The Role And Molecular Mechanism Of Orbitofrontal Cortex-dorsal Striatum Neural Circuit In Memory Retrieval Of Methamphetamine Addiction

BackgroundMethamphetamine(MA)is a synthetic psychoactive drug that is highly neurotoxic and addictive.Repeated use of addictive drugs,such as MA,can lead to the generation of associative memories between the rewarding stimulus of the drug and environment.The memories can be retrieved when the abuser is re-exposed to the relevant cues of the drug.The persistence of addiction memory and continuous craving for drugs is the key problem of addiction.Although great progress has been made in the study of neural circuits and molecular mechanisms related to addiction memory,the brain regions and molecular mechanisms involved in MA addiction memory were remains unclear.Hence,exploring the neural circuit basis of MA addiction and searching for specific molecules related to addiction will help to understand the mechanism of addiction and provide new treatment strategies.It is generally considered that the orbitofrontal cortex(OFC)regulates reward prediction and behavioral decision making.The reckless drug-seeking behavior of abuser is similar to the behavior of individual with OFC dysfunction.Functional imaging studies have shown that the OFC neurons in cocaine abusers are significantly activated when exposed to drug-related environment.Additionally,the OFC undergoes persistent metabolic and neurochemical changes,suggesting OFC plays an important role in the retrieval of addictive memories.Anatomical research has found the projection from the OFC to the striatum,which can be divided into two subregions:the ventral striatum(VS)and the dorsal striatum(DS).Studies indicated that there is a shift from dominance of the VS to dominance of the DS,during the progression from initial drug use to compulsive drug-seeking.Studies on cocaine addiction indicated that dopamine release in the DS is positively correlated with the severity of addiction,indicating a close association between the DS and addiction.Research has found that optogenetic stimulation of the ventral tegmental area(VTA)enhances synaptic transmission in the OFC-DS projection neuron in the simulative compulsive drug seeking model.Furthermore,the OFC-DS circuit mediates the formation of alcohol addiction memories,suggesting its potential importance in drug addiction.However,the role of OFC-DS in the retrieval of MA addiction memories remains unclear,particularly the molecular mechanisms.In the previous study,RNA sequencing analysis is performed on the OFC region of MA addiction mice.Among the differentially expressed molecules obtained,Tsc22d3 is significant upregulation.Tsc22d3 is a member of the transforming growth factor beta induction gene 22(TSC22)family,which is widely expressed in mammalian brain tissue.Studies have indicated that low expression of Tsc22d3 is positively correlated with reduced hippocampal volume in cases with depression.Knocking down Tsc22d3 in the hippocampus and cortical neurons leads to reduced dendritic complexity and loss of mushroom spines,indicating that Tsc22d3 regulates synaptic plasticity,which are the structural basis of addiction memory.Importantly,recent research has also found significant upregulation of Tsc22d3 expression in the striatal of morphine-addicted mice,suggesting that Tsc22d3 may be a specifically expressed molecule in the OFC-DS circuit in addiction.However,does Tsc22d3 regulate MA addiction behavior through the OFC-DS circuit?If so,what could be the possible mechanisms?It is crucial to address these questions and elucidate the role and potential mechanisms of Tsc22d3 and the OFC-DS neural circuitry in MA addiction.Based on the above background,this study aims to establish a conditioned place preference(CPP)paradigm to explore the role and mechanism of the OFC-DS neural circuit and Tsc22d3 in MA addiction.This study is divided into two parts:(1)The role and mechanism of OFC-DS neural circuit in MA addiction memory;(2)The role and mechanism of Tsc22d3 in the OFCDS circuit in MA addictive memory.Objectives1.To clarify the role of OFC-DS circuit in memory retrieval of MA addiction.2.RNA transcriptome sequencing analysis is performed on the OFC region,and identify the expression of