【作者】 樊双义；

【导师】 胡志安；

【作者基本信息】 第三军医大学 ， 生理学， 2010， 博士

【摘要】 当今社会,越来越多的人不得不面对长时间连续作业,甚至牺牲自然睡眠(即睡眠剥夺)连续作业,如连续军事作业,连续机动车(船)驾驶等。长时间连续作业往往导致脑疲劳。已有证据表明,脑疲劳对作业者认知功能和行为反应能力有严重损害作用,而神经元活动代谢产物---腺苷对神经元活动的抑制在脑疲劳过程中,扮演着重要的角色。揭示连续作业与睡眠剥夺对认知功能影响的机制,将有助于对抗连续作业所致的疲劳,提高连续作业能力。本实验在观察连续作业和睡眠剥夺对人认知功能影响的基础上,首先使用脑功能核磁共振成像(functional magnetic resonance imaging,fMRI)技术分析了睡眠剥夺前后数字记忆编码、维持和提取过程的相关激活脑区出现的激活变化规律,然后运用离体脑片膜片钳全细胞记录技术,在可视条件下研究了活动依赖的细胞代谢产物腺苷对内嗅皮层II层星形神经元的兴奋性调节作用及其离子通道机制和突触机制。主要结果如下:1.连续体力作业和睡眠剥夺对人脑认知功能的影响通过数字划销测试、数字搜索测试和数字符号转换测试三个量表,检测连续体力作业3天,30名作业人员,以及睡眠剥夺48小时,6名受试人员注意力和记忆力等脑认知功能的变化。(1)连续体力作业对人脑认知功能的影响数字划销测试结果显示,连续作业第1天划对数为7.73±2.41,第3天下降至4.36±1.92,净分从第1天的4.99±2.03,降至第3天2.42±2.32,差异显著(n=30, P<0.05)。连续体力作业第1天划错数是0.13±1.17、失误率是32.43±15.65%,第3天划错数为0.33±1.06,失误率达60.73±25.67%。数字搜索测试结果显示,失误率在连续作业第1天为37.54±25.90%,低于第3天(48.91±30.40%),差异显著(n=30, P<0.05)。连续体力作业第1天划对数为8.23±3.42、漏划数为6.77±3.42,第3天划对数为8.47±2.91、漏划数是6.53±2.91。第1天净分为6.6±3.83,第3天分值下降至5.27±4.17。上述两项测试结果显示,随着连续体力作业时间延长,作业人员的注意力和短期记忆力等脑认知功能均呈现显著的降低。(2)睡眠剥夺对人脑认知功能的影响数字划销测试结果显示,睡眠剥夺8小时后,剥夺组划对数(18.33±1.37)和净分(17.50±2.05)都低于对照组划对数(18.60±2.61)和净分(17.90±3.91)。剥夺16小时后,剥夺组划对数和净分分别是17.43±2.30、16.14±3.45,对照组划对数是18.50±0.58,净分为17.75±0.87。剥夺组的失误率8.38±2.02%,对照组4.09±1.69%。数字符号转换测试结果显示,睡眠剥夺8小时后,得分24.67±1.51,剥夺至32小时后下降至22.71±1.80,睡眠剥夺48小时后,降至最低20.14±1.21。同一时间段比较,睡眠剥夺组得分均低于对照组,睡眠剥夺16小时后,剥夺组得分23.43±7.93,对照组得分为24.00±1.83;剥夺40小时后,剥夺组得分20.00±2.23,对照组得分为23.71±1.97。以上结果提示,睡眠剥夺使受试者的注意力和记忆力受损,且随着剥夺时间增加,这种认知功能下降程度增加。2.睡眠剥夺影响数字记忆编码、维持和提取的脑功能磁共振成像研究睡眠剥夺受试者数字记忆测试中,错误率从剥夺前8.2±5.4上升到剥夺后13.7±10.1(n=6),反应时间增加,剥夺前738.0±82.1ms,剥夺后824.3±52.3(n=6, P<0.05)。SD状态下记忆编码阶段在睡眠剥夺前后差异的激活区为灰质区,左侧边缘叶海马旁回Brodmann 30,左侧颞叶颞上回42,左侧岛叶41,额叶回6;SD前后维持阶段差异的激活区为灰质区,左侧颞上回Brodmann 38,左侧颞中回21,左侧海马旁回及杏仁,左侧额中回47,左侧豆状核及丘脑,右侧豆状核,左边缘叶扣带后回30,右边缘叶扣带后回30,双侧扣带回24,双侧额中回、额内侧回6;睡眠剥夺前后工作记忆提取阶段机体为了维持清醒状态,大脑双侧海马、右侧杏仁核、右侧顶小叶、左侧楔前叶丘脑出现了过度激活状态,表现为睡眠剥夺前后工作记忆提取阶段阴性激活;睡眠剥夺前后工作记忆提取阶段阳性激活,激活区为左侧颞中回Brodmann 21,双侧扣带回24,左侧额下回47,左顶下小叶19,左侧额中回9。3.稳态因子腺苷调节大鼠内嗅皮层II层星形神经元的离子通道机制和突触机制(1)腺苷抑制星形神经元Ih电流在电流钳模式下,给与100μM腺苷能够明显抑制跃阶超极化电流刺激(-350 ~ -150 pA,50 pA)诱发的电压sag值,给予腺苷,电压sag比值(电压峰改变值/电压稳定改变值)减小到对照时的66±9%(对照:13.0±5.9 mV;腺苷:8.8±4.2 mV;P < 0.001;n = 11)。加入腺苷后Ih电流幅度明显减小,差异显著。(2)腺苷减少自发性谷氨酸释放到星形神经元在灌流液中加入1μM河豚毒素阻断电压依赖的Na+通道和10μM荷包牡丹碱阻断离子通道型GABAA受体,分离出mEPSCs。给予100μM腺苷明显抑制mEPSCs频率(对照的55±9 %;n = 16;P < 0.001),却不影响mEPSCs的幅度(对照的98±6 %;n = 16;P = 0.33)。给予腺苷受体1拮抗剂DPCPX(3μM),腺苷对mEPSCs的抑制效应被阻断(n=10;P = 0.35),而腺苷受体2拮抗剂DMPX(10μM)无影响(n=6;P < 0.001)。提示,腺苷诱发的自发性谷氨酸释放减少是由突触前A1受体介导的。(3)腺苷抑制自发性GABA释放到星形神经元在灌流ACSF中加入1μM河豚毒素、10μM CNQX和50μM AP-V阻断离子通道型谷氨酸受体,分离出mIPSCs。100μM腺苷增加mIPSCs事件间的时间间隔,但不影响mIPSCs的幅度累积曲线,即频率降低到对照的51±6 % (n = 16;P < 0.001),平均幅度没有改变(对照的98±4 %,n = 16; P = 0.07)。给予3μM DPCPX,腺苷不影响mIPSCs活动(n = 6; P = 0.47)。但在10μM DMPX存在的情况下,腺苷降低mIPSCs的频率到53±11 % (n = 10; P < 0.001),提示,腺苷诱发的自发性GABA释放减少也是通过突触前A1受体介导的。(4)电压门控Ca2+通道和胞外Ca2+介导腺苷抑制突触传递的效应灌流液中加入电压门控钙通道(VDCCs)阻断剂Cd2+ (100μM),能明显降低所记录神经元的mEPSCs和mIPSCs的频率至对照的44±11 % (n = 8;P< 0.001)和56±11 % (n = 7;P < 0.001)。用Cd2+处理至少5 min后,给与100μM腺苷,不能降低mEPSCs (n = 8;P = 0.30)和mIPSCs (n = 7;P= 0.20)的频率。胞外无Ca2+也能明显降低基础mEPSCs和mIPSCs的频率至对照45±19 % (n = 8;P < 0.001)和51±15 % (n = 9;P < 0.001)。且,加入腺苷也不能明显改变mEPSCs (n = 8;P = 0.13)和mIPSCs (n = 9;P = 0.38)的频率。上述结果说明,腺苷A1受体介导的抑制自发性谷氨酸和GABA释放与胞外Ca2+通过VDCCs内流有关。综上所述,本研究结果表明,连续体力作业和睡眠剥夺可以使人脑认知功能下降。睡眠剥夺前后,数字记忆编码、维持和提取过程中脑区激活发生差异变化。内源性促睡眠因子--腺苷通过直接抑制HCN通道电流而降低内嗅皮层星形神经元兴奋性;或间接与突触前A1受体结合,抑制电压门控钙通道钙内流,降低兴奋性谷氨酸能和抑制性GABA能突触活动的传入。更多还原

