Effect of Killing or Removing GABA from NPY/AgRP Neurons

描述(申请人提供)：下丘脑弓状区域的一小部分神经元(~5000个细胞)使神经肽Y(NPY)和刺鼠相关蛋白(AgRP)成为成年小鼠生存所必需的。我们将白喉毒素受体(DTR)靶向到这些NPY/AgRP神经元，以允许通过给予白喉毒素(DT)来消融它们。在成年小鼠身上消融这些神经元会导致饥饿。然而，在新生小鼠身上，它们的消融导致了基于回路的代偿，使小鼠几乎正常生长。虽然新生的DT损伤小鼠长到正常大小，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发挥作用。因此，我们建议将编码生物合成酶的两个基因GAD1和GAD2制成条件等位基因，使其在NPY/AgRP神经元中失活，并通过Cre重组酶的作用进行时间控制。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代偿，从而使这些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会促进饥饿。已知NPY/AgRP神经元抑制弓状核中产生前阿片黑素皮质素(POMC)的邻近神经元和它们在室旁核中表达黑素皮质素-4受体的靶神经元。POMC神经元的激活抑制了摄食。因此，我们包括实验来直接测试NPY/AgRP神经元突然消融后黑素皮质素信号通路的上调是否与饥饿表型有关。然而，由于NPY/AgRP神经元也投射到其他几个大脑区域，我们包括了一些实验，以解决这些投射区域中哪些受影响最大，以及它们是否对进食行为起作用。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.