Tsc22d3 in the OFC-DS circuit.3.Investigate the role of Tsc22d3 on the excitability and synaptic transmission in the OFC-DS circuit;explore the role of Tsc22d3 in MA addiction behavior and possible mechanisms.Methods and ResultsPart 1:The role and mechanisms of the OFC-DS circuit in MA addiction memory1.1 MA addiction memory retrival activated OFC-DS circuit neurons(1)C57BL/6J mice were divided into MA group and Saline group,and intraperitoneally with MA(2 mg/kg)or saline to establish CPP model.The expression of c-Fos in OFC and DS was detected by immunofluorescence staining,and the changes of Ca2+level were analyzed by Ca2+ indicator GCaMP6s.The results showed that the number of c-Fos positive neurons significantly increased in MA group,and the fluorescence signal of Ca2+ was also significantly enhanced in OFC and DS.(2)The retrobeads was used to label the projection neurons in OFC-DS circuit,combining c-Fos immunofluorescence staining found that the number of c-Fos positive cells in OFC-DS circuit was significantly increased in MA mice.1.2 Cre-loxp combined with chemogenetics selectively inhibited the OFC-DS circuit reduced CPP scoreCre-loxp technology was used to manipulate the OFC-DS circuit.Cre-expressing retrograde virus was injected into the DS and hM4Di-expressing virus was into the OFC to selectively inhibited the OFC-DS circuit.In addition,AAV-hM4Di-mCherry virus was injected into the OFC region,and intracerebral cannula implantation was performed in the DS.Clozapine Noxide(CNO)was injected into the DS region through the cannula to specifically inhibit the OFC-DS circuit.Behavioral results showed a significant reduction in MA-induced CPP scores,indicating the suppression of OFC-DS circuit inhibited addiction behavior.1.3 MA increased the spontaneous and evoked action potential of the OFC-DS projection neuronThe projection neuron from OFC to DS was labeled by retrobeads,and then spontaneous firing and evoked action potential were analyzed by patch-clamp recording.The results showed that MA increased the frequency of spontaneous current in OFC-DS neurons.The evoked action potential frequency was significantly higher in MA group stimulated by 100 pA current,indicating that MA significantly increased the excitability of neurons in OFC-DS circuit.1.4 Effect of MA on DS spontaneous excitatory postsynaptic current(sEPSC)The anterograde transneuronal virus pscAAV2/1-hSyn-CRE was injected into the OFC,and AAV2/9-CMV-DIO-EGFP was injected into the DS to label the next level neurons contacted with the projection neuron from the OFC to DS.The spontaneous excitatory postsynaptic current(sEPSC)of labeled neurons in DS was recorded by patch clamp.The results showed that sEPSC frequency and amplitude were significantly increased in the MA group compared with the saline group.1.5 MA promoted the synaptic plasticity of OFC and DS regions(1)Sparse labeling virus rAAV-NCSP-YFP-2E5 was used to label neurons in the OFC and DS.The results showed that total dendritic spine density,especially mushroom spines and thin spines,increased in the MA model group.(2)TEM was applied to detect the ultrastructure of OFC.The results demonstrated increased synaptic density,synaptic vesicle docking density,and the thickness and length of postsynaptic density in OFC in MA group,suggesting structural plasticity changes induced by MA.1.6 MA promoted the release of DA transmitter in OFCStudies have reported that OFC directly received the projection of VTA DA neuron,so does the release of DA transmitter in OFC increase by MA?What effective detection means can achieve the detection purpose?In this study,the carbon fiber electrode modified by single atom nanase was used to detect the release of DA transmitter in OFC.The results showed that MA significantly increased the release of DA in OFC,suggesting that MA may promote the release of DA,and then mediate the regulation of MA addiction by the OFC-DS circuit.Part Two:The role and mechanism of Tsc22d3 in regulating MA addiction memory in OFC-DS circuit2.1 Differential gene expression was analyzed by RNA sequencing and verifiedRNA-Seq analysis results revealed that 107 genes were increased,including Tsc22d3 and 19 genes were decreased in OFC.The expression of Tsc22d3 was further confirmed by