Preoptic Nucleus Research Articles

Effect of cyclo‑oxygenase inhibition on embryonic microglia and the sexual differentiation of the brain and behavior of Japanese quail (Coturnix japonica)

Enduring sex differences in the brain are established during a developmental process known as brain sexual differentiation and are mainly driven by estrogens during a critical period. In rodents, the masculinization of the preoptic area by estrogens derived from the central aromatization of testosterone depends in part on the interaction between microglia and prostaglandin E2 (PGE2), a pro-inflammatory hormone of the prostanoid subclass. In contrast, in birds, estrogens produced by females induce a demasculinization, but whether an interaction with the neuro-immune system is involved in this process is unknown. This study addressed this question by testing the effects of blockade of cyclo‑oxygenases (COX), the rate-limiting enzymes for prostanoid synthesis, on embryonic microglia and the sexual differentiation of brain and behavior using the Japanese quail as an animal model. The results show that COX inhibition does not affect the behavior of females, but impairs male sexual behavior and suppresses the sex difference in microglial profiles at embryonic day 12 (E12) in the medial preoptic nucleus by increasing the number of microglia in males only. However, neither prostanoid concentrations nor PGE2 receptors differed between sexes in the hypothalamus and preoptic area (HPOA) during development. Overall, these results uncovered a potential role of prostanoids in the demasculinization of Japanese quail. Moreover, the parallel effect of COX inhibition on behavior and microglia suggests an interaction between prostanoids and microglia in brain demasculinization, thus fueling the hypothesis of a conserved role of the neuroimmune system in the organization of the brain by estrogens.

The hemodynamic stability intraoperatively and postoperatively in dexmedetomidine group compared to the one who were only infused with normal saline: Brachial plexus block

The α2 agonists act through the endogenous sleep-promoting pathways to exert their sedative effect. Dexmedetomidine produces a decrease in activity of the projections of the locus ceruleus to the ventrolateral preoptic nucleus. This increases GABAergic release in the tuberomammillary nucleus, producing a decrease in histamine release in cortical and sub cortical projections. All patients underwent a thorough pre-anaesthetic checkup which included history taking, general examination, systemic examination and local neurological examination. Routine investigations like Haemogram, blood sugar, blood urea, serum creatinine, Chest X-Ray, ECG were carried out for all patients. All patients were taught about pain scale regarding VAS scale during pre-operative visit. Mean blood pressure in group D was lower compared to group P since immediately after induction upto 20 minutes after induction, which were statistically significant. There was no statistically significant difference in mean blood pressure of the patients between the two groups after 20 minutes.

AT1a-dependent GABAA inhibition in the MnPO following chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) is associated with diurnal hypertension, increased sympathetic nerve activity (SNA), and increases in circulating angiotensin II (ANG II). In rats, CIH increases angiotensin type 1 (AT1a) receptor expression in the median preoptic nucleus (MnPO), and pharmacological blockade or viral knockdown of this receptor prevents CIH-dependent increases in diurnal blood pressure. The current study investigates the role of AT1a receptor in modulating the activity of MnPO neurons following 7 days of CIH. Male Sprague-Dawley rats received MnPO injections of an adeno-associated virus with an shRNA against the AT1a receptor or a scrambled control. Rats were then exposed to CIH for 8 h a day for 7 days. In vitro, loose patch recordings of spontaneous action potential activity were made from labeled MnPO neurons in response to brief focal application of ANG II or the GABAA receptor agonist muscimol. In addition, MnPO K-Cl cotransporter isoform 2 (KCC2) protein expression was assessed using Western blot. CIH impaired the duration but not the magnitude of ANG II-mediated excitation in the MnPO. Both CIH and AT1a knockdown also impaired GABAA-mediated inhibition, and CIH with AT1a knockdown produced GABAA-mediated excitation. Recordings using the ratiometric Cl- indicator ClopHensorN showed CIH was associated with Cl- efflux in MnPO neurons that was associated with decreased KCC2 phosphorylation. The combination of CIH and AT1a knockdown attenuated reduced KCC2 phosphorylation seen with CIH alone. The current study shows that CIH, through the activity of AT1a receptors, can impair GABAA-mediated inhibition in the MnPO and contribute to sustained hypertension.

Presynaptic inputs to vasopressin neurons in the hypothalamic supraoptic nucleus and paraventricular nucleus in mice

Arginine vasopressin (AVP) neurons in the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN) are involved in important physiological behaviors, such as controling osmotic stability and thermoregulation. However, the presynaptic input patterns governing AVP neurons have remained poorly understood due to their heterogeneity, as well as intermingling of AVP neurons with other neurons both in the SON and PVN. In the present study, we employed a retrograde modified rabies-virus system to reveal the brain areas that provide specific inputs to AVP neurons in the SON and PVN. We found that AVP neurons of the SON and PVN received similar input patterns from multiple areas of the brain, particularly massive afferent inputs from the diencephalon and other brain regions of the limbic system; however, PVNAVP neurons received relatively broader and denser inputs compared to SONAVP neurons. Additionally, SONAVP neurons received more projections from the median preoptic nucleus and organum vasculosum of the lamina terminalis (a circumventricular organ), compared to PVNAVP neurons, while PVNAVP neurons received more afferent inputs from the bed nucleus of stria terminalis and dorsomedial nucleus of the hypothalamus, both of which are thermoregulatory nuclei, compared to those of SONAVP neurons. In addition, both SONAVP and PVNAVP neurons received direct afferent projections from the bilateral suprachiasmatic nucleus, which is the master regulator of circadian rhythms and is concomitantly responsible for fluctuations in AVP levels. Taken together, our present results provide a comprehensive understanding of the specific afferent framework of AVP neurons both in the SON and PVN, and lay the foundation for further dissecting the diverse roles of SONAVP and PVNAVP neurons.

