arterial pressure and improved autonomic function, cardiac and vascular dysfunction in aging SHR with heart failure, but not in aging WKY rats. Knockdown of salusin β significantly reduced paraventricular nucleus PICs levels, MAPK and NF-κB activity, and ROS levels in aging SHR with heart failure., Conclusion: These data demonstrate that in aging SHR, the heart failure that was developed during the end stage of hypertension could be ameliorated by silencing salusin β., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: Prolonged treatment with neuroleptics has been shown to induce FosB/ΔFosB expression in several brain regions including the medial prefrontal cortex, dorsomedial and dorsolateral striatum, ventrolateral and dorsolateral septum, nucleus accumbens shell and core, and the hypothalamic paraventricular nucleus (PVN). Some of these regions are known to be also stress responsive. This study was designed to determine whether repeated clozapine (CLZ) administration for 7 consecutive days to Wistar rats may modify FosB/ΔFosB expression in the above-mentioned brain areas induced by acute stress or novel stressor that followed 13-day chronic mild stress preconditioning., Methods: Following experimental groups were used: unstressed animals treated with vehicle/ CLZ for 7 days; 7-day vehicle/CLZ-treated animals on the last day exposed to acute stress - forced swimming (FSW); and animals preconditioned with stress for 13 days treated from the 8th day with vehicle/CLZ and on the 14th day exposed to novel stress - FSW., Results: In the unstressed animals CLZ markedly increased FosB/ΔFosB immunoreactivity in the ventrolateral septum and PVN. FSW elevated FosB/ΔFosB expression in the medial prefrontal cortex, striatum, and septum. CLZ markedly potentiated the effect of the FSW on FosB/ΔFosB expression in the PVN, but suppressed it in the dorsomedial striatum. Novel stress with stress preconditioning increased FosB/ΔFosB immunoreactivity in the prefrontal cortex, striatum, ventrolateral septum, and the PVN. In the nucleus accumbens the effect of the novel stressor was potentiated by CLZ., Conclusion: Our data indicate that CLZ may modulate the acute as well as novel stress effects on FosB/ΔFosB expression but its effect differs within the individual brain regions.

The hypothalamus is essential for regulation of energy homeostasis and metabolism. Feeding hypercaloric, high-fat (HF) diet induces hypothalamic arcuate nucleus injury and alters metabolism more severely in male than in female mice. The site(s) and extent of hypothalamic injury in male and female mice are not completely understood. In the paraventricular nucleus (PVN) of the hypothalamus, single-minded family basic helix-loop helix transcription factor 1 (Sim1) neurons are essential to control energy homeostasis. We tested the hypothesis that exposure to HF diet induces injury to Sim1 neurons in the PVN of male and female mice. Mice expressing membrane-bound enhanced green fluorescent protein (mEGFP) in Sim1 neurons (Sim1-Cre:Rosa-mEGFP mice) were generated to visualize the effects of exposure to HF diet on these neurons. Male and female Sim1-Cre:Rosa-mEGFP mice exposed to HF diet had increased weight, hyperleptinemia, and developed hepatosteatosis. In male and female mice exposed to HF diet, expression of mEGFP was reduced by > 40% in Sim1 neurons of the PVN, an effect paralleled by cell apoptosis and neuronal loss, but not by microgliosis. In the arcuate nucleus of the Sim1-Cre:Rosa-mEGFP male mice, there was decreased alpha-melanocyte-stimulating hormone in proopiomelanocortin neurons projecting to the PVN, with increased cell apoptosis, neuronal loss, and microgliosis. These defects were undetectable in the arcuate nucleus of female mice exposed to the HF diet. Thus, injury to Sim1 neurons of the PVN is a shared feature of exposure to HF diet in mice of both sexes, while injury to proopiomelanocortin neurons in arcuate nucleus is specific to male mice. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/., (© 2019 International Society for Neurochemistry.)

