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心理健康障碍，我们必须了解大脑中这些不同层面发生的复杂相互作用。 本研究计划的长期目标是增加对经验依赖性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，这项建议的实验将有助于确定：（1）感觉信息如何影响海马结构（HF）中的立即早期基因（IEG）转录，以及（2）任务需求如何影响HF中定义的神经元群体中的IEG转录。 虽然这项研究的中心焦点是在系统神经生物学和行为的水平，从拟议的研究结果将提供重要的主要信息，为未来重点研究的分子机制，控制IEG表达在不同的生理条件下。我们将使用我们最近开发的改良荧光原位杂交（FISH）协议来可视化脑组织中多种不同的IEG RNA。 该FISH测定非常灵敏，能够检测脑神经元中活性IEG转录的推定位点（转录灶或TF）。 我们已经在大鼠中进行了一系列实验，以表明一些IEG的TF可以在电刺激或探索新环境的2分钟内可视化。 此外，在行为和电刺激后30分钟，IEG TF处于基线水平时，这种诱导的转录是瞬时的，并且在该时间点发现新生mRNA分布在细胞质中。 由于细胞核与细胞质RNA积累的时间过程不同，因此通过共聚焦显微镜测定的RNA的亚细胞分布可用于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通过FISH或catFISH对时间活动进行功能性脑成像细胞区室分析的方法。我们已经开始使用IEG catFISH来研究离散的感觉信息对HF中IEG转录激活模式的作用。 在许多这些研究中，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体的转录模式。 我们的早期数据表明，两个群体的CA1神经元诱导IEG弧的转录后，探索两个环境，而探索相同的环境两次只激活转录的弧RNA在一个群体的神经元。 这提供了令人信服的证据表明，IEG转录在CA1神经元与信息处理，而不是由广义或非特异性的影响。 拟议研究的数据将为记忆形成期间经验依赖性IEG表达的作用以及动物处理不同类型信息时在HF内发生的神经元群体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.