Cells In Paraventricular Nucleus Research Articles

Co-expression of interleukin (IL)-17RA and IL-36γ in the hypothalamic paraventricular nucleus of the stress-induced hypertension rats

Objectives: The up-regulation of proinflammatory cytokines in the hypothalamic paraventricular nucleus (PVN) is well demonstrated to be involved in the development of neurogenic hypertension, including stress-induced hypertension (SIH). IL-17A has been found to be increased in the PVN of several hypertensive animal models and has been shown to play a key role in the development of hypertension. Although IL-36γ was found to be expressed in spinal neurons, its role in hypertension remains elusive. Here, we investigated the co-expression of IL-17 receptor A (IL-17RA) and IL-36γ in the PVN cells of SIH rats. Methods: The electric foot shock combined with buzzer noise stressors were used to make hypertensive rat model. The immunochemical staining or immunofluorescence staining was used to reveal cells as requested. The Western blot was used to detect the related protein levels. Results and Conclusion: In the PVN of the SIH rats, the number of CD3+CD4+ T cells was significantly increased by the immunochemical staining. Additionally, the protein levels of RORγt and IL-17A were significantly upregulated by Western blot, confirming the infiltration of CD4+ T cells and differentiation into Th17 cells in the PVN of SIH rats. Immunofluorescence staining revealed abundant expression of IL-17RA in PVN neurons, with relatively less expression in astrocytes or microglia. Furthermore, IL-36γ positive cells and protein expression of IL-36R were significantly increased. Notably, this study demonstrates for the first time that most IL-36γ cells were strongly colocalized with IL-17RA positive cells in the PVN of SIH rats, and the colocalized cells were significantly higher in SIH rats. This suggests that IL-17A secreted by infiltrated Th17 cells may stimulate PVN neurons to produce IL-36γ via IL-17RA, indicating that the combination of IL-17 and IL-36γ might produce strong pro-inflammatory effects in the PVN of SIH rats.

Glutamate Spillover Dynamically Strengthens Gabaergic Synaptic Inhibition of the Hypothalamic Paraventricular Nucleus.

The hypothalamic paraventricular nucleus (PVN) is strongly inhibited by γ-aminobutyric acid (GABA) from the surrounding peri-nuclear zone (PNZ). Because glutamate mediates fast excitatory transmission and is substrate for GABA synthesis, we tested its capacity to dynamically strengthen GABA inhibition. In PVN slices from male mice, bath glutamate applied during ionotropic glutamate receptor blockade increased PNZ-evoked inhibitory postsynaptic currents (eIPSCs) without affecting GABA-A receptor agonist currents or single-channel conductance, implicating a presynaptic mechanism(s). Consistent with this interpretation, bath glutamate failed to strengthen IPSCs during pharmacological saturation of GABA-A receptors. Presynaptic analyses revealed that glutamate did not affect paired-pulse ratio, peak eIPSC variability, GABA vesicle recycling speed, or readily releasable pool (RRP) size. Notably, glutamate-GABA strengthening (GGS) was unaffected by metabotropic glutamate receptor blockade and graded external Ca2+ when normalized to baseline amplitude. GGS was prevented by pan- but not glial-specific inhibition of glutamate uptake and by inhibition of glutamic acid decarboxylase (GAD), indicating reliance on glutamate uptake by neuronal excitatory amino acid transporter 3 (EAAT3) and enzymatic conversion of glutamate to GABA. EAAT3 immunoreactivity was strongly localized to presumptive PVN GABA terminals. High bath K+ also induced GGS, which was prevented by glutamate vesicle depletion, indicating that synaptic glutamate release strengthens PVN GABA inhibition. GGS suppressed PVN cell firing, indicating its functional significance. In sum, PVN GGS buffers neuronal excitation by apparent "over-filling" of vesicles with GABA synthesized from synaptically released glutamate. We posit that GGS protects against sustained PVN excitation and excitotoxicity while potentially aiding stress adaptation and habituation.

Inhibition of cGAS in Paraventricular Nucleus Attenuates Hypertensive Heart Injury Via Regulating Microglial Autophagy.

