Journal of Neuroendocrinology

Neural circuits in female rats are exposed to estradiol and sequential progesterone to regulate the luteinizing hormone (LH) surge and thus ovulation. Estradiol induces progesterone receptors (PGRs) in rostral periventricular region of the third ventricle (RP3V) kisspeptin neurons, and positive feedback estradiol concentrations induce neuroprogesterone (neuroP) synthesis in hypothalamic astrocytes that signal to PGRs expressed in kisspeptin neurons to trigger the LH surge. We tested the hypothesis that neuroP-PGR signals through Src family kinase (Src) to trigger the LH surge. As in vitro, PGR and Src are co-expressed in RP3V neurons. Estradiol treatment increased the number of PGR immunopositive cells and PGR and Src colocalization. Infusion of the Src inhibitor (PP2) into the RP3V, attenuated the LH surge measured by ELISA in trunk blood collected 53 hours post-EB injection. While PP2 reduced the LH surge in 50 g EB treated ovariectomized/adrenalectomized (ovx/adx) rats, activation of either PGR or Src in 2g EB primed animals significantly elevated LH concentrations compared with DMSO treated ovx/adx rats. These results support the importance of Src in the estradiol and neuroP triggering of the LH surge.

Ghrelin is a gut-derived hormone that is secreted during conditions of negative caloric balance and acts as a key modulator of feeding, increasing food intake and affecting several physiological systems such as metabolism, behavior and the control of endocrine and autonomic functions. Previous studies showed that ghrelin participates in the stress response, acting on hypothalamic paraventricular nucleus neurons that express corticotropin-releasing hormone (PVNCRH neurons). In the present study, we investigated the effects of ghrelin administration on the behavioral responses to restraint stress in mice. In their homecage, C57Bl6 mice in basal conditions expressed the behaviors of surveying, walking, rearing, grooming and, to a lesser extent, digging, climbing and freezing. Restraint stress increased the time spent in grooming without significant changes in other behaviors. Ghrelin administration did not affect behavior in control mice, but it reversed the effect of restraint stress on grooming. Chemogenetic activation of PVNCRH neurons by clozapine N-Oxide (CNO) administration in hM3Dq DREADD mice increased grooming, while ghrelin mitigated this effect. In addition, CNO administration decreased walking and rearing, both in the presence or absence of ghrelin. Food intake was increased by ghrelin administration, however, it was not affected by stress or CNO. These results indicate that ghrelin decreases the activity of PVNCRH neurons, partially preventing the behavioral effects of restraint stress. The inhibitory input to PVNCRH neurons probably arrives from other nuclei, since GABAergic neurons were not identified in the PVN neurons of these mice.

1.Corticotropin-releasing hormone (CRH) neurons in the paraventricular hypothalamic nucleus (Pa) are in the position to integrate stress-related information and initiate adaptive neuroendocrine-, autonomic-, metabolic- and behavioral responses. In addition to hypophyseotropic cells, CRH is widely expressed in the CNS, however their involvement in organization of the stress response is not fully understood. In these experiments, we took the advantage of recently available Crh-IRES-Cre; Ai9 mouse line to study the recruitment of hypothalamic and extrahypothalamic CRH neurons in categorically distinct, acute stress reactions. Reporter mice were exposed to restraint, ether, high salt and lipopolisacharide stress. The induced activation of CRH neurons was detected by colocalization of immediate early gene c-Fos in Td tomato expressing cells. We found differential activation of CRH neurons in central amygdaloid nucleus (Ce), bed nucleus stria terminalis lateral division, ventral posterior (BSTLVP), medial preoptic nucleus (MPO), ventromedial hypothalamic nucleus (VMH), premammillary nucleus (PM) and prepositus hypoglossal nucleus (Pr) in response to physiological (ether, high salt and LPS) and psychological (restraint) stressors. CRH positive cells in Pa became activated, however in the Barringtons nucleus and locus coeruleus no actiovation could be observed due to most tested stressors. Furthermore no CRH neuron activation occured in dorsal (BSTLD) and posterior (BSTLP) region of lateral division of bed nucleus stria terminalis after restraint stress. In the inferior olive only ether exposition resulted in CRH neuron activation. These results confirm activation of CRH neurons in the Pa, reveals new subset of stress-related CRH cells through the mouse brain and disprove the recruitment of CRH cells in the SOL and in the Barringtons nucleus to acute psychological stress in mice.

