Csaba Vastagh

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. Ca2+-imaging revealed a ghrelin-triggered increase of the Ca2+-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 Ca2+-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.

N-Methyl-d-aspartate (NMDA) Receptor Composition Modulates Dendritic Spine Morphology in Striatal Medium Spiny Neurons

Background: An interplay between dopamine (DA) and NMDA receptors in striatum is essential to drive motor behavior. Results: NR2A antagonist induces an increase of spine head width as induced by D1 activation. Conclusion: NMDA receptor subunit composition regulates dendritic spine morphology in MSNs. Significance: Therapies targeted to modulate NMDA receptor subunits may lead to a morphological outcome in dendritic spines of MSNs. Dendritic spines of medium spiny neurons represent an essential site of information processing between NMDA and dopamine receptors in striatum. Even if activation of NMDA receptors in the striatum has important implications for synaptic plasticity and disease states, the contribution of specific NMDA receptor subunits still remains to be elucidated. Here, we show that treatment of corticostriatal slices with NR2A antagonist NVP-AAM077 or with NR2A blocking peptide induces a significant increase of spine head width. Sustained treatment with D1 receptor agonist (SKF38393) leads to a significant decrease of NR2A-containing NMDA receptors and to a concomitant increase of spine head width. Interestingly, co-treatment of corticostriatal slices with NR2A antagonist (NVP-AAM077) and D1 receptor agonist augmented the increase of dendritic spine head width as obtained with SKF38393. Conversely, NR2B antagonist (ifenprodil) blocked any morphological effect induced by D1 activation. These results indicate that alteration of NMDA receptor composition at the corticostriatal synapse contributes not only to the clinical features of disease states such as experimental parkinsonism but leads also to a functional and morphological outcome in dendritic spines of medium spiny neurons.

Postnatal development of unipolar brush cells in the cerebellar cortex of cat

The postnatal developmental distribution pattern of metabotropic glutamate receptor (mGluR1a) immunoreactive unipolar brush cells (UBCs) was studied in the cerebellar cortex of kittens. On the day of birth (P0) UBCs are already present in the white matter in lobule X of the vermis, but only a few of these cell seemed to migrate to the deeper region of the internal granular layer. By the end of the first week (P8) UBCs were seen to invade the white matter + internal granular layer of lobules IX, VIII, I, and II of the vermis, and they spread further in the transitory area medio‐laterally from the vermis toward the cerebellar hemispheres. By P15, UBCs appeared in lobules III and VII of the vermis, as well as in corresponding lobules of the neocerebellum, with especially high numbers in lobule VII. By P22, UBCs migrated further after their medio‐lateral course in the neocerebellum, and began to invade lobules V and VI. At P62 the amount of UBCs in midsagittal planes of early developing vermal lobules (I, II, VII–X) resembled the P132 or adult pattern. The medio‐lateral migration and incorporation of UBCs into the late‐developing cerebellar lobules V and VI was completed only by P132, when the spatial distribution of UBCs in both the vermal and neocerebellar lobules was comparable to that seen in the 1 year old young adult cat. Although by P132 the postnatal migration of the vast majority of UBCs seemed to be completed, in the cerebellum of adult cats a few migrating UBCs could still be observed in the white matter of the cerebellar lobules, and beneath the ependyma of the fourth ventricle. It is concluded that during ontogenesis the migration course of UBCs follows essentially the developmental sequence of cerebellar lobules, although the incorporation of UBCs into the internal granular layer continues until 4 months postnatally, i.e., much beyond the apparent completion (about two months postnatally) of cytoarchitectonic built up of the cerebellar cortex of kittens. J. Neurosci. Res. 61:107–115, 2000.

Estradiol and isotype-selective estrogen receptor agonists modulate the mesocortical dopaminergic system in gonadectomized female rats

The mesocortical dopaminergic pathway projecting from the ventral tegmental area (VTA) to the prefrontal cortex (PFC) contributes to the processing of reward signals. This pathway is regulated by gonadal steroids including estradiol. To address the putative role of estradiol and isotype-selective estrogen receptor (ER) agonists in the regulation of the rodent mesocortical system, we combined fMRI, HPLC-MS and qRT-PCR techniques. In fMRI experiments adult, chronically ovariectomized rats, treated with either vehicle, estradiol, ERα agonist 16α-lactone-estradiol (LE2) or ERβ agonist diarylpropionitrile (DPN), received a single dose of d-amphetamine-sulphate (10mg/kg, i.p.) and BOLD responses were monitored in the VTA and the PFC. Ovariectomized rats showed no significant response to amphetamine. In contrast, the VTA of ER agonist-substituted ovariectomized rats showed robust amphetamine-evoked BOLD increases. The PFC of estradiol-replaced animals was also responsive to amphetamine. Mass spectroscopic analysis of dopamine and its metabolites revealed a two-fold increase in both dopamine and 3,4-dihydroxyphenylacetic acid content of the PFC in estradiol-replaced animals compared to ovariectomized controls. qRT-PCR studies revealed upregulation of dopamine transporter and dopamine receptor in the VTA and PFC, respectively, of ER agonist-treated ovariectomized animals. Collectively, the results indicate that E2 and isotype-selective ER agonists can powerfully modulate the responsiveness of the mesocortical dopaminergic system, increase the expression of key genes related to dopaminergic neurotransmission and augment the dopamine content of the PFC. In a broader sense, the findings support the concept that the manifestation of reward signals in the PFC is dependent on the actual estrogen milieu of the brain.

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, 17β-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.

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 N5-[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.

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.

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.

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.

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.