||Impact of prenatal stress on hippocampal neuroplasticity
||Institute of Basic Medical Sciences
我們發現到孕期壓力對出生子代所造成的影響是來自於神經突觸塑性的改變，並不影響個體的成長曲線。在受孕期壓力的子代海馬迴 CA1 區域的腦薄片電生理記錄長期增益現象 (LTP) 是有被抑制的，然而長期抑制現象 (LTD) 是延遲至子代大鼠五周時，仍有表現。以調控代謝型麩胺酸受體的選擇性促進劑(RS)-3,5-dihydroxyphenylglycine (DHPG) 形式誘發的長期抑制現象並不受影響。經由細分不同的細胞分子蛋白質組成成份，初步觀察孕期壓力及對照組的NMDA 接受體組成次單元 NR1、NR2A、NR2B 都沒有顯著的改變。值得注意的是在我們實驗中發現到，受孕期壓力的組別，pro-BDNF 有顯著的增加及 tPA 有減少的情形。這結果暗示 pro-BDNF 轉變成 mBDNF 的過程受到抑制，而這過程可能是日後孕期壓力造成海馬迴神經突觸塑性的影響。雖然我們並沒有觀察到孕期壓力對於 tPA 基因有表觀遺傳上的調控。但在受孕期壓力的子代大鼠上，以 ChIP 分析看到磷酸化的 CREB 結合至 tPA 的轉錄子的量有減少的情形，造成的相關機制需進一步研究釐清。暸解此等相關的機制有助於開發更有效的治療策略及方法來緩解孕期壓力所造成的精神病理疾病發生。
The environment in early life can have a major impact on later life. Extensive evidence from animal and human studies suggests that maternal stress may impede normal brain development and functions of their offspring. A significant number of stress exposed offspring show selective impairments of attention, arousal and information processing, which appear early in lifetime and later go on to develop into major cognitive deficits and affective disorders later in life. Although the biological consequence and underlying mechanisms by which prenatal stress exposure causes the cognitive deficits remain essentially unknown, it has been proposed to be the results of alterations in the anatomical organization or function of the hippocampus, a brain region critically involved in the control of cognitive functions. The overall hypothesis to be evaluated is that exposure to stress in utero alters synaptic plasticity in the hippocampus and these aberrant regulations may result in maladaptive changes in neural circuitry that enhance the risk of developing psychopathology later in life.
We found that the prenatal stress on the impact of the offspring came from the change of synaptic plasticity without effects on the growth curve. Electrophysiological recording on hippocampal CA1 brain slices from prenatal stressed offspring showed impairment in the induction of long term potentiation (LTP). However, the induction of long term depression (LTD) was prolonged to express in 5-weeek-old offspring by prenatal stress. The induction of LTD by metabotropic glutamate receptor (RS)-3,5-dihydroxyphenylglycine (DHPG) was not affected by prenatal stress. By separating different subcellular fractionation, the expression of NMDA receptor subunit NR1, NR2A, NR2B showed no significant difference between prenatal stress and control group. Notably, we found that prenatal stress induced a significant increase in the levels of pro-BDNF and the decrease in the levels of tissue plasminogen activator (tPA). These results suggest that an inhibition of the converting process of pro-BDNF to mBDNF may be account, at least in part, to the effect of prenatal stress on the subsequent induction of hippocampal synaptic plasticity in later life. Although we did not observe a role for epigenetic regulation of tPA gene in the effect of prenatal stress on tPA expression. However, the phosphorylation of CREB (p-CREB) binding to tPA promoter was decreased in PS-treated rats by ChIP assay and associated mechanism needs further experiment to elucidate. Understanding the biochemical substrates and the underlying mechanisms of the cognitive deficits induced by prenatal stress exposure will facilitate the development of more effective intervention strategies target these pathways to treat mental illnesses.
Abstract in Chinese I
Table content VII
Figure contents VIII
I. Introduction 1
1.1. Impact of prenatal stress on fetus’s outcomes 1
1.2. Hypothalamus-pituitary-adrenal (HPA) axis 1
1.3. Brain-derived neurotrophic factor (BDNF) 3
1.4. LTP and LTD 4
1.5. tPA and CREB 4
Specific Aims 6
II. Materials and Methods 7
2.1. Animals 7
2.2. Prenatal stress procedure 7
2.3. Plasma cortisosterone assay 8
2.4. Hippocampal slice preparations and electrophysiology 8
2.5. Preparation of synaptoneurosomes 10
2.6. Preparation of subcellular fractions 10
2.7. Quantitative real-time PCR (qRT-PCR) 10
2.8. Western blotting 11
2.9. tPA zymography 12
2.10. Matrix metalloprotease zymography 13
2.11. tPA activity assay 13
2.12. MMP activity assay 14
2.13. Histology and quantification 14
2.14. Golgi impregnation 15
2.15. Chromatin immunoprecipitation (ChIP) assay 16
2.16. Bisulfite conversion and Pyrosequencing 17
2.17. Preparation of primary hippocampal cultured neuron 18
2.18. pharmacological treatment 19
2.19. Data analysis 19
III. Results 20
3.1. Effect of PS on fetal somatic growth 20
3.2. Effect of PS on glutamatergic synaptic transmission 20
3.3. PS impairs LTP but enhances LTD induction 21
3.4. PS does not alter dendritic morphology of hippocampal CA1 pyramidal neurons 24
3.5. PS does not affect the expression of NMDA receptor subunits 25
3.6. PS inhibits the proteolytic conversion of pro-BDNF to mBDNF 26
3.7. Regulation of tPA gene expression 29
3.8. Mimic the PS effect in culture neuron system 30
IV. Discussion 31
V. Conclusion 38
VI. References 40
VII. Publications 76
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