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Title: Involvement of PKA and CREB in memory consolidation/reconsolidation after single-trial reward classical conditioning in Lymnaea stagnalis
Author: Michel, Maximilian
ISNI:       0000 0001 3621 827X
Awarding Body: University of Sussex
Current Institution: University of Sussex
Date of Award: 2008
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This thesis presents the first set of evidence for a genuine PKA catalytic subunit in the central nervous system of the pond snail Lymnaea stagnalis, a well-established experimental system for the analysis of the molecular mechanisms of associative memory. The kinase is funCtionally conserved in that it can be visualized with anti-human PKA antibodies, it can phosphorylate a peptide designed for PKA and it can be inhibited by two commonly used specific inhibitors of PKA. Furthermore, the catalytic subunit was purified utilizing the holoenzyme's biochemical properties followed by fragmentation and identification with mass-spectrometry. This data concurs with cloning data obtained by sequencing the mRNA for the catalytic subunit. Subsequent sequence analysis showed that the Lymnaea catalytic subunit has only diverged in residues not intrinsically linked to enzymatic function. After confirming the presence of PKA mRNA and protein in neurons of the feeding neuronal circuitry to be conditioned, the PKA inhibitor KT5720 was used to analyze the component role played by PKA in an established paradigm for Lymnaea single-trial associative reward-memory formation. There was a prolonged dependence of 24 hour recall upon PKA activity (until about three hours) with a severely reduced window of sensitivity to the inhibitor for recall at six hours post-conditioning. This dependence coincided with a heightened PKA activity after learning for at least one hour which was not present any longer at either six or 24 hours. Conditioning furthermore resulted in phosphorylation of the constitutive transcription factor CREB by six hours which was specific to the learning group and cannot be observed in any of five learning control groups. Preliminary evidence is presented to show that this CREB phosphorylation is invivo dependent upon PKA activity, inhibition of which abolishes the observed phosphorylation. This paradigm further exhibits a bona-fide reconsolidation effect because translational inhibition with anisomycin - if administered after a recall stimulus either at six or 24 hours after learning - induces amnesia. This finding was extended by showing that inhibition of PKA has a similar amnesia inducing effect if administered after a reminder session at six hours but not at 24 hours post-conditioning. Analogous to the observed differential time-window of PKA requirement for the six and�· 24 hour memory traces, this result shows a differential susceptibility to amnestic effects after recall, i.e. dependent on the time post-conditioning different intracellular cascades remain partially active thereby rendering the memory trace amenable to disruptive treatments. Taken together, this work provides the first comprehensive identification of PKA as a key signalling molecule of memory consolidation and reconsolidation after single-trial conditioning in Lymnaea.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available