IntroductionProkineticin 2 (PK2) signaling to gonadotropin-releasing hormone (GnRH) neurons is essential for their embryonic migration from the nasal placode to the brain, and its disruption results in hypogonadotropic hypogonadism. Although PK2 has been implicated in the regulation of reproductive function in adult rodents, its direct cellular targets remain insufficiently defined. Here, we investigated whether GnRH neurons in adult female mice receive and functionally respond to PK2 signals.MethodsWhole-cell patch-clamp recordings were obtained from acute forebrain slices prepared from adult female GnRH-GFP mice. The effects of PK2 and prokineticin receptor antagonists on spontaneous firing and miniature postsynaptic currents (mPSCs) were assessed. Expression of PK2 receptor (PKR2) in GnRH neurons was examined using RNAscope in situ hybridization for simultaneous detection of PKR2 and GnRH mRNAs, followed by confocal laser microscopic analysis.ResultsPK2 increased action potential firing and mPSC frequency in 42.9% (firing) and 42.1% (mPSC) of GnRH neurons. These effects were prevented by intracellular G-protein inhibition with GDP-β-S and by pharmacological blockade of prokineticin receptors using PKRA7 or PC27. Inhibition of nitric oxide synthase by NPLA also eliminated the PK2-induced elevation of mPSC frequency. RNAscope analysis revealed PKR2 mRNA expression in approximately one-third of GnRH neurons.DiscussionThese findings demonstrate that PK2 directly enhances the excitability of a subset of GnRH neurons through PKR2-dependent mechanisms, identifying these neurons as functional targets of PK2 signaling in the mature female mouse brain.ConclusionPK2 serves as a direct modulator of the reproductive neuroendocrine axis in adult female mice by activating PKR2 expressed in a subpopulation of GnRH neurons.

Introduction:Environmental estrogenic chemicals can cross the maternal-fetal barrier and disrupt endocrine and metabolic regulation in the developing embryo/fetus. Bisphenol A (BPA) and 17α-ethinylestradiol (EE2) are widely present in the environment and have been linked to increased cardio-metabolic disease risk. Purpose:This study investigated the effects of maternal BPA and EE2 exposure on metabolic function and circadian energy regulation in male offspring. Methods:Pregnant and lactating dams were chronically administered BPA (20 µg/kg bw/day) or EE2 (0.01 µg/kg bw/day) via osmotic minipumps from gestational day 9 to postnatal day 21 to mimic environmental exposure. Adult male offspring (60-80 days old) were assessed for body composition, fasting glucose, and metabolic and activity parameters using the TSE Phenomaster system. Results:BPA-exposed offspring exhibited reduced lean body mass, fat mass, fat ratio, and 24-hour fasting glucose levels compared to controls and EE2-exposed offspring. Both BPA- and EE2-exposed groups showed altered circadian patterns of locomotor activity, food intake, energy expenditure, and respiratory exchange ratio, with effects predominantly occurring during the night phase. Conclusions:Maternal exposure to environmentally relevant doses of BPA or EE2 can alter the development and function of metabolic regulatory systems, producing distinct disruptions in circadian energy homeostasis in adult offspring. These differential effects likely reflect the partially overlapping yet distinct organizational and activational pathways through which these endocrine-disrupting chemicals act during the perinatal period.

