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Astaxanthin (AST) has a variety of biochemical effects, including anti-inflammatory, antioxidative, and antihypertensive functions. The aim of the present study was to determine whether AST ameliorates blood pressure in salt-induced prehypertensive rats by ROS/MAPK/NF-κB pathways in hypothalamic paraventricular nucleus. To explore the central effects of AST on the development of blood pressure, prehypertensive rats were induced by a high-salt diet (HS, 8% NaCl) and its control groups were treated with normal-salt diet (NS, 0.3% NaCl). The Dahl salt-sensitive (S) rats with HS diet for 6 weeks received AST or vehicle by gastric perfusion for 6 weeks. Compared to those with NS diet, rats with HS diet exhibited increased mean arterial pressure (MAP) and heart rate (HR). These increases were associated with higher plasma level of norepinephrine (NE), interleukin 1β (IL-1β), and interleukin 6 (IL-6); elevated PVN level of reactive oxygen species (ROS), NOX2, and NOX4, that of IL-1β, IL-6, monocyte chemotactic protein 1 (MCP-1), tyrosine hydroxylase (TH), phosphorylation extracellular-signal-regulated kinase (p-ERK1/2), phosphorylation Jun N-terminal kinases (p-JNK), nuclear factor-kappa B (NF-κB) activity; and lower levels of IL-10, superoxide dismutase (SOD), and catalase (CAT) in the PVN. In addition, our data demonstrated that chronic AST treatment ameliorated these changes in the HS but not NS diet rats. These data suggested that AST could alleviate prehypertensive response in HS-induced prehypertension through ROS/MAPK/NF-κB pathways in the PVN.

Maternal care, including by non-biological parents, is important for offspring survival1–8. Oxytocin1,2,9–15, which is released by the hypothalamic paraventricular nucleus (PVN), is a critical maternal hormone. In mice, oxytocin enables neuroplasticity in the auditory cortex for maternal recognition of pup distress15. However, it is unclear how initial parental experience promotes hypothalamic signalling and cortical plasticity for reliable maternal care. Here we continuously monitored the behaviour of female virgin mice co-housed with an experienced mother and litter. This documentary approach was synchronized with neural recordings from the virgin PVN, including oxytocin neurons. These cells were activated as virgins were enlisted in maternal care by experienced mothers, who shepherded virgins into the nest and demonstrated pup retrieval. Virgins visually observed maternal retrieval, which activated PVN oxytocin neurons and promoted alloparenting. Thus rodents can acquire maternal behaviour by social transmission, providing a mechanism for adapting the brains of adult caregivers to infant needs via endogenous oxytocin., Behavioural studies and neural recordings in mice show that virgin mice can acquire maternal behaviour through an oxytocin-dependent mechanism.

Corticotropin releasing hormone (CRH) neurons located in the nucleus of hippocampal commissure (NHpC) have been proposed to be involved in the avian neuroendocrine regulation of stress and appeared to respond prior to CRH neurons in the hypothalamic paraventricular nucleus (PVN) when food deprivation stress was applied. Since the response of the NHpC was rapid and short-lived, was it regulated differentially from CRH neurons in the PVN? We, therefore, applied immobilization stress to test whether the NHpC response was stressor specific. Gene expression of CRH and stress-related genes in the NHpC, PVN, anterior pituitary (APit) as well as plasma corticosterone (CORT) were determined. Furthermore, brain derived neurotrophic factor (BDNF) and glucocorticoid receptor (GR) were examined regarding their possible roles in the regulation of CRH neurons. Data showed that rapid activation of CRH mRNA in the NHpC occurred and preceded a slower gene activation in the PVN, upregulation of proopiomelanocortin (POMC) transcripts in the APit and significant increases of CORT concentrations. Results suggested BDNF's role in negative feedback between CRH and CRHR1 in the NHpC and positive feedback between CRH and CRHR1 in the PVN. In the APit, V1bR activation appeared responsible for sustaining CORT release when stress persisted. Overall, data suggest that the NHpC functions as part of the HPA axis of birds and perhaps a comparable extra-hypothalamic structure occurs in other vertebrates.Lay SummaryThe nucleus of the hippocampal commissure, a structure outside of the hypothalamus, shows rapidly increased neural gene expression that appears to contribute to the early activation of the traditional hypothalamic-pituitary-adrenal (HPA) axis responsible for the production of stress hormones.

Social stressors produce neurobiological and emotional consequences in social species. Environmental interventions, such as environmental enrichment and exercise, may modulate physiological and behavioral stress responses. The present study investigated the benefits of environmental enrichment and exercise against social stress in the socially monogamous prairie vole. Female prairie voles remained paired with a sibling (control) or were isolated from a sibling for 4 weeks. The isolated groups were separated into isolated sedentary, isolated with environmental enrichment, and isolated with both enrichment and exercise conditions. Behaviors related to depression, anxiety, and sociality were investigated using the forced swim test (FST), elevated plus maze (EPM), and a social crowding stressor (SCS), respectively. cFos expression was evaluated in stress-related circuitry following the SCS. Both enrichment and enrichment with exercise protected against depression-relevant behaviors in the FST and social behavioral disruptions in the SCS, but only enrichment with exercise protected against anxiety-related behaviors in the EPM and altered cFos expression in the hypothalamic paraventricular nucleus in isolated prairie voles. Enrichment may alleviate emotion-related and social behaviors, however physical exercise may be an important component of environmental strategies for protecting against anxiety-related behaviors and reducing neural activation as a function of social stress.

Activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling in cardiovascular regulatory regions of the brain contributes to sympathetic excitation in myocardial infarction (MI)-induced heart failure (HF) by increasing brain renin-angiotensin system (RAS) activity, neuroinflammation, and endoplasmic reticulum (ER) stress. The mechanisms eliciting brain ERK1/2 signaling in HF are still poorly understood. We tested the involvement of the epidermal growth factor receptor (EGFR) which, upon activation, stimulates ERK1/2 activity. Adult male Sprague-Dawley rats received bilateral microinjections of a lentiviral vector encoding a small interfering RNA (siRNA) for EGFR, or a scrambled siRNA, into the hypothalamic paraventricular nucleus (PVN), a recognized source of sympathetic overactivity in HF. One week later, coronary artery ligation was performed to induce HF. Four weeks later, the EGFR siRNA-treated HF rats, compared with the scrambled siRNA-treated HF rats, had lower mRNA and protein levels of EGFR, lower levels of phosphorylated (p-) EGFR and p-ERK1/2 and lower mRNA levels of the inflammatory mediators TNF-α, IL-1β and cyclooxygenase-2, the RAS components angiotensin-converting enzyme and angiotensin II type 1a receptor and the ER stress markers BIP and ATF4 in the PVN. They also had lower plasma and urinary norepinephrine levels and improved peripheral manifestations of HF. Additional studies revealed that p-EGFR was increased in the PVN of HF rats, compared with sham-operated control rats. These results suggest that activation of EGFR in the PVN triggers ERK1/2 signaling, along with ER stress, neuroinflammation and RAS activity, in MI-induced HF. Brain EGFR may be a novel target for therapeutic intervention in MI-induced HF.

Neurons in the paraventricular nucleus of the thalamus (PVT) respond to emotionally salient events and project densely to subcortical regions known to mediate adaptive behavioral responses. The areas of the forebrain most densely innervated by the PVT include striatal-like subcortical regions that consist of the shell of the nucleus accumbens (NAcSh), the dorsolateral region of the bed nucleus of the stria terminalis (BSTDL) and the lateral-capsular division of the central nucleus of the amygdala (CeL). A recent tracing experiment demonstrated that the PVT is composed of two intermixed populations of neurons that primarily project to either the dorsomedial (dmNAcSh) or ventromedial region of the NAcSh (vmNAcSh) with many of the vmNAcSh projecting neurons providing collateral innervation of the BSTDL and CeL. The present study used triple injections of the retrograde tracer cholera toxin B to provide a detailed map of the location of PVT neurons that provide collaterals to the vmNAcSh, BSTDL and CeL. These neurons were intermixed throughout the PVT and did not form uniquely localized subpopulations. An intersectional viral anterograde tracing approach was used to demonstrate that regardless of its presumed target of innervation (dmNAcSh, vmNAcSh, BSTDL, or CeL), most neurons in the PVT provide collateral innervation to a common set of forebrain regions. The paper shows that PVT-dmNAcSh projecting neurons provide the most divergent projection system and that these neurons express the immediate early gene product cFos following an aversive incident. We propose that the PVT may regulate a broad range of responses to physiological and psychological challenges by simultaneously influencing functionally diverse regions of the forebrain that include the cortex, striatal-like regions in the basal forebrain and a number of hypothalamic nuclei.

