We tested a number of different inhibitors of cell signalling pathways to elucidate the pathways that lead from mAChR activation to AMPAR internalisation. In many cases we obtained negative results but this is not due to ineffective inhibition of the target compound. Not only were the inhibitors applied directly to the postsynaptic cell via the patch pipette, at concentrations known to be effective in other experiments, but in most cases we found, during parallel experiments, that the same compounds were effective on other forms of synaptic plasticity (e.g. [
54]).
Compared to DHPG-LTD very little is known about the downstream signalling during mAChR-LTD. Classically, stimulation of M1 receptors leads to activation of PKC and the release of Ca
2+ from intracellular stores. However, we found no evidence that either limb of this pathway was involved in mAChR-LTD. The lack of effects of PKC inhibitors agree with previous studies of LTD induced by carbachol [
14] and DHPG [
55,
56]. The effect of interfering with Ca
2+ stores is less clear, since a partial inhibition by CPA of CCh-LTD was observed in perirhinal cortex [
16]. This might reflect a difference in brain region. In the present study, the LTD studied was also unaffected by BAPTA. This insensitivity to the chelation of intracellular Ca
2+ has also been reported for DHPG-LTD [
57], and suggests that the signalling pathways involved in these Gq-dependent forms of synaptic plasticity can be Ca
2+-independent. Previous work has implicated protein synthesis in mAChR-LTD. In two of these studies the effect of protein translation inhibitors were apparent rapidly but were only partially effective [
16,
20] whilst in another study these same inhibitors only affected mAChR-LTD after a delay of more than an hour [
14]. In agreement with the latter report, we found no effect of protein translation inhibitors on mAChR-LTD during the duration of our experiments. A similar dichotomy has been reported with mGluR-LTD, with reports of both protein synthesis dependence [
49] and independence [
23], for reasons that are not clear. In terms of treatments that were effective, we did find that inhibition of PTPs completely prevented the induction of mAChR-LTD. This observation, together with the insensitivity to a serine/threonine protein phosphatase, again highlights similarities between mAChR-LTD and mGluR-LTD [
22,
23]. In summary, we can conclude that activation of M1 receptors results in the loss of surface AMPARs and the generation of LTD via a Ca
2+-independent signalling cascade that involves one or more types of PTP.