MEMORY CONSOLIDATION: HIPPOCAMPUS & GENE EXPRESSION

记忆是分子、细胞和大脑系统相互作用的结果。为了更好地治疗和诊断影响记忆和认知的精神健康障碍，我们必须了解大脑中这些不同层次上发生的复杂相互作用。这项研究计划的长期目标是增加对经验依赖的神经元基因表达在长期记忆（LTM）形成中的作用的理解。在这个广泛的框架内，本提案的实验将有助于确定：(1)感觉信息如何影响海马形成（HF）中的即时早期基因（IEG）转录；(2)任务需求如何影响HF中特定神经元群体的IEG转录。虽然本研究的中心焦点是在系统神经生物学和行为水平上，但所提出的研究结果将为未来在不同生理条件下控制IEG表达的分子机制的研究提供重要的初步信息。我们将使用我们最近开发的一种改进的荧光原位杂交（FISH）方案来可视化脑组织中多种不同的IEG rna。这种FISH检测非常敏感，能够检测脑神经元中活性IEG转录的假定位点（转录灶，或TF）。我们在大鼠身上进行了一系列的实验，表明在电刺激或探索新环境的2分钟内，许多脑电图的TF都可以被可视化。此外，这种诱导的转录是短暂的，在行为刺激和电刺激后30分钟的IEG TF处于基线水平，并且在这个时间点发现的新生mRNA分布在细胞质中。由于细胞核和细胞质RNA积累的时间过程是不同的，共聚焦显微镜测定的RNA的亚细胞分布可以用来推断单个神经元在两个不同时间的活动历史。我们将这种方法称为功能性脑成像细胞室分析的时间活动FISH，或catFISH。我们已经开始使用IEG catFISH来检查离散感觉信息在HF中IEG转录激活模式中的作用。在许多这样的研究中，我们利用catFISH的时间分辨率特性来观察离散的行为体验（在时间上相隔20分钟）如何影响不同神经元群的转录模式。我们的早期数据表明，两个CA1神经元群体在探索两个环境后会诱导IEG Arc的转录，而在同一环境中探索两次只会激活一个神经元群体中Arc RNA的转录。这提供了令人信服的证据，证明CA1神经元中的IEG转录与信息处理有关，而不是由一般或非特异性影响驱动的。这些研究的数据将为经验依赖的IEG表达在记忆形成过程中的作用以及在动物处理不同类型信息时心衰内发生的神经元群相互作用提供新的见解。

Memory is the result of molecular, cellular, and brain system interactions. To better treat and diagnose mental health disorders that affect memory and cognition, we must understand the complex interactions that occur at these different levels in the brain. The long-term objective of this research program is to increase understanding on the role of experience-dependent neuronal gene expression in the formation of long-term memory (LTM). Within this broad framework, the experiments of this proposal will help determine: (1) how sensory information affects immediate-early gene (IEG) transcription in the hippocampal formation (HF) and (2) how task demands influence IEG transcription in defined neuronal populations in the HF. While the central focus of this research is at the level of systems neurobiology and behavior, the findings from the proposed studies will provide important primary information for future focused studies on the molecular mechanisms that control IEG expression under different physiologic conditions. We will use a modified fluorescent in situ hybridization (FISH) protocol that we developed recently to visualize multiple different IEG RNAs in brain tissue. This FISH assay is extremely sensitive and is capable of detecting the putative sites of active IEG transcription (transcription foci, or TF) in brain neurons. We have performed a series of experiments in rats to show that TF for a number of IEGs can be visualized within 2 minutes of either an electrical stimulus or by exploration of a new environment. Moreover, this induced transcription is transient with IEG TF at baseline levels by 30 minutes after behavioral and electrical stimulus, and the nascent mRNA found distributed in the cytoplasm at this time point. Because the time course of nuclear versus cytoplasmic RNA accumulation is distinct, the subcellular distribution of RNA, determined by confocal microscopy, can be used to infer the activity history of individual neurons at two different times. We have termed this approach to functional brain imaging cellular compartment analysis of temporal activity by FISH, or catFISH. We have begun to use IEG catFISH to examine the role that discrete sensory information has on patterns of IEG transcriptional activation in the HF. In many of these studies, we exploit the temporal resolution properties of catFISH to see how discrete behavioral experiences, separated in time by 20 minutes, influence transcriptional patterns in different neuronal populations. Our early data show that two populations of CA1 neurons induce transcription of the IEG Arc following exploration of two environments, whereas exploration of the same environment twice only activates transcription of Arc RNA in one population of neurons. This provides compelling evidence that IEG transcription in CA1 neurons is linked to information processing, and is not driven by generalized or nonspecific influences. Data from the proposed studies will provide new insights into the role of experience-dependent IEG expression during memory formation, and in the neuronal population interactions that occur within the HF as an animal processes different types of information.