Neuropeptide Y Neurons Research Articles

Sympathetic neuropeptide Y protects from obesity by sustaining thermogenic fat.

Human mutations in neuropeptide Y (NPY) have been linked to high body mass index but not altered dietary patterns1. Here we uncover the mechanism by which NPY in sympathetic neurons2,3 protects from obesity. Imaging of cleared mouse brown and white adipose tissue (BAT and WAT, respectively) established that NPY+ sympathetic axons are a smallersubset that mostly maps to the perivasculature; analysis of single-cell RNA sequencing datasets identified mural cells as the main NPY-responsive cells in adipose tissues. We show that NPY sustains the proliferation ofmural cells, which are a source of thermogenic adipocytes in both BAT and WAT4-6. We found that diet-induced obesity leads to neuropathy of NPY+ axons and concomitant depletion of mural cells. This defect was replicated in mice with NPY abrogated from sympathetic neurons. The loss of NPY in sympathetic neurons whitened interscapularBAT, reducing itsthermogenic ability and decreasing energy expenditure before the onset of obesity. It also caused adult-onset obesity of mice fed on a regular chow diet and rendered them more susceptible to diet-induced obesity without increasing food consumption. Our results indicate that, relative to central NPY, peripheral NPY produced by sympathetic nerves has the opposite effect on body weight by sustaining energy expenditure independently of food intake.

Lineage-tracing reveals an expanded population of NPY neurons in the inferior colliculus.

Growing evidence suggests that neuropeptide signaling shapes auditory computations. We previously showed that neuropeptide Y (NPY) is expressed in the inferior colliculus (IC) by a population of GABAergic stellate neurons and that NPY regulates the strength of local excitatory circuits in the IC. NPY neurons were initially characterized using the NPY-hrGFP mouse, in which humanized renilla green fluorescent protein (hrGFP) expression indicates NPY expression at the time of assay, i.e., an expression-tracking approach. However, studies in other brain regions have shown that NPY expression can vary based on several factors, suggesting that the NPY-hrGFP mouse might miss NPY neurons not expressing NPY on the experiment date. Here, we hypothesized that neurons with the ability to express NPY represent a larger population of IC GABAergic neurons than previously reported. To test this hypothesis, we used a lineage-tracing approach to irreversibly tag neurons that expressed NPY at any point prior to the experiment date. We then compared the physiological and anatomical features of neurons labeled with this lineage-tracing approach to our prior data set, revealing a larger population of NPY neurons than previously found. In addition, we used optogenetics to test the local connectivity of NPY neurons and found that NPY neurons provide inhibitory synaptic input to other neurons in the ipsilateral IC. Together, our data expand the definition of NPY neurons in the IC, suggest that NPY expression might be dynamically regulated in the IC, and provide functional evidence that NPY neurons form local inhibitory circuits in the IC.NEW & NOTEWORTHY Across brain regions, neuropeptide Y (NPY) expression is dynamic and influenced by extrinsic and intrinsic factors. We previously showed that NPY is expressed by a class of inhibitory neurons in the auditory midbrain. Here, we find that this neuron class also includes neurons that previously expressed NPY, suggesting that NPY expression is dynamically regulated in the auditory midbrain. We also provide functional evidence that NPY neurons contribute to local inhibitory circuits in the auditory midbrain.

Estrogen receptor-α signaling in tanycytes lies at the crossroads of fertility and metabolism

