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.

Oestrogen engages brain MC4R signalling to drive physical activity in female mice.

Oestrogen depletion in rodents and humans leads to inactivity, fat accumulation and diabetes1,2, underscoring the conserved metabolic benefits of oestrogen that inevitably decrease with age. In rodents, the preovulatory surge in 17ß-oestradiol (E2) temporarily increases energy expenditure to coordinate increased physical activity with peak sexual receptivity. Here we report that a subset of oestrogen-sensitive neurons in the ventrolateral ventromedial hypothalamic nucleus (VMHvl)3-7 projects to arousal centres in the hippocampus and hindbrain, and enables oestrogen to rebalance energy allocation in female mice. Surges in E2 increase melanocortin-4 receptor (MC4R) signalling in these VMHvl neurons by directly recruiting oestrogen receptor-α (ERα) to the Mc4r gene. Sedentary behaviour and obesity in oestrogen-depleted female mice were reversed after chemogenetic stimulation of VMHvl neurons expressing both MC4R and ERα. Similarly, a long-term increase in physical activity is observed after CRISPR-mediated activation of this node. These data extend the effect of MC4R signalling - the most common cause of monogenic human obesity8 - beyond the regulation of food intake and rationalize reported sex differences in melanocortin signalling, including greater disease severity of MC4R insufficiency in women9. This hormone-dependent node illuminates the power of oestrogen during the reproductive cycle in motivating behaviour and maintaining an active lifestyle in women.

Spike Activity in the Ventromedial Nucleus of Rat Hypothalamus during Aging.

Spike activity of neurons in the ventromedial nucleus (VMN) of the hypothalamus in adult (6-8 months) and aged (2 years) male rats was studied by the in vivo extracellular method using stereotaxic insertion of microelectrodes. In all animals, firing frequency of most VMN neurons increased in response to glucose administration. However, in aged rats, the mean baseline and glucose-induced spike frequencies of VMN neurons were lower than in adult animals. These results support the hypothesis that aging is associated with a decrease in the functional activity of hypothalamic neurons.

A hypothalamic-thalamostriatal circuit that controls approach-avoidance conflict in rats.

Survival depends on a balance between seeking rewards and avoiding potential threats, but the neural circuits that regulate this motivational conflict remain largely unknown. Using an approach-food vs. avoid-predator threat conflict test in rats, we identified a subpopulation of neurons in the anterior portion of the paraventricular thalamic nucleus (aPVT) which express corticotrophin-releasing factor (CRF) and are preferentially recruited during conflict. Inactivation of aPVTCRF neurons during conflict biases animal's response toward food, whereas activation of these cells recapitulates the food-seeking suppression observed during conflict. aPVTCRF neurons project densely to the nucleus accumbens (NAc), and activity in this pathway reduces food seeking and increases avoidance. In addition, we identified the ventromedial hypothalamus (VMH) as a critical input to aPVTCRF neurons, and demonstrated that VMH-aPVT neurons mediate defensive behaviors exclusively during conflict. Together, our findings describe a hypothalamic-thalamostriatal circuit that suppresses reward-seeking behavior under the competing demands of avoiding threats.

Cross-species analysis defines the conservation of anatomically segregated VMH neuron populations.

The ventromedial hypothalamic nucleus (VMH) controls diverse behaviors and physiologic functions, suggesting the existence of multiple VMH neural subtypes with distinct functions. Combing translating ribosome affinity purification with RNA-sequencing (TRAP-seq) data with single-nucleus RNA-sequencing (snRNA-seq) data, we identified 24 mouse VMH neuron clusters. Further analysis, including snRNA-seq data from macaque tissue, defined a more tractable VMH parceling scheme consisting of six major genetically and anatomically differentiated VMH neuron classes with good cross-species conservation. In addition to two major ventrolateral classes, we identified three distinct classes of dorsomedial VMH neurons. Consistent with previously suggested unique roles for leptin receptor (Lepr)-expressing VMH neurons, Lepr expression marked a single dorsomedial class. We also identified a class of glutamatergic VMH neurons that resides in the tuberal region, anterolateral to the neuroanatomical core of the VMH. This atlas of conserved VMH neuron populations provides an unbiased starting point for the analysis of VMH circuitry and function.

Decoding neuronal composition and ontogeny of individual hypothalamic nuclei.

