The behavior of female rats changes profoundly as they become mothers. The brain region that plays a central role in this regulation is the preoptic area, and lesions in this area eliminates maternal behaviors in rodents. The molecular background of the behavioral changes has not been established yet; therefore, in the present study, we applied proteomics to compare protein level changes associated with maternal care in the rat preoptic area. Using 2-dimensional fluorescence gel electrophoresis followed by identification of altered spots with mass spectrometry, 12 proteins were found to be significantly increased, and 6 proteins showed a significantly reduced level in mothers. These results show some similarities with a previous proteomics study of the maternal medial prefrontal cortex and genomics approaches applied to the preoptic area. Gene ontological analysis suggested that most altered proteins are involved in glucose metabolism and neuroplasticity. These proteins may support the maintenance of increased neuronal activity in the preoptic area, and morphological changes in preoptic neuronal circuits are known to take place in mothers. An increase in the level of alpha-crystallin B chain (Cryab) was confirmed by Western blotting. This small heat shock protein may also contribute to maintaining the increased activity of preoptic neurons by stabilizing protein structures. Common regulator and target analysis of the altered proteins suggested a role of prolactin in the molecular changes in the preoptic area. These results first identified the protein level changes in the maternal preoptic area. The altered proteins contribute to the maintenance of maternal behaviors and may also be relevant to postpartum depression, which can occur as a molecular level maladaptation to motherhood.

The macroscale neuronal connections of the lateral preoptic area (LPO) and the caudally adjacent lateral hypothalamic area anterior region (LHAa) were investigated in mice by anterograde and retrograde axonal tracing. Both hypothalamic regions are highly and diversely connected, with connections to >200 gray matter regions spanning the forebrain, midbrain, and rhombicbrain. Intrahypothalamic connections predominate, followed by connections with the cerebral cortex and cerebral nuclei. A similar overall pattern of LPO and LHAa connections contrasts with substantial differences between their input and output connections. Strongest connections include outputs to the lateral habenula, medial septal and diagonal band nuclei, and inputs from rostral and caudal lateral septal nuclei; however, numerous additional robust connections were also observed. The results are discussed in relation to a current model for the mammalian forebrain network that associates LPO and LHAa with a range of functional roles, including reward prediction, innate survival behaviors (including integrated somatomotor and physiological control), and affect. The present data suggest a broad and intricate role for LPO and LHAa in behavioral control, similar in that regard to previously investigated LHA regions, contributing to the finely tuned sensory-motor integration that is necessary for behavioral guidance supporting survival and reproduction.

The ventral tegmental area (VTA) has been proposed to play a role in pain, but the brain structures modulating VTA activity in response to nociceptive stimuli remain unclear. Here, we demonstrate that the lateral preoptic area (LPO) glutamate neurons relay nociceptive information to the VTA. These LPO glutamatergic neurons synapsing on VTA neurons respond to nociceptive stimulation and conditioned stimuli predicting nociceptive stimulation and also mediate aversion. In contrast, LPO GABA neurons synapsing in the VTA mediate reward. By ultrastructural quantitative synaptic analysis, ex vivo electrophysiology, and functional neuroanatomy we identify a complex circuitry between LPO glutamatergic and GABAergic neurons and VTA dopaminergic, GABAergic, and glutamatergic neurons. We conclude that LPO glutamatergic neurons play a causal role in the processing of nociceptive stimuli and in relaying information about nociceptive stimuli. The pathway from LPO glutamatergic neurons to the VTA represents an unpredicted interface between peripheral nociceptive information and the limbic system.