BackgroundEstrogen secretion by the ovaries regulates the hypothalamic-pituitary-gonadal axis during the reproductive cycle, influencing gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, and also plays a role in regulating metabolism. Here, we establish that hypothalamic tanycytes—specialized glia lining the floor and walls of the third ventricle—integrate estrogenic feedback signals from the gonads and couple reproduction with metabolism by relaying this information to orexigenic neuropeptide Y (NPY) neurons. MethodsUsing mouse models, including mice floxed for Esr1 (encoding estrogen receptor alpha, ERα) and those with Cre-dependent expression of designer receptors exclusively activated by designer drugs (DREADDs), along with viral-mediated, pharmacological and indirect calorimetric approaches, we evaluated the role of tanycytes and tanycytic estrogen signaling in pulsatile LH secretion, cFos expression in NPY neurons, estrous cyclicity, body-weight changes and metabolic parameters in adult females. ResultsIn ovariectomized mice, chemogenetic activation of tanycytes significantly reduced LH pulsatile release, mimicking the effects of direct NPY neuron activation. In intact mice, tanycytes were crucial for the estrogen-mediated control of GnRH/LH release, with tanycytic ERα activation suppressing fasting-induced NPY neuron activation. Selective knockout of Esr1 in tanycytes altered estrous cyclicity and fertility in female mice and affected estrogen's ability to inhibit refeeding in fasting mice. The absence of ERα signaling in tanycytes increased Npy transcripts and body weight in intact mice and prevented the estrogen-mediated decrease in food intake as well as increase in energy expenditure and fatty acid oxidation in ovariectomized mice. ConclusionsOur findings underscore the pivotal role of tanycytes in the neuroendocrine coupling of reproduction and metabolism, with potential implications for its age-related deregulation after menopause. Significance statementOur investigation reveals that tanycytes, specialized glial cells in the brain, are key interpreters of estrogen signals for orexigenic NPY neurons in the hypothalamus. Disrupting tanycytic estrogen receptors not only alters fertility in female mice but also impairs the ability of estrogens to suppress appetite. This work thus sheds light on the critical role played by tanycytes in bridging the hormonal regulation of cyclic reproductive function and appetite/feeding behavior. This understanding may have potential implications for age-related metabolic deregulation after menopause.

Chemogenetic activation of arcuate nucleus NPY and NPY/AgRP neurons increases feeding behaviour in mice

Neuropeptide Y (NPY) plays a crucial role in controlling energy homeostasis and feeding behaviour. The role of NPY neurons located in the arcuate nucleus of the hypothalamus (Arc) in responding to homeostatic signals has been the focus of much investigation, but most studies have used AgRP promoter-driven models, which do not fully encompass Arc NPY neurons. To directly investigate NPY-expressing versus AgRP-expressing Arc neurons function, we utilised chemogenetic techniques in NPY-Cre and AgRP-Cre animals to activate Arc NPY or AgRP neurons in the presence of food and food-related stimuli. Our findings suggest that chemogenetic activation of the broader population of Arc NPY neurons, including AgRP-positive and AgRP-negative NPY neurons, has equivalent effects on feeding behaviour as activation of Arc AgRP neurons. Our results demonstrate that these Arc NPY neurons respond specifically to caloric signals and do not respond to non-caloric signals, in line with what has been observed in AgRP neurons. Activating Arc NPY neurons significantly increases food consumption and influences macronutrient selection to prefer fat intake.

A brainstem to hypothalamic arcuate nucleus GABAergic circuit drives feeding

The obesity epidemic is principally driven by the consumption of more calories than the body requires. It is therefore essential that the mechanisms underpinning feeding behavior are defined. Neurons within the brainstem dorsal vagal complex (DVC) receive direct information from the digestive system and project to second-order regions in the brain to regulate food intake. Although γ-aminobutyric acid is expressed in the DVC (GABADVC), its function in this region has not been defined. In order to discover the unique gene expression signature of GABADVC cells, we used single-nucleus RNA sequencing (Nuc-seq), and this revealed 19 separate clusters. We next probed the function of GABADVC cells and discovered that the selective activation of GABADVC neurons significantly controls food intake and body weight. Optogenetic interrogation of GABADVC circuitry identified GABADVC → hypothalamic arcuate nucleus (ARC) projections as appetite suppressive without creating aversion. Electrophysiological analysis revealed that GABADVC → ARC stimulation inhibits hunger-promoting neuropeptide Y (NPY) neurons via GABA release. Adopting an intersectional genetics strategy, we clarify that the GABADVC → ARC circuit curbs food intake. These data identify GABADVC as a new modulator of feeding behavior and body weight and a controller of orexigenic NPY neuron activity, thereby providing insight into the neural underpinnings of obesity.