Rationale: Increased microglial activation and neuroinflammation within autonomic brain regions have been implicated in sustained hypertension, and their inhibition by minocycline-an anti-inflammatory antibiotic-produces beneficial effects. These observations led us to propose a dysfunctional brain-gut communication hypothesis for hypertension. However, it has been difficult to reconcile whether an anti-inflammatory or antimicrobial action is the primary beneficial effect of minocycline in hypertension. Accordingly, we utilized chemically modified tetracycline-3 (CMT-3)-a derivative of tetracycline that has potent anti-inflammatory activity-to address this question., Objective: Test the hypothesis that central administration of CMT-3 would inhibit microglial activation, attenuate neuroinflammation, alter selective gut microbial communities, protect the gut wall from developing hypertension-associated pathology, and attenuate hypertension., Methods and Results: Rats were implanted with radiotelemetry devices for recording mean arterial pressure. Ang II (angiotensin II) was infused subcutaneously using osmotic mini-pumps to induce hypertension. Another osmotic mini-pump was surgically implanted to infuse CMT-3 intracerebroventricularly. Intracerebroventricular CMT- 3 infusion was also investigated in SHR (spontaneously hypertensive rats). Physiological, pathological, immunohistological parameters, and fecal microbiota were analyzed. Intracerebroventricular CMT-3 significantly inhibited Ang II-induced increases in number of microglia, their activation, and proinflammatory cytokines in the paraventricular nucleus of hypothalamus. Further, intracerebroventricular CMT-3 attenuated increased mean arterial pressure, normalized sympathetic activity, and left ventricular hypertrophy in Ang II rats, as well as in the SHR. Finally, CMT-3 beneficially restored certain gut microbial communities altered by Ang II and attenuated pathological alterations in gut wall., Conclusions: These observations demonstrate that inhibition of microglial activation alone was sufficient to induce significant antihypertensive effects. This was associated with unique changes in gut microbial communities and profound attenuation of gut pathology. They suggest, for the first time, a link between microglia and certain microbial communities that may have implications for treatment of hypertension.

Increases in sodium concentrations ([Na + ]) in body fluids elevate blood pressure (BP) by enhancing sympathetic nerve activity (SNA). However, the mechanisms by which information on increased [Na + ] is translated to SNA have not yet been elucidated. We herein reveal that sympathetic activation leading to BP increases is not induced by mandatory high salt intakes or the intraperitoneal/intracerebroventricular infusions of hypertonic NaCl solutions in Na x -knockout mice in contrast to wild-type mice. We identify Na x channels expressed in specific glial cells in the organum vasculosum lamina terminalis (OVLT) as the sensors detecting increases in [Na + ] in body fluids and show that OVLT neurons projecting to the paraventricular nucleus (PVN) are activated via acid-sensing ion channel 1a (ASIC1a) by H + ions exported from Na x -positive glial cells. The present results provide an insight into the neurogenic mechanisms responsible for salt-induced BP elevations., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The lack of precise therapies for stress-induced hypertension highlights the need to explore the process of blood pressure changes. Studies have shown that neuroinflammation in the central nervous system is associated with hypertension, although the mechanisms remain elusive. Microglia, are known to play dualistic protective and destructive roles, representing logical but challenging targets for improving stress-induced hypertension. Here, as a model, we used rats with stress-induced hypertension, and found that a switch from an immunoregulatory (M2) to a pro-inflammatory (M1) dominant response occurred in microglia during development of stress-induced hypertension. Administration of minocycline, which is commonly used to inhibit microglial M1 polarisation, attenuated the increase in activated microglia and M1 microglial markers expression in the hypothalamic paraventricular nucleus of rats with stress-induced hypertension. To shed further light on development of stress-induced hypertension, we examined changes in pro- and anti-inflammatory cytokines, and found increased expression of M2 microglial markers during early pathogenesis. Based on these results, we propose the possibility that M1/M2 microglia are related to development of stress-induced hypertension. Consequently, a target molecule that skews M2 polarisation of microglia may be a beneficial therapy for this disease., (Copyright © 2018. Published by Elsevier B.V.)