BACKGROUND N-Methyl-d-aspartate receptor (NMDAR) in the hypothalamic paraventricular nucleus (PVN) plays critical roles in regulating sympathetic outflow. Studies showed that acute application of the antagonists of NMDAR or its subunits would reduce sympathetic nerve discharges. However, little is known about the effect of long-term management of NMDAR in hypertensive animals. METHODS PEAQX, the specific antagonist of NMDAR subunit 2A (GluN2A) was injected into both sides of the PVN of two-kidney, one-clip (2K1C) renal hypertensive rats and control (normotensive rats) for 3 weeks. RESULTS Three weeks of PEAQX infusion significantly reduced the blood pressure of the 2K1C rats. It managed to resume the balance between excitatory and inhibitory neural transmitters, reduce the level of proinflammatory cytokines and reactive oxygen species in the PVN, and reduce the level of norepinephrine in plasma of the 2K1C rats. PEAQX administration also largely reduced the transcription and translation levels of GluN2A and changed the expression levels of NMDAR subunits 1 and 2B (GluN1 and GluN2B). In addition, NMDAR was known to function through activating the extracellular regulated protein kinases (ERK) or phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways. In our study, we found that in the PVN of 2K1C rats treated with PEAQX, the phosphorylation levels of mitogen-activated protein kinase kinase (MEK), ERK1/2, and cAMP-response element-binding protein (CREB) significantly reduced, while the phosphorylation level of PI3K did not change significantly. CONCLUSIONS Chronic blockade of GluN2A alleviates hypertension through suppression of MEK/ERK/CREB pathway.

It is apparent that the c-Fos and FosB/ΔFosB immunohistochemistry has generally become a useful tool for determining the different antipsychotic (AP) drugs activities in the brain. It is also noteworthy that there are no spatial limits, while to the extent of their identification over the whole brain axis. In addition, they can be in a parallel manner utilized in the unmasking of the brain cell phenotype character activated by APs and by this way also to identify the possible brain circuits underwent to the APs action. However, up to date, the number of APs involved in the extra-striatal studies is still limited, what prevents the possibility to fully understand their extra-striatal effects as a complex as well as differentiate their extra-striatal impact in qualitative and quantitative dimensions. Actually, it is very believable that more and more anatomical/functional knowledge might bring new insights into the APs extra-striatal actions by identifying new region-specific activities of APs as well as novel cellular targets affected by APs, which might reveal more details of their possible side effects of the extra-striatal origin.

Multiple sequential actions, performed during parental behaviors, are essential elements of reproduction in mammalian species. We showed that neurons expressing melanin concentrating hormone (MCH) in the lateral hypothalamic area (LHA) are more active in rodents of both sexes when exhibiting parental nursing behavior. Genetic ablation of the LHA-MCH neurons impaired maternal nursing. The post-birth survival rate was lower in pups born to female mice with congenitally ablated MCH neurons under control of tet-off system, exhibiting reduced crouching behavior. Virgin female and male mice with ablated MCH neurons were less interested in pups and maternal care. Chemogenetic and optogenetic stimulation of LHA-MCH neurons induced parental nursing in virgin female and male mice. LHA-MCH GABAergic neurons project fibres to the paraventricular hypothalamic nucleus (PVN) neurons. Optogenetic stimulation of PVN induces nursing crouching behavior along with increasing plasma oxytocin levels. The hypothalamic MCH neural relays play important functional roles in parental nursing behavior in female and male mice.

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.

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.

Blood pressure is controlled by endocrine, autonomic, and behavioral responses that maintain blood volume and perfusion pressure at levels optimal for survival. Although it is clear that central angiotensin type 1a receptors (AT1aR; encoded by theAgtr1agene) influence these processes, the neuronal circuits mediating these effects are incompletely understood. The present studies characterize the structure and function of AT1aR neurons in the lamina terminalis (containing the median preoptic nucleus and organum vasculosum of the lamina terminalis), thereby evaluating their roles in blood pressure control. Using maleAgtr1a-Cre mice, neuroanatomical studies reveal that AT1aR neurons in the area are largely glutamatergic and send projections to the paraventricular nucleus of the hypothalamus (PVN) that appear to synapse onto vasopressin-synthesizing neurons. To evaluate the functionality of these lamina terminalis AT1aR neurons, we virally delivered light-sensitive opsins and then optogenetically excited or inhibited the neurons while evaluating cardiovascular parameters or fluid intake. Optogenetic excitation robustly elevated blood pressure, water intake, and sodium intake, while optogenetic inhibition produced the opposite effects. Intriguingly, optogenetic excitation of these AT1aR neurons of the lamina terminalis also resulted in Fos induction in vasopressin neurons within the PVN and supraoptic nucleus. Further, within the PVN, selective optogenetic stimulation of afferents that arise from these lamina terminalis AT1aR neurons induced glutamate release onto magnocellular neurons and was sufficient to increase blood pressure. These cardiovascular effects were attenuated by systemic pretreatment with a vasopressin-1a-receptor antagonist. Collectively, these data indicate that excitation of lamina terminalis AT1aR neurons induces neuroendocrine and behavioral responses that increase blood pressure.SIGNIFICANCE STATEMENTHypertension is a widespread health problem and risk factor for cardiovascular disease. Although treatments exist, a substantial percentage of patients suffer from “drug-resistant” hypertension, a condition associated with increased activation of brain angiotensin receptors, enhanced sympathetic nervous system activity, and elevated vasopressin levels. The present study highlights a role for angiotensin Type 1a receptor expressing neurons located within the lamina terminalis in regulating endocrine and behavioral responses that are involved in maintaining cardiovascular homeostasis. More specifically, data presented here reveal functional excitatory connections between angiotensin-sensitive neurons in the lamina terminals and vasopressin neurons in the paraventricular nucleus of the hypothalamus, and further indicate that activation of this circuit raises blood pressure. These neurons may be a promising target for antihypertensive therapeutics.

Arginine vasopressin (AVP) is synthesized in parvocellular- and magnocellular neuroendocrine neurons in the paraventricular nucleus (PVN) of the hypothalamus. Whereas magnocellular AVP neurons project primarily to the posterior pituitary, parvocellular AVP neurons project to the median eminence (ME) and to extrahypothalamic areas. The AVP gene encodes pre-pro-AVP that comprises the signal peptide, AVP, neurophysin (NPII), and a copeptin glycopeptide. In the present study, we used an N-terminal copeptin antiserum to examine copeptin expression in magnocellular and parvocellular neurons in the hypothalamus in the mouse, rat, and macaque monkey. Although magnocellular NPII-expressing neurons exhibited strong N-terminal copeptin immunoreactivity in all three species, a great majority (~90%) of parvocellular neurons that expressed NPII was devoid of copeptin immunoreactivity in the mouse, and in approximately half (~53%) of them in the rat, whereas in monkey hypothalamus, virtually all NPII-immunoreactive parvocellular neurons contained strong copeptin immunoreactivity. Immunoelectron microscopy in the mouse clearly showed copeptin-immunoreactivity co-localized with NPII-immunoreactivity in neurosecretory vesicles in the internal layer of the ME and posterior pituitary, but not in the external layer of the ME. Intracerebroventricular administration of a prohormone convertase inhibitor, hexa-d-arginine amide resulted in a marked reduction of copeptin-immunoreactivity in the NPII-immunoreactive magnocellular PVN neurons in the mouse, suggesting that low protease activity and incomplete processing of pro-AVP could explain the disproportionally low levels of N-terminal copeptin expression in rodent AVP (NPII)-expressing parvocellular neurons. Physiologic and phylogenetic aspects of copeptin expression among neuroendocrine neurons require further exploration.