Neuroinflammation in the cardiovascular center plays a critical role in the progression of hypertensive heart disease. And microglial autophagy is involved in the regulation of neuroinflammation. Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, senses mitochondrial DNA (mtDNA) and regulates autophagy. The detailed mechanisms of central cGAS affects neuroinflammatory response in hypertensive heart disease via regulating autophagy remain unknown. Angiotensin II (Ang II, 1.5 mg·kg−1·12 h−1, 2 weeks) was intraperitoneally injected to induce hypertension in mice. The cGAS-STING pathway was activated in the paraventricular nucleus (PVN) of Ang II-induced hypertensive mice. The contractile dysfunction of heart was alleviated in Ang II-induced hypertensive cGAS−/− mice. To observe the central effects of cGAS on regulating hypertensive heart disease, the RU.521 (a cGAS inhibitor) was intracisternally infused in hypertensive mice. Intracisternal infusion of the RU.521-alleviated myocardial interstitial fibrosis, cardiomyocyte hypertrophy, and the contractile dysfunction in Ang II-induced hypertensive mice. Intracisternal infusion of RU.521 attenuated the microglial activation, neuroinflammation, sympathetic/parasympathetic activity ratio, and lowered blood pressure. The autophagic flux in the PVN cells was blocked, while intracisternal infusion of RU.521 alleviated this effect in the Ang II-induced hypertensive mice. In vitro, it was found that cGAS-STING activation-induced autophagic flux blockage, while when the impaired autophagic flux was facilitated by rapamycin, an autophagy inducer, the microglial M1 polarization was decreased correspondingly. In conclusion, cGAS induces the inflammatory phenotype of microglia via impairing autophagic flux, thereby participating in neuroinflammation, which leads to sympathetic overactivation in hypertension and further caused hypertensive myocardial injury.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-022-02994-1.

Conditional ablation of vasopressin-synthesizing neurons in transgenic rats.

Vasopressin‐synthesizing neurons are located in several brain regions, including the hypothalamic paraventricular nucleus (PVN), supraoptic nucleus (SON) and suprachiasmatic nucleus (SCN). Vasopressin has been shown to have various functions in the brain, including social recognition memory, stress responses, emotional behaviors and circadian rhythms. The precise physiological functions of vasopressin‐synthesizing neurons in specific brain regions remain to be clarified. Conditional ablation of local vasopressin‐synthesizing neurons may be a useful tool for investigation of the functions of vasopressin neurons in the regions. In the present study, we characterized a transgenic rat line that expresses a mutated human diphtheria toxin receptor under control of the vasopressin gene promoter. Under a condition of salt loading, which activates the vasopressin gene in the hypothalamic PVN and SON, transgenic rats were i.c.v. injected with diphtheria toxin. Intracerebroventricular administration of diphtheria toxin after salt loading depleted vasopressin‐immunoreactive cells in the hypothalamic PVN and SON, but not in the SCN. The number of oxytocin‐immunoreactive cells in the hypothalamus was not significantly changed. The rats that received i.c.v. diphtheria toxin after salt loading showed polydipsia and polyuria, which were rescued by peripheral administration of 1‐deamino‐8‐d‐arginine vasopressin via an osmotic mini‐pump. Intrahypothalamic administration of diphtheria toxin in transgenic rats under a normal hydration condition reduced the number of vasopressin‐immunoreactive neurons, but not the number of oxytocin‐immunoreactive neurons. The transgenic rat model can be used for selective ablation of vasopressin‐synthesizing neurons and may be useful for clarifying roles of vasopressin neurons at least in the hypothalamic PVN and SON in the rat.

The effect of amisulpride, olanzapine, quetiapine, and aripiprazole single administration on c-Fos expression in vasopressinergic and oxytocinergic neurons of the rat hypothalamic paraventricular nucleus

Antipsychotics, including amisulpride (AMI), quetiapine (QUE), aripiprazole (ARI), and olanzapine (OLA), are used to treat mental illnesses associated with psychotic symptoms. The effect of these drugs on c-Fos expression in vasopressinergic (AVP) and oxytocinergic (OXY) neurons was studied in the hypothalamic paraventricular nucleus (PVN) of rats. The presence of c-Fos in AVP and OXY perikarya was investigated in seven PVN cells segregations: the anterior (Ant), dorsal cup (Dc), wing-shaped (Wi), periventricular zone (Pe), circle-shaped core (Co) and shell of core (Sh), and the posterior (pPVN) after an acute treatment with AMI-20 mg/kg, QUE-15 mg/kg, ARI-10 mg/kg, and OLA-5 mg/kg/bw in rats. Ninety min after treatments, the animals were sacrificed by transcardial perfusion with fixative and the PVN area sliced into 35 μm thick coronal sections for immunohistochemistry. The c-Fos was processed by avidin-biotin-peroxidase complex intensified with nickel-enhanced 3,3′-diaminobenzidine tetrahydrochloride. Visualization of AVP- and OXY-synthesizing neurons was achieved by a fluorescent marker Alexa Flour 568. The c-Fos-AVP and c-Fos-OXY colocalizations were evaluated from c-Fos stained sections merged with AVP or OXY ones. AMI, QUE, ARI, and OLA, single administration distinctly increased the c-Fos expression in each of the PVN cells segregations. QUE induced the highest magnitude of activation of AVP and OXY neurons, while OLA and AMI had only moderate effects. Incontestable variabilities detected in c-Fos expression in PVN AVP and OXY neurons extend the knowledge of selected antipsychotics extra-striatal actions and may also be helpful in a presumption of their possible functional impact.