Putative identification of proopiomelanocortin and neuropeptide-Y neurons of the arcuate nucleus by their response to leptin: in vivo electrophysiology study in male and female rats.

The arcuate nucleus (ARN) of the hypothalamus is involved in multiple biological functions, such as feeding, sexual activity, and the regulation of the cardiovascular system. It was reported that leptin increased c-Fos expression in the proopiomelanocortin (POMC)- and decreased it in the neuropeptide-Y (NPY)-positive neurons of the ARN, suggesting that it stimulates the former, and inhibits the later. This study aimed at the direct electrophysiological examination of the effect of leptin on ARN neurons and to investigate potential sex-dimorphic changes. Wistar rats were anesthetized with urethane and the electrodes were inserted into the ARN. After a spontaneous active neuron was recorded for at least one minute, leptin was administered intravenously, and the firing activity of the same neuron was recorded for two additional minutes. It was found that approximately half of the ARN neurons had an excitatory, and another half an inhibitory response to the leptin administration. The excitability of the neurons with excitatory response to leptin was not different between the sexes. The average firing rate of the neurons with inhibitory response to leptin in females was, however, significantly lower comparing to the males. The obtained results demonstrate that the ARN neurons with stimulatory response to leptin are POMC and those with inhibitory response are NPY neurons. NPY Y1 receptor be might responsible, at least in part, for the sex differences in the excitability of the neurons putatively identified as NPY neurons.

Mechanical and chemical itch regulated by neuropeptide Y-Y1 signaling.

Itch is a somatosensory sensation to remove potential harmful stimulation with a scratching desire, which could be divided into mechanical and chemical itch according to diverse stimuli, such as wool fiber and insect biting. It has been reported that neuropeptide Y (NPY) neurons, a population of spinal inhibitory interneurons, could gate the transmission of mechanical itch, with no effect on chemical itch. In our study, we verified that chemogenetic activation of NPY neurons could inhibit the mechanical itch as well as the chemical itch, which also attenuated the alloknesis phenomenon in the chronic dry skin model. Afterwards, intrathecal administration of NPY1R agonist, [Leu31, Pro34]-NPY (LP-NPY), showed the similar inhibition effect on mechanical itch, chemical itch and alloknesis as chemo-activation of NPY neurons. Whereas, intrathecal administration of NPY1R antagonist BIBO 3304 enhanced mechanical itch and reversed the alloknesis phenomenon inhibited by LP-NPY treatment. Moreover, selectively knocking down NPY1R by intrathecal injection of Npy1r siRNA enhanced mechanical and chemical itch behavior as well. These results indicate that NPY neurons in spinal cord regulate mechanical and chemical itch, and alloknesis in dry skin model through NPY1 receptors.

Possible role of neuropeptide Y on zymosan- and lipopolysaccharide-induced change in gastrointestinal feed passage via the medulla oblongata in chicks

Zymosan is a fungi-derived pathogen-associated molecular pattern. It activates the immune system and induces the reduction of feed passage rate in the gastrointestinal tract of vertebrates including birds. However, the mechanism mediating the zymosan-induced inhibition of feed passage in the gastrointestinal tract remains unknown. Since the medulla oblongata regulates the digestive function, it is plausible that the medulla oblongata is involved in the zymosan-induced inhibition of feed passage. The present study was performed to identify the genes that were affected by zymosan within the medulla oblongata of chicks (Gallus gallus) using an RNA sequencing approach. We found that mRNAs of several bioactive molecules including neuropeptide Y (NPY) were increased with an intraperitoneal (IP) injection of zymosan. The increase of mRNA expression of NPY in the medulla oblongata was also observed after the IP injection of lipopolysaccharide, derived from gram-negative bacteria. These results suggest that medullary NPY is associated with physiological changes during fungal and bacterial infection. Furthermore, we found that intracerebroventricular injection of NPY and its receptor agonists reduced the feed passage from the crop. Additionally, the injection of NPY reduced the feed passage from the proventriculus to lower digestive tract. NPY also suppressed the activity of duodenal activities of amylase and trypsin. The present study suggests that fungi- and bacteria-induced activation of the immune system may activate the NPY neurons in the medulla oblongata and thereby reduce the digestive function in chicks.

