Neurons In Hypothalamic Paraventricular Nucleus Research Articles

Maternal Limosilactobacillus reuteri rescues social and emotional recognition behavior in an environmental mouse model of autism

Introduction: Polybrominated diphenyl ethers (PBDEs) are thyroid disrupting chemicals with adverse neurodevelopmental effects in children. Our group has previously shown that PBDEs produce autism (ASD)-like traits (Kozlova et al. 2022 Arch Tox; PMID: 34687351) in combination with reduced hypothalamic oxytocin expression in developmentally exposed female mice offspring. Limosilactobacillus reuteri (LR) is a beneficial probiotic that can increase oxytocin and thyroid hormones (THs) in other ASD mouse models. Therefore, we hypothesized that LR would ameliorate PBDE-induced deficits in social behavior, oxytocin and thyroid status. Methods: C57BL/6N dams (n=8/group) were dosed with a commercial mixture of PBDE congeners, DE-71, at 0.1 mg/kg/d (DE-71), or corn oil vehicle control (VEH/CON) during preconception, gestation and lactation). Dams were gavaged with L. reuteri (LR) ATCC-PTA-6475 at 107-108 CFU/mL (gift of Biogaia, Sweden). Cohort 1 pups (C1) received LR via dam until weaning (PND 21). Cohort 2 pups (C2) continued to receive gavage of LR post-weaning, daily, through 6 mo of age. Results: Fecal microbiome analysis confirmed colonization of LR in dams after 25 doses and their offspring at postnatal day 22 and 40. Beta diversity PCoA using unweighted Unifrac revealed differences in microbial communities in 6 month old female (p<.05-.01) and male offspring (apparent) between DE-71 and VEH/CON (C2). LR treatment normalized DE-71-induced abnormal social novelty preference (30 min retention) in adult male (p<.01) and females (p<.05) and long-term social recognition memory (24 h retention) in adult C2 females (p<.001). Using an emotional recognition task DE-71-exposed C2 male and female offspring were unable to distinguish between emotional state of stimulus mice but LR supplementation normalized the DE-71-induced deficits in C2 males (p<.01) and females (p<.05). On a marble burying test DE-71 exposed offspring showed abnormally elevated scores compared to VEH/CON, which were normalized by LR treatment only in C1 females (p<.05). In females, maternal LR treatment ameliorated DE-71-induced locomotor hyperactivity in C1. Since social behavior involves discrimination between social odors we also investigated the effects of DE-71 and LR treatment on olfactory discrimination ability. LR treatment ameliorated DE-71 effects compared to VEH/CON, i.e., reduced dishabituation from social odor 1 to 2 in males and female (p<.0001). Single molecule in situ hybridization performed on paraventricular hypothalamic nucleus (PVN) sections revealed upregulation of thyroid hormone transporter monocarboxylate transporter 8 (Mct8) and downregulation of iodothyronine deiodinase 3 (Dio3) on oxytocin-positive neurons in DE-71 vs VEH/CON in C2 females. Dio3 expression was normalized in DE-71+LR. Conclusions: These results indicate that early life exposure to PBDEs alters thyroid hormone system genes in PVN neurons that express oxytocin, suggesting that PBDEs may alter ASD-relevant behavior via brain thyroid signaling. In addition, maternal LR supplementation provides normalization in a sex-dependent manner. This work is supported by a Danone North America Gut Microbiome, Yogurt and Probiotics Fellowship Grant and a University of California President's Pre-Professoriate Fellowship to E.V.K and an NIH/NIGMS R35GM124724 to A.H. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Dexmedetomidine Inhibits Paraventricular Corticotropin-releasing Hormone Neurons that Attenuate Acute Stress-induced Anxiety-like Behavior in Mice.

Dexmedetomidine has repeatedly shown to improve anxiety, but the precise neural mechanisms underlying this effect remain incompletely understood. This study aims to explore the role of corticotropin-releasing hormone-producing hypothalamic paraventricular nucleus (CRHPVN) neurons in mediating the anxiolytic effects of dexmedetomidine. A social defeat stress mouse model was used to evaluate the anxiolytic effects induced by dexmedetomidine through the elevated plus maze, open-field test, and measurement of serum stress hormone levels. In vivo Ca2+ signal fiber photometry and ex vivo patch-clamp recordings were used to determine the excitability of CRHPVN neurons and investigate the specific mechanism involved. CRHPVN neuron modulation was achieved through chemogenetic activation or inhibition. Compared with saline, dexmedetomidine (40 µg/kg) alleviated anxiety-like behaviors. Additionally, dexmedetomidine reduced CRHPVN neuronal excitability. Chemogenetic activation of CRHPVN neurons decreased the time spent in the open arms of the elevated plus maze and in the central area of the open-field test. Conversely, chemogenetic inhibition of CRHPVN neurons had the opposite effect. Moreover, the suppressive impact of dexmedetomidine on CRHPVN neurons was attenuated by the α2-receptor antagonist yohimbine. The results indicate that the anxiety-like effects of dexmedetomidine are mediated via α2-adrenergic receptor-triggered inhibition of CRHPVN neuronal excitability in the hypothalamus.