The hypothalamus plays crucial roles in regulating endocrine, autonomic, and behavioral functions via its diverse nuclei and neuronal subtypes. The developmental mechanisms underlying ontogenetic establishment of different hypothalamic nuclei and generation of neuronal diversity remain largely unknown. Here, we show that combinatorial T-box 3 (TBX3), orthopedia homeobox (OTP), and distal-less homeobox (DLX) expression delineates all arcuate nucleus (Arc) neurons and defines four distinct subpopulations, whereas combinatorial NKX2.1/SF1 and OTP/DLX expression identifies ventromedial hypothalamus (VMH) and tuberal nucleus (TuN) neuronal subpopulations, respectively. Developmental analysis indicates that all four Arc subpopulations are mosaically and simultaneously generated from embryonic Arc progenitors, whereas glutamatergic VMH neurons and GABAergic TuN neurons are sequentially generated from common embryonic VMH progenitors. Moreover, clonal lineage-tracing analysis reveals that diverse lineages from multipotent radial glia progenitors orchestrate Arc and VMH-TuN establishment. Together, our study reveals cellular mechanisms underlying generation and organization of diverse neuronal subtypes and ontogenetic establishment of individual nuclei in the mammalian hypothalamus.

Hypoglycemia-Sensing Neurons of the Ventromedial Hypothalamus Require AMPK-Induced Txn2 Expression but Are Dispensable for Physiological Counterregulation.

The ventromedial nucleus of the hypothalamus (VMN) is involved in the counterregulatory response to hypoglycemia. VMN neurons activated by hypoglycemia (glucose-inhibited [GI] neurons) have been assumed to play a critical although untested role in this response. Here, we show that expression of a dominant negative form of AMPK or inactivation of AMPK α1 and α2 subunit genes in Sf1 neurons of the VMN selectively suppressed GI neuron activity. We found that Txn2, encoding a mitochondrial redox enzyme, was strongly downregulated in the absence of AMPK activity and that reexpression of Txn2 in Sf1 neurons restored GI neuron activity. In cell lines, Txn2 was required to limit glucopenia-induced reactive oxygen species production. In physiological studies, absence of GI neuron activity after AMPK suppression in the VMN had no impact on the counterregulatory hormone response to hypoglycemia or on feeding. Thus, AMPK is required for GI neuron activity by controlling the expression of the antioxidant enzyme Txn2. However, the glucose-sensing capacity of VMN GI neurons is not required for the normal counterregulatory response to hypoglycemia. Instead, it may represent a fail-safe system in case of impaired hypoglycemia sensing by peripherally located glucose detection systems that are connected to the VMN.

Exposure to prenatal PCBs shifts the timing of neurogenesis in the hypothalamus of developing rats.

The developing brain is highly sensitive to the hormonal milieu, with gonadal steroid hormones involved in neurogenesis, neural survival, and brain organization. Limited available evidence suggests that endocrine-disrupting chemicals (EDCs) may perturb these developmental processes. In this study, we tested the hypothesis that prenatal exposure to a mixture of polychlorinated biphenyls (PCBs), Aroclor 1221, would disrupt the normal timing of neurogenesis in two hypothalamic regions: the ventromedial nucleus (VMN) and the preoptic area (POA). These regions were selected because of their important roles in the control of sociosexual behaviors that are perturbed in adulthood by prenatal EDC exposure. Pregnant Sprague-Dawley rats were exposed to PCBs from Embryonic Day 8 (E8) to E18, encompassing the period of neurogenesis of all hypothalamic neurons. To determine the birth dates of neurons, bromo-2-deoxy-5-uridine (BrdU) was administered to dams on E12, E14, or E16. On the day after birth, male and female pups were perfused, brains immunolabeled for BrdU, and numbers of cells counted. In the VMN, exposure to PCBs significantly advanced the timing of neurogenesis compared to vehicle-treated pups, without changing the total number of BrdU+ cells. In the POA, PCBs did not change the timing of neurogenesis nor the total number of cells born. This is the first study to show that PCBs can shift the timing of neurogenesis in the hypothalamus, specifically in the VMN but not the POA. This result has implications for functions controlled by the VMN, especially sociosexual behaviors, as well as for sexual selection more generally.

