Laboratory of Neurochemistry, National Institute of Neurological Disease and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS One. 2013 Jul 24;8(7):e69407. doi: 10.1371/journal.pone.0069407. Print 2013.
The oxytocin (Oxt) and vasopressin (Avp) magnocellular neurons (MCNs) in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON), and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM), and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV) vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun) are differentially expressed in the Oxt and Avp MCNs.
下丘脑的催产素 (Oxt) 和加压素 (Avp) 大细胞神经元 (MCN) 是仅存在于视上核 (SON) 中的神经元表型，其特征是它们对 Oxt 或 Avp 基因具有强大而选择性的表达。在本文中，我们利用这些神经肽基因的差异表达，通过激光捕获显微切割 (LCM) 从大鼠 SON 中鉴定和分离这两种单个表型，并通过 qRT-PCR 分析它们的几个转录因子 mRNA 的差异表达。我们通过立体定向注射包含细胞类型特异性 Oxt 或 Avp 启动子的重组腺相关病毒 (rAAV) 载体来鉴定这些神经元表型，该启动子选择性地在 Oxt 或 Avp MCN 中驱动 EGFP 的表达。然后使用本文中描述的新型 Cap Road Map 协议通过 LCM 对荧光 MCN 进行切割，并提取纯化的 MCNs 进行 RNA 提取。这些 RNA 的 qRT-PCR 显示，一些转录因子 (RORA 和 c-jun) 在 Oxt 和 Avp MCN 中差异表达。
