| Background:Drug addiction,a chronic recurring neuropsychiatric disorder caused by the consumption of psychoactive drugs,has become a global public health and social problem.Uncovering the biological basis of addiction and finding effective treatments is one of the major scientific challenges recognized by scientific communities around the world.When abusers repeatedly use psychoactive drugs,such as methamphetamine(METH),pathological memories are generated when the reward stimulation of the drug is associated with the environment.Once further exposed to drug-related stimulation,negative emotions such as stress and relapse can be induced.Currently,it is broadly accepted that the persistence of addiction memory and craving for drugs are the basic problems of addiction.However,the molecular mechanisms underlying the storage and retrieval of addiction memory remain unclear.Lactate has long been considered a product of energy metabolism.In recent years,more evidence has shown that lactate is not only an energy molecule but also a signaling molecule that is widely involved in many physiological processes such as the immune response,neural development,and synaptic plasticity.Lactate is produced mainly through glycogenolysis and aerobic glycolysis in astrocytes in the brain,transported to the extracellular space via lactate transporter monocarboxylate transporter 1(MCT1)and MCT4,and then enters neurons via MCT2.Studies have shown that lactate plays an important role in regulating the formation of memory.Firstly,glycogenolysis inhibitors such as 1,4-dideoxy-1,4-imino-D-arabinitol(DAB)inhibited glycogenolysis to block lactate production,which completely disrupted long-term memory formation for inhibitory avoidance(IA)behavior in C57BL/6J mice.This process restored memory by supplementing lactate instead of an equivalent amount of glucose,which suggests that lactate has an energy-independent regulatory effect on memory formation.Secondly,lactate significantly increased the expression of Zif268,c-Fos and activity regulated cytoskeleton associated protein(Arc)associated with synaptic plasticity in cultured neurons,while glucose and pyruvic acid had no such effects.Thirdly,knockdown of astrocyte MCT4 by antisense oligodeoxynucleotide(ODN)technology inhibited lactate transport to extracellular fluid,and knockdown of neuronal MCT2 inhibited lactate transport to neurons,both of which can impair consolidation and storage of inhibitory avoidance memory in mice.Lactate injected into the hippocampus only remediated the memory impairment caused by knockdown of MCT4,while there was no such effect for knockdown of MCT2.This finding strongly indicates that lactate transportation between astrocytes and neurons plays a key regulatory role in memory formation.Recently,researchers injected DAB into the basolateral amygdala(BLA)and significantly damaged cocaine-induced memory consolidation.Lactate has an obvious remedial effect by significantly altering the expression of synaptic plasticity related genes in the BLA.The hippocampus was thought to be the earliest region to study memory.The dorsal hippocampus(dHPC)mainly encodes spatial,temporal and conditional information and facilitates the regulation of environment-related reward memory,whereas the ventral hippocampus(vHPC)primarily regulates emotional responses and abstracted information.It is known that dHPC plays an important regulatory role in reward memory associated with the environment.However,the role of lactate in hippocampal neuronal plasticity and METH induced addictive memory has not been reported,especially the underlying mechanism of regulation of addictive memory is still unclear.Nicotinamide adenine dinucleotide(NADH)is an important biological hydrogen carrier and electron donor in living organisms.It is transformed from lactate through the oxidation process of lactate dehydrogenase(LDH)1 and is primarily involved in cellular energy metabolism.The vast majority of NADH is produced in glycolysis and the tricarboxylic acid(TCA)cycle and oxidative phosphorylation is carried out on the mitochondrial membrane to transfer energy to adenosine triphosphate(ATP).Recently,it was found that NADH can simulate the process of lactate in primary cultured cortical neurons,which can promote the expression of the synaptic plasticity related molecules Arc and c-Fos via the N-methyl-D-aspartate receptor(NMDAR)and extracellular signal regulated kinase(ERK)1/2 pathway.This result suggested that NADH can enhance synaptic plasticity.While,the oxidant 5,5’-dithiobis2-nitrobenzoic acid(DTNB)blocked the enhancement of NMDAR signaling by