CXCR7 Regulates Excitatory Synaptic Transmission In Hippocampal Dentate Granule Cells And Modifies Seizures Activity In Vivo

Part one: CXCR7 regulates dendritic spine and synaptic plasticity in hippocampal dentate granule cellsObjective: Here we investigate the effects of CXCR7 on the growth and morphology of dendritic spines and synaptic plasticity in hippocampal dentate granule cells.Methods: 1.Immunofluorescence analysis was used to detect the expression and distribution of CXCR7 in the hippocampal dentate gyrus(DG)of C57BL/6 mouse.2.Adeno-associated virus(AAV)carrying CXCR7-sh RNA and AAV carrying CXCR7-CDS were constructed.To knock-down and increase the expression of CXCR7 in the hippocampal DG,AAV-CXCR7-sh RNA and AAV-CXCR7-CDS were stereotactically injected into hippocampal DG of C57BL/6 mice,respectively.The expression pattern of CXCR7 was detected by immunofluorescence and western blot.3.Golgi staining was used to detect apical dendritic spines of granular cells in hippocampal DG.4.The excitatory synapses(ESs)and its postsynaptic density(PSD)in hippocampal DG were detected by transmission electron microscope.Results: 1.In hippocampal DG,CXCR7 was mainly expressed in Neu N+ cells in the granular layer.2.Knock-down of CXCR7 inhibited the growth and maturation of dendritic spines: the reductions in number of dendritic spines and in the proportion of mushroom dendritic spines;over-expression of CXCR7 promoted the growth and maturation of dendritic spines: the increases in number of dendritic spines and in the proportion of mushroom dendritic spines.In rescue experiment,the negative effects of CXCR7-sh RNA on the growth and maturation of dendritic spines were attenuated by CXCR7 over-expression.3.Knock-down of CXCR7 reduced the number of excitatory synapses(ESs)and the length and thickness of PSD.CXCR7 over-expression increased the number of ESs and the length and thickness of PSD.In rescue experiment,the negative effects of CXCR7-sh RNA on the number of ESs and the length and thickness of PSD were rescued by CXCR7 over-expression.Conclusion: CXCR7 regulates the growth and morphology of dendritic spines,also the synaptogenesis and synaptic plasticity in hippocampal dentate granule cells.Part two: CXCR7 regulates excitatory synaptic transmission in hippocampal dentate granule cellsObjective: To investigate the effects of CXCR7 on excitatory synaptic transmission in hippocampal dentate granule cells.Methods: 1.To knock-down and increase the expression of CXCR7 in the hippocampal DG,AAV-CXCR7-sh RNA and AAV-CXCR7-CDS were stereotactically injected into hippocampal DG of C57BL/6 mice,respectively.2.Whole-cell patch-clamp recording was used to detect m EPSCs and PP-evoked e EPSCs in hippocampal dentate granular cells.3.Western blot was used to detect expression levels of NR2 A and NR2 B in hippocampus.Results: 1.Knock-down of CXCR7 reduced the frequency and amplitude of NMDAR m EPSCs;over-expression of CXCR7 increased the frequency and amplitude of NMDAR m EPSCs.In rescue experiment,the negative effects of CXCR7-sh RNA on NMDAR m EPSCs were attenuated by CXCR7 over-expression.Neither knock-down of CXCR7 nor over-expression of CXCR7 changed the amplitude and frequency of AMPAR m EPSCs.2.Knock-down of CXCR7 reduced the amplitude of NMDAR e EPSCs;over-expression of CXCR7 increased the amplitude of NMDAR e EPSCs.In rescue experiment,the negative effect of CXCR7-sh RNA on NMDAR e EPSCs was attenuated by CXCR7 over-expression.Neither knock-down of CXCR7 nor over-expression of CXCR7 changed the amplitude of AMPAR e EPSCs.3.Knock-down of CXCR7 reduced the amplitude of NR2 A e EPSCs;over-expression of CXCR7 increased the amplitude of NR2 A e EPSCs.In rescue experiment,the negative effect of CXCR7-sh RNA on NR2 A e EPSCs was attenuated by CXCR7 over-expression.Neither knock-down nor over-expression of CXCR7 changed the amplitude of NR2 B e EPSCs.4.Knock-down of CXCR7 reduced the surface expression of NR2 A and had no effect on the total expression of NR2 A.Over-expression of CXCR7 increased the surface expression of NR2 A and also had no effect