Effects Of NMDAR And CaMKⅡα On Learning And Memory Impairment In Depressed Rats With Different Charge By Modified Electroconvulsive Shock

BackgroundIn 2017,322 million people worldwide suffered from depression.In2019,the prevalence of depression in China ranged from 3.0 percent to 4.3percent,which is mainly manifested by an increase in negative thinking（such as low mood）and a decrease in positive emotions（such as anhedonia）.Modified electroconvulsive therapy（MECT）,as one of the causes of overworked depression,has the highest response rate and causes rapid results.However,the learning and memory function is impaired,which seriously affects the postoperative life of the patients and aggravates the psychological burden.The stimulus charge of MECT is one of the most significant factors affecting cognitive function.Clinical observation found that gender,age and previous times of treatment were all factors that caused the difference in the threshold of convulsion,and some patients needed to increase the charge to achieve the antidepressant effect,which would lead to more serious memory impairment.Therefore,it is urgent to explore the mechanism of learning and memory impairment of MECT charge dependence in order to find appropriate protective measures to improve the quality of life of patients after treatment.Previous studies have shown that learning and memory processes are dependent on synaptic plasticity in CA1 region of hippocampus,and changes in synaptic excitability can affect LTP induction threshold,in which Ca²⁺exchange plays an important role.N-methyl-D-aspartic acid receptor（NMDAR）can change Ca²⁺permeability by regulating the components of each subunit.Ca²⁺/calmodulin dependent protein kinase IIα（Ca MKⅡα）activates and phosphorylates Ca MKⅡα,thereby regulating the induction and maintenance of LTP.However,it is still unknown whether different charge of MECT can cause different degrees of learning and memory impairment by affecting NMDAR structure and Ca MKⅡαactivation level.ObjectiveMECS（modified electroconvulsive shock,MECT experimental animal model）was worked with gradients of increased charge.To observe the effects of novelty exploration behavior and spatial learning and memory ability in depressed rats,and to explore the internal mechanism of synaptic plasticity related to Ca MKⅡαand NMDAR,in order to find the molecular mechanism of power-related learning and memory impairment,and to provide a basis for the protection of spatial learning dysfunction after high-charge MECS.MethodsHealthy Sprague-Dawley（SD）rats aged 2～3 months（180～250g）were fed in specific pathogen free（SPF）environment.After 7 days of adaptive feeding,all the rats were treated with chronic unpredictable mild stress（CUMS）to establish the depression model.During the modeling period,the rats were weighed on days 0,7,14,21 and 28.After 28 days of modeling,the sucrose preference test（SPT）and the open field test（OFT）were used to evaluate depression status.Rats with sucrose preference percentage（SPP）≤65%were randomly divided into 5 groups（n=12）,namely group M0,group M60,group M120,group M180,and group M240for corresponding charge MECS.The process is as follows,intraperitoneal injection（I.P.）of propofol（80mg/kg）was used for anesthesia.After the recalibration reflex disappeared,A biphasic rectangular wave with amplitude of 0.8A,wave width of 1.5ms and 125 pulses/s was used.The MECS stimulus of 0m C,60m C,120m C,180m C and 240m C was generated with the duration of 0s,0.4s,0.8s,1.2s and 1.6s,respectively,and was processed continuously for 7 days.After MECS,SPT and OFT were used to assess the differences in depressive behavior improvement,and the Morris water maze test（MWM）was used to assess spatial learning and memory ability.Immediately after the end of the behavioral experiment,the rats were sacrificed under pentobarbital sodium deep anesthesia to obtain hippocampal tissue.Then the protein expressions of Ca MKⅡα,p-Thr305-Ca MKⅡα,N-methyl-D-aspartate Receptor 1（Glu N1）,Glu N2A and Glu N2B in hippocampus were detected by Western blot.Co-immunoprecipitation（CO-IP）was used to detect the binding amount of Ca MKⅡαand Glu N2B subunit in hippocampus after MECS.Results（1）Body weight On Day 0,Day 7,Day 14 and Day 21,body weight of rats in each group increased gradually;On the 28th day（i.e.,the 4th week of modeling）,the body weight of each group decreased compared with the 21st day（P<0.05）,showing a negative growth trend,and there was no statistically significant difference between groups（P>0.05）.