Journal of Neuroendocrinology
Watanabe, Y;Fisher, L;Campbell, R;Jasoni, C;
| DOI: 10.1111/jne.13302
Polycystic ovary syndrome (PCOS) is a female endocrine disorder that is associated with prenatal exposure to excess androgens. In prenatally androgenized (PNA) mice that model PCOS, GABAergic neural transmission to and innervation of GnRH neurons is increased. Evidence suggests that elevated GABAergic innervation originates in the arcuate nucleus (ARC). We hypothesised that GABA-GnRH circuit abnormalities are a direct consequence of PNA, resulting from DHT binding to androgen receptor (AR) in the prenatal brain. However, whether prenatal ARC neurons express AR at the time of PNA treatment is presently unknown. We used RNAScope _in situ_ hybridization to localize AR mRNA (_Ar_)-expressing cells in healthy gestational day (GD) 17.5 female mouse brains and to assess co-expression levels in specific neuronal phenotypes. Our study revealed that less than 10% of ARC GABA cells expressed _Ar_. In contrast, we found that ARC kisspeptin neurons, critical regulators of GnRH neurons, were highly co-localised with _Ar_. Approximately 75% of ARC _Kiss1_-expressing cells also expressed _Ar_ at GD17.5, suggesting that ARC kisspeptin neurons are potential targets of PNA. Investigating other neuronal populations in the ARC we found that approximately 50% of pro-opiomelanocortin (_Pomc_) cells, 22% of tyrosine hydroxylase (_Th_) cells, 8% of agouti-related protein (_Agrp_) cells and 8% of somatostatin (_Sst_) cells express _Ar_. Lastly, RNAscope in coronal sections showed _Ar_ expression in the medial preoptic area (mPOA), and the ventral part of the lateral septum (vLS). These _Ar_-expressing regions were highly GABAergic, and 22% of GABA cells in the mPOA and 25% of GABA cells in the vLS also expressed _Ar_. Our findings identify specific neuronal phenotypes in the ARC, mPOA and vLS that are androgen sensitive in late gestation. PNA-induced functional changes in these neurons may be related to the development of impaired central mechanisms associated with PCOS-like features.
The Journal of physiology
Peltekian, L;Gasparini, S;Fazan, FS;Karthik, S;Iverson, G;Resch, JM;Geerling, JC;
PMID: 37291801 | DOI: 10.1113/JP283169
In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue-specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. KEY POINTS: Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain-specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double-deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver-specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin-deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.
Liu Y, Huang Y, Liu T, Wu H, Cui H, Gautron L.
PMID: 27111742 | DOI: -
While Agouti-related peptide (AgRP) neurons play a key role in the regulation of food intake, their contribution to the anorexia caused by pro-inflammatory insults has yet to be identified. Using a combination of neuroanatomical and pharmacogenetics experiments, this study sought to investigate the importance of AgRP neurons and downstream targets in the anorexia caused by the peripheral administration of a moderate dose of lipopolysaccharide (LPS; 100 μ g/kg, ip). First, in the C57/Bl6 mouse, we demonstrated that LPS induced c-fos in select AgRP-innervated brain sites involved in feeding, but not in any arcuate proopiomelanocortin neurons. Double immunohistochemistry further showed that LPS selectively induced c-Fos in a large subset of melanocortin 4 receptor-expressing neurons in the lateral parabrachial nucleus. Secondly, we used pharmacogenetics to stimulate the activity of AgRP neurons during the course of LPS-induced anorexia. In AgRP-Cre mice expressing the designer receptor hM3Dq-Gq only in AgRP neurons, the administration of the designer drug clozapine-N-oxide (CNO) induced robust food intake. Strikingly, CNO-mediated food intake was rapidly and completely blunted by the coadministration of LPS. Neuroanatomical experiments further indicated that LPS did not interfere with the ability of CNO to stimulate c-Fos in AgRP neurons. In summary, our findings combined together support the view that the stimulation of select AgRP-innervated brain sites and target neurons, rather than the inhibition of AgRP neurons themselves, is likely to contribute to the rapid suppression of food intake observed during acute bacterial endotoxemia.
