Stress during pubertal development affects female sociosexual behavior in mice.

Puberty is a crucial phase for the development of female sexual behavior. Growing evidence suggests that stress during this period may interfere with the development of sexual behavior. However, the neural circuits involved in this alteration remain elusive. Here, we demonstrated in mice that pubertal stress permanently disrupted sexual performance without affecting sexual preference. This was associated with a reduced expression and activation of neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Fiber photometry revealed that VMHvl nNOS neurons are strongly responsive to male olfactory cues with this activation being substantially reduced in pubertally stressed females. Finally, treatment with a NO donor partially restored sexual performance in pubertally stressed females. This study provides insights into the involvement of VMHvl nNOS in the processing of olfactory cues important for the expression of female sexual behavior. In addition, exposure to stress during puberty disrupts the integration of male olfactory cues leading to reduced sexual behavior.

A dedicated hypothalamic oxytocin circuit controls aversive social learning.

To survive in a complex social group, one needs to know who to approach and, more importantly, who to avoid. In mice, a single defeat causes the losing mouse to stay away from the winner for weeks1. Here through a series of functional manipulation and recording experiments, we identify oxytocin neurons in the retrochiasmatic supraoptic nucleus (SOROXT) and oxytocin-receptor-expressing cells in the anterior subdivision of the ventromedial hypothalamus, ventrolateral part (aVMHvlOXTR) as a key circuit motif for defeat-induced social avoidance. Before defeat, aVMHvlOXTR cells minimally respond to aggressor cues. During defeat, aVMHvlOXTR cells are highly activated and, with the help of an exclusive oxytocin supply from the SOR, potentiate their responses to aggressor cues. After defeat, strong aggressor-induced aVMHvlOXTR cell activation drives the animal to avoid the aggressor and minimizes future defeat. Our study uncovers a neural process that supports rapid social learning caused by defeat and highlights the importance of the brain oxytocin system in social plasticity.

Dynamic, sex- and diet-specific pleiotropism in the PAC1 receptor-mediated regulation of arcuate proopiomelanocortin and Neuropeptide Y/Agouti related peptide neuronal excitability by anorexigenic ventromedial nucleus PACAP neurons.

This study furthers the investigation of how pituitary adenylate cyclase activating polypeptide (PACAP) and the PAC1 receptor (PAC1R) regulate the homeostatic energy balance circuitry. We hypothesized that apoptotic ablation of PACAP neurones in the hypothalamic ventromedial nucleus (VMN) would affect both energy intake and energy expenditure. We also hypothesized that selective PAC1R knockdown would impair the PACAP-induced excitation in anorexigenic proopiomelanocortin (POMC) neurones and inhibition of orexigenic neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurones in the hypothalamic arcuate nucleus (ARC). The results show CASPASE-3-induced ablation of VMN PACAP neurones leads to increased energy intake and meal frequency as well as decreased energy expenditure in lean animals. The effects were more robust in obese males, whereas we saw the opposite effects in obese females. We then utilized visualized whole-cell patch clamp recordings in hypothalamic slices. PAC1R knockdown in POMC neurones diminishes the PACAP-induced depolarization, increase in firing, decreases in energy intake and meal size, as well as increases in CO2 production and O2 consumption. Similarly, the lack of expression of the PAC1R in NPY/AgRP neurones greatly attenuates the PACAP-induced hyperpolarization, suppression of firing, decreases in energy intake and meal frequency, as well as increases in energy expenditure. The PACAP response in NPY/AgRP neurones switched from predominantly inhibitory to excitatory in fasted animals. Finally, the anorexigenic effect of PACAP was potentiated when oestradiol was injected into the ARC in ovariectomized females. This study demonstrates the critical role of anorexigenic VMN PACAP neurones and the PAC1R in exciting POMC and inhibiting NPY/AgRP neurons to control homeostatic feeding.

Sex-dependent endozepinergic regulation of ventromedial hypothalamic nucleus glucose counter-regulatory neuron aromatase protein expression in the adult rat.

