| Epidemiological studies have demonstrated that people with intrauterine growth retardation (IUGR) are more likely to develop metabolic disorders (e.g. obesity, hypertension and type 2 diabetes) in adulthood. This association between IUGR and adult metabolic disorders isdescribed as "programming" hypothesis that adverse factors during gestation could permanently change the structures and functions of the multiple fetal tissues then cause dysfunctions and diseases in adulthood. The hypothalamic-pituitary-adrenal (HPA) axis is an important neuroendocrine axis involved in metabolism and stress response. Intrauterine programming of the HPA axis might have life-long effects on neuroendocrine functions. Epidemiological, clinical and animal studies have consistently shown that an adverse intrauterine environment usually results in a low baseline and enhanced sensitivity of the HPA axis in adulthood. Furthermore, several studies have revealed that the low baseline and enhanced sensitivity of the HPA axis is linked to the development of metabolic or mental diseases (e.g. diabetes, non-alcoholic fatty liver disease and depression) mediated by intrauterine events.Alcohol consumption is common in daily life. A recent survey showed an obvious increasing trend of alcohol abuse in young women over recent decades and prenatal ethanol exposure (PEE) rate for newborns is increasing each year. Approximately 50% of women on reproductive age are heavy drinkers in North America and 10%-15% of pregnant women drink alcoholic beverages. Alcohol consumption during pregnancy is a definitive cause for fetal developmental toxicity. Numerous studies have shown that PEE increases the incidence of diseases in the offspring, including IUGR, neuropsychiatric disorders and adult metabolic syndromes. Our previous work have introduced a mechanism of "HPA axis-associated neuroendocrine metabolic programming alteration" to explain the increased susceptibility to metabolic diseases found in IUGR offspring rats with PEE. We also found that PEE causes IUGR by inhibiting the development of fetal HPA axis and altering the peripheral glucose and lipid metabolism. These changes were maintained until adulthood and are characterized by low baseline and enhanced sensitivity of the HPA axis in adult offspring rats, meanwhile, the dysfunction of the HPA axis can further cause adult metabolic disease by interfering with peripheral glucose and lipid metabolism under high-fat diet. However, the specific mechanism responsible for the intrauterine programming of hypersensitivity of the HPA axis in the PEE offspring rats remains unclear.The hypothalamic paraventricular nucleus (PVN) controls the activity of the HPA axis.The hippocampus has the feedback regulation on PVN so that suppresses the HPA axis. Balance between excitatory neurotransmitter glutamate (Glu) and inhibitory neurotransmitter gamma aminobutyric acid (GABA) has an important role in the regulation of activities of both hypothalamus and hippocampus. L-glutamic acid decarboxylase (GAD) plays a vital role in the regulation of Glu and GABA. Remodeling of glutamatergic and GABAergic afferent inputs contributes as an underlying mechanism to the altered excitatory/inhibitory balance in the PVN.Binding of GC to corticoid receptor (GR) is not only related to the GC levels in the corticoid, but also to the expression levels of the 11β-hydroxysteroid dehydrogenase (11β-HSDs), which modulates GC metabolism in the fetal tissues. Studies reported that GC can inhibit the expression and secretion of insulin-like growth factor 1 (IGF1). IGF1 signaling pathway is involved in the regulation of the development, proliferation, differentiation, synapse formation, neuronal protection, inhibiting neuronal apoptosis, neural regeneration and energy metabolism of the tissues in the uterus. Previous studies have demonstrated that prenatal ethanol exposure leads to fetal over-exposure to maternal GC, thus PEE may induce synaptic plasticity and function-change of hippocampus and hypothalamus by affecting GC metabolism system (11βHSDs/GR/C/EBPa) and IGF1 signaling pathway.In this