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.
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