Essential and sex-specific effects of mGluR5 in ventromedial hypothalamus regulating estrogen signaling and glucose balance.

The ventromedial hypothalamus (VMH) plays chief roles regulating energy and glucose homeostasis and is sexually dimorphic. We discovered that expression of metabotropic glutamate receptor subtype 5 (mGluR5) in the VMH is regulated by caloric status in normal mice and reduced in brain-derived neurotrophic factor (BDNF) mutants, which are severely obese and have diminished glucose balance control. These findings led us to investigate whether mGluR5 might act downstream of BDNF to critically regulate VMH neuronal activity and metabolic function. We found that mGluR5 depletion in VMH SF1 neurons did not affect energy balance regulation. However, it significantly impaired insulin sensitivity, glycemic control, lipid metabolism, and sympathetic output in females but not in males. These sex-specific deficits are linked to reductions in intrinsic excitability and firing rate of SF1 neurons. Abnormal excitatory and inhibitory synapse assembly and elevated expression of the GABAergic synthetic enzyme GAD67 also cooperate to decrease and potentiate the synaptic excitatory and inhibitory tone onto mutant SF1 neurons, respectively. Notably, these alterations arise from disrupted functional interactions of mGluR5 with estrogen receptors that switch the normally positive effects of estrogen on SF1 neuronal activity and glucose balance control to paradoxical and detrimental. The collective data inform an essential central mechanism regulating metabolic function in females and underlying the protective effects of estrogen against metabolic disease.

TrkB-expressing paraventricular hypothalamic neurons suppress appetite through multiple neurocircuits.

The TrkB receptor is critical for the control of energy balance, as mutations in its gene (NTRK2) lead to hyperphagia and severe obesity. The main neural substrate mediating the appetite-suppressing activity of TrkB, however, remains unknown. Here, we demonstrate that selective Ntrk2 deletion within paraventricular hypothalamus (PVH) leads to severe hyperphagic obesity. Furthermore, chemogenetic activation or inhibition of TrkB-expressing PVH (PVHTrkB) neurons suppresses or increases food intake, respectively. PVHTrkB neurons project to multiple brain regions, including ventromedial hypothalamus (VMH) and lateral parabrachial nucleus (LPBN). We find that PVHTrkB neurons projecting to LPBN are distinct from those to VMH, yet Ntrk2 deletion in PVH neurons projecting to either VMH or LPBN results in hyperphagia and obesity. Additionally, TrkB activation with BDNF increases firing of these PVH neurons. Therefore, TrkB signaling is a key regulator of a previously uncharacterized neuronal population within the PVH that impinges upon multiple circuits to govern appetite.

Hierarchical Representations of Aggression in a Hypothalamic-Midbrain Circuit.

Although the ventromedial hypothalamus ventrolateral area (VMHvl) is now well established as a critical locus for the generation of conspecific aggression, its role is complex, with neurons responding during multiple phases of social interactions with both males and females. It has been previously unclear how the brain uses this complex multidimensional signal and coordinates a discrete action: the attack. Here, we find a hypothalamic-midbrain circuit that represents hierarchically organized social signals during aggression. Optogenetic-assisted circuit mapping reveals a preferential projection from VMHvlvGlut2 to lPAGvGlut2 cells, and inactivation of downstream lPAGvGlut2 populations results in aggression-specific deficits. lPAG neurons are selective for attack action and exhibit short-latency, time-locked spiking relative to the activity of jaw muscles during biting. Last, we find that this projection conveys male-biased signals from the VMHvl to downstream lPAGvGlut2 neurons that are sensitive to features of ongoing activity, suggesting that action selectivity is generated by a combination of pre- and postsynaptic mechanisms.

Effects of acute versus recurrent insulin-induced hypoglycemia on ventromedial hypothalamic nucleus metabolic-sensory neuron AMPK activity: Impact of alpha1-adrenergic receptor signaling.