Local administration of bicuculline into the ventrolateral and medial preoptic nuclei modifies sleep and maternal behavior in lactating rats.

The preoptic area (POA) is a brain structure classically involved in a wide variety of animal behavior including sleep and maternal care. In the current study, we evaluate the specific effect of disinhibition of two specific regions of the POA, the medial POA nucleus (mPOA) and the ventrolateral POA area (VLPO) on sleep and maternal behavior in lactating rats. For this purpose, mother rats on postpartum day 1 (PPD1) were implanted for polysomnographic recordings and with bilateral cannulae either in the mPOA or in the VLPO. The rats were tested for sleep and maternal behavior on PPD4–8 after the infusion of the GABA-A antagonist, bicuculline (0, 10 or 30 ng/0.2 µl/side). Infusion of bicuculline into the mPOA augmented retrieving and nest building behaviors and reduced both nursing and milk ejections but had almost no effect on sleep. When bicuculine was microinjected into the VLPO, the rats significantly increase the number of retrievings and mouthings and reduced the nursing time without changes in milk ejections, which was associated with an increase in wakefulness and a reduction in light sleep.Our results show that disinhibition of the mPOA, a key area in the control of maternal behavior, increased active maternal behaviors and reduced nursing without affecting wakefulness or sleep time. In contrast, the enhancement of some active maternal behaviors when the drug was infused into the VLPO, a sleep-promoting area, with a concomitant increase in wakefulness suggests that mother rats devote this additional waking time in the active maternal care of the pups. We hypothesize that maternal behavior changes after bicuculine microinjection into the VLPO are caused by a reduction in the sleep drive, rather than a direct effect on maternal behavior.

Sexually dimorphic perineuronal nets in the rodent and primate reproductive circuit.

Perineuronal nets are extracellular glycoprotein structures that have been found on some neurons in the central nervous system and that have been shown to regulate their structural plasticity. Until now work on perineuronal nets has been focused on their role in cortical structures where they are selectively expressed on parvalbumin-positive neurons and are reported to restrict the experience-dependent plasticity of inhibitory afferents. Here, we examined the expression of perineuronal nets subcortically, showing that they are expressed in several discrete structures, including nuclei that comprise the brain network controlling reproductive behaviors (e.g., mounting, lordosis, aggression, and social defense). In particular, perineuronal nets were found in the posterior dorsal division of the medial amygdala, the medial preoptic nucleus, the posterior medial bed nucleus of the stria terminalis, the ventrolateral ventromedial hypothalamus and adjacent tuberal nucleus, and the ventral premammillary nucleus in both the mouse and primate brain. Comparison of perineuronal nets in male and female mice revealed a significant sexually dimorphic expression, with expression found prominently on estrogen receptor expressing neurons in the medial amygdala. These findings suggest that perineuronal nets may be involved in regulating neural plasticity in the mammalian reproductive system.

Distribution of Vasopressin and Oxytocin Neurons in the Basal Forebrain and Midbrain of Spiny Mice (Acomys cahirinus)

The nonapeptides vasopressin (VP) and oxytocin (OT) are present in some form in most vertebrates. VP and OT play critical roles in modulating physiology and are well-studied for their influences on a variety of social behaviors, ranging from affiliation to aggression. Their anatomical distributions have been mapped for numerous species across taxa, demonstrating relatively strong evolutionary conservation in distributions throughout the basal forebrain and midbrain. Here we examined the distribution of VP-immunoreactive (-ir) and OT-ir neurons in a gregarious, cooperatively breeding rodent species, the spiny mouse (Acomys cahirinus), for which nonapeptide mapping does not yet exist. Immunohistochemical techniques revealed VP-ir and OT-ir neuronal populations throughout the hypothalamus and amygdala of males and females that are consistent with those of other rodents. However, a novel population of OT-ir neurons was observed in the median preoptic nucleus of both sexes, located dorsally to the anterior commissure. Furthermore, we found widespread sex differences in OT neuronal populations, with males having significantly more OT-ir neurons than females. However, we observed a sex difference in only one VP cell group – that of the bed nucleus of the stria terminalis (BST), a VP neuronal population that exhibits a phylogenetically widespread sexual dimorphism. These findings provide mapping distributions of VP and OT neurons in Acomys cahirinus. Spiny mice lend themselves to the study of mammalian cooperation and sociality, and the nonapeptide neuronal mapping presented here can serve as a basic foundation for the study of nonapeptide-mediated behavior in a group of highly social rodents.