Interoceptive feedback to the brain regarding the bodys physiological state plays an important role in guiding motivated behaviors. For example, a state of negative energy balance tends to increase exploratory/food-seeking behaviors while reducing avoidance behaviors. We recently reported that overnight food deprivation reduces conditioned passive avoidance behavior in male (but not female) rats. Since fasting increases circulating levels of ghrelin, we hypothesized that ghrelin signaling contributes to the ability of fasting to reduce conditioned avoidance. To test this, ad libitum-fed male rats were trained in a passive avoidance procedure using mild footshock. Later, following overnight food deprivation, the same rats were pretreated with ghrelin receptor antagonist (GRA) or saline vehicle 30 min before avoidance testing. GRA restored passive avoidance in fasted rats as measured both by latency to enter and time spent in the shock-paired context. In addition, compared to vehicle-injected fasted rats, fasted rats that received GRA before re-exposure to the shock-paired context displayed more activation of prolactin-releasing peptide (PrRP)-positive noradrenergic neurons in the caudal nucleus of the solitary tract, accompanied by more activation of downstream neurons in the bed nucleus of the stria terminalis and paraventricular nucleus of the hypothalamus. These results support the view that ghrelin signaling contributes to the inhibitory effect of fasting on learned passive avoidance behavior, perhaps by suppressing recruitment of PrRP-positive noradrenergic neurons and their downstream hypothalamic and limbic forebrain targets.

In recent years, our understanding of the physiological role of kisspeptin (Kiss1) has evolved significantly, revealing its pivotal role in shaping sexual differentiation within the brain. Predominantly expressed in the hypothalamus, Kiss1 plays a crucial role in regulating release of the gonadotrophin-releasing hormone (GnRH). Kisspeptin 1 receptor (Kiss1R) exhibits sex-dependent expression in various brain regions, including the medial amygdala and preoptic area (POA) of the hypothalamus, regions underlying sexual development. Importantly, in the critical period for the sexual development in mammals occurs a synchronized surge of Kiss1 and testosterone, highlighting its importance in sexual dimorphism. It has been suggested that the neonatal Kiss1 surge is associated with the buildup of gender-linked social development. We investigated this hypothesis by testing the effect of neonatal blockage of Kiss1 signaling on the sex-like phenotype in rats. We postulate that Kiss1 actions are both time and region-dependent, particularly during the neonatal period, having lasting impact on social interactions. For that we injected intracerebroventricular (ICV) a Kiss1R antagonist, Kp234, in the first 24h of life. Our data showed, that neonatal Kiss1 is determinant in modulating physiological responses, such as weight and testis development in males (N: males Veh = 10; males Kp234 = 10). Also, we document, for the first time, that Kiss1 modulates the testosterone surge in males and inhibits it in females (N: males Vehicle = 3; males Kp234 = 4; females Vehicle = 3; females Kp234 = 4). Furthermore, neonatal Kiss1 blockade modulated the sexual drive in, while the sexual performance in both sexes is maintained males (N: males Vehicle = 8; males Kp234 = 9; females Vehicle = 7; females Kp234 = 8). In conclusion, this study underscores the significance of Kiss1 during the neonatal period, a critical phase for sexual differentiation and neuroplasticity. Neonatal Kiss1 emerges as a key player in the development of a sex-like phenotype in rats. Additionally, our findings show dimorphic patterns in Kiss1 influence. This work raises the intriguing possibility that the early influence of Kiss1 in brain circuitry is pivotal in the modulation of gender and plays a role in the social construction of the bimodality of gender identity.

