Transcriptional analysis of adult newborn hippocampal neurons

描述（由申请人提供）：新生神经元在一生中都在海马体中产生，可能有助于特定类型的学习和记忆。据推测，这需要将它们的功能集成到已有的电路中。成人海马神经发生随着年龄的增长而减少，但这种下降可以通过体育锻炼、生长因子和某些类型的环境刺激来减轻。我们假设成年新生海马神经元成熟的每个阶段都与一个特征的转录谱有关，新生神经元具有独特的转录反应，并且这些细胞整合到功能回路中从根本上改变了它们的基因表达模式。解决这些假设需要新的工具来确定完整大脑中一小部分细胞的转录谱。我们将使用表达刚地弓形虫尿嘧啶磷酸核糖基转移酶（UPRT）的逆转录病毒在其出生时特异性标记成年新生儿海马神经元群。只有这些细胞能够将核苷酸类似物-硫脲嘧啶（4-TU）结合到新生的RNA转录物中。tu标记的rna将被生物素化，纯化，并通过RNASeq分析。研究将描述小鼠成年新生海马神经元在不同成熟阶段和暴露于运动、磷酸二酯酶抑制剂、罗立平治疗和TrkB信号激活后的转录谱，并将这些谱与已有神经元的转录谱进行比较。将分析成年新生神经元转录本，以确定增强神经发生和成熟的治疗是否刺激或抑制可能构成其特征可塑性的特定信号通路。逆转录病毒表达的shrna将用于测试所选基因产物对新生神经元成熟的形态和功能特征的贡献。使用野生型和NMDA受体缺乏的成年新生海马神经元进行tu标记将使我们能够确定在功能整合时发生的兴奋性突触活动如何改变其转录程序。特定转录本的变化将与形态学成熟相关，使用共聚焦显微镜评估，并通过电生理学确定功能特性。选定的转录本和蛋白产物将在逆转录病毒标记后的特定时间通过单细胞PCR和免疫组织化学进行监测，以确定其精确的表达起点。针对选定转录本的逆转录病毒表达shrna将用于测试它们是否干扰整合神经元的功能特性。该项目利用了古德曼和韦斯特布鲁克实验室在基因组学、转录和海马体回路方面的综合专业知识。1

DESCRIPTION (provided by applicant): Newborn neurons are produced in the hippocampus throughout life and may contribute to specific types of learning and memory. Presumably, this requires their functional integration into pre-existing circuits. Adult hippocampal neurogenesis decreases with age, but this decline can be lessened by physical exercise, growth factors, and certain types of environmental stimuli. We hypothesize that each stage of adult newborn hippocampal neuron maturation is associated with a characteristic transcriptional profile, that newborn neurons are uniquely responsive transcriptionally, and that integration of these cells into functional circuits fundamentally alters their patterns of gene expression. Addressing these hypotheses requires new tools to determine the transcriptional profiles of a small population of cells within the intact brain. We will use retroviruses expressing Toxoplasma gondii uracil phosphoribosyltransferase (UPRT) to label adult newborn hippocampal neurons population specifically at their birth. Only these cells will be able to incorporate the nucleotide analogue, -thiouracil (4-TU), into nascent RNA transcripts. TU-tagged RNAs will be biotinylated, purified, and analyzed by RNASeq. Studies will characterize the transcriptional profiles of adult newborn hippocampal neurons in mice at various stages of maturation and after exposure to exercise, treatment with the phosphodiesterase inhibitor, Rollipam, and activation of TrkB signaling, comparing these profiles to those of pre-existing neurons. Adult newborn neuron transcripts will be analyzed to determine whether treatments that augment neurogenesis and maturation stimulate or repress particular signaling pathways that could underlie their characteristic plasticity. Retrovirally-expressed shRNAs will be utilized to test the contributions of selected gene products to morphological and functional features of newborn neuron maturation. TU-tagging performed using wild type and NMDA receptor- deficient adult newborn hippocampal neurons will allow us to determine how the excitatory synaptic activity that occurs upon functional integration changes their transcriptional program. Changes in specific transcripts will be correlated with morphological maturation, assessed using confocal microscopy, and functional properties, determined electrophysiologically. Selected transcripts and protein products will be monitored by single-cell PCR and immunohistochemistry at defined times after retroviral marking to determine their precise onset of expression. Retrovirally-expressed shRNAs directed against selected transcripts will be utilized to test whether they perturb functional properties of the integrated neurons. This project takes advantage of the combined expertise of the Goodman and Westbrook labs in genomics, transcription, and hippocampal circuitry. 1