Abstract Objective Glucagon-like peptide-1 (GLP-1), a peptide neurotransmitter in the brain, is synthesized from proglucagon, encoded by the glucagon gene ( Gcg ). Besides medullary GLP-1 neurons, Gcg -expressing neuron populations were identified in the olfactory bulb and basolateral amygdala. However, several lines of evidence suggest that additional Gcg neuron populations might exist. Methods We conducted a brain-wide mapping of Gcg -expressing cells by fluorescent in situ hybridization in C57BL/6J and FVB/Ant mice. Proglucagon and GLP-1 expression were studied with immunofluorescence. We characterized a Gcg -Cre;tdTomato mouse line and studied the expression of proglucagon-processing enzymes in Gcg -expressing neuron populations. We used adeno-associated virus-mediated tracing in Gcg -Cre mice to map the projections of hypothalamic Gcg neurons. Results Gcg -expressing neuron populations were identified in the olfactory bulb, claustrum, piriform cortex, basolateral amygdala, posterior hippocampus, posterior hypothalamic nucleus (PH), periaqueductal gray/dorsal raphe, and dorsal nucleus of the lateral lemniscus. These neurons express lower Gcg mRNA levels than medullary GLP-1 neurons. Proglucagon and GLP-1-immunoreactivity (C-terminus) were detected in almost all Gcg -expressing neuron populations, along with the mRNAs for prohormone convertases 1/3 and 2, enzymes generating GLP-1 or glucagon, respectively. Fasting markedly increased Gcg mRNA, proglucagon and GLP-1 synthesis in the PH. PH Gcg neurons project densely to the ventral and intermediate lateral septum, preoptic region, ventrolateral preoptic nucleus, lateral hypothalamus and zona incerta, establishing close contacts with both GLP-1 receptor-positive and -negative neurons. Conclusions Proglucagon is expressed in 9 distinct neuron populations. Feeding status regulates GLP-1 synthesis in PH neurons that likely control feeding- or energy balance-related functions.

Aging affects the reproductive system, although its impact on GnRH neurons is mainly unexplored. Thus, we compared the transcriptome and electrophysiology of GnRH neurons obtained from female middle-aged (MA, 400-430 d) and young (Y, 70 d), diestrous mice, respectively. Transcriptomic analysis revealed reproductive senescence-related molecular changes in one-third of the MA mice. The upregulated genes (n = 225) were linked to immune signaling, olfactory- and vomeronasal receptors. The downregulated genes (n = 233) were related to mRNA processing, G-protein-coupled receptors, oxidative phosphorylation, electron transport, and estrogen signaling. In addition, ion channel (Na, K, Ca), neurotransmitter (ACh, GABA, glutamate), and various neuropeptide receptor-coding genes showed differential expression indicating functional alterations of the cells. Accordingly, whole-cell patch-clamp recordings revealed a twofold increase in spontaneous firing frequency of MA-GnRH neurons. Significant changes were also observed in characteristics of action potentials and afterhyperpolarization amplitudes. Conspicuously, miniature postsynaptic currents were absent in 72% of MA-GnRH neurons, and pharmacological blockade of GABAA and glutamate receptors didn't affect the firing rate. However, administration of their ligands evoked inward currents and facilitated firing in both animal groups, although with a decreased efficacy in MA animals. MA-GnRH neurons sustained responsiveness to estradiol, G-protein inhibition, and kisspeptin (KP) like those of young animals. While KP receptor antagonist, KP-234, diminished firing frequency of MA-GnRH neurons, it had no effect on young GnRH cells. Collectively, these findings revealed that both the transcriptome and electrophysiological activity of GnRH neurons change at middle age and the explored alterations are hallmarks of early phase of reproductive senescence.

GnRH-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms haven't been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and the ACh receptor (AChR) agonist carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of mPSCs, indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) ACh receptors. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both miniature postsynaptic currents (mPSCs) and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked co-transmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system immensely regulates GnRH neurons and activates the HPG-axis via ACh and ACh/GABA neurotransmissions in male mice. Significance statement Cholinergic drugs influence reproduction centrally, although the exact neuronal targets and regulatory mechanisms remain unsettled. We found that pharmacogenetic activation of the cholinergic system in vivo evoked an augmented LH release in male mice. The study also identified cholinergic cell groups in the mouse forebrain that innervate gonadotropin-releasing hormone (GnRH) neurons, the main hypothalamic regulators of reproduction. We also determined the subtypes of nicotinic and muscarinic receptors involved in neuronal information transfer and explored how their ligands affect the electrophysiological activity of GnRH neurons. A subset of cholinergic neurons was found to co-transmit GABA which excites GnRH cells via GABA-A receptors. The findings suggest a novel, cholinergic regulation of the GnRH system activating the pituitary-gonadal axis in adult male mice.

