The Role And Mechanism Of Kv7 Potassium Channel And Prelimbic Cortex To Nucleus Accumbens Core Pathway In The Regulation Of Methamphetamine Addiction

Methamphetamine(METH),as a psychoactive drug,has been extensively abused and is known to be neurotoxic,which leads to the addiction after a long-term use.With repetitive use of drugs like METH,individuals can easily develop associative memories between rewarding stimuli and environmental cues.These persistent drug memories and cravings are the essential characteristics resulting in addiction.However,the key neural pathway and the molecular mechanisms by which addiction is associated with memory remain unclear.Previous studies have reported that the prelimbic cortex(PrL)to nucleus accumbens core(NAcC)pathway is closely associated with cognition,emotion,and addiction.Moreover,various physiological functions such as neural excitability,synaptic plasticity,and learning memory are some of the functions that Kv7 potassium channel regulates.In our study,we aim to demonstrate whether Kv7 can be shaped by the PrL-NAcC neural pathway and thus what its role is in the acquisition of METH memory.Objective(1)To illustrate the role of PrL-NAcC pathway in METH-induced conditioned place preference(CPP)in mice model.(2)To investigate the effect of Kv7 potassium channel in the PrL-NAcC pathway from addiction with METH.Methods and Results(1)METH activated neurons locating in the PrL-NAcC pathway:C57BL/6J mice were intraperitoneally injected with 2 mg/kg of METH to establish the METH addiction model with the conditioned place preference(CPP)diagram.Immunofluorescence staining was used to identify the expression of c-Fos in the PrL and the NAcC region.Furthermore,adenoviruses containing the Ca2+ indicator GCaMP6s was microinjected into the PrL and the NAcC to examine the level of intracellular Ca2+.The results revealed that both the number of the c-Fospositive cells and the fluorescence intensity of the Ca2+level were increased significantly in METH-addicted mice.Moreover,the retrobeads was injected into NAcC to label neurons projected from the PrL to the NAcC region.The findings above can be concluded that METH could activate the projection neurons in the PrL-NAcC pathway.(2)Cre-Loxp virus combined with chemogenetics could selectively inhibit the PrL-NAcC pathway and decrease the CPP score:Firstly,the PrL-NAcC pathway was specifically inhibited by Cre-Loxp virus.The retrograde tracer virus carrying Cre recombinase was injected into the NAcC while the Cre-dependent human muscarinic cholinergic receptor M4(hM4Di)adenovirus was injected into the PrL.Secondly,induced by Clozapine N-oxide(CNO),the PrLNAcC pathway was selectively inhibited and the results demonstrated that the METH-induced CPP score was decreased.(3)The expression of Kv7 potassium channel was decreased by METH in the PrL region:Kv7.2 and Kv7.3 homomeric channels control cellular excitability.The expression level of Kv7.2 and Kv7.3 in the PrL was dramatically decreased after the METH management from detection by immunofluorescence staining and real-time quantitative PCR.In addition,retrograde tracer and immunofluorescence staining also showed that the expression of Kv7.2 and Kv7.3 was decreased in the PrL neurons projecting to the NAcC.(4)Enhanced Kv7’s function could inhibit the CPP scores:Retigabine,a selective Kv7/M channel agonist,was injected into the PrL region to enhance the function of the Kv7.The results indicated that the CPP scores were significantly reduced.Kv7.3 can resulted in expression in Kv7.2 and is found as heteromers(Kv7.2/Kv7.3).Furthermore,the overexpression of Kv7.3 was established in the PrL-NAcC pathway by Cre-Loxp virus from which the CPP scores were also significantly reduced.(5)Enhanced Kv7’s function could inhibit the neuronal excitability:Three groups were established(the control group,METH group,and retigabine group).The retrobeads was injected into the NAcC to label the projection neurons.We utilized the patch clamp to document the M currents and spontaneous firing of labeled neurons in the PrL.The findings demonstrated that METH could enhance the frequency and amplitude of spontaneous firing while decrease the M current.Moreover,the activation of the Kv7 potassium channel could obviously alleviate the neuronal excitability induced by METH.(6)The changes of synaptic plasticity:The sparsely labeled virus rAAV-NCSP-YFP-2E5 was injected into the PrL region to analyze the complexity of neural dendrite and the morphological change of dendritic spine.The expression of activity-regulated cytoskeletal protein(Arch)was detected by the immunostaining analysis.The findings suggested that METH could enhance the density of dendritic spines in the PrL region,especially the mushroom and thin dendritic spines.Additionally,the expression of Arch was also considerably elevated by METH.However,the changes of synaptic plasticity above could be decreased after the activation of Kv7 channel.(7)The DA content in NAcC detected by electrochemical method:Electrochemical methods can be used to detect the level of dopamine(DA)in the NAcC.The results showed that DA level in the NAcC was significantly increased in METH-induced mice model.However,the change of DA level above could be inversed following the activation of Kv7 channels in the PrL.(8)METH increased the Kv7.3 sumoylation:Immunoprecipitation was used to determine the level of Kv7.3 sumoylation in the PrL.And the results showed that METH could reduce the expression of SENP2,which is a de-SUMO protease,and increase the expression of SUMO-2,which is a ubiquitination-like modification molecule.In addition,after the injection of the sumoylation modification inhibitor 2D08 in the PrL,the CPP scores were significantly decreased.Conclusion(1)The projection neurons from the PrL-NAcC pathway were activated in the METH addiction,from which the METH-induced CPP scores could be decreased by selective inhibition of the pathway.(2)The expression of the Kv7.3 channel was decreased in METH-induced mice model.However,the enhanced function of the Kv7.3 could reduce the neuronal excitability,the synaptic plasticity,and decreased the DA levels in the NAcC region and the CPP scores.(3)The Kv7.3 channel in the PrL region may be involved in the regulation of METH memory by increasing the level of its sumoylation.