Mechanisms that underlie metabolic sensor acclimation to recurring insulin-induced hypoglycemia (RIIH) are unclear. Norepinephrine (NE) regulates ventromedial hypothalamic nucleus (VMN) gluco-stimulatory nitric oxide (NO) and gluco-inhibitory γ-aminobutryic acid (GABA) neuron signaling. Current research addressed the hypothesis that during RIIH, NE suppresses 5'-AMP-activated protein kinase (AMPK) reactivity in both populations and impedes counter-regulation. The brain is postulated to utilize non-glucose substrates, e.g. amino acids glutamine (Gln), glutamate (Glu), and aspartate (Asp), to produce energy during hypoglycemia. A correlated aim investigated whether NE controls pyruvate recycling pathway marker protein (glutaminase, GLT; malic enzyme, ME-1) expression in either metabolic-sensory cell population. Male rats were injected subcutaneously with vehicle or insulin on days 1-3, then pretreated on day 4 by intracerebroventricular delivery of the alpha1-adrenergic receptor (α1-AR) reverse-agonist prazocin (PRZ) or vehicle before final insulin therapy. PRZ prevented acute hypoglycemic augmentation of AMPK activation in each cell group. Antecedent hypoglycemic repression of sensor activity was reversed by PRZ in GABA neurons. During RIIH, nitrergic neurons exhibited α1-AR - dependent up-regulated GLT and α2-AR profiles, while GABA cells showed down-regulated α1-AR. LC-ESI-MS analysis documented a decline in VMN Glu, Gln, and Asp concentrations during acute hypoglycemia, and habituation of the former two profiles to RIIH. PRZ attenuated glucagon and corticosterone secretion during acute hypoglycemia, but reversed decrements in output of both hormones during RIIH. Results implicate adjustments in impact of α1-AR signaling in repressed VMN metabolic-sensory AMPK activation and counter-regulatory dysfunction during RIIH. Antecedent hypoglycemia may up-regulate NO neuron energy yield via α1-AR - mediated up-regulated pyruvate recycling.

Effects of Intracerebroventricular Glycogen Phosphorylase Inhibitor CP-316,819 Infusion on Hypothalamic Glycogen Content and Metabolic Neuron AMPK Activity and Neurotransmitter Expression in Male Rat.

Brain glycogen is a vital energy source during metabolic imbalance. Metabolic sensory neurons in the ventromedial hypothalamic nucleus (VMN) shape glucose counter-regulation. Insulin-induced hypoglycemic (IIH) male rats were infused icv with the glycogen breakdown inhibitor CP-316,819 (CP) to investigate whether glycogen-derived fuel controls basal and/or hypoglycemic patterns of VMN gluco-regulatory neuron energy stability and transmitter signaling. CP caused dose-dependent amplification of basal VMN glycogen content and either mobilization (low dose) or augmentation (high dose) of this depot during IIH. Drug treatment also prevented hypoglycemic diminution of tissue glucose in multiple structures. Low CP dose caused IIH-reversible augmentation of AMPK activity and glutamate decarboxylase (GAD) protein levels in laser-microdissected VMN GABA neurons, while the higher dose abolished hypoglycemic adjustments in these profiles. VMN steroidogenic factor-1 (SF-1) neurons exhibited suppressed (low CP dose) or unchanged (high CP dose) basal SF-1 expression and AMPK refractoriness of hypoglycemia at each dose. CP caused dose-proportionate augmentation of neuronal nitric oxide synthase protein and enhancement (low dose) or diminution (high dose) of this profile during IIH; AMPK activity in these cells was decreased in high dose-pretreated IIH rats. CP exerted dose-dependent effects on basal and hypoglycemic patterns of glucagon, but not corticosterone secretion. Results verify that VMN GABA, SF-1, and nitrergic neurons are metabolic sensory in function and infer that these populations may screen unique aspects of neurometabolic instability. Correlation of VMN glycogen augmentation with attenuated hypoglycemic VMN gluco-regulatory neuron AMPK activity implies that expansion of this fuel reservoir preserves cellular energy stability during this metabolic threat.

Emergent decision-making behaviour and rhythm generation in a computational model of the ventromedial nucleus of the hypothalamus.