The hypothalamic brain cell types that produce estradiol from testosterone remain unclear. Aromatase inhibition affects ventromedial hypothalamic nucleus (VMN) glucose-stimulatory nitric oxide (NO) and glucose-inhibitory γ-aminobutyric acid (GABA) transmission during insulin (INS)-induced hypoglycemia (IIH). Pure GABA and NO nerve cell samples acquired by laser-catapult-microdissection from consecutive rostro-caudal segments of the VMN were analyzed by Western blot to investigate whether regional subpopulations of each cell type contain machinery for neuro-estradiol synthesis. Astrocyte endozepinergic signaling governs brain steroidogenesis. Pharmacological tools were used here to determine if the glio-peptide octadecaneuropeptide (ODN) controls aromatase expression in GABA and NO neurons during eu- and/or hypoglycemia. Intracerebroventricular administration of the ODN G-protein coupled-receptor antagonist cyclo(1-8)[DLeu5]OP (LV-1075) decreased (male) or enhanced (female) VMN GABAergic neuron aromatase expression, but increased or reduced this profile in nitrergic neurons in a region-specific manner in each sex. IIH suppressed aromatase levels in GABA neurons located in the middle segment of the male VMN or distributed throughout this nucleus in the female. This inhibitory response was altered by the ODN isoactive surrogate octapeptide (OP) in female, but was refractory to OP in male. NO neuron aromatase protein in hypoglycemic male (middle and caudal VMN) and female (rostral and caudal VMN) rats, but was normalized in OP- plus INS-treated rats of both sexes. Results provide novel evidence that VMN glucose-regulatory neurons may produce neuro-estradiol, and that the astrocyte endozepine transmitter ODN may impose sex-specific control of baseline and/or hypoglycemic patterns of aromatase expression in distinct subsets of nitrergic and GABAergic neurons in this neural structure.

GHRH Neurons from the Ventromedial Hypothalamic Nucleus Provide Dynamic and Sex-Specific Input to the Brain Glucose-Regulatory Network.

The ventromedial hypothalamic nucleus (VMN) controls glucose counter-regulation, including pituitary growth hormone (GH) secretion. VMN neurons that express the transcription factor steroidogenic factor-1/NR5A1 (SF-1) participate in glucose homeostasis. Research utilized in vivo gene knockdown tools to determine if VMN growth hormone-releasing hormone (Ghrh) regulates hypoglycemic patterns of glucagon, corticosterone, and GH outflow according to sex. Intra-VMN Ghrh siRNA administration blunted hypoglycemic hypercorticosteronemia in each sex, but abolished elevated GH release in males only. Single-cell multiplex qPCR showed that dorsomedial VMN (VMNdm) Ghrh neurons express mRNAs encoding Ghrh, SF-1, and protein markers for glucose-inhibitory (γ-aminobutyric acid) or -stimulatory (nitric oxide; glutamate) neurotransmitters. Hypoglycemia decreased glutamate decarboxylase67 (GAD67) transcripts in male, not female VMNdm Ghrh/SF-1 neurons, a response that was refractory to Ghrh siRNA. Ghrh gene knockdown prevented, in each sex, hypoglycemic down-regulation of Ghrh/SF-1 nerve cell GAD65 transcription. Ghrh siRNA amplified hypoglycemia-associated up-regulation of Ghrh/SF-1 neuron nitric oxide synthase mRNA in male and female, without affecting glutaminase gene expression. Ghrh gene knockdown altered Ghrh/SF-1 neuron estrogen receptor-alpha (ERα) and ER-beta transcripts in hypoglycemic male, not female rats, but up-regulated GPR81 lactate receptor mRNA in both sexes. Outcomes infer that VMNdm Ghrh/SF-1 neurons may be an effector of SF-1 control of counter-regulation, and document Ghrh modulation of hypoglycemic patterns of glucose-regulatory neurotransmitter along with estradiol and lactate receptor gene transcription in these cells. Co-transmission of glucose-inhibitory and -stimulatory neurochemicals of diverse chemical structure, spatial, and temporal profiles may enable VMNdm Ghrh neurons to provide complex dynamic, sex-specific input to the brain glucose-regulatory network.

mGluR5 in Astrocytes in the Ventromedial Hypothalamus Regulates Pituitary Adenylate Cyclase-Activating Polypeptide Neurons and Glucose Homeostasis.