Paraventricular Mast Cell-Derived Histamine Activates CRH Neurons to Mediate Adult Visceral Hypersensitivity Induced by Neonatal Maternal Separation.

Neonatal maternal separation (NMS) is an early-life stress (ELS) that can result in adult visceral hypersensitivity, which is usually manifested as chronic visceral pain. Although mast cells and corticotropin-releasing hormone (CRH) neurons are involved in stress response, whether there is an interaction between mast cells and CRH neurons in hypothalamic paraventricular nucleus (PVN) during the ELS-induced visceral hypersensitivity remains elusive. Herein, we established an NMS model by separating neonatal mice from their mothers, and observed that these mice presented visceral hypersensitivity in adulthood, as indicated by elevated abdominal withdrawal reflex and lowered visceral pain threshold. The NMS-induced adult visceral hypersensitivity was accompanied by activation of mast cells and CRH neurons in PVN. Also, NMS increased the histamine content (an inflammatory mediator mainly released by mast cells) and histamine H2 receptor (H2R) expression of CRH neurons in PVN. Remarkably, intra-PVN administration with mast cell stabilizer attenuated the NMS-induced CRH neuronal activation and adult visceral pain, while histamine administration showed the opposite effects. Moreover, intra-PVN injection with H2R antagonist alleviated the NMS-induced CRH neuronal activation, PKA and CREB phosphorylation, and importantly, adult visceral pain. Together, our findings revealed a role of an interaction between paraventricular mast cells and CRH neurons in NMS-induced adult visceral hypersensitivity, thereby providing a perspective for the management of visceral pain.

Energy deficiency promotes rhythmic foraging behavior by activating neurons in paraventricular hypothalamic nucleus.

Dysregulation of feeding behavior leads to a variety of pathological manifestations ranging from obesity to anorexia. The foraging behavior of animals affected by food deficiency is not fully understood. Home-Cage system was used to monitor the behaviors. Immunohistochemical staining was used to monitor the trend of neuronal activity. Chemogenetic approach was used to modify neuronal activity. We described here a unique mouse model of foraging behavior and unveiled that food deprivation significantly increases the general activities of mice with a daily rhythmic pattern, particularly foraging behavior. The increased foraging behavior is potentiated by food cues (mouthfeel, odor, size, and shape) and energy deficit, rather than macronutrient protein, carbohydrate, and fat. Notably, energy deficiency increases nocturnal neuronal activity in paraventricular hypothalamic nucleus (PVH), accompanying a similar change in rhythmic foraging behavior. Activating neuronal activity in PVH enhances the amplitude of foraging behavior in mice. Conversely, inactivating neuronal activity in PVH decreases the amplitude of foraging behavior and impairs the rhythm of foraging behavior. These results illustrate that energy status and food cues regulate the rhythmic foraging behavior via PVH neuronal activity. Understanding foraging behavior provides insights into the underlying mechanism of eating-related disorders.

Kisspeptin neuron projections to oxytocin neurons are not necessary for parturition in the mouse

Oxytocin is synthesized by hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) neurons and is released from the posterior pituitary gland to trigger uterine contractions during parturition. In rats, oxytocin neuron innervation by periventricular nucleus (PeN) kisspeptin neurons increases over pregnancy and intra-SON kisspeptin administration excites oxytocin neurons only in late pregnancy. To test the hypothesis that kisspeptin neurons excite oxytocin neurons to trigger uterine contractions during birth in C57/B6J mice, double-label immunohistochemistry for kisspeptin and oxytocin first confirmed that kisspeptin neurons project to the SON and PVN. Furthermore, kisspeptin fibers expressed synaptophysin and formed close appositions with oxytocin neurons in the mouse SON and PVN before and during pregnancy. Stereotaxic viral delivery of caspase-3 into the AVPV/PeN of Kiss-Cre mice before mating reduced kisspeptin expression in the AVPV, PeN, SON and PVN by > 90% but did not affect the duration of pregnancy or the timing of delivery of each pup during parturition. Therefore, it appears that AVPV/PeN kisspeptin neuron projections to oxytocin neurons are not necessary for parturition in the mouse.

