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.

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.

[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.

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.

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.

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.

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.

Ovariectomy Alters Gene Expression of the Hippocampal Formation in Middle-Aged Rats.

Ovarian hormones regulate the transcriptome of the hippocampus and modulate its functions. During menopause this complex signaling declines, leading to impaired learning and memory. This study was undertaken to clarify the effects of long-term, surgical ovariectomy (OVX) on the rat hippocampal transcriptome. At age of 13 months, intact control and ovariectomized groups were formed. All animals were killed 5 weeks after gonadectomy; hippocampal formations were dissected and processed for transcriptome analysis. Microarray and polymerase chain reaction studies identified 252 and 61 genes, respectively, whose expression was altered in the lack of ovarian hormones. Pathway analysis revealed impact on neuroactive ligand-receptor interaction, endocannabinoid, and estrogen signaling, among others. Network and interaction analyses of proteins encoded by OVX-regulated genes revealed upregulation of growth/troph/transcription factor signaling assembly (Mdk, Fgf1, Igf2, Ngf, Ngfr, Ntf3, Ntrk1, Otx2, Hif1a, Esr1, Nr4a3), peptides/peptide receptors (Cartpt, Kl, Ttr, Gnrhr), neurotransmission (Grm1, Gria4, Gls, Slc18a2, Kcnj6), and genes serving immune functions (C3, Ccl2, Itgam, Il1b). Downregulated clusters included neuropeptides and their receptors (Adcyap1, Cbln2, Cck, Cckbr, Crhr1 and 2, Oprd1, Nts, Penk, Sstr1, Vip), neurotransmitter signaling (Htr2c, Chrna3, Chrm4, Grm8, Hrh3, Slc17a6), and potassium channels (Kcnk9, Kcnj9, Kcnma1, Kcnc2). Several transcription factors (Rxra, Thrb), solute carriers and defense molecules (Apitd1, Bcl2, C1ql3, Ilr3a, Sod1, Sncb) also underwent downregulation. The findings indicate that surgical gonadectomy carried out at middle-age robustly changes the hippocampal transcriptome that alters neurogenesis, synaptic plasticity, immune modulation, causing cognitive dysfunctions.

Altered Expression of Genes Encoding Neurotransmitter Receptors in GnRH Neurons of Proestrous Mice.

Gonadotropin-releasing hormone (GnRH) neurons play a key role in the central regulation of reproduction. In proestrous female mice, estradiol triggers the pre-ovulatory GnRH surge, however, its impact on the expression of neurotransmitter receptor genes in GnRH neurons has not been explored yet. We hypothesized that proestrus is accompanied by substantial changes in the expression profile of genes coding for neurotransmitter receptors in GnRH neurons. We compared the transcriptome of GnRH neurons obtained from intact, proestrous, and metestrous female GnRH-GFP transgenic mice, respectively. About 1500 individual GnRH neurons were sampled from both groups and their transcriptome was analyzed using microarray hybridization and real-time PCR. In this study, changes in mRNA expression of genes involved in neurotransmitter signaling were investigated. Differential gene expression was most apparent in GABA-ergic (Gabbr1, Gabra3, Gabrb3, Gabrb2, Gabrg2), glutamatergic (Gria1, Gria2, Grin1, Grin3a, Grm1, Slc17a6), cholinergic (Chrnb2, Chrm4) and dopaminergic (Drd3, Drd4), adrenergic (Adra1b, Adra2a, Adra2c), adenosinergic (Adora2a, Adora2b), glycinergic (Glra), purinergic (P2rx7), and serotonergic (Htr1b) receptors. In concert with these events, expression of genes in the signaling pathways downstream to the receptors, i.e., G-proteins (Gnai1, Gnai2, Gnas), adenylate-cyclases (Adcy3, Adcy5), protein kinase A (Prkaca, Prkacb) protein kinase C (Prkca) and certain transporters (Slc1a4, Slc17a6, Slc6a17) were also changed. The marked differences found in the expression of genes involved in neurotransmitter signaling of GnRH neurons at pro- and metestrous stages of the ovarian cycle indicate the differential contribution of these neurotransmitter systems to the induction of the pre-ovulatory GnRH surge, the known prerequisite of the subsequent hormonal cascade inducing ovulation.