lactate,suggesting that lactate altered redox-state-dependent NMDAR activity during NADH production and thereby promoted the expression of synaptic plasticity related molecules.However,there is currently no report on NADH directly regulating memory,especially the role of signaling molecules associated with lactate and NADH in METH addiction.NADH is likely to be a key downstream molecule in mediating the regulatory effect of lactate on addiction memory,and further studies are urgently needed.Based on the above background,this study aims to establish a conditioned place preference(CPP)paradigm to explore the role and mechanism of lactate and NADH in METH addiction memory.This study is divided into two parts:1)The effect of lactate transport on METH addiction memory and synaptic plasticity;2)Lactate regulates synaptic plasticity and METH addiction memory through NADH and NMDAR-ERK1/2 signaling pathways.Part Ⅰ:Effects of lactate transport on METH addiction memory and hippocampal synaptic plasticityObjective:To investigate the role of lactate transportation in METH addiction memory and to elucidate the role of lactate in the regulation of lactate on synaptic plasticity and memory-related gene expression in the dHPC.This experiment will provide a new perspective on the mechanisms of METH addiction.Methods:C57BL/6J mice were selected and subjected to three cycles of METH treatment(intraperitoneal injection of 2 mg/kg)during the CPP training period(2 d,4 d and 6 d).A behavioral test was conducted on the 7th day to establish a CPP model.The experimental animals were assigned to the control group,METH model group,DAB group and DAB+lactate group.The cannula was embedded in the dHPC of mice and DAB(300 μM,1 μL)or DAB+lactate(100 mM,1 μL)was injected into the cannula during memory consolidation.Firstly,we analyzed the effects of glycogenolysis inhibition performed by DAB and lactate supplementation on METH addiction memory and behavioral sensitization.Then,the effect of lactate on the synaptic plasticity of dHPC was analyzed.The level of Ca2+in neurons was analyzed by intracerebral injection of adenoviral vector containing GCaMP6s,a Ca2+indicator.Golgi staining and transmission electron microscopy were used to observe structural plasticity changes in dHPC neurons at the cellular and ultrastructural levels.Brain slices containing dHPC were obtained by vibrating sections,and the changes in micro excitatory postsynaptic current(mEPSC)and micro inhibitory postsynaptic current(mIPSC)were recorded by patch clamp.The effect of lactate transportation on the expression of genes related to synaptic plasticity was further explored.The expression changes in Npas4,Arc and c-Fos in the dHPC during memory retrieval were detected by real-time qPCR,western blotting and immunofluorescence staining.Finally,MCT1-ODN,MCT2-ODN and MCT4-ODN were injected into the dHPC to knock down MCT1,MCT2 and MCT4,respectively.Then,the corresponding lactate was given.The effects of MCT knockdown on lactate transport inhibition,addictive behaviors and synaptic plasticity related gene expression after lactate supplementation were analyzed.Results:1.1 Inhibition of lactate production in the dHPC disrupts plasticity related memory retrieval,consolidation and reactivationThe CPP test results showed that METH induced mice obviously preferred the METH injection box,and CPP experimental model was successfully prepared.lactate concentration analysis results showed that METH increased the lactate concentration in dHPC during CPP training,and DAB inhibited glycogen decomposition significantly reduced the lactate concentration.DAB was injected into dHPC with intracapsular tube to inhibit lactate production,which significantly reduced CPP scores induced by METH in memory consolidation,extraction and reactivation,and CPP preferences were completely restored after lactate treatment.At the same time,a behavioral sensitization model was prepared in this experiment.The analysis results of autonomous activity video system showed that the spontaneous activity distance of DAB group was significantly reduced compared with the model group,and the blocking effect of DAB was completely reversed in DAB+Lactate group after supplementing with lactate.1.2 Lactate promotes synaptic plasticity of dHPC neuronsGolgi staining and Sholl analysis showed that METH significantly increased the total dendrite count,length and spinous density of dHPC neurons compared with the control group.The inhibition of lactate production by DAB blocked the above structural plasticity changes induced by METH,while supplementation of lactate significantly