The ventromedial hypothalamus (VMH) is a functionally heterogeneous nucleus critical for systemic energy, glucose, and lipid balance. We showed previously that the metabotropic glutamate receptor 5 (mGluR5) plays essential roles regulating excitatory and inhibitory transmission in SF1+ neurons of the VMH and facilitating glucose and lipid homeostasis in female mice. Although mGluR5 is also highly expressed in VMH astrocytes in the mature brain, its role there influencing central metabolic circuits is unknown. In contrast to the glucose intolerance observed only in female mice lacking mGluR5 in VMH SF1 neurons, selective depletion of mGluR5 in VMH astrocytes enhanced glucose tolerance without affecting food intake or body weight in both adult female and male mice. The improved glucose tolerance was associated with elevated glucose-stimulated insulin release. Astrocytic mGluR5 male and female mutants also exhibited reduced adipocyte size and increased sympathetic tone in gonadal white adipose tissue. Diminished excitatory drive and synaptic inputs onto VMH Pituitary adenylate cyclase-activating polypeptide (PACAP+) neurons and reduced activity of these cells during acute hyperglycemia underlie the observed changes in glycemic control. These studies reveal an essential role of astrocytic mGluR5 in the VMH regulating the excitatory drive onto PACAP+ neurons and activity of these cells facilitating glucose homeostasis in male and female mice.SIGNIFICANCE STATEMENT Neuronal circuits within the VMH play chief roles in the regulation of whole-body metabolic homeostasis. It remains unclear how astrocytes influence neurotransmission in this region to facilitate energy and glucose balance control. Here, we explored the role of the metabotropic glutamate receptor, mGluR5, using a mouse model with selective depletion of mGluR5 from VMH astrocytes. We show that astrocytic mGluR5 critically regulates the excitatory drive and activity of PACAP-expressing neurons in the VMH to control glucose homeostasis in both female and male mice. Furthermore, mGluR5 in VMH astrocytes influences adipocyte size and sympathetic tone in white adipose tissue. These studies provide novel insight toward the importance of hypothalamic astrocytes participating in central circuits regulating peripheral metabolism.

Glycogen phosphorylase isoenzyme GPbb versus GPmm regulation of ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and counter-regulatory hormone profiles during hypoglycemia: Role of L-lactate and octadecaneuropeptide.

Glucose accesses the brain primarily via the astrocyte cell compartment, where it passes through the glycogen shunt before catabolism to the oxidizable fuel L-lactate. Glycogen phosphorylase (GP) isoenzymes GPbb and GPmm impose distinctive control of ventromedial hypothalamic nucleus (VMN) glucose-regulatory neurotransmission during hypoglycemia, but lactate and/or gliotransmitter involvement in those actions is unknown. Lactate or the octadecaneuropeptide receptor antagonist cyclo(1-8)[DLeu5] OP (LV-1075) did not affect gene product down-regulation caused by GPbb or GPmm siRNA, but suppressed non-targeted GP variant expression in a VMN region-specific manner. Hypoglycemic up-regulation of neuronal nitric oxide synthase was enhanced in rostral and caudal VMN by GPbb knockdown, yet attenuated by GPMM siRNA in the middle VMN; lactate or LV-1075 reversed these silencing effects. Hypoglycemic inhibition of glutamate decarboxylase65/67 was magnified by GPbb (middle and caudal VMN) or GPmm (middle VMN) knockdown, responses that were negated by lactate or LV-1075. GPbb or GPmm siRNA enlarged hypoglycemic VMN glycogen profiles in rostral and middle VMN. Lactate and LV-1075 elicited progressive rostral VMN glycogen augmentation in GPbb knockdown rats, but stepwise-diminution of rostral and middle VMN glycogen after GPmm silencing. GPbb, not GPmm, knockdown caused lactate or LV-1075 - reversible amplification of hypoglycemic hyperglucagonemia and hypercorticosteronemia. Results show that lactate and octadecaneuropeptide exert opposing control of GPbb protein in distinct VMN regions, while the latter stimulates GPmm. During hypoglycemia, GPbb and GPmm may respectively diminish (rostral, caudal VMN) or enhance (middle VMN) nitrergic transmission and each oppose GABAergic signaling (middle VMN) by lactate- and octadecaneuropeptide-dependent mechanisms.

Anoctamin 4 channel currents activate glucose-inhibited neurons in the mouse ventromedial hypothalamus during hypoglycemia.