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and the ACh receptor (AChR) agonist carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of mPSCs, indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) ACh receptors. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both miniature postsynaptic currents (mPSCs) and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked co-transmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system immensely regulates GnRH neurons and activates the HPG-axis via ACh and ACh/GABA neurotransmissions. Significance statement Cholinergic drugs influence reproduction centrally, although the exact neuronal targets and regulatory mechanisms remain unsettled. We found that pharmacogenetic activation of the cholinergic system in vivo evoked an augmented LH release. The study also identified cholinergic cell groups in the mouse forebrain that innervate gonadotropin-releasing hormone (GnRH) neurons, the main hypothalamic regulators of reproduction. We also determined the subtypes of nicotinic and muscarinic receptors involved in neuronal information transmission and explored how their ligands affect the electrophysiological activity of GnRH neurons. A subset of cholinergic neurons was found to co-transmit GABA, which excites GnRH cells via GABA-A receptors. The findings suggest a novel, cholinergic regulation of the adult GnRH system in male mice that activates the pituitary-gonadal axis.

Orexin neurons are involved in homeostatic regulatory processes, including arousal and feeding, and provide a major input from the hypothalamus to the ventral tegmental area (VTA) of the midbrain. VTA neurons are a central hub processing reward and motivation and target the medial prefrontal cortex (mPFC) and the shell part of nucleus accumbens (NAcs). We investigated whether subpopulations of dopamine (DA) neurons in the VTA projecting either to the mPFC or the medial division of shell part of nucleus accumbens (mNAcs) receive differential input from orexin neurons and whether orexin exerts differential electrophysiological effects upon these cells. VTA neurons projecting to the mPFC or the mNAcs were traced retrogradely by Cav2-Cre virus and identified by expression of yellow fluorescent protein (YFP). Immunocytochemical analysis showed that a higher proportion of all orexin-innervated DA neurons projected to the mNAcs (34.5%) than to the mPFC (5.2%). Of all sampled VTA neurons projecting either to the mPFC or mNAcs, the dopaminergic (68.3 vs. 79.6%) and orexin-innervated DA neurons (68.9 vs. 64.4%) represented the major phenotype. Whole-cell current clamp recordings were obtained from fluorescently labeled neurons in slices during baseline periods and bath application of orexin A. Orexin similarly increased the firing rate of VTA dopamine neurons projecting to mNAcs (1.99 ± 0.61 Hz to 2.53 ± 0.72 Hz) and mPFC (0.40 ± 0.22 Hz to 1.45 ± 0.56 Hz). Thus, the hypothalamic orexin system targets mNAcs and to a lesser extent mPFC-projecting dopaminergic neurons of the VTA and exerts facilitatory effects on both clusters of dopamine neurons.

Rising serum estradiol triggers the surge release of gonadotropin-releasing hormone (GnRH) at late proestrus leading to ovulation. We hypothesized that proestrus evokes alterations in peptidergic signaling onto GnRH neurons inducing a differential expression of neuropeptide-, growth factor-, and orphan G-protein-coupled receptor (GPCR) genes. Thus, we analyzed the transcriptome of GnRH neurons collected from intact, proestrous and metestrous GnRH-green fluorescent protein (GnRH-GFP) transgenic mice using Affymetrix microarray technique. Proestrus resulted in a differential expression of genes coding for peptide/neuropeptide receptors including Adipor1, Prokr1, Ednrb, Rtn4r, Nmbr, Acvr2b, Sctr, Npr3, Nmur1, Mc3r, Cckbr, and Amhr2. In this gene cluster, Adipor1 mRNA expression was upregulated and the others were downregulated. Expression of growth factor receptors and their related proteins was also altered showing upregulation of Fgfr1, Igf1r, Grb2, Grb10, and Ngfrap1 and downregulation of Egfr and Tgfbr2 genes. Gpr107, an orphan GPCR, was upregulated during proestrus, while others were significantly downregulated (Gpr1, Gpr87, Gpr18, Gpr62, Gpr125, Gpr183, Gpr4, and Gpr88). Further affected receptors included vomeronasal receptors (Vmn1r172, Vmn2r-ps54, and Vmn1r148) and platelet-activating factor receptor (Ptafr), all with marked downregulation. Patch-clamp recordings from mouse GnRH-GFP neurons carried out at metestrus confirmed that the differentially expressed IGF-1, secretin, and GPR107 receptors were operational, as their activation by specific ligands evoked an increase in the frequency of miniature postsynaptic currents (mPSCs). These findings show the contribution of certain novel peptides, growth factors, and ligands of orphan GPCRs to regulation of GnRH neurons and their preparation for the surge release.

