Ventral Hypothalamus Research Articles

Melatonin influences reproduction in the sapphire devil Chrysiptera cyanea via regulation of dopamine 2b receptor transcription

Melatonin and dopamine participate in the regulation of reproduction-related hormone/peptide synthesis and secretion at the hypothalamic–pituitary–gonadal axis in fish. Here, we report a unique reproduction-related interaction of melatonin and dopamine in the brain of the sapphire devil Chrysiptera cyanea, a tropical damselfish with a long-day preference. We examined the expression of arylalkylamine N-acetyltransferase 2 (aanat2)—a rate-limiting enzyme of melatonin, dopamine 2b receptor (d2b), gonadotropin-releasing hormone (gnrh1), and β-subunit of follicle-stimulating hormone (fshβ) and luteinizing hormone (lhβ)—in the brain of the sapphire devil. During the reproductive season, mature females were divided into the early vitellogenesis (EV), late vitellogenesis (LV), and post-spawning (PS) stages; a day-low and night-high profile of aanat2 was observed during EV and LV but not during PS. There were nocturnal increases in gnrh1 during EV and LV as well as d2b during LV, suggesting that melatonin has a positive effect on the levels of gnrh1 and d2b transcripts in mature fish. When the brains of females in the non-breeding season were sampled at 4-h intervals, nocturnal increases in the levels of fshβ and lhβ transcripts were observed at 00:00 and 04:00, respectively. The immersion of immature fish in melatonin-containing seawater for 6 h resulted in the upregulation of fshβ and lhβ, as well as downregulation of d2b, in the brain. Additionally, in situ hybridization analysis showed that melatonin treatment lowered the signals of d2b transcripts in the ventral hypothalamus, rostral pars distalis, proximal pars distalis, and pars intermedia, suggesting that melatonin has a negative impact on the levels of d2b transcripts in the hypothalamus and pituitary of immature females; the opposite effect is likely concerning the levels of fshβ and lhβ transcripts in the pituitary. In conclusion, melatonin positively and negatively acted on the hypothalamus and pituitary in the sapphire devil; these opposite effects were related to differences in gonadal development status.

Direct comparison of Hoxb8-driven reporter distribution in the brains of four transgenic mouse lines: towards a spinofugal projection atlas.

Hox genes govern rostro-caudal identity along the developing spinal cord, which has a well-defined division of function between dorsal (sensory) and ventral (motor) halves. Here we exploit developmental Hoxb8 expression, normally restricted to the dorsal cord below the obex, to genetically label spinal cord-to-brain ("spinofugal") axons. We crossed two targeted (knock-in) and two non-targeted recombinase-expressing lines (Hoxb8-IRES-Cre and Hoxb8-T2AFlpO; Hoxb8-Cre and Hoxb8-FlpO, respectively) with appropriate tdtomato-expressing reporter strains. Serial sectioning, confocal and superresolution microscopy, as well as light-sheet imaging was used to reveal robust labeling of ascending axons and their terminals in expected and unexpected regions. This strategy provides unprecedented anatomical detail of ascending spinal tracts anterior to the brainstem, and reveals a previously undescribed decussating tract in the ventral hypothalamus (the spinofugal hypothalamic decussating tract, or shxt). The absence of Hoxb8-suppressing elements led to multiple instances of ectopic reporter expression in Hoxb8-Cre mice (retinal ganglion and vomeronasal axons, anterior thalamic nuclei and their projections to the anterior cingulate and retrosplenial cortices and subiculum, and a population of astrocytes at the cephalic flexure) and Hoxb8-FlpO mice (Cajal-Retzius cells of the dentate gyrus, and mesenchymal cells of the choroid plexus). While targeted transgenic lines were similar in terms of known spinofugal projections, Hoxb8-IRES-Cre reporters had an additional projection to the core of the facial motor nucleus, and more abundant Hoxb8-lineage microglia scattered throughout the brain than Hoxb8-T2A-FlpO (or any other) mice, suggesting dysregulated Hoxb8-driven reporter expression in one or both lines. This work complements structural and connectivity atlases of the mouse central nervous system, and provides a platform upon which their reactions to injury or disease can be studied. Ectopic Hoxb8-driven recombinase expression may also be a useful tool to study structure and function of other cell populations in non-targeted lines.

Apparent absence of hypothalamic cholinergic neurons in the common ostrich and emu: Implications for global brain states during sleep.