Glucose is the basic fuel essential for maintenance of viability and functionality of all cells. However, some neurons - namely, glucose-inhibited (GI) neurons - paradoxically increase their firing activity in low-glucose conditions and decrease that activity in high-glucose conditions. The ionic mechanisms mediating electric responses of GI neurons to glucose fluctuations remain unclear. Here, we showed that currents mediated by the anoctamin 4 (Ano4) channel are only detected in GI neurons in the ventromedial hypothalamic nucleus (VMH) and are functionally required for their activation in response to low glucose. Genetic disruption of the Ano4 gene in VMH neurons reduced blood glucose and impaired counterregulatory responses during hypoglycemia in mice. Activation of VMHAno4 neurons increased food intake and blood glucose, while chronic inhibition of VMHAno4 neurons ameliorated hyperglycemia in a type 1 diabetic mouse model. Finally, we showed that VMHAno4 neurons represent a unique orexigenic VMH population and transmit a positive valence, while stimulation of neurons that do not express Ano4 in the VMH (VMHnon-Ano4) suppress feeding and transmit a negative valence. Together, our results indicate that the Ano4 channel and VMHAno4 neurons are potential therapeutic targets for human diseases with abnormal feeding behavior or glucose imbalance.

Astrocyte Glycogen Is a Major Source of Hypothalamic Lactate in Rats With Recurrent Hypoglycemia.

Lactate is an important metabolic substrate for sustaining brain energy requirements when glucose supplies are limited. Recurring exposure to hypoglycemia (RH) raises lactate levels in the ventromedial hypothalamus (VMH), which contributes to counterregulatory failure. However, the source of this lactate remains unclear. The current study investigates whether astrocytic glycogen serves as the major source of lactate in the VMH of RH rats. By decreasing the expression of a key lactate transporter in VMH astrocytes of RH rats, we reduced extracellular lactate concentrations, suggesting excess lactate was locally produced from astrocytes. To determine whether astrocytic glycogen serves as the major source of lactate, we chronically delivered either artificial extracellular fluid or 1,4-dideoxy-1,4-imino-d-arabinitol to inhibit glycogen turnover in the VMH of RH animals. Inhibiting glycogen turnover in RH animals prevented the rise in VMH lactate and the development of counterregulatory failure. Lastly, we noted that RH led to an increase in glycogen shunt activity in response to hypoglycemia and elevated glycogen phosphorylase activity in the hours following a bout of hypoglycemia. Our data suggest that dysregulation of astrocytic glycogen metabolism following RH may be responsible, at least in part, for the rise in VMH lactate levels. ARTICLE HIGHLIGHTS: Astrocytic glycogen serves as the major source of elevated lactate levels in the ventromedial hypothalamus (VMH) of animals exposed to recurring episodes of hypoglycemia. Antecedent hypoglycemia alters VMH glycogen turnover. Antecedent exposure to hypoglycemia enhances glycogen shunt activity in the VMH during subsequent bouts of hypoglycemia. In the immediate hours following a bout of hypoglycemia, sustained elevations in glycogen phosphorylase activity in the VMH of recurrently hypoglycemic animals contribute to sustained elevations in local lactate levels.

Sexual differentiation of estrogen receptor alpha subpopulations in the ventromedial nucleus of the hypothalamus.

Estrogen receptor (ER) α-expressing neurons in the ventrolateral area of the ventromedial hypothalamus (VMHvl) are implicated in the control of many behaviors and physiological processes, some of which are sex-specific. Recently, three sex-differentiated ERα subpopulations have been discovered in the VMHvl marked by co-expression with tachikinin1 (Tac1), reprimo (Rprm), or prodynorphin (Pdyn), that may subserve specific functions. These markers show sex differences in adulthood: females have many more Tac1/Esr1 and Rprm/Esr1 co-expressing cells, while males have more Pdyn/Esr1 cells. In this study, we sought to understand the development of these sex differences and pinpoint the sex-differentiating signal. We examined developmental changes in the number of Esr1 cells co-expressing Tac1, Rprm or Pdyn using single-molecule in situ hybridization. We found that both sexes have similarly high numbers of Tac1/Esr1 and Rprm/Esr1 cells at birth, but newborn males have many more Pdyn/Esr1 cells than females. However, the number of cells with Tac1/Esr1 and Rprm/Esr1 co-expression markedly decreases by weaning in males, but not females, leading to sex differences in neurochemical expression. Female mice administered testosterone at birth have expression patterns akin to male mice. Thus, a substantial neurochemical reorganization of the VMHvl occurs in males between birth and weaning that likely underlies the previously reported sex differences in behavioral and physiological responses to estrogens in adulthood.