Hedonic eating, obesity, and addiction result from increased neuropeptide Y in the nucleus accumbens during human brain evolution

The nucleus accumbens (NAc) is central to motivation and action, exhibiting one of the highest densities of neuropeptide Y (NPY) in the brain. Within the NAc, NPY plays a role in reward and is involved in emotional behavior and in increasing alcohol and drug addiction and fat intake. Here, we examined NPY innervation and neurons of the NAc in humans and other anthropoid primates in order to determine whether there are differences among these various species that would correspond to behavioral or life history variables. We quantified NPY-immunoreactive axons and neurons in the NAc of 13 primate species, including humans, great apes, and monkeys. Our data show that the human brain is unique among primates in having denser NPY innervation within the NAc, as measured by axon length density to neuron density, even after accounting for brain size. Combined with our previous finding of increased dopaminergic innervation in the same region, our results suggest that the neurochemical profile of the human NAc appears to have rendered our species uniquely susceptible to neurophysiological conditions such as addiction. The increase in NPY specific to the NAc may represent an adaptation that favors fat intake and contributes to an increased vulnerability to eating disorders, obesity, as well as alcohol and drug dependence. Along with our findings for dopamine, these deeply rooted structural attributes of the human brain are likely to have emerged early in the human clade, laying the groundwork for later brain expansion and the development of cognitive and behavioral specializations.

Morphological study of neuropeptide Y expression in human and mouse anterior insular cortex: Overexpression in the insular cortex and nucleus accumbens in obese mice on a long-term obesogenic diet

BackgroundThe anterior lobe of the insular cortex (aINS) is a cortical region that has reciprocal connections with limbic centers such as the anterior cingulate cortex, prefrontal cortex, amygdala and nucleus accumbens (NAc). In fact, the aINS has been involved in the integration of autonomic information for emotional and motivational functions. The compulsive consumption of drugs or high-fat foods induces alterations at both behavioural and brain levels. Brain reward circuits are altered in response to continued intake, in particular the dopaminergic projections from the ventral tegmental area (VTA) to the NAc. The aINS has multiple connections with the components of this system. In recent years, efforts have been made to better understand the fundamental role of the aINS in addiction, making it one of the key centres of interest for research into new treatments for addiction. ObjectivesThe present work focuses on studying 1.- whether the human aINS expresses orexigenic peptides such as neuropeptide Y (NPY), a peptide known to induce hyperphagia, and which has been implicated in the onset and development of obesity, 2.- the long-term effect of an obesogenic diet on NPY expression in the aINS and NAc of C57BL/6 mice. MethodsA total of 17 female C57BL/6 J mice were used in this study. Female mice were fed ad libitum with water and, either a standard diet (SD) or a high-fat diet (HFD) to induce obesity. There were seven female mice on the SD and ten on the HFD. The duration of the experiment was 180 days. We also studied 3 human adult brains (1 male and 2 females, mean age 55.7 ± 5.2 years). The morphological study was performed using immunohistochemistry and double immunofluorescence techniques to study the neurochemical profile of NPY neurons of the aINS and NAc of humans and mice. ResultsOur morphological analysis demonstrates for the first time the basal expression of NPY in different layers of the human cortex (II, III, IV, V/VI), in a pattern similar to previous studies in other species. Furthermore, we observed an increase in the number of NPY-positive cells and their intracytoplasmic signal in the aINS and NAc of the obese mice subjected to a long-term obesogenic diet. ConclusionsTo our knowledge, this is the first study to show the distribution and expression of NPY in the human INS and how its expression is altered after prolonged treatment with an obesogenic diet in obese mice. Our findings may contribute to the understanding of the pathophysiological mechanisms underlying obesity in regions related to the reward system and associated with uncontrolled intake of high-fat foods, thus facilitating the identification of novel therapeutic targets.