Glucagon-like peptide-1 (GLP-1) regulates reproduction centrally, although, the neuroanatomical basis of the process is unknown. Therefore, the putative networking of the central GLP-1 and gonadotropin-releasing hormone (GnRH) systems was addressed in male mice using whole mount immunocytochemistry and optogenetics. Enhanced antibody penetration and optical clearing procedures applied to 500–1000 µm thick basal forebrain slices allowed the simultaneous visualization of the two distinct systems in the basal forebrain. Beaded GLP-1-IR axons innervated about a quarter of GnRH neurons (23.2 ± 1.4%) forming either single or multiple contacts. GnRH dendrites received a more intense GLP-1 innervation (64.6 ± 0.03%) than perikarya (35.4 ± 0.03%). The physiological significance of the innervation was examined by optogenetic activation of channelrhodopsin-2 (ChR2)-expressing axons of preproglucagon (GCG) neurons upon the firing of GnRH neurons by patch clamp electrophysiology in acute brain slices of triple transgenic mice (Gcg-cre/ChR2/GFP-GnRH). High-frequency laser beam stimulation (20 Hz, 10 ms pulse width, 3 mW laser power) of ChR2-expressing GCG axons in the mPOA increased the firing rate of GnRH neurons (by 75 ± 17.3%, p = 0.0007). Application of the GLP-1 receptor antagonist, Exendin-3-(9-39) (1 μM), prior to the photo-stimulation, abolished the facilitatory effect. In contrast, low-frequency trains of laser pulses (0.2 Hz, 60 pulses) had no effect on the spontaneous postsynaptic currents of GnRH neurons. The findings indicate a direct wiring of GLP-1 neurons with GnRH cells which route is excitatory for the GnRH system. The pathway may relay metabolic signals to GnRH neurons and synchronize metabolism with reproduction.

Objective The lateral parabrachial nucleus (lPBN) in the brainstem has emerged as a key area involved in feeding control that is targeted by several circulating anorexigenic hormones. Here, the objective was to determine whether the lPBN is also a relevant site for the orexigenic hormone ghrelin, inspired by studies in mice and rats showing that there is an abundance of ghrelin receptors in this area. Methods This study first explored whether iPBN cells respond to ghrelin involving Fos mapping and electrophysiological studies in rats. Next, rats were injected acutely with ghrelin, a ghrelin receptor antagonist, or vehicle into the lPBN to investigate feeding-linked behaviors. Results Curiously, ghrelin injection (intracerebroventricular or intravenous) increased Fos protein expression in the lPBN yet the predominant electrophysiological response was inhibitory. Intra-lPBN ghrelin injection increased chow or high-fat diet intake, whereas the antagonist decreased chow intake only. In a choice paradigm, intra-lPBN ghrelin increased intake of chow but not lard or sucrose. Intra-lPBN ghrelin did not alter progressive ratio lever pressing for sucrose or conditioned place preference for chocolate. Conclusions The lPBN is a novel locus from which ghrelin can alter consummatory behaviors (food intake and choice) but not appetitive behaviors (food reward and motivation).