Trigeminal nerve stimulation restores hippocampal dopamine deficiency to promote cognitive recovery in traumatic brain injury

Cognitive impairment (CI) is a common neurological disease resulting from traumatic brain injury (TBI). Trigeminal nerve stimulation (TNS) is an emerging, non-invasive, and effective neuromodulation therapy especially for patients suffering from brain function disorders. However, the treatment and recovery mechanisms of TNS remain poorly understood. By using combined advanced technologies, we revealed here that the neuroprotective potential of TNS to improve CI caused by TBI. The study results found that 40 Hz TNS treatment has the ability to improve CI in TBI mice and communicates with central nervous system via the trigeminal ganglion (TG). Transsynaptic virus experiments revealed that TG is connected to the hippocampus (HPC) through the corticotropin-releasing hormone (CRH) neurons of paraventricular hypothalamic nucleus (PVN) and the dopamine transporter (DAT) neurons of substantia nigra pars compacta/ventral tegmental area (SNc/VTA). Mechanistically, the data showed that TNS can increase the release of dopamine in the HPC by activating the following neural circuit: TG→CRH+ PVN→DAT+ SNc/VTA → HPC. Bulk RNA sequencing confirmed changes in the expression of dopamine-related genes in the HPC. This work preliminarily explains the efficacy and mechanism of TNS and adds to the increasing evidence demonstrating that nerve stimulation is an effective method to treat neurological diseases.

Hypothalamic TRH mediates anorectic effects of serotonin in rats.

Among the modulatory functions of thyrotropin-releasing hormone (TRH), an anorectic behavior in rodents is observed when centrally injected. Hypothalamic PVN neurons receive serotonergic inputs from dorsal raphe nucleus and express serotonin (5HT) receptors such as 5HT1A, 5HT2A/2C, 5HT6, which are involved in 5HT-induced feeding regulation. Rats subjected to dehydration-induced anorexia (DIA) model show increased PVN TRH mRNA expression, associated with their decreased food intake. We analyzed whether 5HT input is implicated in the enhanced PVN TRH transcription that anorectic rats exhibit, given that 5HT increases TRH expression and release when studied in vitro By using mHypoA-2/30 hypothalamic cell cultures, we found that 5HT stimulated TRH mRNA, pCREB and pERK1/2 levels. By inhibiting basal PKA or PKC activities or those induced by 5HT, pCREB or pERK1/2 content did not increase suggesting involvement of both kinases in their phosphorylation. 5HT effect on TRH mRNA was not affected by PKA inhibition, but it diminished in the presence of PKCi suggesting involvement of PKC in 5HT-induced TRH increased transcription. This likely involves 5HT2A/2C and the activation of alternative transduction pathways than those studied here. In agreement with the in vitro data, we found that injecting 5HT2A/2C antagonists into the PVN of DIA rats reversed the increased TRH expression of anorectic animals, as well as their decreased food intake; also, the agonist reduced food intake of hungry restricted animals along with elevated PVN TRH mRNA levels. Our results support that the anorectic effects of serotonin are mediated by PVN TRH in this model.Significance statementInteraction between brain peptides and neurotransmitters' pathways regulates feeding behavior, but when altered it could lead to the development of eating disorders, such as anorexia. An abnormal increased TRH expression in hypothalamic PVN results in dehydration-induced anorectic rats, associated to their low food intake. The role of neurotransmitters in that alteration is unknown, and since serotonin inhibits feeding and has receptors in PVN, we analyzed its participation in increasing TRH expression and reducing feeding in anorectic rats. By antagonizing PVN serotonin receptors in anorectic rats, we identify decreased TRH expression and increased feeding, suggesting that the anorectic effects of serotonin are mediated by PVN TRH. Elucidating brain networks involved in feeding regulation would help to design therapies for eating disorders.

Central Nucleus of Amygdala Mediate Pressor Response Elicited by Microinjection of Angiotensin II into the Parvocellular Paraventricular Nucleus in Rats.