We examined the presence/absence and parcellation of cholinergic neurons in the hypothalami of five birds: a Congo grey parrot (Psittacus erithacus), a Timneh grey parrot (P. timneh), a pied crow (Corvus albus), a common ostrich (Struthio camelus), and an emu (Dromaius novaehollandiae). Using immunohistochemistry to an antibody raised against the enzyme choline acetyltransferase, hypothalamic cholinergic neurons were observed in six distinct clusters in the medial, lateral, and ventral hypothalamus in the parrots and crow, similar to prior observations made in the pigeon. The expression of cholinergic nuclei was most prominent in the Congo grey parrot, both in the medial and lateral hypothalamus. In contrast, no evidence of cholinergic neurons in the hypothalami of either the ostrich or emu was found. It is known that the expression of sleep states in the ostrich is unusual and resembles that observed in the monotremes that also lack hypothalamic cholinergic neurons. It has been proposed that the cholinergic system acts globally to produce and maintain brain states, such as those of arousal and rapid-eye-movement sleep. The hiatus in the cholinergic system of the ostrich, due to the lack of hypothalamic cholinergic neurons, may explain, in part, the unusual expression of sleep states in this species. These comparative anatomical and sleep studies provide supportive evidence for global cholinergic actions and may provide an important framework for our understanding of one broad function of the cholinergic system and possible dysfunctions associated with global cholinergic neural activity.

Mate calling alters expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain of male frog Microhyla nilphamariensis

Croaking is a unique component of reproductive behaviour in amphibians which plays a key role in intraspecies communication and mate evaluation. While gonadal hormones are known to induce croaking, central regulation of sound production is less studied. Croaking is a dramatic, transient activity that sets apart an animal from non-croaking individuals. Herein, we aim at examining the profile of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in actively croaking and non-croaking frog Microhyla nilphamariensis. In anurans, this peptide is widely expressed in the areas inclusive of acoustical nuclei as well as areas relevant to reproduction. CART immunoreactivity was far more in the preoptic area (POA), anteroventral tegmentum (AV), ventral hypothalamus (vHy), pineal (P) and pituitary gland of croaking frog compared to non-croaking animals. On similar lines, tissue fragments collected from the mid region of the brain inclusive of POA, vHy, AV, pineal and pituitary gland of croaking frog showed upregulation of CART mRNA. However, CART immunoreactivity in the neuronal perikarya of raphe (Ra) was completely abolished during croaking activity. The data suggest that CART signaling in the brain may be an important player in mediating croaking behaviour in the frog.

Dynamic modulation of genomic enhancer elements in the suprachiasmatic nucleus, the site of the mammalian circadian clock.

The mammalian suprachiasmatic nucleus (SCN), located in the ventral hypothalamus, synchronizes and maintains daily cellular and physiological rhythms across the body, in accordance with environmental and visceral cues. Consequently, the systematic regulation of spatiotemporal gene transcription in the SCN is vital for daily timekeeping. So far, the regulatory elements assisting circadian gene transcription have only been studied in peripheral tissues, lacking the critical neuronal dimension intrinsic to the role of the SCN as central brain pacemaker. By using histone-ChIP-seq, we identified SCN-enriched gene regulatory elements that associated with temporal gene expression. Based on tissue-specific H3K27ac and H3K4me3 marks, we successfully produced the first-ever SCN gene-regulatory map. We found that a large majority of SCN enhancers not only show robust 24-h rhythmic modulation in H3K27ac occupancy, peaking at distinct times of day, but also possess canonical E-box (CACGTG) motifs potentially influencing downstream cycling gene expression. To establish enhancer-gene relationships in the SCN, we conducted directional RNA-seq at six distinct times across the day and night, and studied the association between dynamically changing histone acetylation and gene transcript levels. About 35% of the cycling H3K27ac sites were found adjacent to rhythmic gene transcripts, often preceding the rise in mRNA levels. We also noted that enhancers encompass noncoding, actively transcribing enhancer RNAs (eRNAs) in the SCN, which in turn oscillate, along with cyclic histone acetylation, and correlate with rhythmic gene transcription. Taken together, these findings shed light on genome-wide pretranscriptional regulation operative in the central clock that confers its precise and robust oscillation necessary to orchestrate daily timekeeping in mammals.