Macronutrient intake is associated with cardiometabolic health, ageing and longevity, but the mechanisms underlying its regulation have remained unclear. Most rodents increase carbohydrate selection under certain physiological and pathological conditions such as fasting. When presented with a choice between a basally preferable high-fat diet (HFD) and a high-carbohydrate diet (HCD) such as a high-sucrose diet, fasted mice first eat the HFD and then switch to the HCD during the first few hours of refeeding and continue to eat the HCD up to 24 h in the two-diet choice approach. Such consumption of an HCD after fasting reverses the fasting-induced increase in the plasma concentration of ketone bodies more rapidly than does refeeding with an HFD alone. 5'-AMP-activated protein kinase (AMPK)-regulated neurons in the paraventricular nucleus of the hypothalamus (PVH) that express corticotropin-releasing hormone (CRH) are necessary and sufficient for the fasting-induced selection of carbohydrate over an HFD in mice. These neurons appear to contribute to a fasting-induced increase in the positive valence of carbohydrate without affecting the preference for more palatable and energy-dense diets such as an HFD. Identification of the neural circuits in which AMPK-regulated CRH neurons in the PVH of mice are embedded should shed new light on the physiological and molecular mechanisms responsible for macronutrient selection.

Defective rhythmic metabolism is associated with high-fat high-caloric diet (HFD) feeding, ageing and obesity; however, the neural basis underlying HFD effects on diurnal metabolism remains elusive. Here we show that deletion of BMAL1, a core clock gene, in paraventricular hypothalamic (PVH) neurons reduces diurnal rhythmicity in metabolism, causes obesity and diminishes PVH neuron activation in response to fast-refeeding. Animal models mimicking deficiency in PVH neuron responsiveness, achieved through clamping PVH neuron activity at high or low levels, both show obesity and reduced diurnal rhythmicity in metabolism. Interestingly, the PVH exhibits BMAL1-controlled rhythmic expression of GABA-A receptor γ2 subunit, and dampening rhythmicity of GABAergic input to the PVH reduces diurnal rhythmicity in metabolism and causes obesity. Finally, BMAL1 deletion blunts PVH neuron responses to external stressors, an effect mimicked by HFD feeding. Thus, BMAL1-driven PVH neuron responsiveness in dynamic activity changes involving rhythmic GABAergic neurotransmission mediates diurnal rhythmicity in metabolism and is implicated in diet-induced obesity., Defective rhythmic metabolism is associated with high-fat diet feeding and obesity. The authors show that the clock gene BMAL1 drives paraventricular hypothalamic neuron activity via rhythmic GABAergic neurotransmission, and that this mediates diurnal metabolism and diet-induced obesity.

The neuropeptide arginine-vasopressin (AVP) has long been implicated in the regulation of social behavior and communication in diverse taxa, but the source of AVP release relevant for behavior has not been precisely determined. Potential sources include hypothalamic cell populations such as the paraventricular (PVN), supraoptic, and suprachiasmatic nuclei, as well as extrahypothalamic cell groups in the extended amygdala. To address if AVP-expressing cells in the PVN are important for mouse social communication, we deleted PVN AVP-expressing cells using viral-mediated delivery of Cre-dependent caspase-9 cell death construct into the PVN of AVP-Cre-positive mice (expressing Cre-recombinase under the control of the AVP promoter) or AVP-Cre-negative littermate controls, and assessed their levels of social investigation, social communication, anxiety, sex behavior, and aggressive behavior. We found that these lesions increased social investigation in females, but not in males. However, in males but not in females, these lesions increased non-social anxiety-related behaviors in the elevated-plus maze. These results therefore point at differential involvement of PVN AVP-expressing cells in the context of social and emotional behavior in the two sexes, which may contribute to sex differences in social communication and anxiety disorders.

Binge-eating disorder is the most common eating disorder. Various neuropeptides play important roles in the regulation of feeding behavior, including relaxin-3 (RLN3), which stimulates food intake in rats through the activation of the relaxin-family peptide-3 receptor (RXFP3). Here we demonstrate that a likely mechanism underlying the orexigenic action of RLN3 is RXFP3-mediated inhibition of oxytocin- and arginine-vasopressin-synthesizing paraventricular nucleus (PVN) magnocellular neurosecretory cells. Moreover, we reveal that, in male and female rats, this action depends on M-like potassium conductance. Notably, higher intra- and peri-PVN RLN3 fiber densities were observed in females, which may constitute an anatomic substrate for observed sex differences in binge-eating disorder. Finally, in a model of binge-eating in female rats, RXFP3 blockade within the PVN prevented binge-eating behavior. These data demonstrate a direct RLN3/RXFP3 action in the PVN of male and female rats, identify the associated ionic mechanisms, and reveal that hypothalamic RLN3/RXFP3 signaling regulates binge-eating behavior. SIGNIFICANCE STATEMENT Binge-eating disorder is the most common eating disorder worldwide, affecting women twice as frequently as men. Various neuropeptides play important roles in the regulation of feeding behavior, including relaxin-3, which acts via the relaxin-family peptide-3 receptor (RXFP3). Using a model of binge-eating, we demonstrated that relaxin-3/RXFP3 signaling in the hypothalamic paraventricular nucleus (PVN) is necessary for the expression of binge-eating behavior in female rats. Moreover, we elucidated the neuronal mechanism of RLN3/RXFP3 signaling in PVN in male and female rats and characterized sex differences in the RLN3 innervation of the PVN. These findings increase our understanding of the brain circuits and neurotransmitters involved in binge-eating disorder pathology and identify RXFP3 as a therapeutic target for binge-like eating disorders.

Background Sympathetic overactivation after acute myocardial infarction (AMI) contributes to ventricular arrhythmia (VA). Paraventricular nucleus (PVN) of the hypothalamus may play an important role on this context, however, the mechanisms remain unknown. In this study, we investigated whether inhibition of activated astrocytes in the PVN could reduce VA in rats with AMI. Methods The anesthetized rats were randomly divided into four groups of sham-operated, AMI, AMI + vehicle and AMI + fluorocitrate (FCA). Electrocardiogram was continuously recorded. RNA sequencing, sympathetic nerve activity (heart rate variability and norepinephrine levels) and ventricular electrical instability (ventricular effective refractory period and ventricular fibrillation inducibility) were measured. Furthermore, brain tissues were extracted to detect expression of inflammatory cytokines (IL-6, and TNF-α), astrocyte and neuro activation. Results RNA sequencing analysis showed that functions of differentially expressed genes in the PVN of AMI rats were significantly enriched in immune system- and neuroactive-related pathways, along with enhance expression of cytokines and Glial fibrillary acidic protein (GFAP). We further characterized that astrocytes were activated in PVN and intervention of activation astrocytes by FCA significantly inhibited sympathetic nerve activity and decreased the incidence of VA and ventricular electrical instability in rats with AMI. Moreover, FCA significantly attenuated neurons activation and downregulated expression of inflammatory cytokines in the PVN. Conclusions Inhibition of activated astrocytes in the PVN could reduce VA occurrence and improve ventricular electrical instability in AMI rats by central neuro-immune pathway. These findings suggest that astrocytes are a potential target for prevention and treatment of VA complicating AMI.

Elucidating molecular pathways regulating neuroimmune communication is critical for therapeutic interventions in conditions characterized by overactive immune responses and dysfunctional autonomic nervous system. We generated a bone marrow-specific adrenergic beta 1 and beta 2 knockout mouse chimera (AdrB1.B2 KO) to determine how sympathetic drive to the bone affects transcripts and miRNAs in the hypothalamic paraventricular nucleus (PVN). This model has previously exhibited a dampened systemic immune response and decreased blood pressure compared with control animals. Reduced sympathetic responsiveness of the bone marrow hematopoietic cells of AdrB1.B2 KO chimera led to suppression of transcriptional networks that included leukocyte cell adhesion and migration and T cell-activation and recruitment. Transcriptome responses related to IL-17a signaling and the renin-angiotensin system were also suppressed in the PVN. Based on the transcriptome response, we next computationally predicted miRNAs in the PVN that may underscore the reduced sympathetic responsiveness of the bone marrow cells. These included miR-27b-3p, miR-150, miR-223-3p, and miR-326. Using real-time PCR, we measured a downregulation in the expression of miR-150-5p, miR-205-5p, miR-223-3p, miR-375-5p, miR-499a-5p, miR-27b-3p, let-7a-5p, and miR-21a-5p in the PVN of AdrB1.B2 KO chimera, confirming computational predictions that these miRNAs are associated with reduced neuro-immune responses and the loss of sympathetic responsiveness in the bone marrow. Intriguingly, directional responses of the miRNA corresponded to mRNAs, suggesting complex temporal or circuit-dependent posttranscriptional control of gene expression in the PVN. This study identifies molecular pathways involved in neural-immune interactions that may act as targets of therapeutic intervention for a dysfunctional autonomic nervous system.

