| Background:Prenatal nicotine exposure(PNE)is associated with the abnormal behavior of their offsprings,and the mechanism has remianed unclear.The hippocampus,located in the limbic system of the brain,plays an important role in learning,memory,and emotional regulation.At present,it has not been systematic reported that the effects of PNE on the multiple neurocytes at hippocampus developmental stages.Objective:To explore the relationship between PNE-induced behavioral abnormalities in the offspring with the changes of different neurocytes,so as to discover the sensitive time window and sensitive cell population of PNE in the development of the offspring hippocampus.The research provides a theoretical basis for exploring its internal mechanism and explaining the effects of PNE on the offsprings behavior changes.Methods:60 C57BL/6 mice(50 females and 10 males)were randomly caged according to the male to female ratio of 2:1 after one week of adaptive feeding.The day was calculated as gestation 0(G0)days when the vaginal plug was found.From the start G9 day to delivery,nicotine was given with 1.5 mg/kg(twice a day,8:30 and 20:30)by subcutaneous injection,and the control group was given isometric normal saline.The brain samples of offspring mice were collected at postnatal(P)1,7,14,21,and 90 days for HE staining,immunohistochemistry,and Golgi staining experiments,and behavioral experiments were performed before P90 day.The experimental animals were given sufficient food and water at room temperature,natural light,and naturally circulated day and night.Results:(1)Compared with the control group,in the nicotine group,there were no significantly changes of the weight increase rate of pregnant mice and the weight and somatic feature of offspring mice.(2)In behavioral experiments,compared with the control group,in the nicotine group,the escape time was increased(p<0.05,p<0.01),the time of exploring in the quadrant that the platform located was decreased(p<0.01),the nunber of across the reversal platform was decreased(p<0.05);the index of discrimination was decreased(p<0.05);the immobility latency was shortened and the immobility time was increased in forced swimming test(p<0.05,p<0.01);the immobility time was increased in tail suspended test(p<0.05);the activity distance and stand number were increased(p<0.05,p<0.01),and grooming number was reduced(p<0.01);the percentage sucrose preference was decreased(p<0.05).(3)HE staining of hippocampal showed that the size,form and arrangement of cells in the hippocampal region of the nicotine group mice were normal,the development process was continuous,and no obvious pathological changes were observed in the nicotine group.(4)Golgi staining:compared with P7 day,the complexity of neuron dendrites in hippocampal DG,CA1,and CA3 regions were increased gradually in the control and nicotine groups mice,and the peak of dendritic development was occurred at P14-21 days;compared with the control group,in offspring mice of the nicotine group,the number of dendritic intersections and total dendritic length of neurons in the DG region were decreased on P21 day(p<0.05,p<0.01),density of dendritic spines in basal secondary dendrites of neurons was decreased on P7,P14,and P21 days(p<0.05,p<0.01);the number of dendritic intersections and total dendritic length of neurons in CA1 region were decreased at P14 and P21 days(p<0.05,p<0.01),density of dendritic spines in basal secondary dendrites of neurons was decreased on P7,P14,and P21 days(p<0.05,p<0.01),density of dendritic spines in apcial secondary dendrites of neurons was decreased on P21 and P90 days(p<0.05,p<0.01);the number of neuron dendritic intersections in the CA3 region of mice decreased on P21 day(p<0.05),and the total dendritic length of neurons was decreased on P14 and P21 days(p<0.01),density of dendritic spines in basal secondary dendrites of neurons was decreased on days P7,P14,and P21(p<0.05,p<0.01).(5)Compared with P1 day,the proliferating cell marker protein Ki67 in the DG,CA1 and CA3 regions in the hippocampus of the control and nicotine groups were gradually decreased(p<0.01).Compared with the control group,in the nicotine group,the number of Ki67-positive cells in the hippocampal DG region was increased on P1 and P7 days(p<0.01),while decreased on P14,P21,and P90 days(p<0.05,p<0.01);the number of Ki67-positive cells in CA1 region was increased on P1 and P7 days(p<0.05,p<0.01),while decreased on P14 day(p<0.05);the number of Ki67-positive cells in CA3 region was increased on P7 day(p<0.01),while decreased on P14 day(p<0.01).