Postnatal AVP treatments prevent social deficit in adolescence of valproic acid-induced rat autism model

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.

Corticosteroid receptor rebalancing alleviates critical illness-related corticosteroid insufficiency after traumatic brain injury by promoting paraventricular nuclear cell survival via Akt/CREB/BDNF signaling

BackgroundWe 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.MethodsBased 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.ResultsWe 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.ConclusionThe 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.

Maternal separation changes maternal care, anxiety-like behaviour and expression of paraventricular oxytocin and corticotrophin-releasing factor immunoreactivity in lactating rats.

The early postnatal period is a time of tremendous change for the dam and her offspring. During this time, environmental insults such as repeated stress exposure can have detrimental effects. In research that has focused on the effect of postnatal stress exposure on the dams, conflicting changes in maternal care and anxiety-like behaviour have been reported. Additionally, changes to hypothalamic neuropeptides that are crucially involved in the transition to motherhood and stress regulation, namely oxytocin and corticotrophin-releasing factor (CRF), have not been examined. Accordingly, the present study aimed to determine (i) whether repeated postpartum stress increases engagement in maternal care behaviours and anxiety-like behaviour and (ii) whether these behavioural changes correspond with changes to CRF- or oxytocin-immunoreactive (-IR) cells in the paraventricular nucleus (PVN) of the hypothalamus. A non-lactating group was also included to control for the effects of lactation on anxiety and the hypothalamic neuroendocrine system. Following the birth of their litters, Long-Evans dams were separated from their pups from postnatal day (PND) 1 to PND21 for either 15minutes (maternal separation [MS]15) or 6hours (MS360). Maternal behaviours were recorded for 30minutes on select PNDs following the separation. On PND22, dams were exposed to the elevated plus maze, brains were collected, and immunofluorescence analysis of PVN oxytocin- and CRF-IR cells was conducted. Our findings demonstrate that prolonged maternal separation altered typical maternal behaviours and reduced anxiety relative to MS15 dams. At the cellular level, oxytocin-IR cells in the caudal PVN were reduced in MS360 dams to a level similar to that in non-lactating controls, and PVN CRF-IR cells were reduced relative to both MS15 and non-lactating controls. Taken together, these data reveal the behavioural and neuronal changes that occur in the mother dam following repeated postnatal stress exposure.

Detection and Characterization of Estrogen Receptor Beta Expression in the Brain with Newly Developed Transgenic Mice

Two types of nuclear estrogen receptors, ERα and ERβ, have been shown to be differentially involved in the regulation of various types of behaviors. Due to a lack of tools for identifying ERβ expression, detailed anatomical distribution and neurochemical characteristics of ERβ expressing cells and cellular co-expression with ERα remain unclear. We have generated transgenic mice ERβ-RFPtg, in which RFP was inserted downstream of ERβ BAC promotor. We verified RFP signals as ERβ by confirming: (1) high ERβ mRNA levels in RFP-expressing cells collected by fluorescence-activated cell sorting; and (2) co-localization of ERβ mRNA and RFP proteins in the paraventricular nucleus (PVN). Strong ERβ-RFP signals were found in the PVN, medial preoptic area (MPOA), bed nucleus of the stria terminalis, medial amygdala (MeA), and dorsal raphe nucleus (DRN). In the MPOA and MeA, three types of cell populations were identified; those expressing both ERα and ERβ, and those expressing exclusively either ERα or ERβ. The majority of PVN and DRN cells expressed only ERβ-RFP. Further, ERβ-RFP positive cells co-expressed oxytocin in the PVN, and tryptophan hydroxylase 2 and progesterone receptors in the DRN. In the MeA, some ERβ-RFP positive cells co-expressed oxytocin receptors. These findings collectively suggest that ERβ-RFPtg mice can be a powerful tool for future studies on ERβ function in the estrogenic regulation of social behaviors.

The ageing PVN: cardiovascular implications for reduced TRPV4 expression?

