| BackgroundMethamphetamine(MA)is one of the widely abused psychoactive drugs in the world,easily leading to addiction and causing neurotoxicity.MA addiction has become a global public health and social problem.It is urgently needed to reveal the neurobiological mechanisms of MA addiction and find effective intervention strategies.The neural projection from the ventral tegmental area(VTA)dopamine(DA)neurons to the nucleus accumbens(NAc)plays a key role in addiction induced by MA and other drugs.Different subgroups of DA neurons in the VTA project to the NAc core,NAc medial shell(NAcMed),and NAc lateral shell(NAcLat),forming more refined neural circuits with different biological functions.Compared to the NAc core,addictive drugs such as cocaine and morphine preferentially increase DA levels in the NAc shell of rats.The projection of VTA DA neurons to NAcLat plays a dominant role in predicting reward cues,while the projection of VTA DA neurons to NAcMed does not participate in this process.These studies suggest that the VTA-NAcLat circuit may play a more important role in addiction than the VTA-NAcMed circuit.However,there is currently little research on the role of VTA-NAcLat circuit in MA addiction and the molecular mechanisms that can effectively intervene in MA addiction are still unknown.Different subgroups of VTA neurons projecting to different target areas are mixed together,making it a great challenge to isolate DA neuron subgroups in the VTA and analyze their molecular characteristics.Patch-seq is a new technique which combines whole-cell patch-clamp recording and high-quality single-cell RNA sequencing(RNA-seq),integrating cellular morphology,physiology,and gene expression at the single-cell level.Patch-seq generates a comprehensive profile of neuronal cell types,thereby determining the potential relationship between gene expression and neuronal function.In the preliminary experiments of this study,we accurately obtained the DA neurons in the VTA-NAcLat circuit of mice in the control group and the MA-treated group by combining patch-clamp recording and circuit tracing.Single-cell RNA-seq was conducted and differentially expressed gene KCNQ3(Kv7.3),which is related to cell excitability,was further analyzed for its mechanism.KCNQ family genes encode Kv7.1-Kv7.5 subunits,which form the homomeric or heteromeric Kv7 channels.All Kv7 channel isoforms are activated at subthreshold potentials of approximately-60 mV and their activation stabilizes the neuronal resting potential.Kv7 channels are voltage-dependent K+channels,mostly via Kv7.2/3 heteromers,are involved in the formation of M currents.Studies have shown that Kv7 channels stabilize neuronal resting membrane potential,reduce neurotransmitter release,and decrease synaptic plasticity.It has reported that Kv7.3 is associated with learning and memory function.Specific knockdown of Kv7.3 in hippocampal pyramidal neurons reduces spontaneous excitatory postsynaptic currents(sEPSCs).Moreover,in the study of alcohol addiction,it was found that activation of Kv7.2/3 reduces alcohol intake in rats.This suggests that Kv7 channels play an important role in regulating synaptic plasticity and drug addiction.However,there is little research on the role of Kv7 channels in MA addiction,especially the mechanisms by which they regulate MA addiction in the VTA-NAcLat circuit remain unknown.Unveiling the role of Kv7 channels in MA addiction and its mechanisms will provide a new perspective for understanding drug addiction mechanisms and finding potential intervention molecules.Objectives1.Identify the types of VTA neurons in the VTA-NAcLat circuit and analyze the role of this circuit in MA addiction.2.Obtain VTA neuron subgroups in the VTA-NacLat circuit through retrograde labeling and Patch-seq technology,and analyze the gene expression changes in this circuit induced by MA using RNA sequencing.3.Uncover the mechanism of differentially expressed molecule Kv7 channels in MA addiction through the VTA-NAcLat circuit.Methods1.Detection indicators for MA addiction behaviorC57BL/6J mice were randomly divided into control and MA-treated groups.The MA-treated group was injected with MA intraperitoneally(2 mg/kg),while the control group received an equivalent saline treatment.Conditioned place preference(CPP)and behavioral sensitization experiments were conducted to measure the MA addiction behavior.2.Determine the distribution and neuron types of VTA neurons in the VTA-NAcLat circuit(1)Retrograde labeling tracer,retrobeads,was injected into NAcLat to label VTA neurons projecting to NAcLat.Immunofluorescence staining of tyrosine hydroxylase(TH)was used to determine the types and distribution of VTA neurons in this circuit and calculate the proportion of DA neurons.