Agrp-negative arcuate NPY neurons drive feeding under positive energy balance via altering leptin responsiveness in POMC neurons

Neuropeptide Y (NPY) in the arcuate nucleus (ARC) is known as one of the most critical regulators of feeding. However, how NPY promotes feeding under obese conditions is unclear. Here, we show that positive energy balance, induced by high-fat diet (HFD) or in genetically obese leptin-receptor-deficient mice, leads to elevated Npy2r expression especially on proopiomelanocortin (POMC) neurons, which also alters leptin responsiveness. Circuit mapping identified a subset of ARC agouti-related peptide (Agrp)-negative NPY neurons that control these Npy2r expressing POMC neurons. Chemogenetic activation of this newly discovered circuitry strongly drives feeding, while optogenetic inhibition reduces feeding. Consistent with that, lack of Npy2r on POMC neurons leads to reduced food intake and fat mass. This suggests that under energy surplus conditions, when ARC NPY levels generally drop, high-affinity NPY2R on POMC neurons is still able to drive food intake and enhance obesity development via NPY released predominantly from Agrp-negative NPY neurons.

Exogenous Melatonin Regulates Puberty and the Hypothalamic GnRH-GnIH System in Female Mice.

In recent years, the age of children entering puberty is getting lower and the incidence of central precocious puberty is increasing. It is known that melatonin plays an increasingly important role in regulating animal reproduction, but the specific role and mechanism of melatonin in regulating the initiation of puberty remain unclear. The purpose of the current study was to investigate the effect of subcutaneous melatonin injection on pubertal development in female mice and its mechanism of action. Female mice that were 22 days old received 1 mg/kg doses of melatonin subcutaneously every day for 10, 15 and 20 days. The vaginal opening was checked daily. Hematoxylin and eosin (HE) stain was used to determine the growth of the uterus and ovaries. Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of follicle-stimulating hormone (FSH), gonadotropin-inhibiting hormone (GnIH), and gonadotropin-releasing hormone (GnRH) in serum. By using RT-PCR and Western blotting, the mRNA and protein expression of the hypothalamus GnRH, GnIH, Kisspeptin (Kp), Proopiomelanocortin (POMC), Neuropeptide Y (NPY), as well as G protein-coupled receptor 147 (GPR147) were identified. The findings demonstrated that melatonin could suppress ovarian follicle and uterine wall growth as well as delay vaginal opening, decrease serum levels of GnRH and FSH and increase levels of GnIH. Melatonin increased GnIH and GPR147 expression in the hypothalamus in comparison to the saline group, while decreasing the expression of GnRH, Kisspeptin, POMC, and NPY. In conclusion, exogenous melatonin can inhibit the onset of puberty in female mice by modulating the expression of hypothalamic GnRH, GnIH, Kisspeptin, POMC and NPY neurons and suppressing the hypothalamic-pituitary-gonadal axis.

D-Allulose Inhibits Ghrelin-Responsive, Glucose-Sensitive and Neuropeptide Y Neurons in the Arcuate Nucleus and Central Injection Suppresses Appetite-Associated Food Intake in Mice.