Background: The Paraventricular Hypothalamic Nucleus (PVN) coordinates autonomic and neuroendocrine systems to maintain homeostasis. Microinjection of angiotensin II (AngII) into the PVN has been previously shown to produce pressor and bradycardia responses. Anatomical evidence has indicated that a substantial proportion of PVN neurons is connected with the neurons in the central amygdala (CeA). The present study aimed to examine the possible contribution of the CeA in cardiovascular responses evoked by microinjection of AngII into the parvocellular portion of PVN (PVNp) before and after microinjection of cobalt chloride (CoCl2) into the CeA.Methods: The experiments were conducted at the Department of Physiology of Shiraz University of Medical Sciences, from April 2019 to November 2019. There were two groups of 21 eight-week-old urethane anesthetized male rats, namely saline (n=9 rats) and AngII (n=12 rats) groups. Drugs (100 nL) were microinjected via a single-glass micropipette into the PVNp and CeA. Their blood pressure (BP) and heart rate (HR) were recorded throughout the experiments. The mean arterial pressure (MAP) and heart rate (HR) were compared to the pre-injection values using paired t test, and to those of the saline group using independent t test.Results: Microinjection of AngII into the PVNp produced pressor response (P<0.0001) with no significant changes in HR (P=0.70). Blockade of CeA with CoCl2 attenuated the pressor response to microinjection of AngII into the PVNp (P<0.001).Conclusion: In the PVNp, Ang II increased the rats’ blood pressure. This response was in part mediated by the CeA. Our study suggested that these two nuclei cooperate to perform their cardiovascular functions.

A short note on oxytocin and stress attenuation.

Stress is integral part of life and it initiates appropriate response at times of adversities to promise survival. Stress could be either physiological or psychogenic. Stress is often psychogenic in nature and it induces the release of cortisol from adrenal cortex into circulation by activating Hypo thalamo-pituitary-adrenal axis (HPA). Cortisol thus released mediates the stress response by its catabolic effects to enhance the activity of vital organs during emergency. However, prolonged activation of the HPA axis can lead to physical and mental illness as an outcome of persistent stress. Nature has bestowed the biological system with an array of endogenous mechanisms to buffer stress. Oxytocin, a nano-peptide released by the magno-cellular neurons of hypothalamic paraventricular nucleus (PVN) is an efficient stress buffering neuro-peptide. This hormone mediates many physiological and behavioural functions get released during stress. It attenuates the stress axis initiated by the release of corticotropin releasing hormone (CRH) from the parvocellular neurons of the same hypothalamic nucleus. Oxytocin released by PVN exerts an inhibitory effect on the release of CRH by down-regulating the expression of the gene that transcribes for this hypothalamic hormone. Thus, it inhibits the release of adreno cotico trophic hormone (ACTH) and cortisol, exerting an overall suppressive modulation of the stress axis and attenuates stress.

Experiences affect social behaviors via altering neuronal morphology and oxytocin system

Life experiences, such as maternal deprivation (MD) and environment enrichment (EE), affect social behaviors in the adult. But, the underlying mechanism remains unclear. In the present study, we determined whether neonatal MD induces social deficits, whether postweaning EE restores the deficits, and their effects on neuron morphology and oxytocin (OT)-oxytocin receptor (OTR) system. We found that MD induced repetitive behavior and deficits in novel object recognition and sociability, and EE alleviated these deficits. MD decreased oxytocinergic neurons in the magnocellular hypothalamic paraventricular nucleus (mPVH), which was parallel to the increased OTR levels and dendritic branches of projection neurons in the basolateral amygdala (BLA). EE increased the OTR levels in the prelimbic cortex (PL) and the oxytocinergic neurons in the parvocellular PVH (vPVH), which were parallel to the increased dendritic branches of small pyramidal neurons in the PL and synaptic connections marked with synaptophysin and postsynaptic density protein 95 in the BLA and PL. Together, the results suggest that postweaning EE alleviates the social impairments induced by neonatal MD and OT-OTR system are experience-dependent and associated with social behaviors and neuron morphology.

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.

Sex and age influence gonadal steroid hormone receptor distributions relative to estrogen receptor β-containing neurons in the mouse hypothalamic paraventricular nucleus.

