| Part one The effects of running on the changes of depressive-like behaviors and parvalbumin interneuronsin hippocampus of depression modelmiceObjective:Depression is a kind of mental disorder which has a serious impact on human health and social development.At present,the conventional antidepressant therapy still has some short comings,such as slow onset and low response rate.Running exercise has been proved to improve and prevent depressive-like behavior of patients,but its mechanism is still unclear.A great quantity of literatures have pointed out that the attack of depression is strongly linked to?-aminobutyric acid(GABA)systems and parvalbumin(PV~+)interneuronsin hippocampus.Therefore,the purpose of this study is to explore whether running exercise can improve depressive symptoms through protecting PV~+interneurons in hippocampus of depression model mice induced by chronic unpredictable stress(CUS),and to provide data support for finding the target that running exercise treats depression.Methods:Male C57 mice(6-8 weeks old)were raised for 1 week to make them familiar with the new environment.During this period,the health of mice was observed,and the mice with congenital disability were excluded.After the mice were familiar with the new living environment,the baseline of sucrose preference was adjusted and the mice without a preference for sucrose were excluded.Finally,18 mice in the Control group and 134 mice in the CUS group were obtained.The mice in the CUS group were subjected to CUS stress for 5 weeks,and the mice in the CUS group were subjected to the sucrose preference test(SPT)at the same time of each week.At the end of the fifth week,the mice in the CUS group were subjected to Forced swimming test(FST)and Tail suspension test(TST).Behavioral results showed that a total of 42 mice successfully showed depressive-like symptoms.These mice were divided into two groups:20 mice in the CUS Standard group and 22 mice in the CUS+Running group.Mice in CUS+Running group were trained to run on treadmill for 2 weeks.Mice run at a speed of 5 m/min during the first week,and the speed was increased to 8 m/min in the next week after the mice were adapted.The mice ran for 10 min at 5:00 pm from Monday to Friday,and the sucrose preference was detected at the same time every week.During the whole process,5 mice in the Control group were fed in a cage(except the behavioral experiment),and the mice in the CUS Standard group and the CUS+Running group were fed in a single cage.After the end of running intervention,5 mice in each group were anesthetized,and the brain tissue was carefully taken after perfusion and fixation with paraformaldehyde.The hemispheres of mice were dehydrated with sucrose solution of gradient concentrations.The embedded hemispheres were cut into 50-?m-thick serial sections with a cryostatmicrotome.The brain sections containing hippocampal tissue were put into 5 EP tubes in turn to obtain 5 series of equidistant and continuous hippocampal tissue sections.GABA~+cells and PV~+cells were stained with immunohistochemistry.The numbers of PV~+interneurons and the proportion of PV~+interneurons in GABA~+interneurons(PV~+/GABA~+)in the hippocampus were calculated with the stereological method.In addition,another 5 mice were selected in each group.Fresh brain tissue was quickly taken from one hemisphere to peel off the hippocampus in low temperature environment after the mice were sacrificed.Then the tissues of hippocampus were added into the RIPA lysate with PMSF and the tissues were crushed by ultrasonic homogenizer and centrifuged at low temperature.The protein concentration of the supernatant was determined and balanced with the minimum concentration in the samples.Mix the balanced protein solution with 1/4 volume of protein loading buffer,and boiled the protein solution for 8 minutes.The protein levels of PV,GAD65,which is expressed in the synapses of GABA interneuron,and GAD67,which is expressed in the cytoplasm of GABA interneuron in 3 groups of protein solution were detected by Western blot.Results:1.Behavioral tests results.There was no significant difference in percentage of sucrose preference between the CUS groupand the Control group in the first week(p>0.05).After 5 weeks of CUS stress,compared with the Control group,the percentage of sucrose preference of the CUS group was decreased significantly(p<0.001).In FST,compared with the Control group,the immobility time of mice in the CUS group was increased significantly(p<0.001).In TST,compared with the Control group,the immobility time of mice in the CUS group was also increased significantly(p=0.005).After 2 weeks of running,compared with the Control group,the percentage of sucrose preference of the CUS Standard group was decreased significantly(p=0.008).The percentage of sucrose preference of the CUS+Running group was increased significantly when compared with the CUS Standard group(p=0.045).2.Results of stereological counting.Compared with the Control group,the number of PV~+interneurons in the mouse hippocampus of the CUS Standard group was decreased significantly(p=0.013),but there was no significant difference in the number of PV~+interneuronsin the mouse hippocampus between the CUS Standard group and the CUS+Running group(p>0.05).Compared with the Control group,PV~+/GABA~+in the mouse hippocampus of the CUS Standard group was decreased significantly(p=0.014).Compared with the CUS Standard group,PV~+/GABA~+in the mouse hippocampus of the CUS+Running group was increased