Objective We aimed to investigate the mechanism of paraventricular nucleus (PVN) and ventral tegmental area (VTA) circuit in the pathogenesis of visceral pain-depression with a rat model induced by neonatal and adult colorectal distension (CRD). Methods Neonate male Sprague-Dayley (SD) rats underwent CRD on postnatal days 8, 10, and 12, and when matured, were tested for adult abdominal withdrawal reflex (AWR) scores to assess visceral hypersensitivity. The forced swimming test was employed to evaluate depression-like behaviors. The rats exhibiting visceral pain-depressive behaviors underwent lidocaine injection in the VTA to explore the relationship between VTA and visceral pain. Moreover, double immunofluorescence was employed to evaluate the qualitative and quantitative expression of dopamine/ c-Fos in CRD rats. After verifying the existed fiber projection from PVN to VTA, the intra-PVN microinjection of CRH-RNAi lentivirus to inhibit corticotropin-releasing hormone (CRH) expression, behavioral changes were assessed by AWR score and FST. Thereafter, with the sacrifice of the rats, the variations of TH protein in rats were evaluated by immunofluorescence and Western blot. Results Intra-VTA microinjection of lidocaine increased the pain threshold of CRD group. After intra-VTA microinjection of green retrograde tracer, immunofluorescence photomicrographs visualized the PVN with a typical green retrograde tracer. Intra-PVN microinjection of CRH-RNAi lentivirus alleviated the visceral pain-depression behaviors and decreased the TH protein expression in the VTA. Conclusion These data demonstrated that the VTA played a functional role in chronic visceral pain and depression, and the CRH-containing neurons in hypothalamic PVN may be implicated in the onset and maintenance of the chronic visceral pain and depression via the activation of dopamine in the VTA.

Food intake ensures energy resources sufficient for basic metabolism, immune system and reproductive investment. It is already known that food-seeking performances, which are crucially controlled by orexins (ORXs), may be under the influence of environmental factors including pollutants. Among these, mancozeb (mz) is becoming an environmental risk for neurodegenerative diseases. Due to few studies on marine fish exposed to mz, it was our intention to correlate feeding latency, food intake and feeding duration to potential neurodegenerative processes in key diencephalic sites and expression changes of the ORX neuroreceptor (ORXR) in the ornate wrasses (Thalassoma pavo). Hence, fish exposed for 4 days (d) to mz 0.2 mg/l (deriving from a 0.07, 0.14, 0.2, 0.3 mg/l screening test) displayed a significant reduction (p < 0.05) of food intake compared to controls as early as 1d that became more evident (p < 0.01) after 3d. Moreover, significant enhancements of feeding latency were reported after 1d up to 3d (p < 0.001) and even feeding duration was enhanced up to 3d (p < 0.001), which instead moderately increased after 4d (p < 0.05). A reduction (-120%; p < 0.001) of mean body weight was also detected at the end of exposure. Likewise, a notable (p < 0.001) activation of ORXR protein occurred together with mRNA up-regulations in diencephalic areas such as the diffuse nucleus of the inferior lobe (+48%) that also exhibited evident degenerative neuronal fields. Overall, these results highlight an ORX role as a vital component of the neuroprotective program under environmental conditions that interfere with feeding behaviors., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Developing novel therapeutics for bipolar disorder (BD) has been hampered by limited mechanistic knowledge how sufferers switch between mania and depression-how the same brain can switch between extreme states-described as the "holy grail" of BD research. Strong evidence implicates seasonally-induced switching between states, with mania associated with summer-onset, depression with winter-onset. Determining mechanisms of and sensitivity to such switching is required. C57BL/6J and dopamine transporter hypomorphic (DAT-HY 50% expression) mice performed a battery of psychiatry-relevant behavioral tasks following 2-week housing in chambers under seasonally relevant photoperiod extremes. Summer-like and winter-like photoperiod exposure induced mania-relevant and depression-relevant behaviors respectively in mice. This behavioral switch paralleled neurotransmitter switching from dopamine to somatostatin in hypothalamic neurons (receiving direct input from the photoperiod-processing center, the suprachiasmatic nucleus). Mice with reduced DAT expression exhibited hypersensitivity to these summer-like and winter-like photoperiods, including more extreme mania-relevant (including reward sensitivity during reinforcement learning), and depression-relevant (including punishment-sensitivity and loss-sensitivity during reinforcement learning) behaviors. DAT mRNA levels switched in wildtype littermate mice across photoperiods, an effect not replicated in DAT hypomorphic mice. This inability to adjust DAT levels to match photoperiod-induced neurotransmitter switching as a homeostatic control likely contributes to the susceptibility of DAT hypormophic mice to these switching photoperiods. These data reveal the potential contribution of photoperiod-induced neuroplasticity within an identified circuit of the hypothalamus, linked with reduced DAT function, underlying switching between states in BD. Further investigations of the circuit will likely identify novel therapeutic targets to block switching between states.