d-allulose, a rare sugar, has sweetness with few calories. d-allulose regulates feeding and glycemia, and ameliorates hyperphagia, obesity and diabetes. All these functions involve the central nervous system. However, central mechanisms underlying these effects of d-allulose remain unknown. We recently reported that d-allulose activates the anorexigenic neurons in the hypothalamic arcuate nucleus (ARC), the neurons that respond to glucagon-like peptide-1 and that express proopiomelanocortin. However, its action on the orexigenic neurons remains unknown. This study investigated the effects of d-allulose on the ARC neurons implicated in hunger, by measuring cytosolic Ca2+ concentration ([Ca2+]i) in single neurons. d-allulose depressed the increases in [Ca2+]i induced by ghrelin and by low glucose in ARC neurons and inhibited spontaneous oscillatory [Ca2+]i increases in neuropeptide Y (NPY) neurons. d-allulose inhibited 10 of 35 (28%) ghrelin-responsive, 18 of 60 (30%) glucose-sensitive and 3 of 8 (37.5%) NPY neurons in ARC. Intracerebroventricular injection of d-allulose inhibited food intake at 20:00 and 22:00, the early dark phase when hunger is promoted. These results indicate that d-allulose suppresses hunger-associated feeding and inhibits hunger-promoting neurons in ARC. These central actions of d-allulose represent the potential of d-allulose to inhibit the hyperphagia with excessive appetite, thereby counteracting obesity and diabetes.

The arcuate nucleus: A site of synergism between Angiotensin II and leptin to increase sympathetic nerve activity and blood pressure in rats

The action of leptin in brain to increase sympathetic nerve activity (SNA) and blood pressure depends upon functional Angiotensin II (AngII) type 1a receptors (AT1aR); however, the sites and mechanism of interaction are unknown. Here we identify one site, the hypothalamic arcuate nucleus (ArcN), since prior local blockade of AT1aR in the ArcN with losartan or candesartan in anesthetized male rats essentially eliminated the sympathoexcitatory and pressor responses to ArcN leptin nanoinjections. Unlike mice, in male and female rats, AT1aR and LepR rarely co-localized, suggesting that this interdependence occurs indirectly, via a local interneuron or network of neurons. ArcN leptin increases SNA by activating pro-opiomelanocortin (POMC) inputs to the PVN, but this activation requires simultaneous suppression of tonic PVN Neuropeptide Y (NPY) sympathoinhibition. Because AngII-AT1aR inhibits ArcN NPY neurons, we propose that loss of AT1aR suppression of NPY blocks leptin-induced increases in SNA; in other words, ArcN-AngII-AT1aR is a gatekeeper for leptin-induced sympathoexcitation. With obesity, both leptin and AngII increase; therefore, the increased AT1aR activation could open the gate, allowing leptin (and insulin) to drive sympathoexcitation unabated, leading to hypertension.

Inhibitory NPY Neurons Provide a Large and Heterotopic Commissural Projection in the Inferior Colliculus.

Located in the midbrain, the inferior colliculus (IC) plays an essential role in many auditory computations, including speech processing and sound localization. The right and left sides of the IC are interconnected by a dense fiber tract, the commissure of the IC (CoIC), that provides each IC with one of its largest sources of input (i.e., the contralateral IC). Despite its prominence, the CoIC remains poorly understood. Previous studies using anterograde and retrograde tract-tracing showed that IC commissural projections are predominately homotopic and tonotopic, targeting mirror-image locations in the same frequency region in the contralateral IC. However, it is unknown whether specific classes of neurons, particularly inhibitory neurons which constitute ~10%–40% of the commissural projection, follow this pattern. We, therefore, examined the commissural projections of Neuropeptide Y (NPY) neurons, the first molecularly identifiable class of GABAergic neurons in the IC. Using retrograde tracing with Retrobeads (RB) in NPY-hrGFP mice of both sexes, we found that NPY neurons comprise ~11% of the commissural projection. Moreover, focal injections of Retrobeads showed that NPY neurons in the central nucleus of the IC exhibit a more divergent and heterotopic commissural projection pattern than non-NPY neurons. Thus, commissural NPY neurons are positioned to provide lateral inhibition to the contralateral IC. Through this circuit, sounds that drive activity in limited regions on one side of the IC likely suppress activity across a broader region in the contralateral IC.

Interactions between Brainstem Neurons That Regulate the Motility to the Stomach.

