Effect of Killing or Removing GABA from NPY/AgRP Neurons

描述（由申请方提供）：下丘脑弓形区的一小群神经元（约5000个细胞），使神经肽Y（NPY）和刺豚鼠相关蛋白（AgRP）成为成年小鼠生存所必需的。我们将白喉毒素受体（DTR）靶向这些NPY/AgRP神经元，以允许通过白喉毒素（DT）的给药对其进行消融。成年小鼠中这些神经元的消融导致饥饿。然而，它们在新生小鼠中的消融导致基于回路的补偿，使得小鼠几乎正常生长。尽管脑内DT损伤的小鼠生长到正常大小，我们通过实验探索它们是否表现出通常依赖于NPY/AgRP神经元的生理反应的缺陷。我们建议确定由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皮质素信号通路或其他回路发挥作用。关键信号分子的主要候选者是？-氨基丁酸（GABA）;因此，我们建议制备编码生物合成酶的两个基因GAD 1和GAD 2的条件等位基因，使得它们可以在NPY/AgRP神经元中失活，并且通过Cre重组酶的作用以时间控制的方式失活。如果GABA是关键的信号分子，那么我们预测新生小鼠的NPY/AgRP神经元中GABA生物合成的失活将导致补偿，使得细胞不再是生存所必需的。相反，成年小鼠NPY/AgRP神经元中GABA生物合成的失活可能促进饥饿。已知NPY/AgRP神经元抑制弓状核中产生前阿黑皮素（POMC）的相邻神经元和其室旁核中表达黑皮质素-4受体的靶神经元。POMC神经元的激活抑制进食。因此，我们包括实验，直接测试是否上调这一黑皮质素信号通路后，突然消融的NPY/AgRP神经元是至关重要的参与饥饿表型。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，以解决这些投射区域中受影响最大的区域，以及它们是否有助于进食行为。这些实验应该可以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.