Neurexin-3 (Nrxn3) has been genetically associated with obesity, but the underlying neural mechanisms remain poorly understood. This study aimed to investigate the role of Nrxn3 in the paraventricular nucleus of the hypothalamus (PVN) in regulating energy balance and glucose homeostasis. We found that Nrxn3 expression in the PVN was upregulated in response to metabolic stressors, including cold exposure and fasting. Using Cre-loxP technology, we selectively ablated Nrxn3 in CaMKIIα-expressing neurons of the PVN in male mice. This genetic manipulation resulted in marked weight gain attributable to increased adiposity and impaired glucose tolerance, without affecting food intake. Our findings identify PVN CaMKIIα-expressing neurons as a critical locus where Nrxn3 modulates energy balance by regulating adipogenesis and glucose metabolism, independently of appetite. These results reveal a novel neural mechanism potentially linking Nrxn3 dysfunction to obesity pathogenesis, suggesting that targeting PVN Nrxn3-dependent neural pathways may inform new therapeutic approaches for obesity prevention and treatment.

Dexmedetomidine (DEX) is a highly selective α2-adrenoceptor agonist with sedative effects on sleep homeostasis. Oxytocin-expressing (OXT) neurons in the paraventricular nucleus (PVN) of the hypothalamus (PVNOXT) regulate sexual reproduction, drinking, sleep-wakefulness, and other instinctive behaviors. To investigate the effect of DEX on the activity and signal transmission of PVNOXT in regulating the sleep-wakefulness cycle. Here, we employed OXT-cre mice to selectively target and express the designer receptors exclusively activated by designer drugs (DREADD)-based chemogenetic tool hM3D(Gq) in PVNOXT neurons. Combining chemogenetic methods with electroencephalogram (EEG) /electromyogram (EMG) recordings, we found that cannula injection of DEX in PVN significantly increased the duration of non-rapid eye movement (NREM) sleep in mice. Furthermore, the chemogenetic activation of PVNOXT neurons using i.p. injection of clozapine N-oxide (CNO) after cannula injection of DEX to PVN led to a substantial increase in wakefulness. Electrophysiological results showed that DEX decreased the frequency of action potential (AP) and the spontaneous excitatory postsynaptic current (sEPSC) of PVNOXT neurons through α2-adrenoceptors. Therefore, these results identify that DEX promotes sleep and maintains sleep homeostasis by inhibiting PVNOXT neurons through the α2-adrenoceptor.

To ensure the unrestricted expression of maternal behaviour peripartum, activity of the corticotropin-releasing factor (CRF) system needs to be minimised. CRF binding protein (CRF-BP) might be crucial for this adaptation, as its primary function is to sequester freely available CRF and urocortin1, thereby dampening CRF receptor (CRF-R) signalling. So far, the role of CRF-BP in the maternal brain has barely been studied, and a potential role in curtailing activation of the stress axis is unknown. We studied gene expression for CRF-BP and both CRF-R within the paraventricular nucleus (PVN) of the hypothalamus. In lactating rats, Crh-bp expression in the parvocellular PVN was significantly higher and Crh-r1 expression in the PVN significantly lower compared to virgin rats. Acute CRF-BP inhibition in the PVN with infusion of CRF(6-33) increased basal plasma corticosterone concentrations under unstressed conditions in dams. Furthermore, while acute intra-PVN infusion of CRF increased corticosterone secretion in virgin rats, it was ineffective in vehicle (VEH)-pre-treated lactating rats, probably due to a buffering effect of CRF-BP. Indeed, pre-treatment with CRF(6-33) reinstated a corticosterone response to CRF in lactating rats, highlighting the critical role of CRF-BP in maintaining attenuated stress reactivity in lactation. To our knowledge, this is the first study linking hypothalamic CRF-BP activity to hypothalamic-pituitary-adrenal axis regulation in lactation. In terms of behaviour, acute CRF-BP inhibition in the PVN under non-stress conditions reduced blanket nursing 60 min and licking/grooming 90 min after infusion compared to VEH-treated rats, while increasing maternal aggression towards an intruder. Lastly, chronic intra-PVN inhibition of CRF-BP strongly reduced maternal aggression, with modest effects on maternal motivation and care. Taken together, intact activity of the CRF-BP in the PVN during the postpartum period is essential for the dampened responsiveness of the stress axis, as well as for the full expression of appropriate maternal behaviour.

