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April 23, 2014
Canadian Excellence

Time course of Arc mRNA induction

Compartment Analysis of Temporal Activity by Flourescence in situ Hyridization (catFISH)

Monitoring the activity of neuronal ensembles (i.e., distributed groups of neurons that are functionally connected) provides insights into global functions that are not apparent from recordings obtained from one cell at a time, or from macroscopic measures related to net activity. Thus, there is a critical need for a method that permits discrimination of the activity of functional ensembles across a greater number of cells during multiple epochs of behaviour with cellular resolution.

In fact, a sensitive technique combining fluorescence in situ hybridization and high resolution confocal microscopy has been developed that permits just such discrimination. This novel method, termed cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH), can map the distribution of neurons activated during two discrete behaviours by visualizing the sub-cellular localization of immediate early gene (IEG) mRNA. Thus, the catFISH method permits the activity history of individual cells to be determined at two different time points (Fig. 1), and generates estimates of the numbers of neurons active during a distinct behavioural episode that match electrophysiological estimates under comparable conditions, but with unlimited sampling capability. Thus, catFISH represents a powerful new tool for the study of the neurobiology of aging.

The goal of the proposed research program is to exploit this innovative methodology and recent findings to explore the cellular bases of behaviourally-induced plasticity, its relationship to the creation of long-lasting memory, and how these processes are altered in aging.

Fig. 1, right: Time course of Arc mRNA induction. Representative images from hippocampal CA1 are shown in which Arc mRNA staining is indicated by CY3 (red) and nuclei are stained with DAPI (blue). Caged control animals (A, top) were sacrificed from their colony cages; A, immediate were sacrificed immediately after a 5 min exploration (B, mid); A, delay were sacrificed 25 min following exploration (C, below). Quantitative data demonstrate that, despite low Arc expression in caged control animals, ~40% of cells express Arc following spatial exploration, which then migrates from the nucleus to the cytoplasm (A, delay). If the same environment is visited twice , the same proportion of cells express Arc in both compartments. If different environments are visited, statistically independent populations of cells are active, as predicted from electrophysiological recordings (from Guzowski et al., 1999).