EA-induced In Vivo LTD Governs Cerebral Ischemic Tolerance

Ischemic pretreatment is the phenomenon whereby brief periods of sublethal ischemia protect against a subsequent,prolonged,ischemic insult.Our previous study confirms that electroacupuncture?EA?plays a neuroprotective role as a non-ischemic pretreatment^[13].In recent years,many studies have been conducted on how EA pretreatment produces the neuroprotective effects^[2-9],but the exact molecular mechanism underlying this phenomenon has yet to be fully understood.Synaptic long-term depression?LTD?is likely involved in ischemic neuronal death caused by ischemic stroke.It is possible that in vivo LTD alone could mimic ischemic pretreatment to induce endogenous neuroprotection^[10,11].In a study published in Cell in 2012,our Canadian cooperator demonstrates that exogenous cannabinoid induces LTD in dorsal hippocampus?dHPC?CA1^[12].Our previous study demonstrates that endocannabinoids?eCBs?are involved in EA-induced neuroprotection^[13].Therefore,we proposed the hypothesis that EA pretreatment-induced in vivo LTD mediates cerebral ischemic tolerance.In this study,we showed that EA pretreatment induced in vivo LTD at dHPC CA3-CA1 synapses.Moreover,CB₁R antagonist AM281 or global GABAergic CB₁R deletion both blocked EA-elicited LTD and EA-induced neuroprotection against transient global cerebral ischemia.These observations indicate that EA induces in vivo LTD at dHPC CA3-CA1 synapses and neuroprotection against global ischemia through GABAergic CB₁R.Experiment 1:EA pretreatment-induced in vivo LTD at dHPC CA3-CA1Objective:To examine whether EA pretreatment could induce in vivo LTD at dHPC CA3-CA1 synapses.Methods:Male adult C57BL/6 mice were randomly divided into EA and sham EA two groups.EA or sham EA was given for 30min,then in vivo recordings of field excitatory postsynaptic potentials?fEPSP?at CA3-CA1 synapses in anesthetized mice.Results:The in vivo electrophysiological results showed that EA pretreatment induced a decrease of fEPSP slope in dHPC CA3-CA1 compared with baseline levels at 2 h post EA preatreatment?P<0.05?,indicating that EA preatreatment can induce in vivo LTD,whereas sham EA pretreatment for 30 min didn't have this effect.Conclusions:EA pretreatment induces in vivo LTD at dHPC CA3-CA1 synapses.Experiment 2:EA pretreatment-induced in vivo LTD at dHPC CA3-CA1 synapses is required for ischemic toleranceObjective:To explore whether EA pretreatment-induced in vivo LTD at dHPC CA3-CA1synapses could mediate ischemic tolerance in global cerebral ischemia.Methods:Male adult C57BL/6 mice were randomly divided into four groups:Control,GCI,EA+GCI and Sham EA+GCI,20 min bilateral common carotid arteries occlusion,the positive cells of NeuN and Fluoro-Jade B?FJB?staining were detected respectively in the bilateral dHPC CA1 of animals 72 h after reperfusion.Results:NeuN and FJB staining and counting results showed that EA pretreatment significantly increased the number of NeuN-positive surviving neurons?P<0.01?and reduced the number of FJB-positive degeneration neurons?P<0.01?compared with the group of Ischemia and Sham EA+Ischemia.Conclusions:EA pretreatment-induced in vivo LTD at dHPC CA3-CA1 synapses mediates ischemic tolerance in dHPC CA1 in global cerebral ischemia.Experiment 3:The CB₁R antagonist AM281 blocks EA pretreatment-induced in vivo LTD at dHPC CA3-CA1Objective:To investigate whether CB₁R mediates the in vivo LTD at dHPC CA3-CA1induced by EA pretreatment.Methods:Male adult C57BL/6 mice were randomly divided into Vehicle+EA and AM281+EA two groups.We injected the selective CB₁R antagonist AM281 or vehicle to anesthetized mice 15 min prior to EA pretreatment,then in vivo recordings of fEPSP at dHPC CA3-CA1 synapses in anesthetized mice.Results:The in vivo electrophysiological results showed that EA pretreatment-induced in vivo LTD at dHPC CA3-CA1 was almost completely blocked by AM281?P<0.05?,but not affected by the vehicle.Conclusions:EA pretreatment induces in vivo LTD at dHPC CA3-CA1 by activating CB₁R.Experiment 4:The CB₁R antagonist AM281 blocks ischemic tolerance of EA pretreatment-induced in vivo LTD at dHPC CA3-CA1 in global cerebral ischemiaObjective:To investigate whether CB₁R invloves in EA pretreatment-induced in vivo LTD at dHPC CA3-CA1 synapses mediates ischemic tolerance in global cerebral ischemia.Methods:Male adult C57BL/6 mice were randomly divided into Vehicle+EA and AM281+EA two groups,20 min bilateral common carotid arteries occlusion,the positive cells of NeuN and FJB staining were detected in the bilateral dHPC CA1 of animals 72 h after reperfusion.Results:NeuN and FJB staining and counting results showed that AM281 significantly decreased the number of NeuN-positive surviving neurons?P<0.01?and increased the number of FJB-positive degeneration neurons?P<0.01?compared with the group of Vehicle+EA,that is,CB₁R antagonist AM281 blocked EA pretreatment-induced neuroprotective in global cerebral ischemia.Conclusions:CB₁R invloves in EA pretreatment-induced in vivo LTD at dHPC CA3-CA1synapses mediates ischemic tolerance in global cerebral ischemia.Experiment 5:GABAergic CB₁R mediates EA pretreatment-induced in vivo LTD at dHPC CA3-CA1Objective:To identify the cell specific of CB₁R in EA pretreatment-elicited in vivo LTD at dHPC