BackgroundKisspeptinergic signaling is well-established as crucial for regulation of reproduction, but its potential broader role in brain function is less understood. This study investigates the distribution and chemotyping of kisspeptin-expressing neurons within the mouse brain. MethodsRNAscope singleplex, duplex and multiplex in situ hybridization methods were used to assess kisspeptin mRNA (Kiss1) expression and its co-expression with other neuropeptides, excitatory and inhibitory neurotransmitter markers, and sex steroid receptors in intact and gonadectomized young adult mice. ResultsSeven distinct kisspeptin neuronal chemotypes were characterized, including within two novel Kiss1-expressing groups described here for the first time: the ventral premammillary nucleus, and the nucleus of the solitary tract. Kiss1 mRNA was also localized in the soma, and within the dendritic compartment, of hypothalamic neurons. Altered Kiss1 expression following gonadectomy suggests a previously unappreciated role for androgen receptors in regulating kisspeptin signaling. ConclusionThis study provides a detailed chemoanatomical map of kisspeptin-expressing neurons in the brain, highlighting their potential functional diversity. The discovery of new kisspeptin-expressing neuronal populations, and gonadectomy-induced changes in Kiss1 expression patterns, provide a basis for further exploration of non-endocrine roles for kisspeptin in brain function.

Puberty is a critical transition period to achieve fertility and reproductive capacity in all mammalian species. At puberty, the hypothalamic-pituitary-gonadal (HPG) is activated by neuroendocrine changes in the brain. Central to this are Kiss1 neurons that produce kisspeptin, a neuropeptide which is a potent stimulator of gonadotropin releasing hormone (GnRH) secretion. Kiss1 neurons in the arcuate region of the hypothalamus (Kiss1ARC) increase pulsatile secretion of GnRH at puberty. Other developmental maturational changes in the brain are often accompanied by neuronal plasticity changes but this has not been studied in Kiss1 neurons. Electrophysiological characterisation of Kiss1ARC neurons from female mice shows that these neurons undergo profound intrinsic plasticity at puberty with a critical window between 3 and 4 weeks. Immature Kiss1ARC neurons cannot sustain depolarisation-evoked firing for even 500 ms and instead fire a brief burst of high frequency spikes before falling silent. This would make them unsuitable for the sustained activity that is needed to activate GnRH neurons and trigger LH secretion in the HPG axis. After puberty, sustained firing can be maintained, which endows post-puberty Kiss1ARC neurons with a mature physiological phenotype that is amenable to neuropeptide modulation for generation of burst firing and pulsatile release of kisspeptin. There is a corresponding decrease in the threshold for action potential initiation, a more hyperpolarised post-spike trough and a larger medium after-hyperpolarisation (mAHP). Gene expression analysis showed a significant decrease in Scn2a (Nav1.2 channel), Kcnq2 (Kv7.2 channel) and Lrrc55 (BK channel auxiliary {gamma}3-subunit) expression and an increase in Hcn1 (hyperpolarization activated cyclic nucleotide-gated potassium channel) expression which may contribute to the observed electrophysiological changes. Ovariectomy and {beta}-estradiol replacement defined a window of estrogen-dependent plasticity of action potential firing at puberty, such that post-puberty Kiss1ARC neurons achieve a mature physiological phenotype for activation of the HPG axis.

Vasopressin (AVP), a nonapeptide synthesized predominantly by magnocellular hypothalamic neurons, is conveyed to the posterior pituitary via the pituitary stalk, where AVP is secreted into the circulation. Known to regulate blood pressure and water homeostasis, it also modulates diverse social behaviors, such as pair-bonding, social recognition and cognition in mammals including humans. Importantly, AVP modulates social behaviors in a sex-specific manner, perhaps, due to sex differences in the distribution in the brain of AVP and its main receptor AVPR1a. There is a corpus of integrative studies for the expression of AVP and AVPR1a in various brain regions, and their functions in modulating central and peripheral actions. In order to purposefully address sexually dimorphic and novel roles of AVP on central and peripheral functions through its AVPR1a, we utilized RNAscope to map Avp and Avpr1a single transcript expression in the mouse brain. As the most comprehensive atlas of AVP and AVPR1a in the mouse brain, this compendium highlights the importance of newly identified AVP/AVPR1a neuronal nodes that may stimulate further functional studies.

