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 Kiss1ARH 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 Ca2+ signaling. In brain slices, photostimulation depolarized and increased firing of Kiss1ARH 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 Kiss1ARH network depends on activation dynamics, reconciles inconsistencies, and establishes photopharmacology as a powerful approach for probing neuroendocrine timing mechanisms.