(2)Patch-clamp recording was performed on retrobeads-labeled VTA neurons to induce the dopamine neuron-specific "sag" characteristic by applying a-300pA current,thus determining the proportion of DA neurons in this circuit.3.Investigate the role of the VTA-NAcLat circuit in MA addiction using chemogenetic strategies(1)A chemogenetic virus,pAAV-EF1a-hM4Di-mCherry-WPRE was bilaterally injected into the VTA using stereotactic surgery,while a cannula was implanted into the NAcLat.Local injections of clozapine-N-oxide(CNO)specifically inhibited the VTA-NAcLat circuit.The effects of inhibiting this circuit on MA-induced CPP and behavioral sensitization were assessed.(2)Bilateral NAcLat injections of retrograde viral vector pAAV-hSyn-EGFP-P2A-NLS-Cre-WPRE(retro)were performed simultaneously with VTA injections of Cre-dependent virus pAAV-EF1a-DIO-hM4Di-mCherry-WPRE.Combined with Cre-loxP and chemogenetic techniques,the specific inhibition of the VTA-NAcLat circuit was further verified,and its effects on MA-induced CPP and behavioral sensitization were determined.4.Analyze the molecular characteristics of the VTA-NAcLat circuit using retrograde labeling and Patch-seq(1)Retrobeads were used to label VTA neurons projecting to NAcLat in both MA-treated and control mice.Retrobeads-labeled VTA neurons were extracted for single-cell RNA-seq.(2)The most significantly differentially expressed 32 genes from the sequencing results were validated using single-cell PCR to identify KCNQ3(Kv7.3),a significantly downregulated gene,for further functional analysis.5.Expression changes of KCNQ2-5(Kv7.2-5)in the VTA-NAcLat circuit of MA-treated mice(1)Retrobeads-labeled VTA neurons were extracted from live brain slices for single-cell PCR experiments to detect the expression of KCNQ2-5 in the VTA-NAcLat circuit of MA-treated mice.(2)Immunofluorescence staining was performed on brain slices after retrobeads labeling of VTA-NAcLat circuit neurons to detect the expression of Kv7.2-5 in the VTA-NAcLat circuit of MA-treated mice.6.The effect of Kv7 channel activation on MA-induced CPP and behavioral sensitization Using stereotactic surgery,cannulas were implanted bilaterally into the VTA,and activators of Kv7.2/3(ICA069673,0.1 pg/200 nL),Kv7.4(fasudil,0.1 μg/200 nL),and Kv7(retigabine,0.1 μg/200 nL)were locally injected.The effects of Kv7 channel activation on MA-induced CPP and behavioral sensitization were analyzed.7.The effect of Kv7 channel activation on the excitability of VTA neurons in the VTA-NAcLat circuit(1)Retrobeads were used to label VTA neurons in the VTA-NAcLat circuit.Patch-clamp technique was used to stimulate the labeled neurons with voltage steps ranging from-20mV to-60mV,in order to investigate the effect of Kv7.2/3(ICA069673)and Kv7.4(fasudil)channel activation on the M-current in the VTA-NAcLat circuit in MA-treated mice.(2)Whole-cell patch-clamp recording mode was used to examine the effect of Kv7.2/3 and Kv7.4 activators on the spontaneous action potentials of VTA neurons in the VTA-NAcLat circuit in MA-treated mice.(3)Labeled VTA neurons were stimulated with step currents ranging from 50pA to 150pA using the patch-clamp to investigate the effect of Kv7.2/3 and Kv7.4 channel activators on the evoked action potentials of VTA neurons in the VTA-NAcLat circuit in MA-treated mice.8.The effect of Kv7.2/3 channel activation on the activity of VTA and NAcLat neurons Since Kv7.3 was identified as a significantly differentially expressed molecule in the preliminary experiments and the heterotetramer formed by Kv7.3 and Kv7.2 is the primary basis for controlling the M-current,the subsequent experiments focused on analyzing the effect of Kv7.2/3 on the VTA-NAcLat circuit.(1)Local field potential(LFP)recordings were used to analyze the changes in delta bands(1-4 Hz),theta bands(4-8 Hz),alpha bands(8-13 Hz),beta bands(13-30 Hz),and gamma bands(30-100 Hz)in the VTA and NAcLat of MA-treated mice and control mice after treatment with Kv7.2/3 activator(ICA069673).