Cholinergic interneurons (ChIs) act as master regulators of striatal output, finely tuning neurotransmission to control motivated behaviors. ChIs are a cellular target of many peptide and hormonal neuromodulators, including corticotropin releasing factor, opioids, insulin and leptin, which can influence an animals behavior by signaling stress, pleasure, pain and nutritional status. However, little is known about how sex hormones via estrogen receptors influence the function of these other neuromodulators. Here, we performed in situ hybridization on mouse striatal tissue to characterize the effect of sex and sex hormones on choline acetyltransferase (Chat), estrogen receptor alpha (Esr1), and corticotropin releasing factor type 1 receptor (Crhr1) expression. Although we did not detect sex differences in ChAT protein levels in the striatum, we found that female mice have more Chat mRNA-expressing neurons than males. At the population level, we observed a sexually dimorphic distribution of Esr1- and Crhr1-expressing ChIs in the ventral striatum that demonstrates an antagonistic correlational relationship, which is abolished by ovariectomy. Only in the NAc did we find a significant population of ChIs that co-express Crhr1 and Esr1. At the cellular level, Crhr1 and Esr1 transcript levels were negatively correlated only during estrus, indicating that changes in sex hormones levels can modulate the interaction between Crhr1 and Esr1 mRNA levels. Together, these data provide evidence for the unique expression and interaction of Esr1 and Crhr1 in ventral striatal ChIs, warranting further investigation into how these transcriptomic patterns might underlie important functions for ChIs at the intersection of stress and reproductive behaviors.

Growth hormone (GH) receptor (GHR), expressed in different brain regions, is known to participate in the regulation of whole-body energy homeostasis and glucose metabolism. However, GH activation of these GHR-expressing neurons is less studied. We have generated a novel GHR-driven Cre recombinase transgenic mouse line (GHRcre) in combination with the floxed tdTomato reporter mouse line we tracked and activated GHR-expressing neurons in different regions of the brain. We focused on neurons of the hypothalamic arcuate nucleus (ARC) where GHR was shown to elicit a negative feedback loop that regulates GH production. We found that ARCGHR+ neurons are co-localized with AgRP, GHRH, and somatostatin neurons, which were activated by GH stimulation. Using designer receptors exclusively activated by designer drugs (DREADDs) to control GHRARC neuronal activity, we revealed that activation of GHRARC neurons was sufficient in regulating distinct aspects of energy balance and glucose metabolism. Overall, our study provides a novel mouse model to study in vivo regulation and physiological function of GHR-expressing neurons in various brain regions. Furthermore, we identified for the first time specific neuronal population that responds to GH and directly linked it to metabolic responses in vivo.

Hyperactivity of the basolateral amygdaloid nuclear complex (BLA) is a hallmark of anxiety-related disorders in humans. Excitation of BLA projection neurons (PN) is fine-tuned by inhibitory interneurons (INs). Monoaminergic afferents to the BLA modulate PN and IN activity. In the present study, BLA-INs immunoreactive(ir) for parvalbumin (PV) or neuropeptide Y (NPY) and their interrelations with serotonergic and catecholaminergic afferents were analyzed in wildtype (WT) and serotonin transporter knockout (5-HTT KO) mice, a model for anxiety- and stress-related disorders. In WT mice, PV- and NPY-ir INs fall into morphological subgroups which possess perisomatic appositions by serotonergic and tyrosine hydroxylase-ir afferents. Dual immunolabeling shows no colocalization of PV and NPY. NPY/somatostatin(SOM) dual labeling documents colocalization of the peptides in some neurons, and single labeling for NPY or SOM in others. These features appear largely preserved in 5-HTT KO mice. However, quantification of PV- and NPY-ir neurons documents a reduction in number and density of NPY-ir neurons throughout the rostrocaudal extent of the amygdala in 5-HTT KO mice. PV-ir neurons remain unchanged. Quantitative PCR shows increased expression of Npy receptor 2, Som receptor 4, and corticotropin releasing factor receptor 1 in the BLA of 5-HTT KO mice. mRNA for the three peptides is unchanged, indicating that it may be NPY propeptide translation which is reduced in 5-HTT KO mice. Taken together, the results document an effect of life-long serotonin imbalance on the BLA NPY-system, which may contribute to previously observed morphological alterations in BLA PNs and increased anxiety-like behavior in 5-HTT KO mice.