Absztrakt: A colorectalis carcinoma (CRC) az egyik leggyakrabban előforduló daganatos megbetegedés világszerte. A sporadikus vastagbélrák incidenciája ötvenéves kor alatt alacsonyabb, majd az életkor előrehaladtával nő, továbbá jellegzetes klinikai, lokalizáció szerinti és molekuláris eltérést mutathat a nők és a férfiak között. Epidemiológiai és molekuláris biológiai kutatások eredményei szerint az ösztradiol (E2) által szabályozott jelútrendszer meghatározó szerepet játszik a CRC kialakulásában és prognosztikájában, döntően a vastagbélhámban domináns ösztrogénreceptor-bétán (ERβ) keresztül. Az ösztradiol emésztőrendszeri hatásai igen sokrétűek, az ép és tumoros vastagbélhámsejtekre gyakorolt hatását in vitro és in vivo vizsgálatok egyaránt igazolták. Az ösztrogénreceptor-alfával (ERα) ellentétben az ERβ aktivációja a sejtosztódást gátolja és az apoptózist fokozza, a béta-receptor kifejeződése ugyanakkor mind az élettani öregedés, mind a vastagbél kórállapotaiban megváltozhat. Az ösztradiol ERβ által közvetített daganatellenes hatása a sejtproliferáció gátlása, az apoptózis serkentése, az áttétképzés gátlása és gyulladáscsökkentő hatása révén valósulhat meg. Sejtkultúra- és állatkísérletes kutatások eredményei alapján az ösztrogénreceptor-bétára szelektíven ható receptormodulátorok (szelektív ösztrogénreceptor-modulátor [SERM]) és a fitoösztrogének új, hozzáadott kezelési lehetőséget jelenthetnek az idült gyulladással és a kóros sejtproliferációval jellemezhető colorectalis megbetegedésekben. Orv Hetil. 2020; 161(14): 532–543.

The subsequent isolation, purification and sequencing of this factor revealed that it was a decapeptide that could stimulate secretion of both luteinizing hormone (LH) and follicle-stimulating hormone from the pituitary. Due to these properties, this peptide was termed LH-releasing hormone (LHRH) or gonadotropin-releasing hormone. The feasibility of obtaining immortalized LHRH neuronal cell lines became apparent following two major developments in transgenic mouse technology. First, Mason and colleagues had successfully targeted LHRH neurons for gene therapy in hypogonadal mice. A second development relates to ability to target expression of the simian virus-40 T-antigen oncogene to a given cell such that it becomes immortalized. When the anterior hypothalamic tumor cells were dispersed and grown in culture, they were very heterogeneous. Due to this property, these immortalized neurons can be co-cultured with other cell lines or with primary cells from the pituitary, olfactory placode, hypothalamus, or other regions of the brain to study cell-cell interactions.

Colorectal cancer (CRC) is one of the most common types of cancers worldwide. The incidence of sporadic CRC is lower in individuals below 50 years and increases with age, furthermore, it shows typical clinical, macroscopic and molecular differences between females and males. According to the results of epidemiological and molecular biology studies, the estradiol-regulating signaling pathway plays an important role in the development and prognosis of CRC, predominantly through estrogen receptor beta (ER beta), which is dominant in the colonic epithelium. Estradiol has multiple gastrointestinal effects, which were confirmed by in vitro and in vivo studies on histologically intact and cancerous cells as well. In contrast to estrogen receptor alpha (ER alpha), the activation of ER beta inhibits cell proliferation and enhances apoptosis, nevertheless, the expression of estrogen receptor beta can change both during physiological ageing and in colorectal disorders. The ER beta-mediated antitumour effects of estradiol may be exerted through inhibition of cell proliferation, stimulation of apoptosis, inhibition of metastasis formation and its anti-inflammatory activity. Based on the results of cell culture and animal studies, selective modulators of estrogen receptor beta (selective estrogen receptor modulator [SERM]) and phytoestrogens can be new, additional therapeutic options in the treatment of colorectal diseases characterized by chronic inflammation and uncontrolled cell proliferation.