antagonized the inhibitory effect caused by DAB.The results of transmission electron microscopy showed that,compared with the model group,DAB significantly reduced the number of synapses,docking vesicle density and postsynaptic density length of dHPC neurons,while supplementation of lactate antagonized the inhibitory effect of DAB.1.3 Lactate upregulates the levels of Ca2+in dHPC neuronsAfter injecting an adenovirus vector containing the Ca2+indicator GCaMP6 into the dHPC and analyzing the changes in Ca2+levels in neurons.The results showed that the Ca2+fluorescence intensity of dHPC neurons in METH model mice was significantly higher than that of the control group.DAB reduced METH’s induction effect on calcium ion fluorescence intensity by inhibiting lactate production,while lactate injection significantly restored the fluorescence signal intensity.1.4 Effects of lactate on miniature excitatory postsynaptic currents(mEPSCs)and miniature inhibitory postsynaptic currents(mIPSCs)in dHPC neuronsThe clamp voltage was holding at-70 mV,and the recording solution was added with the GABA receptor antagonist bicuculline with a final concentration of 30 μM and TTX with a concentration of 0.4 μM to record the small excitatory postsynaptic current(mEPSC).The results showed that METH increased the amplitude and frequency of mEPSC,DBA group decreased the amplitude and frequency of mEPSC compared with METH group,and DAB+Lactate group supplemented with lactate reversed the inhibitory effect of DAB.In addition,mIPSC was recorded by adding a final concentration of 10 μM CNQX(AMPA/kainate receptor blockers),50 μM D-AP5(NMDA receptor blocker),and 1 μM TTX to the recording solution.The results showed that the amplitude and frequency of mIPSC were not affected by inhibiting glycogenolysis and blocking the production of lactate by DAB,and the amplitude and frequency of mIPSC were not significantly different from those of DAB group after supplemting lactate.1.5 Lactate promotes the expression of synaptic plasticity related genes in METH treated miceqPCR and Western blot results showed that METH significantly increased the mRNA and protein expression of Npas4,Arc and c-Fos in the dHPC during memory consolidation(2 d,4 d)and memory retrieval(7 d).DAB injected into the dHPC completely inhibited the increased expression of Npas4,Arc and c-Fos,while supplementation with lactate in the dHPC reversed the inhibitory effect of DAB and caused the mRNA and protein expression of Npas4,Arc and c-Fos to rise to the level of the METH group.1.6 Knockdown of MCT1 or MCT4 on CPP preference and the expression of synaptic plasticity related genesUsing antisense oligonucleotide technology,MCT1-ODN and MCT4-ODN were injected into the dHPC to knock down MCT1 and MCT4,and inhibit the transportation of lactate to astrocytes,which significantly reduced the plasticity related CPP score and significantly reduced the mRNA and protein levels of Npas4,Arcand c-Fos.Moreover,CPP preference was restored after the injection of lactate,and the expression of Npas4,Arcand c-Fos was increased.1.7 Knockdown of MCT2 on CPP preference and the expression of synaptic plasticity related genesInjecting the antisense oligonucleotide MCT2-ODN targeting MCT2 into the dHPC inhibited lactate transport from extracellular fluid to neurons and reduced the plasticity related CPP score.qPCR and Western blot results also showed that MCT2-ODN reduced the mRNA and protein expression levels of Npas4,Arc and c-Fos.However,lactate injection did not antagonize the decrease in CPP score or the expression of Npas4,Arc and c-Fos caused by MCT2 knockdown.These results convincingly suggest that lactate transport between astrocytes and neurons plays an important role in regulating METH addiction memory.Conclusion:Both inhibition of lactate production in the dHPC and knockdown of MCTs to block lactate transport inhibited METH addiction memory.Lactate promoted the structural and functional synaptic plasticity of dHPC neurons and increased the expression of plasticity related genes.This part of our study clarified the relationship between lactate and METH addiction memory.Part Ⅱ:Lactate regulates synaptic plasticity and METH addiction through NADH and the NMDAR-ERK1/2 pathwayObjective:To explore the molecular mechanisms by which lactate regulates addiction memory,to elucidate the role of NMDAR-ERK1/2 in lactate-mediated regulation of METH addiction memory and to investigate the role and mechanisms of NADH,a downstream molecule of lactate,in the regulation of METH addiction memory.This study may help to understand the mechanisms of METH addiction