Ghrelin Decreases Firing Activity of Gonadotropin-Releasing Hormone (GnRH) Neurons in an Estrous Cycle and Endocannabinoid Signaling Dependent Manner.

The orexigenic peptide, ghrelin is known to influence function of GnRH neurons, however, the direct effects of the hormone upon these neurons have not been explored, yet. The present study was undertaken to reveal expression of growth hormone secretagogue receptor (GHS-R) in GnRH neurons and elucidate the mechanisms of ghrelin actions upon them. Ca(2+)-imaging revealed a ghrelin-triggered increase of the Ca(2+)-content in GT1-7 neurons kept in a steroid-free medium, which was abolished by GHS-R-antagonist JMV2959 (10µM) suggesting direct action of ghrelin. Estradiol (1nM) eliminated the ghrelin-evoked rise of Ca(2+)-content, indicating the estradiol dependency of the process. Expression of GHS-R mRNA was then confirmed in GnRH-GFP neurons of transgenic mice by single cell RT-PCR. Firing rate and burst frequency of GnRH-GFP neurons were lower in metestrous than proestrous mice. Ghrelin (40nM-4μM) administration resulted in a decreased firing rate and burst frequency of GnRH neurons in metestrous, but not in proestrous mice. Ghrelin also decreased the firing rate of GnRH neurons in males. The ghrelin-evoked alterations of the firing parameters were prevented by JMV2959, supporting the receptor-specific actions of ghrelin on GnRH neurons. In metestrous mice, ghrelin decreased the frequency of GABAergic mPSCs in GnRH neurons. Effects of ghrelin were abolished by the cannabinoid receptor type-1 (CB1) antagonist AM251 (1µM) and the intracellularly applied DAG-lipase inhibitor THL (10µM), indicating the involvement of retrograde endocannabinoid signaling. These findings demonstrate that ghrelin exerts direct regulatory effects on GnRH neurons via GHS-R, and modulates the firing of GnRH neurons in an ovarian-cycle and endocannabinoid dependent manner

A kisagy születés utáni fejlődésének plaszticitása = Postnatal development plasticity of the cerebellum

Vizsgáltuk a kisagy különböző sejttípusainak születés utáni vándorlását és integrálódását. A sejtek tér-időbeli eloszlásának részletes vizsgálatából következtettünk a kisagykéregre jellemző sejtes szerkezet kialakulásának dinamikájára. Megállapítottuk, hogy a Purkinje sejtek mintegy egyharmada, a Lugaro sejtek, az unipoláris ecsetsejtek és a molekuláris réteg interneuronjai csak a peri-és posztnatális fejlődési periódusban, és sajátságos, elhúzódó, többnyire hetekig tartó időintervallumban jelennek meg végleges helyükön. A kisagykéreg szerkezetének születés utáni kialakulása igen érzékeny különböző endogén vagy exogén ártalmakra, és jelentős mértékben megzavarható brómdezoxi uridin (BrdU) szisztémás alkalmazásával. A BrdU-t 50 ug/g testsúly dózisban alkalmazva a születés után 6 illetve 12 napon keresztül, a szomatikus és idegrendszeri fejlődés retardációja, illetve az idegrendszer károsodása figyelhető meg C56Bl6 egereken. A szemcsesejtek - BrdU össz-dózisától függő hánya - elpusztul, a Purkinje sejtek pedig nem képesek a ganglionáris monolayer-be rendeződni. Zavart szenved a szemcsesejtek, továbbá a kisagykéreg molekuláris rétegében található interneuronok születés utáni vándorlása is. A kisagykéreg morfológiai károsodását a mozgáskoordináció zavarai kísérik. Posztantális BrdU kezelés - kisebb mértékben - retardálja a szomatoszenzoros kéreg fejlődését is, ami a kérgi sejtek hiperexcitabilitásában, és fokozott "seizure" érzékenységében nyilvánul meg. | We have investigated the postnatal migration and integration of several cerebellocortical neuronal types, based on detailed spatio-temporal analysis of the distribution of the different neuronal populations. We concluded, that about one third of the Purkinje cells, the Lugaro cells, the unipolar brush cells as well as the interneurons of the molecular layer occupy their place in the cerebellocortical cytoarchitectonics during a prolonged perinatal-postnatal developmental period, which is characteristic of the given neuron. The postnatal build up of the cerebellar cortex is rather sensitive to different endogenous or exogenous noxas, and can be disturbed by systemic application of 5-bromo-2'- deoxyuridine (BrdU). Postnatal BrdU treatment by 50 ug/g b.w. BrdU for 6 and 12 days after birth results in the retardation of somatic and neuronal developmnet in C57Bl6 mice. Dose dependent proportion of granule cells is going to die, high number of the Purkinje cells remain in ectopic position and many more are not able to align into the ganglionic monolayer. Postnatal migration of cerebellar granule cells and the interneurons of the molecular layer are also disturbed. The morphological damage of the cerebellar cortex is followed by a disturbed movement-coordination. Postnatal BrdU application, in a lesser extent, results in the retardation of the development of somatosensory cortex too, followed by increased hyperexcitability and enhanced seizure susceptibility

The spatio-temporal segregation of GAD forms defines distinct GABA signaling functions in the developing mouse olfactory system and provides novel insights into the origin and migration of GnRH neurons.

