||The memory-associated alterations in protein and epigenetic signature
||Institute of Basic Medical Sciences
Conditioned place preference
Early life stress
長期記憶的形成，需合成所體驗對應相關的蛋白質。利用二維凝膠電泳與液相層析質譜學的分析方法，鑑別提取利用古柯鹼建立的小鼠制約場地偏好(CPP)時，表現在伏隔核(nucleus accumbens, NAc)的相關記憶候選蛋白質群。其中，經由西方墨點分析以及活體內質體轉殖蛋白表現系統證實，14-3-3ζ蛋白表現會影響提取古柯鹼建立的制約場地偏好記憶。在蛋白質體學分析所得到的記憶候選蛋白質中，針對記憶提取為負調控的14-3-3ζ蛋白，分析它在古柯鹼制約場地偏好記憶的功能。結果顯示，在小鼠伏隔核區表現14-3-3ζ蛋白(14-3-3蛋白亞型之一)時，制約線索調節的古柯鹼相關記憶會受到不可逆的削減。然而，這種在伏隔核表現14-3-3ζ蛋白質方式所得到的結果，並不影響小鼠的活動能力或改變局部多巴胺(dopamine)的新陳代謝。相反的，在小鼠伏隔核區表現14-3-3ε蛋白(另一種14-3-3蛋白亞型)時，制約線索調節的古柯鹼相關記憶提取並不會受到影響。意味著，伏隔核內的14-3-3ε蛋白質無論在儲存古柯鹼誘發的制約場地偏好記憶，或提取相對應的記憶，都比較次要。綜合以上的實驗結果，過多的14-3-3ζ蛋白表現在伏隔核會降低提取制約線索所主導的古柯鹼相關記憶。此外，在生命早期所遭受的壓力經驗，會透過表觀基因體機制影響爾後的學習和記憶。以恐懼增益的驚嚇方式模擬受創傷的過程，藉由所記取的恐懼程度，評估幼年時遭受壓力事件的母鼠，於成年後再次遭遇創傷，其反應程度會有何種改變。結果發現，在出生後連續八天 (D2-9PP)，每天經歷一小時隔離程序的母鼠，會降低成年後的恐懼增益驚跳反應；但仍保有正常的聽覺驚嚇反應。檢測後評估實驗中所建立的隔離方式並不影響Corticosterone (CORT) 的基準值及面臨壓力時的分泌量，卻會造成血液中Estradiol(E2) 濃度的增加；經由表觀基因體印記分析，此隔離方式並未改變母鼠前額葉皮質組織內組蛋白3離胺酸9 位置 (Histone 3 lysine 9, H3K9)的乙醯化(acetylation)，取而代之的是降低該位置的單-及三-甲基化程度(mono- and tri-methylation)。在隔離過程中給予幼鼠二種不同劑量的5-aza-2’-deoxycytidine (AZA)治療，無影響隔離造成的成年恐懼增益驚跳反應程度降低。但分別給予幼鼠高、中及低劑量的 valproic acid (VPA)，卻可回復因隔離造成的成年恐懼增益驚跳反應降低的現象。在不影響幼鼠體重的VPA劑量治療下，成年後的母鼠，並不會改變因幼時隔離所增加的血清Estradiol分泌量，但可回復因隔離所降低的前額葉皮質組織內H3K9位置的單-及三-甲基化程度。然而，當幼時經歷隔離的母鼠，成熟後(D28PP)再接受連續八天的VPA或AZA的治療；對隔離所降低的恐懼增益驚跳反應、血液中增加的Estradiol濃度、以及前額葉皮質組織內組蛋白3離胺酸9 位置的單-及三-甲基化成度的降低，並沒有實質的效果。所以，前額葉皮質組織內組蛋白3離胺酸9 位置的單-及三-甲基化程度降低，可能與幼時隔離所造成的恐懼記憶形成減退有關。早期立即給予VPA 的治療，對於幼時受壓力抑制的線索恐懼制約會比後期有效果。
The formation of long term memories requires de novo protein synthesis shortly after the relevant experience. Two-dimensional gel electrophoresis was used to identify accumbal candidate proteins involved in the retrieval of a cue-mediated memory. Among the identified candidate proteins, a downregulated 14-3-3ζ protein was chosen and confirmed by Western immunoblotting. A polymer-mediated plasmid DNA delivery system was then used to overexpress 14-3-3ζ protein in the mouse nucleus accumbens before the CPP retrieval tests. Overexpression of accumbal 14-3-3ζ protein was found to diminish conditioned cue/context-mediated cocaine-induced CPP. Overexpression of accumbal 14-3-3ζ protein did not produce motor activity-impairing effect or alter local dopamine metabolism. Moreover, overexpression of accumbal 14-3-3ζ protein did not affect food-induced CPP. These results indicated that overexpressed accumbal 14-3-3ζ protein specifically decreased conditioned cue/context-mediated cocaine memory. In addition, epigenetic processes influence stress effects of early-life experience on learning and memory. In this study, a fear-potentiated startle was used to model the acquisition of traumatic event-related memory in female rats experiencing early life stress. We found that a 1 hr/day maternal and sibling separation throughout day 2-9 postpartum (D2-9PP) caused a decrease in the fear-potentiated startle, but not acoustic startle baseline, in female adult rats. The separation procedure did not affect baseline and acute stress-stimulated corticosterone (CORT) secretion but produced an increase in serum estradiol concentration. Moreover, the separation procedure did not affect histone 3 lysine 9 (H3K9) acetylation but decreased H3K9 mono- and tri-methylation in frontal cortices. Treatment with 5-aza-2’-deoxycytidine (AZA), an H3K9 acetylation enhancer, at 2 variant dosing regimens did not affect the separation-decreased fear-potentiated startle. Treatment with valproic acid (VPA), a histone deacetylase inhibitor, at 3 dosing regimens prior to daily separation reversed such a decrease in fear-potentiated startle. An effective VPA dosing regimen did not affect early life separation-produced increase in serum E2 secretion but reversed separation-decreased H3K9 mono- and tri-methylation in frontal cortices. Eight consecutive days of VPA and AZA treatments starting at D28PP were ineffective in altering the separation-decreased fear-potentiated startle, -increased E2 concentration, or -decreased frontal cortical mono-, tri-methylation. We suggest that decreased H3K9 mono- and tri-methylation in frontal cortex may be involved in early life separation-decreased fear memory formation. Early, but not late, VPA treatment is effective in treating early life stress-produced decrease in cued fear conditioning.
