Optical control of the hypothalamic arcuate nucleus kisspeptin neuronal network with a photoswitchable peptide that drives luteinizing hormone release in female mice

Pulsatile gonadotropin‐releasing hormone (GnRH) and luteinizing hormone (LH) secretion are essential for reproductive function and are thought to be governed by arcuate nucleus kisspeptin Kiss1 ARH neurons via neurokinin B (NKB) signaling through TacR3. However, discrepancies between ex vivo and in vivo pharmacology limit mechanistic understanding of how TacR3 activation regulates pulsatile hormone release. We developed and synthesized a photoswitchable TacR3 agonist ( azo ‐senktide) and characterized its photochemical and pharmacological properties using UV–Vis spectroscopy, TacR3‐expressing HEK293T cells, electrophysiology in mouse hypothalamic slices, and in vivo optofluidic delivery combined with light stimulation to assess LH secretion in freely moving mice. Azo ‐senktide reversibly photoisomerized between inactive (trans) and more potent (cis) conformations. In cells, light activation enhanced TacR3‐mediated Ca 2+ signaling. In brain slices, photostimulation depolarized and increased firing of Kiss1 ARH neurons through a mechanism consistent with the established TacR3–TRPC5 signaling pathway. In vivo, optical activation of azo ‐senktide in the arcuate nucleus elicited a time‐locked LH pulse at an intermediate dose, whereas lower or higher doses were ineffective, indicating a nonlinear, dose‐dependent response. These findings demonstrate that temporally gated TacR3 activation via photoswitchable azo ‐senktide evokes an increase in LH, revealing that neurokinin B signaling in the Kiss1 ARH network depends on activation dynamics, reconciles inconsistencies, and establishes photopharmacology as a powerful approach for probing neuroendocrine timing mechanisms.