Several changes in cardiovascular, metabolic and autonomic function associate with ageing and given the increase in the average human lifespan, a greater understanding of age‐related changes in the physiological control of homeostasis is needed. For example, cardiovascular control by the autonomic nervous system is one of the most vulnerable functions in the aging process, however, the underlying mechanisms are not fully understood.The paraventricular nucleus (PVN) of the hypothalamus is an important autonomic control centre in the brain with many homeostatic roles. PVN neurones project directly to the spinal cord and can modulate cardiovascular function via the sympathetic system. Age‐related changes to the PVN remain largely unknown: PVN cell numbers remain unaltered but little is known about changes in the expression profile of the ion channels. It is hypothesised that any changes in PVN ion channels is likely to result in perturbed physiological processes and/or declined cardiovascular function.In this study, we compared the cardiovascular function of younger (3 months) and older (26 months) adult male Wistar rats. We measured the heart rate, blood pressure (BP) and tail blood volume using non‐invasive tail plethysmography. In addition, we determined the gene expression profiles in the PVN with RNA deep sequencing (RNA‐seq). Results are given as mean ± SEM; significances were assessed by one‐way ANOVA or Student’s t‐tests where appropriate.We found no differences in baseline measurements of blood pressure, heart rate or tail blood volume between adult and aged rats (n=6, p>0.05). We found that slightly higher‐than‐thermoneutral temperatures of 34 °C were required to achieve stable readings from the aged animals. In adult rats, intraperitoneal (i.p) administration of the TRPV4 agonist GSK 1016790A (50 μg/kg) resulted in increased blood pressure and tail blood volume (n=6, p<0.05), whereas this response was absent in aged rats.Our RNA‐seq data showed profound differences in the gene profile of the adult PVN compared to the aged PVN. Bioinformatic analysis showed 3.5‐fold decreased expression of TRPV4 (p<0.05) and the most greatly enriched KEGG pathway was the “Neuroactive Ligand Receptor Interaction” pathway (p<0.5×106) including several important homeostatic control receptors and peptides such as tachykinin 3.Our data illustrates that activation of TRPV4 results in increased blood pressure and tail blood volume in adult rodents, and in aged rodents where there is widespread change of receptor expression, this effect is ameliorated. These experiments were conducted in higher than‐thermoneutral temperatures and TRPV4 has known thermoregulatory roles, therefore further experiments are required to understand the relationship between reduced expression of TRPV4 in ageing and the decline in both cardiovascular and thermoregulatory function via the PVN.Support or Funding InformationThis work was funded by the BBSRC and King Abdulaziz University

Effect of electroacupuncture at "Shenmen"(HT7) and "Sanyinjiao"(SP6) on energy metabolism in paraventricular nucleus of hypothalamus of insomnia rats

To observe the effect of electroacupuncture(EA) at "Shenmen"(HT7) and "Sanyinjiao"(SP6)on energy metabolism in paraventricular nucleus (PVN) of hypothalamus in insomnia rats, so as to explore its mechanism underlying improvement of insomnia. A total of 66 SD rats (half male and half female) were randomized into 3 groups：normal control, model and EA groups(n＝22 per group). The insomnia model was established by binding the rat for at least 4 h (step increase of 30 min per day), once daily for 15 days. EA (5 Hz /25 Hz, 0.5－1.0 mA) was applied to unilateral HT7 and SP6 for 15 min, once daily for 5 days. The rats' spontaneous activities during day and night were recorded by using the ClockLab Data Collection and Analysis System, and the duration of exhausted swimming was detected by using load-bearing endurance swimming test. The expression of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) of PVN tissue was assayed by Western blot, and the contents of acetyl coenzyme A (Ac-CoA) and Na＋-K＋adenosine triphosphatase (Na＋-K＋-ATPase) in the PVN tissue, and corticosterone (CORT) in plasma were assayed by ELISA. Changes of the ultrastructure of PVN cells were observed by transmission electron microscope. After modeling, the rats' daytime and nocturnal locomotor activities were significantly increased and decreased, respectively (P<0.05), and the duration of exhausted swimming was considerably shortened in the model group compared with that of the normal control group (P<0.05). The expression level of AMPK protein in the PVN was obviously up-regulated (P<0.05), and the contents of Ac-CoA and Na＋-K＋-ATPase in PVN and CORT in plasma were markedly decreased in the model control group relevant to the normal group (P<0.05). After EA intervention, the increased daytime locomotion and the decreased nocturnal activities, the shortened duration of exhausted swimming, the up-regulated expression of AMPK, and the decreased Ac-CoA, Na＋-K＋-ATPase and CORT contents were all reversed in the EA treated rats relevant to those of the insomnia rats (all P<0.05). Moreover, ultrastructural observation showed mitochondrial swelling and disappearance of partial ribosomes in the plasma of PVN cells in the model group, while in the EA group, only mild swelling of some mitochondria was found, being with basically normal nuclear membrane, mitochondria, rough endoplasmic reticulum, Golgi complex and ribosomes. EA at HT7 and SP6 has a positive effect in improving insomnia and insomnia-induced fatigue in insomnia rats, which may be associated with its effects in restraining the expression of AMPK protein, and up-regulating the contents of Ac-CoA and Na＋-K＋-ATPase in PVN and CORT in plasma.