Oxytocin (OT) is critical for the expression of social behavior across a wide array of species; however, the role of this system in the encoding of socially relevant information is not well understood. In the present study, we show that chemogenetic activation of OT neurons within the paraventricular nucleus of the hypothalamus (PVH) of male mice (OT-Ires-Cre) enhanced social investigation during a social choice test, while chemogenetic inhibition of these neurons abolished typical social preferences. These data suggest that activation of the OT system is necessary to direct behavior preferentially toward social stimuli. To determine whether the presence of a social stimulus is sufficient to induce activation of PVH-OT neurons, we performed the first definitive recording of OT neurons in awake mice using two-photon calcium imaging. These recordings demonstrate that social stimuli activate PVH-OT neurons and that these neurons differentially encode social and nonsocial stimuli, suggesting that PVH-OT neurons may act to convey social salience of environmental stimuli. Finally, an attenuation of social salience is associated with social disorders, such as autism. We therefore also examined possible OT system dysfunction in a mouse model of autism,Shank3bknock-out (KO) mice. MaleShank3bKO mice showed a marked reduction in PVH-OT neuron number and administration of an OT receptor agonist improved social deficits. Overall, these data suggest that the presence of a social stimulus induces activation of the PVH-OT neurons to promote adaptive social behavior responses.SIGNIFICANCE STATEMENTAlthough the oxytocin (OT) system is well known to regulate a diverse array of social behaviors, the mechanism in which OT acts to promote the appropriate social response is poorly understood. One hypothesis is that the presence of social conspecifics activates the OT system to generate an adaptive social response. Here, we selectively recorded from OT neurons in the paraventricular hypothalamic nucleus (PVH) to show that social stimulus exposure indeed induces activation of the OT system. We also show that activation of the OT system is necessary to promote social behavior and that mice with abnormal social behavior have reduced numbers of PVH-OT neurons. Finally, aberrant social behavior in these mice was rescued by administration of an OT receptor agonist.

Sex and ovarian function contribute to hypertension susceptibility, however, the mechanisms are not well understood. Prior studies show that estrogens and neurogenic factors, including hypothalamic glutamatergic NMDA receptor plasticity, play significant roles in rodent hypertension. Here, we investigated the role of sex and ovarian failure on AMPA receptor plasticity in estrogen-sensitive paraventricular nucleus (PVN) neurons in naïve and angiotensin II (AngII) infused male and female mice and female mice at early and late stages of accelerated ovarian failure (AOF). High-resolution electron microscopy was used to assess the subcellular distribution of AMPA GluA1 in age-matched male and female estrogen receptor beta (ERβ) enhanced green fluorescent protein (EGFP) reporter mice as well as female ERβ-EGFP mice treated with 4-vinylcyclohexene diepoxide. In the absence of AngII, female mice at a late stage of AOF displayed higher levels of GluA1 on the plasma membrane, indicative of functional protein, in ERβ-expressing PVN dendrites when compared to male, naïve female and early stage AOF mice. Following slow-pressor AngII infusion, males, as well as early and late stage AOF females had elevated blood pressure. Significantly, only late stage-AOF female mice infused with AngII had an increase in GluA1 near the plasma membrane in dendrites of ERβ-expressing PVN neurons. In contrast, prior studies reported that plasmalemmal NMDA GluN1 increased in ERβ-expressing PVN dendrites in males and early, but not late stage AOF females. Together, these findings reveal that early and late stage AOF female mice display unique molecular signatures of long-lasting synaptic strength prior to, and following hypertension.

Body weight (BW) is regulated in age-dependent manner; it continues to increase during growth period, and reaches a plateau once reaching adulthood. However, its underlying mechanism remains unknown. Regarding such mechanisms in the brain, we here report that neural circuits from the hypothalamus (paraventricular nucleus: PVN) to the brainstem (dorsal vagal complex: DVC) suppress late-onset BW gain without affecting food intake. The genetic suppression of the PVN-DVC circuit induced BW increase only in aged rats, indicating that this circuit contributes to suppress the BW at a fixed level after reaching adulthood. PVN neurons in the hypothalamus were inactive in younger rats but active in aged rats. The density of neuropeptide Y (NPY) terminal/fiber is reduced in the aged rat PVN area. The differences in neuronal activity, including oxytocin neurons in the PVN, were affected by the application of NPY or its receptor inhibitor, indicating that NPY is a possible regulator of this pathway. Our data provide new insights into understanding age-dependent BW regulation.

Background Thyrotropin-releasing hormone (TRH) was the first hypothalamic hormone isolated that stimulates pituitary thyroid-stimulating hormone (TSH) secretion. TRH was also later found to be a stimulator of pituitary prolactin and distributed throughout the brain, gastrointestinal tract, and pancreatic β cells. We previously reported the development of TRH null mice (conventional TRHKO), which exhibit characteristic tertiary hypothyroidism and impaired glucose tolerance due to insufficient insulin secretion. Although in the last five decades many investigators, us included, have attempted to determine the hypothalamic nucleus responsible for the hypothalamo-pituitary-thyroid (HPT) axis, it remained obscure because of the broad expression of TRH. Methods To determine the part of the hypothalamic region functionally responsible for the hypothalamo-pituitary-thyroid (HPT) axis, we established paraventricular nucleus (PVN)-specific TRH knock-out (PVN-TRHKO) mice by mating Trh floxed mice and Sim1-Cre transgenic mice. We originally confirmed that most Sim1 was expressed in the paraventricular nucleus using Sim1-Cre/tdTomato mice. Results These PVN-TRHKO mice exhibited tertiary hypothyroidism similar to conventional TRHKO mice, however, they did not show the impaired glucose tolerance observed in the latter, suggesting that TRH from non-PVN sources is essential for glucose regulation. In addition, a severe reduction in prolactin expression was observed in the pituitary of PVN-TRHKO mice compared with that in TRHKO mice. Conclusions These findings are conclusive evidence that the PVN is the center of the HPT axis for regulation of serum levels of thyroid hormones, and that the serum TSH levels are not decreased in tertiary hypothyroidism. We also noted that TRH from the PVN regulated prolactin, whereas TRH from non-PVN sources regulated glucose metabolism.

Made available in DSpace on 2022-04-28T19:44:51Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The paraventricular nucleus of the hypothalamus (PVN) is an important site for autonomic control, which integrates thermoregulation centers and sympathetic outflow to thermoeffector organs. PVN neurons express the neuronal isoform of nitric oxide synthase (nNOS) whose expression is locally upregulated by physical exercise. Thus, the aim of the present study was to evaluate the role of nNOS in the PVN in the exercise-induced hyperthermia. Seven days after surgery, male Wistar rats received bilateral intra-PVN microinjections of the selective nNOS inhibitor Nw-Propyl-L-Arginine (NPLA) or vehicle (saline) and were submitted to an acute progressive exercise session on a treadmill until fatigue. Abdominal and tail skin temperature (Tabd and Ttail, respectively) were measured, and the threshold (Hthr; °C) and sensitivity (Hsen) for heat dissipation calculated. Performance variables were also collected. During the progressive exercise protocol, all animals displayed an increase in the Tabd. However, compared to vehicle group, the microinjection of NPLA in the PVN attenuated the exercise-induced hyperthermia. There was no difference in Ttail or Hthr between NPLA and control rats. In contrast, Hsen was increased in the NPLA group compared to vehicle. In addition, heat storage was lower in NPLA-treated animals. Despite the temperature differences, inhibition of nNOS in the PVN did not affect running performance on the treadmill. These results suggest that nitrergic signaling within the PVN, under nNOS activation, drives the increase of body temperature, being necessary for the proper thermal regulatory mechanisms during progressive exercise-induced hyperthermia. Department of Physiology and Biophysics Institute of Biological Science Federal University of Minas Gerais (ICB/UFMG) Department of Drugs and Medicine School of Pharmaceutical Sciences São Paulo State University (UNESP) Department of Drugs and Medicine School of Pharmaceutical Sciences São Paulo State University (UNESP) CNPq: #403769/2016–7 CNPq: PQ-2 #311516/2020–3

Decades of studies have revealed molecular and neural circuit bases for body weight homeostasis. Neural hormone oxytocin (Oxt) has received attention in this context because it is produced by neurons in the paraventricular hypothalamic nucleus (PVH), a known output center of hypothalamic regulation of appetite. Oxt has an anorexigenic effect, as shown in human studies, and can mediate satiety signals in rodents. However, the function of Oxt signaling in the physiological regulation of appetite has remained in question, because whole-body knockout (KO) of Oxt or Oxt receptor (Oxtr) has little effect on food intake. We herein show that acute conditional KO (cKO) of Oxt selectively in the adult PVH, but not in the supraoptic nucleus, markedly increases body weight and food intake, with an elevated level of plasma triglyceride and leptin. Intraperitoneal administration of Oxt rescues the hyperphagic phenotype of the PVH Oxt cKO model. Furthermore, we show that cKO of Oxtr selectively in the posterior hypothalamic regions, especially the arcuate hypothalamic nucleus, a primary center for appetite regulations, phenocopies hyperphagic obesity. Collectively, these data reveal that Oxt signaling in the arcuate nucleus suppresses excessive food intake.