The impact of stress on reproductive function is significant. Hypothalamic paraventricular nucleus (PVN) corticotrophin-releasing hormone (CRH) plays a major role in regulating the stress response. Understanding how the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis interact is crucial for comprehending how stress can lead to reproductive dysfunction. However, whether stress influences reproductive function via modulating PVN CRH or HPA sequelae is not fully elucidated.
In this study, we investigated the impact of chemogenetic activation of PVN CRH neurons on reproductive function. We chronically and selectively stimulated PVN CRH neurons in female CRH-Cre mice using excitatory designer receptor exclusively activated by designer drugs (DREADDs) viral constructs, which were bilaterally injected into the PVN. The agonist compound-21 (C21) was delivered through the drinking water. We determined the effects of DREADDs activation of PVN CRH neurons on the estrous cycles, LH pulse frequency in diestrus and metestrus and LH surge in proestrus mice. The effect of long-term C21 administration on basal corticosterone secretion and the response to acute restraint stress during metestrus was also examined. Additionally, computer simulations of a mathematical model were used to determine the effects of DREADDs activation of PVN CRH neurons, simulating chronic stress, on the physiological parameters examined experimentally.
As a result, and consistent with our mathematical model predictions, the length of the estrous cycle was extended, with an increase in the time spent in estrus and metestrus, and a decrease in proestrus and diestrus. Additionally, the frequency of LH pulses during metestrus was decreased, but unaffected during diestrus. The occurrence of the preovulatory LH surge during proestrus was disrupted. The basal level of corticosterone during metestrus was not affected, but the response to acute restraint stress was diminished after long-term C21 application.
These data suggest that PVN CRH neurons play a functional role in disrupting ovarian cyclicity and the preovulatory LH surge, and that the activity of the GnRH pulse generator remains relatively robust during diestrus but not during metestrus under chronic stress exposure in accordance with our mathematical model predictions.

Entering pregnancy with a history of adversity, including adverse childhood experiences and racial discrimination stress, is a predictor of negative maternal and fetal health outcomes. Little is known about the biological mechanisms by which preconception adverse experiences are stored and impact future offspring health outcomes. In our maternal preconception stress (MPS) model, female mice underwent chronic stress from postnatal days 28-70 and were mated 2 weeks post-stress. Maternal preconception stress dams blunted the pregnancy-induced shift in the circulating extracellular vesicle proteome and reduced glucose tolerance at mid-gestation, suggesting a shift in pregnancy adaptation. To investigate MPS effects at the maternal:fetal interface, we probed the mid-gestation placental, uterine, and fetal brain tissue transcriptome. Male and female placentas differentially regulated expression of genes involved in growth and metabolic signaling in response to gestation in an MPS dam. We also report novel offspring sex- and MPS-specific responses in the uterine tissue apposing these placentas. In the fetal compartment, MPS female offspring reduced expression of neurodevelopmental genes. Using a ribosome-tagging transgenic approach we detected a dramatic increase in genes involved in chromatin regulation in a PVN-enriched neuronal population in females at PN21. While MPS had an additive effect on high-fat-diet (HFD)-induced weight gain in male offspring, both MPS and HFD were necessary to induce significant weight gain in female offspring. These data highlight the preconception period as a determinant of maternal health in pregnancy and provides novel insights into mechanisms by which maternal stress history impacts offspring developmental programming.

The paraventricular nucleus (PVN) of the hypothalamus plays a vital role in maintaining homeostasis and modulates cardiovascular function via autonomic pre-sympathetic neurones. We have previously shown that coupling between transient receptor potential cation channel subfamily V Member 4 (Trpv4) and small-conductance calcium-activated potassium channels (SK) in the PVN facilitate osmosensing, but since TRP channels are also thermosensitive, in this report we investigated the temperature sensitivity of these neurones. Methods: TRP channel mRNA was quantified from mouse PVN with RT-PCR and thermosensitivity of Trpv4-like PVN neuronal ion channels characterised with cell-attached patch-clamp electrophysiology. Following recovery of temperature-sensitive single-channel kinetic schema, we constructed a predictive stochastic mathematical model of these neurones and validated this with electrophysiological recordings of action current frequency. Results: 7 thermosensitive TRP channel genes were found in PVN punches. Trpv4 was the most abundant of these and was identified at the single channel level on PVN neurones. We investigated the thermosensitivity of these Trpv4-like channels; open probability (Po) markedly decreased when temperature was decreased, mediated by a decrease in mean open dwell times. Our neuronal model predicted that PVN spontaneous action current frequency (ACf) would increase as temperature is decreased and in our electrophysiological experiments, we found that ACf from PVN neurones was significantly higher at lower temperatures. The broad-spectrum channel blocker gadolinium (100 µM), was used to block the warm-activated, Ca2+-permeable Trpv4 channels. In the presence of gadolinium (100 µM), the temperature effect was largely retained. Using econazole (10 µM), a blocker of Trpm2, we found there were significant increases in overall ACf and the temperature effect was inhibited. Conclusion: Trpv4, the abundantly transcribed thermosensitive TRP channel gene in the PVN appears to contribute to intrinsic thermosensitive properties of PVN neurones. At physiological temperatures (37°C), we observed relatively low ACf primarily due to the activity of Trpm2 channels, whereas at room temperature, where most of the previous characterisation of PVN neuronal activity has been performed, ACf is much higher, and appears to be predominately due to reduced Trpv4 activity. This work gives insight into the fundamental mechanisms by which the body decodes temperature signals and maintains homeostasis.