Nicotine is a neuroteratogenic component of tobacco smoke, e-cigarettes, and other products and can exert sex-specific effects in the developing brain, likely mediated through sex hormones. Estradiol modulates expression of nicotinic acetylcholine receptors in rats, and plays critical roles in neurodevelopmental processes, including sexual differentiation of the brain. Here, we examined the effects of developmental nicotine exposure on the sexual differentiation of the preoptic area (POA), a brain region that normally displays robust structural sexual dimorphisms and controls adult mating behavior in rodents. Using a rat model of gestational exposure, developing pups were exposed to nicotine (2 mg/kg/day) via maternal osmotic minipump (subcutaneously, sc) throughout the critical window for brain sexual differentiation. At postnatal day (PND) 4, a subset of offspring was analyzed for epigenetic effects in the POA. At PND40, all offspring were gonadectomized, implanted with a testosterone-releasing capsule (sc), and assessed for male sexual behavior at PND60. Following sexual behavior assessment, the area of the sexually dimorphic nucleus of the POA (SDN-POA) was measured using immunofluorescent staining techniques. In adults, normal sex differences in male sexual behavior and in the SDN-POA area were eliminated in nicotine-treated animals. Using novel analytical approaches to evaluate overall masculinization of the adult POA, we identified significant masculinization of the nicotine-treated female POA. In neonates (PND4), nicotine exposure induced trending alterations in methylation-dependent masculinizing gene expression and DNA methylation levels at sexually-dimorphic differentially methylated regions, suggesting that developmental nicotine exposure is capable of triggering masculinization of the rat POA via epigenetic mechanisms.

Homeotherms maintain a stable internal body temperature despite changing environments. During energy deficiency, some species can cease to defend their body temperature and enter a hypothermic and hypometabolic state known as torpor. Recent advances have revealed the medial preoptic area (MPA) as a key site for the regulation of torpor in mice. The MPA is estrogen-sensitive and estrogens also have potent effects on both temperature and metabolism. Here, we demonstrate that estrogen-sensitive neurons in the MPA can coordinate hypothermia and hypometabolism in mice. Selectively activating estrogen-sensitive MPA neurons was sufficient to drive a coordinated depression of metabolic rate and body temperature similar to torpor, as measured by body temperature, physical activity, indirect calorimetry, heart rate, and brain activity. Inducing torpor with a prolonged fast revealed larger and more variable calcium transients from estrogen-sensitive MPA neurons during bouts of hypothermia. Finally, whereas selective ablation of estrogen-sensitive MPA neurons demonstrated that these neurons are required for the full expression of fasting-induced torpor in both female and male mice, their effects on thermoregulation and torpor bout initiation exhibit differences across sex. Together, these findings suggest a role for estrogen-sensitive MPA neurons in directing the thermoregulatory and metabolic responses to energy deficiency.

Body temperature control is essential for survival. In mammals, thermoregulation is mediated by the preoptic area of anterior hypothalamus (POA), with ∼30% of its neurons sensitive to brain temperature change. It is still unknown whether and how these temperature-sensitive neurons are involved in thermoregulation, because for eight decades they have only been identified via electrophysiological recording. By combining single-cell RNA-seq with whole-cell patch-clamp recordings, we identified Ptgds as a genetic marker for temperature-sensitive POA neurons. Then, we demonstrated these neurons' role in thermoregulation via chemogenetics. Given that Ptgds encodes the enzyme that synthesizes prostaglandin D2 (PGD2), we further explored its role in thermoregulation. Our study revealed that rising temperature of POA alters the activity of Ptgds-expressing neurons so as to increase PGD2 production. PGD2 activates its receptor DP1 and excites downstream neurons in the ventral medial preoptic area (vMPO) that mediates body temperature decrease, a negative feedback loop for thermoregulation.

Delineating the basic cellular components of cortical inhibitory circuits remains a fundamental issue in order to understand their specific contributions to microcircuit function. It is still unclear how current classifications of cortical interneuron subtypes relate to biological processes such as their developmental specification. Here we identified the developmental trajectory of neurogliaform cells (NGCs), the main effectors of a powerful inhibitory motif recruited by long-range connections. Using in vivo genetic lineage-tracing in mice, we report that NGCs originate from a specific pool of 5-HT3AR-expressing Hmx3+ cells located in the preoptic area (POA). Hmx3-derived 5-HT3AR+ cortical interneurons (INs) expressed the transcription factors PROX1, NR2F2, the marker reelin but not VIP and exhibited the molecular, morphological and electrophysiological profile of NGCs. Overall, these results indicate that NGCs are a distinct class of INs with a unique developmental trajectory and open the possibility to study their specific functional contribution to cortical inhibitory microcircuit motifs.