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.

The endocannabinoid system is an important regulator of the hormonal and behavioral stress responses, which critically involve corticotropin-releasing factor (CRF) and its receptors. While it has been shown that CRF and the cannabinoid type 1 (CB1) receptor are co-localized in several brain regions, the physiological relevance of this co-expression remains unclear. Using double in situ hybridization, we confirmed co-localization in the piriform cortex, the lateral hypothalamic area, the paraventricular nucleus, and the Barrington's nucleus, albeit at low levels. To study the behavioral and physiological implications of this co-expression, we generated a conditional knockout mouse line that selectively lacks the expression of CB1 receptors in CRF neurons. We found no effects on fear and anxiety-related behaviors under basal conditions nor after a traumatic experience. Additionally, plasma corticosterone levels were unaffected at baseline and after restraint stress. Only acoustic startle responses were significantly enhanced in male, but not female, knockout mice. Taken together, the consequences of depleting CB1 in CRF-positive neurons caused a confined hyperarousal phenotype in a sex-dependent manner. The current results suggest that the important interplay between the central endocannabinoid and CRF systems in regulating the organism's stress response is predominantly taking place at the level of CRF receptor-expressing neurons.

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.

Objective The paraventricular nucleus of hypothalamus (PVN), an integrative center in the brain, orchestrates a wide range of physiological and behavioral responses. While the PVN melanocortin 4 receptor (MC4R) signaling (PVNMC4R+) is involved in feeding regulation, the neuroanatomical organization of PVNMC4R+ connectivity and its role in other physiological regulations are incompletely understood. Here we aimed to better characterize the input–output organization of PVNMC4R+ neurons and test their physiological functions beyond feeding. Methods Using a combination of viral tools, we mapped PVNMC4R+ circuits and tested the effects of chemogenetic activation of PVNMC4R+ neurons on thermoregulation, cardiovascular control, and other behavioral responses beyond feeding. Results We found that PVNMC4R+ neurons innervate many different brain regions that are known to be important not only for feeding but also for neuroendocrine and autonomic control of thermoregulation and cardiovascular function, including but not limited to the preoptic area, median eminence, parabrachial nucleus, pre-locus coeruleus, nucleus of solitary tract, ventrolateral medulla, and thoracic spinal cord. Contrary to these broad efferent projections, PVNMC4R+ neurons receive monosynaptic inputs mainly from other hypothalamic nuclei (preoptic area, arcuate and dorsomedial hypothalamic nuclei, supraoptic nucleus, and premammillary nucleus), the circumventricular organs (subfornical organ and vascular organ of lamina terminalis), the bed nucleus of stria terminalis, and the parabrachial nucleus. Consistent with their broad efferent projections, chemogenetic activation of PVNMC4R+ neurons not only suppressed feeding but also led to an apparent increase in heart rate, blood pressure, and brown adipose tissue temperature. These physiological changes accompanied acute transient hyperactivity followed by hypoactivity and resting-like behavior. Conclusions Our results elucidate the neuroanatomical organization of PVNMC4R+ circuits and shed new light on the roles of PVNMC4R+ pathways in autonomic control of thermoregulation, cardiovascular function, and biphasic behavioral activation., Highlights • PVNMC4R+ neurons innervate brain regions involved in feeding and autonomic regulation. • Activation of PVNMC4R+ neurons increases BAT thermogenesis. • Activation of PVNMC4R+ neurons increases blood pressure and heart rate. • Activation of PVNMC4R+ neurons lead to transient hyperactivity followed by hypoactivity and resting-like behavior.

Adipocytokine chemerin is a biologically active molecule secreted from adipose tissue. Chemerin elicits a variety of functions via chemokine-like receptor 1 (CMKLR1). The cardiovascular center in brain that regulates blood pressure (BP) is involved in pathophysiology of systemic hypertension. Thus, we explored the roles of brain chemerin/CMKLR1 on regulation of BP in spontaneously hypertensive rats (SHR). For this aim, we examined effects of intracerebroventricular (i.c.v.) injection of CMKLR1 small interfering (si)RNA on both systemic BP as measured by tail cuff system and protein expression in paraventricular nucleus (PVN) of SHR as determined by Western blotting. We also examined both central and peripheral protein expression of chemerin by Western blotting. Systolic BP of SHR but not normotensive Wistar Kyoto rats (WKY) was decreased by CMKLR1 siRNA. The decrease of BP by CMKLR1 siRNA persisted for 3 days. Protein expression of CMKLR1 in PVN of SHR tended to be increased compared with WKY, which was suppressed by CMKLR1 siRNA. Protein expression of chemerin in brain, peripheral plasma, and adipose tissue was not different between WKY and SHR. In summary, we for the first time revealed that the increased protein expression of CMKLR1 in PVN is at least partly responsible for systemic hypertension in SHR.

Both rodents and primates have evolved to orchestrate food intake to maintain thermal homeostasis in coping with ambient temperature challenges. However, the mechanisms underlying temperature-coordinated feeding behavior are rarely reported. Here we find that a non-canonical feeding center, the anteroventral and periventricular portions of medial preoptic area (apMPOA) respond to altered dietary states in mice. Two neighboring but distinct neuronal populations in apMPOA mediate feeding behavior by receiving anatomical inputs from external and dorsal subnuclei of lateral parabrachial nucleus. While both populations are glutamatergic, the arcuate nucleus-projecting neurons in apMPOA can sense low temperature and promote food intake. The other type, the paraventricular hypothalamic nucleus (PVH)-projecting neurons in apMPOA are primarily sensitive to high temperature and suppress food intake. Caspase ablation or chemogenetic inhibition of the apMPOA→PVH pathway can eliminate the temperature dependence of feeding. Further projection-specific RNA sequencing and fluorescence in situ hybridization identify that the two neuronal populations are molecularly marked by galanin receptor and apelin receptor. These findings reveal unrecognized cell populations and circuits of apMPOA that orchestrates feeding behavior against thermal challenges.

Arginine vasopressin (AVP) is expressed in both hypothalamic and extra-hypothalamic neurons. The expression and role of AVP exhibit remarkable divergence between these two neuronal populations. Polysynaptic pathways enable these neuronal groups to regulate each other. AVP neurons in the paraventricular nucleus of the hypothalamus increase the production of adrenal stress hormones by stimulating the hypothalamic–pituitary–adrenal axis. Outside the hypothalamus, the medial amygdala also contains robust amounts of AVP. Contrary to the hypothalamic counterpart, the expression of extra-hypothalamic medial amygdala AVP is sexually dimorphic, in that it is preferentially transcribed in males in response to the continual presence of testosterone. Male gonadal hormones typically generate a negative feedback on the neuroendocrine stress axis. Here, we investigated whether testosterone-responsive medial amygdala AVP neurons provide negative feedback to hypothalamic AVP, thereby providing a feedback loop to suppress stress endocrine response during periods of high testosterone secretion. Contrary to our expectation, we found that AVP overexpression within the posterodorsal medial amygdala increased the recruitment of hypothalamic AVP neurons during stress, without affecting the total number of AVP neurons or the number of recently activated neurons following stress. These observations suggest that the effects of testosterone on extra-hypothalamic AVP facilitate stress responsiveness through permissive influence on the recruitment of hypothalamic AVP neurons. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00850-2.

