Detection of Immediate Early Genes by Fluorescence in Situ Hybridization and Nuclear Counterstaining

Expression of immediate early genes (IEG) is induced in brain neurons by synaptic activity and is frequently used to monitor neuronal activation. This unit describes a technique (cellular compartment analysis of temporal activity by fluorescence in situ hybridization or catFISH) that uses fluorescence in situ hybridization (FISH) of IEG RNAs to slide-mounted tissue sections, and confocal microscopy to identify neuronal populations activated at two distinct times. The temporal resolution properties of catFISH derive from the time course of IEG transcriptional activation (and subsequent termination) and of the translocation of the nascent mRNA to the cytoplasm. Cells active shortly before the sacrifice of an animal (∼2 to 15 min) are distinguished by the presence of one or two areas of intense intranuclear fluorescence; these foci represent the sites of RNA transcription for that particular IEG. The intranuclear foci typically disappear within 20 min and the mRNA becomes prominent within the cytoplasm. This cytoplasmic RNA signal is used to identify neurons active ∼30 to 45 min earlier. Thus, the subcellular location of an IEG RNA signal can be used to discriminate cells activated by two distinct experiences separated by ∼30 min.