Prenatal Dexamethasone Exposure Induced Alterations In Neurobehavior And Hippocampal Glutamatergic System Balance In Female Offspring Rats

Objective: In the developing brain,glutamatergic system is involved in cell survival and differentiation,maintaining the balance of the neurotransmitters,the conduction of signals between synapses and the occurrence of neurobehavior.Epidemiological investigations have suggested that periodic use of dexamethasone during pregnancy is a risk factor for abnormal behavior in offspring,but the potential mechanism remains unclear.In this study,we investigated the changes in the glutamatergic system and neurobehavior in female offspring with prenatal dexamethasone exposure(PDE),and explored the intrauterine programming mechanisms.Methods:(1)Animal experiments: Pregnant Wistar rats were subcutaneously injected with 0.2mg/kg·d dexamethasone from gestational day(GD)9 to GD20.One group of pregnant rats was sacrificed at GD20 to collect fetal hippocampus,the other pregnant rats were naturally delivered.At postnatal week(PW)26,the behavior of two groups of animals were tested,including elevated plus maze,the open field test and the Morris water maze,the animals was subsequently anesthetized and sacrificed.Morphological changes in the whole brain and hippocampus were observed by hematoxylin-eosin(H&E)staining.The ultrastructural changes of hippocampus were observed by transmission electron microscope.According to Golgi staining,the changes of the branching and length of the dendrites and dendritic spines in the cornu ammonis 1(CA1)and dentate gyrus(DG)of the hippocampus were observed.Concentrations of glutamate(Glu)and γ-aminobutyric acid(GABA)in serum and hippocampus were detected by biochemical kit.We used Glu labeled the excitatory transmitter glutamate,glutamate decarboxylase 67(GAD67)labeled the GABA,neuronal nuclear antigen(Neu N)labeled neurons,the co-localization expression and quantitative analysis of immunofluorescence were used to observe the changes of Glu and GABA in the hippocampus of PDE offspring.Further,the changes of glucocorticoid receptor(GR)/histone deacetylase 2(HDAC2)/brain-derived neurotrophic factor(BDNF)pathway and the genes expression of glutamatergic system were observed by real-time quantitative polymerase chain reaction(RT-q PCR),Western blotting and immunofluorescence.In addition,the histones acetylation level of HDAC2 was observed using Chromatin immunoprecipitation(Ch IP).(2)In vitro cell line experiments: Different concentrations of dexamethasone(0.02,0.1,0.5 μM)were used to treat rat fetal hippocampal H19-7 neuron cell lines for three days,or coadministered 0.5μM dexamethasone with BDNF,GR antagonists RU486,HDAC2 inhibitor Rom for three days.HE staining and flow cytometry were used to observe the cell apoptosis of dexamethasone treated with different concentrations.RT-q PCR,Ch IP,Western blotting and immunofluorescence were used to observe the changes of GR/HDAC2/BDNF pathway,the leval of HDAC2 histones acetylation and the change of glutamatergic system in cells treated with 0.5 μM dexamethasone or different interventions.Results:(1)The behavior of adult offspring rats: In the elevated plus maze,time spent in the open arms and the number of entries into the open arms were obviously lower in the PDE group,while the duration of time in the closed arms was obviously prolonged(P < 0.05,P < 0.01);In the open field test,female rats in the PDE group spent significantly less time and travelled less distance in the central area,as well as exhibiting fewer total hindlimb rearings,which suggested poor motor ability and weakened activity(P < 0.05,P < 0.01).Compared with the control group,in the Morris water maze(MWM),female adult rats in the PDE group showed a prolonged escape latency on days3-5 and an increased distance travelled on days 4-5 during the training stage(P < 0.05,P < 0.01),while during the hidden platform test,they showed weakened spatial localization ability and prolonged escape latency and increased movement distance(P < 0.05,P < 0.01).In the probe test,the localization ability of the PDE rats was weakened,and the movement distance was not obviously changed,but the number of platform crossings and the time spent in the target quadrant were both significantly decreased(P < 0.05,P < 0.01).