CA3-CA1.Methods:Three cell-specific CB₁R conditional knockout mice were used.CB₁R-floxed mice were crossed either with the GAD2-iCreERT2,VGLUT1-iCreERT2 and GFAP-CreERT2 mutant mice to generate the GAD2-CB₁R-KO,VGLUT1-CB₁R-KO and GFAP-CB₁R-KO mouse line respectively.EA pretreatment was given for 30 min,then in vivo fEPSP was record at dHPC CA3-CA1 synapses in anesthetized mice.Results:The in vivo electrophysiological results showed that the specific deletion of GABAergic CB₁R abolished EA pretreatment-induced in vivo LTD at dHPC CA3-CA1?P<0.01?,but specific deletion of glutamatergic and astroglial CB₁R didn't influence EA pretreatment-induced in vivo LTD.Conclusions:EA pretreatment-induced in vivo LTD at dHPC CA3-CA1 requires GABAergic CB₁R but not glutamatergic or astroglial CB₁R.Experiment 6:EA pretreatment-induced in vivo LTD mediates ischemic tolerance through GABAergic CB₁R at dHPC CA3-CA1Objective:To investigate whether EA pretreatment-induced in vivo LTD mediates neuroprotection by activating GABAergic CB₁R at dHPC CA3-CA1.Methods:Male adult GABAergic CB₁R knockout mice and wild-type littermates were used?GAD2-CB₁R-KO and GAD2-CB₁R-WT?.After 30 min EA pretreatment,20 min bilateral common carotid arteries occlusion was induced,the positive cells of NeuN and FJB staining were detected in the bilateral dHPC CA1 of animals 72 h after reperfusion.Results:NeuN and FJB staining and counting results showed that the number of NeuN-positive surviving neurons decreased?P<0.01?and the number of FJB-positive degeneration neurons increased?P<0.01?after EA pretreatment 30 min in GAD2-CB₁R-KO animals compared with the group of GAD2-CB₁R-WT animals.These results showed that systemic knockout GABAergic CB₁R blocked the neuroprotection of EA pretreatment-induced in vivo LTD at dHPC CA3-CA1.Conclusions:GABAergic CB₁R participates in the ischemic tolerance of EA pretreatment-induced in vivo LTD at dHPC CA3-CA1.Experiment 7:EA pretreatment induces increase of GABA in dHPC CA1Objective:To investigate the effect of EA pretreatment on GABA level in dHPC CA1region.Methods:Male adult C57BL/6 mice were randomly divided into sham EA and EA two group.After 30 min sham EA or EA pretreatment,in vivo microdialysis was performed to detect extracellular GABA content in the dHPC CA1.Results:The results of in vivo microdialysis showed that EA pretreatment gradually increased the content of GABA in dPHC CA1 region and reached the maximum at the end of EA 30 min?P<0.01?,then the content of GABA gradually decreased to the baseline level within 30 min after the end of EA,however,there was no significant change in the GABA content in hippocampal CA1 region of the group of sham EA.Conclusions:EA pretreatment increases the content of GABA in hippocampal CA1region.Experiment 8:Knockout GABAergic CB₁R blocks induction of GABA increase in dHPC CA1 induced by EA pretreatmentObjective:To investigate whether EA pretreatment change the level of GABA through GABAergic CB₁R in dHPC CA1.Methods:Male adult GABAergic CB₁R knockout mice and wild-type littermates were used?GAD2-CB₁R-KO and GAD2-CB₁R-WT?.After 30 min EA pretreatment,in vivo microdialysis technique was applied to detect extracellular GABA content in the dHPC CA1.Results:The results of in vivo microdialysis showed that EA pretreatment-induced the increase of GABA in dHPC CA1 disappeared in GAD2-CB₁R-KO animals compared with the group of GAD2-CB₁R-WT animals?P<0.01?.Conclusions:EA pretreatment increases the content of GABA through GABAergic CB₁R in dHPC CA1.Experiment 9:EA pretreatment induces in vivo LTD without altering spatial working memoryObjective:To explore whether EA pretreatment-induced in vivo LTD impairs spatial working memory.Methods:Male adult rats were randomly divided into the following three groups:Control,Sham EA and EA,T-maze test was applied after 30 min sham EA or EA administration to detect spatial working memory in rats.Results:The results of T-maze test showed that there was no significant difference in the accuracy of the target arm selection between the three groups of rats?P>0.05?.Conclusions:EA pretreatment induces in vivo LTD without impairing spatial working memory in rats.Experiment 10:Systemic GABAergic CB₁R deletion doesn't affect spontaneous motor function and anxiety levelsObjective:To investigate whether GABAergic CB₁R deletion affects spontaneous motor and anxiety levels in mice.Methods:Male adult GABAergic CB₁R knockout mice and wild-type littermates were used?GAD2-CB₁R-KO and GAD2-CB₁R-WT?,the open field and elevated plus behavior test were applied to detect spontaneous motor function and anxiety levels in mice.Results:1.The results of open field behavior test showed that there was no significant difference in the number of line cross,the percentage of time entering the open arm/total time and the number of entering the central area in the two groups?P>0.05?;2.The results of elevated plus behavior test showed that there was no significant difference in the percentage of time entering the open arm/total time,the percentage of time entering the central area/total time,and the number of entering the arm opening and the central area?P>0.05?.Conclusions:Systemic knockout GABAergic CB₁R doesn't significant influence spontaneous motor function and anxiety levels in mice.