Supplementary Material for: Kisspeptin Neuron-Specific and Self-Sustained Calcium Oscillation in the Hypothalamic Arcuate Nucleus of Neonatal Mice: Regulatory Factors of its Synchronization

<b><i>Introduction:</i></b> Synchronous and pulsatile neural activation of kisspeptin neurons in the arcuate nucleus (ARN) are important components of the gonadotropin-releasing hormone pulse generator, the final common pathway for central regulation of mammalian reproduction. However, whether ARN kisspeptin neurons can intrinsically generate self-sustained synchronous oscillations from the early neonatal period and how they are regulated remain unclear. <b><i>Objective:</i></b> This study aimed to examine the endogenous rhythmicity of ARN kisspeptin neurons and its neural regulation using a neonatal organotypic slice culture model. <b><i>Methods:</i></b> We monitored calcium (Ca²⁺) dynamics in real-time from individual ARN kisspeptin neurons in neonatal organotypic explant cultures of <i>Kiss1</i>-IRES-Cre mice transduced with genetically encoded Ca²⁺ indicators. Pharmacological approaches were employed to determine the regulations of kisspeptin neuron-specific Ca²⁺ oscillations. A chemogenetic approach was utilized to assess the contribution of ARN kisspeptin neurons to the population dynamics. <b><i>Results:</i></b> ARN kisspeptin neurons in neonatal organotypic cultures exhibited a robust synchronized Ca²⁺ oscillation with a period of approximately 3 min. Kisspeptin neuron-specific Ca²⁺ oscillations were dependent on voltage-gated sodium channels and regulated by endoplasmic reticulum-dependent Ca²⁺ homeostasis. Chemogenetic inhibition of kisspeptin neurons abolished synchronous Ca²⁺ oscillations, but the autocrine actions of the neuropeptides were marginally effective. Finally, neonatal ARN kisspeptin neurons were regulated by N-methyl-D-aspartate and gamma-aminobutyric acid receptor-mediated neurotransmission. <b><i>Conclusion:</i></b> These data demonstrate that ARN kisspeptin neurons in organotypic cultures can generate synchronized and self-sustained Ca²⁺ oscillations. These oscillations controlled by multiple regulators within the ARN are a novel ultradian rhythm generator that is active during the early neonatal period.