Cholinergic Control of GnRH Neuron Physiology and Luteinizing Hormone Secretion in Male Mice: Involvement of ACh/GABA Cotransmission.

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 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 miniature postsynaptic currents (mPSCs), indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4ß2, α3ß4, and α7) and muscarinic (M1-M5) AChRs. 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 mPSCs and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system regulates GnRH neurons and activates the pituitary-gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.

Characterization of orexin input to dopamine neurons of the ventral tegmental area projecting to the medial prefrontal cortex and shell of nucleus accumbens.

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.

Insulin-Like Growth Factor 1 Increases GABAergic Neurotransmission to GnRH Neurons via Suppressing the Retrograde Tonic Endocannabinoid Signaling Pathway in Mice.

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.

Networking of glucagon-like peptide-1 axons with GnRH neurons in the basal forebrain of male mice revealed by 3DISCO-based immunocytochemistry and optogenetics.

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.

Ghrelin Receptor Stimulation of the Lateral Parabrachial Nucleus in Rats Increases Food Intake but not Food Motivation.

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).

[Potential role of estrogens in colorectal tumour development]. / Az ösztrogének lehetséges szerepe a vastagbéldaganatok kialakulásában.

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 (ERß), 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 (ERα), the activation of ERß inhibits cell proliferation and enhances apoptosis, nevertheless, the expression of estrogen receptor beta can change both during physiological ageing and in colorectal disorders. The ERß-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. Orv Hetil. 2020; 161(14): 532-543.

Gonadal Cycle-Dependent Expression of Genes Encoding Peptide-, Growth Factor-, and Orphan G-Protein-Coupled Receptors in Gonadotropin- Releasing Hormone Neurons of Mice.

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.

Blunted leptin sensitivity during hedonic overeating can be reinstated by activating galanin 2 receptors (Gal2R) in the lateral hypothalamus.

AIM: Since foods with high hedonic value are often consumed in excess of energetic needs, this study was designed to identify the mechanisms that may counter anorexigenic signalling in the presence of hedonic foods in lean animals. METHODS: Mice, in different states of satiety (fed/fasted, or fed/fasted and treated with ghrelin or leptin, respectively), were allowed to choose between high-fat/high-sucrose and standard foods. Intake of each food type and the activity of hypothalamic neuropetidergic neurons that regulate appetite were monitored. In some cases, food choice was monitored in leptin-injected fasted mice that received microinjections of galanin receptor agonists into the lateral hypothalamus. RESULTS: Appetite-stimulating orexin neurons in the lateral hypothalamus are rapidly activated when lean, satiated mice consume a highly palatable food (PF); such activation (upregulated c-Fos expression) occurred even after administration of the anorexigenic hormone leptin and despite intact leptin signalling in the hypothalamus. The ability of leptin to restrain PF eating is restored when a galanin receptor 2 (Gal2R) agonist is injected into the lateral hypothalamus. CONCLUSION: Hedonically-loaded foods interrupt the inhibitory actions of leptin on orexin neurons and interfere with the homeostatic control of feeding. Overeating of palatable foods can be curtailed in lean animals by activating Gal2R in the lateral hypothalamus.

Hypothalamic dopamine signaling regulates brown fat thermogenesis.

Dopamine signaling is a crucial part of the brain reward system and can affect feeding behavior. 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 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 CNS to regulate energy balance.

MCH Regulates SIRT1/FoxO1 and Reduces POMC Neuronal Activity to Induce Hyperphagia, Adiposity, and Glucose Intolerance.

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.

Secretin Regulates Excitatory GABAergic Neurotransmission to GnRH Neurons via Retrograde NO Signaling Pathway in Mice.

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-ß-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.

Proestrus Differentially Regulates Expression of Ion Channel and Calcium Homeostasis Genes in GnRH Neurons of Mice.

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.

Interleukin-6 in the central amygdala is bioactive and co-localised with glucagon-like peptide-1 receptor.

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.

Long-term selective estrogen receptor-beta agonist treatment modulates gene expression in bone and bone marrow of ovariectomized rats.

Gonadal hormones including 17ß-estradiol exert important protective functions in skeletal homeostasis. However, numerous details of ovarian hormone deficiency in the common bone marrow microenvironment have not yet been revealed and little information is available on the tissue-specific acts either, especially those via estrogen receptor beta (ERß). The aim of the present study was therefore to examine the bone-related gene expression changes after ovariectomy (OVX) and long-term ERß agonist diarylpropionitrile (DPN) administration. We found that OVX produced strong and widespread changes of gene expression in both femoral bone and bone marrow. In the bone out of 22 genes, 20 genes were up- and 2 were downregulated after OVX. It is noteworthy that DPN restored mRNA expression of 10 OVX-induced changes (Aldh2, Col1a1, Daam1, Fgf12, Igf1, Il6r, Nfkb1, Notch1, Notch2 and Psen1) suggesting a modulatory role of ERß in bone physiology. In bone marrow, out of 37 categorized genes, transcription of 25 genes were up- and 12 were downregulated indicating that the marrow is highly responsive to gonadal hormones. DPN modestly affected transcription, only expression of two genes (Nfatc1 and Tgfb1) was restored by DPN action. The PI3K/Akt signaling pathway was the most affected gene cluster following the interventions in bone and bone marrow, as demonstrated by canonical variates analysis (CVA). We suggested that our results contribute to a deeper understanding of alterations in gene expression of bone and bone marrow niche elicited by ERß and selective ERß analogs.