study, we will focus on the development of HPA axis in IUGR offspring rats induced by PEE before and after birth with/without chronic stress (CS) and explore the intrauterine programming mechanism via measuring theglutamatergic and GABAergic neurons differentiation of hypothalamus and the feedback capacity of hippocampus. This study is of important theoretical value and practical significance to further clarify the development toxicity mechanism of ethanol, and to better understand the intrauterine programming of chronic adult diseases (such as nervous and mental disease, metabolic diseases)-associated with the dysfunction of HPA axis, and improve the quality of life of the population.PART ONEIntrauterine origin of low baseline and enhanced sensitivity of HPA axis in IUGR offspring induced by prenatal ethanol exposureObjective:To demonstrate PEE lead to the low baseline and enhanced sensitivity of HPA axis originated in utero on PEE-induced IUGR rat model.Methods:Pregnant rats were randomly divided into control and PEE groups. From GD9 to GD20, rats in the PEE group were administered ethanol 4 g/kg day by oral gavage. Rats in control group were given the same volume of distilled water. On GD20, some pregnant rats were sacrificed and live fetuses were quickly removed to weigh and distinguish gender. IUGR rates were also calculated. Fetal blood corticosterone (CORT) concentrations were measured by enzyme linked immunosorbent assay (ELISA) kit. Real-time PCR was used to quantify hypothalamic mRNA expressions of genes, including corticotrophin (CRH), arginine vasopressin (AVP), paired box 6 (Pax6), T-box brain protein 2 (Tbr2), vesicular glutamate transporter 2 (VGluT2), PSD95 (post-synaptic density-95), Ca2+/calmodulin-dependent protein kinase II-a (aCaMKII), mammalian achaete-scute homolog-1 (Mashl), GAD65 and reelin (Reln). The other pregnant rats were subjected to spontaneously deliver. The postnatal growth trajectories of offspring rats were recorded and body weight gain rates were calculated. The offspring rats were fed by normal diet after weaning. On postnatal week (PW) 11, each groups were randomly divided into swimming and non-swimming groups. The swimming groups were all exposed to a 2 weeks cold water swimming at 5 to 7℃ for 5 min. In PW13, the offspring were sacrificed to collect blood and hypothalamus tissues rapidly. The serum adrenocorticotrophic hormone (ACTH) concentration was measured by radioimmunoassay kit following the manufacturer’s protocol. The serum CORT concentration was determined by ELISA assay. Additionally, real-time PCR was used to quantify the mRNA expression of hypothalamic CRH, AVP, VGluT2, PSD95, aCaMKII, GAD65 and Reln.Results:Male offspring:(1) Adult offspring rats: ①Body weights and IUGR rates:The mean bodyweights of offspring in the PEE group were lower than control on postnatal day (PD) 1 (P<0.01). The bodyweights of offspring from PEE group were also lower than control (P<0.01) with no obvious change of bodyweight gain rates during PW1-PW12. ②HPA axisactivity:The serum ACTH, CORT levels and the hypothalamic AVP mRNA expression of adult PEE rats before CS from the PEE group were significantly lower than control (P<0.05). However, serum ACTH, CORT concentrations and hypothalamic CRH, AVP mRNA expression levels were all significantly increased in the PEE group when compared with control after CS (P<0.05,P<0.01). ③Hypothalamic glutamatergic/GABAergic neuronal proteins:Both hypothalamic GAD65 and Reln were markedly decreased in the PEE group relative to control before and after CS (P<0.05). Furthermore, the VGluT2/GAD65 expression ratios were increased in the PEE group when compared with control before and after CS (P<0.05). However, the expression levels of VGluT2, PSD95, and aCaMKII were not changed after PEE. (2) Fetal rats:①Body weights and serum CORT concentrations:When compared to control, the mean body weights were significantly decreased (P<0.01), while the IUGR rates and the serum CORT concentrations were significantly increased (P<0.01) in the PEE group.