Department of Physiology and Pharmacology, Oregon National Primate Research Center, Oregon Health and Science University, Portland, Oregon 97239-3098, USA.
Endocrinology. 2011 Apr;152(4):1503-14. doi: 10.1210/en.2010-1285. Epub 2011 Feb 1.
Hypothalamic kisspeptin neurons are critical for driving reproductive function, but virtually nothing is known about their endogenous electrophysiological properties and the effects of leptin on their excitability. Therefore, we used the slice preparation from female guinea pigs to study the endogenous conductances and the effects of leptin on kisspeptin neurons. We targeted the arcuate kisspeptin neurons using visualized-patch whole-cell recording and identified kisspeptin neurons using immuocytochemical staining for kisspeptin or single cell RT-PCR. We also harvested dispersed arcuate neurons for analysis of expression of channel transcripts. Kisspeptin neurons exhibited a relatively negative resting membrane potential, and eighty percent of the neurons expressed a pacemaker current (h-current) and a T-type Ca(2+) current. Furthermore, the glutamate receptor agonist N-methyl D-aspartic acid depolarized and induced burst firing in kisspeptin neurons. Leptin activated an inward current that depolarized kisspeptin neurons and increased (burst) firing, but leptin hyperpolarized NPY neurons. Lanthanum, a TRPC-4,-5 channel activator, potentiated the leptin-induced inward current by 170%. The leptin-activated current reversed near -15 mV and was abrogated by the relatively selective TRPC channel blocker 2-APB. The leptin effects were also blocked by a Janus kinase inhibitor, a phosphatidylinositol 3 kinase inhibitor, and a phospholipase Cγ inhibitor. In addition, the majority of these neurons expressed TRPC1 and -5 and phospholipase Cγ1 based on single cell RT-PCR. Therefore, guinea pig kisspeptin neurons express endogenous pacemaker currents, and leptin excites these neurons via activation of TRPC channels. The leptin excitatory effects on kisspeptin neurons may be critical for governing the excitatory drive to GnRH neurons during different nutritional states.
下丘脑 kisspeptin 神经元对于驱动生殖功能至关重要，但实际上人们对其内在的电生理特性以及瘦素对其兴奋性的影响知之甚少。因此，我们使用雌性豚鼠的切片制备物来研究内源性电导和瘦素对 kisspeptin 神经元的影响。我们使用可视化贴片全细胞记录来靶向弓状 kisspeptin 神经元，并使用 kisspeptin 的免疫细胞化学染色或单细胞 RT-PCR 来鉴定 kisspeptin 神经元。我们还收获分散的弓状神经元用于分析通道转录本的表达。Kisspeptin 神经元表现出相对负的静息膜电位，并且 80%的神经元表达起搏电流（h 电流）和 T 型 Ca(2+)电流。此外，谷氨酸受体激动剂 N-甲基-D-天冬氨酸使 kisspeptin 神经元去极化并诱导爆发式放电。瘦素激活内向电流，使 kisspeptin 神经元去极化并增加（爆发）放电，但瘦素使 NPY 神经元超极化。镧，一种 TRPC-4、-5 通道激活剂，使瘦素诱导的内向电流增强了 170%。瘦素激活的电流在约 -15 mV 处反转，并且被相对选择性的 TRPC 通道阻滞剂 2-APB 阻断。Janus 激酶抑制剂、磷脂酰肌醇 3 激酶抑制剂和磷脂酶 Cγ 抑制剂也阻断了瘦素的作用。此外，基于单细胞 RT-PCR，这些神经元中的大多数表达 TRPC1 和 -5 和磷脂酶 Cγ1。因此，豚鼠 kisspeptin 神经元表达内源性起搏电流，瘦素通过激活 TRPC 通道来兴奋这些神经元。瘦素对 kisspeptin 神经元的兴奋作用对于在不同营养状态下控制 GnRH 神经元的兴奋驱动可能至关重要。
