FRI277 Temporal Coordination Of Female Reproductive Axis Activity By Vasoactive Intestinal Polypeptide (VIP) Expressing Cells In The Suprachiasmatic Nucleus

Abstract

Abstract Disclosure: C. Phumsatitpong: None. S.R. Bever: None. S.L. Hu: None. M.C. Ambrose: None. L.J. Kriegsfeld: None. Circadian rhythms orchestrated by a master clock in the suprachiasmatic nucleus (SCN) are essential for ovulation in spontaneously-ovulating rodents. SCN signaling integrates with estradiol positive feedback to generate the preovulatory luteinizing hormone (LH) surge. Additionally, subordinate clocks in reproductively-relevant SCN targets temporally gate responsiveness to upstream signaling to coordinate ovulation with the active time of day. We have previously shown that the SCN communicates directly to kisspeptin (Kp) and RFamide-related peptide-3 (RFRP-3) neurons to temporally balance key stimulatory and inhibitory input to GnRH neurons required for LH surge generation. Because VIP-expressing neurons in the SCN have been implicated in the LH surge, we employed a cell-specific chemogenetic approach to determine the functional role of this cell phenotype in female reproductive axis regulation required for surge generation. We hypothesized that temporal gating by subordinate cellular clocks will prevent morning stimulation of VIP-expressing cells from increasing Kp and GnRH, and decreasing RFRP-3, cellular activity. In the afternoon, we expected inhibition of VIP-expressing cells to decrease Kp and GnRH, and increase RFRP-3, cellular activity. Specifically, female VIP-Cre mice were stereotaxically injected with either AAV-hM3Dq or hM4Di to express activating (3Dq) or inhibitory (4Di) DREADDs in VIP-expressing SCN cells. Following recovery, mice were injected clozapine n-oxide (CNO) either in the morning, prior to the LH surge (3Dq experiment), or the afternoon, around the time of the LH surge (4Di experiment). Brains were collected to examine neuronal activation in Kp, GnRH, and RFRP-3 neurons using FOS immunofluorescence as a marker. Morning stimulation of SCN VIP neurons decreased RFRP-3 neuronal activation, whereas Kp and GnRH neurons were unaffected at this time, suggesting a temporal gating mechanism at GnRH and/or Kp cells prevents premature ovulation. In contrast, afternoon inhibition of SCN VIP-expressing neurons led to an increase in neuronal activation in RFRP-3 neurons concomitant with a decrease in GnRH and Kp neurons, suggesting that VIP stimulation is required to remove RFRP-3 inhibition on the GnRH and Kp systems. We confirmed this possibility by localizing afternoon injections of CNO to RFRP-3 cells, which led to comparable decreases in GnRH and Kp cellular activity. These data indicate the importance of SCN VIP neurons in the temporally coordinated disinhibition of RFRP-3 neuronal activity required for stimulation of GnRH and Kp neurons around the time of LH surge. Supported by NIH Grant HD-050470. Presentation: Friday, June 16, 2023