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.

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.

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.

Characterization of ventromedial hypothalamus activity during exposure to innate and conditioned threats.

In the face of imminent predatory danger, animals quickly detect the threat and mobilize key survival defensive actions, such as escape and freezing. The dorsomedial portion of the ventromedial hypothalamus (VMH) is a central node in innate and conditioned predator-induced defensive behaviours. Prior studies have shown that activity of steroidogenic factor 1 (sf1)-expressing VMH cells is necessary for such defensive behaviours. However, sf1-VMH neural activity during exposure to predatory threats has not been well characterized. Here, we use single-cell recordings of calcium transients from VMH cells in male and female mice. We show this region is activated by threat proximity and that it encodes future occurrence of escape but not freezing. Our data also show that VMH cells encoded proximity of an innate predatory threat but not a fear-conditioned shock grid. Furthermore, chemogenetic activation of the VMH increases avoidance of innate threats, such as open spaces and a live predator. This manipulation also increased freezing towards the predator, without altering defensive behaviours induced by a shock grid. Lastly, we show that optogenetic VMH activation recruited a broad swath of regions, suggestive of widespread changes in neural defensive state. Taken together, these data reveal the neural dynamics of the VMH during predator exposure and further highlight its role as a critical component of the hypothalamic predator defense system.

An approximate line attractor in the hypothalamus encodes an aggressive state.

The hypothalamus regulates innate social behaviors, including mating and aggression. These behaviors can be evoked by optogenetic stimulation of specific neuronal subpopulations within MPOA and VMHvl, respectively. Here, we perform dynamical systems modeling of population neuronal activity in these nuclei during social behaviors. In VMHvl, unsupervised analysis identified a dominant dimension of neural activity with a large time constant (>50 s), generating an approximate line attractor in neural state space. Progression of the neural trajectory along this attractor was correlated with an escalation of agonistic behavior, suggesting that it may encode a scalable state of aggressiveness. Consistent with this, individual differences in the magnitude of the integration dimension time constant were strongly correlated with differences in aggressiveness. In contrast, approximate line attractors were not observed in MPOA during mating; instead, neurons with fast dynamics were tuned to specific actions. Thus, different hypothalamic nuclei employ distinct neural population codes to represent similar social behaviors.

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.

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.

A developmental switch between electrical and neuropeptide communication in the ventromedial hypothalamus.

Dissecting neural connectivity patterns within local brain regions is an essential step to understanding the function of the brain.1 Neural microcircuits in brain regions, such as the neocortex and the hippocampus, have been extensively studied.2 By contrast, the microcircuit in the hypothalamus remains largely uncharacterized. The hypothalamus is crucial for animals' survival and reproduction.3 Knowledge of how different hypothalamic nuclei coordinate with each other and outside brain regions for hypothalamus-related functions has been significantly advanced.4-9 Although there are limited studies on the neural microcircuit in the lateral hypothalamus (LHA)10,11 and the suprachiasmatic nucleus (SCN),12,13 the patterns of neural microcircuits in most of the given hypothalamic nuclei remain largely unknown. This study applied combinatory approaches to address the local neural circuit pattern in the ventromedial hypothalamus (VMH) and other hypothalamic nuclei. We discovered a unique neural circuit design in the VMH. Neurons in the VMH were electrically coupled at the early postnatal stage like ones in the neocortex.14 However, unlike neocortical neurons,14,15 they developed very few chemical synapses after the disappearance of electrical synapses. Instead, VMH neurons communicated with neuropeptides. The similar scarceness of synaptic connectivity found in other hypothalamic nuclei further indicated that the lack of synaptic connections is a unique feature for local neural circuits in most adult hypothalamic nuclei. Thus, our findings provide a solid synaptic basis at the cellular level to understand hypothalamic functions better.

A single vomeronasal receptor promotes intermale aggression through dedicated hypothalamic neurons.

The pheromonal information received by the vomeronasal system plays a crucial role in regulating social behaviors such as aggression in mice. Despite accumulating knowledge of the brain regions involved in aggression, the specific vomeronasal receptors and the exact neural circuits responsible for pheromone-mediated aggression remain unknown. Here, we identified one murine vomeronasal receptor, Vmn2r53, that is activated by urine from males of various strains and is responsible for evoking intermale aggression. We prepared a purified pheromonal fraction and Vmn2r53 knockout mice and applied genetic tools for neuronal activity recording, manipulation, and circuit tracing to decipher the neural mechanisms underlying Vmn2r53-mediated aggression. We found that Vmn2r53-mediated aggression is regulated by specific neuronal populations in the ventral premammillary nucleus and the ventromedial hypothalamic nucleus. Together, our results shed light on the hypothalamic regulation of male aggression mediated by a single vomeronasal receptor.

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.

Apelin-13 facilitates lordosis behavior following infusions to the ventromedial hypothalamus or preoptic area in ovariectomized, estrogen-primed rats.

In normal hormonal conditions, increased neuronal activity in the ventromedial hypothalamus (VMH) induces lordosis whereas activation of the preoptic area (POA) exerts an opposite effect. In the present work, we explored the effect of bilateral infusion of different doses of the apelin-13 (0.37, 0.75, 1.5, and 15 µg) in both brain areas on the expression of lordosis behavior. Lordosis quotient and lordosis reflex score were performed at 30, 120, and 240 min. Weak lordosis was observed following the 0.37 µg dose of apelin-13 at 30 min in the VMH of EB-primed rats; however, the rest of the doses induced significant lordosis relative to the control group. At 120 min, all doses induced lordosis behavior, while at 240 min, the highest dose of 15 µg did not induce significant differences. Interestingly, only the 0.75 µg infusion of apelin in the POA induced significant lordosis at 120 and 240 min. These results indicate that apelin-13 acts preferably in HVM and slightly in POA to initiate lordosis behavior in estrogen-primed rats.