Organization of the corticotropin-releasing hormone and corticotropin-releasing hormone-binding protein systems in the central nervous system of the sea lamprey Petromyzon marinus.

The expression of the corticotropin-releasing hormone (PmCRH) and the CRH-binding protein (PmCRHBP) mRNAs was studied by in situ hybridization in the brain of prolarvae, larvae, and adults of the sea lamprey Petromyzon marinus. We also generated an antibody against the PmCRH mature peptide to study the distribution of PmCRH-immunoreactive cells and fibers. PmCRH immunohistochemistry was combined with antityrosine hydroxylase immunohistochemistry, PmCRHBP in situ hybridization, or neurobiotin transport from the spinal cord. The most numerous PmCRH-expressing cells were observed in the magnocellular preoptic nucleus-paraventricular nucleus and in the superior and medial rhombencephalic reticular formation. PmCRH expression was more extended in adults than in larvae, and some cell populations were mainly (olfactory bulb) or only (striatum, ventral hypothalamus, prethalamus) observed in adults. The preopto-paraventricular fibers form conspicuous tracts coursing toward the neurohypophysis, but many immunoreactive fibers were also observed coursing in many other brain regions. Brain descending fibers in the spinal cord mainly come from cells located in the isthmus and in the medial rhombencephalic reticular nucleus. The distribution of PmCRHBP-expressing neurons was different from that of PmCRH cells, with cells mainly present in the septum, striatum, preoptic region, tuberal hypothalamus, pretectum, pineal complex, isthmus, reticular formation, and spinal cord. Again, expression in adults was more extended than in larvae. PmCRH- and PmCRHBP-expressing cells are different, excluding colocalization of these substances in the same neuron. Present findings reveal a complex CRH/CRHBP system in the brain of the oldest extant vertebrate group, the agnathans, which shows similarities but important divergences with that of mammals.

Social status mediated variation in hypothalamic transcriptional profiles of male mice

Animals of different social status exhibit variation in aggression, territorial and reproductive behavior as well as activity patterns, feeding, drinking and status signaling. This behavioral and physiological plasticity is coordinated by underlying changes in brain gene transcription. Using Tag-based RNA sequencing (Tag-seq), we explore RNA transcriptomes from the medial preoptic area (mPOA) and ventral hypothalamus (vHYP) of male mice of different social ranks in a dominance hierarchy and detect candidate genes and cellular pathways that underlie status-related plasticity. Within the mPOA, oxytocin (Oxt) and vasopressin (Avp) are more highly expressed in subdominant mice compared to other ranks, while nitric oxide synthase (Nos1) has lower expression in subdominant mice. Within the vHYP, we find that both orexigenic and anorexigenic genes involved in feeding behavior, including agouti-related peptide (Agrp), neuropeptide-Y (Npy), galanin (Gal), proopiomelanocortin (Pomc), and Cocaine- and Amphetamine-Regulated Transcript Protein prepropeptide (Cartpt), are less expressed in dominant animals compared to more subordinate ranks. We suggest that this may represent a reshaping of feeding circuits in dominant compared to subdominant and subordinate animals. Furthermore, we determine several genes that are positively and negatively associated with the level of despotism (aggression) in dominant males. Ultimately, we identify hypothalamic genes controlling feeding and social behaviors that are differentially transcribed across animals of varying social status. These changes in brain transcriptomics likely support phenotypic variation that enable animals to adapt to their current social status.

Cocaine and amphetamine regulated transcript peptide (CART) in the tadpole brain: Response to different energy states

Cocaine- and amphetamine-regulated transcript peptide (CART) is an anorexigenic neuropeptide known to play a key role in energy homeostasis across the vertebrate phyla. In the current study, we have investigated the response of the CART immunoreactive system to varying energy states in the brain of a tadpole model. The pro-metamorphic tadpoles of Euphlyctis cyanophlyctis were fasted, or intracranially injected with glucose or 2-deoxy-d-glucose (2DG; an antagonist to glucose inducing glucoprivation) and the response of the CART containing system in various neuroanatomical areas was studied using immunohistochemistry. Glucose administration increased the CART immunoreactivity in the entopeduncular neurons (EN), preoptic area (POA), ventral hypothalamus (vHy) and the Edinger Westphal nucleus (EW) while CART positive cells decrease in response to fasting and glucoprivation. A substantial decrease in CART was noted in the EW nucleus of tadpoles injected with 2DG. These regions might contain the glucose-sensing neurons and regulate food intake in anurans. Therefore, we speculate that the function of central CART and its antagonistic action with NPY in food and feeding circuitry of anurans is evolutionary conserved and might be responsible for glucose homeostasis.