significantly(p=0.005).3.Results of related protein levels.There were no significant difference in the expression of PV,GAD65 and GAD67 proteins in the mouse hippocampus among the three groups(p>0.05).Conclusions:1.The decrease of percentage of sucrose preference in CUS model mice was significantly alleviated by 2 weeks of running.2.The decrease of PV~+interneuronsin hippocampus might be one of the structural bases of the depressive-like behavior of CUS model mice.3.The increased PV~+/GABA~+indicated that the increase of PV~+interneurons might be one of the mechanisms for the antidepressant effect of running exercise.Part two Running exercise enhances plasticity of PV~+ interneurons through PGC-1 ? in hippocampus to improve the depressive-like behaviors of miceObjective:The studies in the first part confirmed that running exercise could increase the PV+ interneurons in hippocampus might be one of the structural basises to relieve the depressive-like behaviors of CUS model mice,but the mechanism is still unclear.PGC-1? is a transcriptional coactivator of PPAR?,which plays an important role in signal transduction of energy related pathways.Some studies have indicated that running exercise can increase the expression of PGC-1? in hippocampus,and PGC-1? has a potential causal relationship with the onset and treatment of depression.Other studies have shown that PGC-1? plays an important role in regulating PV+ interneurons.The study will explore internal mechanism of running regulating PV+ interneurons in hippocampus in the treatment of depressive-like symptoms in mice,in order to provide an important theoretical basis for the mechanism of running in the treatment of depressive-like symptoms.Methods:1.Detection of PGC-1? protein content in hippocampus of CUS model mice.Five mice were randomly selected from each of the three groups in the first part of the experiment.Fresh brain tissue was quickly taken from one hemisphere to peel off the hippocampus in low temperature environment after the mice were sacrificed.Then the tissues of hippocampus were added into the RIPA lysate with PMSF and the tissues were crushed by ultrasonic homogenizer and centrifuged at low temperature.The protein concentration of the supernatant was determined and balanced with the minimum concentration in the samples.The PGC-1? protein content in the protein solutions of 3 group mice was detected using ELISA kit.2.Steps and follow-up experiments of PGC-1?-knockdown in hippocampus of mice.Male C57 mice(6-8 weeks old)were raised for 1 week to make them familiar with the new environment.During this period,the health of mice was observed,and the mice with congenital disability were excluded.After the mice were familiar with the new living environment,the baseline of sucrose preference was adjusted and the mice without a preference for sucrose were excluded.Finally,30 mice in the Control-N group and 31 mice in the PGC-1?-KD group were obtained.2?l adeno-associated empty virus vector was injected into the bilateral hippocampus of mice in the Control-N group respectively and 2 ?l of adeno-associated knockdown PGC-1? virus was injected into the bilateral hippocampus of mice in the PGC-1?-KD group respectively.The recovery of mice was closely monitored after operation.Mice were fed routinely for 4 weeks after virus injection,and SPT was conducted at a fixed time every week.After the virus was successfully transfected,FST and TST were carried out to detect the depressive-like behavior of mice.Then,half of the mice from the Control-N group and the PGC-1?-KD group were regrouped separately,15 mice in the Control-N+R group and 16 mice in the PGC-1?-KD+R group were obtained.The mice in the Control-N+R group and the PGC-1?-KD+R group were trained to run on treadmill for 4 weeks.Mice run at a speed of 5 m/min during the first week,and the speed was increased to 8 m / min in the next week after the mice were adapted,and the speed was increased to 10 m / min in the third and forth week.The mice ran for 10 min at 5:00 pm from Monday to Friday,and their sucrose preference were detected at the same time every week.The other two groups of mice were fed routinely.After 4 weeks,FST and TST were carried out to detect the depressive-like behaviors of mice in each group.Then,according to the method of part one,the equidistant and continuous hippocampal tissue sections were obtained,and PV immunohistochemical staining was performed.PV+ interneurons in the total and subfields of the mouse hippocampus in the 4 groups were accurately quantified with the stereological method.In addition,a series of sections were arranged in order and sampled again according to 1 / 2 sampling fraction.The sections were put into two EP tubes,one of which was labeled with c Fos/PV(activity of PV+interneurons)by immunofluorescence method,and the numbers of c Fos+/PV+ cells in different subfields of hippocampus were counted.In addition,another 5 mice were selected in each group.Fresh brain tissue was quickly taken from one hemisphere to peel off the hippocampus in low temperature environment after the mice were sacrificed.RNA was extracted from the hippocampus randomly and transcribed into c DNA.The gene expression levels of PV,GAD65 and GAD67 in 4 groups of samples were detected by quantitative PCR.The other side of hippocampus was made into protein solution according to the first part method andthe protein levels of PV,GAD65 and GAD67 in 4 groups of protein solutions were detected by Western blot.Results:1.Results of PGC-1? protein level in hippocampus of 3 groups of mice.Compared with the Control group,the PGC-1 ? proteinlevel in the mouse hippocampus of CUS Standard group was decreased significantly(p = 0.045).After 2 weeks of running,compared with the CUS Standard group,the PGC-1 ? protein level in the mouse hippocampus of CUS + Running group was increased significantly(p < 0.001).2.Behavioral results of mice in 4 groups.There was no significant difference in percentage of sucrose preference between the PGC-1?