The role of the dorsomedial and ventromedial hypothalamus in regulating behaviorally coupled and resting autonomic drive.

Nearly a century ago it was reported that stimulation of the hypothalamus could evoke profound behavioral state changes coupled with altered autonomic function. Since these initial observations, further studies in animals have revealed that two hypothalamic regions-the dorsomedial and ventromedial hypothalamic nuclei-are critical for numerous behaviors, including those in response to psychological stressors. These behaviors are coupled with changes in autonomic functions, such as altered blood pressure, heart rate, sympathetic nerve activity, resetting of the baroreflex and changes in pituitary function. There is also growing evidence that these two hypothalamic regions play a critical role in thermogenesis, and suggestions they could also be responsible for the hypertension associated with obesity. The aim of this chapter is to review the anatomy, projection patterns, and function of the dorsomedial and ventromedial hypothalamus with a particular focus on their role in autonomic regulation. While most of what is known about these two hypothalamic regions is derived from laboratory animal experiments, recent human studies will also be explored. Finally, we will describe recent human brain imaging studies that provide evidence of a role for these hypothalamic regions in setting resting sympathetic drive and their potential role in conditions such as hypertension.

Ventromedial Nucleus of the Hypothalamus Neurons Under the Magnifying Glass.

The ventromedial nucleus of the hypothalamus (VMH) is a complex brain structure that is integral to many neuroendocrine functions, including glucose regulation, thermogenesis, and appetitive, social, and sexual behaviors. As such, it is of little surprise that the nucleus is under intensive investigation to decipher the mechanisms which underlie these diverse roles. Developments in genetic and investigative tools, for example the targeting of steroidogenic factor-1-expressing neurons, have allowed us to take a closer look at the VMH, its connections, and how it affects competing behaviors. In the current review, we aim to integrate recent findings into the literature and contemplate the conclusions that can be drawn.

Phosphorylation of STAT3 in hypothalamic nuclei is stimulated by lower doses of leptin than are needed to inhibit food intake.

This experiment investigated which hypothalamic nuclei were activated by a dose of leptin that inhibited food intake. Foodnot intake, energy expenditure, respiratory exchange ratio (RER), and intrascapular brown adipose tissue (IBAT) temperature were measured in male and female Sprague Dawley rats for 36 h following an intraperitoneal injection of 0, 50, 200, 500, or 1,000 µg leptin/kg with each rat tested with each dose of leptin in random order. In both males and females, RER and 12-h food intake were inhibited only by 1,000 µg leptin/kg, but there was no effect on energy expenditure or IBAT temperature. At the end of the experiment, phosphorylated signal transducer and activator of transcription 3 (pSTAT3) immunoreactivity was measured 1 h after injection of 0, 50, 500, or 1,000 µg leptin/kg. In male rats, the lowest dose of leptin produced a maximal activation of STAT3 in the Arc and nucleus of the solitary tract (NTS). There was no response in the dorsomedial hypothalamus, but there was a progressive increase in ventromedial nucleus of the hypothalamus (VMH) pSTAT3 with increasing doses of leptin. In female rats, there was no significant change in Arc and pSTAT3 NTS activation was maximal with 500 mg leptin/kg, but only the highest dose of leptin increased VMH pSTAT3. These results suggest that the VMH plays an important role in the energetic response to elevations of circulating leptin but do not exclude the possibility that multiple nuclei provide the appropriate integrated response to hyperleptinemia.NEW & NOTEWORTHY The results of this experiment show that doses of leptin too small to inhibit food intake produce a maximal response to leptin in the arcuate nucleus. By contrast the VMH shows a robust response that correlates with inhibition of food intake. This suggests that the VMH plays an important role in the energetic response to hyperleptinemia.

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.

Rap1 in the VMH regulates glucose homeostasis.

The hypothalamus is a critical regulator of glucose metabolism and is capable of correcting diabetes conditions independently of an effect on energy balance. The small GTPase Rap1 in the forebrain is implicated in high-fat diet-induced (HFD-induced) obesity and glucose imbalance. Here, we report that increasing Rap1 activity selectively in the medial hypothalamus elevated blood glucose without increasing the body weight of HFD-fed mice. In contrast, decreasing hypothalamic Rap1 activity protected mice from diet-induced hyperglycemia but did not prevent weight gain. The remarkable glycemic effect of Rap1 was reproduced when Rap1 was specifically deleted in steroidogenic factor-1-positive (SF-1-positive) neurons in the ventromedial hypothalamic nucleus (VMH) known to regulate glucose metabolism. While having no effect on body weight regardless of sex, diet, and age, Rap1 deficiency in the VMH SF1 neurons markedly lowered blood glucose and insulin levels, improved glucose and insulin tolerance, and protected mice against HFD-induced neural leptin resistance and peripheral insulin resistance at the cellular and whole-body levels. Last, acute pharmacological inhibition of brain exchange protein directly activated by cAMP 2, a direct activator of Rap1, corrected glucose imbalance in obese mouse models. Our findings uncover the primary role of VMH Rap1 in glycemic control and implicate Rap1 signaling as a potential target for therapeutic intervention in diabetes.

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.