【摘要】： 目的通過建立小鼠恐懼記憶能力的實驗動物模型，探索恐懼記憶形成后海馬CA1區(qū)NMDA受體亞單位表達的變化，從而尋找其分子作用機制。 方法選用昆明種(KM)和C57BL／6小鼠，將足底電擊刺激的訓練與聲音相結合，觀察同一品種小鼠用不同電擊強度以及不同品種小鼠用相同條件訓練時，恐懼性記憶建立的差異。以訓練后24 h測試時出現(xiàn)木僵反應(stupor response)，作為恐懼性記憶形成的表現(xiàn)。以Flou-3／AM作為熒光探針，用同源共聚焦顯微鏡測定腦片和培養(yǎng)海馬神經(jīng)細胞內(nèi)鈣離子濃度變化；取腦組織，冷凍切片，用免疫組化方法測定海馬CA1區(qū)NMDA受體亞單位及c-fos表達，高效液相色譜法測定血漿皮質(zhì)酮水平，將以上結果與行為學結果進行相關性分析。 結果將聲音分別結合0.4mA、0.6mA、0.9mA足底電擊，24 h后在KM小鼠引出的木僵反應時間分別為60.00±57.77s，110.00±129.61s，101.99±20.38s，強度0.6mA組的合格率最高(30％)，組間比較無顯著性差異(P＞0.05)。C57BL／6小鼠在聲音結合0.4mA電擊條件下，木僵反應時間為275.62±36.99s，顯著長于KM小鼠的時間(P＜0.01)，其合格率為100％，遠高于KM小鼠的7.69％(0.4mA)。KM小鼠對照組和訓練組血漿皮質(zhì)酮水平分別為(101.083±70.266)μg/L、(215.453±60.276)μg/L，兩者比較差異顯著(P＜0.01)；訓練組血漿皮質(zhì)酮水平與訓練結果木僵反應時間(36.00±29.292)s之間無相關性(r=-0.273，P＞0.05)。C57BL／6小鼠對照組和訓練組血漿皮質(zhì)酮水平分別為(108.009±20.452)μg/L、(176.933±38.202)μg/L，兩者比較差異顯著(P＜0.01)；訓練組血漿皮質(zhì)酮水平與訓練結果木僵反應時間(257.50±36.450s)之間無相關性(r=-0.462，P＞0.05)。兩種小鼠的對照組、訓練組比較，均為P＞0.05，差異無顯著性意義。 C57BL／6小鼠訓練組海馬組織CA1區(qū)神經(jīng)細胞c-fos表達水平明顯高于對照組(P＜0.05)，前者陽性細胞數(shù)平均值為185.18±31.131，后者為145.39±54.383；前者平均灰度值為12583.02±3543.554，后者為8436.851±3434.484，兩者比較差異有統(tǒng)計學顯著意義(P＜0.01)。NR2B的檢測，陽性細胞數(shù)平均值訓練組為(287.75±19.992)、對照組為(254.26±31.897)，兩者比較差異有統(tǒng)計學顯著意義(P＜0.01)；前者平均灰度值為47703.90±11152.912，，后者為39009.06±11586.680，兩者比較差異有統(tǒng)計學顯著意義(P＜0.05)。 結論①C57BL／6小鼠在聲音70dB，0.4mA電刺激下，24h后恐懼木僵反應時間滿足實驗要求，成功率達95％以上；②血漿皮質(zhì)酮水平與訓練成績無明顯相關性，但C57BL／6小鼠相關系數(shù)r為-0.462，|r|大于0.3，小于0.5，呈低度負相關。兩種小鼠訓練前后血漿皮質(zhì)酮水平無明顯差異，訓練效果的差異與激素水平高低無關；是否遺傳背景的差異影響記憶保持的分子機制，有待進一步研究；③海馬CA1區(qū)神經(jīng)元在恐懼記憶形成后，NR2B、c-fos表達明顯增多，提示應激時血漿內(nèi)皮質(zhì)酮濃度升高，可能通過細胞內(nèi)基因表達增強，引起海馬CA1腦區(qū)LTP的易化和固化，從而使恐懼記憶得以較長時間保留。
[Abstract]:Objective To study the changes of the expression of NMDA receptor subunits in the CA1 region of the hippocampus after the formation of the fear memory by establishing an experimental animal model of the mouse's fear and memory ability. Methods Kunming (KM) and C57BL/ 6 mice were selected to combine the training and the sound of the stimulation of the electric shock of the sole. The difference between memory and memory was recalled. The post-training 24-hour test showed a stupor response as a memory of fear. The changes of calcium ion concentration in the brain and the cultured hippocampal neurons were measured by using Flou-3/ AM as a fluorescent probe, and the concentration of calcium in the neurons of the hippocampus was measured by using a homologous confocal microscope. The NMDA receptor subunits and c in the CA1 region of the hippocampus were determined by immunohistochemistry.-fos expression, high performance liquid chromatography to measure the plasma corticosterone level, and the above results are compared with the behavior results. The results showed that the reaction time was 60. 00, 57. 77s, 110. 00, 129. 61s, 101. 99, 20. 38s, and the intensity of 0. 6mA group was the highest (30%) after 24 h, respectively. There was a significant difference (P> 0.05). In C57BL/ 6 mice, the reaction time was 275.62-36.99s, which was significantly longer than that of KM mice (P <0.01). The qualified rate was 100%, which was much higher than that of KM mice (P <0.01). The levels of plasma corticosterone in the control group and the training group of KM mice were (101.08). 3 (70. 266). mu. g/ L, (215. 453, 60. 276). mu. g/ L, the difference was significant (P <0.01), and there was no correlation between the level of plasma corticosterone in the training group (36. 00-292.292) s (r =-). The levels of the plasma corticosterone in the control group and the training group of the C57BL/ 6 mice were (108.009-20.452) and (176.933-382.202). m/ L, respectively (P <0.01), and the plasma corticosterone levels in the training group were compared with the training results (257.50-360.450s). No correlation (r =-The control group and the training group of the two mice were P> The expression of c-fos in the hippocampal tissue of the training group of C57BL/ 6 mice was significantly higher than that in the control group (P <0.05). The average positive cell number was 185.18-31.131, the latter was 145.39-543.383; the average gray-scale value of the former was 12583.02-3543. 554, the latter being 8436,851, 3434. 484, both The difference was statistically significant (P <0.01). The mean value of the positive cells was (282875-19.992), and the control group was (252.26-31.897), and the difference was statistically significant (P <0.01). The average gray-scale value of the former was 47703. 90-11152.912. The person is 39009. 06/ 11586. 680, both Conclusion The results showed that in C57BL/ 6 mice, the reaction time of fear wood after 24 h was higher than 95% after 24 h under the electric stimulation of 70dB and 0.4mA. However, the level of corticosterone in plasma was not related to the results of training, but C57 The correlation coefficient r of BL/ 6 mice was-There was no significant difference in plasma corticosterone levels before and after training in both mice. The difference of the training effect was not related to the level of the hormone. The expression of NR2B and c-fos increased significantly after the formation of the fear and memory in the CA1 region of the hippocampus, suggesting that the concentration of the corticosterone in the plasma during the stress could be enhanced by the expression of the gene in the cells, causing the CA1 of the hippocampus.
【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R338

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