Contextual fear conditioning induced differentially expressed genes in the rat mid-temporal brain

In the contextual fear conditioning (CFC), a form of associative learning, the animals learn to score a new environment because of its temporal association with an aversive unconditioned stimulus (US), usually a foot shock. This learning process requires functional integrity and activation of several sub-cortical and cortical sites within the mid-temporal brain, such as hippocampus, amygdala and perirhinal cortex. Studies performed in our laboratory on the Rattus norvegicus have show that the freezing response was still exhibited one month after acquisition of CFC and that a clear correlation was found between the increase of hippocampal excitability, up to the 7-day delay, and freezing response consolidation. Long-term memory for CFC requires the synthesis of new proteins, likely as a result of the induction of genes. In order to verify this hypothesis, two different libraries of cDNA have been generated with the Suppression Subtractive Hybridisation (SSH) method. The former (forward library) contained transcripts positively conditioning-induced while the latter (reverse library) contained the negatively conditioning-modulated transcripts. The SSH method allows to isolate even the more rare transcripted genes, otherwise impossible to obtain with the other available techniques used to analyse the patterns of genic expression. Two groups of animals were employed: (i) rats subjected to a CFC paradigm training entailing the administration of aversive electrical foot shocks (8 min exposure) (conditioned group) and (ii) naïve rats never placed in the apparatus (control group). After evaluating subtraction efficiency, cDNA was directly cloned and subjected to screening. The total amount of obtained clones is 600 for the forward library and 1200 for the reverse library. In this abstract data concerning the screening of about 100 clones are reported. The analysis of the remaining clones is still in progress. Sequences of differential clones, obtained by automatic sequencer, have been analysed by comparison with sequences present in GenBank and EMBL, using program such as FASTA and BLASTN. Analysis of most interesting cDNA of the forward library have showed identity with the coding sequences for the “Complexin 1”, the “Phosphoprotein enriched in astrocytes”, the “Mithocondrial ATPase Subunit 6”, the “Tripartite motif protein 32”, the “Proteasome maturation protein” and the “Type-Iα regulatory subunit of cAMP dependent protein kinase”. In order to let evident the really differentially expressed genes a relative RT-PCR, with total RNA from control and conditioned brain, has been carried out.