abstract
ABSTRACT
The mechanisms of long-term learning and memory are the issue of this thesis. Techniques in molecular biology have been used to analyze changes of gene expression which might occur in events of learning and memory, in the central nervous system of the rat. The experimental work can be split in two parts. In the first part, the molecular mechanisms underlying memory induced by contextual fear conditional (CFC) have been studied by means of transcriptomics techniques. Rats subjected to CFC show freezing all times they are put again in the conditioning apparatus starting from 0 up to 28 days after CFC.
Previous experiments carried out on slices from hippocampus of animals subjected to CFC showed an increase of the excitability in CA1 region up to 7 days after the training. These long-lasting behavioural and electrophysiological changes suggested that a modification of the gene expression can occur. At the aim of identifying genes whose expression was modulated by CFC we have compared cDNA obtained from medium-temporal regions of the brains of rats subjected 48 hours before to CFC with cDNA isolated from control rats which never entered into the conditioning apparatus. The suppression subtractive hybridisation technique has been used. This method generates and equalizes representation of differentially expressed genes irrespective of their relative abundance and it is based on the construction of forward and reverse libraries. The libraries obtained have been cloned. A differential screening has been performed for all collected clones. The positive clones have been sequenced and the obtained sequences have been compared in GenBank, using software like FASTA and BLAST. The analysis of expression of isolated genes has been done by means RT-
abstract
real-time PCR. In the second part of the thesis we have analyzed more in detail the effects of 2 mM Acetyl-L-carnitine (ALC) on the gene expression in the brain of rats chronically treated for 21 days. ALC is a natural molecule known to improve the cognitive performance and to have neuroprotective action. We have used a proteomics approach such as Western Blot to both confirm and quantify the levels of protein expression for genes resulted differentially expressed after the ALC treatment. This at the aim to single out genes involved in processes underlying memory and neuroprotection which can be modulated by ALC treatments.