(2)The morphology and ultrastructure of adult offspring hippocampus: Compared with the control group,H&E staining showed that there was no significant change in the overall brain area of the PDE group,but under × 200 magnification with a light microscope,partial neurons in hippocampal CA1 and CA3 regions were triangular and irregularly shaped,had hyperchromatic nuclei and pyknosis,as well as uneven staining.Transmission electron microscopy(TEM)showed that mitochondria were mildly swollen,the Golgi apparatus was slightly hypertrophic and the endoplasmic reticulum was expanded in the PDE group.Furthermore,Golgi staining showed that there were significant changes in the representative images of neurons in the PDE group.The total lengths of the dendrites in the CA1 and DG regions of the hippocampus were decreased,and the number of dendritic branches and dendritic spines in the CA1 region were also significantly reduced in the PDE group(P < 0.05,P < 0.01).(3)The hippocampal glutamatergic system and GR/HDAC2/BDNF pathway in adult offspring rats: Compared with the control group,serum and hippocampal Glu and GABA contents were significantly increased in PDE female adult offspring(P < 0.05,P < 0.01,P < 0.001),however,the m RNA expression of hippocampal Glu decarboxylase 67(GAD67)and vesicle-associated membrane protein(VAMP)1/2 was significantly increased,and the m RNA expression of vesicular glutamate transporter 1(VGlu T1)and excitatory amino acid transporter 3(EAAT3)was significantly reduced(P < 0.05,P < 0.01).Immunofluorescence results showed that the expression of Glu and GAD67 in the CA1 region of the hippocampus were significantly increased in the PDE group(P < 0.05,P <0.01).In addition,the m RNA expression of HDAC1,BDNF exon I(BDNF I)and BDNF exon VI(BDNF VI)were not significantly changed,but the m RNA expression of HDAC2 was significantly increased,and total BDNF and BDNF IV were significantly reduced(P < 0.05,P < 0.01).The results of immunofluorescence and quantitative analysis showed that the expression of GR and HDAC2 in the CA1 region of the hippocampus in PDE group was significantly increased(P < 0.01),and the expression of BDNF was significantly reduced(P < 0.05).The results of chromatin immunoprecipitation quantitative polymerase chain reaction(Ch IP-PCR)showed that the histone H3 lysine 14 acetylation(H3K14ac)level of BDNF IV in PDE group was decreased,and the levels of H3K9 ac and H3K27 ac were not significantly changed(P < 0.01).(4)The hippocampal morphology,glutamatergic system and GR/HDAC2/BDNF pathway in fetal rats: Compared with the control group,the overall brain and hippocampal areas of female fetal rats in the PDE group decreased significantly,under × 200 magnifications with a light microscope,we observed some hemorrhagic damage in hippocampal CA1 and DG areas of female fetal rats with PDE.The hippocampal Glu and GABA contents were significantly increased in the PDE group,and the m RNA expression of GAD67 and VAMP1/2 were significantly increased,while the expression of VGlu T1 and EAAT3 were significantly reduced(P < 0.05,P < 0.01).The results of immunofluorescence showed that the expression of Glu and GAD67 in the CA1 region of the fetal hippocampus was significantly increased in the PDE group(P < 0.05,P < 0.01).Further,the m RNA expression of GR and HDAC2 was significantly increased,the m RNA expression of HDAC1,BDNF I and BDNF VI was not significantly changed,and the m RNA expression of BDNF and BDNF IV was significantly reduced in female fetuses in the PDE group(P < 0.05,P < 0.01,P < 0.001).The results of immunohistochemistry showed that the expression of HDAC2 in the CA1 and DG areas of the fetal hippocampus in PDE group was significantly increased,and the expression of BDNF was significantly reduced(P < 0.05,P < 0.01).The results of immunofluorescence showed a significant increase in the expression of GR and HDAC2 in the fetal hippocampal CA1 region of the PDE group(P < 0.05,P < 0.01),while the expression of BDNF was significantly reduced(P < 0.01).The results of Western blotting showed that GR and HDAC2 protein levels significantly increased,while the protein level of fetal hippocampal BDNF significantly decreased in the PDE group(P < 0.05,P <0.01).The H3K14 ac level of BDNF IV was decreased,and the levels of H3K9 ac and H3K27 ac were not significantly changed(P < 0.05).