Mice display robust, stereotyped behaviours towards pups: virgin males typically attack pups, whereas virgin females and sexually experienced males and females display parental care. Here we show that virgin males genetically impaired in vomeronasal sensing do not attack pups and are parental. Furthermore, we uncover a subset of galanin-expressing neurons in the medial preoptic area (MPOA) that are specifically activated during male and female parenting, and a different subpopulation that is activated during mating. Genetic ablation of MPOA galanin neurons results in marked impairment of parental responses in males and females and affects male mating. Optogenetic activation of these neurons in virgin males suppresses inter-male and pup-directed aggression and induces pup grooming. Thus, MPOA galanin neurons emerge as an essential regulatory node of male and female parenting behaviour and other social responses. These results provide an entry point to a circuit-level dissection of parental behaviour and its modulation by social experience.

The preoptic area-anterior hypothalamus (POA-AH) is characterized by sexually dimorphic features in a number of vertebrates and is a key region of the forebrain for regulating physiological responses and sexual behaviours. Using live-cell fluorescence video microscopy with organotypic brain slices, the current study examined sex differences in the movement characteristics of neurons expressing yellow fluorescent protein (YFP) driven by the Thy-1 promoter. Cells in slices from embryonic day 14 (E14), but not E13, mice displayed significant sex differences in their basal neuronal movement characteristics. Exposure to 10 nm estradiol-17beta (E2), but not 100 nm dihydrotestosterone, significantly altered cell movement characteristics within minutes of exposure, in a location-specific manner. E2 treatment decreased the rate of motion of cells located in the dorsal POA-AH but increased the frequency of movement in cells located more ventrally. These effects were consistent across age and sex. To further determine whether early-developing sex differences in the POA-AH depend upon gonadal steroids, we examined cell positions in mice with a disruption of the steroidogenic factor-1 gene, in which gonads do not form. An early-born cohort of cells were labelled with the mitotic indicator bromodeoxyuridine (BrdU) on E11. More cells were found in the POA-AH of females than males on the day of birth (P0) regardless of gonadal status. These results support the hypothesis that estrogen partially contributes to brain sexual dimorphism through its influence on cell movements during development. Estrogen's influence may be superimposed upon a pre-existing genetic bias.

Neural networks that control reproduction must integrate social and hormonal signals, tune motivation, and coordinate social interactions. However, the neural circuit mechanisms for these processes remain unresolved. The medial preoptic area (mPOA), an essential node for social behaviors, comprises molecularly diverse neurons with widespread projections. Here we identify a steroid-responsive subset of neurotensin (Nts)-expressing mPOA neurons that interface with the ventral tegmental area (VTA) to form a socially engaged reward circuit. Using in vivo two-photon imaging in female mice, we show that mPOANts neurons preferentially encode attractive male cues compared to nonsocial appetitive stimuli. Ovarian hormone signals regulate both the physiological and cue-encoding properties of these cells. Furthermore, optogenetic stimulation of mPOANts-VTA circuitry promotes rewarding phenotypes, social approach and striatal dopamine release. Collectively, these data demonstrate that steroid-sensitive mPOA neurons encode ethologically relevant stimuli and co-opt midbrain reward circuits to promote prosocial behaviors critical for species survival.

Sexual experience in male rodents, induced by a first exposure to a receptive female, improves efficiency of following copulations. In mice, the mechanisms supporting this improvement are poorly understood. We characterized molecular modifications of the mouse hypothalamic medial preoptic area (mPOA), the main integrative structure for male sexual behaviour, after a single mating event. This paradigm induced long-lasting behavioural improvements and mPOA morphological changes, evidenced by dendritic spine maturation and an increase in the acetylated and tri-methylated forms of histone H3. Ejaculation affected testosterone, progesterone and corticosterone levels in both naive and experienced mice, but sexual experience did not modify basal plasma or hypothalamic levels of steroids. In contrast to studies carried out in rats, no changes were observed, either in the nitrergic system, or in sex steroid receptor levels. However, levels of glutamate- and calcium-associated proteins, including PSD-95, calbindin and the GluN1 subunit of the NMDA receptor, were increased in sexually experienced male mice. The Iba-1 microglial marker was up-regulated in these animals suggesting multicellular interactions induced within the mPOA by sexual experience. In conclusion, plasticity mechanisms induced by sexual experience differ between rat and mouse, even if in both cases they converge to potentiation of the mPOA network.

