The Essential Roles Of Protein Kinase A In Scopolamine Induced Synaptic Plasticity And Antidepressant Responses

Background:It is predicted that depression will place a heavy burden on public health in future.the current therapy approaches such as serotonin reuptake inhibitors and tricyclic antidepressant are not ideal with uncomplete understanding of the biological mechanism of depression.The nonselective cholinergic receptor(mAchR)antagonist scopolamine has mainly been used in the treatment of motion sickness,Parkinson’s disease,and pregnancy-related vomiting in the past.Similar to ketamine,it has been discovered that a single intravenous injection of a subclinical dose of scopolamine produces a rapid(within 12 h)and potent anti-depressant response without severe adverse effects such as hallucination and addiction.Thus,scopolamine is better tolerated in clinical applications.A recent study revealed that scopolamine activates the mammalian target of rapamycin(mTOR),the key signaling protein that triggers the subsequent synaptic plasticity and rapid anti-depressant response.This process requires the participation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(AMPAR).the current theory of scopolamine’s action on AMPAR is indistinct.c AMP-dependent protein kinase A(PKA)signaling is a ubiquitous pathway that induces AMPAR phosphorylation on ser845,which promotes membrane insertion and channel conductance of GluA1-containing AMPARs.Given that M2 cholinergic receptors are negatively coupled to the adenylate cyclase(AC)-c AMP pathway,blockade of M2 receptors may directly induce the PKA pathway through AC-c AMP disinhibition.The current study was undertaken to determine the possible mechanism by which PKA participates in scopolamine-induced AMPAR reinforcement and counteraction of depression-like symptoms.Objective:We used a combination of electrophysiological techniques,Western blotting,membrane protein biotinylation assays,learned helplessness model for depression and behavioral experiments,to identify the potential M2-PKA mechanism underlying scopolamine-induced AMPA receptor potentiation,compare the distinct regulation between Glu A1 expression and Ser845 phosphorylation and their contribution in mTOR pathway activation and counteraction of depression-like symptoms.This investigation may be helpful in enhancing complete understanding of the pharmacological mechanism of the anti-depressant activity of scopolamine,provide experimental evidence for novel anti-depressant strategy.Methods:1.We made depressed SD rats model according to a previous reported learned helplessness paradigm,and validate the LH model by behavioral assays including FST,SPT,and OFT.2.We treated the slices with different dose of scopolamine(0,0.01,0.1,1,10 μM)to test if scopolamine’s effect on Glu A1 and mTOR is dose dependent.3.After APV(100 μM)pretreatment and perfusion to minimize interference from NMDA receptors,Field excitatory postsynaptic potentials(fEPSPs)at the stratum radiatum invoked by CA3-CA1 Schaffer collateral stimulating was then record to comfirm scopolamine induced synaptic transmission onset,and if PKA is requirement in this process.4.Western blot was performed to detect Glu A1 and Glu A2 expression in membrane surface protein sub-dissected from hippocampal slices perfused by scopolamine or H89.5.We inspected scopolamine’s action on hippocampal slices after pretreated with picrotoxin(GABAa inhibitor)to investigate whether GABA receptors disinhibition can occlude scopolamine-induced Glu A1 phosphorylation and expression,as well as mTOR activation.6.The rats are intracerebroventricularly injected with H89 before scopolamine or methoctramine administration.Then we operate the open field test and forced swimming test to assess their depression-like behavior and Western blot technique to detect the expression and phosphorylation of Glu A1,as well as mTOR pathway activation.Results:1.We made the learned helplessness Rat model of depression and validate them by behavioral assays.2.From 0 to 10μM,scopolamine dose dependently increased the phosphorylation of Glu A1 at serine 845;otherwise,significant mTOR activation only obseved in the concentration of 1μM..3.scopolamine potentiated excitatory synaptic transmission at SC-CA1 synapses in the presence of APV,and this effect could be blocked by PKA inhibitor-H89.4.scopolamine rapidly increased membrane surface Glu A1 expression in a PKA dependent manner,while Glu A2 was not apparently affected.5.The actions of scopolamine-induced enhancement of Glu A1 phosphorylation and potentiation of mTOR activation were not occluded by GABAergic disinhibition,While scopolamine failed to further increase Glu A1 expression in the presence of picrotoxin.6.Intraperitoneal scopolamine administration resulted in higher ser845 and ser2448 phosphorylation levels in a PKA dependent manner.However,the increased Glu A1 expression in the CA1 area is independent of PKA pathway.7.We found that,similar to scopolamine,the selective M2 receptor antagonist,methoctramine also produced Glu A1 ser845 and mTOR ser2448 phosphorylation onset,while there was no apparent effect on Glu A1 expression.Furthermore,these effects on ser845 and ser2448 were abolished by a pre-injection with H89.8.We confirmed that administration of both scopolamine and methoctramine reduced the duration of immobility in a subsequent FST,while the locomotor activity estimated by the OFT remains unchanged.These anti-depressant effects were abolished by H89.Conclusions:1.PKA is required for scopolamine induced AMPAR reinforcement and m TOR activation,as well as its rapid anti-depressant.PKA-induced phosphorylation at Ser845 may be involved in scopolamine-induced AMPAR trafficking and subsequent synaptic plasticity.2.Scopolamine induced Glu A1 expression do not depend on PKA but regulated by GABAergic disinhibition.3.Selective M2 antagonist produces a phosphorylation and behavioral response similar to that of scopolamine and indicated M2 blockade can be a triggering event for subsequent PKA activation that involved in synaptic remodeling.4.Selective M2 antagonist do not promote Glu A1 expression,which means M2-PKA mechanism is distinct from Glu A1 expression in the early stage of scopolamine’s action and sufficient for subsequent counteraction effect of depression-like symptoms.