Catecholaminergic (CA) neurons of the medial extended amygdala, preoptic region and adjacent alar hypothalamus have been involved in different aspects of social behavior. Previous data suggested that at least some CA neurons of the medial extended amygdala could originate in a hypothalamic embryonic domain that expresses the transcription factor Otp. To investigate this, we used Otp-eGFP mice to analyze coexpression of GFP and tyrosine hydroxylase (TH) throughout ontogenesis by way of double immunofluorescence. Our results showed that some TH cells coexpress GFP in the medial extended amygdala preoptic region, and alar and basal hypothalamus. However, the presence of TH/GFP double-labeled cells in the extended amygdala is transient, as they are not seen in adults. Based on previous data, the Otp-related CA cells of the medial extended amygdala might derive from the telencephalon-opto-hypothalamic domain, and those of the central paraventricular and supraoptic hypothalamic nuclei likely derive from the supraopto-paraventricular hypothalamic core domain. Taken together, these data provide new evidence for several Otp-related CA subpopulations in centers of the social brain network. The results open interesting questions about the role of these catecholaminergic subpopulations during the development and in different aspects of social behavior.

BackgroundNeurons in the oval subnucleus of the bed nucleus of the stria terminalis (ovBNST) integrate stress and reward signals to regulate motivated behaviors, including food consumption. However, the contribution of specific ovBNST neuronal subpopulations remains poorly understood. Here, we investigated vasoactive intestinal peptide receptor 2 (Vipr2) expressing ovBNST neurons using chemogenetics, immunohistochemistry, and viral circuit mapping. Methods and ResultsUsing stimulatory hM3Dq designer receptors exclusively activated by designer drugs (DREADDs), we found that chemogenetic activation of ovBNSTVipr2neurons significantly increased food intake. We then quantified cFos activation in Vipr2-tdTomato reporter mice following several unique feeding-related manipulations, finding that food restriction (FR) robustly activated ovBNSTVipr2 neurons. Further analysis revealed decreased vasoactive intestinal peptide (VIP) innervation of the ovBNST following FR, in which reduced VIP expression was significantly associated with greater ovBNSTVipr2 cFos activation. Given previous reports of reduced food intake following stimulation of ovBNST neurons expressing protein kinase C delta (PKC{delta}), we used immunostaining to uncover that Vipr2 and PKC{delta} mark largely non-overlapping ovBNST neuronal subpopulations, aligning with their opposing effects on food intake. Finally, Cre-dependent anterograde viral tracing revealed that ovBNSTVipr2 neurons project prominently to the parasubthalamic nucleus (PSTN) and paraventricular nucleus of the hypothalamus (PVN), two feeding-related regions. ConclusionsTogether, these results identify ovBNSTVipr2neurons as a functionally distinct BNST subpopulation that promotes feeding, is activated by food restriction, and links ovBNST neuropeptide signaling to hypothalamic feeding centers.

To induce ovulation, neural circuits are sequentially activated by estradiol and progesterone. In female rodents, estradiol-induced neuroprogesterone, synthesized in astrocytes, is essential for the luteinizing hormone (LH) surge and subsequently, ovulation. However, the gonadotropin-releasing hormone (GnRH) neurons do not express the steroid receptors necessary for reproduction: progesterone receptors (PGR) or estrogen receptor- (ER). Steroid information is transduced by rostral periventricular (RP3V) kisspeptin neurons that express ER and PGR and innervate GnRH neurons in the diagonal band of Broca (DBB) and the medial septum. In this study, we tested the hypothesis that estradiol induced neuroprogesterone needed for the LH surge is mediated by kisspeptin. Neuroprogesterone synthesis was inhibited with aminoglutethimide (AGT; s.c.) in 17{beta}-estradiol benzoate (EB)-primed, ovariectomized (ovx) and adrenalectomized (adx) rats. Kisspeptin-10 (20 nmol/{micro}l) was infused into the DBB, trunk blood was collected 53 hours post-EB injection, and serum LH levels were analyzed by ELISA. AGT inhibition of neuroprogesterone synthesis blocked the EB-induced LH surge. Subsequent treatment with either progesterone or DBB kisspeptin-10 infusion restored the LH surge. Kisspeptin restored the LH surge, which was blocked by DBB infusion of kisspeptin receptor (GPR54) antagonist (kisspeptin-234). Finally, knockdown of kisspeptin protein levels in the RP3V with kisspeptin antisense oligodeoxynucleotide (ODN) significantly lowered LH levels in EB-primed rats compared to scrambled ODN, demonstrating the importance of endogenous RP3V kisspeptin for the LH surge. These results support the hypothesis that neuroprogesterone induces both kisspeptin release from RP3V neurons impacting the LH surge.