【Abstract】 In the modern society, more and more people have to face a long time continuous operation, even at the expense of natural sleep (ie, sleep deprivation) continuous operation, such as the continuous military operations, continuous vehicle driving. Working for long hours often leads to mental fatigue. There is evidence that mental fatigue is serious damage to cognitive function and behavior of the operator, and the metabolite of neuronal activity---adenosine inhibition of neuronal activity plays an important role during fatigue in the brain. Revealing the mechanism of continuous operation and sleep deprivation on cognitive function, will help combat the fatigue caused by continuous operation, continuous work and improve capacity of operators.In the present study, we firstly used a combined behavioral and functional magnetic resonance imaging (fMRI) design, allowing for a whole-brain, systems-level approach, to explore the ability of the human brain to form new digital memories in the absence of prior sleep. And then we investigated the electrophysiological effects of adenosine on stellate neurons in live brain slices of the EC using whole-cell patch-clamp recordings, observing the ionic and synaptic mechanisms involved in adenosine. The results show as follow: 1. Detrimental influence of continuous physical work and sleep deprivation on cognitive function in the human brainThe cognitive function of 30 continuous physical working people (3 days) and 6 sleep deprivation (SD) subjects (48h) were evaluated with Number cancellation test (NCT), Number searching test (NST) and Digit symbol substitution (DSST). The results of NCT showed that scores on accurate cancellation and total score had significant difference in the 1st and 3rd continuous physical work of people (n=30, P<0.05), and scores on false cancellation and error rate in 1st work (0.13±1.17/32.43±15.65%) was lower than that in 3rd continuous physical work (0.33±1.06/60.73±25.67%). NST also demonstrated that scores on total score in 1st work (6.6±3.83) was higher than 3rd continuous physical work (5.27±4.17), while scores on error rate on people in 1st work (37.54±25.90%) was significant lower than that in 3rd continuous physical work (48.91±30.40%) (n=30, P<0.05). NCT presented that after SD for 8h, the scores on both accurate cancellation and total score in SD subjects was lower than that in controls The same as SD for 16h, the scores on accurate cancellation and total score in SD subjects were 17.43±2.30 and 17.50±2.05, lower than that in controls (18.50±0.58/17.75±0.87). Experiment on DSST showed that the scores after SD for 8h, 32h and 48h decreased gradually, 24.67±1.51, 22.71±1.80 and 20.14±1.21, respectively. And the scores on SD for 16h and 40h were 23.43±7.93, 20.00±2.23, lower than that in controls (24.00±1.83/23.71±1.97). These results suggest that continuous physical work and sleep deprivation have an obvious detrimental influence on the cognitive function in human brain.2. Impairment of digital memory retrieval after 48h sleep deprivation6 subjects were awake during day 1, night 1, day 2 and night 2, accumulating approximately 48h of total sleep deprivation before the encoding session. Subjects underwent a digital memory encoding, maintenance and retrieval session during fMRI scanning in which they viewed a series of number (0 ~ 9). The results showed that the error rate (13.7±10.1) after SD was higher than that before SD (8.2±5.4), while the response time increased from 738.0±82.1 ms to 824.3±52.3ms after SD. During encoding trials different fMRI regions of significant activation (relative to fixation baseline) between sleep control and sleep deprivation are left Brodmann 30, left Brodmann 42, left Brodmann 41 and left Brodmann 6. During maintenance trials different fMRI regions of significant activation are left Brodmann 38, left Brodmann 21, left parahippocampus and amygdala, left Brodmann 47, left lentiform nucleus and thalamus, right lentiform nucleus, left Brodmann 30, right Brodmann 30, bilateral Brodmann 24 and bilateral Brodmann 6. During retrieval trials different fMRI regions of significantly negative activation are bilateral hippocampus, right amygdale, left precuneus, left thalamus. During retrieval trials different fMRI regions of significantly positive activation are left inferior frontal gyrus, left middle frontal gyrus, left middle temporal gyrus, bilateral cingulate gyrus, left inferior parietal lobule, Brodmann 21, Brodmann 24, Brodmann 47, Brodmann 19 and Brodmann 9.3. Inhibitory effects of adenosine on stellate neurons in EC 3.1 Adenosine inhibits Ih currents of stellate neuronsApplication of adenosine produced a reduction of voltage sag in recording neurons in response to hyperpolarizing current steps (from -350 to -150 pA, 50 pA, step), the voltage sag (peak voltage change-voltage change at steady state) decreased to 66±9% of control in the presence of adenosine (control, 13.0±5.9 mV; post-adenosine, 8.8±4.2 mV; n = 11; P < 0.001). In addition, adenosine produced a significant decrease in the amplitude of Ih currents evoked by the voltage step protocol (from -70 to -120 mV; -10 mV; step; 1000 ms). The suppression by adenosine on Ih currents was displayed in almost all hyperpolarized voltage steps except -70 mV. Together, these data indicate that adenosine inhibits the excitability of stellate neurons in the EC involved its inhibition on HCN channels.3.2 Adenosine activates presynaptic A1 receptors to decrease spontaneous glutamate release on to stellate neuronsIn the presence of bicuculline (10μM) and TTX (1μM) mEPSCs were recorded. Application of adenosine (100μM) significantly decreased the frequency (55±9 % of control; n = 16, p < 0.001), but not the amplitude (98±6 % of control; n = 16; p = 0.33) of mEPSCs. Application of adenosine A1 receptor antagonist DPCPX (3μM) completely blocked adenosine mediated decrease