②HPA axis activity and hypothalamic glutamatergic/GABAergic differentiation: The mRNA expression levels of hypothalamic AVP, GAD65, Reln and Mashl, but not CRH and VGluT2, were decreased from PEE group than that in control (P<0.05). While that of Pax6 (P=0.078), Tbr2 (P=0.079), PSD95 (P=0.070) and αCaMKⅡ (p=0.082) had decline tendency. The expression ratios of VGluT2/GAD65 were significantly increased in the PEE group (P<0.05).Female offspring:(1) Adult offspring rats:①Body weights and IUGR rates:The mean bodyweights of offspring in the PEE group were lower than control on PD1 (P<0.01). The bodyweights of offspring from PEE group were also lower than control with no obvious change of bodyweight gain rates during PW1-PW12 (P<0.01).②HPA axis activity:The serum levels of ACTH and CORT, as well as the mRNA expression levels of hypothalamic CRH and AVP in adult PEE rats before CS were significantly lower than control (P<0.05, P<0.01). However, the serum levels of ACTH and CORT, as well as the mRNA expression levels of hypothalamic CRH and AVP were all significantly increased in the PEE group when compared to control after CS (P<0.05, P<0.01).③Hypothalamic glutamatergic/GABAergic neuronal proteins:When compared with control, both hypothalamic GAD65 and Reln expression levels were markedly decreased in the PEE group before and after CS (P<0.05). Furthermore, the expression ratios of VGluT2/GAD65 were increased from the PEE group when compared to control before and after CS (P<0.05). However, the expressions of VGluT2, PSD95, and aCaMKII were unchanged after PEE. (2) Fetal rats: ①Body weights and serum CORT concentrations:When compared with control, the mean body weights were significantly decreased (P<0.01), while the IUGR rates and the serum CORT concentrations were significantly increased (P<0.01) in the PEE group. ②HPA axis activity and hypothalamic glutamatergic/GABAergic differentiation:PEE significantly decreased the expression levels of GABAergic transcriptional factor Mashl in fetal hypothalamus, as well as the GAD65 and Reln (P<0.05, P<0.01), but not Pax6, Tbr2, VGluT2, PSD95 and α-CaMKII. The expression ratios of VGluT2/GAD65 were significantly increased in the PEE group when compared to control (P<0.05).Conclusion:The low baseline and enhanced sensitivity of the HPA axis in both male and female IUGR offspring rats induced by PEE were characterized by intrauterine programming, presenting inhibition of development (decreased CRH and AVP) and increased potential excitatory ability (imbalance of glutamatergic and GABAergic differentiation and increased VGluT2/GAD65 ratio) of fetal hypothalamus. All of the above would contribute to the low baseline (shown by decreased levels of serum ACTH as well as hypothalamic CRH and AVP expression) and enhanced sensitivity (shown by increased levels of serum ACTH and CORT as well as hypothalamic CRH and AVP expression, imbalance of glutamatergic and GABAergic differentiation and increased VGluT2/GAD65 expression ratio) of the HPA axis in adult offspring.PART TWOAn intrauterine programming mechanism of low baseline of the hypothalamic-pituitary-adrenal axis in IUGR offspring rats induced by prenatal ethanol exposureObjective:To observe and compare the metabolic activation of GC, the expression levels of GAD67 and the balance of excitatory/inhibitory neurotransmitters, IGF1 signaling pathway, synaptic plasticity and morphologyin hypothalamus of IUGR offspring induced by PEE before and after birth, and to investigate the intrauterine programming mechanism of low baseline of HPA axis in PEE offspring rats.Methods:The pregnant rats treatment was the same as Part one. On GD20, some pregnant rats were sacrificed and live fetuses were quickly removed to weigh and distinguish gender. Three whole brains from each group were fixed. Real-time PCR was used to quantify fetal hypothalamic 11βHSD1,11βHSD2, GR, CCAAT enhancer binding protein (C/EBPa), GAD67, IGF1, IGF1R, protien kinase B (AKT1), synaptotagmin 1, synapsin 1, gamma-aminobutyric acid Aβ1 receptors (GABA-A-β1R) and GABA-A-β2R mRNA expression. Fetal hypothalamic histology and ultrastructure were examined by Hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). The