GABA (gamma-aminobutyric acid) has a dual role as an inhibitory neurotransmitter in the adult central nervous system (CNS) and as a signaling molecule exerting largely excitatory actions during development. The rate-limiting step of GABA synthesis is catalyzed by two glutamic acid decarboxylase isoforms GAD65 and GAD67 co-expressed in the GABAergic neurons of the CNS. Here we report that the two GADs show virtually non-overlapping expression patterns consistent with distinct roles in the developing peripheral olfactory system. GAD65 is expressed exclusively in undifferentiated neuronal progenitors confined to the proliferative zones of the sensory vomeronasal and olfactory epithelia. In contrast GAD67 is expressed in a subregion of the non-sensory epithelium/vomeronasal organ epithelium containing the putative GnRH progenitors and GnRH neurons migrating from this region through the frontonasal mesenchyme (FNM) into the basal forebrain. Only GAD67+, but not GAD65+ cells accumulate detectable GABA. We further demonstrate that GAD67 and its embryonic splice variant EGAD concomitant with GnRH are dynamically regulated during GnRH neuronal migration in vivo and in two immortalized cell lines representing migratory (GN11) and post-migratory (GT1-7) stage GnRH neurons, respectively. Analysis of GAD65/67 single and double knock-out (KO) embryos revealed that the two GADs play complementary (inhibitory) roles in GnRH migration ultimately modulating the speed and/or direction of GnRH migration. Our results also suggest that GAD65 and GAD67/EGAD characterized by distinct subcellular localization and kinetics have disparate functions during olfactory system development mediating proliferative and migratory responses putatively through specific subcellular GABA pools. (c) 2014 Wiley Periodicals, Inc. Develop Neurobiol, 2014

Altered Gene Expression Profile of the Hypothalamic Arcuate Nucleus of Male Mice Suggests 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. Altered peptidergic and amino acidergic neurotransmission accompanying pubertal maturation of the ARC are 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 day-14 infantile and day-60 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 were compared between the two timepoints. Adult animals showed increased mRNA levels encoding cocaine- and amphetamine-regulated transcript, galanin-like peptide, dynorphin, kisspeptin, proopiomelanocortin, proenkephalin and galanin and 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 (5-fold) increase. Melanocortin receptor-3 and the Y1 and Y5 neuropeptide Y receptors increased 1.5-1.8-fold, whereas delta-opioid receptor and neurotensin receptor-1 transcripts were reduced by 27 and 21%, respectively. Androgen-, progesterone- and alpha-estrogen receptor transcripts increased by 54-72%. The mRNAs of glutamic acid decarboxylase 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 reduced inhibitory and/or enhanced excitatory neuropeptidergic drive on fertility in adult animals. (c) 2015 S. Karger AG, Basel

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

Glucagon-Like Peptide-1 Excites Firing and Increases GABAergic Miniature Postsynaptic Currents (mPSCs) in Gonadotropin-Releasing Hormone (GnRH) Neurons of the Male Mice via Activation of Nitric Oxide (NO) and Suppression of Endocannabinoid Signaling Pathways

Glucagon-like peptide-1 (GLP-1), a metabolic signal molecule, regulates reproduction, although, the involved molecular mechanisms have not been elucidated, yet. Therefore, responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the GLP-1 analog Exendin-4 and elucidation of molecular pathways acting downstream to the GLP-1 receptor (GLP-1R) have been challenged. Loose patch-clamp recordings revealed that Exendin-4 (100 nM-5 μM) elevated firing rate in hypothalamic GnRH-GFP neurons of male mice via activation of GLP-1R. Whole-cell patch-clamp measurements demonstrated increased excitatory GABAergic miniature postsynaptic currents (mPSCs) frequency after Exendin-4 administration, which was eliminated by the GLP-1R antagonist Exendin-3(9-39) (1 μM). Intracellular application of the G-protein inhibitor GDP-β-S (2 mM) impeded action of Exendin-4 on mPSCs, suggesting direct excitatory action of GLP-1 on GnRH neurons. Blockade of nitric-oxide (NO) synthesis by Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME; 100 μM) or N(5)-[Imino(propylamino)methyl]-L-ornithine hydrochloride (NPLA; 1 μM) or intracellular scavenging of NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO; 1 mM) partially attenuated the excitatory effect of Exendin-4. Similar partial inhibition was achieved by hindering endocannabinoid pathway using cannabinoid receptor type-1 (CB1) inverse-agonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl) pyrazole-3-carboxamide (AM251; 1 μM). Simultaneous blockade of NO and endocannabinoid signaling mechanisms eliminated action of Exendin-4 suggesting involvement of both retrograde machineries. Intracellular application of the transient receptor potential vanilloid 1 (TRPV1)-antagonist 2E-N-(2, 3-Dihydro-1,4-benzodioxin-6-yl)-3-[4-(1, 1-dimethylethyl)phenyl]-2-Propenamide (AMG9810; 10 μM) or the fatty acid amide hydrolase (FAAH)-inhibitor PF3845 (5 μM) impeded the GLP-1-triggered endocannabinoid pathway indicating an anandamide-TRPV1-sensitive control of 2-arachidonoylglycerol (2-AG) production. Furthermore, GLP-1 immunoreactive (IR) axons innervated GnRH neurons in the hypothalamus suggesting that GLP-1 of both peripheral and neuronal sources can modulate GnRH neurons. RT-qPCR study confirmed the expression of GLP-1R and neuronal NO synthase (nNOS) mRNAs in GnRH-GFP neurons. Immuno-electron microscopic analysis revealed the presence of nNOS protein in GnRH neurons. These results indicate that GLP-1 exerts direct facilitatory actions via GLP-1R on GnRH neurons and modulates NO and 2-AG retrograde signaling mechanisms that control the presynaptic excitatory GABAergic inputs to GnRH neurons

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-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

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 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-GFP 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 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 the better understanding the differences in cellular mechanisms of GnRH neurons in the two sexes. (c) 2015 S. Karger AG, Basel