A long-lasting (no less than 7 days) increase of hippocampal

abstract

ABSTRACT

The aim of this experimental work is to analyse the large-scale transcriptional responses that occur in the amygdala and hippocampus of rat after contextual fear conditioning (CFC).

Emotional memories tend to be long-lasting and play an important role in regulating the behavioural responses of animals. Most of the current knowledge of emotional memories derives from the studies of classical fear conditioning It is believed that CFC is dependent on the structural integrity of the hippocampus and amygdala, whereas contextual fear conditioning is hippocampal-indipendent, but requires the amygdala. “Conditioned” rats exhibited long-lasting freezing (no less than 4 weeks) when placed in the apparatus again (retrieval test).

A long-lasting (no less than 7 days) increase of hippocampal

excitability was measured in hippocampal slices prepared from the

brains of “conditioned” rats. These findings are suggesting that CFC

might exert its effects with changes of gene expression. Two different

libraries of cDNA have been generated with the Suppression

subtractive hybridisation (SSH) method to verify this hipothesis. The

SSH method efficiently identifies genes with a differential expression

due to treatments or pathological conditions. The SSH method allows

to isolate even the more rare transcripted genes, otherwise impossible

to obtain with the other available techniques, which analyse the

patterns of genic expression. In this experimental work, a group of rats

has been subjected to CFC (“conditioned”), whereas another group

(“naïve”) was never placed in the apparatus. After 48 hours from

training, the brains of both groups were rapidly removed and dissected

into 3 sections: an anterior, a medio-temporal (containing amygdala

abstract

and hippocampus) and cerebellum. RNA poliA

isolated from medio-

temporal sections has been used to apply SSH method. After

evaluating subtraction efficiency, cDNA was directly cloned and

subjected to screening. Sequences of differential clones, obtained by

automatic sequencer, have been analysed by comparison with

sequences present in gene data bank using program such as FASTA,

BLASTN. Analysis of most interesting differentially expressed cDNA

has shown identity with the coding sequences for the “Tripartite motif

protein 32”, the “Proteasome maturation protein” and the “Type-Iα

regulatory subunit of cAMP dependent protein kinase”. In order to

further confirm the really differentially expressed genes a relative RT-

PCR, with total RNA from “naïve” and “conditioned” brains has been

carried out.