Journal of Neuroendocrinology
Decourt, C;Connolly, G;Ancel, C;Inglis, M;Anderson, G;
| DOI: 10.1111/jne.13190
Agouti-related peptide (AgRP) neurons are thought to indirectly regulate the activity of hypothalamic gonadotrophin-releasing hormone neurons which control fertility. AgRP neurons also drive caloric intake and are modulated by metabolically-relevant hormones, providing a link to the hypothalamic-pituitary-gonadal axis. In mice expressing Cre-dependant designer receptors (DREADDs) in AgRP neurons, we activated or silenced these neurons in vivo using the synthetic ligand clozapine-N-oxide (CNO) to observe the effect of AgRP neuron activity on timing of puberty. To validate these animals, we chronically treated both stimulatory (hM3Dq) and inhibitory (hM4Di) DREADD × AgRP-Cre mice with CNO, observing a pronounced increase and decrease of food intake, respectively, consistent with the known orexigenic effects of these neurons. RNAscope was performed to visually confirm the activation of AgRP neurons. Puberty onset was assessed in males and females. There was no effect on preputial separation in males or vaginal opening and first oestrus in females after CNO treatment from day 26 to 30 to chronically modulate AgRP neurons. Next, to determine whether the delay in puberty onset occurring in response to neonatal underfeeding could be overcome by inhibiting AgRP neuronal activity, mice were raised in large (neonatally underfed) or normal litter sizes. The delay in puberty from underfeeding was completely reversed in CNO-treated AgRP-hM4Di male mice. These data highlight the inhibitory role of AgRP neurons to delay puberty onset when undernutrition occurs during the neonatal period, at least in male mice.
Journal of cellular physiology
Zhang, CL;Lin, YZ;Wu, Q;Yan, C;Wong, MW;Zeng, F;Zhu, P;Bowes, K;Lee, K;Zhang, X;Song, ZY;Lin, S;Shi, YC;
PMID: 35312067 | DOI: 10.1002/jcp.30719
Chronic high salt intake is one of the leading causes of hypertension. Salt activates the release of the key neurotransmitters in the hypothalamus such as vasopressin to increase blood pressure, and neuropepetide Y (NPY) has been implicated in the modulation of vasopressin levels. NPY in the hypothalamic arcuate nucleus (Arc) is best known for its control in appetite and energy homeostasis, but it is unclear whether it is also involved in the development of salt-induced hypertension. Here, we demonstrate that wild-type mice given 2% NaCl salt water for 8 weeks developed hypertension which was associated with marked downregulation of NPY expression in the hypothalamic Arc as demonstrated in NPY-GFP reporter mice as well as by in situ hybridization analysis. Furthermore, salt intake activates neurons in the hypothalamic paraventricular nucleus (PVN) where mRNA expression of brain-derived neurotrophic factor (BDNF) and vasopressin was found to be upregulated, leading to elevated serum vasopressin levels. This finding suggests an inverse correlation between the Arc NPY level and expression of vasopressin and BDNF in the PVN. Specific restoration of NPY by injecting AAV-Cre recombinase into the Arc only of the NPY-targeted mutant mice carrying a loxP-flanked STOP cassette reversed effects of salt intake on vasopressin and BDNF expression, leading to a normalization of salt-dependent blood pressure. In summary, our study uncovers an important Arc NPY-originated neuronal circuitry that could sense and respond to peripheral electrolyte signals and thereby regulate hypertension via vasopressin and BDNF in the PVN.
Gupta, R;Wang, M;Ma, Y;Offermanns, S;Whim, MD;
PMID: 35595517 | DOI: 10.1210/endocr/bqac077
During fasting, increased sympatho-adrenal activity leads to epinephrine release and multiple forms of plasticity within the adrenal medulla including an increase in the strength of the preganglionic → chromaffin cell synapse and elevated levels of AgRP, a peptidergic co-transmitter in chromaffin cells. Although these changes contribute to the sympathetic response, how fasting evokes this plasticity is not known. Here we report these effects involve activation of GPR109A (HCAR2). The endogenous agonist of this G protein-coupled receptor is β-hydroxybutyrate, a ketone body whose levels rise during fasting. In wild type animals, 24 hr fasting increased AgRP-ir in adrenal chromaffin cells but this effect was absent in GPR109A knockout mice. GPR109A agonists increased AgRP-ir in isolated chromaffin cells through a GPR109A- and pertussis toxin-sensitive pathway. Incubation of adrenal slices in nicotinic acid, a GPR109A agonist, mimicked the fasting-induced increase in the strength of the preganglionic → chromaffin cell synapse. Finally, RT-PCR experiments confirmed the mouse adrenal medulla contains GPR109A mRNA. These results are consistent with the activation of a GPR109A signaling pathway located within the adrenal gland. Because fasting evokes epinephrine release, which stimulates lipolysis and the production of β-hydroxybutyrate, our results indicate that chromaffin cells are components of an autonomic-adipose-hepatic feedback circuit. Coupling a change in adrenal physiology to a metabolite whose levels rise during fasting is presumably an efficient way to co-ordinate the homeostatic response to food deprivation.