TABLE OF CONTENTS vii
LIST OF FIGURES x
Chapter 1 Introduction 1
1.1 Accumbal 14-3-3ζ protein downregulation is associated with cocaine-conditioned memory 1
1.1.1 Summary 1
1.1.2 Conditioned cue/context-mediated drug memory retrieval 2
1.1.3 NAc has been implicated in conditioned cue-elicited drug seeking behavior 3
1.2 Neonatal isolation decreases cued fear conditioning and frontal cortical histone 3 lysine 9 methylation in adult female rats 3
1.2.1 Summary 3
1.2.2 Early life stress is through to enhance adult susceptibility to stress and stress-related mood disorders, especially post-traumatic stress disorder 5
1.2.3 The acoustic startle response is modulated by limbic systems implicated in PTSD 5
1.2.4 Early life stress can make adult subjects susceptible to acquiring negative emotion-supported memory 6
1.2.5 Epigenetic modifications are involved in the processes by which early-life experience 7
1.3 Objective 8
Chapter 2 Materials and Methods 9
2.1 Animals for CPP 9
2.2 Cocaine-induced CPP training, test and cocaine priming test 9
2.3 2-DE and candidate protein determination 10
2.4 Mouse 14-3-3ζ and 14-3-3ε plasmid DNA construction 11
2.5 Preparation of the PEI-mediated plasmid DNA delivery system 11
2.6 Stereotaxic surgery and cannula implantation 12
2.7 Locomotor activity 13
2.8 Dopamine (DA) and 3, 4-dihydroxyphenylacetic acid (DOPAC) determination 13
2.9 Maternal and sibling separation 14
2.10 Acoustic startle baseline and cued fear conditioning 15
2.11 Histone H3K9 acetylation and methylation assay in frontal cortex 16
2.12 Drug treatment 17
2.13 Quantification of serum corticosterone (CORT) and estradiol (E2) 18
2.14 Statistical analysis 18
Chapter 3 Results 20
3.1 Cocaine-induced CPP is reliably established in the Paired group 20
3.2 Down-regulated 14-3-3ζ protein in the NAc is identified and confirmed in the Paired group Cocaine-induced CPP is reliably established in the Paired group 20
3.3 Mouse 14-3-3ζ DNA is delivered into the NAc neurons to express 14-3-3ζ protein 21
3.4 Expression of accumbal 14-3-3ζ protein irreversibly mitigates conditioned cue/context-mediated cocaine memory 22
3.5 Accumbal 14-3-3ε expression does not affect conditioned cue/context-mediated cocaine memory 24
3.6 Expression of accumbal 14-3-3ζ protein did not affect food-Induced CPP 25
3.7 Early life maternal and sibling separation did not affect acoustic startle baseline, but decrease cued fear startle in adult female rats 25
3.8 Early life maternal and sibling separation did not affect histone H3K9 acetylation, but decrease H3K9 methylation in adult frontal cortical tissues 26
3.9 Early life maternal and sibling separation did not affect baseline CORT or stress-challenged CORT secretions, while decreased serum E2 concentration in female rats 26
3.10 Treatment with VPA reversed the decreases in cued fear startle and H3K9 mono-methylation in frontal cortices produced by early life separation procedure 26
3.11 Late VPA treatment did not reverse the early life separation-produced decreases in cued light-potentiated startle and H3K9 mono-, tri-methylation in frontal cortices 27
Chapter 4 Discussion 29
Chapter 5 Conclusion 36
Chapter 6 References 37
Chapter 7 Figures 41
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