Anoxia at Birth Induces Behaviorally-Relevant Changes in alpha2-Noradrenergic Receptor Binding in the Adult Rat

Evidence suggests that Caesarean section birth in the rat, with or without an additional period of anoxia, results in long-term changes in brain catecholamine levels as well as reactivity to stress. The purpose of the present investigation was to determine whether Caesarean birth plus anoxia alters (alpha)2-noradrenergic receptor binding and sensitivity to the (alpha)2 receptor agonist, clonidine, in the Porsolt forced swim test. Sprague Dawley rat dams were decapitated and the uteruses were removed by Caesarean section. Pups were then delivered either immediately (Caesarean Only group), or were immersed in a saline bath for approximately 15 minutes (Caesarean plus Anoxia group) before delivery of the pups. A third group of animals born vaginally served as controls (Vaginally-born group). Four to five months postnatally, the expression of (alpha)2 receptors was measured by receptor autoradiography using [3H]-Rauwolscine binding. Receptor binding was increased in the area of the ventral hypothalamus and decreased in the CA1 region of the hippocampus in animals subjected to Caesarean plus Anoxia at birth. These animals also displayed a subsensitive response to the immobilizing effects of clonidine (100 micrograms/kg, i.p.) in the Porsolt forced swim test. Specifically these data show that Caesarean birth produces long-term changes in (alpha)2 receptor density and that, in animals subjected to Caesarean plus anoxia, these changes are reflected in a behavioral subsensitivity to the (alpha)2 agonist, clonidine. The findings reported here provide further experimental support for the hypothesis that birth complications may contribute to the pathophysiology of disorders such as schizophrenia that involve central catecholamine dysfunction.

Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

Lorcaserin Alters Serotonin and Noradrenaline Tissue Content and Their Interaction With Dopamine in the Rat Brain.

Lorcaserin is a preferential serotonin2C receptor (5-HT2CR) agonist effective to treat obesity that has also recently been proposed to treat addiction and epilepsy. Central dopamine (DA) mechanisms are likely involved in the lorcaserin mechanism of action, but other monoamines 5-HT and noradrenaline (NA) contents or their interaction with DA might account for its effects. Here we showed that lorcaserin at 3, but not 0.3 mg/kg enhanced 5-HT content in the insular cortex, the core of the nucleus accumbens, and ventral hypothalamus. Without affecting the metabolite 5-hydroxy indole acetic acid, lorcaserin reduced the indirect index of 5-HT turnover in the hippocampus, substantia nigra, and habenula. Lorcaserin at 3 mg/kg increased NA content in the orbitofrontal cortex, the central amygdala (also at 0.3 mg/kg), the ventral hypothalamus, and the shell of the nucleus accumbens. A correlative analysis of the tissue contents between pairs of brain regions revealed that 0.3 mg/kg lorcaserin enhanced the number of correlations for 5-HT, its metabolism, and NA to a lower extent. The correlation profiles were very different between saline, 0.3 and 3 mg/kg lorcaserin. Lorcaserin enhanced the correlations established between NA or 5-HT at 0.3 and 3 mg/kg and reduced the number of correlations established between the index of the turnover for DA and 5-HT. These results show that lorcaserin modulates the biochemistry of NA and 5-HT systems in a subset of brain regions. Qualitatively, they reveal, oppositely to the DA changes, that lorcaserin at 0.3, but not 3 mg/kg, enhanced the number of correlations of 5-HT content between brain regions.

