Effect of Killing or Removing GABA from NPY/AgRP Neurons

描述（由申请人提供）：下丘脑弓状区的一小群神经元（约5000个细胞）产生神经肽Y （NPY）和针刺相关蛋白（AgRP），对成年小鼠的生存至关重要。我们将白喉毒素受体（DTR）靶向这些NPY/AgRP神经元，通过白喉毒素（DT）的施用使其消融。成年小鼠这些神经元的消融会导致饥饿。然而，它们在新生小鼠中的消融导致了基于电路的补偿，使小鼠几乎正常生长。虽然新生dt损伤小鼠生长到正常大小，但我们通过实验探索它们是否表现出通常依赖于NPY/AgRP神经元的生理反应缺陷。我们建议确定NPY/AgRP神经元产生的关键信号分子，以及它们是否主要通过调节黑素皮质素信号通路或其他电路起作用。关键信号分子的主要候选者是？氨基丁酸（GABA）；因此，我们提出在NPY/AgRP神经元中制备编码生物合成酶的GAD1和GAD2两个基因的条件等位基因，使其在Cre重组酶的作用下失活并暂时控制。如果GABA是关键的信号分子，那么我们预测新生小鼠NPY/AgRP神经元中GABA生物合成的失活将导致细胞不再需要生存的补偿。相反，成年小鼠NPY/AgRP神经元GABA生物合成失活可能促进饥饿。已知NPY/AgRP神经元可抑制邻近弓状核中产生POMC的神经元及其室旁核中表达黑素皮质素-4受体的靶神经元。活化POMC神经元抑制摄食。因此，我们通过实验直接测试NPY/AgRP神经元突然消融后黑素皮质素信号通路的上调是否在饥饿表型中起关键作用。然而，由于NPY/AgRP神经元也投射到其他几个大脑区域，我们包括实验，以解决哪些投射区域受影响最大，以及它们是否有助于摄食行为。这些实验应该能让我们深入了解对进食至关重要的神经回路和信号分子。

DESCRIPTION (provided by applicant): A small population of neurons (~ 5000 cells) in the arcuate region of hypothalamus that make neuropeptide Y (NPY) and agouti-related protein (AgRP) become essential for survival in adult mice. We targeted the diphtheria toxin receptor (DTR) to these NPY/AgRP neurons to allow their ablation by administration of diphtheria toxin (DT). Ablation of these neurons in adult mice results in starvation. However, their ablation in neonatal mice results in circuit-based compensation such that mice grow almost normally. Although neonatally DT-lesioned mice grow to normal size, we experimentally explore whether they manifest deficits in physiological responses that normally depend on NPY/AgRP neurons. We propose to determine the critical signaling molecules made by NPY/AgRP neurons and whether they function primarily by regulating the melanocortin-signaling pathway or other circuits. A prime candidate for the critical signaling molecule is ?-aminobutyric acid (GABA); thus, we propose to make conditional alleles of the two genes, GAD1 and GAD2 encoding the biosynthetic enzymes such that they can be inactivated in NPY/AgRP neurons and temporally controlled manner by the action of Cre recombinase. If GABA is the critical signaling molecule, then we predict that inactivation of GABA biosynthesis in NPY/AgRP neurons of neonatal mice will lead to compensation such that the cells are no longer necessary for survival. In contrast, inactivation of GABA biosynthesis in NPY/AgRP neurons of adult mice may promote starvation. NPY/AgRP neurons are known to inhibit neighboring neurons in the arcuate nucleus that make proopiomelanocortin (POMC) and their target neurons in the paraventricular nucleus that express melanocortin-4 receptor. Activation of POMC neurons inhibits feeding. Thus, we include experiments to directly test whether up-regulation of this melanocortin-signaling pathway after sudden ablation of NPY/AgRP neurons is critically involved in the starvation phenotype. However, because NPY/AgRP neurons also project to several other brain regions, we include experiments that address which of these projection regions are affected the most and whether they contribute to feeding behavior. These experiments should provide insight into the neural circuits and signaling molecules that are critical for feeding.