N6-methyladenosine (m6A) is the most prevalent post-translational modification in eukaryotic mRNA. Recently, m6A editing modified by methyltransferase-like enzyme 3 (METTL3), the core m6A methyltransferase, has been demonstrated to be involved in cardiac sympathetic hyperactivity. This study aimed to clarify the effects and underlying mechanisms of METTL3 in the paraventricular nucleus (PVN) in mediating sympathetic activity following myocardial infarction (MI).
We established rat MI models by left anterior descending coronary artery ligation. m6A quantification was performed.The expression of METTL3 and its downstream gene, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), were determined. The functional role of METTL3 in sympathetic hyperactivity and electrical conduction stability were verified by assessing renal sympathetic nerve activity (RSNA), norepinephrine (NE) levels, and programmed electrical stimulation. Rescue experiments were also conducted. The mechanism by which m6A is involved in mitochondrial reactive oxygen species (mROS) production, mediated by TRAF6/ECSIT pathway, was explored in lipopolysaccharide (LPS) treated primary microglial cells.
METTL3 was predominantly localized in the microglia and significantly increased within the PVN at 3 days post-MI. Inhibition of METTL3 decreased m6A levels, TRAF6 expression, and mROS production; downregulated sympathoexcitation, indicated by attenuated NE concentration and RSNA; decreased the incidence of ventricular tachycardia or fibrillation; and improved cardiac function. Mechanistically, downregulation of METTL3 prevented TRAF6 translocation to the mitochondria in the microglia and subsequent TRAF6/ECSIT pathway activation, resulting in decreased mROS production.
This study demonstrates that METTL3-mediated m6A modification promotes sympathetic hyperactivity through TRAF6/ECSIT pathway and mitochondrial oxidative stress in the PVN, thereby leading to ventricular arrhythmias post-MI.

Oxytocin-expressing paraventricular hypothalamic neurons (PVNOT neurons) integrate afferent signals from the gut, including cholecystokinin (CCK), to adjust whole-body energy homeostasis. However, the molecular underpinnings by which PVNOT neurons orchestrate gut-to-brain feeding control remain unclear. Here, we show that mice undergoing selective ablation of PVNOT neurons fail to reduce food intake in response to CCK and develop hyperphagic obesity on a chow diet. Notably, exposing wild-type mice to a high-fat/high-sugar (HFHS) diet recapitulates this insensitivity toward CCK, which is linked to diet-induced transcriptional and electrophysiological aberrations specifically in PVNOT neurons. Restoring OT pathways in diet-induced obese (DIO) mice via chemogenetics or polypharmacology sufficiently re-establishes CCK\'s anorexigenic effects. Last, by single-cell profiling, we identify a specialized PVNOT neuronal subpopulation with increased κ-opioid signaling under an HFHS diet, which restrains their CCK-evoked activation. In sum, we document a (patho)mechanism by which PVNOT signaling uncouples a gut-brain satiation pathway under obesogenic conditions.

The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.

Stress-related psychiatric disorders and the stress system show prominent differences between males and females, as well as strongly divergent transcriptional changes. Despite several proposed mechanisms, we still lack the understanding of the molecular processes at play. Here, we explore the contribution of cell types to transcriptional sex dimorphism using single-cell RNA sequencing. We identify cell-type-specific signatures of acute restraint stress in the paraventricular nucleus of the hypothalamus, a central hub of the stress response, in male and female mice. Further, we show that a history of chronic mild stress alters these signatures in a sex-specific way, and we identify oligodendrocytes as a major target for these sex-specific effects. This dataset, which we provide as an online interactive app, offers the transcriptomes of thousands of individual cells as a molecular resource for an in-depth dissection of the interplay between cell types and sex on the mechanisms of the stress response.