Corticotropin-releasing hormone (CRH) cells are the dominant neuronal population responsive to the growth hormone (GH) in the paraventricular nucleus of the hypothalamus (PVH). However, the physiological importance of GH receptor (GHR) signaling in CRH neurons is currently unknown. Thus, the main objective of the present study was to investigate the consequences of GHR ablation in CRH-expressing cells of male and female mice. GHR ablation in CRH cells did not cause significant changes in body weight, body composition, food intake, substrate oxidation, locomotor activity, glucose tolerance, insulin sensitivity, counterregulatory response to 2-deoxy-D-glucose and ghrelin-induced food intake. However, reduced energy expenditure was observed in female mice carrying GHR ablation in CRH cells. The absence of GHR in CRH cells did not affect anxiety, circadian glucocorticoid levels or restraint-stress-induced corticosterone secretion and activation of PVH neurons in both male and female mice. In summary, GHR ablation, specifically in CRH-expressing neurons, does not lead to major alterations in metabolism, hypothalamic–pituitary–adrenal axis, acute stress response or anxiety in mice. Considering the previous studies showing that central GHR signaling regulates homeostasis in situations of metabolic stress, future studies are still necessary to identify the potential physiological importance of GH action on CRH neurons.

SummaryPulsatile release of the hormone oxytocin (OT) mediates uterine contraction during parturition and milk ejection during lactation1–3. These pulses are generated by unique activity patterns of the central neuroendocrine OT neurons located in the paraventricular and supraoptic hypothalamus. Classical studies have characterized putative OT neurons by in vivo extracellular recording techniques in rats and rabbits under anesthesia1, 4–7 or awake8–10. Due to technical limitations, however, the identity of OT neurons in these previous studies was speculative based on their electrophysiological characteristics and axonal projection to the posterior pituitary, not on OT gene expression. To pinpoint OT neural activities among other hypothalamic neurons that project to the pituitary11, 12 and make better use of cell-type-specific neuroscience toolkits13, a mouse model needs to be developed for studies of parturition and lactation. We herein introduce viral genetic approaches in mice to characterize the maternal activities of OT neurons by fiber photometry. During lactation, a sharp photometric peak of OT neurons appeared at approximately 520 s following simultaneous suckling stimuli from three pups. The amplitude of the peaks increased as the mother mice experienced lactation, irrespective of the age of the pups, suggesting the intrinsic plasticity of maternal OT neurons. Based on a mono-synaptic input map to OT neurons, we pharmacogenetically activated the inhibitory neurons in the bed nucleus of the stria terminalis and found suppression of the activities of OT neurons. Collectively, our study illuminates temporal dynamics in the maternal neural activities of OT neurons and identifies one of its modulatory circuits.Highlights- Pulsatile activities of genetically-defined OT neurons in mother mice were recorded in vivo.- The maternal experience-dependent plasticity of the OT neural activities was found.- Input-mapping of OT neurons in mother mice was performed by rabies-mediated trans-synaptic tracing.- Photometric peaks of OT neurons were suppressed by the activation of BST inhibitory neurons.

The paraventricular nucleus of the thalamus (PVT) is a midline thalamic brain region that has emerged as a critical circuit node in the regulation of behaviors across domains of affect and motivation, stress responses, and alcohol- and drug-related behaviors. The influence of the PVT in this diverse array of behaviors is a function of its ability to integrate and convey information about salience and valence through its connections with cortical, hypothalamic, hindbrain, and limbic brain regions. While understudied to date, recent studies suggest that several PVT efferents play critical and complex roles in drug and alcohol-related phenotypes. The PVT is also the site of signaling for many neuropeptides released from the synaptic terminals of distal inputs and local neuropeptidergic neurons within. While there is some evidence that neuropeptides including orexin, neurotensin, substance P, and cocaine and amphetamine-related transcript (CART) signal in the PVT to regulate alcohol/drug intake and reinstatement, there remains an overall lack of understanding of the roles of neuropeptides in the PVT in addiction-related behaviors, especially in a circuit-specific context. In this review, we present the current status of preclinical research regarding PVT circuits and neuropeptide modulation of the PVT in three aspects of the addiction cycle: reward/acquisition, withdrawal, and relapse, with a focus on alcohol, opioids (particularly morphine), and psychostimulants (particularly cocaine). Given the PVT's unique position within the broader neural landscape, we further discuss the potential ways in which neuropeptides may regulate these behaviors through their actions upon PVT circuits. This article is part of the special Issue on ‘Neurocircuitry Modulating Drug and Alcohol Abuse'.

The tightly localized noradrenergic neurons (NA) in the locus coeruleus (LC) are well recognized as essential for focused arousal and novelty-oriented responses, while many children with autism spectrum disorder (ASD) exhibit diminished attention, engagement and orienting to exogenous stimuli. This has led to the hypothesis that atypical LC activity may be involved in ASD. Oxytocin (OXT) neurons and receptors are known to play an important role in social behavior, pair bonding and cognitive processes and are under investigation as a potential treatment for ASD. However, little is known about the neurotransmission from hypothalamic paraventricular (PVN) OXT neurons to LC NA neurons. In this study, we test, in male and female rats, whether PVN OXT neurons excite LC neurons, whether oxytocin is released and involved in this neurotransmission, and whether activation of PVN OXT neurons alters novel object recognition. Using “oxytocin sniffer cells” (CHO cells that express the human oxytocin receptor and a Ca indicator) we show that there is release of OXT from hypothalamic PVN OXT fibers in the LC. Optogenetic excitation of PVN OXT fibers excites LC NA neurons by co-release of OXT and glutamate, and this neurotransmission is greater in males than females. In male, but not in female animals, chemogenetic activation of PVN OXT neurons increases attention to novel objects.

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

Oxytocin and vasopressin secretion from the posterior pituitary gland are required for normal pregnancy and lactation. Oxytocin secretion is relatively low and constant under basal conditions but becomes pulsatile during birth and lactation to stimulate episodic contraction of the uterus for delivery of the fetus and milk ejection during suckling. Vasopressin secretion is maintained in pregnancy and lactation despite reduced osmolality (the principal stimulus for vasopressin secretion) to increase water retention to cope with the cardiovascular demands of pregnancy and lactation. Oxytocin and vasopressin secretion are determined by the action potential (spike) firing of magnocellular neurosecretory neurons of the hypothalamic supraoptic and paraventricular nucleus. In addition to synaptic input activity, spike firing depends on intrinsic excitability conferred by the suite of channels expressed by the neurons. Therefore, we analysed oxytocin and vasopressin neuron activity in anaesthetised non-pregnant, late-pregnant and lactating rats to test the hypothesis that intrinsic excitability of oxytocin and vasopressin neurons is increased in late pregnancy and lactation to promote oxytocin and vasopressin secretion required for successful pregnancy and lactation. Hazard analysis of spike firing revealed a higher incidence of post-spike hyperexcitability immediately following each spike in oxytocin neurons, but not in vasopressin neurons, in late pregnancy and lactation, which is expected to facilitate high frequency firing during bursts. Despite lower osmolality in late-pregnant and lactating rats, vasopressin neuron activity was not different between non-pregnant, late-pregnant and lactating rats, and blockade of osmosensitive ΔN-TRPV1 channels inhibited vasopressin neurons to a similar extent in non-pregnant, late-pregnant and lactating rats. Furthermore, supraoptic nucleus ΔN-TRPV1 mRNA expression was not different between non-pregnant and late-pregnant rats, suggesting that enhanced activity of ΔN-TRPV1 channels might maintain vasopressin neuron activity to increase water retention during pregnancy and lactation.

Disturbed activation or regulation of the stress response through the hypothalamic-pituitary-adrenal (HPA) axis is a fundamental component of multiple stress-related diseases, including psychiatric, metabolic, and immune disorders. The FK506 binding protein 51 (FKBP5) is a negative regulator of the glucocorticoid receptor (GR), the main driver of HPA axis regulation, and FKBP5 polymorphisms have been repeatedly linked to stress-related disorders in humans. However, the specific role of Fkbp5 in the paraventricular nucleus of the hypothalamus (PVN) in shaping HPA axis (re)activity remains to be elucidated. We here demonstrate that the deletion of Fkbp5 in Sim1+ neurons dampens the acute stress response and increases GR sensitivity. In contrast, Fkbp5 overexpression in the PVN results in a chronic HPA axis over-activation, and a PVN-specific rescue of Fkbp5 expression in full Fkbp5 KO mice normalizes the HPA axis phenotype. Single-cell RNA sequencing revealed the cell-type-specific expression pattern of Fkbp5 in the PVN and showed that Fkbp5 expression is specifically upregulated in Crh+ neurons after stress. Finally, Crh-specific Fkbp5 overexpression alters Crh neuron activity, but only partially recapitulates the PVN-specific Fkbp5 overexpression phenotype. Together, the data establish the central and cell-type-specific importance of Fkbp5 in the PVN in shaping HPA axis regulation and the acute stress response.