Effects of neurokinin 3 receptor antagonist fezolinetant on hot flash-like symptoms in ovariectomized rats

The majority of women experience vasomotor symptoms (VMS), such as hot flashes and night sweats, during the menopausal transition. Recent evidence strongly suggests a connection between neurokinin 3 (NK3) receptor signaling and VMS associated with menopause. The NK3 receptor antagonist fezolinetant is currently in phase 3 development for treatment of moderate to severe VMS associated with menopause. We investigated the pharmacological effects of repeated administration of fezolinetant on levels of sex hormones and gonadotropins, neuronal activity in the hypothalamus, and skin temperature as an index of hot flash-like symptoms in ovariectomized rats as a model of menopause. Ovariectomized rats exhibited several typical menopausal symptoms: hyperphagia, increased body weight, significantly decreased plasma estradiol levels, increased luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels, and significantly increased skin temperature. Increased c-Fos expression (an indirect marker of neuronal activity) in median preoptic nucleus (MnPO) hypothalamic neurons was also observed in ovariectomized rats. Repeated oral administration of fezolinetant (1–10 mg/kg, twice daily) for 1 week dose-dependently reduced plasma LH levels without affecting estradiol or FSH levels, inhibited the activation of MnPO neurons, and attenuated hot flash-like symptoms. In addition, fezolinetant dose-dependently reduced hyperphagia and weight gain in ovariectomized rats. These preclinical findings suggest that fezolinetant attenuates hot flash-like symptoms via inhibition of neuronal activity in the MnPO of ovariectomized rats and provides further support for the ongoing clinical development of fezolinetant for the treatment of VMS associated with menopause.

Effects of propofol on sleep architecture and sleep–wake systems in rats

Previous studies have shown that the synchronization of electroencephalogram (EEG) signals is found during propofol-induced general anesthesia, which is similar to that of slow-wave sleep (SWS). However, a complete understanding is lacking in terms of the characteristics of EEG changes in rats after propofol administration and whether propofol acts through natural sleep circuits. Here, we examined the characteristics of EEG patterns induced by intraperitoneal injection of propofol in rats. We found that high (10 mg/kg) and medium (5 mg/kg) doses of propofol induced a cortical EEG of low-frequency, high-amplitude activity with rare electromyographic activity and markedly shortened sleep latency. The high dose of propofol increased deep slow-wave sleep (SWS2) to 4 h, as well as the number of large SWS2 bouts (>480 s), their mean duration and the peak of the power density curve in the delta range of 0.75–3.25 Hz. After the medium dose of propofol, the total number of wakefulness, light slow-wave sleep (SWS1) and SWS2 episodes increased, whereas the mean duration of wakefulness decreased. The high dose of propofol significantly increased c-fos expression in the ventrolateral preoptic nucleus (VLPO) sleep center and decreased the number of c-fos-immunoreactive neurons in wake-related systems including the tuberomammillary nucleus (TMN), perifornical nucleus (PeF), lateral hypothalamic nucleus (LH), ventrolateral periaqueductal gray (vPAG) and supramammillary region (SuM). These results indicated that the high dose of propofol produced high-quality sleep by increasing SWS2, whereas the medium dose produced fragmented and low-quality sleep by disrupting the continuity of wakefulness. Furthermore, sleep-promoting effects of propofol are correlated with activation of the VLPO cluster and inhibition of the TMN, PeF, LH, vPAG and SuM.

QPLOT Neurons-Converging on a Thermoregulatory Preoptic Neuronal Population.

The preoptic area of the hypothalamus is a homeostatic control center. The heterogeneous neurons in this nucleus function to regulate the sleep/wake cycle, reproduction, thirst and hydration, as well as thermogenesis and other metabolic responses. Several recent studies have analyzed preoptic neuronal populations and demonstrated neuronal subtype-specific roles in suppression of thermogenesis. These studies showed similar thermogenesis responses to chemogenetic modulation, and similar synaptic tracing patterns for neurons that were responsive to cold, to inflammatory stimuli, and to violet light. A reanalysis of single-cell/nucleus RNA-sequencing datasets of the preoptic nucleus indicate that these studies have converged on a common neuronal population that when activated, are sufficient to suppress thermogenesis. Expanding on a previous name for these neurons (Q neurons, which reflect their ability to promote quiescence and expression of Qrfp), we propose a new name: QPLOT neurons, to reflect numerous molecular markers of this population and to capture its broader roles in metabolic regulation. Here, we summarize previous findings on this population and present a unified description of QPLOT neurons, the excitatory preoptic neuronal population that integrate a variety of thermal, metabolic, hormonal and environmental stimuli in order to regulate metabolism and thermogenesis.