Within the hypothalamic paraventricular nucleus (PVN), estrogen receptor (ER) β and other gonadal hormone receptors play a role in central cardiovascular processes. However, the influence of sex and age on the cellular and subcellular relationships of ERβ with ERα, G-protein ER (GPER1), as well as progestin and androgen receptors (PR and AR) in the PVN is uncertain. In young (2- to 3-month-old) females and males, ERβ-enhanced green fluorescent protein (EGFP) containing neurons were approximately four times greater than ERα-labeled and PR-labeled nuclei in the PVN. In subdivisions of the PVN, young females, compared to males, had: (1) more ERβ-EGFP neurons in neuroendocrine rostral regions; (2) fewer ERα-labeled nuclei in neuroendocrine and autonomic projecting medial subregions; and (3) more ERα-labeled nuclei in an autonomic projecting caudal region. In contrast, young males, compared to females, had approximately 20 times more AR-labeled nuclei, which often colocalized with ERβ-EGFP in neuroendocrine (approximately 70%) and autonomic (approximately 50%) projecting subregions. Ultrastructurally, in soma and dendrites, PVN ERβ-EGFP colocalized primarily with extranuclear AR (approximately 85% soma) and GPER1 (approximately 70% soma). Aged (12- to 24-month-old) males had more ERβ-EGFP neurons in a rostral neuroendocrine subregion compared to aged females and females with accelerated ovarian failure (AOF) and in a caudal autonomic subregion compared to post-AOF females. Late-aged (18- to 24-month-old) females compared to early-aged (12- to 14-month-old) females and AOF females had fewer AR-labeled nuclei in neuroendrocrine and autonomic projecting subregions. These findings indicate that gonadal steroids may directly and indirectly influence PVN neurons via nuclear and extranuclear gonadal hormone receptors in a sex-specific manner.

Sympathoexcitatory input from hypothalamic paraventricular nucleus neurons projecting to rostral ventrolateral medulla is enhanced after myocardial infarction.

Elevated sympathetic vasomotor tone seen in heart failure (HF) may involve dysfunction of the hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN-RVLM neurons). This study aimed to elucidate the role of PVN-RVLM neurons in the maintenance of resting renal sympathetic nerve activity (RSNA) after myocardial infarction (MI). In male rats, the left coronary artery was chronically ligated to induce MI. The rats received PVN microinjections of an adeno-associated viral (AAV) vector encoding archaerhodopsin T (ArchT) with the reporter yellow fluorescence protein (eYFP). The ArchT rats had abundant distributions of eYFP-labeled, PVN-derived axons in the RVLM. In anesthetized ArchT rats with MI (n = 12), optogenetic inhibition of the PVN-RVLM pathway achieved by 532-nm-wavelength laser illumination to the RVLM significantly decreased RSNA. This effect was not found in sham-operated ArchT rats (n = 6). Other rat groups received RVLM microinjections of a retrograde AAV vector encoding the red light-drivable halorhodopsin Jaws (Jaws) with the reporter green fluorescence protein (GFP) and showed expression of GFP-labeled cell bodies and dendrites in the PVN. Laser illumination of the PVN at a 635 nm wavelength elicited significant renal sympathoinhibition in Jaws rats with MI (n = 9) but not in sham-operated Jaws rats (n = 8). These results indicate that sympathoexcitatory input from PVN-RVLM neurons is enhanced after MI, suggesting that this monosynaptic pathway is part of the central nervous system circuitry that plays a critical role in generating an elevated sympathetic vasomotor tone commonly seen with HF.NEW & NOTEWORTHY Using optogenetics in rats, we report that sympathoexcitatory input from hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla is enhanced after myocardial infarction. It is suggested that this monosynaptic pathway makes up a key part of central nervous system circuitry underlying sympathetic hyperactivation commonly seen in heart failure.

Anatomical Characterization Of Gαi2 Expressing Hypothalamic Paraventricular Nucleus Neurons

