Pulsatile secretion of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) is indispensable for follicle development and spermatogenesis in mammalian species.  KNDy neurons, expressing kisspeptin, neurokinin B (NKB) and dynorphin, are considered to be a central pacemaker regulating pulsatile GnRH secretion.  A synchronous discharge of KNDy neurons might be obligatory for pulsatile GnRH secretion.  The present study aimed to determine the cellular mechanism regulating the synchronized activities of KNDy neurons.  KNDy-GFP cells were collected from the mediobasal hypothalamus of the transgenic mice expressing GFP under the control of the Kiss1 promoter at day 17-18 of the embryonic age and cultured on a glass-base dish for 2 to 6 weeks.  Intracellular Ca²⁺concentrations were measured in individual KNDy-GFP cells using the fluorescent Ca²⁺ indicator Fura-PE3.  Senktide, a selective agonist for NKB receptor (NK3R), increased the frequency of the Ca²⁺ oscillations in cultured KNDy-GFP cells.  The senktide-induced Ca²⁺ oscillations were synchronized in the neighboring KNDy-GFP cells.  Such Ca²⁺oscillations were abolished by chelating extracellular Ca²⁺, suggesting that the Ca²⁺ oscillations are caused by an influx of extracellular Ca²⁺ through the calcium channels in KNDy-GFP cells.  Further, 18β-glycyrrhetinic acid, an inhibitor of neuron-to-astrocyte gap junction, and mefloquine, an inhibitor of neuron-to-neuron gap junction, attenuated senktide-induced Ca²⁺ oscillations in KNDy-GFP cells.  These results indicate that NKB-NK3R signaling is required for synchronized activities in neighboring KNDy neurons in an autocrine/paracrine manner and that both neuron-to-neuron and neuron-to-astrocyte communications via gap junctions may play a role in controlling synchronized activities of KNDy neurons.  This work was supported by the Research Program on Innovative Technologies for Animal Breeding, Reproduction, and Vaccine Development