Aberrant DNA methylation of hypothalamic angiotensin receptor in prenatal programmed hypertension.

Maternal malnutrition, which causes prenatal exposure to excessive glucocorticoid, induces adverse metabolic programming, leading to hypertension in offspring. In offspring of pregnant rats receiving a low-protein diet or dexamethasone, a synthetic glucocorticoid, mRNA expression of angiotensin receptor type 1a (Agtr1a) in the paraventricular nucleus (PVN) of the hypothalamus was upregulated, concurrent with reduced expression of DNA methyltransferase 3a (Dnmt3a), reduced binding of DNMT3a to the Agtr1a gene, and DNA demethylation. Salt loading increased BP in both types of offspring, suggesting that elevated hypothalamic Agtr1a expression is epigenetically modulated by excessive glucocorticoid and leads to adult-onset salt-sensitive hypertension. Consistent with this, dexamethasone treatment of PVN cells upregulated Agtr1a, while downregulating Dnmt3a, and decreased DNMT3a binding and DNA demethylation at the Agtr1a locus. In addition, Dnmt3a knockdown upregulated Agtr1a independently of dexamethasone. Hypothalamic neuron-specific Dnmt3a-deficient mice exhibited upregulation of Agtr1a in the PVN and salt-induced BP elevation without dexamethasone treatment. By contrast, dexamethasone-treated Agtr1a-deficient mice failed to show salt-induced BP elevation, despite reduced expression of Dnmt3a. Thus, epigenetic modulation of hypothalamic angiotensin signaling contributes to salt-sensitive hypertension induced by prenatal glucocorticoid excess in offspring of mothers that are malnourished during pregnancy.

Regional distribution of hypothalamic paraventricular nucleus neurons activated by acute intermittent hypoxia

Upon returning to normoxic ventilation following exposure to acute intermittent hypoxia (AIH), sympathetic nerve activity (SNA) is observed to progressively increase – a response termed sympathetic long term facilitation (sLTF). We recently reported that inhibition of the hypothalamic paraventricular nucleus (PVN) prior to AIH attenuated post‐AIH sLTF in anesthetized rats. Although this study implicated PVN neuronal activity in post‐AIH sLTF, the specific PVN cell groups involved and their neurochemical phenotypes were not determined. Here we hypothesized that AIH activates parvocellular subregions of the PVN enriched with sympathetic regulatory neurons, and tested this using c‐Fos IHC to identify neurons activated by 10 cycles of AIH in awake rats. Separate groups were exposed to AIH in which FIO2 was reduced every 6 minutes to nadirs of 10%, 8%, or 6%, with each nadir lasting ~75 s. Compared to control rats exposed to sham AIH (n=6), the number of Fos+ nuclei in the medial parvocellular (MP) region of the PVN was progressively greater in rats exposed to AIH to 10% (367%, P&lt;0.01, n=4), 8% (566%, P&lt;0.01, n=5) and 6% (1054%, P&lt;0.01, n=4) FIO2. Fos+ counts increased in the dorsal parvocellular and central magnocellular regions of the PVN as well, but only in response to AIH to 6% FIO2 (P&lt;0.05, n=4–5). No level of AIH increased Fos+ nuclei in the caudally located lateral parvocellular area (P&gt;0.05, n=4–5). Neurochemical phenotypes of neurons activated by AIH to 8% FIO2 were examined by co‐immunostaining for corticotropin‐releasing hormone (CRH) or arginine vasopressin (AVP), which are stress‐responsive neurons enriched in the MP region. AIH to 8% FIO2 was selected for this analysis because it induced robust Fos expression without eliciting escape behavior as was observed in rats exposed to AIH to 6% FIO2. Whereas CRH+ neurons (~772) outnumbered AVP+ neurons (~50), the population size of each was similar between sham treated and AIH exposed rats. A significantly greater fraction of CRH+ neurons (~14%, P&lt;0.05) and AVP+ neurons (~13%, P&lt;0.05) was Fos+ in AIH exposed rats (n=4) compared to sham treated controls (CRH+, ~3%; AVP+, ~0%; n=4). Further analysis revealed that ~30% of AIH‐activated PVN neurons were CRH+ (110/371) while only ~2% (7/371) were AVP+. Thus a sizable fraction of AIH‐activated PVN neurons were CRH‐containing, while the neurochemical phenotype(s) of the majority of activated neurons (~68%) remains to be identified. These data suggest that responses to AIH are unlikely to be mediated by PVN‐AVP neurons, and raise the possibility that CRH neurons could contribute to endocrine and/or autonomic responses to AIH, including post‐AIH sLTF. Studies are needed to test the latter possibility and to define the role of AIH‐activated PVN neurons whose chemical phenotypes remain unknown. Determining intrinsic and synaptic mechanisms leading to AIH activation of PVN neurons could provide insight into mechanisms of persistent sympathetic activation in cardiovascular and metabolic diseases.Support or Funding InformationSupport: NIH HL088052 &amp; AHA 25710176 (GMT)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Central Orexin‐A in Restraint Stress‐induced Gastric Dysmotility in Rats

