A combined method of laser capture microdissection and X-Gal histology to analyze gene expression in c-Fos-specific neurons

c-Fos is a member of the activator protein 1 family that regulates transcription of target genes. c-Fos is transiently induced in specific regions of the brain after a variety of external stimuli including learning and memory formation. Analysis of gene expression in c-Fos-expressing cells of the brain may help identify target genes that play important roles in synaptic strength or neuronal morphology. In the present study, we developed a combined method of laser capture microdissection and 5-bromo-4-chloro-3-indoly-β-d-galactopyranosidase (X-Gal) histology to analyze gene expression in stimulus-induced c-Fos-positive cells. Using transgenic mice carrying a c-fos-lacZ fusion gene, c-Fos-positive cells were easily identified by measuring of β-galactosidase (β-Gal) activity. To establish the fidelity of the reporter transgene, the time course of endogenous c-Fos and the c-fos-lacZ transgene expression in the amygdala induced by LiCl administration was investigated by immunohistochemistry and X-Gal staining. LiCl increased the numbers of c-Fos- and β-Gal-positive cells in the central and basolateral amygdala of the transgenic mice. To ensure that RNA was preserved in X-Gal stained tissue sections, different fixations were examined, with the conclusion that ethanol fixation was best for both RNA preservation and X-Gal staining quality. Finally, in combining X-Gal staining, single-cell LCM and RT-PCR, we confirmed mRNA expression of endogenous c-fos and β-actin genes in LiCl-induced β-Gal-positive cells in the CeA, cortex and hippocampus. Combining LCM and transgenic reporter genes provides a powerful tool with which to investigate tissue- or cell-specific gene expression.