The ventromedial nucleus of the hypothalamus (VMN) has an important role in diverse behaviours. The common involvement in these of sex steroids, nutritionally-related signals, and emotional inputs from other brain areas, suggests that, at any given time, its output is in one of a discrete number of possible states corresponding to discrete motivational drives. Here we explored how networks of VMN neurons might generate such a decision-making architecture. We began with minimalist assumptions about the intrinsic properties of VMN neurons inferred from electrophysiological recordings of these neurons in rats in vivo, using an integrate-and-fire based model modified to simulate activity-dependent post-spike changes in neuronal excitability. We used a genetic algorithm based method to fit model parameters to the statistical features of spike patterning in each cell. The spike patterns in both recorded cells and model cells were assessed by analysis of interspike interval distributions and of the index of dispersion of firing rate over different binwidths. Simpler patterned cells could be closely matched by single neuron models incorporating a hyperpolarising afterpotential and either a slow afterhyperpolarisation or a depolarising afterpotential, but many others could not. We then constructed network models with the challenge of explaining the more complex patterns. We assumed that neurons of a given type (with heterogeneity introduced by independently random patterns of external input) were mutually interconnected at random by excitatory synaptic connections (with a variable delay and a random chance of failure). Simple network models of one or two cell types were able to explain the more complex patterns. We then explored the information processing features of such networks that might be relevant for a decision-making network. We concluded that rhythm generation (in the slow theta range) and bistability arise as emergent properties of networks of heterogeneous VMN neurons.

Connectional architecture of a mouse hypothalamic circuit node controlling social behavior.

Type 1 estrogen receptor-expressing neurons in the ventrolateral subdivision of the ventromedial hypothalamus (VMHvlEsr1) play a causal role in the control of social behaviors, including aggression. Here we use six different viral-genetic tracing methods to systematically map the connectional architecture of VMHvlEsr1 neurons. These data reveal a high level of input convergence and output divergence ("fan-in/fan-out") from and to over 30 distinct brain regions, with a high degree (∼90%) of bidirectionality, including both direct as well as indirect feedback. Unbiased collateralization mapping experiments indicate that VMHvlEsr1 neurons project to multiple targets. However, we identify two anatomically distinct subpopulations with anterior vs. posterior biases in their collateralization targets. Nevertheless, these two subpopulations receive indistinguishable inputs. These studies suggest an overall system architecture in which an anatomically feed-forward sensory-to-motor processing stream is integrated with a dense, highly recurrent central processing circuit. This architecture differs from the "brain-inspired," hierarchical feed-forward circuits used in certain types of artificial intelligence networks.

Divergent medial amygdala projections regulate approach-avoidance conflict behavior.

Avoidance of innate threats is often in conflict with motivations to engage in exploratory approach behavior. The neural pathways that mediate this approach-avoidance conflict are not well resolved. Here we isolated a population of dopamine D1 receptor (D1R)-expressing neurons within the posteroventral region of the medial amygdala (MeApv) in mice that are activated either during approach or during avoidance of an innate threat stimulus. Distinct subpopulations of MeApv-D1R neurons differentially innervate the ventromedial hypothalamus and bed nucleus of the stria terminalis, and these projections have opposing effects on investigation or avoidance of threatening stimuli. These projections are potently modulated through opposite actions of D1R signaling that bias approach behavior. These data demonstrate divergent pathways in the MeApv that can be differentially weighted toward exploration or evasion of threats.

Norepinephrine control of ventromedial hypothalamic nucleus glucoregulatory neurotransmitter expression in the female rat: Role of monocarboxylate transporter function.

The ventromedial hypothalamic nucleus (VMN) is a critical component of the neural circuitry that regulates glucostasis. Astrocyte glycogen is a vital reserve of glucose and its oxidizable metabolite L-lactate. In hypoglycemic female rats, estradiol-dependent augmentation of VMN glycogen phosphorylase (GP) protein requires hindbrain catecholamine input. Research here investigated the premise that norepinephrine (NE) regulation of VMN astrocyte metabolism shapes local glucoregulatory neurotransmitter signaling in this sex. Estradiol-implanted ovariectomized rats were pretreated by intra-VMN administration of the monocarboxylate transporter inhibitor alpha-cyano-4-hydroxy-cinnamic acid (4CIN) or vehicle before NE delivery to that site. NE caused 4CIN-reversible reduction or augmentation of VMN glycogen synthase and phosphorylase expression. 4CIN prevented NE stimulation of gluco-inhibitory (glutamate decarboxylase65/67) and suppression of gluco-stimulatory (neuronal nitric oxide synthase) neuron marker proteins. These outcomes imply that effects of noradrenergic stimulation of VMN astrocyte glycogen depletion on glucoregulatory transmitter signaling may be mediated, in part, by glycogen-derived substrate fuel provision. NE control of astrocyte glycogen metabolism may involve down-regulated adrenoreceptor (AR), e.g. alpha1 and alpha2, alongside amplified beta1 AR and estrogen receptor-beta signaling. Noradrenergic hypoglycemia was refractory to 4CIN, implying that additional NE-sensitive VMN glucoregulatory neurochemicals may be insensitive to monocarboxylate uptake. Augmentation of circulating free fatty acids by combinatory NE and 4CIN, but not NE alone implies that acute hypoglycemia induced here is an insufficient stimulus for mobilization of these fuels, but is adequate when paired with diminished brain monocarboxylate fuel availability.

