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

Abstract

GnRH-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms haven't been clarified. Here, we report thatin vivopharmacogenetic 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 the ACh receptor (AChR) agonist 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 mPSCs, indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (α4β2, α3β4, and α7) and muscarinic (M1-M5) ACh receptors. 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 miniature postsynaptic currents (mPSCs) and firing. 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. These findings confirm that the central cholinergic system immensely regulates GnRH neurons and activates the HPG-axis via ACh and ACh/GABA neurotransmissions in male mice.Significance statementCholinergic drugs influence reproduction centrally, although the exact neuronal targets and regulatory mechanisms remain unsettled. We found that pharmacogenetic activation of the cholinergic systemin vivoevoked an augmented LH release in male mice. The study also identified cholinergic cell groups in the mouse forebrain that innervate gonadotropin-releasing hormone (GnRH) neurons, the main hypothalamic regulators of reproduction. We also determined the subtypes of nicotinic and muscarinic receptors involved in neuronal information transfer and explored how their ligands affect the electrophysiological activity of GnRH neurons. A subset of cholinergic neurons was found to co-transmit GABA which excites GnRH cells via GABA-A receptors. The findings suggest a novel, cholinergic regulation of the GnRH system activating the pituitary-gonadal axis in adult male mice.