The preoptic area (POA) has long been recognized as a sleep center, first proposed by von Economo. The POA, especially the medial POA (MPOA), is also involved in the regulation of various innate functions such as sexual and parental behaviors. Consistent with its many roles, the MPOA is composed of subregions that are identified by different gene and protein expressions. This review addresses the current understanding of the molecular and cellular architecture of POA neurons in relation to sleep and reproductive behavior. Optogenetic and pharmacogenetic studies have revealed a diverse group of neurons within the POA that exhibit different neural activity patterns depending on vigilance states and whose activity can enhance or suppress wake, non-rapid eye movement (NREM) sleep, or rapid eye movement (REM) sleep. These sleep-regulating neurons are not restricted to the ventrolateral POA (VLPO) region but are widespread in the lateral MPOA and LPOA as well. Neurons expressing galanin also express gonadal steroid receptors and regulate motivational aspects of reproductive behaviors. Moxd1, a novel marker of sexually dimorphic nuclei (SDN), visualizes the SDN of the POA (SDN-POA). The role of the POA in sleep and other innate behaviors has been addressed separately; more integrated observation will be necessary to obtain physiologically relevant insight that penetrates the different dimensions of animal behavior.

Rapid-eye-movement sleep (REMs) is characterized by activated electroencephalogram (EEG) and muscle atonia, accompanied by vivid dreams. REMs is homeostatically regulated, ensuring that any loss of REMs is compensated by a subsequent increase in its amount. However, the neural mechanisms underlying the homeostatic control of REMs are largely unknown. Here, we show that GABAergic neurons in the preoptic area of the hypothalamus projecting to the tuberomammillary nucleus (POAGAD2→TMN neurons) are crucial for the homeostatic regulation of REMs. POAGAD2→TMN neurons are most active during REMs, and inhibiting them specifically decreases REMs. REMs restriction leads to an increased number and amplitude of calcium transients in POAGAD2→TMN neurons, reflecting the accumulation of REMs pressure. Inhibiting POAGAD2→TMN neurons during REMs restriction blocked the subsequent rebound of REMs. Our findings reveal a hypothalamic circuit whose activity mirrors the buildup of homeostatic REMs pressure during restriction and that is required for the ensuing rebound in REMs.

The role of the hypothalamic preoptic area (POA) in arousal state regulation has been studied since Constantin von Economo first recognized its importance in the early twentieth century. Over the intervening decades, the POA has been shown to modulate arousal in both natural (sleep and wake) as well as drug-induced (anesthetic-induced unconsciousness) states. While the POA is well known for its role in sleep promotion, populations of wake-promoting neurons within the region have also been identified. However, the complexity and molecular heterogeneity of the POA has made distinguishing these two populations difficult. Though multiple lines of evidence demonstrate that general anesthetics modulate the activity of the POA, the region's heterogeneity has also made it challenging to determine whether the same neurons involved in sleep/wake regulation also modulate arousal in response to general anesthetics. While a number of studies show that sleep-promoting POA neurons are activated by various anesthetics, recent work suggests this is not universal to all arousal-regulating POA neurons. Technical innovations are making it increasingly possible to classify and distinguish the molecular identities of neurons involved in sleep/wake regulation as well as anesthetic-induced unconsciousness. Here, we review the current understanding of the POA's role in arousal state regulation of both natural and drug-induced forms of unconsciousness, including its molecular organization and connectivity to other known sleep and wake promoting regions. Further insights into the molecular identities and connectivity of arousal-regulating POA neurons will be critical in fully understanding how this complex region regulates arousal states.

