Liposits, Z. | bioRxiv Science

bioRxiv2026

Brain-wide mapping of proglucagon expression in mice identifies fasting-responsive GLP-1 neurons in the posterior hypothalamic nucleus

ObjectiveGlucagon-like peptide-1 (GLP-1), a peptide neurotransmitter in the brain, is synthesized from proglucagon, encoded by the glucagon gene (Gcg). Besides medullary GLP-1 neurons, Gcg-expressing neuron populations were identified in the olfactory bulb and basolateral amygdala. However, several lines of evidence suggest that additional Gcg neuron populations might exist. MethodsWe conducted a brain-wide mapping of Gcg-expressing cells by fluorescent in situ hybridization in C57BL/6J and FVB/Ant mice. Proglucagon and GLP-1 expression were studied with immunofluorescence. We characterized a Gcg-Cre;tdTomato mouse line and studied the expression of proglucagon-processing enzymes in Gcg-expressing neuron populations. We used adeno-associated virus-mediated tracing in Gcg-Cre mice to map the projections of hypothalamic Gcg neurons. ResultsGcg-expressing neuron populations were identified in the olfactory bulb, claustrum, piriform cortex, basolateral amygdala, posterior hippocampus, posterior hypothalamic nucleus (PH), periaqueductal gray/dorsal raphe, and dorsal nucleus of the lateral lemniscus. These neurons express lower Gcg mRNA levels than medullary GLP-1 neurons. Proglucagon and GLP-1-immunoreactivity (C-terminus) were detected in almost all Gcg-expressing neuron populations, along with the mRNAs for prohormone convertases 1/3 and 2, enzymes generating GLP-1 or glucagon, respectively. Fasting markedly increased Gcg mRNA, proglucagon and GLP-1 synthesis in the PH. PH Gcg neurons project densely to the ventral and intermediate lateral septum, preoptic region, ventrolateral preoptic nucleus, lateral hypothalamus and zona incerta, establishing close contacts with both GLP-1 receptor-positive and -negative neurons. ConclusionsProglucagon is expressed in 9 distinct neuron populations. Feeding status regulates GLP-1 synthesis in PH neurons that likely control feeding- or energy balance-related functions.

neuroscience↗

bioRxiv2023

Central cholinergic regulation of the GnRH system: involvement of ACh/GABA co-transmission

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 clarified. 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 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 (4{beta}2, 3{beta}4, and 7) 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. Significance statementCholinergic drugs influence reproduction centrally, although the exact neuronal targets and regulatory mechanisms remain unsettled. We found that pharmacogenetic activation of the cholinergic system in vivo evoked an augmented LH release. 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 transmission 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 adult GnRH system in male mice that activates the pituitary-gonadal axis.

physiology↗