Porniece Kumar, M;Cremer, AL;Klemm, P;Steuernagel, L;Sundaram, S;Jais, A;Hausen, AC;Tao, J;Secher, A;Pedersen, TÅ;Schwaninger, M;Wunderlich, FT;Lowell, BB;Backes, H;Brüning, JC;
PMID: 34931084 | DOI: 10.1038/s42255-021-00499-0
Insulin acts on neurons and glial cells to regulate systemic glucose metabolism and feeding. However, the mechanisms of insulin access in discrete brain regions are incompletely defined. Here we show that insulin receptors in tanycytes, but not in brain endothelial cells, are required to regulate insulin access to the hypothalamic arcuate nucleus. Mice lacking insulin receptors in tanycytes (IR∆Tan mice) exhibit systemic insulin resistance, while displaying normal food intake and energy expenditure. Tanycytic insulin receptors are also necessary for the orexigenic effects of ghrelin, but not for the anorexic effects of leptin. IR∆Tan mice exhibit increased agouti-related peptide (AgRP) neuronal activity, while displaying blunted AgRP neuronal adaptations to feeding-related stimuli. Lastly, a highly palatable food decreases tanycytic and arcuate nucleus insulin signalling to levels comparable to those seen in IR∆Tan mice. These changes are rooted in modifications of cellular stress responses and of mitochondrial protein quality control in tanycytes. Conclusively, we reveal a critical role of tanycyte insulin receptors in gating feeding-state-dependent regulation of AgRP neurons and systemic insulin sensitivity, and show that insulin resistance in tanycytes contributes to the pleiotropic manifestations of obesity-associated insulin resistance.
Feng J, Xian Q, Guan T, Hu J, Wang M, Huang Y, So KF, Evans SM, Chai G, Goffinet AM, Qu Y, Zhou L.
PMID: 27170656 | DOI: -
Celsr3 and Fzd3 regulate the development of reciprocal thalamocortical projections independently of their expression in cortical or thalamic neurons. To understand this cell non autonomous mechanism further, we tested whether Celsr3 and Fzd3 could act via Isl1-positive guidepost cells. Isl1-positive cells appear in the forebrain at embryonic day (E) 9.5-E10.5 and, from E12.5, they form 2 contingents in ventral telencephalon and prethalamus. In control mice, corticothalamic axons run in the ventral telencephalic corridor in close contact with Isl1-positive cells. When Celsr3 or Fzd3 is inactivated in Isl1-expressing cells, corticofugal fibers stall and loop in the ventral telencephalic corridor of high Isl1 expression, and thalamic axons fail to cross the diencephalon-telencephalon junction (DTJ). At E12.5, before thalamic and cortical axons emerge, pioneer projections from Isl1-positive cells cross the DTJ from both sides in control but not mutant embryos. These early projections appear to act like a bridge to guide later growing thalamic axons through the DTJ. Our data suggest that Celsr3 and Fzd3 orchestrate the formation of a scaffold of pioneer neurons and their axons. This scaffold extends from prethalamus to ventral telencephalon and subcortex, and steers reciprocal corticothalamic fibers.
Claflin KE, Sandgren JA, Lambertz AM, Weidemann BJ, Littlejohn NK, Burnett CM, Pearson NA, Morgan DA, Gibson-Corley KN, Rahmouni K, Grobe JL.
PMID: 28263184 | DOI: 10.1172/JCI88641
Leptin contributes to the control of resting metabolic rate (RMR) and blood pressure (BP) through its actions in the arcuate nucleus (ARC). The renin-angiotensin system (RAS) and angiotensin AT1 receptors within the brain are also involved in the control of RMR and BP, but whether this regulation overlaps with leptin's actions is unclear. Here, we have demonstrated the selective requirement of the AT1A receptor in leptin-mediated control of RMR. We observed that AT1A receptors colocalized with leptin receptors (LEPRs) in the ARC. Cellular coexpression of AT1A and LEPR was almost exclusive to the ARC and occurred primarily within neurons expressing agouti-related peptide (AgRP). Mice lacking the AT1A receptor specifically in LEPR-expressing cells failed to show an increase in RMR in response to a high-fat diet and deoxycorticosterone acetate-salt (DOCA-salt) treatments, but BP control remained intact. Accordingly, loss of RMR control was recapitulated in mice lacking AT1A in AgRP-expressing cells. We conclude that angiotensin activates divergent mechanisms to control BP and RMR and that the brain RAS functions as a major integrator for RMR control through its actions at leptin-sensitive AgRP cells of the ARC.