Chronic Amyloid ß Oligomer Infusion Evokes Sustained Inflammation and Microglial Changes in the Rat Hippocampus via NLRP3.

Microglia are instrumental for recognition and elimination of amyloid ß1-42 oligomers (AßOs), but the long-term consequences of AßO-induced inflammatory changes in the brain are unclear. Here, we explored microglial responses and transciptome-level inflammatory signatures in the rat hippocampus after chronic AßO challenge. Middle-aged Long Evans rats received intracerebroventricular infusion of AßO or vehicle for 4 weeks, followed by treatment with artificial CSF or MCC950 for the subsequent 4 weeks. AßO infusion evoked a sustained inflammatory response including activation of NF-κB, triggered microglia activation and increased the expression of pattern recognition and phagocytic receptors. Aß1-42 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 AßO-evoked microglia reactivity, restored expression of neuronal inhibitory ligands, reversed downregulation of ERα, and abolished memory impairments. Furthermore, MCC950 abrogated AßO-invoked reduction of serum IL-10. These findings provide evidence that in response to AßO infusion microglia change their phenotype, but the resulting inflammatory changes are sustained for at least one month after the end of AßO challenge. Lasting NLRP3-driven inflammatory alterations and altered hippocampal gene expression contribute to spatial memory decline.

GnRH Neurons Provide Direct Input to Hypothalamic Tyrosine Hydroxylase Immunoreactive Neurons Which Is Maintained During Lactation.

Gonadotropin releasing hormone (GnRH) neurons provide neuronal input to the preoptic area (POA) and the arcuate nucleus (Arc), two regions involved critically in the regulation of neuroendocrine functions and associated behaviors. These areas contain tyrosine hydroxylase immunoreactive (TH-IR) neurons, which play location-specific roles in the neuroendocrine control of both the luteinizing hormone and prolactin secretion, as well as, sexually motivated behaviors. Concerning changes in the activity of GnRH neurons and the secretion pattern of GnRH seen under the influence of rising serum estrogen levels and during lactation, we tested the hypothesis that the functional state of GnRH neurons is mediated via direct synaptic connections to TH-IR neurons in the POA and Arc. In addition, we examined putative changes of these inputs in lactating mice and in mothers separated from their pups. Confocal microscopic and pre-embedding immunohistochemical studies on ovariectomized mice treated with 17ß-estradiol (OVX+E2) provided evidence for direct appositions and asymmetric synapses between GnRH-IR fiber varicosities and TH-IR neurons in the POA and the Arc. As TH co-localizes with kisspeptin (KP) in the POA, confocal microscopic analysis was continued on sections additionally labeled for KP. The TH-IR neurons showed a lower level of co-labeling for KP in lactating mice compared to OVX+E2 mice (16.1 ± 5% vs. 57.8 ± 4.3%). Removing the pups for 24 h did not alter significantly the KP production in TH-IR neurons (17.3 ± 4.6%). The mean number of GnRH-IR varicosities on preoptic and arcuate TH cells did not differ in the three animal models investigated. This study shows evidence that GnRH neurons provide direct synaptic inputs to POA and Arc dopaminergic neurons. The scale of anatomical connectivity with these target cells was unaltered during lactation indicating a maintained GnRH input, inspite of the altered hormonal condition.

Endocannabinoid and nitric oxide systems of the hypothalamic paraventricular nucleus mediate effects of NPY on energy expenditure.

OBJECTIVE: Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN. METHODS: Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis. RESULTS: We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure. CONCLUSION: Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.

Estradiol Increases Glutamate and GABA Neurotransmission into GnRH Neurons via Retrograde NO-Signaling in Proestrous Mice during the Positive Estradiol Feedback Period.

Surge release of gonadotropin-releasing hormone (GnRH) is essential in the activation of pituitary gonadal unit at proestrus afternoon preceded by the rise of serum 17ß-estradiol (E2) level during positive feedback period. Here, we describe a mechanism of positive estradiol feedback regulation acting directly on GnRH-green fluorescent protein (GFP) neurons of mice. Whole-cell clamp and loose patch recordings revealed that a high physiological dose of estradiol (200 pM), significantly increased firing rate at proestrus afternoon. The mPSC frequency at proestrus afternoon also increased, whereas it decreased at metestrus afternoon and had no effect at proestrus morning. Inhibition of the estrogen receptor ß (ERß), intracellular blockade of the Src kinase and phosphatidylinositol 3 kinase (PI3K) and scavenge of nitric oxide (NO) inside GnRH neurons prevented the facilitatory estradiol effect indicating involvement of the ERß/Src/PI3K/Akt/nNOS pathway in this fast, direct stimulatory effect. Immunohistochemistry localized soluble guanylate cyclase, the main NO receptor, in both glutamatergic and GABAergic terminals innervating GnRH neurons. Accordingly, estradiol facilitated neurotransmissions to GnRH neurons via both GABAA-R and glutamate/AMPA/kainate-R. These results indicate that estradiol acts directly on GnRH neurons via the ERß/Akt/nNOS pathway at proestrus afternoon generating NO that retrogradely accelerates GABA and glutamate release from the presynaptic terminals contacting GnRH neurons. The newly explored mechanism might contribute to the regulation of the GnRH surge, a fundamental prerequisite of the ovulation.