protein expression levels of GAD67 in the hypothalamus were measured by immunohistochemistry. The activity of hypothalamic Glu/GABA neurons was examined by immunofluorescence microscopy. The other pregnant rats were subjected to spontaneously delivery. The offspring rats were all fed with normal diet. On PD85, the offspring rats were sacrificed to collect the hypothalamus tissues rapidly. Three whole brains from each group were fixed. Real-time PCR was used to quantify hypothalamic 11βHSD1,11βHSD2, GR, C/EBPa, GAD67, IGF1, IGF1R, AKT1, synaptotagmin 1, synapsin 1, GABA-A-β1R and GABA-A-β2R mRNA expression. Biochemical assay and ELISA kit were applied to measure hypothalamic neurotransmitter as Glu and GABA contents, respectively. Hypothalamic histology was examined by HE staining. The protein expression levels of GAD67 in the hypothalamus were measured by immunohistochemistry. The activity of hypothalamic Glu/GABA neurons was examined by immunofluorescence microscopy.Results:Male offspring:(1) Adult offspring rats:① Hypothalamic GC-metabolic activation system:When compared with control, the expression ratios of 11βHSD1/11βHSD2 in hypothalamus were significantly increased (P<0.05), while the mRNA expression levels of hypothalamic C/EBPa were significantly decreased (P<0.05), but GR showed no obvious change in the PEE groups. ②Hypothalamic GAD67 expression and neurotransmitter contents:The mRNA and protein expression levels of hypothalamic GAD67 were significantly increased in PEE group when compared with control (P<0.05, P<0.01). The contents of neurotransmitter Glu were significantly reduced (P<0.05), while the contents of GABA were increased significantly (P<0.05) in PEE group than that of control. ③ Hypothalamic IGF1 signaling, synaptic plasticity and morphology:When compared with control, the serum IGF1 levels present an increasing trend (P=0.08), while the mRNA expression levels of hypothalamic IGF1, AKT1, synaptotagmin 1, synapsin 1, GABA-A-β1R were increased significantly (P<0.05), but there were no obvious change of hypothalamic morphology in the PEE group. (2) Fetal rats:①Hypothalamic GC-metabolic activation system:When compared with control, the mRNA expression levels of hypothalamic 11βHSD2, C/EBPa was increased significantly (P<0.01) and GR showed increasing tendency (P=0.062), the mRNA expression levels of 11βHSD1 and the expression ratios of 11βHSD1/11βHSD2 had no obvious change in the PEE groups. ②Hypothalamic GAD67 expression and glutamatergic/GABAergic differentiation:The mRNA and protein expression levels of hypothalamic GAD67 were significantly increased in PEE group when compared with control (P<0.05, P<0.01). The activity of glutamatergic neurons were significantly reduced (P<0.05), while the activity of GABAergic neurons were increased significantly (P<0.01) in PEE group than that of control.③Hypothalamic IGF1 signaling, synaptic plasticity and morphology:When compared with control, the levels of serum IGF1 and hypothalamic IGF1, IGF1R, AKT1, synaptotagmin 1, synapsin 1, GABA-A-β1R, GABA-A-β2R mRNA expression were increased significantly in the PEE group (P<0.05, P<0.01). There were no obvious morphologic change but visible dilatation of the endoplasmic reticulum was observed in neurons of fetal hypothalamus in the PEE group.Female offspring:(1) Adult offspring rats: ①Hypothalamic GC-metabolic activation system:When compared to control, the GR mRNA expression levels and the 11βHSD1/11βHSD2 ratios were significantly increased (P<0.05), while the mRNA expression levels of PEE hypothalamic 11βHSD1 had decline tendency (P=0.060). ② Hypothalamic GAD67 expression and neurotransmitter contents:The mRNA and protein expression levels of hypothalamic GAD67 were significantly increased in PEE group when compared to the control (P<0.05). The contents of neurotransmitter Glu were significantly reduced (P<0.05), while the contents of GABA were increased significantly (P<0.05) from PEE group than that in control. ③Hypothalamic IGF1 signaling, synaptic plasticity and morphology. When compared with control, the serum IGF1 levels were decreased significantly (P<0.05), while the mRNA expression levels of hypothalamic IGF1, AKT1, synaptotagmin 1, synapsin 1, GABA-A-β1R, GABA-A-β2R and the morphology of hypothalamus presented no obvious change in the PEE group. (2) Fetal rats:①Hypothalamic GC-metabolic activation system:When compared with control, the mRNA expression levels of hypothalamic 11βHSD1, GR, C/EBPα and the expression ratios of 11βHSD1/11βHSD2 were observed increased significantly in the PEE group (P<0.05,P<0.01).