Identification of Cell Types that Express Dio3 Deiodinase, a Thyroid Hormone-Inactivating Enzyme, Using a Dio3-CreERT2 Reporter System

Background: Thyroid hormone promotes development, growth and metabolism. The level of thyroid hormone ligand (triiodothyronine, T3) in tissues depends not only on circulating levels but also upon tight regulation by activating and inactivating deiodinases within tissues. Type 3 deiodinase (Dio3) inactivates T3 and its precursor thyroxine (T4) and mediates many functions including in neurodevelopmental, sensory and reproductive systems. Dio3 is subject to genomic imprinting. Despite its critical functions, Dio3 is often expressed transiently and at low levels in restricted cell populations making it difficult to detect in natural tissues. Methods: To visualize Dio3 expression at cellular resolution, we derived a Dio3-CreERt2 knockin allele that expresses tamoxifen-dependent Cre recombinase from the endogenous Dio3 gene. When crossed with Ai6 reporter mice, Dio3-CreERt2-positive cells display fluorescent signals. When tamoxifen-treated at neonatal ages, Dio3-CreERt2 recapitulates endogenous Dio3 expression as previously reported in brain: in the bed nucleus of the stria terminalis and preoptic nuclei. In addition, we uncovered several positive cell groups in the hypothalamus, brain stem, pituitary and other tissues. Drastic differences were observed for Dio3-CreERt2 as a paternally versus maternally inherited allele, revealing imprinting effect in specific cell types. Dio3-CreERT2 activity is enhanced by T3 administration, in accordance with Dio3 as a T3-indicible gene. Conclusion: The Dio3-CreERT2 model sensitively reveals Dio3-expressing cell types in tissues. The model is useful for studying expression patterns, imprinting and lineage tracing of Dio3-positive cells during development and homeostatic challenges.

066 Noradrenaline and acetylcholine inhibit sleep-promoting neurons of ventrolateral preoptic area through a local GABAergic circuit

Abstract Introduction The ventrolateral preoptic (VLPO) nucleus is a key area involved in the initiation and maintenance of sleep. During wakefulness, sleep-promoting galanin neurons in the VLPO are directly inhibited by arousal signals including noradrenaline and acetylcholine. We have found that while these neurotransmitters directly inhibit VLPO galanin neurons, they also activate GABAergic neurons in the VLPO that do not express galanin. We propose that when activated by monoaminergic and cholinergic inputs, these local VLPO GABAergic neurons provide an additional inhibition of the VLPO galanin sleep-promoting neurons. We tested this model in brain slices in mice. Methods We studied VLPO galanin neurons in mouse brain slices using patch-clamp recordings. We recorded from fluorescently labeled VLPO galanin neurons following the injection of a cre-dependent AAV encoding for mCherry, into the VLPO of Gal-cre mice. For the optogenetic studies we expressed channelrhodopsin-2 (ChR-2) in VLPO VGAT neurons and mCherry in galanin neurons by injecting a flp-dependent and a cre-dependent AAV encoding respectively for ChR2 and mCherry into the VLPO of VGAT-flp::Gal-cre mice. We photo-stimulated local GABAergic neurons and recorded from labeled VLPO galanin neurons. Noradrenaline, carbachol and receptor antagonists were bath-applied. Results Noradrenaline and carbachol inhibited VLPO galanin neurons by alpha-2 and muscarinic receptors and these effects were maintained in the presence of tetrodotoxin (TTX) indicating, as previously proposed, a direct inhibitory effect of noradrenaline and carbachol on VLPO galanin neurons. In addition, both noradrenaline and carbachol increased the frequency of spontaneous inhibitory post-synaptic currents (sIPSCs) of VLPO galanin neurons, suggesting an additional inhibitory action on VLPO galanin neurons. Finally, optogenetic stimulation of local VLPO GABAergic neurons produced short latency, TTX-resistant, opto-evoked IPSCs in VLPO galanin neurons. Both noradrenaline and carbachol increased the amplitude of these opto-evoked IPSCs by the activation of alpha-1 and muscarinic receptors. Conclusion Our results demonstrate that noradrenaline and acetylcholine inhibit VLPO galanin neurons directly and indirectly. Both noradrenaline and acetylcholine increase GABAergic afferent inputs to VLPO galanin neurons by activating local GABAergic neurons. We propose that during wakefulness this feedforward inhibition provides additional inhibition of VLPO galanin sleep-promoting neurons. Support (if any) NS091126 and HL149630

Exposure to Iodoacetic Acid, a Water Disinfection Byproduct, Leads to Abnormal Expression of Key Reproductive Axis Genes in the Hypothalamus and Pituitary