Stress exposure stimulates the production of corticotropin‐releasing factor (CRF) in hypothalamic paraventricular nucleus (PVN) which in turn influences gastrointestinal (GI) motor functions by altering autonomic outflow in brain. In rodents, acute restraint stress (ARS) has been shown to delay gastric emptying (GE) by impairing the postprandial coordination of of antro‐pyloric contractions through a CRF‐dependent pathway. Besides its well‐defined orexigenic function, the hypothalamic neuropeptide orexin‐A (OXA) is also known to alter GI motor functions through central autonomic pathways. OXA‐producing cells reside mainly in lateral hypothalamic area (LHA), while its receptor OX1R is found in PVN. Accumulating evidence indicate that OXA‐containing neurons send intra‐hypothalamic projections to the CRF‐producing cells in PVN. Furthermore, central administration of OXA has been shown to enhance CRF‐induced neuroendocrine functions suggesting that central OXA may contribute to the CRF‐mediated impairment of GI motor functions under stressed conditions. The aim of the present study was to investigate the involvement of orexin‐A in CRF‐induced gastric motor dysfunction under stressed conditions. Central (icv) drug administrations were performed through an injection cannula stereotaxically placed into the lateral ventricle. Solid GE was measured following an overnight fasting in non‐stressed (NS) and ARS‐loaded rats following central pretreatment of CRF and OXA antagonists α‐helical‐CRF9,41 (10 nmol, 5 μl) and SB‐334867 (100 nmol, 5 μl), respectively. For recording of gastric motility, two miniature strain gages were implanted onto the serosal surface of antrum and pylorus. Postprandial antro‐pyloric contractions were monitored in conscious and freely‐moving rats simultaneously with microdialysis. To perform microdialysis, a probe with high molecular cut‐off membrane was placed into the PVN. The intra‐PVN microinjection of vehicle or selective SB‐334867 (10 nmol, 100 nL) was performed prior to the perfusion of the probe with artificial cerebro‐spinal fluid (aCSF) containing high KCl (100 mmol/L). CRF concentrations in microdialysis samples were quantified by enzyme immunoassay. Compared with NS rats, ARS significantly (p&lt;0.01) delayed GE. The ARS‐induced delayed GE was partially attenuated both by pretreatment of SB‐334867 (p&lt;0.05) and α‐helical‐CRF9,41 (p&lt;0.05), whereas it was completely restored (p&lt;0.01) when both antagonists were administered together. Perfusion with high KCl‐containing aCSF significantly increased the CRF level in microdialysates which was significantly attenuated by intra‐PVN microinjection of SB‐334867. Perfusion of KCl impaired the coordination of postprandial antro‐pyloric contractions. Compared with vehicle‐injected rats, KCL‐induced alterations were remarkably abolished by SB‐334867. These results suggest that central OXA plays a role in stress‐induced alterations in gastric motor functions through OX1R. Therefore, OX1R antagonism appears to be a candidate for the treatment of the stress‐related gastric motor dysfunctions.