②Hypothalamic GAD67 expressions and glutamatergic/GABAergic differentiation:The mRNA and protein expression levels of hypothalamic GAD67 were significantly increased in PEE group when compared with control (P<0.05). The activity of glutamatergic neurons were significantly reduced (P<0.05) while the activity of GABAergic neurons were increased significantly (P<0.05) in PEE group than that of control. ③Hypothalamic IGF1 signaling, synaptic plasticity and morphology:When compared with control, the levels of serum IGF1 and the mRNA expression levels of hypothalamic IGF1, synaptotagmin 1, synapsin 1 were decreased significantly in the PEE group. The hypothalamic AKT1 mRNA expression levels had decline tendency (P=0.067), but the mRNA expression levels of hypothalamic GABA-A-β1R and GABA-A-β2R showed no obvious change. There were no obvious morphologic change but visible hypertrophy of the Golgi body and dilatation of the endoplasmic reticulum were observed in neurons from fetal hypothalamus of the PEE group.Conclusion:PEE caused fetal higher GC level and induced gender specific-effect on hypothalamic 11βHSDs/GR/C/EBPs system:for male offspring, metabolic activation and inactivation of GC common existed in fetal hypothalamic neurons, enhanced expression-programming of GAD67 was mediated by highly-expressed GR in hypothalamus and increased the conversion of Glu into GABA, thus inhibiting the activity and function-development of hypothalamic CRH neurons; for female offspring, enhanced expression-programming of GAD67 and decreased expression-programming of IGF1 signaling mediated by metabolic activation of GC inhibited the function and development of hypothalamic CRH neurons. These changes finally lead to decreased expression levels of hypothalamic CRH, AVP and low baseline of HPA axis in PEE offspring.PART THREEAn intrauterine programming mechanism of enhanced sensitivity of the hypothalamic-pituitary-adrenal axis in offspring rats inducedby prenatal ethanol exposureObjective:To observe and compare the metabolic activation of GC, the expression levels of GAD67 and the balance of excitatory/inhibitory neurotransmitters, IGF1 signaling pathway, synaptic plasticity and morphology in hippocampus of IUGR offspring induced by PEE before and after birth, and to investigate the intrauterine programming mechanism of enhanced sensitivity of HPA in PEE offspring rats.Methods:The pregnant rats manipulation was the same as Part one. On GD20, some pregnant rats were sacrificed and live fetuses were quickly removed to weigh and distinguish gender. Three whole brains from each group were fixed. Real-time PCR was used to quantify fetal hippocampal 110HSD1,11(3HSD2, C/EBPa, GAD67, GR, IGF1, IGF1R, AKT1, synaptotagmin 1, synapsin 1, NR1, NR2A and NR2B mRNA expression. Use bisulfite genomic sequencing PCR (BSP) to detect and analysis the methylation pattern of GAD67 promoter. Fetal hippocampus histology and ultrastructure were examined by HE staining and TEM. The protein expression levels of hippocampal GAD67 were measured by immunohistochemistry assay. The activity of hippocampal glutamatergic/GABAergic neurons were examined by immunofluorescence microscopy. The other pregnant rats were subjected to spontaneously deliver. The offspring rats were all fed with normal fiet. On PD85, all offspring rats were sacrificed to collect the hippocampus tissues rapidly one hour after chronic stress. Three whole brains from each group were fixed. Real-time PCR was used to quantify hippocampal 11βHSD1, 11βHSD2, C/EBPa, GAD67, GR, IGF1, IGF1R, AKT1, synaptotagmin 1, synapsin 1, NR1, NR2A and NR2B mRNA expression. Hippocampal