GPCRs in Behaviour and Neurodegeneration
Our interest lies primarily in the role that the muscarinic receptor play in learning and memory and in particular in the role that receptor phosphorylation plays in regulating memory and learning.
It has been known for some time that muscarinic receptors are important in mediating hippocampal based memory and learning. Base largely on pharmacological studies the Muscarinic subtype thought to mediate this response was the M1-muscarinic receptor. However recent gene knockout studies and the use of specific pharmacological agents have suggested that M1-muscarinic receptors impact memory and learning through actions on the prefrontal cortex rather than directly on the hippocampus.
We set out to investigate the possibility that the M3-muscarinic receptor could mediate memory and learning by employing an M3R-KO mice lacking the gene for the M3-muscarinic receptor. These mice were found to have a deficit in hippocampal (contextual) based memory and learning.
Fear Conditioning Test
Fear Conditioning test: Mice were placed into a new cage and subjected to a tone followed by a mild foot shock. This training was repeated twice. The animals were then returned to the training cage 24 hours later and the time the animals spent frozen was a measure of the contextual -hippocampal based memory. 24 after the contextual test the animals were played the tone and the amount of time spent frozen was a measure of the amygdala based – cued learning.
To test if the process of memory and learning required the phosphorylation and arrestin-dependent signalling we employed a transgenic mouse where a mutant M3-muscarinic receptor containing S-A substitutions in the phospho-acceptor sites in the third intracellular loop was knockin to the wild type M3-muscarinic receptor gene locus. This mutant receptor was expressed normally at the cell surface and in tissues were the receptor was normally found. The receptor was shown to couple normally to Gq/11-signalling but was uncoupled from phosphorylation dependent signalling such as arrestin recruitment and receptor internalisation.
Fear Conditioning in M3R- KO Mice
M3R - KO Mice showed a deficiency in fear conditioning memory and learning
Fear Conditioning in M3R-KI Mice
M3R-KI Mice showed a deficiency in fear conditioning memory and learning
In conclusion, the deficit in fear conditioning shown by the M3R-KO mice implicates this receptor in cholinergic-mediated learning and memory. The fact that the mutant mice expressing a phosphorylation-deficient M3-muscarinic receptor showed a similar fear conditioning deficit to the M3R-KO mice demonstrates the importance of receptor phosphorylation, and potentially arrestin recruitment, in the mechanism of action of this receptor in learning and memory. In light of these findings it would seem plausible that biased ligands that direct the signalling of the M3-muscarinic receptor through phosphorylation/arrestin pathways might be of clinical benefit in the treatment of cognitive disorders.
This work can be found in the following reference:
Benoit Poulin, Adrian Butcher, Julie-Myrtille Bourgognon, Robert Pawlak, ,Kok Choi Kong, Phillip McWilliams, Andrew Bottrill+, Jurgen Wess, Fadi F.Hamdan, Tim Werry, Hannah A.Cragg, Alison, D.Smart, Andrew B. Tobin*. (2010) .The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner. Proc. Natl. Acad. Sci. 107, 9440–9445.