Orthopedia (Otp) transcription factor is a critical determinant in the development of the neuroendocrine hypothalamus, and its embryonic deletion results in lethality. Although Otp expression is maintained throughout life, its physiological function in adulthood is not well understood. Here, we generated a forebrain-specific, tamoxifen-inducible, conditional knockout mouse model to investigate the roles of Otp beyond development. Conditional deletion of Otp in two-month-old mice resulted in impaired stress responses, characterized by increased depressive-like behavior and elevated stress-induced cortisol levels. It also led to various metabolic changes, including reduced thyroid hormone levels and body temperature, a higher percentage of fat mass, and diminished responsiveness to ghrelin without affecting food intake, energy expenditure, or body weight. This composite metabolic phenotype was associated with reduced hypothalamic neuropeptides TRH, CRH, AgRP, and NPY expression. Our findings highlight the role of Otp in adult physiological functions as a key neuroendocrine integrator of adaptive stress response and energy balance.

Melanin-concentrating hormone (MCH) is a ubiquitous vertebrate neuropeptide predominantly synthesized by neurons of the diencephalon that can act through two G protein-coupled receptors, called MCHR1 and MCHR2. The expression of Mchr1 has been investigated in both rats and mice, but its synthesis remains poorly described. After identifying an antibody that detects MCHR1 with high specificity, we employed immunohistochemistry to map the distribution of MCHR1 in the CNS of rats and mice. Multiple neurochemical markers were also employed to characterize some of the neuronal populations that synthesize MCHR1. Our results show that MCHR1 is abundantly found in a sensory subcellular structure called the neuronal primary cilium, which has been associated with the detection of free neurochemical agents released to act through volume transmission. Ciliary MCHR1 was found in a wide range of areas, including the olfactory bulb, cortical mantle, striatum, hippocampal formation, amygdala, midline thalamic nuclei, periventricular hypothalamic nuclei, and midbrain areas. No differences were observed between male and female mice, and rats and mice diverged in two key areas: the caudate-putamen nucleus and the subgranular zone of the dentate gyrus. Ciliary MCHR1 was found in close association to several neurochemical markers, including tyrosine hydroxylase, calretinin, kisspeptin, estrogen receptor, oxytocin, vasopressin, and corticotropin-releasing factor. Given the role of neuronal primary cilia in sensing free neurochemical messengers in the extracellular fluid, the widespread distribution of ciliary MCHR1, and the diverse neurochemical populations who synthesize MCHR1, our data indicates that volume transmission may play a prominent role in the normal function of the MCH system.

The Rab3A and Rab3gaps are essential to the Ca+2-dependent neuronal exocytosis in the hypothalamus. The arcuate nucleus of the hypothalamus (ARC) controls food intake and energy expenditure. We have earlier described that the high-fat diet (HFD) feeding induces an obesity phenotype with high leptin production and alteration of proteins related to endosome sorting, and ubiquitination in the ARC of mice. In this study, real-time PCR data analysis revealed that HFD feeding decreases significantly Rab3a, Rab3gap1, and Rab3gap2 transcript levels in the ARC when compared to the group receiving a control diet. The decrease of Rab3gap1/2 transcript levels in the ARC was strongly associated with an increase in plasma leptin. Altogether, our studies demonstrate that HFD feeding could be altering the general network of endosome compartmentalization in the ARC of mice, contributing to a failure in exocytosis and receptor recycling. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

Kisspeptin-expressing neurons in the rostral periventricular region of the third ventricle (RP3V) play an essential role in female reproduction. However, adult male mice were reported to have very few Kisspeptin-expressing neurons in the RP3V compared to females. This led to the hypothesis that Kiss1RP3V neurons are responsible for the ability of females, but not males, to generate a surge of LH, triggering ovulation and steroid synthesis in the female. Using mouse genetics and cell type-specific gene expression analysis, we show that male mice harbor almost as many Kiss1RP3V neurons as the female and that gene expression in these neurons is very similar. Specific activation of male Kiss1RP3V neurons expressing viral-encoded hM3Dq caused a surge in serum testosterone levels. These results demonstrate that Kiss1RP3V neurons are present in the adult male and fully capable of regulating the hypothalamic/pituitary/gonadal axis. We suggest that these neurons may continue to play a role in reproductive behavior in adult male mice.