Hypothalamic kappa opioid receptor mediates both diet-induced and melanin concentrating hormone-induced liver damage through inflammation and endoplasmic reticulum stress.

UNLABELLED: The opioid system is widely known to modulate the brain reward system and thus affect the behavior of humans and other animals, including feeding. We hypothesized that the hypothalamic opioid system might also control energy metabolism in peripheral tissues. Mice lacking the kappa opioid receptor (κOR) and adenoviral vectors overexpressing or silencing κOR were stereotaxically delivered in the lateral hypothalamic area (LHA) of rats. Vagal denervation was performed to assess its effect on liver metabolism. Endoplasmic reticulum (ER) stress was inhibited by pharmacological (tauroursodeoxycholic acid) and genetic (overexpression of the chaperone glucose-regulated protein 78 kDa) approaches. The peripheral effects on lipid metabolism were assessed by histological techniques and western blot. We show that in the LHA κOR directly controls hepatic lipid metabolism through the parasympathetic nervous system, independent of changes in food intake and body weight. κOR colocalizes with melanin concentrating hormone receptor 1 (MCH-R1) in the LHA, and genetic disruption of κOR reduced melanin concentrating hormone-induced liver steatosis. The functional relevance of these findings was given by the fact that silencing of κOR in the LHA attenuated both methionine choline-deficient, diet-induced and choline-deficient, high-fat diet-induced ER stress, inflammation, steatohepatitis, and fibrosis, whereas overexpression of κOR in this area promoted liver steatosis. Overexpression of glucose-regulated protein 78 kDa in the liver abolished hypothalamic κOR-induced steatosis by reducing hepatic ER stress. CONCLUSIONS: This study reveals a novel hypothalamic-parasympathetic circuit modulating hepatic function through inflammation and ER stress independent of changes in food intake or body weight; these findings might have implications for the clinical use of opioid receptor antagonists. (Hepatology 2016;64:1086-1104).

Long-Term Estrogen Receptor Beta Agonist Treatment Modifies the Hippocampal Transcriptome in Middle-Aged Ovariectomized Rats.

Estradiol (E2) robustly activates transcription of a broad array of genes in the hippocampal formation of middle-aged ovariectomized rats via estrogen receptors (ERα, ERß, and G protein-coupled ER). Selective ERß agonists also influence hippocampal functions, although their downstream molecular targets and mechanisms are not known. In this study, we explored the effects of long-term treatment with ERß agonist diarylpropionitrile (DPN, 0.05 mg/kg/day, sc.) on the hippocampal transcriptome in ovariectomized, middle-aged (13 month) rats. Isolated hippocampal formations were analyzed by Affymetrix oligonucleotide microarray and quantitative real-time PCR. Four hundred ninety-seven genes fulfilled the absolute fold change higher than 2 (FC > 2) selection criterion. Among them 370 genes were activated. Pathway analysis identified terms including glutamatergic and cholinergic synapse, RNA transport, endocytosis, thyroid hormone signaling, RNA degradation, retrograde endocannabinoid signaling, and mRNA surveillance. PCR studies showed transcriptional regulation of 58 genes encoding growth factors (Igf2, Igfb2, Igf1r, Fgf1, Mdk, Ntf3, Bdnf), transcription factors (Otx2, Msx1), potassium channels (Kcne2), neuropeptides (Cck, Pdyn), peptide receptors (Crhr2, Oprm1, Gnrhr, Galr2, Sstr1, Sstr3), neurotransmitter receptors (Htr1a, Htr2c, Htr2a, Gria2, Gria3, Grm5, Gabra1, Chrm5, Adrb1), and vesicular neurotransmitter transporters (Slc32a1, Slc17a7). Protein-protein interaction analysis revealed networking of clusters associated with the regulation of growth/troph factor signaling, transcription, translation, neurotransmitter and neurohormone signaling mechanisms and potassium channels. Collectively, the results reveal the contribution of ERß-mediated processes to the regulation of transcription, translation, neurogenesis, neuromodulation, and neuroprotection in the hippocampal formation of ovariectomized, middle-aged rats and elucidate regulatory channels responsible for DPN-altered functional patterns. These findings support the notion that selective activation of ERß may be a viable approach for treating the neural symptoms of E2 deficiency in menopause.