Pulmonary hypertension (PH) is a devastating disease and its successful treatment remains to be accomplished despite recent advances in pharmacotherapy. It has been proposed that PH be considered as a systemic disease, rather than primarily a disease of the pulmonary vasculature. Consequently, an investigation of the intricate interplay between multiple organs such as brain, vasculature, and lung in PH could lead to the identification of new targets for its therapy. However, little is known about this interplay. This study was undertaken to examine the concept that altered autonomic-pulmonary communication is important in PH pathophysiology. Therefore, we hypothesize that activation of microglial cells in the paraventricular nucleus of hypothalamus and neuroinflammation is associated with increased sympathetic drive and pulmonary pathophysiology contributing to PH. We utilized the monocrotaline rat model for PH and intracerebroventricular administration of minocycline for inhibition of microglial cells activation to investigate this hypothesis. Hemodynamic, echocardiographic, histological, immunohistochemical, and confocal microscopic techniques assessed cardiac and pulmonary function and microglial cells. Monocrotaline treatment caused cardiac and pulmonary pathophysiology associated with PH. There were also increased activated microglial cells and mRNA for proinflammatory cytokines (IL [interleukin]-1β, IL-6, and TNF [tumor necrosis factor]-α) in the paraventricular nucleus. Furthermore, increased sympathetic drive and plasma norepinephrine were observed in rats with PH. Intracerebroventricular infusion of minocycline inhibited all these parameters and significantly attenuated PH. These observations implicate a dysfunctional autonomic-lung communication in the development and progression of PH providing new therapeutic targets, such as neuroinflammation, for PH therapy., (© 2018 American Heart Association, Inc.)

Neurotransmitter switching in the adult mammalian brain occurs following photoperiod-induced stress, but the mechanism of regulation is unknown. Here, we demonstrate that elevated activity of dopaminergic neurons in the paraventricular nucleus of the hypothalamus (PaVN) in the adult rat is required for the loss of dopamine expression after long-day photoperiod exposure. The transmitter switch occurs exclusively in PaVN dopaminergic neurons that coexpress vesicular glutamate transporter 2 (VGLUT2), is accompanied by a loss of dopamine type 2 receptors (D2Rs) on corticotrophin-releasing factor (CRF) neurons, and can lead to increased release of CRF. Suppressing activity of all PaVN glutamatergic neurons decreases the number of inhibitory PaVN dopaminergic neurons, indicating homeostatic regulation of transmitter expression in the PaVN., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

In response to an acute threat to homeostasis or well-being, the hypothalamic-pituitary-adrenocortical (HPA) axis is engaged. A major outcome of this HPA axis activation is the mobilization of stored energy, to fuel an appropriate behavioral and/or physiological response to the perceived threat. Importantly, the extent of HPA axis activity is thought to be modulated by an individual's nutritional environment. In this study, we report that nutritional manipulations signaling a relative depletion of dietary carbohydrates, thereby inducing nutritional ketosis, acutely and chronically activate the HPA axis. Male rats and mice maintained on a low-carbohydrate high-fat ketogenic diet (KD) exhibited canonical markers of chronic stress, including increased basal and stress-evoked plasma corticosterone, increased adrenal sensitivity to adrenocorticotropin hormone, increased stress-evoked c-Fos immunolabeling in the paraventricular nucleus of the hypothalamus, and thymic atrophy, an indicator of chronic glucocorticoid exposure. Moreover, acutely feeding medium-chain triglycerides (MCTs) to rapidly induce ketosis among chow-fed male rats and mice also acutely increased HPA axis activity. Lastly, and consistent with a growing literature that characterizes the hepatokine fibroblast growth factor-21 (FGF21) as both a marker of the ketotic state and as a key metabolic stress hormone, the HPA response to both KD and MCTs was significantly blunted among mice lacking FGF21. We conclude that dietary manipulations that induce ketosis lead to increased HPA axis tone, and that the hepatokine FGF21 may play an important role to facilitate this effect., (Copyright © 2018 Endocrine Society.)