and search for targeted intervention molecules.Methods:In vivo,C57BL/6J mice were selected and assigned to the control group,METH group,MK801/U0126 group and MK801/U0126+lactate/NADH group.On one hand,we injected ERK1/2 signaling pathway inhibitor U0126 or NMD A receptor antagonist MK801 into dHPC through cannulars,as well as Lactate or NADH,and analyzed the changes in CPP scores after inhibition of NMDAR and ERK1/2 and supplementation with lactate or NADH.On other hand,the expression changes of synaptic plasticity molecules Npas4,c-Fos and Arc were analyzed by qPCR,Western blot and immunofluorescence staining of dHPC.In vitro,neonatal mouse hippocampal tissue was extracted and purified for cultivation of hippocampal neurons.They were assigned to the control group,lactate/NADH group,MK801/U0126 group and MK801/U0126+lactate/NADH group.MK801(40 μM)or U0126(10 μM)was added to the culture medium to inhibit the activity of NMDAR and ERK1/2,respectively.The expression levels of Npas4,c-Fos,and Arc were detected using qPCR and Western blot.We also analyzed whether lactate(20 mM)and its downstream molecule NADH(20 mM)can regulate the expression of synaptic plasticity molecules through the NMDAR/ERK1/2 pathway.Results:2.1 Lactate regulates plasticity related CPP performance through the NMDAR/ERK1/2 pathwayThe CPP score was significantly reduced,and lactate injection could not reverse this alteration after injecting MK801 into the dHPC to inhibit NMDAR.Similarly,lactate did not restore the decreased plasticity related CPP score caused by U0126-mediated inhibition of ERK1/2.This suggests that lactate regulates addiction memory retrieval through the NMDAR/ERK1/2 pathway.2.2 Lactate promotes the expression of synaptic plasticity related genes through the NMDAR/ERK1/2 pathwayqPCR and Western blot results showed that compared to the control group,METH increased the mRNA and protein levels of Npas4,Arc and c-Fos.MK801 inhibited NMDAR and U0126 inhibited ERK1/2,both of which reduced the promoting effect of METH on the expression of Npas4,Arc and c-Fos.Lactate supplementation failed to increase the expression of these three synaptic plasticity molecules.The results of immunofluorescence staining were consistent with the mRNA and protein detection results.It is suggested that NMDAR-ERK1/2 is a downstream signaling pathway of lactate regulating addiction behavior and synaptic plasticity.2.3 The downstream molecule NADH of lactate promotes the molecular expression of addictive memory and synaptic plasticityIn the lactate remediation experiment,NADH was used instead of lactate for co-injection.CPP behavior analysis results showed that DAB blocked the CPP preference induced by METH during memory extraction,and NADH supplementation significantly restored CPP score,about 88%of CPP score of METH group.qPCR and Western blot results showed that NADH also simulated the blocking effect of lactate antagonistic DAB on the expression of Npas4,Arc and c-Fos.It is suggested that NADH,as a downstream signaling molecule,mimics the action effect of lactate.2.4 NADH promotes addiction memory retrieval and the expression of synaptic plasticity related genes through the NMDAR/ERK1/2 pathwayDoes NADH mimic the action of lactate?Does NMDAR/ERK1/2 promote addictive memory retrieval and synaptic plasticity molecular expression in model mice?Behavioral,qPCR and Western blot results showed that inhibition of NMDAR by MK801 or ERK1/2 by U0126 blocked the role of METH in enhancing memory extraction and the expression of Npas4,c-Fos and Arc molecules after the addition of NADH into dHPC through cannula.The addition of NADH did not restore CPP scores and molecular expression levels.The results of immunofluorescence staining were consistent with the results of qPCR and Western blot.MK801 or U0126 could reduce the fluorescence staining intensity of METH induced above molecules,and the addition of NADH could not improve the fluorescence intensity.These results suggest that NADH mimics lactate and promotes METH addiction memory extraction and synaptic plasticity molecular expression through NMDAR/ERK1/2.2.5 Lactate promotes the expression of Npas4,Arc and c-Fos in a concentration-and time-dependent mannerIn primary cultured hippocampal neurons,on one hand,the effects of different concentrations of lactate were observed.qPCR results showed that the mRNA expression levels of Npas4,c-Fos and Arc gradually increased with the increase of lactate concentration,and the expression levels of three synaptic plasticity molecules were significantly increased with 10 mM and 20 mM lactate.Among them,the expression