GABA-containing (GABAergic) interneurons play an important role in the function of the cerebral cortex. Through mostly inhibitory mechanisms, interneurons control hyperexcitability and synchronize and shape the spatiotemporal dynamics of cortical activity underlying various brain functions. Studies over the past 10 years have demonstrated that, in most mammals, interneurons originate during development from the subcortical telencephalon--the subpallium--and reach the cerebral cortex through tangential migration. Until now, interneurons have been demonstrated to derive exclusively from two subpallial regions, the medial ganglionic eminence and the caudal ganglionic eminence. Here, we show that another subpallial structure, the preoptic area, is a novel source of cortical GABAergic interneurons in the mouse. In utero labeling and genetic lineage-tracing experiments demonstrate that neurons born in this region migrate to the neocortex and hippocampus, where they differentiate into a distinct population of GABAergic interneurons with relatively uniform neurochemical, morphological, and electrophysiological properties.

The lateral preoptic area (LPO) is a hypothalamic region whose function has been largely unexplored. Its direct and indirect projections to the ventral tegmental area (VTA) suggest that the LPO could modulate the activity of the VTA and the reward-related behaviors that the VTA underlies. We examined the role of the LPO on reward taking and seeking using operant self-administration of cocaine or sucrose. Rats were trained to self-administer cocaine or sucrose and then subjected to extinction, whereby responding was no longer reinforced. We tested if stimulating the LPO pharmacologically with bicuculline or chemogenetically with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) modifies self-administration and/or seeking. In another set of experiments, we tested if manipulating the LPO influences cocaine self-administration during and after punishment. To examine the functional connectivity between the LPO and VTA, we used in vivo electrophysiology recordings in anesthetized rats. We tested if stimulating the LPO modifies the activity of GABA and dopamine neurons of the VTA. We found that stimulating the LPO reinstated cocaine and sucrose seeking behavior but had no effect on reward intake. Furthermore, both stimulating and inhibiting the LPO prevented the sustained reduction in cocaine intake seen after punishment. Finally, stimulating the LPO inhibited the activity of VTA GABA neurons while enhancing that of VTA dopamine neurons. These findings indicate that the LPO has the capacity to drive reward seeking, modulate sustained reductions in self-administration following punishment, and regulate the activity of VTA neurons. Taken together, these findings implicate the LPO as a previously overlooked member of the reward circuit.

The antero-ventral periventricular zone (AVPV) and medial preoptic area (MPOA) have been recognized as gonadal hormone receptive regions of the rodent brain that-via wiring to gonadotropin-releasing hormone (GnRH) neurons-contribute to orchestration of the preovulatory GnRH surge. We hypothesized that neural genes regulating the induction of GnRH surge show altered expression in proestrus. Therefore, we compared the expression of 48 genes obtained from intact proestrous and metestrous mice, respectively, by quantitative real-time PCR (qPCR) method. Differential expression of 24 genes reached significance (p < 0.05). Genes upregulated in proestrus encoded neuropeptides (kisspeptin (KP), galanin (GAL), neurotensin (NT), cholecystokinin (CCK)), hormone receptors (growth hormone secretagogue receptor, μ-opioid receptor), gonadal steroid receptors (estrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR)), solute carrier family proteins (vesicular glutamate transporter 2, vesicular monoamine transporter 2), proteins of transmitter synthesis (tyrosine hydroxylase (TH)) and transmitter receptor subunit (AMPA4), and other proteins (uncoupling protein 2, nuclear receptor related 1 protein). Proestrus evoked a marked downregulation of genes coding for adenosine A2a receptor, vesicular gamma-aminobutyric acid (GABA) transporter, 4-aminobutyrate aminotransferase, tachykinin precursor 1, NT receptor 3, arginine vasopressin receptor 1A, cannabinoid receptor 1, ephrin receptor A3 and aldehyde dehydrogenase 1 family, member L1. Immunocytochemistry was used to visualize the proteins encoded by Kiss1, Gal, Cck and Th genes in neuronal subsets of the AVPV/MPOA of the proestrous mice. The results indicate that gene expression of the AVPV/MPOA is significantly modified at late proestrus including genes that code for neuropeptides, gonadal steroid hormone receptors and synaptic vesicle transporters. These events support cellular and neuronal network requirements of the positive estradiol feedback action and contribute to preparation of the GnRH neuron system for the pre-ovulatory surge release.

