Electrophysiological recordings made on "slices" of the hippocampus of

abstract  

ABSTRACT

This thesis is part of an experimental research project based on the study of mechanisms of learning and long-term memory after contextual fear conditioning in rats. In classical conditioning to fear, or 'classical fear conditioning, it is known that the activation of a large part of the amygdala in response to a stimulus of fear leads to a type of behavioral response. In “contextual fear conditioning” (CFC), in addition to the activation of the amygdala also the hippocampus is involved. In this conditions the animal are not conditioned only by the stimulus of fear but also by the environment in which it is placed. Once submitted to the CFC in fact, the animals show the typical response of "freezing" if repositioned in the apparatus of conditioning (retrieval tests). This behavioral response is observed up to 28 days after training.

Electrophysiological recordings made on "slices" of the hippocampus of

rats have also revealed an increase of conditional excitability in layer

CA1 up to 7 days by the training. This long-lasting experimental

evidence have suggested the possibility of a different gene expression in

the nervous system of rats conditioned respect to unconditioned controls

(naïve), resulting in differential synthesis of mRNA and protein. To

identify differentially expressed genes we used the Suppression

Subtractive Hybridization method (SSH) that allows to isolate rare

transcripts scarcely or no detectable using other techniques. Starting

from poliA

mRNA extracted from mid-temporal regions of the brain of

naïve and conditioned rats libraries have been built forward and reverse

abstract  

subtractive cDNA. Having assessed the efficiency of subtraction, the

cDNA sequences were cloned and screened. The sequences of

differential clones were compared with those deposited in databases

using software such as FASTA and BLAST. The analysis of expression

of genes isolated was performed by RT real-time PCR. It was

subsequently quantified with Western blot technique, and the differential

synthesis of proteins of the genes sequenced. In particular, in this thesis

it has been assessed the changes in protein synthesis of the following

genes: Stathmin, Amphiphysin, Profilin 2. Based on the physiological

function of proteins synthesized, clones were divided into several

functional categories: mitochondrial energy metabolism, training,

transportation and release of synaptic vesicles, mechanism of signal

transduction and protein turnover.