histology was examined by HE staining. Biochemical assay and ELISA assay were applied to measure hippocampal neurotransmitters as Glu and GABA contents, respectively. The protein expression levels of hippocampal GAD67 were measured by immunohistochemistry assay. The activity of hippocampal glutamatergic/GABAergic neurons was examined by immunofluorescence microscopy.Results:Male offspring:(1) Adult offspring rats: ① Hippocampal GC-metabolic activation system:When compared with control, hippocampal GC-metabolic activation system was observed no obvious change in the PEE group before CS. Hippocampal 11βHSD2, GR and C/EBPa expression levels were increased significantly in the PEE group than that of control (P<0.05, P<0.01). ②Hippocampal GAD67 expressions, epigenetic modifications, neurotransmitter contents and the activity of glutamatergic/GABAergic neurons:Before CS, the mRNA levels of GAD67 in hippocampus and protein expression levels of GAD67 in dentate gyrus (DG) and cornu ammonis (CA3) subfields were significantly increased in PEE group than that of control (P<0.05, P<0.01); BSP results showed that, when comparing with control, the total methylation rate of -219～-4 bp region of GAD67 promoter was significantly increased in the PEE group (P<0.01); The contents of neurotransmitter Glu, GABA in PEE hippocampus were shown no obvious change relative to control. After CS, the expression levels of GAD67 and the protein levels of GAD67 in DG and CA3 subfields were significantly increased in PEE group than that of control (P<0.05,P<0.01); When compared with control, the content of neurotransmitter Glu and the activity of hippocampal glutamatergic neurons was decreased significantly, while the content of neurotransmitter GABA and the activity of hippocampal GABAergic neurons was increased significantly in the PEE group (P<0.05). ③Hippocampal IGF1 signaling, synaptic plasticity expression and morphology:Before CS, the mRNA expression levels of hippocampal IGF1R, AKT1 and synapsinl were increased significantly (P<0.05, P<0.01) while the mRNA expression levels of hippocampal NR1, NR2A and NR2B were decreased significantly (P<0.05, P<0.01), but there were no obvious change of hippocampal IGF1 mRNA expression levels in the PEE group. There were only a few nuclei of neurons in the CA3 of PEE group that were dense and darkly stained. After CS, the mRNA expression levels of hippocampal IGF1, IGF1R and AKT1 were decreased while synapsinl was increased significantly in the PEE group (P<0.05). There were still only a few nuclei of neurons in the CA3 subfield of PEE group that were dense and darkly stained. (2) Fetal rats: ①Hippocampal GC-metabolic activation system: When compared with control, hippocampal 11βHSD2, GR, C/EBPα were increased significantly (P<0.05, P<0.01), while the 11βHSD1/11βHSD2 ratios were decreased significantly (P<0.05) in the PEE group. ② Hippocampal GAD67 expression, neurotransmitter contents and the activity of glutamatergic/GABAergic neurons:The mRNA levels of GAD67 in hippocampus and protein expression levels of GAD67 in DG and CA3 subfields were significantly increased in PEE group than that of control (P<0.05). Furthermore, BSP results showed that, when comparing with control, the total methylation rate of -219～4 bp region of GAD67 promoter was significantly increased in the PEE group (P<0.05). When compared with control, the activity of hippocampal glutamatergic neurons was decreased significantly (P<0.05), while the activity of hippocampal GABAergic neurons was increased significantly (P<0.05) in the PEE group. ③Hippocampal IGF1 signaling, synaptic plasticity and morphology:When compared with control, the mRNA expression levels of hippocampal IGF1, IGF1R, AKT1, synapsinl, NR1, NR2A and NR2B were increased significantly (P<0.05, P<0.01) in the PEE group. There were only a few nuclei of neurons in the CA3 of PEE group that were dense and darkly stained. After CS, the mRNA expression levels of hippocampal IGF1, IGF1R and AKT1 were decreased while synapsin1 was increased significantly in the PEE group (P<0.05, P<0.01). The morphology of the PEE hippocampus showed no obvious change, but visible hypertrophy of the Golgi body and dilatation of the endoplasmic reticulum were observed in a few neurons from fetal hypothalamus of the PEE group.Female offspring:(1) Adult offspring rats: ①Hippocampal GC-metabolic activation system:When compared with control, hippocampal 11βHSD1/11βHSD2 ratios, but not GR and C/EBPa, were decreased significantly in the PEE group before CS (P<0.05). After CS, hippocampal 11βHSD1, GR, C/EBPa expression levels and 11βHSD1/11βHSD2 ratios were increased significantly in PEE group than those of control (P<0.05, P<0.01).