Sex-dimorphic hindbrain lactate regulation of ventromedial hypothalamic nucleus glucoregulatory neuron 5'-AMP-activated protein kinase activity and transmitter marker protein expression.

BACKGROUND: The oxidizable glycolytic end-product L-lactate is a gauge of nerve cell metabolic fuel stability that metabolic-sensory hindbrain A2 noradrenergic neurons impart to the brain glucose-regulatory network. Current research investigated the premise that hindbrain lactate deficiency exerts sex-specific control of energy sensor and transmitter marker protein responses to hypoglycemia in ventromedial hypothalamic nucleus (VMN) glucose-regulatory nitrergic and γ-aminobutyric acid (GABA) neurons. METHODS: Nitric oxide synthase (nNOS)- or glutamate decarboxylase65/67 (GAD)-immunoreactive neurons were laser-catapult-microdissected from male and female rat VMN after subcutaneous insulin injection and caudal fourth ventricular L-lactate or vehicle infusion for Western blot protein analysis. RESULTS: Hindbrain lactate repletion reversed hypoglycemia-associated augmentation (males) or inhibition (females) of nitrergic neuron nNOS expression, and prevented up-regulation of phosphorylated AMPK 5'-AMP-activated protein kinase (pAMPK) expression in those neurons. Hypoglycemic suppression of GABAergic neuron GAD protein was averted by exogenous lactate over the rostro-caudal length of the male VMN and in the middle region of the female VMN. Lactate normalized GABA neuron pAMPK profiles in hypoglycemic male (caudal VMN) and female (all VMN segments) rats. Hypoglycemic patterns of norepinephrine (NE) signaling were lactate-dependent throughout the male VMN, but confined to the rostral and middle female VMN. CONCLUSIONS: Results document, in each sex, regional VMN glucose-regulatory transmitter responses to hypoglycemia that are controlled by hindbrain lactate status. Hindbrain metabolic-sensory regulation of hypoglycemia-correlated nitric oxide or GABA release may entail AMPK-dependent mechanisms in specific VMN rostro-caudal segments in each sex. Additional effort is required to examine the role of hindbrain lactoprivic-sensitive VMN neurotransmitters in lactate-mediated attenuation of hypoglycemic hyperglucagonemia and hypercorticosteronemia in male and female rats.

Populational heterogeneity and partial migratory origin of the ventromedial hypothalamic nucleus: genoarchitectonic analysis in the mouse.

The ventromedial hypothalamic nucleus (VMH) is one of the most distinctive hypothalamic tuberal structures, subject of numerous classic and modern functional studies. Commonly, the adult VMH has been divided in several portions, attending to differences in cell aggregation, cell type, connectivity, and function. Consensus VMH partitions in the literature comprise the dorsomedial (VMHdm), and ventrolateral (VMHvl) subnuclei, which are separated by an intermediate or central (VMHc) population (topographic names based on the columnar axis). However, some recent transcriptome analyses have identified a higher number of different cell types in the VMH, suggesting additional subdivisions, as well as the possibility of separate origins. We offer a topologic and genoarchitectonic developmental study of the mouse VMH complex using the prosomeric axis as a reference. We analyzed genes labeling specific VMH subpopulations, with particular focus upon the Nkx2.2 transcription factor, a marker of the alar-basal boundary territory of the prosencephalon, from where some cells seem to migrate dorsoventrally into VMH. We also identified separate neuroepithelial origins of a Nr2f1-positive subpopulation, and a new Six3-positive component, as well as subtle differences in origin of Nr5a1 positive versus Nkx2.2-positive cell populations entering dorsoventrally the VMH. Several of these migrating cell types are born in the dorsal tuberal domain and translocate ventralwards to reach the intermediate tuberal domain, where the adult VMH mass is located in the adult. This work provides a more detailed area map on the intrinsic organization of the postmigratory VMH complex, helpful for deeper functional studies of this basal hypothalamic entity.

G protein-coupled lactate receptor GPR81 control of ventrolateral ventromedial hypothalamic nucleus glucoregulatory neurotransmitter and 5'-AMP-activated protein kinase expression.