A circuit from hippocampal CA2 to lateral septum disinhibits social aggression.

Although the hippocampus is known to be important for declarative memory, it is less clear how hippocampal output regulates motivated behaviours, such as social aggression. Here we report that pyramidal neurons in the CA2 region of the hippocampus, which are important for social memory, promote social aggression in mice. This action depends on output from CA2 to the lateral septum, which is selectively enhanced immediately before an attack. Activation of the lateral septum by CA2 recruits a circuit that disinhibits a subnucleus of the ventromedial hypothalamus that is known to trigger attack. The social hormone arginine vasopressin enhances social aggression by acting on arginine vasopressin 1b receptors on CA2 presynaptic terminals in the lateral septum to facilitate excitatory synaptic transmission. In this manner, release of arginine vasopressin in the lateral septum, driven by an animal's internal state, may serve as a modulatory control that determines whether CA2 activity leads to declarative memory of a social encounter and/or promotes motivated social aggression.

Optogenetic Study of Anterior BNST and Basomedial Amygdala Projections to the Ventromedial Hypothalamus.

The basomedial amygdala (BM) influences the ventromedial nucleus of the hypothalamus (VMH) through direct glutamatergic projections as well as indirectly, through the anterior part of the bed nucleus of the stria terminalis (BNSTa). However, BM and BNSTa axons end in a segregated fashion in VMH. BM projects to the core of VMH, where VMH's projection cells are located, whereas BNSTa projects to the shell of VMH, where GABAergic cells that inhibit core neurons are concentrated. However, the consequences of this dual regulation of VMH by BM and BNSTa are unknown. To study this question, we recorded the responses of VMH's shell and core neurons to the optogenetic activation of BM or BNSTa inputs in transgenic mice that selectively express Cre-recombinase in glutamatergic or GABAergic neurons. Glutamatergic BM inputs fired most core neurons but elicited no response in GABAergic shell neurons. Following BM infusions of AAV-EF1α-DIO-hChR2-mCherry in Vgat-ires-Cre-Ai6 mice, no anterograde labeling was observed in the VMH, suggesting that GABAergic BM neurons do not project to the VMH. In contrast, BNSTa sent mostly GABAergic projections that inhibited both shell and core neurons. However, BNSTa-evoked IPSPs had a higher amplitude in shell neurons. Since we also found that activation of GABAergic shell neurons causes an inhibition of core neurons, these results suggest that depending on the firing rate of shell neurons, BNSTa inputs could elicit a net inhibition or disinhibition of core neurons. Thus, the dual regulation of VMH by BM and BNSTa imparts flexibility to this regulator of defensive and social behaviors.

PACAP neurons in the ventral premammillary nucleus regulate reproductive function in the female mouse.

Pituitary adenylate cyclase activating polypeptide (PACAP, Adcyap1) is a neuromodulator implicated in anxiety, metabolism and reproductive behavior. PACAP global knockout mice have decreased fertility and PACAP modulates LH release. However, its source and role at the hypothalamic level remain unknown. We demonstrate that PACAP-expressing neurons of the ventral premamillary nucleus of the hypothalamus (PMVPACAP) project to, and make direct contact with, kisspeptin neurons in the arcuate and AVPV/PeN nuclei and a subset of these neurons respond to PACAP exposure. Targeted deletion of PACAP from the PMV through stereotaxic virally mediated cre- injection or genetic cross to LepR-i-cre mice with Adcyap1fl/fl mice led to delayed puberty onset and impaired reproductive function in female, but not male, mice. We propose a new role for PACAP-expressing neurons in the PMV in the relay of nutritional state information to regulate GnRH release by modulating the activity of kisspeptin neurons, thereby regulating reproduction in female mice.