BackgroundHypertension is a global health burden, and excess dietary salt is an established cause of hypertension. The hypothalamic paraventricular nucleus (PVN) is an essential component of the neuronal network that regulates sodium homeostasis and blood pressure. Previously, we have shown PVN specific GPCR‐coupled Gαi2 subunit proteins are essential to counter the development of salt‐sensitive hypertension by mediating the sympathoinhibitory and natriuretic responses to increased dietary sodium intake to maintain sodium homeostasis and normotension. However, the cellular localization and identity of PVN Gαi2 expressing neurons is currently unknown.ObjectiveTo determine the anatomical characterization of Gαi2 expressing PVN neurons.MethodologyThree‐month‐old male (n=3) and female (n=3) Sprague Dawley rats were subjected to transcardial perfusion, and brains were post fixed in 4% paraformaldehyde and 30% sucrose. 20μm frozen brain slices were collected. Using in‐situ hybridization (ACD RNAscope kits) neuronal expression of Gαi2, vGLUT2, GAD67, corticotropin‐releasing hormone (CRH), tyrosine hydroxylase (TH), oxytocin, and arginine vasopressin (AVP) in hypothalamic PVN neurons was determined. The visualization and evaluation of co‐localization of Gαi2 expressing PVN parvocellular neurons with vGLUT2, GAD67, CRH, TH, oxytocin and AVP was done using a Keyence Bz‐9000e microscope and Image J respectively.ResultsWe observed no differences among male and female rats, therefore, all data is presented as n=6. Our data show that neurons containing Gαi2 mRNA are localized in parvocellular but not magnocellular region of hypothalamic PVN. In the parvocellular region Gαi2 mRNA co‐localized with 87.5 ± 3.0% of GABA expressing neurons and 25.1 ± 2.7 % of vGLUT2 neurons, signifying that the Gαi2 mRNA is highly co‐localized with GABAergic neurons. Additionally, Gαi2 co‐localized with 76.3 ± 5.2% of CRH neurons and 33.5 ± 1.8% of TH expressing parvocellular neurons. We observed no localization of Gαi2 neurons with oxytocin or AVP expressing magnocellular neurons.ConclusionGiven 1) our prior data demonstrate PVN Gαi2 proteins play a critical role in mediating sympathoinhibition and, 2) that the GABAB receptor signals predominantly through a Gαi2 pathway we speculate, given the high co‐localization of Gαi2 mRNA with GABAergic neurons, that Gαi2 proteins modulate GABAergic signaling to impact sympathetic outflow and blood pressure.Support or Funding InformationT32 HL007224 to PCR01 HL139867 to RDWR01 HL141406 to RDWR56 AG057687 to RDW

Integration of energy homeostasis and stress by parvocellular neurons in rat hypothalamic paraventricular nucleus.

Central regulation of energy homeostasis and stress are believed to be reciprocally regulated, i.e. excessive food intake suppresses, while prolonged hunger exacerbates, stress responses in vivo. This relationship may be mediated by neuroendocrine parvocellular corticotropin-releasing hormone (CRH) neurons in the hypothalamic paraventricular nucleus that receive both stress- and feeding-related input. We find that hunger strongly and selectively potentiates, while re-feeding suppresses, a cellular analogue of a stress response induced by acute glucopenia in CRH neurons in rat hypothalamic slices. Neuronal activation in response to glucopenia was mediated synaptically, via the relative enhancement of glutamate over GABA input. These results illustrate how acute stress responses may be initiated in vivo and show that it is reciprocally integrated with energy balance via local hypothalamic mechanisms acting at the level of CRH neurons and their afferent terminals. Increased food intake is a common response to help cope with stress, implying the existence of a previously postulated but imperfectly understood, inverse relationship between the regulation of feeding and stress. We have identified components of the neural circuitry that can integrate these homeostatic responses. Prior fasting (∼24h) potentiates, and re-feeding suppresses, excitatory responses to acute glucopenia in about half of the corticotropin releasing hormone (CRH)-expressing, putatively neurosecretory, stress-related neurons in the paraventricular nucleus of the hypothalamus studied. Glucoprivation stress ex vivo resulted from a preferential relative increase in excitatory (glutamatergic) over inhibitory (GABAergic) inputs. Putative preautonomic cells were less sensitive to fasting, and showed a predominant inhibition to acute glucopenia. We conclude that hunger may sensitize hypothalamic stress responses by acting via local mechanisms, at the level of CRH neurons and their presynaptic inputs. Those mechanisms involve neither presynaptic ATP-sensitive potassium channels nor postsynaptic ATP levels.

Hypothalamic paraventricular nucleus neurons activated by estrogen GPER1 receptors promote anti-inflammation effects in the early stage of colitis.

The hypothalamus-pituitary-adrenal (HPA) axis is known to mediate gut-brain interaction, and the pathological inflammatory process in the intestine can induce HPA axis involved 'fight or flight' response to suppress or facilitate intestinal inflammation. Hypothalamic paraventricular nucleus (PVN) neurons are responsible for controlling the HPA axis activity, but their exact role in modulating intestinal inflammation remains unclear. In this study, we used the dextran sulfate sodium (DSS)-induced mice colitis model, gene editing, and RNA interference to determine the effects of PVN neurons on intestinal inflammation. We found that at the early stage (third day) after DSS treatment, there was a mild inflammation in the colorectal area and an increased neuron activation in the PVN but not in the adjacent area. At the same time, ~80% of activated PVN neurons also expressed novel estrogen GPER1 receptor. The colitis noticeably worsened in GPER1-knockout mice and local PVN GPER1-knockdown mice. These results indicated that PVN GPER1 positive neurons potentially have a protective function during the early stages of DSS-induced colitis, and this may be a mechanism by which the central nervous system attempts to suppress intestinal inflammation to achieve self-protection.