Introduction: Hypophysiotropic gonadotropin-releasing hormone (GnRH) neurons orchestrate various physiological events that control the onset of puberty. Previous studies showed that insulin-like growth factor 1 (IGF-1) induces the secretion of GnRH and accelerates the onset of puberty, suggesting a regulatory role of this hormone upon GnRH neurons. Methods: To reveal responsiveness of GnRH neurons to IGF-1 and elucidate molecular pathways acting downstream to the IGF-1 receptor (IGF-1R), in vitro electrophysiological experiments were carried out on GnRH-GFP neurons in acute brain slices from prepubertal (23–29 days) and pubertal (50 days) male mice. Results: Administration of IGF-1 (13 nM) significantly increased the firing rate and frequency of spontaneous postsynaptic currents and that of excitatory GABAergic miniature postsynaptic currents (mPSCs). No GABAergic mPSCs were induced by IGF-1 in the presence of the GABAA-R blocker picrotoxin. The increase in the mPSC frequency was prevented by the use of the IGF-1R antagonist, JB1 (1 µM), or the intracellularly applied PI3K blocker (LY294002, 50 µM), showing involvement of IGF-1R and PI3K in the mechanism. Blockade of the transient receptor potential vanilloid 1, an element of the tonic retrograde endocannabinoid machinery, by AMG9810 (10 µM) or antagonizing the cannabinoid receptor type-1 by AM251 (1 µM) abolished the effect. Discussion/Conclusion: These findings indicate that IGF-1 arrests the tonic retrograde endocannabinoid pathway in GnRH neurons, and this disinhibition increases the release of GABA from presynaptic terminals that, in turn, activates GnRH neurons leading to the fine-tuning of the hypothalamo-pituitary-gonadal axis.

In mammals, reproduction is regulated by a wide range of metabolic hormones that maintain the proper energy balance. In addition to regulating feeding and energy expenditure, these metabolic messengers also modulate the functional performance of the hypothalamic-pituitary-gonadal (HPG) axis. Secretin, a member of the secretin-glucagon-vasoactive intestinal peptide hormone family, has been shown to alter reproduction centrally, although the underlying mechanisms have not been explored yet. In order to elucidate its central action in the neuroendocrine regulation of reproduction, in vitro electrophysiological slice experiments were carried out on GnRH-GFP neurons in male mice. Bath application of secretin (100 nM) significantly increased the frequency of the spontaneous postsynaptic currents (sPSCs) to 118.0 ± 2.64% compared to the control, and that of the GABAergic miniature postsynaptic currents (mPSCs) to 147.6 ± 19.19%. Resting membrane potential became depolarized by 12.74 ± 4.539 mV after secretin treatment. Frequency of evoked action potentials (APs) also increased to 144.3 ± 10.8%. The secretin-triggered elevation of the frequency of mPSCs was prevented by using either a secretin receptor antagonist (3 μM) or intracellularly applied G-protein-coupled receptor blocker (GDP-β-S; 2 mM) supporting the involvement of secretin receptor in the process. Regarding the actions downstream to secretin receptor, intracellular blockade of protein kinase A (PKA) with KT-5720 (2 μM) or intracellular inhibition of the neuronal nitric oxide synthase (nNOS) by NPLA (1 μM) abolished the stimulatory effect of secretin on mPSCs. These data suggest that secretin acts on GnRH neurons via secretin receptors whose activation triggers the cAMP/PKA/nNOS signaling pathway resulting in nitric oxide release and in the presynaptic terminals this retrograde NO machinery regulates the GABAergic input to GnRH neurons.