Sexual dysfunction is a poorly understood condition that affects up to one-third of men around the world. Existing treatments that target the periphery do not work for all men. Previous studies have shown that central melanocortins, which are released by pro-opiomelanocortin neurons in the arcuate nucleus of the hypothalamus, can lead to male erection and increased libido. Several studies specifically implicate the melanocortin 4 receptor (MC4R) in the central control of sexual function, but the specific neural circuitry involved is unknown. We hypothesized that single-minded homolog 1 (Sim1) neurons play an important role in the melanocortin-mediated regulation of male sexual behavior. To test this hypothesis, we examined the sexual behavior of mice expressing MC4R only on Sim1-positive neurons (tbMC4Rsim1 mice) in comparison with tbMC4R null mice and wild-type controls. In tbMC4Rsim1 mice, MC4R reexpression was found in the medial amygdala and paraventricular nucleus of the hypothalamus. These mice were paired with sexually experienced females, and their sexual function and behavior was scored based on mounting, intromission, and ejaculation. tbMC4R null mice showed a longer latency to mount, a reduced intromission efficiency, and an inability to reach ejaculation. Expression of MC4R only on Sim1 neurons reversed the sexual deficits seen in tbMC4R null mice. This study implicates melanocortin signaling via the MC4R on Sim1 neurons in the central control of male sexual behavior., (Copyright © 2018 Endocrine Society.)

Background: Human African trypanosomiasis is one of the neglected and re-emerging infectious diseases in Africa with over 60 million people being at risk of contracting the disease., Objective: To investigate the effects of Trypanosoma brucei brucei infection on secretion of adrenocorticotropic hormone (ACTH) and histology of the pituitary gland and paraventricular nucleus in rats., Methods: Rats were randomly divided into two groups, control and experimental. Experimental rats were injected intraperitonially with 0.2ml of blood containing 1.0 × 10 4 live T.b.brucei parasites. Tail blood samples were collected weekly for the determination of plasma concentration of ACTH. The pituitary gland and coronal section of brain were processed histologically and observed microscopically., Results: There was a significant difference (p = 0.0190) in plasma ACTH concentration between the control and experimental rats. Histological alterations were observed in both the pituitary and paraventricular nucleus of experimental rats., Conclusion: T.b.brucei infection causes histological changes in both the paraventricular nucleus and pituitary gland in rats. These histological changes could account for the decrease in corticotropin releasing hormone (CRH) and ACTH production in the infected rats.