Iodoacetic acid (IAA) – a water disinfection byproduct (DBP) formed from the reaction between an oxidizing disinfectant, i.e. chlorine, and iodide – is an understudied, yet potentially dangerous environmental toxicant. DBPs have been epidemiologically associated with reproductive dysfunction. In vitro studies have indicated that IAA is one of the most cyto- and genotoxic DBPs. Further, murine ovarian research has shown that IAA exposure significantly inhibits antral follicle growth and reduces estradiol levels. Despite this evidence, little is known about the other components of the reproductive axis: the hypothalamus and pituitary. To address this, we tested the hypothesis that IAA exposure would lead to disrupted expression of key hypothalamic and pituitary genes related to reproductive function. We exposed adult female CD1 mice to 0.5, 10, 100, or 500 mg/L IAA in their drinking water from postnatal day 40 (P40) to their first day in diestrus after P75. From this experiment, we collected whole pituitaries and hypothalamic punches containing the arcuate nucleus (ARC), anteroventral periventricular zone (AVPV), and medial preoptic nucleus (mPOA), and processed them for mRNA analysis. We also exposed pituitary explant cultures to IAA to observe direct effects on gene expression. In vivo, we found that mRNA levels of kisspeptin (Kiss1) are significantly increased in the ARC, the region that controls pulsatile GnRH release, at 0.5 and 10 mg/L IAA concentrations. Kiss1 is unchanged in the AVPV, the neuron population responsible for generating the LH surge. We also measured ARC expression of neurokinin B (Tac2) and dynorphin (Pdyn), neuropeptides secreted by kisspeptin co-expressing neurons to autosynaptically stimulate Kiss1 release. We saw no difference in either. GnRH (Gnrh1) expression was also unchanged. Both in vivo at 10 mg/L IAA and in culture, we found IAA exposure significantly reduced Fshb mRNA. Preliminary immunohistochemistry (IHC) data suggests it also leads to an apparent reduction in FSH-positive cells in vitro (N=2). Lhb and the α-subunit (Cga) were unaltered in vivo, though were significantly reduced with in vitro exposure. In neither context was mRNA expression of the GnRH receptor (Gnrhr) changed. Noting apparent direct effects of IAA on the pituitary, we assessed expression of the cell-cycle inhibitor p21 (Cdkn1a), which has been shown to increase with toxicant exposure. We found Cdkn1a increased in vivo at 500 mg/L IAA, trending at 100 mg/L (p=.070), and in vitro. IHC data in vitro suggests a marked increase in P21-positivity following IAA exposure. These data, together with prior ovarian findings, implicate IAA as a potential reproductive axis disruptor at each major level – through ARC Kiss1 expression, Fshb expression in vivo and in vitro, FSH expression in vitro, and Lhb and Cga in vitro. Further, Cdkn1a/P21 induction indicates IAA toxicity at the level of the pituitary.

Identification of PACAP-Expressing Neurons in the Hypothalamic Origins of Sympathetic Outflow Tracts Terminating in Brown Adipose Tissue of Mice

Abstract Adaptive thermogenesis in brown adipose tissue is stimulated by the sympathetic nervous system (SNS) in response to cold stress. Using retrograde viral transneuronal tract tracers, previous studies have identified that the paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), and median preoptic nucleus (MnPO) contain neurons that are part of sympathetic outflow tracts to brown adipose tissue, presumptively involved in SNS stimulation of interscapular brown adipose tissue (iBAT). Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is a peptide hormone known to regulate energy homeostasis, acting in both the central (CNS) and peripheral nervous system (PNS). Mice lacking PACAP have impaired adrenergic-induced thermogenesis and a cold-sensitive phenotype. In the CNS, PACAP is highly expressed in the VMH, MnPO, and PVN of the hypothalamus. Injection of PACAP into the VMN increased core body temperature and sympathetic nerve activity to brown adipose tissue. While these studies show exogenous PACAP can activate sympathetic outflow tracts to brown adipose tissue, they do not confirm that endogenously expressed PACAP induces sympathetic nerve activity as an adaptive mechanism to cold stress, or if sympathetic outflow tracts originating in the hypothalamus express PACAP. We hypothesize that PACAP is expressed in neurons of sympathetic outflow tracts originating in the hypothalamus. To test this hypothesis, PACAP-eGFP transgenic mice were injected with the retrograde neural tracer, pseudorabies virus tagged with β-galactosidase (β-gal, PRV-BaBlu), in iBAT where postganglionic nerves innervate the tissue. Five-days post-infection, animals were culled, brains removed and cryosectioned. Neurons positive for green fluorescent protein (eGFP) and/or β-gal immunoreactivity (ir) were identified by immunohistochemistry in serial coronal and sagittal brain cryo-sections. Co-occurrence of eGFP-ir and β-gal-ir, inferred PACAP expressing neurons present in sympathetic outflow tracts (ImageJ). Co-occurrence was identified in several structures in the hypothalamus and thalamus. In conclusion, this study presents neuroanatomical evidence for populations of PACAPinergic neurons in the hypothalamus that are part of sympathetic outflow tracts to brown adipose tissue, providing further evidence of a central role for PACAP in regulating energy homeostasis.