Estrogen Receptor Beta and 2-arachidonoylglycerol Mediate the Suppressive Effects of Estradiol on Frequency of Postsynaptic Currents in Gonadotropin-Releasing Hormone Neurons of Metestrous Mice: An Acute Slice Electrophysiological Study.

Gonadotropin-releasing hormone (GnRH) neurons are controlled by 17ß-estradiol (E2) contributing to the steroid feedback regulation of the reproductive axis. In rodents, E2 exerts a negative feedback effect upon GnRH neurons throughout the estrus-diestrus phase of the ovarian cycle. The present study was undertaken to reveal the role of estrogen receptor subtypes in the mediation of the E2 signal and elucidate the downstream molecular machinery of suppression. The effect of E2 administration at low physiological concentration (10 pM) on GnRH neurons in acute brain slices obtained from metestrous GnRH-green fluorescent protein (GFP) mice was studied under paradigms of blocking or activating estrogen receptor subtypes and interfering with retrograde 2-arachidonoylglycerol (2-AG) signaling. Whole-cell patch clamp recordings revealed that E2 significantly diminished the frequency of spontaneous postsynaptic currents (sPSCs) in GnRH neurons (49.62 ± 7.6%) which effect was abolished by application of the estrogen receptor (ER) α/ß blocker Faslodex (1 µM). Pretreatment of the brain slices with cannabinoid receptor type 1 (CB1) inverse agonist AM251 (1 µM) and intracellularly applied endocannabinoid synthesis blocker THL (10 µM) significantly attenuated the effect of E2 on the sPSCs. E2 remained effective in the presence of tetrodotoxin (TTX) indicating a direct action of E2 on GnRH cells. The ERß specific agonist DPN (10 pM) also significantly decreased the frequency of miniature postsynaptic currents (mPSCs) in GnRH neurons. In addition, the suppressive effect of E2 was completely blocked by the selective ERß antagonist PHTPP (1 µM) indicating that ERß is required for the observed rapid effect of the E2. In contrast, the ERα agonist PPT (10 pM) or the membrane-associated G protein-coupled estrogen receptor (GPR30) agonist G1 (10 pM) had no significant effect on the frequency of mPSCs in these neurons. AM251 and tetrahydrolipstatin (THL) significantly abolished the effect of E2 whereas AM251 eliminated the action of DPN on the mPSCs. These data suggest the involvement of the retrograde endocannabinoid mechanism in the rapid direct effect of E2. These results collectively indicate that estrogen receptor beta and 2-AG/CB1 signaling mechanisms are coupled and play an important role in the mediation of the negative estradiol feedback on GnRH neurons in acute slice preparation obtained from intact, metestrous mice.

Altered Gene Expression Profiles of the Hypothalamic Arcuate Nucleus of Male Mice Suggest Profound Developmental Changes in Peptidergic Signaling.

Neuropeptides of the hypothalamic arcuate nucleus (ARC) regulate important homeostatic and endocrine functions and also play critical roles in pubertal development. The altered peptidergic and aminoacidergic neurotransmission accompanying pubertal maturation of the ARC is not fully understood. Here we studied the developmental shift in the gene expression profile of the ARC of male mice. RNA samples for quantitative RT-PCR studies were isolated from the ARC of 14-day-old infantile and 60-day-old adult male mice with laser capture microdissection. The expression of 18 neuropeptide, 15 neuropeptide receptor, 4 sex steroid receptor and 6 classic neurotransmitter marker mRNAs was compared between the two time points. The adult animals showed increased mRNA levels encoding cocaine- and amphetamine-regulated transcripts, galanin-like peptide, dynorphin, kisspeptin, proopiomelanocortin, proenkephalin and galanin and a reduced expression of mRNAs for pituitary adenylate cyclase-activating peptide, calcitonin gene-related peptide, neuropeptide Y, substance P, agouti-related protein, neurotensin and growth hormone-releasing hormone. From the neuropeptide receptors tested, melanocortin receptor-4 showed the most striking increase (5-fold). Melanocortin receptor-3 and the Y1 and Y5 neuropeptide Y receptors increased 1.5- to 1.8-fold, whereas δ-opioid receptor and neurotensin receptor-1 transcripts were reduced by 27 and 21%, respectively. Androgen receptor, progesterone receptor and α-estrogen receptor transcripts increased by 54-72%. The mRNAs of glutamic acid decarboxylases-65 and -67, vesicular GABA transporter and choline acetyltransferase remained unchanged. Tyrosine hydroxylase mRNA increased by 44%, whereas type-2 vesicular glutamate transporter mRNA decreased by 43% by adulthood. Many of the developmental changes we revealed in this study suggest a reduced inhibitory and/or enhanced excitatory neuropeptidergic drive on fertility in adult animals.