(5)The glutamatergic system and GR/HDAC2/BDNF pathway in the H19-7 cell line: Compared with the control group,VAMP1/2 m RNA expression in H19-7 neurons increased in a concentration-dependent manner treated with dexamethasone,and the expression of GAD67 significantly increased.VGlu T1 and EAAT3 expression significantly decreased when cells were treated with 0.5 μM dexamethasone(P < 0.05,P < 0.01);Glu and GABA content in cellular and extracellular fluid was significantly increased(P < 0.05,P < 0.01).Furthermore,GR and HDAC2 m RNA expression was significantly increased with dexamethasone treatment,while BDNF and BDNF IV expression decreased in a concentration-dependent manner(P < 0.05,P < 0.01).The results of immunofluorescence and Western blotting showed that GR and HDAC2 expression significantly increased with 0.5 μM dexamethasone treatment,while the expression of BDNF significantly decreased(P < 0.05,P < 0.01,P < 0.001).The results of Ch IP-PCR showed that the binding of GR or HDAC2 in the BDNF promoter region significantly increased when cells were treated with 0.5 μM dexamethasone,and the H3K14 ac level of BDNF IV decreased,while the levels of H3K9 ac and K3K27 ac showed no significant changes(P < 0.05).(6)Effect of exogenous supplementary BDNF,GR antagonist RU486 or HDAC2 inhibitor Rom on dexamethasone action in the H19-7 cell line: Compared with the control group,cotreatment with BDNF and dexamethasone reversed the decrease in VGlu T1 and EAAT3 and the increase in GAD67 caused by dexamethasone,while treatment with BDNF alone had no significant effect on the expression of the abovementioned genes(P < 0.05,P < 0.01).The results of immunofluorescence showed that dexamethasone treatment significantly increased the expression of Glu and GAD67,while cotreatment with BDNF reversed this increase in Glu and GAD67 expression caused by dexamethasone;treatment with BDNF alone had no significant effect on the expression of Glu and GAD67(P < 0.01,P < 0.001).Furthermore,the cotreatment of RU486 or Rom with dexamethasone reversed the decrease in BDNF and BDNF IV caused by dexamethasone,and cotreatment of RU486 with dexamethasone also reversed the increase in HDAC2 caused by dexamethasone;treatment with RU486 or Rom alone had no significant effect on the expression of the abovementioned genes(P <0.01,P < 0.001).The results of immunofluorescence and Western blotting showed that 0.5 μM dexamethasone treatment significantly increased HDAC2 expression and decreased BDNF expression.Cotreatment with RU486 reversed the increase in HDAC2 and the decrease in BDNF caused by dexamethasone,while cotreatment with Rom reversed the decrease in BDNF caused by dexamethasone;treatment with RU486 or Rom alone had no significant effect on the expression of HDAC2 or BDNF(P < 0.05,P < 0.01).With 0.5 μM dexamethasone treatment,the H3K14 ac level of BDNF IV significantly decreased,and cotreatment with RU486 or Rom could reverse the decrease in H3K14 ac caused by dexamethasone,while treatment with RU486 or Rom alone had no significant effect on the acetylation level(P < 0.05,P < 0.01).Conclusion: PDE can increase the expression of HDAC2 by activating hippocampal GR in offspring,causing reduced the H3K14 ac level of BDNF IV and expression of BDNF.Downregulation of BDNF expression can further induce intracellular and extracellular Glu accumulation by reducing the expression of presynaptic VGlu T1 and postsynaptic EAAT3,and further increases conversion to GABA,this process results in upregulation of GABA,which causes an excitation/inhibition(E/I)signal imbalance and neuronal death in the hippocampus,ultimately resulting in a series of neurobehavioral changes in the adult offspring.The reduction in BDNF signaling and the accumulation of Glu may be the pathological basis for abnormal neurobehavior in adult female offspring with PDE.