Abstract
Neurons synthesizing gonadotropin-inhibitory hormone (GnIH) have been implicated in the control of reproduction, food intake and stress. Serotonin (5-HT) receptors have been shown in GnIH neurons; however, their functional role in the regulation of GnIH neurons remains to be elucidated. In this study, we measured intracellular calcium ion levels following 5-HT treatment to hypothalamic primary cultures of enhanced fluorescent green protein-tagged GnIH (EGFP-GnIH) neurons from Wistar rat pups of mixed sex. Three days after initial seeding of the primary cultures, the test groups were pre-treated with lithium chloride to selectively inhibit glycogen synthase kinase 3 beta to promote intracellular calcium levels, whereas the control groups received culture medium with no lithium chloride treatment. 24 h later, the cultures were incubated with rhodamine-2AM (rhod-2AM) calcium indicator dye for one hour prior to imaging. 5-HT was added to the culture dishes 5 min after commencement of imaging. Analysis of intracellular calcium levels in EGFP-GnIH neurons showed that pre-treatment with lithium chloride before 5-HT treatment resulted in significant increase in intracellular calcium levels, two times higher than the baseline. This suggests that lithium chloride enhances the responsiveness of GnIH neurons to 5-HT.
Highlights
Neurons synthesizing gonadotropin-inhibitory hormone (GnIH) have been implicated in the control of reproduction, food intake and stress
The function of GnIH has been highly conserved across vertebrates, acting to inhibit gonadotropin-releasing hormone (GnRH)-mediated gonadotrophin release with some exceptions found in teleosts[1,4,5,6,7,8]
Despite the presence of 5-HT receptors in GnIH neurons[14], the effect of 5-HT on the internal dynamics of GnIH neurons remains to be investigated, which we hypothesize is through the GSK3β-Wnt signaling pathway
Summary
Neurons synthesizing gonadotropin-inhibitory hormone (GnIH) have been implicated in the control of reproduction, food intake and stress. Analysis of intracellular calcium levels in EGFP-GnIH neurons showed that pre-treatment with lithium chloride before 5-HT treatment resulted in significant increase in intracellular calcium levels, two times higher than the baseline. This suggests that lithium chloride enhances the responsiveness of GnIH neurons to 5-HT. The objective of this study was to treat rat EGFP-GnIH neurons in hypothalamic primary cultures with 5-HT and selectively inhibit GSK3β using lithium chloride (LiCl) to observe change in the activity of EGFP-GnIH neurons using calcium imaging. The elucidation of GSK3β-Wnt signaling pathway, by which 5-HT regulates GnIH, will provide a new avenue for future studies to explore the complexity of stress-induced reproductive dysfunction
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