The mechanisms underlying food anticipatory activity is still not well understood. Here we explored the role of oxytocin (OT) and the protein c-Fos in the supraoptic nucleus (SON) and in the medial (PVNm) and posterior (PVNp) regions of the paraventricular hypothalamic nucleus. Adult rats were assigned to one of four groups: scheduled restricted feeding (RF), Ad libitum (AL), fasting after restricted feeding (RF-F), to explore the possible persistence of oscillations, or Ad libitum fasted (AL-F). In the SON and in the PVNm, OT cells were c-Fos positive after food intake; contrasting, OT cells in the PVNp showed c-Fos activation in anticipation to food access, which persisted in RF-F subjects. We conclude that OT cells of the SON and PVNm may play a role as recipients of the entraining signal provided by food intake, whereas those of the PVNp which contain motor preautonomic cells that project to peripheral organs, may be involved in the hormonal and metabolic anticipatory changes in preparation for food presentation and thus, may be part of a link between central and peripheral oscillators. In addition, due to their persistent activation they may participate in the neuronal network for the clock mechanism that leads to food entrainment.

Activity of the hypothalamic-pituitary-adrenal (HPA) axis is tuned by corticosteroid feedback. Corticosteroids regulate cellular function via genomic and nongenomic mechanisms, which operate over diverse time scales. This review summarizes recent advances in our understanding of how corticosteroid feedback regulates hypothalamic stress neuron function and output through synaptic plasticity, changes in intrinsic excitability, and modulation of neuropeptide production. The temporal kinetics of corticosteroid actions in the brain versus the pituitary have important implications for how organisms respond to stress. Furthermore, we will discuss, some of the technical limitations and missing links in the field, and the potential implications these may have on our interpretations of corticosteroid negative feedback experiments.

Aging is associated with a substantial decline in social behavior, whereas positive social interaction can improve overall health in aged individuals. In laboratory rodents, manipulations of the social environment across the lifespan have been shown to affect social behavior. Therefore, we examined the effects of long-term (5–6 weeks) housing conditions (alone, with one adult, or with two adults) on social behavior and the expression of neuroinflammation-related genes as well as oxytocin receptor (OXTR) gene expression in brain areas associated with social behavior regulation in aged male and female Fischer (F) 344 rats. Single-housed males and females exhibited increased social investigation, relative to pair-housed rats (one aged and one adult). Triple-housed (one aged and two adults) aged males exhibited lower levels of social investigation, relative to triple-housed aged females. Aged females were more socially active that their male counterparts. Although social housing condition significantly affected social behavior in males, it had no impact on cytokine gene expression in the paraventricular nucleus of hypothalamus (PVN), bed nucleus of the stria terminalis (BNST) or medial amygdala (MeA). However, in triple-housed aged females, who exhibited social behavior comparable to their single- and pair-housed counterparts, there was a significant increase in the expression of IL-1β and IL-6 mRNA in the MeA. No changes in cytokine gene expression were observed in the PVN or BNST, indicating that the increased expression of cytokines in the MeA was not a result of a generalized increase in neuroinflammation. Single-housed males and females exhibited elevated OXTR gene expression in the BNST. Taken together, these data indicate that manipulations of the social environment in late aging significantly influenced social interactions with a novel partner and gene expression in social behavior circuits and that these effects are sex-specific.

Corticotropin-releasing factor (CRF) regulates stress responses, and aberrant CRF signals are associated with depressive disorders. Crf expression is responsive to arachidonic acid (AA), where CRF is released from the hypothalamic paraventricular nucleus (PVN) to initiate the hypothalamic-pituitary-adrenal axis, culminating in glucocorticoid stress hormone release. Despite this biological and clinical significance, Crf regulation is unclear. Here, we report that acyloxyacyl hydrolase, encoded by Aoah, is expressed in the PVN, and Aoah regulates Crf through the aryl hydrocarbon receptor (AhR). We previously showed that AOAH-deficient mice mimicked interstitial cystitis/bladder pain syndrome, a condition frequently associated with comorbid anxiety and depression. With the use of novelty-suppressed feeding and sucrose preference assays to quantify rodent correlates of anxiety/depression, AOAH-deficient mice exhibited depressive behaviors. AOAH-deficient mice also had increased CNS AA, increased Crf expression in the PVN, and elevated serum corticosterone, consistent with dysfunction of the hypothalamic-pituitary-adrenal axis. The human Crf promoter has putative binding sites for AhR and peroxisome proliferator-activated receptor (PPARγ). PPARγ did not affect AA-dependent Crf expression in vitro, and conditional Pparγ knockout did not alter the AOAH-deficient depressive phenotype, despite previous studies implicating PPARγ as a therapeutic target for depression. In contrast, Crf induction was mediated by AhR binding sites in vitro and increased by AhR overexpression. Furthermore, conditional Ahr knockout rescued the depressive phenotype of AOAH-deficient mice. Finally, an AhR antagonist rescued the AOAH-deficient depressive phenotype. Together, our results demonstrate that Aoah is a novel genetic regulator of Crf mediated through AhR, and AhR is a therapeutic target for depression.

The central melanocortin system is composed of neurons that express either the proopiomelanocortin (POMC) or the agouti-related protein (AgRP). POMC is cleaved in bioactive peptides, including the α-melanocyte-stimulating hormone (α-MSH). α-MSH activates the melanocortin-4 receptor (MC4R) inducing satiety, whereas AgRP acts as an inverse agonist of MC4R. However, only limited information is available regarding possible area-specific differences in the interaction between α-MSH and AgRP terminals on MC4R-expressing cells. Therefore, the objective of the present study was to compare the distribution pattern of α-MSH and AgRP terminals on the perikarya of MC4R-expressing neurons. We performed a triple-label immunofluorescence reaction in brain series of MC4R-reporter mice to visualize MC4R-expressing neurons together with AgRP and α-MSH terminals. POMC and AgRP neurons project to areas that contain MC4R-expressing cells, although several brain nuclei exhibit AgRP and α-MSH terminals, but they do no express MC4R, while other brain areas contain MC4R-expressing cells and receive no apparent innervation of AgRP and POMC neurons. AgRP terminals make more presumptive appositions than α-MSH on the soma of MC4R-expressing neurons of the medial preoptic area and paraventricular nucleus of the hypothalamus (Pa). Additionally, a higher percentage of MC4R cells receive at least one presumptive apposition from AgRP terminals in the median preoptic nucleus and Pa, compared to α-MSH appositions. Thus, our study revealed area-specific differences in the interaction between α-MSH and AgRP terminals and the soma of MC4R-expressing neurons. These findings provide new insights about the relationship between first- and second-order neurons of the central melanocortin system.

Background The adipose afferent reflex (AAR), a sympatho-excitatory reflex, can promote the elevation of sympathetic nerve activity (SNA) and blood pressure (BP). Inflammation in the paraventricular nucleus (PVN) involves sympathetic abnormality in some cardiovascular diseases such as hypertension. This study was designed to explore the effects of tumor necrosis factor alpha (TNFα) in the PVN on the AAR and SNA in rats with obesity-related hypertension (OH) induced by a high-fat diet for 12 weeks. Methods Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were continuously recorded in anesthetized rats, and their responses to capsaicin (CAP) stimulation of the right inguinal white adipose tissue were used to evaluate the AAR. Results Compared to the control rats, the systolic blood pressure (SBP), plasma norepinephrine (NE, indicating SNA) and TNFα levels, TNFα mRNA and protein levels, reactive oxygen species (ROS) content and NADPH oxidase activity in the PVN were significantly elevated in rats with OH. TNFα in the PVN markedly enhanced sympathoexcitation and AAR. Moreover, the enhancement of AAR caused by TNFα can be significantly strengthened by the pretreatment of diethyldithiocarbamate (DETC), a superoxide dismutase inhibitor, but attenuated by TNF-α receptor antagonist R-7050, superoxide scavenger PEG-SOD and NADPH oxidase inhibitor apocynin (Apo) in rats with OH. Acute microinjection of TNF-α into the PVN significantly increased the activity of NADPH oxidase and ROS levels in rats with OH, which were effectively blocked by R-7050. Furthermore, our results also showed that the increased levels of ROS, TNFα and NADPH oxidase subunits mRNA and protein in the PVN of rats with OH were significantly reversed by pentoxifylline (PTX, 30 mg/kg daily ip; in 10% ethanol) application, a cytokine blocker, for a period of 5 weeks. PTX administration also significantly decreased SBP, AAR and plasma NE levels in rats with OH. Conclusions TNFα in the PVN modulates AAR and contributes to sympathoexcitation in OH possibly through NADPH oxidase-dependent ROS generation. TNFα blockade attenuates AAR and sympathoexcitation that unveils TNFα in the PVN may be a possible therapeutic target for the intervention of OH.