-KD group and the Control-N group in the first week(p > 0.05).After 4 weeks of virus transfection,compared with Control-N group,the percentage of sucrose preference in PGC-1?-KD group was decreased significantly(p < 0.001).In FST and TST,compared with Control-N group,the immobility time of the PGC-1?-KD group was increased significantly(p = 0.024,p = 0.013).After 4 weeks of running intervention,compared with Control-N group,the percentage of sucrose preference of the PGC-1?-KD group was decreased significantly(p = 0.038).Compared with Control-N+R group,the percentage of sucrose preference of the PGC-1?-KD+R group was also decreased significantly(p = 0.012).There was no significant difference in percentage of sucrose preference between the PGC-1?-KD group and the PGC-1?-KD+R group(p > 0.05).In the FST,compared with Control-N group,the immobility time of the PGC-1?-KD group was increased significantly(p = 0.045).Compared with Control-N+R group,the immobility time of the PGC-1?-KD+R group was increased significantly(p = 0.032).There was no significant difference in the immobility time between the PGC-1?-KD group and the PGC-1?-KD+R group(p > 0.05).In TST,compared with Control-N+R group,the immobility time of the PGC-1?-KD+R group was increased significantly(p = 0.028).3.Results of protein and gene levels.After 4 weeks of running intervention,compared with Control-N group,the gene level of PV in the mouse hippocampus of the PGC-1?-KD group was decreased significantly(p = 0.01).Compared with Control-N+R group the gene level of PV in the mouse hippocampus of the PGC-1?-KD+R group was decreased significantly(p = 0.007).There was no significant difference in the gene level of PV in the mouse hippocampus between the PGC-1?-KD group and the PGC-1?-KD+R group(p > 0.05).The protein levels of PV showed no significant difference among the four groups(p > 0.05).There was no significant difference in the protein and gene levels of GAD65 and GAD67 in mouse hippocampus among the four groups(p > 0.05).4.Results of stereological counting.After 4 weeks of running intervention,compared with Control-N group,the number of PV+ interneurons in the mouse hippocampus of the PGC-1?-KD group was decreased significantly(p < 0.001).Compared with Control-N+R group,the number of PV+ interneurons in the mouse hippocampus of the PGC-1?-KD+R group was also decreased significantly(p = 0.002).There was no significant difference in the number of PV+ interneuronsin the mouse hippocampus between the PGC-1?-KD group and the PGC-1?-KD+R group(p > 0.05).In CA1 and CA3 subfields,compared with Control-N group,the numbers of PV+ interneuronsin the mouse hippocampus of the PGC-1?-KD group was decreased significantly(p = 0.001)(p = 0.017).Compared with Control-N+R group,the numbers of PV+ interneurons in the mouse hippocampus of the PGC-1?-KD+R group was also decreased significantly(p = 0.004)(p = 0.047).There was no significant difference in the number of PV+ interneurons in the mouse hippocampus between the PGC-1?-KD group and the PGC-1?-KD+R group(p > 0.05).However,there was no significant difference in the numbers of PV+ interneurons among the four groups in DG area(p > 0.05).5.Results of Immunofluorescence double-standard of c Fos+/PV+ cells.After 4 weeks of running intervention,compared with Control-N group,the number of c Fos+/PV+ cells in CA1 subfield of mouse hippocampus in the PGC-1?-KD group was decreased significantly(p < 0.001).Compared with Control-N+R group,the number of c Fos+/PV+ cells in CA1 subfield of mouse hippocampus in the PGC-1?-KD+R group was decreased significantly(p = 0.011).Compared with Control-N group,the number of c Fos+/PV+ cells in CA3 subfield of mouse hippocampus in the PGC-1?-KD group was decreased significantly(p = 0.002).Compared with Control-N+R group,the number of c Fos+/PV+ cells in CA3 subfield of mouse hippocampus in the PGC-1?-KD+R group was decreased significantly(p = 0.039).There was no significant difference in the number of c Fos+/PV+ cells in CA3 subfield of mouse hippocampus between the PGC-1?-KD group and the PGC-1?-KD+R group(p > 0.05).However,there was no significant difference in the number of c Fos+/PV+ cells among the four groups in the DG subfield(p > 0.05).Conclusions: 1.Knockdown of PGC-1? in the hippocampus of mice caused depressive-like behavior,which could not be alleviated by running exercise.2.Regional knockdown of PGC-1? in hippocampus resulted in the decrease of the gene level of PV and the number and activity of PV+ interneurons,suggesting that the cause of depressive-like symptoms in mice might be the change in PV+ interneurons in the hippocampus induced by the lack of PGC-1?.3.Running exercise could not increase the gene level of PV and the number and activity of PV+ interneurons after regional knockdown of PGC-1? in hippocampus.Therefore,running exercise might regulate PV+ interneurons through PGC-1? in the hippocampus of mice to reverse depressive-like behaviors. |
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