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

摘要： Kisspeptin（由Kiss 1基因编码）和神经激肽B（NKB;由Tac 2基因编码）是关键的 女性生殖轴的调节器。NKB及其受体、NK3R或Kiss 1的失活突变， 其受体Kiss1R导致人类和小鼠不育。Kiss 1神经元有两个主要群体 位于弓状核（ARC）和前腹侧室周核（AVPV）， 促性腺激素释放激素（GnRH）神经元和控制性的负反馈和正反馈 生殖功能所必需的类固醇。在ARC中，Kiss 1神经元共表达NKB，这刺激了 kisspeptin释放，以及抑制kisspeptin释放的强啡肽A 神经元内侧杏仁核（MeA）参与情绪和信息素处理，也包含一个 表达Kiss1和NK3R的神经元的群体;然而，在中枢控制中的功能作用， MeA中这些神经元群体的繁殖仍然不明确。我们实验室的初步实验 发现将一种NK3R特异性激动剂senktide注射到MeA区域， 诱导小鼠LH释放。在特定目标1中，转基因Kiss 1小鼠和腺病毒的组合， 将使用特异性靶向MeA中Kiss 1神经元的相关病毒（AAV）注射来操纵 Kiss1神经元的功能决定了它们在促黄体激素（LH）分泌中的作用（这与脉冲密切相关 GnRH）、生育力和生殖行为。在特定目标2中，将在MeA中表达NK3R的神经元 使用与NK3R激动剂缀合的核糖体失活毒素皂草素特异性消融，以确定 这些MeA神经元在生殖功能中的作用MeA是一个高度互联的大脑区域， 向参与调节的多个脑区发送传入投射，并从多个脑区接收传出投射 但在该区域表达Kiss1和Tac2的神经元之间的神经元投射是 未知此外，虽然Tac2在许多大脑区域表达，包括中央杏仁核（CeM） 和ARC，NKB通过NK3R在MeA中起作用的来源仍有待鉴定。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AAV注射的组合将 绘制MeA与调节Kisspeptin和GnRH释放的区域之间的神经投射， 参与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表达神经元在生殖功能调节中的作用 这些神经元在其他大脑区域的分布。理解MeA在情绪整合中的作用， 与生殖的结合对于理解包括人类在内的许多物种的性行为至关重要， 可以帮助开发治疗中枢生殖系统疾病的新方法。

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.