Ectopic over expression of kiss1 may compensate for the loss of kiss2

Kisspeptin (KISS) is a neuropeptide which plays a central role in the regulation of the hypothalamic-pituitary–gonadal axis, and is essential for sexual maturation and fertility in mammals. Unlike mammals, which possess only one KISS gene, two paralogous genes, kiss1 and kiss2, have been identified in zebrafish and other non-mammalian vertebrates. Previous studies suggest that Kiss2, but not Kiss1, is the reproduction relevant form amongst the two. To better understand the role of each of these isoforms in reproduction, a loss of function approach was applied. Two genetic manipulation techniques—clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN)—were used to generate kiss1 and kiss2 knockout (KO) zebrafish lines, respectively. Examination of these KO lines showed that reproductive capability was not impaired, confirming earlier observations. Further analysis revealed that KO of kiss2 caused a significant increase in expression levels of kiss1, kiss2r and tac3a, while KO of kiss1 had no effect on the expression of any of the examined genes. In situ hybridization analysis revealed that kiss1 mRNA is expressed only in the habenula in wild type brains, while in kiss2 KO fish, kiss1 mRNA-expressing cells were identified also in the ventral telencephalon, the ventral part of the entopeduncular nucleus, and the dorsal and ventral hypothalamus. Interestingly, these regions are known to express kiss2r, and the ventral hypothalamus normally expresses kiss2. These results suggest that a compensatory mechanism, involving ectopic kiss1 expression, takes place in the kiss2 KO fish, which may substitute for Kiss2 activity.

SUN-295 Prevalence of Self-Reported Endocrine Comorbidities in Hypothalamic Hamartoma Patients: Data from the Hope for Hypothalamic Hamartoma Survey

Background: Hypothalamic hamartoma (HH) are rare, congenital, benign mass lesions in the ventral hypothalamus that can be asymptomatic or associated with gelastic seizures and treatment-resistant epilepsy. Central precocious puberty (CPP) is the main endocrine comorbidity (30-80% of cases). Other endocrine comorbidities have also been described that tends to occur after surgery. However, previous studies reporting its prevalence have shown inconsistent results because of the rarity of the disease, variability of follow-up, and lack of long-term endocrinologic assessment. Aims: To evaluate the self-reported prevalence of demographics and endocrine comorbidities in a large cohort of HH patients. Methods: Hope for HH is a volunteer-based nonprofit organization founded by parents of children with HH. This international survey was initiated, translated into multiple languages and distributed by mail and electronically to families of children with HH in the Hope for HH database after concerns were raised that there have been multiple ongoing comorbidities (including endocrine) that continues to be under-recognized. Results: In total, 257 HH patients (132M/125F, mainly between ages 4-35 years and from the US, Russia, UK, Australia, Canada, Germany and Kazakhstan) participated in the survey. Some patients had a secondary diagnosis of Pallister-Hall (7.0%), Lennox-Gastaut (1.95%), Prader-Willi (0.8%) and West (0.8%) syndromes. The majority of patients (n=163, 63.4%) underwent surgery (MRI-guided stereotactic laser ablation [n=61, 37.4%], endoscopic resection [n=31, 19.0%], transcallosal resection [n=30, 18.4%], stereotactic radiofrequency ablation [n=27, 16.6%], orbitozygomatic resection [n=9, 5.5%]) or gamma knife radiosurgery (n=28, 17.2%). After surgery and/or radiation, ~50% of patients were seizure-free but reported unchanged, poor or very poor quality of life (QoL), with fatigue (56.4%), heat intolerance (46.3%) and adipsia (21.8%) being the more common symptoms. Reported endocrine comorbidities include CPP (42%), hypothalamic obesity (35.0%), abnormal body composition (31.5%), central hypothyroidism (19.8%), osteopenia/osteoporosis with low BMD (12.8%), diabetes insipidus (11.3%), GH deficiency (10.5%), central adrenal insufficiency (10.5%), central hypogonadism (5.1%), and delayed puberty (4.7%), and 26.5% of patients were not seeing an endocrinologist. Conclusion: In contrast to previous studies reporting low prevalence, mild and transient endocrine comorbidities in HH patients (2,3), this survey suggests a greater prevalence of other non-CPP endocrine comorbidities with a substantial number of patients reporting unchanged or impaired QoL. Thus, long-term endocrinologic follow-up with the involvement of a multidiscliplinary team is essential to diagnose early and treat these comorbidities in these patients.

Both NPY-Expressing and CART-Expressing Neurons Increase Energy Expenditure and Trabecular Bone Mass in Response to AP1 Antagonism, But Have Opposite Effects on Bone Resorption.