Animals must maintain physiological processes within an optimal temperature range despite changes in their environment. Through behavioral assays, whole-brain functional imaging, and neural ablations, we show that larval zebrafish, an ectothermic vertebrate, achieves thermoregulation through homeostatic navigation-non-directional and directional movements toward the temperature closest to its physiological setpoint. A brain-wide circuit encompassing several brain regions enables this behavior. We identified the preoptic area of the hypothalamus (PoA) as a key brain structure in triggering non-directional reorientation when thermal conditions are worsening. This result shows an evolutionary conserved role of the PoA as principal thermoregulator of the brain also in ectotherms. We further show that the habenula (Hb)-interpeduncular nucleus (IPN) circuit retains a short-term memory of the sensory history to support the generation of coherent directed movements even in the absence of continuous sensory cues. We finally provide evidence that this circuit may not be exclusive for temperature but may convey a more abstract representation of relative valence of physiologically meaningful stimuli regardless of their specific identity to enable homeostatic navigation.

The paraventricular nucleus (PVN) of the hypothalamus in mammals coordinates neuroendocrine, autonomic and behavioral responses pivotal for homeostasis and the stress response. A large amount of studies in rodents has documented that the PVN contains diverse neuronal cell types which can be identified by the expression of distinct secretory neuropeptides. Interestingly, PVN cell types often coexpress multiple neuropeptides whose relative coexpression levels are subject to environment-induced plasticity. Due to their small size and transparency, zebrafish larvae offer the possibility to comprehensively study the development and plasticity of the PVN in large groups of intact animals, yet important anatomical information about the larval zebrafish PVN-homologous region has been missing. Therefore we recently defined the location and borders of the larval neurosecretory preoptic area (NPO) as the PVN-homologous region in larval zebrafish based on transcription factor expression and cell type clustering. To identify distinct cell types present in the larval NPO, we also generated a comprehensive 3D map of 9 zebrafish homologs of typical neuropeptides found in the mammalian PVN (arginine vasopressin (AVP), corticotropin-releasing hormone (CRH), proenkephalin a (penka)/b (penkb), neurotensin (NTS), oxytocin (OXT), vasoactive intestinal peptide (VIP), cholecystokinin (CCK), and somatostatin (SST)). Here we extend this chemoarchitectural map to include the degrees of coexpression of two neuropeptides in the same cell by performing systematic pairwise comparisons. Our results allowed the subclassification of NPO cell types, and differences in variability of coexpression profiles suggest potential targets of biochemical plasticity. Thus, this work provides an important basis for the analysis of the development, function, and plasticity of the primary neuroendocrine brain region in larval zebrafish.

Maternal mammals exhibit heightened motivation to care for offspring, but the underlying neuromolecular mechanisms have yet to be clarified. Here, we report that the calcitonin receptor (Calcr) and its ligand amylin are expressed in distinct neuronal populations in the medial preoptic area (MPOA) and are upregulated in mothers. Calcr+ MPOA neurons activated by parental care project to somatomotor and monoaminergic brainstem nuclei. Retrograde monosynaptic tracing reveals that significant modification of afferents to Calcr+ neurons occurs in mothers. Knockdown of either Calcr or amylin gene expression hampers risk-taking maternal care, and specific silencing of Calcr+ MPOA neurons inhibits nurturing behaviors, while pharmacogenetic activation prevents infanticide in virgin males. These data indicate that Calcr+ MPOA neurons are required for both maternal and allomaternal nurturing behaviors and that upregulation of amylin-Calcr signaling in the MPOA at least partially mediates risk-taking maternal care, possibly via modified connectomics of Calcr+ neurons postpartum.