Activity in the dorsal vagal complex (DVC) is essential to gastric motility regulation. We and others have previously shown that this activity is greatly influenced by local GABAergic signaling, primarily because of somatostatin (SST)-expressing GABAergic neurons. To further understand the network dynamics associated with gastric motility control in the DVC, we focused on another neuron prominently distributed in this complex, neuropeptide-Y (NPY) neurons. However, the effect of these neurons on gastric motility remains unknown. Here, we investigate the anatomic and functional characteristics of the NPY neurons in the nucleus tractus solitarius (NTS) and their interactions with SST neurons using transgenic mice of both sexes. We sought to determine whether NPY neurons influence the activity of gastric-projecting neurons, synaptically interact with SST neurons, and affect end-organ function. Our results using combined neuroanatomy and optogenetic in vitro and in vivo show that NPY neurons are part of the gastric vagal circuit as they are trans-synaptically labeled by a viral tracer from the gastric antrum, are primarily excitatory as optogenetic activation of these neurons evoke EPSCs in gastric-antrum-projecting neurons, are functionally coupled to each other and reciprocally connected to SST neurons, whose stimulation has a potent inhibitory effect on the action potential firing of the NPY neurons, and affect gastric tone and motility as reflected by their robust optogenetic response in vivo. These findings indicate that interacting NPY and SST neurons are integral to the network that controls vagal transmission to the stomach.SIGNIFICANCE STATEMENT The brainstem neurons in the dorsal nuclear complex are essential for regulating vagus nerve activity that affects the stomach via tone and motility. Two distinct nonoverlapping populations of predominantly excitatory NPY neurons and predominantly inhibitory SST neurons form reciprocal connections with each other in the NTS and with premotor neurons in the dorsal motor nucleus of the vagus to control gastric mechanics. Light activation and inhibition of NTS NPY neurons increased and decreased gastric motility, respectively, whereas both activation and inhibition of NTS SST neurons enhanced gastric motility.

NPFF Decreases Activity of Human Arcuate NPY Neurons: A Study in Embryonic-Stem-Cell-Derived Model.

Restoring the control of food intake is the key to obesity management and prevention. The arcuate nucleus (ARC) of the hypothalamus is extensively being studied as a potential anti-obesity target. Animal studies showed that neuropeptide FF (NPFF) reduces food intake by its action in neuropeptide Y (NPY) neurons of the hypothalamic ARC, but the detailed mode of action observed in human neurons is missing, due to the lack of a human-neuron-based model for pharmacology testing. Here, we validated and utilized a human-neural-stem-cell-based (hNSC) model of ARC to test the effects of NPFF on cellular pathways and neuronal activity. We found that in the human neurons, decreased cAMP levels by NPFF resulted in a reduced rate of cytoplasmic calcium oscillations, indicating an inhibition of ARC NPY neurons. This suggests the therapeutic potential of NPFFR2 in obesity. In addition, we demonstrate the use of human-stem-cell-derived neurons in pharmacological applications and the potential of this model to address functional aspects of human hypothalamic neurons.

Chronic Restraint Stress Decreases the Excitability of Hypothalamic POMC Neuron and Increases Food Intake.

Stress activates the hypothalamic-pituitary-adrenal system, and induces the release of glucocorticoids, stress hormones, into circulation. Many studies have shown that stress affects feeding behavior, however, the underlying circuitry and molecular mechanisms are not fully understood. The balance between orexigenic (simulating appetite) and anorexigenic (loss of appetite) signals reciprocally modulate feeding behavior. It is suggested that proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons in the arcuate nucleus (ARC) of the hypothalamus are the first-order neurons that respond to the circulating signals of hunger and satiety. Here, we examined a chronic restraint stress model and observed an increase in food intake, which was not correlated with anhedonia. We investigated whether stress affects the properties of POMC and NPY neurons and found that chronic restraint stress reduced the excitatory inputs onto POMC neurons and increased the action potential threshold. Therefore, our study suggests that chronic stress modulates the intrinsic excitability and excitatory inputs in POMC neurons, leading to changes in feeding behavior.

Arcuate Angiotensin II Increases Arterial Pressure via Coordinated Increases in Sympathetic Nerve Activity and Vasopressin Secretion.

The arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII); however, the cellular mechanisms and downstream neurocircuitry are unclear. Here, we show that ArcN AngII increases AP in female rats via two phases, both of which are mediated via activation of AngII type 1 receptors (AT1aRs): initial vasopressin-induced vasoconstriction, followed by slowly developing increases in sympathetic nerve activity (SNA) and heart rate (HR). In male rats, ArcN AngII evoked a similarly slow increase in SNA, but the initial pressor response was variable. In females, the effects of ArcN AngII varied during the estrous cycle, with significant increases in SNA, HR, and AP occurring during diestrus and estrus, but only increased AP during proestrus. Pregnancy markedly increased the expression of AT1aR in the ArcN with parallel substantial AngII-induced increases in SNA and MAP. In both sexes, the sympathoexcitation relied on suppression of tonic ArcN sympathoinhibitory neuropeptide Y (NPY) inputs, and activation of proopiomelanocortin (POMC) projections, to the paraventricular nucleus (PVN). Few or no NPY or POMC neurons expressed the AT1aR, suggesting that AngII increases AP and SNA at least in part indirectly via local interneurons, which express tyrosine hydroxylase (TH) and VGat (i.e., GABAergic). ArcN TH neurons release GABA locally, and central AT1aR and TH neurons mediate stress responses; therefore, we propose that TH AT1aR neurons are well situated to locally coordinate the regulation of multiple modalities within the ArcN in response to stress.

L-type calcium channel subtypes mediating hypothalamic dysfunction caused by amyloid-beta.

In Alzheimer's disease (AD), non-cognitive manifestations attributable to hypothalamic dysfunction such as systemic metabolic deficits can occur prior to the cognitive decline (Ishii, M. and Iadecola C., Cell Metabolism 22:761,2015). We previously found in Tg2576 mice overexpressing the Swedish mutation of amyloid precursor protein that amyloid-beta pathology can cause dysfunction in hypothalamic arcuate neuropeptide Y (NPY) neurons, associated with low adiposity and low plasma leptin levels prior to memory decline or plaque formation (Ishii, M. et al., J Neuroscience 34:9096,2014). Furthermore, the hypothalamic arcuate NPY neuronal dysfunction was caused by amyloid-beta modulating low-threshold activated L-type calcium channels (Ishii, M. et al., J Neuroscience 39:8816,2019). However, which of the two major brain L-type calcium channel subtypes Cav 1.2 or Cav 1.3 is involved has not been established. To examine the role of Cav 1.2, we used the Cre-lox system in genetic mouse models to knock-out Cav 1.2 in hypothalamic arcuate NPY neurons (Cav 1.2 cKO). Cav 1.2 cKO mice were bred to NPY-GFP and Tg2576 mice. Two to 3-month-old male or female mice were used for all studies. Voltage-gated L-type calcium currents from hypothalamic slices containing arcuate NPY neurons were recorded using whole-cell voltage-clamp. Cytoplasmic Ca2+ levels were measured ratiometrically by loading Fura-2AM in dissociated arcuate NPY neurons and recording the emitted fluorescence using an inverted fluorescent microscope. Exogenous oligomeric amyloid-β42 caused a significant increase in cytoplasmic Ca2+ levels and L-type calcium currents in arcuate NPY neurons to a similar extent in both Cav 1.2 cKO and wild-type (WT) littermate mice. These effects of amyloid-β42 were reversed by the L-type calcium channel blocker nimodipine. In vivo, Cav 1.2 cKO mice bred to Tg2576 mice had similar body weight deficits compared to Tg2576 mice without deletion of Cav 1.2 in the arcuate NPY neurons. These results suggest that Cav 1.2 does not have a significant role in amyloid-beta mediated dysfunction of hypothalamic arcuate NPY neurons and that Cav 1.3 is likely the major target of amyloid-beta in these neurons. Future studies modulating Cav 1.3 in hypothalamic neurons will help elucidate the mechanisms underlying hypothalamic dysfunction caused by amyloid-beta pathology and its therapeutic potential in AD.