Relay of peripheral oxytocin to central oxytocin neurons via vagal afferents for regulating feeding

Oxytocin (Oxt), a neurohormone synthesized in the neurons of hypothalamic paraventricular nucleus (PVN) and supraoptic nucleus induces milk-ejection and uterine contraction and regulates social behavior, stress responses, memory and food intake. Peripheral (intraperitoneal and subcutaneous) infusion of Oxt decreases food intake and body weight in obese animals via mechanisms involving vagal afferent nerves and in obese subjects when administered nasally. Peripherally injected and intracerebroventricularly injected Oxt inhibit food intake to similar extent and with similar time course. Thus, peripheral Oxt mimics the effects of central Oxt, however, underlying mechanisms are unclear. In the present study we explored whether intraperitoneal Oxt activates Oxt neurons in PVN via vagal afferents and whether this pathway is linked to inhibition of feeding. We here show that intraperitoneal Oxt injection induces c-Fos expression in PVN largely in Oxt neurons and inhibits food intake, and these effects are blunted by subdiaphragmatic vagotomy. The intraperitoneal Oxt-induced inhibition of food intake was blunted in Oxt KO mice, by intracerebroventricular injection of Oxt receptor antagonist, and by vagotomy. These results demonstrate that intraperitoneal Oxt injection activates PVN Oxt neurons via vagal afferent nerves, thereby inhibiting food intake. This vagal afferents-mediated Oxt's peripheral-to-central coupling may serve to promote satiety and possibly a series of neural functions of Oxt and to treat their disorders.

Exercise training increases GAD65 expression, restores the depressed GABAA receptor function within the PVN and reduces sympathetic modulation in hypertension.

GABAergic inhibitory input within the paraventricular hypothalamic nucleus (PVN) plays a key role in restraining sympathetic outflow. Although experimental evidence has shown depressed GABAA receptor function plus sympathoexcitation in hypertension and augmented GABA levels with reduced sympathetic activity after exercise training (T), the mechanisms underlying T‐induced effects remain unclear. Here we investigated in T and sedentary (S) SHR and WKY: (1) time‐course changes of hemodynamic parameters and PVN glutamic acid decarboxylase (GAD) isoforms’ expression; (2) arterial pressure (AP) and heart rate (HR) responses, sympathetic/parasympathetic modulation of heart and vessels and baroreflex sensitivity to GABAA receptor blockade within the PVN. SHR‐S versus WKY‐S exhibited higher AP and HR, increased sympathetic reduced parasympathetic modulation, smaller baroreflex sensitivity, and reduced PVN GAD65 immunoreactivity. SHR‐T and WKY‐T showed prompt maintained increase (2–8 weeks) in GAD65 expression (responsible for GABA vesicular pool synthesis), which occurred simultaneously with HR reduction in SHR‐T and preceded MAP fall in SHR‐T and resting bradycardia in WKY‐T. There was no change in GAD67 expression (mainly involved with GABA metabolic pool). Resting HR in both groups and basal MAP in SHR were negatively correlated with PVN GAD65 expression. Normalized baroreflex sensitivity and autonomic control observed only in SHR‐T were due to recovery of GABAA receptor function into the PVN since bicuculline administration abolished these effects. Data indicated that training augments in both groups the expression/activity of GABAergic neurotransmission within presympathetic PVN neurons and restores GABAA receptors′ function specifically in the SHR, therefore strengthening GABAergic modulation of sympathetic outflow in hypertension.

Central nervous regulatory mechanism of myocardial ischemia-reperfusion injury in rats: excitability of neurons in hypothalamic paraventricular nucleus