Neuronal circuits involving the central amygdala (CeA) are gaining prominence as important centres for regulation of metabolic functions. As a part of the subcortical food motivation circuitry, CeA is associated with food motivation and hunger. We have previously shown that interleukin (IL)‐6 can act as a downstream mediator of the metabolic effects of glucagon‐like peptide‐1 (GLP‐1) receptor (R) stimulation in the brain, although the sites of these effects are largely unknown. In the present study, we used the newly generated and validated RedIL6 reporter mouse strain to investigate the presence of IL‐6 in the CeA, as well as possible interactions between IL‐6 and GLP‐1 in this nucleus. IL‐6 was present in the CeA, mostly in cells in the medial and lateral parts of this structure, and a majority of IL‐6‐containing cells also co‐expressed GLP‐1R. Triple staining showed GLP‐1 containing fibres co‐staining with synaptophysin close to or overlapping with IL‐6 containing cells. GLP‐1R stimulation enhanced IL‐6 mRNA levels. IL‐6 receptor‐alpha (IL‐6Rα) was found to a large part in neuronal CeA cells. Using electrophysiology, we determined that cells with neuronal properties in the CeA could be rapidly stimulated by IL‐6 administration in vitro. Moreover, microinjections of IL‐6 into the CeA could slightly reduce food intake in vivo in overnight fasted rats. In conclusion, IL‐6 containing cells in the CeA express GLP‐1R, are close to GLP‐1‐containing synapses, and demonstrate increased IL‐6 mRNA in response to GLP‐1R agonist treatment. IL‐6, in turn, exerts biological effects in the CeA, possibly via IL‐6Rα present in this nucleus.

Microglia are instrumental for recognition and elimination of amyloid beta(1-42) oligomers (A beta Os), but the long-term consequences of A beta O-induced inflammatory changes in the brain are unclear. Here, we explored microglial responses and transciptome-level inflammatory signatures in the rat hippocampus after chronic A beta O challenge. Middle-aged Long Evans rats received intracerebroventricular infusion of A beta O or vehicle for 4 weeks, followed by treatment with artificial CSF or MCC950 for the subsequent 4 weeks. AbO infusion evoked a sustained inflammatory response including activation of NF-kappa B, triggered microglia activation and increased the expression of pattern recognition and phagocytic receptors. A beta(1-42) plaques were not detectable likely due to microglial elimination of infused oligomers. In addition, we found upregulation of neuronal inhibitory ligands and their cognate microglial receptors, while downregulation of Esr1 and Scn1a, encoding estrogen receptor alpha and voltage-gated sodium-channel Na(v) 1.1, respectively, was observed. These changes were associated with impaired hippocampus-dependent spatial memory and resembled early neurological changes seen in Alzheimer's disease. To investigate the role of inflammatory actions in memory deterioration, we performed MCC950 infusion, which specifically blocks the NLRP3 inflammasome. MCC950 attenuated A beta O-evoked microglia reactivity, restored expression of neuronal inhibitory ligands, reversed downregulation of ER alpha, and abolished memory impairments. Furthermore, MCC950 abrogated A beta O-invoked reduction of serum IL-10. These findings provide evidence that in response to AbO infusion microglia change their phenotype, but the resulting inflammatory changes are sustained for at least one month after the end of AbO challenge. Lasting NLRP3-driven inflammatory alterations and altered hippocampal gene expression contribute to spatial memory decline. This article is part of a Special Issue entitled: Microglia-Neuron interactions in health and disease - novel perspectives for translational research. (C) 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Melanin-concentrating hormone (MCH) is an important regulator of food intake, glucose metabolism, and adiposity. However, the mechanisms mediating these actions remain largely unknown. We used pharmacological and genetic approaches to show that the sirtuin 1 (SIRT1)/FoxO1 signaling pathway in the hypothalamic arcuate nucleus (ARC) mediates MCH-induced feeding, adiposity, and glucose intolerance. MCH reduces proopiomelanocortin (POMC) neuronal activity, and the SIRT1/FoxO1 pathway regulates the inhibitory effect of MCH on POMC expression. Remarkably, the metabolic actions of MCH are compromised in mice lacking SIRT1 specifically in POMC neurons. Of note, the actions of MCH are independent of agouti-related peptide (AgRP) neurons because inhibition of γ-aminobutyric acid receptor in the ARC did not prevent the orexigenic action of MCH, and the hypophagic effect of MCH silencing was maintained after chemogenetic stimulation of AgRP neurons. Central SIRT1 is required for MCH-induced weight gain through its actions on the sympathetic nervous system. The central MCH knockdown causes hypophagia and weight loss in diet-induced obese wild-type mice; however, these effects were abolished in mice overexpressing SIRT1 fed a high-fat diet. These data reveal the neuronal basis for the effects of MCH on food intake, body weight, and glucose metabolism and highlight the relevance of SIRT1/FoxO1 pathway in obesity.