Astrocytes store glycogen as energy and promote neurometabolic stability through supply of oxidizable l-lactate. Whether lactate regulates ventromedial hypothalamic nucleus (VMN) glucostatic function as a metabolic volume transmitter is unknown. Current research investigated whether G protein-coupled lactate receptor GPR81 controls astrocyte glycogen metabolism and glucose-regulatory neurotransmission in the ventrolateral VMN (VMNvl), where glucose-regulatory neurons reside. Female rats were pretreated by intra-VMN GPR81 or scramble siRNA infusion before insulin or vehicle injection. VMNvl cell or tissue samples were acquired by laser-catapult- or micropunch microdissection for Western blot protein or uHPLC-electrospray ionization-mass spectrometric glycogen analyses. Data show that GPR81 regulates eu- and/or hypoglycemic patterns of VMNvl astrocyte glycogen metabolic enzyme and 5'-AMP-activated protein kinase (AMPK) protein expression according to VMNvl segment. GPR81 stimulates baseline rostral and caudal VMNvl glycogen accumulation but mediates glycogen breakdown in the former site during hypoglycemia. During euglycemia, GPR81 suppresses the transmitter marker neuronal nitric oxide synthase (nNOS) in rostral and caudal VMNvl nitrergic neurons, but stimulates (rostral VMNvl) or inhibits (caudal VMNvl) GABAergic neuron glutamate decarboxylase65/67 (GAD)protein. During hypoglycemia, GPR81 regulates AMPK activation in nitrergic and GABAergic neurons located in the rostral, but not caudal VMNvl. VMN GPR81 knockdown amplified hypoglycemic hypercorticosteronemia, but not hyperglucagonemia. Results provide novel evidence that VMNvl astrocyte and glucose-regulatory neurons express GPR81 protein. Data identify neuroanatomical subpopulations of VMNvl astrocytes and glucose-regulatory neurons that exhibit differential reactivity to GPR81 input. Heterogeneous GPR81 effects during eu- versus hypoglycemia infer that energy state may affect cellular sensitivity to or postreceptor processing of lactate transmitter signaling.

Contribution of the co-chaperone FKBP51 in the ventromedial hypothalamus to metabolic homeostasis in male and female mice.

OBJECTIVE: Steroidogenic factor 1 (SF1) expressing neurons in the ventromedial hypothalamus (VMH) have been directly implicated in whole-body metabolism and in the onset of obesity. The co-chaperone FKBP51 is abundantly expressed in the VMH and was recently linked to type 2 diabetes, insulin resistance, adipogenesis, browning of white adipose tissue (WAT) and bodyweight regulation. METHODS: We investigated the role of FKBP51 in the VMH by conditional deletion and virus-mediated overexpression of FKBP51 in SF1-positive neurons. Baseline and high fat diet (HFD)-induced metabolic- and stress-related phenotypes in male and female mice were obtained. RESULTS: In contrast to previously reported robust phenotypes of FKBP51 manipulation in the entire mediobasal hypothalamus (MBH), selective deletion or overexpression of FKBP51 in the VMH resulted in only a moderate alteration of HFD-induced bodyweight gain and body composition, independent of sex. CONCLUSIONS: Overall, this study shows that animals lacking and overexpressing Fkbp5 in Sf1-expressing cells within the VMH display only a mild metabolic phenotype compared to an MBH-wide manipulation of this gene, suggesting that FKBP51 in SF1 neurons within this hypothalamic nucleus plays a subsidiary role in controlling whole-body metabolism.

Female sexual behavior is disrupted in a preclinical mouse model of PCOS via an attenuated hypothalamic nitric oxide pathway.

Women with polycystic ovary syndrome (PCOS) frequently experience decreased sexual arousal, desire, and sexual satisfaction. While the hypothalamus is known to regulate sexual behavior, the specific neuronal pathways affected in patients with PCOS are not known. To dissect the underlying neural circuitry, we capitalized on a robust preclinical animal model that reliably recapitulates all cardinal PCOS features. We discovered that female mice prenatally treated with anti-Müllerian hormone (PAMH) display impaired sexual behavior and sexual partner preference over the reproductive age. Blunted female sexual behavior was associated with increased sexual rejection and independent of sex steroid hormone status. Structurally, sexual dysfunction was associated with a substantial loss of neuronal nitric oxide synthase (nNOS)-expressing neurons in the ventromedial nucleus of the hypothalamus (VMH) and other areas of hypothalamic nuclei involved in social behaviors. Using in vivo chemogenetic manipulation, we show that nNOSVMH neurons are required for the display of normal sexual behavior in female mice and that pharmacological replenishment of nitric oxide restores normal sexual performance in PAMH mice. Our data provide a framework to investigate facets of hypothalamic nNOS neuron biology with implications for sexual disturbances in PCOS.

