DEVELOPMENT OF PEPTIDERGIC PROJECTIONS FROM ARCUATE NUCLEUS OF HYPOTHALAMUS

这一系列研究的长期目标是阐明从下丘脑弓状核（ARH）到下丘脑区域的肽能神经通路的组织和发展，已知下丘脑区域介导哺乳动物体内平衡的神经内分泌调节，已经进化为整合控制促性腺激素分泌和摄食行为的神经系统。ARH代表了这些系统之间的关键神经解剖学接口，其对促性腺激素分泌和摄食行为的重要性已被充分证明。然而，这些基本功能背后的详细神经机制仍不清楚，下丘脑的核团在介导促性腺激素分泌和摄食行为中起着关键作用，并与ARH以及下丘脑室旁核（PVH）有着密切的联系，这可能是下丘脑调节能量平衡的最终共同途径。此外，这些核中的每一个都表达黑皮质素和阿片肽的受体，这些受体与摄食行为和神经发育的调节有关。这个建议的总体假设是，黑皮质素和阿片肽表达在从RH到AVPV，PVH和DMH的投射中，并且这些肽直接影响这些核之间连接的发展。将使用顺行轴突运输和组织化学方法，以及体内和体外模型系统，以解决以下具体目标。具体目标1。示踪剂PHA-L的顺行轴突运输将与免疫组织化学一起使用，以证明成年小鼠（C57 B1/6 J）中从ARH到AVPV和DMH的投射中β-内啡肽（β END）和α黑素细胞刺激激素（α MSH）的存在。具体目标2。将使用荧光竞争剂DiI在新生雄性和雌性小鼠中展示从ARH到AVPV、PVH和DMH的项目开发。具体Im 3.将使用原位杂交将新生动物的AVPV、PVH和DMH中的MC 4和μ或δ阿片受体的表达与从ARH到达这些核的输入相关联。具体目标4。DiI还将用于研究缺乏β-内啡肽或MC 4功能性受体的转基因小鼠中从RH到AVPV、PVH和DMH的神经投射的发育。具体目标5.在规定条件下体外维持的器官型下丘脑外植体共培养物中，缺乏β-内啡肽和α-MSH功能性受体的转基因小鼠中的轴突转运和免疫组织化学，所述受体含有从ARH到AVPV、PVH或DMH的投射。这些研究将为下丘脑神经系统调节稳态的细胞机制提供新的见解，也可能为与不育和肥胖相关的神经缺陷的个体发生提供线索。

The long range goal of this line of research is to clarify the organization and development of peptidergic neural pathways from the arcuate nucleus of the hypothalamus (ARH) to hypothalamic regions known to mediate neuroendocrine regulation of mammalian homeostasis have evolved to integrate the neural system that control gonadotropin secretion and ingestive behavior. The ARH represents a key neuroanatomical interface between such systems, and its importance for gonadotropin secretion and feeding behavior is well documented. However, the detailed neurological mechanisms underlying these essential functions remain unclear, and little is known about the nuclei of the hypothalamus play key roles in mediating gonadotropin section and feeding behavior and share strong connections with the ARH, as well as with the paraventricular nucleus of the hypothalamus (PVH), which may represent the final common pathway for hypothalamic regulation of energy balance. In addition, each of the these nuclei express receptors for melanocortin and opiatergic peptides, which have been implicated in the regulation of both ingestive behavior and neural development. The overall hypothesis of this proposal is that melanocortin and opiatergic peptides are expressed in projections from the RH to the AVPV, PVH, and DMH, and that these peptides directly influence the development of connections between these nuclei. Anterograde axonal transport and histochemical methods will be used, together with both in vivo and in vitro model systems, to address the following specific aims. Specific Aim 1. Anterograde axonal transport of the tracer PHA-L will be used together with immunohistochemistry to demonstrate the presence of beta- endorphin (betaEND) and alpha melanocyte stimulating hormone (alphaMSH) in projections from the ARH to the AVPV and DMH in adult mice (C57Bl/6J). Specific Aim 2. The fluorescent racer DiI will be used to demonstrate the development of projects from the ARH to the AVPV, PVH and DMH in neonatal male and female mice. Specific Im 3. In situ hybridization will be used to correlate the expression of MC4 and mu or delta opiate receptors in the AVPV, PVH and DMH of neonatal animals with the arrival of inputs to these nuclei from the ARH. Specific Aim 4. DiI will also be used to study the development of neural projections from the RH to the AVPV, PVH and DMH in transgenic mice that lack functional receptors for beta-endorphin or MC4. Specific Aim 5. Axonal transport and immunohistochemistry in transgenic mice that lack functional receptors for beta-endorphin and alpha MSH containing projections from the ARH to the AVPV, PVH or DMH in organotypic hypothalamic explant co-cultures maintained in vitro under defined conditions. These studies will provide novel insights into cellular mechanisms underling the development of hypothalamic neural systems regulating homeostasis, and may also provide clues about the ontogeny of neurological defects related to infertility and obesity.