Anti-obesity medications (AOMs) have become one of the most prescribed drugs in human medicine. While AOMs are known to impact adult neurogenesis in the hypothalamus, their effects on the functional maturation of hypothalamic neurons remain unexplored. Given that AOMs target neurons in the Medial Basal Hypothalamus (MBH), which play a crucial role in regulating energy homeostasis, we hypothesized that AOMs might influence the functional maturation of these neurons, potentially rewiring the MBH. To investigate this, we exposed hypothalamic neurons derived from human induced pluripotent stem cells (hiPSCs) to Semaglutide and lipidized prolactin-releasing peptide (LiPR), two anti-obesity compounds. Contrary to our expectations, treatment with Semaglutide or LiPR during neuronal maturation did not affect the proportion of anorexigenic, Pro-opiomelanocortin-expressing (POMC+) neurons. Additionally, LiPR did not alter the morphology of POMC+ neurons or the expression of selected genes critical for the metabolism or development of anorexigenic neurons. Furthermore, LiPR did not impact the proportion of adult-generated POMC+ neurons in the mouse MBH. Taken together, these results suggest that AOMs do not influence the functional maturation of anorexigenic hypothalamic neurons.

The neuropeptide arginine-vasopressin (AVP) regulates sexually-differentiated social behaviors, including sexual behavior, aggression, and social communication. Much of AVPs effect on social behavior is mediated by the most widely-expressed AVP receptor in the central nervous system: the vasopressin 1a receptor (V1aR). Dense expression of V1aR is found in males and females in the lateral septum (LS), which also receives heavy input from sexually-differentiated populations of AVP cells in the extended amygdala. In order to access and interrogate the structure and function of V1aR cells, we developed and validated a V1aR-Cre knockin mouse line (Avpr1a-P2A-iCre). V1aR-Cre mice did not differ from their Cre-negative littermates in their health, sensorimotor function, anxiety/motivational behavioral responses, or in their V1aR binding levels in the LS. The distribution of Cre in the brain, as assessed by crosses with Cre-reporter mouse lines and in situ hybridization (ISH), was highly similar to patterns of V1aR binding and demonstrated strong colocalization between Cre and V1aR expression within LS cells. We used this V1aR-Cre mouse to identify the inputs and outputs of V1aR+ cells in the LS, via a monosynaptic retrograde rabies virus as well as anterograde synaptophysin-mRuby AAV tracing approaches. Monosynaptic inputs to V1aR LS cells were observed from the medial preoptic area (MPA), lateral hypothalamus (LH), hippocampus (mostly ventral), medial septum, diagonal band of Broca (DBB), and the supramammillary nucleus (SuM). Synaptophysin labeling revealed outputs to some of these same structures (MS, LH, DBB, SuM) as well as parts of the lateral preoptic area (LPO), anterior hypothalamic area (AHA), and ventral pallidum. Notably, most structures (except for hippocampus) bidirectionally connected to V1aR LS cells also contain populations of V1aR+ cells and are targets for BNST/MeA AVP cells, suggesting an integrated AVP/V1aR circuit. Finally, using ISH, we measured levels of V1aR mRNA expression in subregions of the LS and colocalization of V1aR with oxytocin receptor (OTR) mRNA, which also has a high affinity for AVP, within the LS. We found similar high percentages of cells containing V1aR+ puncta across dorsal and intermediate LS in both males and females. In contrast, the ventral LS contained fewer V1aR+ cells in both males and females. The highest level of co-expression of V1aR and OTR was found in the intermediate LS in both sexes, suggesting the possible location of functional interactions between AVP and oxytocin within the LS. We also sought to identify other phenotypic aspects of V1aR cells using ISH and confirmed that all LS V1aR cells are GABAergic and that most also express corticotropin-releasing hormone receptor 2, suggesting a substrate by which AVP could interact with stress-related systems in the LS. Our characterized V1aR-Cre mouse will be a valuable resource for understanding the role of AVP/V1aR in behavioral and physiological systems. Using this mouse, we have characterized the connectional architecture of V1aR cells in the LS and revealed an interlocking set of structures that may be the substrate through which AVP regulates social and emotional behavior.