Leptin receptor-expressing cells in the ventromedial nucleus of the hypothalamus contribute to enhanced CCK-induced satiety following central leptin injection.

Others have shown that leptin and cholecystokinin (CCK) act synergistically to suppress food intake. Experiments described here tested whether leptin in the ventromedial hypothalamus (VMH) contributes to the synergy with peripheral CCK in male Sprague Dawley rats. A subthreshold injection of 50-ng leptin into the VMH 1 h before a peripheral injection of 1 µg/kg CCK did not change the response to CCK in rats offered chow or low-fat purified diet, but did exaggerate the reduction in intake of high-fat diet 30 min and 1 h after injection in rats that had been food deprived for 8 h. By contrast, deletion of leptin receptor-expressing cells in the VMH using leptin-conjugated saporin (Lep-Sap) abolished the response to peripheral CCK in chow-fed rats. Lateral ventricle injection of 2-µg leptin combined with peripheral CCK exaggerated the inhibition of chow intake for up to 6 h in control rats treated with Blank-saporin, but not in Lep-Sap rats. Blank-Saporin rats offered low- or high-fat purified diet also demonstrated a dose-response inhibition of intake that reached significance with 1 µg/kg of CCK for both diets. CCK did not inhibit intake of Lep-Sap rats in either low- or high-fat-fed rats. Thus, although basal activation of VMH leptin receptors makes a significant contribution to the synergy with CCK, increased leptin activity in the VMH does not exaggerate the response to CCK in intact rats offered low-fat diets, but does enhance the response in those offered high-fat diet.NEW & NOTEWORTHY Leptin is a feedback signal in the control of energy balance, whereas cholecystokinin (CCK) is a short-term satiety signal that inhibits meal size. The two hormones synergize to promote satiety. We tested whether leptin receptors in the ventromedial nucleus of the hypothalamus (VMH) contribute to the synergy. The results suggest that there is a requirement for a baseline level of activation of leptin receptors in the VMH in order for CCK to promote satiety.

Ubiquitin-Specific Protease 2 in the Ventromedial Hypothalamus Modifies Blood Glucose Levels by Controlling Sympathetic Nervous Activation.

Ubiquitin-specific protease 2 (USP2) participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscles. However, the glucoregulatory role of USP2 in the CNS is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH, using a Usp2-specific probe, showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, whereas it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells and subsequently promoted the phosphorylation of AMP-activated protein kinase (AMPK). Consistent with these findings, hypothalamic ML364 treatment stimulated AMPKα phosphorylation in the VMH. Inhibition of hypothalamic AMPK prevented ML364 from increasing serum norepinephrine and blood glucose. Removal of ROS restored the ML364-evoked mitochondrial dysfunction in SH-SY5Y cells and impeded the ML364-induced hypothalamic AMPKα phosphorylation as well as prevented the elevation of serum norepinephrine and blood glucose levels in male mice. These results indicate hypothalamic USP2 attenuates perturbations in blood glucose levels by modifying the ROS-AMPK-sympathetic nerve axis.SIGNIFICANCE STATEMENT Under normal conditions (excluding hyperglycemia or hypoglycemia), blood glucose levels are maintained at a constant level. In this study, we used a mouse model to identify a hypothalamic protease controlling blood glucose levels. Pharmacological inhibition of USP2 in the VMH caused a deviation in blood glucose levels under a nonstressed condition, indicating that USP2 determines the set point of the blood glucose level. Modification of sympathetic nervous activity accounts for the USP2-mediated glucoregulation. Mechanistically, USP2 mitigates the accumulation of ROS in the VMH, resulting in attenuation of the phosphorylation of AMPK. Based on these findings, we uncovered a novel glucoregulatory axis consisting of hypothalamic USP2, ROS, AMPK, and the sympathetic nervous system.

Effect of Propionic Acid on Diabetes-Induced Impairment of Unfolded Protein Response Signaling and Astrocyte/Microglia Crosstalk in Rat Ventromedial Nucleus of the Hypothalamus.