Energy metabolism and bone homeostasis share several neuronal regulatory pathways. Within the ventral hypothalamus (VHT), the orexigenic neurons co-express Agouti-related peptide (AgRP) and neuropeptide Y (NPY) and the anorexigenic neurons co-express, α-melanocyte stimulating hormone derived from proopiomelanocortin (POMC), and cocaine and amphetamine-regulated transcript (CART). These neurons regulate both processes, yet their relative contribution is unknown. Previously, using genetically targeted activator protein (AP1) alterations as a tool, we showed in adult mice that AgRP or POMC neurons are capable of inducing whole-body energy catabolism and bone accrual, with different effects on bone resorption. Here, we investigated whether co-residing neurons exert similar regulatory effects. We show that AP1 antagonists targeted to NPY-producing or CART-producing neurons in adult mice stimulate energy expenditure, reduce body weight gain and adiposity and promote trabecular bone formation and mass, yet again via different effects on bone resorption, as measured by serum level of carboxy-terminal collagen type I crosslinks (CTX). In addition, AP1 antagonists promote neurite expansion, increasing neurite number, length, and surface area in primary hypothalamic neuronal cultures. Overall, our data demonstrate that the orexigenic NPY and anorexigenic CART neurons both have the capacity to stimulate energy burning state and increase bone mass. © 2020 American Society for Bone and Mineral Research.

Uncovering the Dorsal Thalamo-hypothalamic Tract of the Human Limbic System

As a non-limbic structure, the human thalamus is the most important modulator of the limbic system. The hypothalamus plays vital roles in the survival of species by regulating fear, learning, feeding behavior, circadian rhythm, sociosexual and reproductive activities of the limbic system through connections with the thalamus. The detailed anatomy of the pathways responsible for mediating these responses, however, is yet to be determined. The mammillothalamic tract is known as the major direct thalamo-hypothalamic connection in the primates including the human brain connecting the ventral thalamus to the dorsal hypothalamus. Multiple dissection animal studies revealed additional connections specially from the dorsal thalamus to the ventral hypothalamic nuclei. Diffusion weighted imaging may be helpful in better visualizing the surgical anatomy of this additional connectivity noninvasively. This study aimed to investigate the utility of high spatial and high angular resolution diffusion weighted tractography technique for mapping the trajectory of this dorsal thalamic connectivity with the ventral hypothalamus in the human brain. We studied 30 healthy human subjects. Using a high-resolution diffusion weighted tractography technique, for the first time, we were able to delineate and reconstruct the trajectory of the dorsal thalamo-hypothalamic tract (DTH). We further revealed the close relationship of the DTH, fornix and hippocampus in healthy adult human brain.

Spatiotemporal single-cell analysis of gene expression in the mouse suprachiasmatic nucleus.

Mammalian circadian behaviors are orchestrated by the suprachiasmatic nucleus (SCN) in the ventral hypothalamus, but the number of SCN cell types and their functional roles remain unclear. We have used single-cell RNA-sequencing to identify the basic cell types in the mouse SCN and to characterize their circadian and light-induced gene expression patterns. We identified eight major cell types, with each type displaying a specific pattern of circadian gene expression. Five SCN neuronal subtypes, each with specific combinations of markers, differ in their spatial distribution, circadian rhythmicity and light responsiveness. Through a complete three-dimensional reconstruction of the mouse SCN at single-cell resolution, we obtained a standardized SCN atlas containing the spatial distribution of these subtypes and gene expression. Furthermore, we observed heterogeneous circadian gene expression between SCN neuron subtypes. Such a spatiotemporal pattern of gene regulation within the SCN may have an important function in the circadian pacemaker.

Lorcaserin bidirectionally regulates dopaminergic function site-dependently and disrupts dopamine brain area correlations in rats

Lorcaserin, which is a selective agonist of serotonin2C receptors (5-HT2CRs), is a new FDA-approved anti-obesity drug that has also shown therapeutic promise in other brain disorders, such as addiction and epilepsy. The modulation of dopaminergic function might be critical in the therapeutic effect of lorcaserin, but its exact effect is unknown. Here, we studied the effect of the peripheral administration of lorcaserin on the ventral tegmental area (VTA), the substantia nigra pars compacta (SNc) dopaminergic neural activity, dopamine (DA) dialysis levels in the nucleus accumbens and striatum and on DA tissue levels in 29 different rat brain regions. Lorcaserin (5–640 μg/kg, i.v.) moderately inhibited only a subpopulation of VTA DA neurons, but had no effect on the SNc neurons. Lorcaserin (0.3, 3 mg/kg, i.p.) did not change VTA and SNc DA population neural activity but slightly decreased the firing rate and burst firing of the spontaneously active VTA neurons, without altering DA extracellular dialysate levels in both the nucleus accumbens and the striatum. Quantitative analysis of DA and metabolites tissue contents of the 29 areas studied revealed that lorcaserin (0.3 or 3 mg/kg, i.p.) only affected a few brain regions, i.e., increased DA in the central amygdala, ventral hypothalamus and nucleus accumbens core and decreased it in the ventromedial striatum. On the other hand, lorcaserin dramatically changed the direction and reduced the number of correlations of DA tissue content among several brain areas. These effects on DA terminal networks might be significant in the therapeutic mechanism of lorcaserin.This article is part of the special issue entitled ‘Serotonin Research: Crossing Scales and Boundaries’.