Adult Onset Isolated Hypogonadotropic Hypogonadism- a Cause of Secondary Amenorrhea

A 23-year-old African American female was referred for secondary amenorrhea evaluation. She attained menarche at 12 years and had regular menses. At 18 years, she used OCPs for few months, and used plan B, after which her menses stopped. She had hot flashes and sweating. She was placed on progesterone, but never had withdrawal bleeding. Review of systems was positive for intentional weight loss after her menses stopped, hair loss and nipple discharge. She denied any loss of sensation of smell. She has a PMH of asthma, anxiety and OSA. Family history was not significant for any fertility issues. She smoked cannabis after menses stopped. On physical examination, vitals were stable, BMI of 35 kg/meter2, well-developed secondary sexual characteristics, no thyromegaly, acne or hirsutism. Upon work up, CBC, CMP were normal, Urine pregnancy test was negative, gonadotropins were undetectable (FSH- <0.7mIU/ml, LH- <0.2mIU/ml), anti-mullerian hormone was 3.82ng/ml (WNL), Estradiol was also absent (<15pg/ml), with a low Total testosterone (11ng/dl), TSH was 1.17uIU/ml, Free T4 was 1.1ng/dl, ACTH was 9.58pg/ml, Cortisol was 14.8mcg/dl, and Prolactin was 1.5ng/ml. MRI brain was normal with normal pituitary gland, no focal lesion visualized. Pelvic ultrasound showed ovaries 5.9mL and 4.6mL with multiple follicles present bilaterally. Diagnosis of adult-onset isolated hypogonadotropic hypogonadism (IHH) was made. Patient was started on estradiol patches and progesterone. IHH is a genetic disorder of defective production or action of GnRH. IHH when associated with anosmia is called Kallmann syndrome. It was first described by German American geneticist Joseph Kallmann in 1944. GnRH is a decapeptide, produced in arcuate nucleus and pre-optic nucleus of hypothalamus. GnRH stimulates anterior pituitary to secrete FSH and LH. IHH is caused due to impaired migration of GnRH neurons to brain during embryogenesis. It is inherited as autosomal or X-linked dominant or recessive. Gene mutations associated are ANOS-1, FGFR, PROK-2. It is rare in females. IHH has a broad spectrum of clinical presentation from complete absence of sexual development to partial completion of puberty. It presents with microphallus, cryptorchidism, cleft lip/palate, syndactyly, renal aplasia. In childhood presents with anosmia, hearing deficits, dental agenesis, mirror movements, short stature. During puberty, absent pubertal growth spurt, amenorrhea, lack of virilization, no secondary sexual characteristics, infertility. In partial forms, known as Adult onset or acquired form of IHH, patients have slight testicular growth, thelarche, menarche. Goals of treatment are- pubertal induction, maintenance of sexual maturation and restoration of fertility. In females, pre-puberty only estrogen is given, after puberty both estrogen and progesterone are used, for fertility, pulsatile gonadotropins or GnRH analogues are used.

029 Median preoptic dual control over sleep regulation

Abstract Introduction Previous studies suggest that the median preoptic nucleus (MnPO) plays an important role in regulating the wake-sleep cycle and in particular homeostatic sleep drive. However, the precise cellular phenotypes, targets and central mechanisms by which the MnPO neurons regulate the wake-sleep cycle remain unknown. Both glutamatergic (Vglut2+) and GABAergic (Vgat+) MnPO neurons innervate brain regions implicated in sleep promotion and maintenance, suggesting that both cell types may participate on sleep control. Methods In this study, we used two genetically-targeted approaches associated with electroencephalographic (EEG) and electromyographic (EMG) recordings in Vgat-IRES-cre and Vglut2-IRES-cre mice to investigate the role of the MnPOVgat and MnPOVglut2 neurons in modulating wake-sleep behavior. Results First, using a chemogenetic approach, we found that activation of MnPOVgat neurons reduced the latency for the first NREM sleep episode, produced an increase in NREM sleep and reduced wakefulness. Then, to test the role of MnPOVgat and MnPOVglut2 neurons in regulating sleep homeostasis, we recorded EEG and EMG responses in mice that had the Vgat+ or Vglut2+ neurons deleted from the MnPO. After deletion of MnPOVgat neurons, mice showed a reduction of NREM sleep and an increase in wakefulness during the light phase. Deletion of MnPOVgat neurons also reduced sleep recovery after 4 hours of sleep deprivation (SD). On the other hand, deletion of the MnPOVglut2 neurons did not change the wake-sleep cycle during the 24h baseline condition, but prevented the sleep recovery immediately after SD. To understand the underlying mechanism in preventing sleep recovery in both MnPOVglut2- and MnPOVgat-deleted mice groups, we exposed these animals to a psychological stress protocol. In response to a psychological stressor, mice with deletion of glutamatergic, but not GABAergic MnPO neurons, had an exacerbation of the stress-induced insomnia. Conclusion Our results suggest that both neuron populations differentially participate in wake-sleep control, with MnPOVgat neurons being critically involved in sleep homeostasis, and MnPOVglut2 neurons promoting sleep during allostatic (stressful) challenges. Support (if any) NIH Grants NS085477, NS072337, HL095491 and Sleep Research Society Foundation (Award 026-JP-20).