Background: The aim was to investigate the influence of propionic acid (PA) on the endoplasmic reticulum (ER), unfolded protein response (UPR) state, and astrocyte/microglia markers in rat ventromedial hypothalamus (VMH) after type 2 diabetes mellitus (T2DM). Methods: Male Wistar rats were divided: (1) control, (2) T2DM, and groups that received the following (14 days, orally): (3) metformin (60 mg/kg), (4) PA (60 mg/kg), and (5) PA+metformin. Western blotting, RT-PCR, transmission electron microscopy, and immunohistochemical staining were performed. Results: We found T2DM-associated enlargement of ER cisterns, while drug administration slightly improved VMH ultrastructural signs of damage. GRP78 level was 2.1-fold lower in T2DM vs. control. Metformin restored GRP78 to control, while PA increased it by 2.56-fold and metformin+PA-by 3.28-fold vs. T2DM. PERK was elevated by 3.61-fold in T2DM, after metformin-by 4.98-fold, PA-5.64-fold, and metformin+PA-3.01-fold vs. control. A 2.45-fold increase in ATF6 was observed in T2DM. Metformin decreased ATF6 content vs. T2DM. Interestingly, PA exerted a more pronounced lowering effect on ATF6, while combined treatment restored ATF6 to control. IRE1 increased in T2DM (2.4-fold), metformin (1.99-fold), and PA (1.45-fold) groups vs. control, while metformin+PA fully normalized its content. The Iba1 level was upregulated in T2DM (5.44-fold) and metformin groups (6.88-fold). Despite PA treatment leading to a further 8.9-fold Iba1 elevation, PA+metformin caused the Iba1 decline vs. metformin and PA treatment. GFAP level did not change in T2DM but rose in metformin and PA groups vs. control. PA+metformin administration diminished GFAP vs. PA. T2DM-induced changes were associated with dramatically decreased ZO-1 levels, while PA treatment increased it almost to control values. Conclusions: T2DM-induced UPR imbalance, activation of microglia, and impairments in cell integrity may trigger VMH dysfunction. Drug administration slightly improved ultrastructural changes in VMH, normalized UPR, and caused an astrocyte activation. PA and metformin exerted beneficial effects for counteracting diabetes-induced ER stress in VMH.

Prolactin-mediated restraint of maternal aggression in lactation.

Aggressive behavior is rarely observed in virgin female mice but is specifically triggered in lactation where it facilitates protection of offspring. Recent studies demonstrated that the hypothalamic ventromedial nucleus (VMN) plays an important role in facilitating aggressive behavior in both sexes. Here, we demonstrate a role for the pituitary hormone, prolactin, acting through the prolactin receptor in the VMN to control the intensity of aggressive behavior exclusively during lactation. Prolactin receptor deletion from glutamatergic neurons or specifically from the VMN resulted in hyperaggressive lactating females, with a marked shift from intruder-directed investigative behavior to very high levels of aggressive behavior. Prolactin-sensitive neurons in the VMN project to a wide range of other hypothalamic and extrahypothalamic regions, including the medial preoptic area, paraventricular nucleus, and bed nucleus of the stria terminalis, all regions known to be part of a complex neuronal network controlling maternal behavior. Within this network, prolactin acts in the VMN to specifically restrain male-directed aggressive behavior in lactating females. This action in the VMN may complement the role of prolactin in other brain regions, by shifting the balance of maternal behaviors from defense-related activities to more pup-directed behaviors necessary for nurturing offspring.

Hemoglobin in the blood acts as a chemosensory signal via the mouse vomeronasal system.

The vomeronasal system plays an essential role in sensing various environmental chemical cues. Here we show that mice exposed to blood and, consequently, hemoglobin results in the activation of vomeronasal sensory neurons expressing a specific vomeronasal G protein-coupled receptor, Vmn2r88, which is mediated by the interaction site, Gly17, on hemoglobin. The hemoglobin signal reaches the medial amygdala (MeA) in both male and female mice. However, it activates the dorsal part of ventromedial hypothalamus (VMHd) only in lactating female mice. As a result, in lactating mothers, hemoglobin enhances digging and rearing behavior. Manipulation of steroidogenic factor 1 (SF1)-expressing neurons in the VMHd is sufficient to induce the hemoglobin-mediated behaviors. Our results suggest that the oxygen-carrier hemoglobin plays a role as a chemosensory signal, eliciting behavioral responses in mice in a state-dependent fashion.