on the total expression of NR2 A.In rescue experiment,the down-regulated effect of CXCR7-sh RNA on the surface expression of NR2 A was partially reversed by CXCR7 over-expression.CXCR7 had no significant effect on both surface expression and total expression of NR2 B.Conclusion: CXCR7 promotes PP-evoekd NMDAR-mediated excitatory synaptic transmission in hippocampal dentate granule cells,in which the NR2A-mediated excitatory synaptic transmission is mainly involved.CXCR7 may promote NR2A-mediated synaptic transmission by up-regulating the surface expression of NR2 A.Part three: CXCR7 promotes NR2A-mediated excitatory synaptic transmission through activating ERK1/2Objective: To investigate whether CXCR7 promotes NR2A-mediated excitatory synaptic transmission by activating ERK1/2.Methods: 1.AAV-CXCR7-CDS was stereotactically injected to up-regulate the expression of CXCR7 in the hippocampal DG.2.ERK1/2 phosphorylation inhibitor SL327(50 mg/kg)was administered intraperitoneally to inhibit the phosphorylation of ERK1/2 in mice hippocampus.3.Whole-cell patch-clamp recording was used to detect PP-evoked NR2 A e EPSCs in hippocampal dentate granular cells.4.Western blot was used to detect the phosphorylation level of ERK1/2 and the expression levels of NR2 A and NR2 B in hippocampus.Results: 1.The expression level of CXCR7 was positively related to the phosphorylation level of ERK1/2.Inhibition of CXCR7 down-regulated the phosphorylation of ERK1/2;over-expression of CXCR7 up-regulated the phosphorylation of ERK1/2.2.SL327 partially blocked the up-regulated effect of CXCR7 over-expression on the phosphorylation of ERK1/2.3.The positive effects of CXCR7 on the surface expression of NR2 A and NR2 A e EPSCs were attenuated by SL327.Conclusion: CXCR7 promotes PP-evoekd NR2A-mediated excitatory synaptic transmission by activating ERK1/2.Part four: The effect of CXCR7 on seizures susceptibility in kainic acid mice epilepsy model.Objective: The regulatory effect of CXCR7 on NMDAR-mediated excitatory synaptic transmission in hippocampal dentate granule cells suggests that CXCR7 may have an association with seizure activity.In this part,we will investigate the effect of CXCR7 on seizure susceptibility in kainic acid(KA)-induced epileptic mice model.Methods: 1.The temporal neocortex tissues of patients with temporal lobe epilepsy(TLE)and controls were collected in this study.2.Immunofluorescence and western blot were used to detect the expression and distribution of CXCR7.3.To knock-down and increase the expression of CXCR7 in the hippocampal DG,AAV-CXCR7-sh RNA and AAV-CXCR7-CDS were stereotactically injected into hippocampal DG,respectively.4.SL327(50 mg/kg)was administered intraperitoneally to inhibit the phosphorylation of ERK1/2 in mice hippocampus.5.A local field potential recording electrode was implanted into the hippocampus to record EEG.Twenty m M KA(50 nl)was stereotactically injected into dorsal hippocampus to establish KA-induced epileptic mouse model,and mice with spontaneous recurrent seizures(SRSs)were included in the epilepsy group.Results: 1.The expression of CXCR7 was up-regulated in hippocampal tissues of KA-induced epileptic mice group compared with control mice group.2.Nine controls and 9 TLE patients were included in this study.The expression of CXCR7 was up-regulated in brain tissues of TLE group compared with control group.3.Knock-down of CXCR7 prolonged the latency of SRSs,reduced the number of SRSs and the proportion of SRSs in stage 4-5.Over-expression of CXCR7 shortened the latency of SRSs,increased the number of SRSs and the proportion of SRSs in stage 4-5.The protective effects of CXCR7-sh RNA on seizure activity were weakened by CXCR7 over-expression.4.Inhibition of ERK1/2 phosphorylation by SL327 attenuated the aggravated effect of CXCR7 over-expression on seizure activity.Conclusion:CXCR7 promotes seizure susceptibility.Inhibition of ERK1/2 phosphorylation by SL327 significantly attenuates the promoted effect of CXCR7 over-expression on seizures,suggesting that CXCR7 may promote seizure susceptibility by activating ERK1/2.