Cholinergic Control of GnRH Neuron Physiology and Luteinizing Hormone Secretion in Male Mice: Involvement of ACh/GABA Cotransmission

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clari fi ed. Here, we report that in vivo pharmacogenetic 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 identi fi ed 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 carbachol evoked a biphasic effect on the fi ring rate in GnRH neurons, fi rst increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of miniature postsynaptic currents (mPSCs), indicating a direct in fl uence on GnRH cells. RT-PCR and whole -cell patch -clamp studies revealed that GnRH neurons expressed both nicotinic ( alpha 4 (3 2, alpha 3 (3 4, and alpha 7) and muscarinic (M1 - M5) AChRs. The nicotinic AChRs contributed to the nicotine -elicited inward current and the rise in fi ring rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both mPSCs and fi ring. Optogenetic activation of channelrhodopsin-2 - tagged cholinergic axons modi fi ed GnRH neuronal activity and evoked cotransmission of ACh and GABA from a subpopulation of boutons. These fi ndings con fi rm that the central cholinergic system regulates GnRH neurons and activates the pituitary - gonadal axis via ACh and ACh/GABA neurotransmissions in male mice.