(2)Activity-regulated cytoskeleton-associated protein(Arc)is an immediate early gene and is often used as a marker for neuronal activity.The effect of Kv7.2/3 activator on the expression of Arc in VTA and NAcLat neurons was observed using immunofluorescence.9.The effect of Kv7.2/3 channel activation on synaptic plasticity in the VTA-NAcLat circuit(1)rAAV-NCSP-YFP-2E5 were injected into VTA to analyze the changes in dendritic spine morphology of VTA neurons after Kv7.2/3 channel activation,to investigate the effect of Kv7.2/3 channel activation on structural plasticity of VTA neurons.(2)Spontaneous excitatory postsynaptic current(sEPSC)in NAcLat neurons were recorded using the patch-clamp to investigate the effect of Kv7.2/3 activation on the functional plasticity of the VTA-NAcLat circuit.10.The effect of Kv7.2/3 activator on DA release in the VTA-NAcLat circuit Carbon fiber electrodes modified with single-atom nanoenzyme were prepared,which can selectively detect the oxidation current of DA.Electrochemical methods were used to detect the release of DA from VTA neuron axon terminals in NAcLat.11.The effect of selective overexpression of KCNQ3 in the VTA-NAcLat circuit on neuronal excitability and addiction behavior(1)Retrograde AAV virus pAAV-hSyn-mCherry-P2A-NLS-Cre-WPRE was injected into NAcLat,and Cre-dependent pAAV-CMV-DIO-KCNQ3-WPRE was injected into VTA to achieve selective overexpression of Kv7.3 in the VTA-NAcLat circuit.The effect of Kv7.3 overexpression on MA-induced CPP and behavioral sensitization was examined.(2)The effect of Kv7.3 overexpression on VTA neuron M-current and spontaneous action potentials in the VTA-NAcLat circuit was recorded using the patch-clamp.Results1.Detection indicators of MA addiction behavior(1)Results of CPP experiments showed that MA-treated mice showed a significant preference for the white compartment,indicating an increased CPP score compared to the control group.(2)Results of behavioral sensitization experiments showed that MA-treated mice exhibited a gradual increase in spontaneous activity distance on the 1st,5th,and 13th days,indicating an enhanced behavioral response to MA compared to the control group.2.Distribution of VTA neurons in the VTA-NAcLat circuit and neuronal types(1)Retrograde tracing results showed that VTA neurons projecting to NAcLat were mainly distributed in the dorsolateral posterior part of VTA.(2)TH immunofluorescence staining showed that approximately 93.8%of VTA neurons in this circuit were DAergic neurons.3.Specific inhibition of the VTA-NAcLat circuit reduces MA-induced CPP and behavioral sensitization(1)Injection of a virus containing hM4Di into the VTA followed by local injection of CNO to inhibit the VTA-NAcLat circuit effectively reduced the CPP scores and behavioral sensitization in the MA-treated mice.(2)By combining Cre-loxp and chemogenetics,the specific inhibition of the VTA-NAcLat circuit also reduced the CPP scores and behavioral sensitization in the MA-treated mice.Patch-clamp recordings showed that the spontaneous firing current of VTA neurons infected with chemogenetic viruses was significantly reduced.4.Single-cell sequencing analysis of VTA neurons in the VTA-NAcLat circuit(1)Patch-seq results showed that VTA neurons in the VTA-NAcLat circuit were accurately obtained using the patch-clamp and retrograde tracing techniques.After low-input gene amplification and sequencing analysis,95 differentially expressed genes(>1.5-fold)were obtained.(2)Thirty-two of the most differentially expressed genes were selected for single-cell PCR validation,and the results were consistent with the sequencing results.Among them,the differentially expressed gene KCNQ3,which is associated with cell excitability,was significantly downregulated.5.MA inhibits the expression of KCNQ2-5(Kv7.2-5)in the VTA-NAcLat circuit(1)Single-cell PCR results showed that MA significantly inhibited the expression of KCNQ2-5 in VTA neurons in the VTA-NAcLat circuit,with KCNQ3 showing the most significant decrease in expression.