realtime quantitative PCR and Western blot.Immunofluorescence staining was combined with the retrobeads to verify the expression of Tsc22d3 in OFC-DS circuit.The results showed that the mRNA and protein were increased in MA group.And the number of positive cells of Tsc22d3+beads obviously enhanced in MA group,which suggested that the expression of Tsc22d3 in the OFC-DS circuit was significantly increased.2.2 Selectively knocking down Tsc22d3 in the OFC-DS circuit reduced CPP scoresTsc22d3 knockdown viral vectors were constructed by Cre-loxp technology.Cre-expressing retrograde viruses were injected into the DS,and Tsc22d3 knockdown viral vectors(pAAVCMV-DIO-EGFP-Tsc22d3sh)were injected into the OFC.Knocking down of Tsc22d3 in the OFC-DS circuit significantly reduced MA addiction memory retrieval and CPP scores.2.3 Knockdown of Tsc22d3 reduced the frequency of evoked action potential in OFC-DS projection neuronThe projection neuron in the OFC-DS circuit with Tsc22d3 knockdown were recorded by patch clamp.The current stimulation from-50 to-300 pA with a step of 50 pA was given induced a significantly lower frequency of evoked action potentials in the Tsc22d3 knockdown group compared to the Scramble group.2.4 Knockdown of Tsc22d3 reduced burst firing in OFC-DS projection neuronsBurst firing can enhance synaptic transmission and neurotransmitter release probability.Patch clamp recordings showed a significant reduction of burst firings in OFC of the Tsc22d3 knockdown group compared to the Scramble group,indicating a decrease in burst firing in OFC-DS circuit neurons.2.5 Effect of Tsc22d3 knockdown on sEPSC and mEPSC in DSWhole-cell patch-clamp recordings were performed to measure sEPSC and miniature excitatory postsynaptic currents(mEPSC)in DS neurons.Bicuculine and 5 μM CGP52432 were added to the recording solution to block GABA-A receptor and GABA-B receptor,respectively.In addition,the postsynaptic current mEPSC was recorded by adding 10 μM Bicuculine,5 μM CGP52432 and 1 μM TTX to the recording solution.The voltage was holding at-70 mV.The results showed that Tsc22d3 knockdown resulted in reduced frequency of sEPSC and mEPSC in DS neurons.2.6 Effect of Tsc22d3 knockdown on sIPSC and mIPSC in DSWhole-cell patch-clamp recordings were performed to measure spontaneous inhibitory postsynaptic currents(sIPSC)and miniature inhibitory postsynaptic currents(mIPSC)in DS neurons.20 μM CNQX and 10 μM AP-5 were added to the recording solution to block AMPA receptor and NMDA receptor,respectively.mIPSC was recorded after glutamate receptor antagonist 20 μM CNQX,10 μM AP-5 and 1μM TTX were added into the recording solution.The voltage was holding at+10 mV.The results showed that Tsc22d3 knockdown resulted in reduced frequency of sIPSC and mIPSC in DS neurons.2.7 Effect of Tsc22d3 knockdown on synaptic plasticityThe TEM was used to analyze the ultrastructural changes of OFC.The results revealed that Tsc22d3 knockdown in the OFC caused a significant reduction in synaptic density,synaptic vesicle docking density,and postsynaptic density thickness compared to the Scramble group,indicating that Tsc22d3 can regulate the OFC structural plasticity.2.8 MA increased Tsc22d3 SUMOylationStudies have suggested that small ubiquitin-like modifier(SUMO)modification is an important mechanism for Tsc22d3 function.Immunoprecipitation experiments demonstrated that MA significantly increased the level of SUMO-1 modification of Tsc22d3.Local injection of SUMOylation inhibitor 2D08 into the OFC decreased SUMO-1 modification of Tsc22d3 and reduced MA-induced CPP scores,suggesting that Tsc22d3 may participate in the regulation of MA addiction through Tsc22d3 SUMOylation.ConclusionBased on the study,the following conclusions were drawn:(1)This study combined with Cre-loxp and chemogenetics to manipulate the OFC-DS neural circuit,and clarified the influence of OFC-DS neural circuit on MA addiction memory;(2)RNA-Seq sequencing analysis showed that Tsc22d3 up-regulated in MAgroup and Tsc22d3 knockdown in OFC-DS circuit can inhibit the excitability and synaptic transmission effects of projection neurons,meanwhile MA addiction behavior was inhibited;(3)Ubiquititin-like modification of Tsc22d3 may be involved in the regulation of MA addictive memory.