Effects of chronic restraint stress on social behaviors and the number of hypothalamic oxytocin neurons in male rats

Oxytocin (OXT) and vasopressin (AVP) are considered to be related to mammalian social behavior and the regulation of stress responses. The present study investigated the effects of chronic homotypic restraint stress (CHRS) on social behaviors and anxiety, as well as its repercussions on OXT- and AVP-positive neurons in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) nuclei in rat. Male Sprague-Dawley rats receiving CHRS were exposed to repeated restraint stress of 30min per day for 10days. Changes in social approach behaviors were evaluated with the three-chambered social approach task. Changes in anxiety-like behaviors were evaluated in the light-dark box test. The number of neurons expressing oxytocin and/or vasopressin in PVN and SON were examined by immunohistochemistry techniques. The results demonstrated that social approach was increased and anxiety was decreased following 10-day exposure to CHRS. Furthermore, the number of OXT-immunoreactive cells in PVN was increased significantly, whereas no change in SON was seen. The number of AVP immunoreactive cells either in PVN or SON was unaffected. The results of this study suggest that certain types of stress could be effective in the treatment of social dysfunction in persons with mental disorders such as autism, social anxiety disorder. The therapeutic effects may be mediated by changes in the function of OXT neurons in PVN.

Dexamethasone exposure affects paraventricular nucleus and pituitary corticotrophs in female rat fetuses: An unbiased stereological and immunohistochemical study.

The hypothalamic paraventricular nucleus (PVN) drives the stress response by activating the hypothalamo-pituitary-adrenal (HPA) axis, particularly vulnerable to glucocorticoid exposure during development. To evaluate the effects of fetal dexamethasone (Dx) exposure on the stereological features of PVN and HPA axis activity in female rat fetuses, pregnant rats received 0.5mg Dx/kg/b.w./day on days 16, 17 and 18 of pregnancy and 21-day-old fetuses were obtained; controls received the same volume of saline. In an unbiased stereological approach, Cavalieri's principle and an optical fractionator were used for estimating volume and total cell number of the PVN, respectively. The intensity of corticotropin-releasing hormone (CRH) immunoreactivity in the median eminence (ME) was determined by CRH optical density and the adrenocorticotropic hormone (ACTH) relative fluorescence signal intensity (RIF) in pituitary corticotrophs was measured using Image J. Significant reductions (p<0.05) in PVN volume and cell number were found in fetuses exposed to Dx. Additionally, CRH optical density in the ME and ACTH RIF (p<0.05) in the corticotrophs were decreased. The established results suggest that the reduced number of cells in the PVN after maternal Dx administration negatively affects the CRH content in the ME and the ACTH quantity in pituitary corticotrophs in near-term fetuses.

Rapid Nongenomic Glucocorticoid Actions in Male Mouse Hypothalamic Neuroendocrine Cells Are Dependent on the Nuclear Glucocorticoid Receptor.

Corticosteroids act classically via cognate nuclear receptors to regulate gene transcription; however, increasing evidence supports rapid, nontranscriptional corticosteroid actions via activation of membrane receptors. Using whole-cell patch clamp recordings in hypothalamic slices from male mouse genetic models, we tested for nongenomic glucocorticoid actions at glutamate and gamma aminobutyric acid (GABA) synapses in hypothalamic neuroendocrine cells, and for their dependence on the nuclear glucocorticoid receptor (GR). In enhanced green fluorescent protein-expressing CRH neurons of the paraventricular nucleus (PVN) and in magnocellular neurons of the PVN and supraoptic nucleus (SON), dexamethasone activated postsynaptic membrane-associated receptors and G protein signaling to elicit a rapid suppression of excitatory postsynaptic inputs, which was blocked by genetic deletion of type I cannabinoid receptors and a type I cannabinoid receptor antagonist. In magnocellular neurons, dexamethasone also elicited a rapid nitric oxide-dependent increase in inhibitory postsynaptic inputs. These data indicate a rapid, synapse-specific glucocorticoid-induced retrograde endocannabinoid signaling at glutamate synapses and nitric oxide signaling at GABA synapses. Unexpectedly, the rapid glucocorticoid effects on both excitatory and inhibitory synaptic transmission were lost with conditional deletion of GR in the PVN and SON in slices from a single minded-1-cre-directed conditional GR knockout mouse. Thus, the nongenomic glucocorticoid actions at glutamate and GABA synapses on PVN and SON neuroendocrine cells are dependent on the nuclear GR. The nuclear GR, therefore, is responsible for transducing the rapid steroid response at the membrane, or is either a critical component in the signaling cascade or regulates a critical component of the signaling cascade of a distinct membrane GR.