Dopamine signalling is a crucial part of the brain reward system and can affect feeding behaviour. Dopamine receptors are also expressed in the hypothalamus, which is known to control energy metabolism in peripheral tissues. Here we show that pharmacological or chemogenetic stimulation of dopamine receptor 2 (D2R) expressing cells in the lateral hypothalamic area (LHA) and the zona incerta (ZI) decreases body weight and stimulates brown fat activity in rodents in a feeding-independent manner. LHA/ZI D2R stimulation requires an intact sympathetic nervous system and orexin system to exert its action and involves inhibition of PI3K in the LHA/ZI. We further demonstrate that, as early as 3 months after the onset of treatment, patients treated with the D2R agonist cabergoline experience an increase in energy expenditure that persists for one year, leading to total body weight and fat loss through a prolactin-independent mechanism. Our results may provide a mechanistic explanation for how clinically used D2R agonists act in the central nervous system to regulate energy balance.

In proestrus, the changing gonadal hormone milieu alters the physiological properties of GnRH neurons and contributes to the development of the GnRH surge. We hypothesized that proestrus also influences the expression of different ion channel genes in mouse GnRH neurons. Therefore, we performed gene expression profiling of GnRH neurons collected from intact, proestrous and metestrous GnRH-GFP transgenic mice, respectively. Proestrus changed the expression of 37 ion channel and 8 calcium homeostasis-regulating genes. Voltage-gated sodium channels responded with upregulation of three alpha subunits (Scn2a1, Scn3a, and Scn9a). Within the voltage-gated potassium channel class, Kcna1, Kcnd3, Kcnh3, and Kcnq2 were upregulated, while others (Kcna4, Kcnc3, Kcnd2, and Kcng1) underwent downregulation. Proestrus also had impact on inwardly rectifying potassium channel subunits manifested in enhanced expression of Kcnj9 and Kcnj10 genes, whereas Kcnj1, Kcnj11, and Kcnj12 subunit genes were downregulated. The two-pore domain potassium channels also showed differential expression with upregulation of Kcnk1 and reduced expression of three subunit genes (Kcnk7, Kcnk12, and Kcnk16). Changes in expression of chloride channels involved both the voltage-gated (Clcn3 and Clcn6) and the intracellular (Clic1) subtypes. Regarding the pore-forming alpha-1 subunits of voltage-gated calcium channels, two (Cacna1b and Cacna1h) were upregulated, while Cacna1g showed downregulation. The ancillary subunits were also differentially regulated (Cacna2d1, Cacna2d2, Cacnb1, Cacnb3, Cacnb4, Cacng5, Cacng6, and Cacng8). In addition, ryanodine receptor 1 (Ryr1) gene was downregulated, while a transient receptor potential cation channel (Trpm3) gene showed enhanced expression. Genes encoding proteins regulating the intracellular calcium homeostasis were also influenced (Calb1, Hpca, Hpcal1, Hpcal4, Cabp7, Cab 39l, and Cib2). The differential expression of genes coding for ion channel proteins in GnRH neurons at late proestrus indicates that the altering hormone milieu contributes to remodeling of different kinds of ion channels of GnRH neurons, which might be a prerequisite of enhanced cellular activity of GnRH neurons and the subsequent surge release of the neurohormone.