of Npas4 in hippocampal neurons in 20 mM lactate group was 4.9 times higher than that in the group without lactate.c-Fos increased 6.1 times;Arc increased by 4.3 times.Western blot results of protein were consistent with those of mRNA.On other hand,the effects of different action times of lactate were analyzed.The results of qPCR showed that the expressions of Npas4,Arc and c-Fos increased gradually with the prolongation of lactate action time.At 60 min,lactate reached its peak effect,and the expression level of Npas4 increased by 4.7 times.Arc increased by 5.7 times;c-Fos increased 4.3 times.The expression trend of protein was consistent with that of mRNA.These results indicated that the molecular expression level of synaptic plasticity induced by facial lactate showed a concentration-dependent and time-dependent relationship.2.6 Inhibition of the NMDAR/ERK1/2 pathway blocks the expression of synaptic plasticity related genes promoted by lactateThe addition of MK801 or U0126 to cultured hippocampal neurons inhibited the activity of NMDAR and ERK1/2,respectively,and again verified the mechanism of lactate regulating the expression of synaptic plasticity molecules.The results of qPCR and Western blot showed that compared with the control group,the expressions of Npas4,Arc and c-Fos in MK801 and U0126 groups were not significantly different.The expressions of 3 synaptic plasticity molecules were significantly increased in Lactate group.The molecular expression levels of MK801+Lactate group and U0126+Lactate group were significantly decreased.2.7 NADH mimics the role of lactate in promoting the expression of synaptic plasticity related molecules in cultured hippocampal neuronsDoes NADH also mimic the effect of lactate in cultured hippocampal neurons?We conducted the experiment using NADH instead of lactate.qPCR results showed that the mRNA expression of Npas4(increased by approximately 8-fold),Arc(increased by approximately 5.5-fold)and c-Fos(increased by approximately 7.4-fold)were significantly increased compared to the control group 1 h after NADH treatment.However,the MK801+NADH group and U0126+NADH group significantly suppressed the expression of Npas4,Arc and c-Fos after inhibiting NMDAR and ERK1/2.Western blot analysis was consistent with the PCR results,and both MK801 and U0126 antagonized the effect of NADH.It is suggested that NADH,as a downstream signaling molecule of lactate,also promoted the expression of synaptic plasticity related molecules at the cellular level.2.8 Lactate and NADH enhance neuronal calcium signaling through regulating NR2B subunit of NMDARTo explore how lactate and NADH regulate NMDAR and ERK1/2 activities,we add lactate/NADH,and the NMDAR subunit NR2B blocker ifenprodil or oxidant DTNB to the cultured hippocampal neurons.Calcium imaging results showed that lactate significantly increased calcium signaling in neurons,and ifenprodil or DTNB significantly inhibited calcium signaling.NADH,a downstream molecule of lactate,also increased calcium signaling in neurons,and ifenprodil or DTNB also had inhibitory effects.The protein analysis results of pERK1/2 showed that both lactate and NADH significantly increased the expression of pERK1/2 protein level,and ifenprodil or DTNB also significantly blocked the protein expression of pERK1/2.Conclusion:Lactate promotes addiction memory retrieval through the NMDAR-ERK1/2 signaling pathway,and increases the expressions of Npas4,Arc and c-Fos.NADH,a metabolite of lactate,as a downstream signaling molecule,also regulates the expression of addiction memory and synaptic plasticity molecules through NMDAR-ERK1/2.This part of the study clarifies the molecular mechanism of lactate and its downstream signaling molecule NADH in promoting addiction memory.Summary:In our study,we summarized the following:1)astrocyte-neuron lactate transport plays a key role in regulating METH addiction memory and dHPC synaptic plasticity;2)lactate regulates the plasticity of the dHPC and the expression of memory-related molecules through the NMDAR/ERK1/2 pathway to regulate METH addiction memory retrieval;3)NADH,a downstream molecule of lactate,simulates the role of lactate and participates in the regulation of addiction memory.From the perspective of lactate and NADH,this experiment aims to elucidate the role of lactate-NADH in METH addiction memory retrieval and reveals the role of the lactate-N ADH-NMD AR/ERK1/2 pathway in addiction memory.This experiment also provides a new avenue for finding potential molecular biomarkers to intervene in addiction memory. |
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