Defining the Neurocircuit Underlying Autonomic Control of Cardiovascular and Metabolic Functions

The central nervous system (CNS) autonomic network play an important role in energy homeostasis, and cardiovascular regulation with important pathophysiological implications. However, whether the same autonomic network is involved in metabolic and cardiovascular control remain unknown. We hypothesize that divergent neurocircuits are involved in the energy balance and cardiovascular regulation. To test this, we employed pseudorabies viruses expressing a green fluorescent protein (PRV-GFP) to understand the polysynaptic connections between CNS nuclei and metabolic organs (liver and brown adipose tissue [BAT]) vs a key cardiovascular organ (kidney). We identified several nuclei that project to all three organs although they may represent different orders (expression appearing on different days). In the brainstem, the nucleus of the solitary tract (nTS) and area postrema expressed GFP after infection of the three organs while the dorsal vagal nucleus expressed GFP after infection of the liver, but less after inoculation of the kidneys. We found GFP expression in the ventral tegmental area following liver infection and after inoculation of the kidneys or BAT. Projections from cortical and subcortical regions such as the motor cortex, bed nucleus of the striatum, septal nuclei, and preoptic nuclei were also observed from the three organs. We observed that the subfornical organ project to the liver, BAT, and kidneys, whereas a few nuclei such as the nucleus accumbens, and insular cortex project specifically to the liver. GFP expression was noted in several hypothalamic nuclei such as the paraventricular nucleus, arcuate nucleus, lateral hypothalamus, and ventromedial hypothalamus (VMH) for all three organs. To define the specific neuronal populations projecting to various organs, we used mice bearing Cre-mediated expression of a fluorescent protein, td-Tomato. We injected PRV-GFP into the kidneys of animals expressing td-Tomato in the steroidogenic factor-1 (SF-1) neurons located in the VMH. Substantial number of VMH SF-1 neurons were labeled with GFP following PRV inoculation in the kidneys. Moreover, we identified several downstream nuclei that receive SF-1 neuron projections where GFP is expressed including the nTS, periaqueductal grey, and amygdala. Our study supports the existence of an overlap, but also unique, projections that sub-serve the liver, BAT, and kidneys. We also established that SF-1 neurons are part of the renal autonomic network. The relevance of the newly identified autonomic projections to hepatic, BAT, and renal functions is under investigation.

CIH Enhances Glutamatergic Transmission in Median Preoptic Nucleus

Studies have shown that the median preoptic nucleus (MnPO) contributes to hypertension associated with chronic intermittent hypoxia (CIH), a model of the hypoxemia associated with sleep apnea. Virally mediated lesions of MnPO neurons prevent the development of a sustained hypertension associated with CIH. To gain more insight into the properties of excitatory transmission to MnPO neurons develops in CIH, whole cell patch clamp recordings were from MnPO neurons brain slices from rats exposed to 7 days of CIH (6 minute cycles; 3 minutes of 21% oxygen room air pumped in, 90 seconds of nitrogen pumped in to lower the chamber O2 to 10% oxygen, and then 90 seconds of maintenance at 10% oxygen) for 8 h during the light phase or were normoxic controls. Slices were perfused with oxygenates artificial cerebrospinal fluid and the MnPO was visually identified. Miniature excitatory postsynaptic currents (mEPSCs) were recorded in the presences of 25 µm SR95531 (GABAA antagonist) and 1 µm TTX (block Na+ channel-mediated action potentials) using a conventional whole cell patch clamp configuration. The mEPSCs were collected over a 4 min period and amplitude and frequency were compared between cells obtained from slice prepared from normoxic control rats (NORM) or rats exposed to CIH. CIH was associated with a significant increase in mean amplitude (CIH 17.15 ± 0.65 pA, n=19 cells; NORM (15.6 ± 0.6 pA; n=18 cells, P <0.05). Similarly, mean frequency was significantly elevated after CIH (CIH 4.05 ± 0.5 Hz, n=19 vs NORM 2.56 ± 0.37 Hz, n=18; p<0.05) and associated with a significant leftward shift in the cumulative probability of inter event intervals. The results suggest that CIH simultaneously increases enhances spontaneous presynaptic neurotransmitter release while also increasing postsynaptic responsiveness in MnPO. These presynaptic and postsynaptic changes in MnPO that are associated with CIH may represent mechanism contributing to CIH hypertension.

Acute Intermittent Optogenetic Stimulation of Median Preoptic Nucleus (MnPO) Induces Sympathetic Long‐term Facilitation (sLTF)

