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