(2)Immunofluorescence staining results showed that compared to the control group,the expression of Kv7.2-7.5 in VTA neurons in the VTA-NAcLat circuit of MA-treated mice was decreased,with Kv7.3 showing the most significant reduction.6.Activation of Kv7 channels suppresses MA-induced CPP and behavioral sensitization Injection of Kv7.2/3 selective activators ICA069673,Kv7.4 selective activator fasudil,and Kv7 activator retigabine into the VTA significantly reduced MA-induced CPP scores and behavioral sensitization,indicating that local infusion of Kv7 activators or Kv7 subunit channel activators in the VTA effectively reverses addictive behavior in the MA-treated mice.7.Activation of Kv7 channels reduces excitability of VTA neurons in the VTA-NAcLat circuit(1)Whole-cell patch-clamp recording results showed that Kv7.2/3 activators and Kv7.4 activators increased the M-current in VTA neurons in the VTA-NAcLat circuit of MA-treated mice.(2)Selective activation of Kv7.2/3 and Kv7.4 channels significantly reduced the spontaneous firing frequency of VTA neurons in the VTA-NAcLat circuit Stimulation with step current from 50pA to 150pA significantly reduced the frequency of evoked action potentials.8.Effects of Kv7.2/3 channel activation on the activity of VTA and NAcLat neurons(1)LFP recordings showed that compared to the control group,the neuronal activity in the VTA and NAcLat of MA-treated mice was enhanced,with increased delta and theta wave oscillations in VTA and NAcLat,and increased alpha,beta,and gamma oscillations in VTA.Selective activation of Kv7.2/3 with ICA069673 normalized oscillations in the VTA and NAcLat of MA-treated mice.(2)Immunofluorescence results showed that compared to the control group,the expression levels of Arc in the VTA and NAcLat of MA-treated mice were significantly increased.Kv7.2/3 activators restored the expression of Arc to normal levels in MA-treated mice.9.Activation of Kv7.2/3 channels reduces synaptic plasticity in the VTA-NAcLat circuit(1)Sparse labeling virus results showed that compared to the control group,the density of dendritic spines and three subtypes of dendritic spines in VTA neurons of MA-treated mice significantly increased.After selective activation of Kv7.2/3 channels with ICA069673,the density of dendritic spines and three subtypes of dendritic spines in VTA neurons significantly decreased.(2)Patch-clamp recording of sEPSC in NAcLat neurons showed that the average amplitude of sEPSC in NAcLat neurons of MA-treated mice increased compared to the control group.Selective activation of Kv7.2/3 channels significantly reduced the average amplitude of sEPSC in NAcLat neurons of MA-treated mice.10.Effects of Kv7.2/3 agonists on DA release in the VTA-NAcLat circuitElectrochemical results showed that compared to the control group,the amount of DA release in the NAcLat of MA-treated mice significantly increased.However,after administering the Kv7.2/3 agonist ICA069673,the amount of DA release in the NAcLat significantly decreased.11.Selective overexpression of Kv7.3 in the VTA-NAcLat circuit inhibits neuronal excitability and addictive behavior(1)By combining Cre-loxp and specific overexpression of Kv7.3 in the VTA-NAcLat circuit,behavioral tests showed that overexpression of Kv7.3 reduced CPP scores and behavioral sensitization in MA-treatd mice.(2)Patch-clamp recordings of M-current and spontaneous action potentials in Kv7.3 virus-infected neurons showed that overexpression of Kv7.3 significantly increased the M-current in VTA neurons and reduced the frequency of spontaneous action potentials.This indicates that overexpression of Kv7.3 inhibits neuronal excitability in the VTA-NAcLat circuit and reverses addictive behavior.Conclusion1.This study elucidates the important regulatory role of the VTA-NAcLat circuit in MA addiction and shows that inhibiting this circuit reduces MA-induced addictive behavior.2.By using patch-clamp and single-cell RNA-seq techniques,the molecular characteristics of VTA neurons in the VTA-NAcLat circuit were accurately obtained,and differentially expressed gene KCNQ3(Kv7.3)associated with MA addiction was identified.3.The role of Kv7.3 in regulating MA addiction in the VTA-NAcLat circuit was clarified.Selective activation of Kv7.2/3 reduces neuronal excitability and synaptic plasticity in the VTA-NAcLat circuit,decreases DA neurotransmitter release and neural oscillations in this circuit,and thereby reduces MA addiction behavior.Overexpression of Kv7.3 also reduces neuronal excitability and addictive behavior.4.This study reveals the mechanism of MA addiction from the perspective of Kv7 channels and the refined VTA-NAcLat circuit,providing experimental evidence for finding potential molecular targets for intervention in MA addiction. |