Yu, B;Zhang, Q;Lin, L;Zhou, X;Ma, W;Wen, S;Li, C;Wang, W;Wu, Q;Wang, X;Li, XM;
PMID: 36788214 | DOI: 10.1038/s41421-022-00506-y
The amygdala, or an amygdala-like structure, is found in the brains of all vertebrates and plays a critical role in survival and reproduction. However, the cellular architecture of the amygdala and how it has evolved remain elusive. Here, we generated single-nucleus RNA-sequencing data for more than 200,000 cells in the amygdala of humans, macaques, mice, and chickens. Abundant neuronal cell types from different amygdala subnuclei were identified in all datasets. Cross-species analysis revealed that inhibitory neurons and inhibitory neuron-enriched subnuclei of the amygdala were well-conserved in cellular composition and marker gene expression, whereas excitatory neuron-enriched subnuclei were relatively divergent. Furthermore, LAMP5+ interneurons were much more abundant in primates, while DRD2+ inhibitory neurons and LAMP5+SATB2+ excitatory neurons were dominant in the human central amygdalar nucleus (CEA) and basolateral amygdalar complex (BLA), respectively. We also identified CEA-like neurons and their species-specific distribution patterns in chickens. This study highlights the extreme cell-type diversity in the amygdala and reveals the conservation and divergence of cell types and gene expression patterns across species that may contribute to species-specific adaptations.
Central anorexigenic actions of bile acids are mediated by TGR5
Perino, A;Velázquez-Villegas, LA;Bresciani, N;Sun, Y;Huang, Q;Fénelon, VS;Castellanos-Jankiewicz, A;Zizzari, P;Bruschetta, G;Jin, S;Baleisyte, A;Gioiello, A;Pellicciari, R;Ivanisevic, J;Schneider, BL;Diano, S;Cota, D;Schoonjans, K;
PMID: 34031591 | DOI: 10.1038/s42255-021-00398-4
Bile acids (BAs) are signalling molecules that mediate various cellular responses in both physiological and pathological processes. Several studies report that BAs can be detected in the brain1, yet their physiological role in the central nervous system is still largely unknown. Here we show that postprandial BAs can reach the brain and activate a negative-feedback loop controlling satiety in response to physiological feeding via TGR5, a G-protein-coupled receptor activated by multiple conjugated and unconjugated BAs2 and an established regulator of peripheral metabolism3-8. Notably, peripheral or central administration of a BA mix or a TGR5-specific BA mimetic (INT-777) exerted an anorexigenic effect in wild-type mice, while whole-body, neuron-specific or agouti-related peptide neuronal TGR5 deletion caused a significant increase in food intake. Accordingly, orexigenic peptide expression and secretion were reduced after short-term TGR5 activation. In vitro studies demonstrated that activation of the Rho-ROCK-actin-remodelling pathway decreases orexigenic agouti-related peptide/neuropeptide Y (AgRP/NPY) release in a TGR5-dependent manner. Taken together, these data identify a signalling cascade by which BAs exert acute effects at the transition between fasting and feeding and prime the switch towards satiety, unveiling a previously unrecognized role of physiological feedback mediated by BAs in the central nervous system.
El Mehdi, M;Takhlidjt, S;Devère, M;Arabo, A;Le Solliec, MA;Maucotel, J;Bénani, A;Nedelec, E;Duparc, C;Lefranc, B;Leprince, J;Anouar, Y;Prévost, G;Chartrel, N;Picot, M;
PMID: 35476025 | DOI: 10.1007/s00125-022-05706-5
26RFa (pyroglutamilated RFamide peptide [QRFP]) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localised in the hypothalamus. In this study we investigated whether 26RFa neurons are involved in the hypothalamic regulation of glucose homeostasis.26Rfa+/+, 26Rfa-/- and insulin-deficient male C57Bl/6J mice were used in this study. Mice received an acute intracerebroventricular (i.c.v.) injection of 26RFa, insulin or the 26RFa receptor (GPR103) antagonist 25e and were subjected to IPGTTs, insulin tolerance tests, acute glucose-stimulated insulin secretion tests and pyruvate tolerance tests (PTTs). Secretion of 26RFa by hypothalamic explants after incubation with glucose, leptin or insulin was assessed. Expression and quantification of the genes encoding 26RFa, agouti-related protein, the insulin receptor and GPR103 were evaluated by quantitative reverse transcription PCR and RNAscope in situ hybridisation.Our data indicate that i.c.v.-injected 26RFa induces a robust antihyperglycaemic effect associated with an increase in insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26Rfa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26Rfa are mainly localised in the lateral hypothalamic area, that they co-express the gene encoding the insulin receptor and that insulin induces the expression of 26Rfa in these neurons. Concurrently, the central antihyperglycaemic effect of insulin is abolished in the presence of a GPR103 antagonist and in 26RFa-deficient mice. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production.We have identified a novel mechanism in the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system, and we provide evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.