Differential Gene Expression in Gonadotropin-Releasing Hormone Neurons of Male and Metestrous Female Mice.

BACKGROUND: Gonadotropin-releasing hormone (GnRH) neurons play a pivotal role in the regulation of the hypothalamic-pituitary gonadal axis in a sex-specific manner. We hypothesized that the differences seen in reproductive functions of males and females are associated with a sexually dimorphic gene expression profile of GnRH neurons. METHODS AND RESULTS: We compared the transcriptome of GnRH neurons obtained from intact metestrous female and male GnRH-green fluorescent protein transgenic mice. About 1,500 individual GnRH neurons from each sex were sampled with laser capture microdissection followed by whole-transcriptome amplification for gene expression profiling. Under stringent selection criteria (fold change >1.6, adjusted p value 0.01), Affymetrix Mouse Genome 430 PM array analysis identified 543 differentially expressed genes. Sexual dimorphism was most apparent in gene clusters associated with synaptic communication, signal transduction, cell adhesion, vesicular transport and cell metabolism. To validate microarray results, 57 genes were selected, and 91% of their differential expression was confirmed by real-time PCR. Similarly, 88% of microarray results were confirmed with PCR from independent samples obtained by patch pipette harvesting and pooling of 30 GnRH neurons from each sex. We found significant differences in the expression of genes involved in vesicle priming and docking (Syt1, Cplx1), GABAergic (Gabra3, Gabrb3, Gabrg2) and glutamatergic (Gria1, Grin1, Slc17a6) neurotransmission, peptide signaling (Sstr3, Npr2, Cxcr4) and the regulation of intracellular ion homeostasis (Cacna1, Cacnb1, Cacng5, Kcnq2, Kcnc1). CONCLUSION: The striking sexual dimorphism of the GnRH neuron transcriptome we report here contributes to a better understanding of the differences in cellular mechanisms of GnRH neurons in the two sexes.

Hippocampal Gene Expression Is Highly Responsive to Estradiol Replacement in Middle-Aged Female Rats.

In the hippocampus, estrogens are powerful modulators of neurotransmission, synaptic plasticity and neurogenesis. In women, menopause is associated with increased risk of memory disturbances, which can be attenuated by timely estrogen therapy. In animal models of menopause, 17ß-estradiol (E2) replacement improves hippocampus-dependent spatial memory. Here, we explored the effect of E2 replacement on hippocampal gene expression in a rat menopause model. Middle-aged ovariectomized female rats were treated continuously for 29 days with E2, and then, the hippocampal transcriptome was investigated with Affymetrix expression arrays. Microarray data were analyzed by Bioconductor packages and web-based softwares, and verified with quantitative PCR. At standard fold change selection criterion, 156 genes responded to E2. All alterations but 4 were transcriptional activation. Robust activation (fold change > 10) occurred in the case of transthyretin, klotho, claudin 2, prolactin receptor, ectodin, coagulation factor V, Igf2, Igfbp2, and sodium/sulfate symporter. Classification of the 156 genes revealed major groups, including signaling (35 genes), metabolism (31 genes), extracellular matrix (17 genes), and transcription (16 genes). We selected 33 genes for further studies, and all changes were confirmed by real-time PCR. The results suggest that E2 promotes retinoid, growth factor, homeoprotein, neurohormone, and neurotransmitter signaling, changes metabolism, extracellular matrix composition, and transcription, and induces protective mechanisms via genomic effects. We propose that these mechanisms contribute to effects of E2 on neurogenesis, neural plasticity, and memory functions. Our findings provide further support for the rationale to develop safe estrogen receptor ligands for the maintenance of cognitive performance in postmenopausal women.