Converging evidence indicates that abnormal glutamatergic and synaptic systems may be associated with the pathophysiology of depression. Over-activation of corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus (PVN) plays a key role in the hypothalamic-pituitary-adrenal (HPA) axis hyperactivity and is a prominent feature in depression. In this study, we examined the effect of chronic unpredictable stress (CUS) in rats on the expression of AMPA subunit GluR1 protein in the hypothalamic PVN as well as on the expression of GluR1 and several synapse-related proteins in the hypothalamus. We found that CUS in rats induced CRH over-expression accompanied by significant GluR1 up-regulation in the hypothalamic PVN and GluR1 co-localized with CRH in the PVN area. The expression of GluR1 and three synapse-related proteins (PSD95, SYP and MAP2) in the hypothalamus were also increased at both mRNA and protein levels in CUS rats. Correlation analysis showed that the levels of the above proteins are directly correlated with depression-like behaviors of rats in sucrose preference test, open field test and forced swim test. These findings suggest that the augmented glutamatergic and synaptic signaling might be a potential mechanism underlying CRH over-activation in the hypothalamic PVN and contribute to CUS-induced depression-like behaviors in rats., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Glucocorticoids rapidly stimulate endocannabinoid synthesis and modulation of synaptic transmission in hypothalamic neuroendocrine cells via a nongenomic signaling mechanism. The endocannabinoid actions are synapse-constrained by astrocyte restriction of extracellular spatial domains. Exogenous cannabinoids have been shown to modulate postsynaptic potassium currents, including the A-type potassium current ( I A ), in different cell types. The activity of magnocellular neuroendocrine cells is shaped by a prominent I A . We tested for a rapid glucocorticoid modulation of the postsynaptic I K and I A in magnocellular neuroendocrine cells of the hypothalamic paraventricular nucleus (PVN) using whole-cell recordings in rat brain slices. Application of the synthetic glucocorticoid dexamethasone (Dex) had no rapid effect on the I K or I A amplitude, voltage dependence, or kinetics in magnocellular neurons in slices from untreated rats. In magnocellular neurons from salt-loaded rats, however, Dex application caused a rapid suppression of the I A and a depolarizing shift in I A voltage dependence. Exogenously applied endocannabinoids mimicked the rapid Dex modulation of the I A , and CB1 receptor antagonists and agonists blocked and occluded the Dex-induced changes in the I A , respectively, suggesting an endocannabinoid dependence of the rapid glucocorticoid effect. Preincubation of control slices in a gliotoxin resulted in the partial recapitulation of the glucocorticoid-induced rapid suppression of the I A . These findings demonstrate a glucocorticoid suppression of the postsynaptic I A in PVN magnocellular neurons via an autocrine endocannabinoid-dependent mechanism following chronic dehydration, and suggest a possible role for astrocytes in the control of the autocrine endocannabinoid actions.

Fetal exposure to high levels of glucocorticoids programs long-term changes in the physiologic stress response and behaviours. However, it is not known whether effects manifest in subsequent generations of offspring following maternal (MT) or paternal (PT) transmission. We treated pregnant guinea pigs with three courses of saline or synthetic glucocorticoid (sGC) at a clinically relevant dose. Altered cortisol response to stress and behaviours transmitted to juvenile female and male F 2 and F 3 offspring from both parental lines. Behavioural effects of sGC in F 1 -F 3 PT females associated with altered expression of genes in the prefrontal cortex and hypothalamic paraventricular nucleus (PVN). Exposure to sGC programmed large transgenerational changes in PVN gene expression, including type II diabetes, thermoregulation, and collagen formation gene networks. We demonstrate transgenerational programming to F 3 following antenatal sGC. Transmission is sex- and generation-dependent, occurring through both parental lines. Paternal transmission to F 3 females strongly implicates epigenetic mechanisms of transmission.

