Cholinergic control of GnRH neuron physiology and luteinizing hormone secretion: involvement of ACh/GABA co-transmission

Abstract

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. They receive commands from various neuronal networks for regulation of the hypothalamo-pituitary-gonadal (HPG)-axis. Here we report acetylcholine (ACh) as a critical regulator of GnRH neurons and reproduction in mice. In acute brain slice preparation, the acetylcholine receptor (AChR) agonists, ACh and carbachol, evoked a biphasic effect on the firing rate in GnRH neurons, first increasing afterwards diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current followed by a decline in frequency of mPSCs. RT-PCR and whole cell patch-clamp studies revealed that GnRH neurons expressed both muscarinic (M1-M5) and nicotinic (α4β2, α3β4 and α7) ACh receptors. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both miniature postsynaptic currents (mPSCs) and firing. The identified nicotinic AChRs contributed to the nicotine-elicited inward current and the rise of firing rate. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked co-transmission of ACh and GABA from a subpopulation of boutons. 3DISCO immunocytochemistry and electron microscopy revealed innervation of GnRH neurons by cholinergic axons. Rabies retrograde viral labelling started from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for specific cholinergic afferents.In vivopharmacogenetic activation of cholinergic system resulted in an increased secretion of luteinizing hormone (LH) in gonadectomized mice. Our findings collectively corroborate that the central cholinergic system exerts an important regulatory influence upon GnRH neurons and activates the HPG-axis via ACh and ACh/GABA neurotransmissions.Graphical abstract