Congenital pituitary hypoplasia model demonstrates hypothalamic OTX2 regulation of pituitary progenitor cells.

Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone-producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.

1796-P: A Ventral Medial Hypothalamus Estrogen Receptor a Neural Circuit Controlling Energy Expenditure

Multiple estrogen receptor α (ERα) neuron populations in the brain are required to maintain normal body weight. While the metabolic effects of these ERα neuron populations have been investigated, the neural circuit and associated neurotransmitter signaling underlying the regulatory effects of ERα neurons on energy balance are unknown. Here we combined retrograde and anterograde trans-synaptic tracers, chemogenetics, optogenetic circuit mapping, and genetic mouse models to functionally map the downstream neural circuits of ERα neurons. We found that selective activation of ERα neurons in the ventral medial hypothalamus (VMH) stimulated brown adipose tissue (BAT) thermogenesis and physical activity without affecting food intake. We also showed that VMH ERα neurons provide monosynaptic glutamatergic inputs to serotonergic neurons in the dorsal Raphe nuclei (DRN). Inhibition of DRN serotonergic neurons partially attenuated the stimulatory effects of VMH ERα neurons on BAT thermogenesis and physical activity. More intriguingly, specific deletion of ERα or vesicular glutamate transporter 2 (vGluT2) from a subpopulation of VMH neurons projecting to the DRN results in decreased BAT thermogenesis and physical activity with normal food intake. Together, these findings suggest a model that VMH ERα neurons activate BAT thermogenesis and physical activity by stimulating DRN serotonergic neurons. Disclosure P. Xu: None. Y. He: None. C. Wang: None. Y. Yang: None. X. Cai: None. Y. Xu: None. Funding American Diabetes Association (1-17-PDF-138 to Y.H.); National Institutes of Health (R01DK093587), (R01DK101379 to Y.X.); (R00DK107008 to P.X.); U.S. Department of Agriculture (3092-5-001-059 to Y.X.); American Heart Association (17GRNT32960003 to Y.X.)

Daily expression of a clock gene in the brain and pituitary of the Malabar grouper (Epinephelus malabaricus)

Recent studies have revealed that, in addition to regulating the circadian system, clock genes such as cryptochrome (Cry) genes are involved in seasonal and lunar rhythmicity in fish. This study clarified the transcriptional characteristics of a Cry subtype (mgCry2) in the brain of the Malabar grouper, Epinephelus malabaricus, which is an important aquaculture species that spawns around the new moon. The cDNA sequence of mgCry2 showed high identity (97–99%) with fish Cry2 and had an open reading frame encoding a protein with 170 amino acids. Phylogenetic analyses revealed that mgCRY2 had high identity with CRY in other fish species. Real-time quantitative polymerase chain reaction (qPCR) showed the widespread distribution of mgCry2 in neural (brain, pituitary, and retina) and peripheral (heart, liver, kidney, spleen, gill, intestine, and ovary) tissues. When immature Malabar groupers were reared under a light-dark cycle (LD = 12:12) and the amounts of mgCry2 mRNA in the telencephalon and diencephalon were measured at 4-h intervals, the levels increased during photophase and decreased during scotophase. Day–night variation in mgCry2 mRNA abundance was also observed in the pituitary. These daily profiles suggest that mgCry2 is a light-responsive gene in neural tissues. In situ hybridization analyses showed that mgCry2 was strongly transcribed in the nucleus lateralis tuberis of the ventral hypothalamus, peripheral area of the proximal pars distalis, and the pars intermedia of the pituitary. We conclude that clock genes expressed in the pituitary and diencephalon play a role in entraining the endocrine network of the Malabar grouper to periodic changes in external cues.