Glycinergic Input to the Mouse Basal Forebrain Cholinergic Neurons.

The basal forebrain (BF) receives afferents from brainstem ascending pathways, which has been implicated first by Moruzzi and Magoun (1949) to induce forebrain activation and cortical arousal/waking behavior; however, it is very little known about how brainstem inhibitory inputs affect cholinergic functions. In the current study, glycine, a major inhibitory neurotransmitter of brainstem neurons, and gliotransmitter of local glial cells, was tested for potential interaction with BF cholinergic (BFC) neurons in male mice. In the BF, glycine receptor α subunit-immunoreactive (IR) sites were localized in choline acetyltransferase (ChAT)-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs (sIPSCs; 0.81 ± 0.25 × 10-1 Hz) recorded in whole-cell conditions. Potential neuronal as well as glial sources of glycine were indicated in the extracellular space of cholinergic neurons by glycine transporter type 1 (GLYT1)- and GLYT2-IR processes found in apposition to ChAT-IR cells. Ultrastructural analyses identified synapses of GLYT2-positive axon terminals on ChAT-IR neurons, as well as GLYT1-positive astroglial processes, which were localized in the vicinity of synapses of ChAT-IR neurons. The brainstem raphe magnus was determined to be a major source of glycinergic axons traced retrogradely from the BF. Our results indicate a direct effect of glycine on BFC neurons. Furthermore, the presence of high levels of plasma membrane glycine transporters in the vicinity of cholinergic neurons suggests a tight control of extracellular glycine in the BF.SIGNIFICANCE STATEMENT Basal forebrain cholinergic (BFC) neurons receive various activating inputs from specific brainstem areas and channel this information to the cortex via multiple projections. So far, very little is known about inhibitory brainstem afferents to the BF. The current study established glycine as a major regulator of BFC neurons by (1) identifying glycinergic neurons in the brainstem projecting to the BF, (2) showing glycine receptor α subunit-immunoreactive (IR) sites in choline acetyltransferase (ChAT)-IR neurons, (3) demonstrating glycine transporter type 2 (GLYT2)-positive axon terminals synapsing on ChAT-IR neurons, and (4) localizing GLYT1-positive astroglial processes in the vicinity of synapses of ChAT-IR neurons. The effect of glycine on BFC neurons was demonstrated by bicuculline-resistant, strychnine-sensitive spontaneous IPSCs recorded in whole-cell conditions.

Impact of Proestrus on Gene Expression in the Medial Preoptic Area of Mice.

The antero-ventral periventricular zone (AVPV) and medial preoptic area (MPOA) have been recognized as gonadal hormone receptive regions of the rodent brain that-via wiring to gonadotropin-releasing hormone (GnRH) neurons-contribute to orchestration of the preovulatory GnRH surge. We hypothesized that neural genes regulating the induction of GnRH surge show altered expression in proestrus. Therefore, we compared the expression of 48 genes obtained from intact proestrous and metestrous mice, respectively, by quantitative real-time PCR (qPCR) method. Differential expression of 24 genes reached significance (p < 0.05). Genes upregulated in proestrus encoded neuropeptides (kisspeptin (KP), galanin (GAL), neurotensin (NT), cholecystokinin (CCK)), hormone receptors (growth hormone secretagogue receptor, µ-opioid receptor), gonadal steroid receptors (estrogen receptor alpha (ERα), progesterone receptor (PR), androgen receptor (AR)), solute carrier family proteins (vesicular glutamate transporter 2, vesicular monoamine transporter 2), proteins of transmitter synthesis (tyrosine hydroxylase (TH)) and transmitter receptor subunit (AMPA4), and other proteins (uncoupling protein 2, nuclear receptor related 1 protein). Proestrus evoked a marked downregulation of genes coding for adenosine A2a receptor, vesicular gamma-aminobutyric acid (GABA) transporter, 4-aminobutyrate aminotransferase, tachykinin precursor 1, NT receptor 3, arginine vasopressin receptor 1A, cannabinoid receptor 1, ephrin receptor A3 and aldehyde dehydrogenase 1 family, member L1. Immunocytochemistry was used to visualize the proteins encoded by Kiss1, Gal, Cck and Th genes in neuronal subsets of the AVPV/MPOA of the proestrous mice. The results indicate that gene expression of the AVPV/MPOA is significantly modified at late proestrus including genes that code for neuropeptides, gonadal steroid hormone receptors and synaptic vesicle transporters. These events support cellular and neuronal network requirements of the positive estradiol feedback action and contribute to preparation of the GnRH neuron system for the pre-ovulatory surge release.