Acute intermittent hypoxia in the rodent model can produce a ramp increase in sympathetic nerve activity (SNA) that progressively increases and is maintained for up to an hour after stimulation. The induction of this increase, termed sympathetic long-term facilitation (sLTF), involves circulating Angiotensin II acting on the subfornical organ (SFO) and the paraventricular nucleus of the hypothalamus (PVN). The median preoptic nucleus (MnPO) receives inputs from the SFO and projects to the PVN and might mediate the induction of sLTF. This study used an intersectional viral approach to test the ability of acute intermittent optogenetic stimulation of PVN-projecting MnPO neurons to induce sLTF. Male Sprague-Dawley rats (250 g) anesthetized with isoflurane were stereotaxically microinjected with CRE-containing retrograde AAV (AAV9.hSyn.HI.eGFP-cre.WPRE.SV40) in PVN and with either Channel rhodopsin 2 (ChR2)-containing AAV (rAAV2/Ef1a-DIO-hchR2(H134R)-mCherry) or control virus (rAAV2/Ef1a-DIO-mCherry) in MnPO. This resulted in CRE-dependent expression of either ChR2 or mCherry in PVN-projecting MnPO neurons. Following a 4-wk recovery, rats underwent acute intermittent optogenetic (AIO) experiments where splanchnic and or renal SNA, blood pressure (BP), and heart rate (HR) were recorded. Each rat was anesthetized with urethane (800mg/kg) and α-chloralose (80 mg/kg) cocktail delivered by intraperitoneal injection, paralyzed with gallamine triethiodide (20 mg/ml, 0.25 ml/h, iv), bilaterally vagotomized, and mechanically ventilated. Noise levels was determined after a bolus injection of hexamethonium (30 mg/kg iv). MnPO terminals in the PVN were stimulated to induce AIO with a train of 10 stimuli, once per 6 minutes. Each stimuli consisted of a duty cycle of 20-ms light pulse delivered at 5 Hz for 1 min. A single optogenetic duty cycle did not alter splanchnic SNA (P=0.216, ChR2 n = 5 vs control n =2), renal SNA (P=0.234, ChR2 n =3 vs control n =2), blood pressure (P=0.928, ChR2 n = 6 vs control n =4), nor heart rate (P=0.174, ChR2 n = 6 vs control n =4). AIO significantly increased splanchnic SNA (P=0.0163) in rats with ChR2 as compared to rats injected with the control vector 1h post-AIO (ChR2 percent baseline: 160.51±20.08%, n= 5 vs control percent baseline: 77.03±11.76%, n = 2). There were no differences in renal SNA (P=0.302, ChR2 n =3 vs control n =2), BP (P=0.339, ChR2 n = 6 vs control n =4), and HR (P=0.731, ChR2 n =3 vs control n =2) between rats injected with ChR2 as compared to those in injected with the control vector 1h post-AIO. These results suggest that intermittent activation of PVN-projecting MnPO neurons may induce sLTF in the splanchnic SNA but not in renal SNA. Further study is needed to clarify the mechanisms responsible for the differential effects of MnPO-PVN AIO.

PVN ‐ projecting MnPO neurons are involved in blood pressure and sympathetic nerve activity regulation in CIH hypertension

Sleep apnea (SA) is characterized by repeated interruptions in breathing resulting in hypoxia, hypercapnia, and increased thoracic pressure. Chronic intermittent hypoxia (CIH) in rats mimics the repetitive hypoxemias that occur with SA leading to a sustained increase in blood pressure associated with increased sympathetic activity and chemoreceptor sensitivity. CIH increases the peripheral renin-angiotensin system (RAS) activating the subfornical organ (SFO) which in turn projects to the hypothalamic paraventricular nucleus (PVN) and the median preoptic nucleus (MnPO). Angiotensin receptor knockdown in SFO or MnPO neurons and virally –mediated caspase lesions PVN-projecting MnPO neurons prevents CIH hypertension. These results suggest that PVN-projecting MnPO neurons play a significant role in blood pressure regulation in CIH hypertension. However, the contribution of MnPO to increased sympathetic nerve activity in CIH hypertension has not been directly tested. Our hypothesis is that the chronic activation of PVN by SFO via MnPO contributes to increased sympathetic outflow sustaining CIH hypertension through the diurnal cycle. In order to selectively inhibit PVN-projecting MnPO neurons, a retrograde Cre-dependent virus was microinjected bilaterally into PVN (AAV9.hSyn.HI.eGFP-Cre.WPRE.SV40) and a Cre-dependent inhibitory DREADD (PAAV.Syn.DIO.hM4D(Gi)-mCherry) was injected into MnPO. Two weeks later, rats were submitted to a 7-day CIH protocol (3 min of 10% oxygen followed by 3 min of 21% oxygen, 8 h/day) or normoxia. Rats were anesthetized (urethane 800 mg/kg and α-chloralose 80 mg/kg ip) and the trachea was cannulated for artificial ventilation. A femoral vein catheter (PE-50 tubing) was implanted for continuous infusion of gallamine triethiodide (20 mg/mL, 0.25 mL/h iv) for neuromuscular blockade. Blood pressure was measured by a Millar catheter system in the femoral artery. The rats were placed in a stereotaxic frame and a craniotomy was performed for intracerebroventricular (ICV) injections. Renal and splanchnic nerves were isolated and placed in a bipolar electrode. Noise levels were determined after a bolus injection of hexamethonium (30 mg/kg iv). Using a pneumatic picopump (WPI) connected to a single-barreled glass micropipette, DREADD agonist CNO (1µg/1µl) was injected ICV and responses were collected after 30 min of the injection. In rats exposed to normoxia, chemogenetic inhibition of PVN-projecting MnPO neurons slightly increased splanchnic SNA (4.9±21, n=3), renal SNA (17± 17, n=3) and increased blood pressure (Normoxia: 4.3±4, n=3; CIH; -0.3± 4, n=3). In rats exposed to CIH, CNO reduced splanchnic SNA (-42.9±37, n=4) and renal SNA (-88±49, n=3). These preliminary results suggest that CIH triggers a neuroadaptation that changes the relationship between the SFO-MnPO-PVN pathway and SNA.