Objective To evaluate the relationship between excitability of neurons in hypothalamic paraventricular nucleus(PVN) and central nervous regulatory mechanism of myocardial ischemia-reperfusion(I/R) injury in rats. Methods Clean-grade healthy adult male Sprague-Dawley rats, in which PVN catheters were successfully implanted, aged 6-8 weeks, weighing 260-300 g, were divided into 4 groups(n=6 each) using a random number table method: sham operation group(S group), myocardial I/R group(I/R group), γ-aminobutyric acid group(GABA group) and L-glutamic acid group(L-Glu group). Myocardial ischemia was induced by occlusion of the left anterior descending coronary artery for 30 min followed by 120-min reperfusion. Normal saline 2.4 μl/h was infused for 50 min via the PVN catheter starting from 10 min before ischemia in S and I/R groups. GABA 30 μmol/L and L-glutamic acid 30 μmol/L were infused for 50 min via the PVN catheter at a rate of 2.4 μl/h starting from 10 min before ischemia in GABA and L-Glu groups. The heart rate(HR) and mean arterial pressure(MAP) were recorded at 10 min before ischemia(T0), beginning of ischemia(T1), 30 min of ischemia(T2) and 120 min of reperfusion(T3). Rate-pressure product(RPP) was calculated. Blood samples were collected at T3 for determination of plasma cardiac troponin I(cTnI) concentrations by chemiluminescence assay. Rats were then sacrificed and myocardial specimens were obtained for measurement of myocardial infarct size(IS) and area at risk(AAS), and IS/AAR percentage was calculated. PVN tissues were taken to detect the expression of c-fos by Western blot. Results No myocardial infarction was found in group S, and myocardial infarction was marked in the other three groups. Compared with group S, the plasma cTnI concentrations were significantly increased, and the expression of c-fos in PVN was up-regulated in I/R, GABA and L-Glu groups(P<0.05), and MAP, HR and RPP were significantly decreased at T1-3 in I/R and GABA groups and at T3 in group L-Glu(P<0.05). Compared with group I/R, IS and IS/AAR percentage were significantly decreased, the plasma cTnI concentrations were decreased, the expression of c-fos in PVN was down-regulated, and HR, MAP and RPP were decreased at T1, 2 in group GABA, and IS and IS/AAR percentage were significantly increased, the plasma cTnI concentrations were increased, the expression of c-fos in PVN was up-regulated, and HR, MAP and RPP were increased at T1, 2 in group L-Glu(P<0.05). Conclusion Excitability of neurons in hypothalamic PVN is involved in central nervous regulatory mechanism of myocardial I/R injury in rats: decreased excitability can attenuate myocardial I/R injury and increased excitability aggravates myocardial I/R injury. Key words: Paraventricular hypothalamic nucleus; Neurons; Myocardial reperfusion injury

Electrophysiological Effects of Ghrelin in the Hypothalamic Paraventricular Nucleus Neurons.

The paraventricular nucleus (PVN) is involved in the control of sympathetic tone and the secretion of hormones, both functions known to be influenced by ghrelin, suggesting direct effect of ghrelin in this nucleus. However, the effects of ghrelin on the excitability of different PVN neuronal populations have not been demonstrated. This study assessed the effects of ghrelin on the activity of PVN neurons, correlating the responses to subpopulations of PVN neurons. We used a 64 multielectrode array to examine the effects of ghrelin administration on extracellular spike frequency in PVN neurons recorded in brain slices obtained from male Sprague-Dawley rats. Bath administration of 10 nM ghrelin increased (29/97, 30%) or decreased (37/97, 38%) spike frequency in PVN neurons. The GABAA and glutamate receptors antagonists abolish the decrease in spike frequency, without changes in the proportion of increases in spike frequency (23/53, 43%) induced by ghrelin. The results indicate a direct effect of ghrelin increasing PVN neurons activity and a synaptic dependent effect decreasing PVN neurons activity. The patch clamp recordings showed similar proportions of PVN neurons influenced by 10 nM ghrelin (33/95, 35% depolarized; 29/95, 30% hyperpolarized). Using electrophysiological fingerprints to identify specific subpopulations of PVN neurons we observed that the majority of pre-autonomic neurons (11/18 -61%) were depolarized by ghrelin, while both neuroendocrine (29% depolarizations, 40% hyperpolarizations), and magnocellular neurons (29% depolarizations, 21% hyperpolarizations) showed mixed responses. Finally, to correlate the electrophysiological response and the neurochemical phenotype of PVN neurons, cell cytoplasm was collected after recordings and RT-PCR performed to assess the presence of mRNA for vasopressin, oxytocin, thyrotropin (TRH) and corticotropin (CRH) releasing hormones. The single-cell RT-PCR showed that most TRH-expressing (4/5) and CRH-expressing (3/4) neurons are hyperpolarized in response to ghrelin. In conclusion, ghrelin either directly increases or indirectly decreases the activity of PVN neurons, this suggests that ghrelin acts on inhibitory PVN neurons that, in turn, decrease the activity of TRH-expressing and CRH-expressing neurons in the PVN.