Melatonin, the receptors for which are abundant in the hypothalamic paraventricular nucleus (PVN), can protect the heart from myocardial ischemia–reperfusion (MI/R) injury. The aim of this study was to determine whether the infusion of melatonin into the PVN protects the heart from MI/R injury by suppressing oxidative stress or regulating the balance between proinflammatory cytokines and anti-inflammatory cytokines in MI/R rats. Male Sprague–Dawley rats were treated with a bilateral PVN infusion of melatonin. MI/R operation was performed 1 week after infusion. At the end of the third week after the infusion, all the rats were euthanized. This was followed by immunohistochemistry and immunofluorescence studies of the rats. MI/R rats showed larger infarct size, increased left ventricular (LV) end-diastolic volume, and decreased LV ejection fraction and LV fractional shortening. Moreover, MI/R rats had a higher level of norepinephrine in the plasma, heart, and PVN; higher PVN levels of reactive oxygen species, NOX2, NOX4, IL-1β, and NF-κB activity; and lower PVN levels of copper/zinc superoxide dismutase (Cu/Zn-SOD) and IL-10 compared with the sham group. Melatonin infusion in PVN reduced LV end-diastolic volume, norepinephrine, reactive oxygen species, NOX2, NOX4, IL-1β, and NF-κB activity, and increased LV ejection fraction, LV fractional shortening, Cu/Zn-SOD, and IL-10. Overall, these results suggest that the infusion of melatonin ameliorates sympathetic nerve activity and MI/R injury by attenuating oxidative stress and inflammatory cytokines in the PVN of MI/R rats.

Sex differences in neural structures are generally believed to underlie sex differences reported in anxiety, depression, and the hypothalamic–pituitary–adrenal axis, although the specific circuitry involved is largely unclear. Using a corticotropin-releasing factor receptor 1 (CRFR1) reporter mouse line, we report a sexually dimorphic distribution of CRFR1 expressing cells within the paraventricular hypothalamus (PVN; males > females). Relative to adult levels, PVN CRFR1-expressing cells are sparse and not sexually dimorphic at postnatal days 0, 4, or 21. This suggests that PVN cells might recruit CRFR1 during puberty or early adulthood in a sex-specific manner. The adult sex difference in PVN CRFR1 persists in old mice (20–24 months). Adult gonadectomy (6 weeks) resulted in a significant decrease in CRFR1-immunoreactive cells in the male but not female PVN. CRFR1 cells show moderate co-expression with estrogen receptor alpha (ERα) and high co-expression with androgen receptor, indicating potential mechanisms through which circulating gonadal hormones might regulate CRFR1 expression and function. Finally, we demonstrate that a psychological stressor, restraint stress, induces a sexually dimorphic pattern of neural activation in PVN CRFR1 cells (males > females) as assessed by co-localization with the transcription/neural activation marker phosphorylated CREB. Given the known role of CRFR1 in regulating stress-associated behaviors and hormonal responses, this CRFR1 PVN sex difference might contribute to sex differences in these functions.

When energy balance is altered by aerobic exercise, starvation, and cold exposure, for example, there appears to be coordination of the responses of skeletal muscle, white adipose (WAT), and brown adipose (BAT) tissues. We hypothesized that WAT, BAT, and skeletal muscle may share an integrated regulation by the central nervous system (CNS); specifically, that neurons in brain regions associated with energy balance would possess neuroanatomical connections to permit coordination of multiple, complementary responses in these downstream tissues. To study this, we used trans-neuronal viral retrograde tract tracing, using isogenic strains of pseudorabies virus (PRV) with distinct fluorescent reporters (either eGFP or mRFP), injected pairwise into male rat gastrocnemius, subcutaneous WAT and interscapular BAT, coupled with neurochemical characterization of specific cell populations for cocaine- and amphetamine-related transcript (CART), oxytocin (OX), corticotrophin releasing hormone (CRH) and calcitonin gene-related peptide (CGRP). Cells in the paraventricular (PVN) and parabrachial (PBN) nuclei and brainstem showed dual projections to muscle + WAT, muscle + BAT, and WAT + BAT. Dual PRV-labeled cells were found in parvocellular, magnocellular and descending/pre-autonomic regions of the PVN, and multiple structural divisions of the PBN and brainstem. In most PBN subdivisions, more than 50% of CGRP cells dually projected to muscle + WAT and muscle + BAT. Similarly, 31–68% of CGRP cells projected both to WAT + BAT. However, dual PRV-labeled cells in PVN only occasionally expressed OX or CRH but not CART. These studies reveal for the first time both separate and shared outflow circuitries among skeletal muscle and subcutaneous WAT and BAT.

Consolation, which entails comforting contact directed toward a distressed party, is a common empathetic response in humans and other species with advanced cognition. Here, using the social defeat paradigm, we provide empirical evidence that highly social and monogamous mandarin voles (Microtus mandarinus) increased grooming toward a socially defeated partner but not toward a partner who underwent only separation. This selective behavioral response existed in both males and females. Accompanied with these behavioral changes, c-Fos expression was elevated in many of the brain regions relevant for emotional processing, such as the anterior cingulate cortex (ACC), bed nucleus of the stria terminalis, paraventricular nucleus (PVN), basal/basolateral and central nucleus of the amygdala, and lateral habenular nucleus in both sexes; in the medial preoptic area, the increase in c-Fos expression was found only in females, whereas in the medial nucleus of the amygdala, this increase was found only in males. In particular, the GAD67/c-Fos and oxytocin (OT)/c-Fos colocalization rates were elevated in the ACC and PVN, indicating selective activation of GABA and OT neurons in these regions. The "stressed" pairs matched their anxiety-like behaviors in the open-field test, and their plasma corticosterone levels correlated well with each other, suggesting an empathy-based mechanism. This partner-directed grooming was blocked by pretreatment with an OT receptor antagonist or a GABAA receptor antagonist in the ACC but not by a V1a subtype vasopressin receptor antagonist. We conclude that consolation behavior can be elicited by the social defeat paradigm in mandarin voles, and this behavior may be involved in a coordinated network of emotion-related brain structures, which differs slightly between the sexes. We also found that the endogenous OT and the GABA systems within the ACC are essential for consolation behavior in mandarin voles.

Ghrelin is a peptide mainly produced and secreted by the stomach. Since its discovery, the impact of ghrelin on the regulation of food intake has been the most studied function of this hormone; however, ghrelin affects a wide range of physiological systems, many of which are controlled by the hypothalamic paraventricular nucleus (PVN). Several pathways may mediate the effects of ghrelin on PVN neurons, such as direct or indirect effects mediated by circumventricular organs and/or the arcuate nucleus. The ghrelin receptor is expressed in PVN neurons, and the peripheral or intracerebroventricular administration of ghrelin affects PVN neuronal activity. Intra-PVN application of ghrelin increases food intake and decreases fat oxidation, which chronically contribute to the increased adiposity. Additionally, ghrelin modulates the neuroendocrine axes controlled by the PVN, increasing the release of vasopressin and oxytocin by magnocellular neurons and corticotropin-releasing hormone by neuroendocrine parvocellular neurons, while possibly inhibiting the release of thyrotropin-releasing hormone. Thus, the PVN is an important target for the actions of ghrelin. Our review discusses the mechanisms of ghrelin actions in the PVN, and its potential implications for energy balance, neuroendocrine, and integrative physiological control.