②Hippocampal GAD67 expression, neurotransmitter contents and the activity of glutamatergic/GABAergic neurons:Before CS, the mRNA and protein expression levels of GAD67, the contents of neurotransmitter and the activity of glutamatergic or GABAergic neurons in hippocampus were observed no obvious change in PEE group relative to control. ③Hippocampal IGF1 signaling, synaptic plasticity and morphology:Before CS, the mRNA expression levels of hippocampal IGF1 and NR1 were decreased significantly (P<0.05), while the mRNA expression levels of hippocampal synapsinl were increased significantly (P<0.05, P<0.01), hippocampal AKT1 and NR2A mRNA expression levels showed a decreasing tendency in the PEE group relative to control (P=0.063, P=0.082). There were only a few nuclei of neurons in the CA3 of PEE group that were dense and darkly stained. After CS, the mRNA expression levels of hippocampal IGF1, IGF1R and AKT1 were decreased significantly, while NR1, NR2B and synapsinl was increased significantly of the PEE group when compared with control (P<0.05). There were still only a few nuclei of neurons in the CA3 subfield of PEE group that were dense and darkly stained. (2) Fetal rats: ①Hippocampal GC-metabolic activation system:When compared to control, hippocampal 11βHSD1, GR, C/EBPa expression levels and 11βHSD1/11βHSD2 expression ratios, but not 11(3HSD2, were increased significantly (P<0.05, P<0.01) in the PEE group. ② Hippocampal GAD67 expressions and the activity of glutamatergic/GABAergic neurons:The mRNA andprotein expression levels of GAD67 in hippocampus showed no obvious change in PEE group relative to control (P<0.05). When compared with control, the activity of hippocampal glutamatergic neurons was decreased significantly in the PEE group (P<0.05), while the activity of hippocampal GABAergic neurons was observed no obvious change in the PEE group. ③Hippocampal IGF1 signaling, synaptic plasticity and morphology:When compared with control, the mRNA expression levels of hippocampal IGF1, IGF1R and AKT1 were decreased significantly (P<0.05, P<0.01), while the mRNA expression levels of hippocampal synapsinl, NR1, NR2A and NR2B were increased significantly (P<0.05, P<0.01) in the PEE group. The morphology of the PEE hippocampus showed no obvious change, but visible hypertrophy of the Golgi body and dilatation of the endoplasmic reticulum were observed in a few neurons from fetal hippocampus of the PEE group.Conclusion:PEE caused fetal higher GC level and induced gender specific-effect on hippocampal 11βHSDs/GR/C/EBPs system:For male offspring, DNA dymethylation within-219～-4 bp region of GAD67 promoter and enhanced expression levels of GAD67 were mediated by activated GR in hippocampus, thus increased amount of Glu were catalyzed to GABA in male PEE hippocampus; For female offspring, the decreased expression levels of hippocampal IGF1 signaling were mediated by GC metabolic activationin female PEE offspring, further impaired the development of the hippocampus and decreased the level of glutamate released from hippocampus; These changes finally lead to the attenuated inhibitory of GABAergic projections to PVN output from the anterior bed nucleus of the stria terminalis (aBST) which is mediated by decreased excitatory of glutamatergic projections from hippocampus, induced the imbalance of hypothalamic excitatory and inhibitory neurons differentiation. All of these changes would contribute to the enhanced sensitivity of the HPA axis in adult offspring. |
PDF Full Text Request