Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca²⁺]_i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1 ≈1 μM) induced increase of [Ca²⁺]_i in a dose-dependent manner. This elevation of [Ca²⁺]_i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca²⁺ stores by the endoplasmic reticulum inhibitor thapsigargin (2 μM), [Ca²⁺]_i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca²⁺-free condition, orexin A failed to induce any changes of Ca²⁺ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca²⁺]_i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 μM) and D609 (10 μM), respectively. Selective blockade of L-type Ca²⁺ channels by nifedipine (5 μM) significantly suppressed the elevation of [Ca²⁺]_i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca²⁺]_i in neurons from deep layers of PFC, which depends on extracellular Ca²⁺ influx via L-type Ca²⁺ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.

中文翻译：

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食欲素 A 诱导的前额叶皮层神经元细胞外钙流入涉及 L 型钙通道。

食欲素是来自下丘脑外侧的新型兴奋性神经肽，与睡眠和觉醒的调节密切相关。在本研究中，我们探讨了食欲素 A 对来自前额叶皮层 (PFC) 的 V 层和 VI 层的新鲜分离神经元的细胞内游离 Ca ²⁺浓度（[Ca ²⁺ ] _i）的影响和机制。[Ca ²⁺ ] _{i 的}变化是用fluo-4/AM 使用共聚焦激光扫描显微镜测量的。结果表明，orexin A (0.1 ≈1 μM) 的应用以剂量依赖性方式诱导了 [Ca ²⁺ ] _{i 的}增加。[Ca ²⁺ ] _{i 的}这种升高被选择性食欲素受体 1 拮抗剂 SB 334867 预处理完全阻断。虽然内质网抑制剂毒胡萝卜素 (2 μM)耗尽细胞内 Ca ²⁺储存，但PFC 神经元中的[Ca ²⁺ ] _i显示对食欲素 A 的反应没有增加. 在细胞外无Ca ²⁺条件下，orexin A 未能在这些急性解离的细胞中诱导Ca ²⁺荧光强度的任何变化。我们的数据进一步表明，食欲素 A 诱导的 [Ca ²⁺ ] _{i 增加}通过分别使用特异性抑制剂 BIS II (1 μM) 和 D609 (10 μM) 抑制细胞内蛋白激酶 C 或磷脂酶 C 的活性，完全消除。L-型Ca的选择性阻断²⁺硝苯地平通道（5μM）显著抑制的[Ca仰角²⁺ ]_我通过食欲肽A.因此诱导，这些研究结果表明，暴露于食欲素A可诱导的[Ca的增加²⁺ ] _i在来自 PFC 深层的神经元中，其依赖于通过 L 型 Ca ²⁺通道通过结合食欲素受体 1 激活细胞内 PLC-PKC 信号通路的细胞外 Ca ²⁺流入。