Microglia, resident immune cells of the CNS are sensitive to various perturbations of the environment, such as stress exposure, and may be involved in translating these changes to behavior. Among the pathways mediating stress-related neuronal cues to microglia, the fractalkine-fractalkine receptor (CX 3 CR1) signaling plays a crucial role. Using mice, in which the CX 3 CR1 gene was deleted, we explored hormonal and behavioral responses to acute and chronic stress along with changes in hypothalamic microglia. CX 3 CR1 -/- animals display active escape in forced swim- and tail suspension tests, exaggerated neuronal activation in the hypothalamic paraventricular nucleus and increased corticosterone release in response to restraint. Analysis of Iba1 immunostaining of hypothalamic sections revealed stress-related reduction of microglia in CX 3 CR1 -/- mice. Because microglia also contribute to energy balance regulation, we characterized metabolic phenotype of CX 3 CR1 -/- mice. Comparison of respiratory exchange ratio did not show genotype effect on fuel preference, however, the energy expenditure was increased in CX 3 CR1 -/- mice, which may be related to their active coping behavior. Microglia and fractalkine signaling has been repeatedly shown to be involved chronic stress-induced depressive state. CX 3 CR1 -/- mice did not become anhedonic in the "two hit" chronic stress paradigm, confirming resistance of these animals to chronic stress-induced mood alterations. However, there was no difference in stress hormone levels, open field performance and hypothalamic microglia distribution between the genotypes. These results highlight differential involvement of microglia fractalkine signaling in controlling/integrating hormonal-, metabolic and behavioral responses to acute and chronic stress challenges., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Aberrant glucocorticoid secretion is implicated in the pathophysiology of stress-related disorders (i.e., depression, anxiety). Glucocorticoids exert biological effects via mineralocorticoid (MR) and glucocorticoid (GR) receptors. Previous data from our laboratory indicate that GR antagonism/modulation (i.e., mifepristone, CORT 108297) regulate endocrine, behavioral, and central stress responses. Because of the dynamic interplay between MR and GR on HPA axis regulation and emotionality, compounds targeting both receptors are of interest for stress-related pathology. We investigated the effects of CORT 118335 (a dual selective GR modulator/MR antagonist) on endocrine, behavioral, and central (c-Fos) stress responses in male rats. Rats were treated for five days with CORT 118335, imipramine (positive control), or vehicle and exposed to restraint or forced swim stress (FST). CORT 118335 dampened corticosterone responses to both stressors, without a concomitant antidepressant-like effect in the FST. Imipramine decreased corticosterone responses to restraint stress; however, the antidepressant-like effect of imipramine in the FST was independent of circulating glucocorticoids. These findings indicate dissociation between endocrine and behavioral stress responses in the FST. CORT 118335 decreased c-Fos expression only in the CA1 division of the hippocampus. Imipramine decreased c-Fos expression in the basolateral amygdala and CA1 and CA3 divisions of the hippocampus. Overall, the data indicate differential effects of CORT 118335 and imipramine on stress-induced neuronal activity in various brain regions. The data also highlight a complex relationship between neuronal activation in stress and mood regulatory brain regions and the ensuing impact on endocrine and behavioral stress responses., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Doe rabbits nurse the litter only once a day, for around 3 min, with circa 24-h periodicity. To explore the participation of the paraventricular nucleus (PVN) in regulating this behavior, we lesioned it bilaterally with kainic acid on lactation day 7. Bilateral lesions, comprising less than 50% of the total PVN volume, abolished nursing behavior (4/8 does) or severely disrupted its normal periodicity (4/8 does). In the latter case, duration of nursing bouts was normal. Body weight, food and water intake were not significantly affected by bilateral PVN lesions. Unilateral lesions of the PVN or lesions located outside this nucleus did not significantly alter nursing periodicity or any other behavioral parameter. Results indicate an important role of the PVN for (i) maintaining maternal behavior, and (ii) the periodic display of nursing across lactation in rabbits., (© 2017 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are environmental pollutants that produce neurotoxicity and neuroendocrine disruption. They affect the vasopressinergic system but their disruptive mechanisms are not well understood. Our group reported that rats perinatally exposed to Aroclor-1254 (A1254) and DE-71 (commercial mixtures of PCBs and PBDEs) decrease somatodendritic vasopressin (AVP) release while increasing plasma AVP responses to osmotic activation, potentially emptying AVP reserves required for body-water balance. The aim of this research was to evaluate the effects of perinatal exposure to A1254 or DE-71 (30mgkg/day) on AVP transcription and protein content in the paraventricular and supraoptic hypothalamic nuclei, of male and female rats, by in situ hybridization and immunohistochemistry. cFOS mRNA expression was evaluated in order to determine neuroendocrine cells activation due to osmotic stimulation. Animal groups were: vehicle (control); exposed to either A1254 or DE-71; both, control and exposed, subjected to osmotic challenge. The results confirmed a physiological increase in AVP-immunoreactivity (AVP-IR) and gene expression in response to osmotic challenge as reported elsewhere. In contrast, the exposed groups did not show this response to osmotic activation, they showed significant reduction in AVP-IR neurons, and AVP mRNA expression as compared to the hyperosmotic controls. cFOS mRNA expression increased in A1254 dehydrated groups, suggesting that the AVP-IR decrease was not due to a lack of the response to the osmotic activation. Therefore, A1254 may interfere with the activation of AVP mRNA transcript levels and protein, causing a central dysfunction of vasopressinergic system., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The vasopressin- and oxytocin-degrading enzyme insulin-regulated aminopeptidase (IRAP) is expressed in various organs including the brain. However, knowledge about its presence in human hypothalamus is fragmentary. Functionally, for a number of reasons (genetic linkage, hydrolysis of oxytocin and vasopressin, its role as angiotensin IV receptor in learning and memory and others) IRAP might play a role in schizophrenia. We studied the regional and cellular localization of IRAP in normal human brain with special emphasis on the hypothalamus and determined numerical densities of IRAP-expressing cells in the paraventricular, supraoptic and suprachiasmatic nuclei in schizophrenia patients and controls. By using immunohistochemistry and Western blot analysis, IRAP was immunolocalized in postmortem human brains. Cell countings were performed to estimate numbers and numerical densities of IRAP immunoreactive hypothalamic neurons in schizophrenia patients and control cases. Shape, size and regional distribution of IRAP-expressing cells, as well the lack of co-localization with the glia marker glutamine synthetase, show that IRAP is expressed in neurons. IRAP immunoreactive cells were observed in the hippocampal formation, cerebral cortex, thalamus, amygdala and, abundantly, hypothalamus. Double labeling experiments (IRAP and oxytocin/neurophysin 1, IRAP with vasopressin/neurophysin 2) revealed that IRAP is present in oxytocinergic and in vasopressinergic neurons. In schizophrenia patients, the numerical density of IRAP-expressing neurons in the paraventricular and the suprachiasmatic nuclei is significantly reduced, which might be associated with the reduction in neurophysin-containing neurons in these nuclei in schizophrenia. The pathophysiological role of lowered hypothalamic IRAP expression in schizophrenia remains to be established.

