Analysis of Gene Expression in the Amygdala during Conditioned Taste Aversion Learning

Conditioned taste aversion (CTA) learning occurs after the pairing of a novel taste with a toxin (e.g. sucrose taste with LiCl toxin). The immediate early gene c-Fos is necessary for CTA learning, but c-Fos alone cannot be sufficient for CTA consolidation. The expression of other activator protein 1 (AP-1) proteins from the Fos (c-Fos, FosB, Fra-1 and Fra-2)- and Jun (c-Jun, JunB and JunD)-families may also be required shortly after conditioning for CTA consolidation. To screen for the expression of AP-1 transcription factors within small subregions, RT-PCR analysis was used after laser capture microdissection (LCM) of the amygdala. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization and immunohistochemistry showed that changes in Fra-2 and c-Fos expression in the BLA and CeA at the time of conditioning, together with constitutive expression of c-Jun and JunD, may contribute to CTA learning. Using double immunolabeling, I confirmed that c-Fos co-localized with Fra-2 in a majority of LiCl-induced c-Fos-positive cells in the CeA. This co-localization of c-Fos and Fra-2 following LiCl suggests that the transcriptional regulation of AP-1 dimeric complexes of c-Fos and Fra-2 may occur in a subset of cells in the CeA. AP-1 family members bind each other to make homo- or hetero-dimers that regulate expression of target genes. For example, c-Fos needs to dimerize with a complementary member of other AP-1 proteins, specially Jun members. Therefore, it can be postulated that there may be transcriptional modulation of a set of AP-1 genes to form a functional AP-1 complex in a neuronal unit that is expressing c-Fos during CTA learning. I examined changes in mRNA expression of immediate early genes including AP-1 family members in c-Fos-specific neurons of the amygdala during CTA learning. Using X-Gal staining, single-cell LCM and RT-PCR, I detected mRNA expression of c-fos and β-actin genes in LiCl-induced lacZ-positive cells in the CeA, cortex and hippocampus of c-fos-lacZ transgenic mice. This result confirmed that endogenous c-fos gene and the c-fos-lacZ transgene were expressed in same cells of the brain. LiCl administration increases c-Fos expression in some brain regions including the CeA. Recent studies show that c-Fos expression in the brain after certain stimuli may be affected by histone modification such as acetylation and phosphorylation. Continuing our studies on gene expression in the amygdala in CTA learning, I investigated if LiCl-induced c-Fos expression in the amygdala is correlated with histone acetylation and phospho-acetylation. LiCl significantly increased the level of acetylated histone H3 (AcH3) in the CeA at 0.5 h and the number of phospho-acetyl-histone H3 (pAcH3)-positive cells in the CeA at 0.5 and 1 h, and the timecourse of LiCl-induced AcH3 and pAcH3 corresponded to LiCl-induced c-Fos timecourse in the CeA. Double immunolabeling results showed that c-Fos co-localized with pAcH3 in a majority of LiCl-induced c-Fos-positive cells in the CeA. These results suggest a possible correlation between LiCl-induced c-Fos expression and modifications of histone H3 in the CeA.