| Hippocampus is an important structure in the limbic system and is related to the abilityof learning,memory and cognitive,especially spatial learning and short-term memory.The hippocampal formation consists of hippocampus,dentate gyrus(DG),inferior support,and hippocampal debris surrounding the carcass.The hippocampus is one of the birthplaces of adult neural stem cells(NSCs).NSCs in the lower dentate gyrus of hippocampal dentate gyrus can generate new progeny cells through proliferating and dividing,integrate into the local neural circuit,and participate in the learning and memory process.The hippocampus can be divided into CA1,CA2,CA3,and CA4 regions based on the differences in cortical development and fiber arrangement.Cytological studies have shown that the hippocampal head is mainly formedby folded CA1 region,while the CA1 region is extremely sensitive to hypoxia and other damage,so the hippocampal CA1 region,also known as the vulnerable area,is the most susceptible to lesions.It has been reported that there is fiber connection between the CA1 and DG regions,but the effect of the CA1 region excitation on the DG region and its mechanism remains unclear.Since the glutamatergic nerve fiber projection is the most important nerve fiber projection from DG region to CA1 region,the aim of this study is:(1)To observe the effect of excitement state of glutamate neurons in CA1 region on proliferation of NSCs in DG region through optical genetic techniques;(2)To study the ability of learning and memory in mice after specific modulation of glutamatergic neuronal excitation in the CA1 region;(3)To study the expression of CXCR4,a key gene in SDF1?/CXCR4/CXCR7 signal pathway,in hippocampus through western blotting.This research may provide the laboratory evidence for the clinical regulation of neural stem cell generation,proliferation,and differentiation.ObjectiveTo study the effect of glutamatergic neurons in hippocampal CA1 region on the proliferation of NSCs in DG region and the ability of learning and memory through optical genetics.Methods1.Experiment animals.One-month-old male C57BL/6NCrl mice.2.Photosensitive virus transfection and grouping.After anesthetization,the viral vectors carrying photosensitive genes ChR2,e NpHR and their controls were injected into the mice based on the CA1 location of the hippocampus CA1.ChR2 group,ChR2-control group,eNpHR group and eNpHR-control group were respectively stimulated or inhibited glutamate neurons in CA1 area.3.Virus expression detection.After 2 weeks of virus injection,frozen sections were continuously taken and the expression of ChR2 virus and eNpHR virus in hippocampal CA1 region were detected under fluorescence microscope.4.Morris water maze concealed platform experiment.Two weeks after the virus injection,the Morris Water Maze Concealed Platform experiment was conducted to train mice to learn to find platforms hidden in water for a total of 6 days.5.Photogenetic stimulation.After the end of the Morris Water Maze Concealed Platform experiment,on the 7th day,optical genetic technology was used to activate photoreactive channel proteins ChR2 and eNpHR with blue and green light,respectively.6.Morris water maze space exploration experiment.After 6 h,12 h,24 h,and 72 h of the photosensitive channel protein activation,the Morris water maze space exploration experiments were performed to record the number of animals entering the target quadrant and the percentage of time spent in the target quadrant as detection indicators of spatial memory ability.7.Immunofluorescence detection of regeneration and proliferation of NSCs in DG region.After the Morris water maze test,the heart was perfused with 4% paraformaldehyde,the brain was separated and frozen sections were taken.Immunofluorescence was used to detect the number of neural stem cells in hippocampal DG region.8.Western blotting detection of CXC chemokine receptor(CXCR4)expression.After Morris water maze test,hippocampal tissue was separated rapidly.The expression of CXCR4,a factor in SDF1?/CXCR4/CXCR7 signaling pathway,was detected by Western blotting.9.Statistical analysis.SPSS21.0 was used for statistical analysis.Data were expressed as mean ± standard error(Mean ± SEM),which were analyzed by one-way ANOVA or independent sample t-test.P < 0.05 is considered statistically significant.Results1.Two weeks after the virus injection,ChR2 virus and eNpHR virus expression in the hippocampal CA1 region was detected under fluorescence microscope,showed that the virus was well expressed,indicating that the light sensitive virus was successfully transfected.2.The experimental results of the Morris water maze concealment platform showed that there was no significant difference in escape latency between the ChR2 group and the eNpHR group compared with the control groups before light stimulation(P > 0.05).3.Morris water maze space exploration experiments were conducted at 6 h,12 h,24 h,and 72 h after the genetic blue light was used to excite glutamatergic neurons in hippocampal CA1 region.At 24 h and 72 h after light stimulation,the number of crossing the platform times of ChR2 group(5.50±1.38,6.67±2.06)significantly increased compared with ChR2-control group(2.67±1.03)(*P < 0.05,***P < 0.001).At 6 h,12 h,24 h,and 72 h after light stimulation,the percentage of ChR2 group in the target quadrant(31.04±7.90%,33.08 ± 7.92%,37.47 ± 7.89%,44.20 ± 8.36%)was significantly increased compared with the ChR2-control group(17.89 ± 5.91%)(*P < 0.05,*P < 0.05,**P < 0.01,***P < 0.001).4.Morris water maze space exploration experiments were conducted at 6 h,12 h,24 h,and 72 h after the genetic green light was used to inhibit glutamatergic neurons in hippocampal CA1 region.The results showed that at 24 h and 72 h after light stimulation the number of crossing the platform times of the eNpHR group(1.0 ± 0.63,0.67 ± 0.52)was significantly lower than that of the eNpHR-control group(2.47 ± 0.93)(**P < 0.01,***P < 0.001).AT 12 h,24 h,and 72 h after light stimulation,the percentage of the eNpHR group in the target quadrant(8.60±2.82%,5.79±2.19%,4.92±2.49%)was significantly lower than that of the eNpHR-control group(18.91±5.79%)(**P < 0.01,***P < 0.001,***P < 0.001).5.When glutamatergic neurons in hippocampal CA1 region were excited,the number of Nestin positive cells detected at 12 h,24 h and 72 h after light stimulation(56.40±16.26,70.51±19.41,111.71±18.33)were significantly increased compared with the ChR2-control group(48.09±9.87)(*P < 0.05,***P < 0.001,***P < 0.001).6.When glutamatergic neurons in hippocampal CA1 region were inhibited,the number of Nestin positive cells detected at 12 h,24 h and 72 h after light stimulation(40.13±15.71,27.79±8.96,14.33±8.81)were significantly decreased compared with the eNpHR-control group(47.04±9.56)(**P < 0.01,***P < 0.001,***P < 0.001).7.When glutamatergic neurons in hippocampal CA1 region were excited,Western blotting results showed that the expression of CXCR4 in ChR2 group at 24 h and 72 h after stimulation was significantly increased than that in ChR2-control group(*P < 0.05,*P < 0.05).8.After inhibiting glutamatergic neurons in hippocampal CA1 region,Western blotting results showed that the expression of CXCR4 in eNpHR group at 24 h and 72 h after stimulation was significantly decreased compared with the e NpHR-control group(*P < 0.05,***P < 0.001).Conclusion1.Specific excitation of CA1 glutamatergic neurons promotes the proliferation of NSCs in the DG region,and increase the ability of learning and memory.2.Specific inhibition of CA1 glutamatergic neurons restrain the proliferation of NSCs in the DG area,and weaken the ability of learning and memory.3.The effect of glutamatergic neurons in hippocampal CA1 region on the proliferation of NSCs in DG region may be related to the receptor protein CXCR4 of stromal cell derived factor(SDF1?). |
PDF Full Text Request