in mEPSCs frequency (n = 10; P = 0.35). The ability of adenosine A2 receptor antagonist DMPX (10μM) to block the effect of adenosine on mEPSCs was also tested. Application of adenosine A2 receptor antagonist DMPX (10μM) failed to change the adenosine-induced shift of the interevent interval distribution (n = 6; P < 0.001) excluding the involvement of adenosine A2 receptors. These results suggest that the adenosine-induced decrease in spontaneous glutamate release is mediated by presynaptic A1 receptors.3.3 Adenosine inhibits the GABAergic drive to stellate neurons by activating presynaptic A1 receptorsIn the presence of TTX (1μM), CNQX (10μM) and AP-V (50μM) mIPSCs were recorded. Application of adenosine (100μM) significantly decreased the frequency (51±6 % of control; n = 16, p < 0.001,), but not the amplitude (98±4 % of control; n = 16; p = 0.07) of mIPSCs. Application of adenosine A1 receptor antagonist DPCPX (3μM) completely blocked adenosine mediated decrease in mIPSCs frequency (n = 6; P = 0.47). Application of adenosine A2 receptor antagonist DMPX (10μM) failed to change the adenosine-induced shift of the interevent interval distribution (n = 10; P < 0.001) excluding the involvement of adenosine A2 receptors. These results suggest that the adenosine- induced decrease in spontaneous glutamate release is mediated by presynaptic A1 receptors.3.4 Inhibition of spontaneous glutamate and GABA release by adenosine A1 receptoractivation is mediated by voltage-dependent Ca2+ channels and extracellular Ca2+ Bath application of voltage-dependent Ca2+ channel (VDCC) blocker Cd2+ (100μM) alone significantly decreased the baseline frequency of both mEPSCs and mIPSCs in all of the neurons tested to 44±11% (n = 8; P < 0.001) and 56±11% (n = 7; P < 0.001; K-S test) of the control, respectively. After at least 5 min pretreatment of Cd2+, adenosine (100μM) failed to decrease mEPSC (n = 8; P = 0.30) and mIPSC frequency (n = 7; P = 0.20). Ca2+-free external solution also markedly decreased basal mEPSC and mIPSC frequency to 45±19% (n = 8; P < 0.001) and 51±15% (n = 9; P < 0.001) of the baseline, respectively. Furthermore, the application of 100μM adenosine did not produce a significant change in the frequency of mEPSCs (n = 8; P = 0.13) and mIPSCs (n = 9; P = 0.38) in the Ca2+-free solution. These results suggest that the adenosine A1 receptor-mediated inhibition of spontaneous glutamate and GABA release is related to the Ca2+ influx passing through presynaptic VDCCs.In summary, our study has indicated that continuous physical work and sleep deprivation have deleterious effect on human cognitive function, and digital memory retrieval was impaired after 48 sleep deprivation. Adenosine inhibits the excitability of stellate neurons in the EC through inhibition on HCN channels. In addition, adenosine-induced decrease in spontaneous glutamate and GABA release is mediated by presynaptic A1 receptors. Furthermore, adenosine A1 receptor-mediated inhibition of spontaneous glutamate and GABA release is related to the Ca2+ influx passing through presynaptic VDCCs.更多还原