The paraventricular nucleus of hypothalamus plays important roles in the regulation of energy balance and fetal growth. However, the molecular mechanisms underlying its formation and function have not been clearly elucidated. Various mutations in the human COUP-TFII gene, which encodes a nuclear receptor, result in growth retardation, congenital diaphragmatic hernia and congenital heart defects. Here, we show that COUP-TFII gene is expressed in the developing hypothalamus in mouse. The ventral forebrain-specific RXCre/+; COUP-TFII F/F mutant mice display growth retardation. The development of the paraventricular nucleus of hypothalamus is compromised in the COUP-TFII mutant mainly because of increased apoptosis and mis-migration of the Brn2 + neurons. Moreover, hypoplastic anterior pituitary with blood cell clusters and shrunken posterior pituitary lacking AVP/OT neuron innervations are observed in the mutant, indicating the failure of formation of the hypothalamic-pituitary axis. Mechanistic studies show that the expression of Bdnf and Nrp1 genes is reduced in the mutant embryo, and that Bdnf is a direct downstream target of the COUP-TFII protein. Thus, our findings provide a novel functional validation that COUP-TFII gene promotes the expression of Bdnf and Nrp1 genes to ensure the appropriate morphogenesis of the hypothalamic-pituitary axis, especially the paraventricular nucleus of hypothalamus, and to prevent growth retardation.

Obesity increases sympathetic nerve activity (SNA) via activation of proopiomelanocortin neurons in the arcuate nucleus (ArcN), and this action requires simultaneous withdrawal of tonic neuropeptide Y (NPY) sympathoinhibition. However, the sites and neurocircuitry by which NPY decreases SNA are unclear. Here, using designer receptors exclusively activated by designer drugs (DREADDs) to selectively activate or inhibit ArcN NPY neurons expressing agouti-related peptide (AgRP) in mice, we have demonstrated that this neuronal population tonically suppresses splanchnic SNA (SSNA), arterial pressure, and heart rate via projections to the paraventricular nucleus (PVN) and dorsomedial hypothalamus (DMH). First, we found that ArcN NPY/AgRP fibers closely appose PVN and DMH presympathetic neurons. Second, nanoinjections of NPY or an NPY receptor Y1 (NPY1R) antagonist into PVN or DMH decreased or increased SSNA, respectively. Third, blockade of DMH NPY1R reversed the sympathoinhibition elicited by selective, DREADD-mediated activation of ArcN NPY/AgRP neurons. Finally, stimulation of ArcN NPY/AgRP terminal fields in the PVN and DMH decreased SSNA. Considering that chronic obesity decreases ArcN NPY content, we propose that the ArcN NPY neuropathway to the PVN and DMH is pivotal in obesity-induced elevations in SNA.