（2）Sucrose preference test After CUMS,SPP of depression model group（M0,M60,M120,M180 and M240 groups）had no difference among all groups（P>0.05）,and significantly decreased compared with before modeling（P<0.05）,and met the assessment criteria of≤65%.After MECS,the SPP of M120,M180 and M240 groups was higher than that of M0 and M60 groups（P<0.05）,and there was no statistical difference among other groups（P>0.05）.（3）Open field test After CUMS,the crossing number and the rearing number of rats in each group had no difference（P>0.05）,which was significantly reduced compared with the two groups before CUMS（P<0.05）.After treatment with MECS,compared with M0 and M60 groups,the crossing number in the other 3 groups was significantly increased（P<0.05）.In terms of rearing number,M120,M180 and M240 group were higher than those in M0 group,and M180 and M240 group were higher than those in M60 group（P<0.05）.There was no significant difference among other groups.（4）Morris water maze test There was no significant difference in swimming speed among the groups.With the extension of learning time,the escape latency（EL）of 5 consecutive days gradually shortened.In addition,EL increased with the increase of charge,but there was no significant difference between M0 group and M60 group,M180 group and M240 group（P<0.05）.After removing the platform on the 6th day,it was observed that the space exploration time（SET）shortened with the increase of charge（P<0.05）,and there was no significant difference between M0and M60,and between M120 and M180.（5）Protein expression of Ca MKⅡαand p-Thr305-Ca MKII in hippocampus Western blot showed that there was no significant difference in the expression of Ca MKⅡαin hippocampus among groups（P>0.05）,the expression of p-Thr305-Ca MKⅡαin hippocampus in M60,M120 and M180 groups was significantly lower than that in M0 and M240groups,and the expression of p-Thr305-Ca MKⅡαin M60 and M120groups was lower than that in M180 group（P<0.05）,and there was no significant difference among other groups.（6）Protein expression of Glu N1,Glu N2A and Glu N2B in hippocampus Western blot analysis showed that the expression of Glu N1in M0 and M60 groups was higher than that in M120 and M180 groups,and the expression of Glu N1 in the latter two groups was higher than that in M240 group（P<0.001）.The expression of Glu N2A and Glu N2B protein in hippocampus showed the same trend.Compared with M0 group,the other four groups were significantly decreased（P<0.05）.The expression of M240 group was the least,and the expression of M180 group was also significantly decreased compared with M60 and M120 groups.There was no significant difference among the other groups.（7）The expression of PSD-95 protein in hippocampus Compared with M0 and M240 groups,the expression of PSD-95 in other three groups was significantly increased,and the expression of PSD-95 in M60 and M120 groups was higher than that in M180 group（P<0.05）.（8）Co-expression of Ca MKⅡαwith Glu N2B subunits in hippocampus The binding amount of Glu N2B and Ca MKⅡαvaries with the charge.The result of CO-IP showed that Ca MKⅡαbinding to Glu N2B was the most in the group of M60 and M120,followed by M180,and the least binding amount was found in M0 and M240（P<0.05）.These results showed that depression can hinder Glu N2B and Ca MKⅡαbinding,and the same effect can be found in MECS with high electric quantity.Conclusions（1）The degree of learning and memory impairment in depressed rats was gradually increased with the increase of MECS charge,which was related to the increase of p-T305-Ca MKII expression in the hippocampus and the inhibition of Ca MKⅡα/Glu N2B complex formation.（2）The aggravation of learning and memory impairment caused by the increase of MECS in rats was regulated by the decrease of Glu N1,Glu N2A and Glu N2B expression levels to varying degrees.