Supplement : Contribution of Kisspeptin and Neurokinin B in the Medial Amygdala to Female Reproductive Function

摘要： Kispeptin(由Kiss1基因编码)和Neurokinin B(NKB；由Tac2基因编码)是关键 女性生殖轴的调节器。NKB及其受体NK3R的失活突变或Kiss1和 它的受体Kiss1R会导致人类和小鼠的不孕。Kiss1神经元有两个主要群体 位于弓状核(ARC)和前腹侧脑室周围核(AVPV)直接接触 促性腺激素释放激素(GnRH)神经元与性别负反馈和正反馈的调控 生殖功能所必需的类固醇。在ARC，Kiss1神经元共同表达NKB，这刺激了 在一组名为KNDy的神经元中，Kispeptin的释放，以及抑制Kispeptin释放的强啡肽A 神经元。杏仁内侧核(Mea)参与情绪和信息素的处理，也包含一个 Kiss1和NK3R表达神经元的数量；然而，在中枢控制中的功能作用 这些神经元群体在MEA中的再生仍然没有明确的定义。我们实验室的初步实验 研究发现，将NK3R的特异性激动剂Senktide注射到MEA的NK3R神经元所在的区域 在小鼠体内定位能诱导黄体生成素的释放。在特定目标1中，转基因Kiss1小鼠和腺病毒载体的组合。 针对MEA中Kiss1神经元的相关病毒(AAV)注射将被用来操纵 Kiss1神经元的功能，以确定它们在黄体生成素(LH)分泌(与脉搏密切对应)中的作用 GnRH)、生育力和生殖行为。在特定的目标2中，在MeA中表达NK3R的神经元将是 用与NK3R激动剂结合的核糖体失活毒素Saporin特异性消融以确定 这些MeA神经元在生殖功能中的作用。MEA是一个高度相互联系的大脑区域， 将传入投射发送到参与调节的多个大脑区域并从其接收传出投射 但Kiss1和Tac2在该区域表达神经元之间的神经元投射是 未知。此外，虽然Tac2在许多大脑区域都有表达，包括杏仁中央内侧核(CEM) 而通过NK3R作用于MEA的NKB的来源ARC仍有待确定。在具体目标3中，a 在Kiss1-Cre或Tac2-Cre转基因小鼠中顺行和逆行注射AAV将 在MeA和调节Kispeptin和GnRH释放的区域之间映射神经投射，以及 涉及生殖行为和信息素处理的区域。这些结果将开始定义角色 MeA、Kiss1和NK3R在生殖功能调节中的表达神经元与人群的比较 这些神经元在大脑的其他区域。理解MEA在情绪和情绪整合中的作用 与生殖的联系对于理解包括人类在内的许多物种的性行为至关重要，而且 可以帮助开发治疗中枢性生殖障碍的新方法。

ABSTRACT: Kisspeptin (encoded by the Kiss1 gene) and neurokinin B (NKB; encoded by the Tac2 gene) are critical regulators of the female reproductive axis. Inactivating mutations to NKB and its receptor, NK3R or to Kiss1 and its receptor Kiss1R lead to infertility in both humans and mice. There are two major populations of Kiss1 neurons located in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV) that directly contact gonadotrophin-releasing hormone (GnRH) neurons and control the negative and positive feedback of sex steroids necessary for reproductive function. In the ARC, Kiss1 neurons co-express both NKB, which stimulates kisspeptin release, and dynorphin A, which inhibits kisspeptin release, in a population of neurons called KNDy neurons. The medial amygdala (MeA), which is involved in emotion and pheromone processing, also contains a population of Kiss1 and NK3R expressing neurons; however, the functional role in the central control of reproduction of these neuron populations in the MeA remains ill-defined. Preliminary experiments from our lab found that injection of senktide, a specific agonist of NK3R, into the area of the MeA where the NK3R neurons are located induces LH release in mice. In Specific Aim 1, a combination of transgenic Kiss1 mice and adeno- associated virus (AAV) injections targeted specifically to Kiss1 neurons in the MeA will be used to manipulate Kiss1 neuron function to determine their role in luteinizing hormone (LH) secretion (which closely mirrors pulses of GnRH), fertility and reproductive behavior. In Specific Aim 2, neurons expressing NK3R in the MeA will be specifically ablated using the ribosomal inactivating toxin saporin conjugated to an agonist for NK3R to determine the role of these MeA neurons in reproductive function. The MeA is a highly interconnected brain region that sends afferent projections to and receives efferent projections from multiple brain areas involved in the regulation of reproduction but the neuronal projections between Kiss1 and Tac2 expressing neurons in this region are unknown. Additionally, while Tac2 is expressed in many brain areas, including the centromedial amygdala (CeM) and the ARC, the source of NKB acting in the MeA through NK3R remains to be identified. In Specific Aim 3, a combination of anterograde and retrograde AAV injections in either Kiss1-Cre or Tac2-Cre transgenic mice will map the neural projections between the MeA and regions that regulate the release of Kisspeptin and GnRH, and regions involved in reproductive behavior and pheromone processing. These results will begin to define the role of MeA Kiss1 and NK3R expressing neurons in the regulation of reproductive function compared to populations of these neurons in other brain regions. Understanding the role of the MeA in the integration of emotions and bonding with reproduction is critical to understanding sexual behavior in many species, including humans, and could aid in the development of new approaches to treat central reproductive disorders.