Coexpression analysis of nine neuropeptides in the neurosecretory preoptic area of larval zebrafish

The paraventricular nucleus (PVN) of the hypothalamus in mammals coordinates neuroendocrine, autonomic and behavioral responses pivotal for homeostasis and the stress response. A large amount of studies in rodents has documented that the PVN contains diverse neuronal cell types which can be identified by the expression of distinct secretory neuropeptides. Interestingly, PVN cell types often coexpress multiple neuropeptides whose relative coexpression levels are subject to environment-induced plasticity. Due to their small size and transparency, zebrafish larvae offer the possibility to comprehensively study the development and plasticity of the PVN in large groups of intact animals, yet important anatomical information about the larval zebrafish PVN-homologous region has been missing. Therefore we recently defined the location and borders of the larval neurosecretory preoptic area (NPO) as the PVN-homologous region in larval zebrafish based on transcription factor expression and cell type clustering. To identify distinct cell types present in the larval NPO, we also generated a comprehensive 3D map of 9 zebrafish homologs of typical neuropeptides found in the mammalian PVN (arginine vasopressin (AVP), corticotropin-releasing hormone (CRH), proenkephalin a (penka)/b (penkb), neurotensin (NTS), oxytocin (OXT), vasoactive intestinal peptide (VIP), cholecystokinin (CCK), and somatostatin (SST)). Here we extend this chemoarchitectural map to include the degrees of coexpression of two neuropeptides in the same cell by performing systematic pairwise comparisons. Our results allowed the subclassification of NPO cell types, and differences in variability of coexpression profiles suggest potential targets of biochemical plasticity. Thus, this work provides an important basis for the analysis of the development, function, and plasticity of the primary neuroendocrine brain region in larval zebrafish.

Female protection from slow-pressor effects of angiotensin II involves prevention of ROS production independent of NMDA receptor trafficking in hypothalamic neurons expressing angiotensin 1A receptors.

Renin–angiotensin system overactivity, upregulation of postsynaptic NMDA receptor function, and increased reactive oxygen species (ROS) production in the hypothalamic paraventricular nucleus (PVN) are hallmarks of angiotensin II (AngII)-induced hypertension, which is far more common in young males than in young females. We hypothesize that the sex differences in hypertension are related to differential AngII-induced changes in postsynaptic trafficking of the essential NMDA receptor GluN1 subunit and ROS production in PVN cells expressing angiotensin Type 1a receptor (AT1aR). We tested this hypothesis using slow-pressor (14-day) infusion of AngII (600 ng/kg/min) in mice, which elicits hypertension in males but not in young females. Two-month-old male and female transgenic mice expressing enhanced green fluorescent protein (EGFP) in AT1aR-containing cells were used. In males, but not in females, AngII increased blood pressure and ROS production in AT1aR–EGFP PVN cells at baseline and following NMDA treatment. Electron microscopy showed that AngII increased cytoplasmic and total GluN1–silver-intensified immunogold (SIG) densities and induced a trend toward an increase in near plasmalemmal GluN1–SIG density in AT1aR–EGFP dendrites of males and females. Moreover, AngII decreased dendritic area and diameter in males, but increased dendritic area of small (<1 µm) dendrites and decreased diameter of large (>1 µm) dendrites in females. Fluorescence microscopy revealed that AT1aR and estrogen receptor β do not colocalize, suggesting that if estrogen is involved, its effect is indirect. These data suggest that the sexual dimorphism in AngII-induced hypertension is associated with sex differences in ROS production in AT1aR-containing PVN cells but not with postsynaptic NMDA receptor trafficking.

Hypothalamic vasopressin systems are more sensitive to the long term effects of social defeat in males versus females.

Vasopressin signaling has important effects on the regulation of social behaviors and stress responses, and is considered a promising pathway to target for new therapeutics of stress-induced psychiatric disorders. Although there is evidence for sex differences in the behavioral effects of arginine vasopressin (AVP), few data have directly compared the effects of stress on endogenous AVP signaling in males and females. We used California mice (Peromyscus californicus) to study the short and long term effects of social defeat stress on AVP immunoreactive cells in the paraventricular nucleus (PVN) and the posteromedial bed nucleus of the stria terminalis (BNSTmp). Acute exposure to defeat increased AVP/c-fos cells in the PVN and SON of both males and females. In contrast, there were sex differences in the long term effects of defeat. Males but not females exposed to defeat had less avp mRNA in the PVN, and in two experiments defeat reduced the number of AVP positive cells in the caudal PVN of males but not females. Interestingly, during relatively benign social encounters with a target mouse, there was a rapid decrease in AVP percent staining (including cell bodies and fibers) in the PVN of males but not females. Defeat reduced AVP percent staining in males, but did not block the socially induced decrease in percent staining. When mice were tested in resident-intruder tests, males exposed to defeat were no less aggressive than control males whereas aggression was abolished in females. However, bouts of aggression were positively correlated with the number of AVP neurons in the BNSTmp of control males but not stressed males, suggesting that different mechanisms mediate aggression in control and stressed males. These data show that while acute AVP responses to defeat are similar in males and females, the long term effects of defeat on AVP are stronger in males.