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.

Farkas, Imre and Vastagh, Csaba and Farkas, Erzsébet and Bálint, Flóra and Skrapits, Katalin and Hrabovszky, Erik and Fekete, Csaba and Liposits, Zsolt (2016) 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. Frontiers in cellular neuroscience, 10. p. 1. ISSN 1662-5102, ESSN: 1662-5102

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Abstract

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.

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