(6)Compared with P1 day,the neuronal precursor cell marker protein DCX in the DG,CA1 and CA3 regions in the hippocampus of the control group were first increases and then decreases with prolonged time(p<0.05,p<0.01),and the peak proliferation period was occurred at P7 day.Compared with the control group,in the nicotine group,the mean optical density of DCX in the hippocampal DG region was increased on P1 day(p<0.01)while decreased on P7,P14,and P21 days(p<0.01),and the number of DCX-positive cells in DG region was decreased at P90 day(p<0.01);the mean optical density of DCX in CAI and CA3 regions was increased on P1 day(p<0.05,P<0.01)and decreased on 14 day(p<0.05,P<0.01).(7)Compared with P1 day,in the control group,the neuronal marker protein NeuN in the DG region was increased with time(p<0.05,p<0.01),in the CA1 region it was first increased then decreased and then increased(p<0.01),and in the CA3 region it was decreased(p<0.01).Compared with the control group,in the nicotine group,the number of NeuN-positive cells in the hippocampal DG region was increased on P1 and P7 days(p<0.05,p<0.01),while decreased on P14 and P21 days(p<0.05,p<0.01);the number of NeuN-positive cells in CA1 and CA3 regions were increased on P1 and P7 days(p<0.01)and decreased on P14 days(p<0.05,P<0.01).(8)Compared with P1 day,the intermediate neuron marker protein CB in the hippocampus of control and nicotine groups were increased in the DG region(p<0.05,p<0.01),while in CA1 and CA3 regions it was decreased(p<0.05,p<0.01).Compared with the control group,in the nicotine group,the number of CB-positive cells in the hippocampal DG region was increased on P7 day(p<0.01),while decreased on P14,P21,and P90 days(p<0.05,p<0.01);the number of CB-positive cells in CA1 region increased on P1 and P7 days(p<0.01),while decreased on P21 and P90 days(p<0.01);the number of CB-positive cells in CA3 region was increased on P1 and P7 days(p<0.01),while decreased on P14 days(p<0.01).(9)Compared with P1 day,the astrocyte marker protein GFAP in the hippocampus of control and nicotine groups were decreased with time in the DG,CA1,and CA3 regions(p<0.05,p<0.01).Compared with the control group,in the nicotine group,the number of GFAP-positive cells in the hippocampal DG region was increased on P1 and P7 days(p<0.05,p<0.01),and in CA1 and CA3 region it was increased on P7 day(p<0.01).(10)Compared with P1 day,the microglia cell marker protein IBal in the hippocampus of the control and nicotine groups were first decreased and then increased in the DG and CA3 regions with time(p<0.05,p<0.01),and in the CA1 region it was increased(p<0.01).Compared with the control group,in the nicotine group,the number of IBal-positive cells in the hippocampal DG region was increased on P1 day(p<0.01)and decreased on P7 day(p<0.01),in CA1 region it was increased on P1 day(p<0.05)and decreased on P14 and P21 days(p<0.05,p<0.01),and in CA3 region it was decreased on 14 day(p<0.01).Conclusion:(1)PNE impairs the learning and memory abilities of adult offspring mice and induces anxiety and depression.(2)PNE continuously inhibits dendritic spines development of hippocampal neurons,the sensitivity time were P14-21 days,and there are regional differences,in CA1 region,the reduced density of dendritic spines in neurons was continued to adulthood,while in DG and CA3 regions it was decreased at P7-21 days and rebacked to normal in adulthood.(3)PNE promotes the nerve proliferation of offspring mice hippocampal in the early postnatal(P1-P7),induces neurogenesis,and subsequently reduces the number of microglia in the CA1 region,impairs neurogenesis in the DG region and reduces the number of interneurons in the CA1 region continuously.(4)PNE may causes emotional and behavioural disorders in adult offspring mice by interference the neuroplasticity of hippocampus in developmental stages,inhibition the neurogenesis in DG region of adult offspring mice,and impairs learning memory function and emotional regulation of hippocampus.(5)The population of sensitive cells in the hippocampus of PNE offspring mice were neuronal precursors,interneurons and microglial cells,and the sensitive periods on changing cell structure and neuron morphology were P7-21 days,which can be used as important object and key periods for the subsequent researches on the signaling pathways,corresponding molecular mechanisms and development of therapeutic drugs of PNE. |
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