吗啡成瘾和正常大鼠前额叶皮质表达的蛋白质组学变化

The prefrontal cortex is involved in the regulation and control of substance addiction-related cognitive,behavioral,and emotional changes.The present study identified prefrontal cortex protein profiles in morphine-addicted rats;these were subsequently compared with normal rats.Results showed 87 protein spots with differentially expressed levels in the morphine addiction group,with the majority located in meta acid zones at pH 4.2-6.8 and having a molecular weight of 30-110 kDa.In addition,2 protein spots were identified as being associated with neurotoxicity(Snap25 isoform β-Snap25 of synaptosomal-associated protein 25 and β-actin).     

大鼠大脑皮质神经元氧糖剥夺/复氧模型的建立

目的 体外大鼠大脑皮质神经元氧糖剥夺/复氧试验模型的建立.方法 取 Wistar 大鼠乳鼠脑组织,采用过滤、消化、离心等技术获取大脑皮质神经元并培养,通过倒置显微镜、免疫细胞化学鉴定,采用MTT 法、酶学检查、光学显微镜等评价该模型.结果 (1)形态学、免疫细胞化学(NSE)鉴定所培养的细胞为神经元;(2)氧糖剥夺/复氧前后细胞形态结构变化显著,MTT 法及LDH释放测定均提示神经元损伤随氧糖剥夺时间呈时间依赖关系,与正常对照组有显著差异(P<0.05).结论 成功地建立体外大脑皮质神经元的氧糖剥夺/复氧模型.     

细胞间黏附分子5在血清剥夺条件下对PAJU细胞的保护作用

目的 探索细胞间黏附分子5（ICAM-5）在血清剥夺条件下对PAJU细胞是否具有保护作用。方法 应用无ICAM-5蛋白表达且转染空载体的PAJU-NEO细胞和转染ICAM-5基因表达ICAM-5蛋白的PAJU-TLN细胞作为神经元模型,应用无血清培养的方法造成细胞“饥饿”（starvation）模拟脑梗死后缺血状态,使用相差显微镜观察无血清培养条件下PAJU细胞的生存变化,采用MTT法检测细胞活力,Hoechst 33258细胞核染色和流式细胞术检测细胞凋亡率。结果 在无血清培养过程中两组细胞均受到了损伤,无血清培养5天后,PAJU-NEO细胞大部分死亡,而PAJU-TLN细胞仅少部分死亡;无血清培养7天后,PAJU-NEO组细胞全部死亡,而PAJU-TLN细胞仍有部分活细胞,直到无血清培养第14天才全部死亡。MTT结果示24h两组活力无明显差异（P=0.7416）,但72h和120h时 PAJU-TLN组细胞的存活率高于PAJU-NEO组细胞的存活率（分别为P=0.0288,P=0.0070）。Hoechst 33258细胞核染色示两组细胞核凋亡率在无血清培养48h时出现差异,在72h后明显增多,PAJU-NEO组细胞核凋亡率显著高于PAJU-TLN组细胞核凋亡率（P<0.05）。流式细胞仪检测显示无血清培养24h时无显著差异,但无血清培养48h时PAJU-TLN组细胞凋亡率明显低于PAJU-NEO组细胞凋亡率（P=0.0463）。 结论 ICAM-5可能具有神经营养因子样作用,是发挥神经元内源性神经保护作用的因素之一,但其具体机制有待进一步研究。     

睡眠剥夺对认知影响的研究进展

现代社会,睡眠剥夺(sleep deprivation,SD)是一种常见现象,困扰着很多人,引起人的日间工作能力降低.近年来的研究结果显示,SD可以使认知功能受损,但是其损害机制尚不清楚.本综述将重点阐述SD对行为和中枢系统的影响,进而阐明SD引起认知功能受损的可能机制,并且展望未来的研究方向.     

黄体酮对大鼠脑微血管内皮细胞氧糖剥夺后的保护作用

目的研究黄体酮(progesterone,PROG)对大鼠脑微血管内皮细胞氧糖剥夺后的保护作用机制。方法体外培养大鼠脑微血管内皮细胞(brain microvascular endothelial cell,BMEC),并构建氧糖剥夺(oxygen and glucose deprivation,OGD)模型。以3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,MTT]法检测BMEC存活率,比色法测定乳酸脱氢酶(lactate dehydrogenase,LDH)漏出率反映细胞损伤程度。为反映BMEC的氧化应激损伤水平,利用生物化学法测定细胞培养液中及细胞内丙二醛(malondialdelyde,MDA)水平及细胞内超氧化物歧化酶(superoxide dismutase,SOD)和谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性。结果 OGD 6h明显降低BMEC的存活率,并增加LDH释放率。OGD 6h可明显增加BMEC培养基中及细胞内MDA含量,减少BMEC内SOD、GSH-Px活性。与氧糖剥夺组相比,黄体酮可明显降低培养基中和细胞内的MDA含量,提高BMEC内SOD、GSH-Px活性。结论 OGD可通过诱导氧化应激等途径损伤BMEC,黄体酮能减少OGD对BMEC造成的损伤。     

天麻素后处理对糖氧剥夺大鼠皮层神经元EphA4表达的影响

目的研究天麻素对糖氧剥夺(oxygen-glucose deprivation,OGD)大鼠皮层神经元损伤的保护性作用及EphA4表达的影响。方法提取新生大鼠大脑皮层神经元体外培养7 d,对4组神经元分别施以60 min的OGD,换回正常培养液培养30 min后分别及给予含终浓度为50、125、250、500μmol/L天麻素的培养液作为后处理措施,培养4 h后换回正常条件培养24 h;采用倒置相差显微镜观察神经元形态学变化,LDH活性测定检测神经元损伤情况,CY3荧光染色检测EphA4表达变化。结果天麻素后处理能降低糖氧剥夺神经元的损伤,降低EphA4表达。结论天麻素对糖氧剥夺神经元损伤有保护作用,该作用与EphA4表达降低有关。     

伏核脑深部刺激对吗啡成瘾心理依赖大鼠的作用研究

目的研究伏核脑深部刺激（DBS）对吗啡成瘾大鼠心理依赖的作用。方法将60只SD大鼠随机等分为三组：假刺激（ShS）组、DBS组、正常对照（NS）组。刺激前后行条件性位置偏爱实验，并观察伏核、海马、前额叶脑组织的形态学变化。结果刺激前DBS组与ShS组大鼠在伴药箱平均停留时间均长于NS组，差异显著（P〈0．01）；刺激后，DBS组较ShS组在伴药箱平均停留时间明显缩短（P〈0．01），而与NS组差异无统计学意义（P〉0．05）。刺激前，DBS组与ShS组大鼠伏核、海马神经元部分丢失，且水肿明显，细胞器减少；刺激后，DBS组较ShS组细胞水肿明显减轻，以伏核变化明显。结论伏核DBS有效治疗了大鼠的吗啡成瘾心理依赖作用。     

头孢曲松钠对大鼠脑皮质神经元氧糖剥夺损伤的保护作用及其机制

目的研究头孢曲松钠对大鼠脑皮质神经元氧糖剥夺(OGD)损伤的保护作用及其可能的机制。方法体外培养大鼠脑皮质神经细胞,分为正常对照组、氧糖剥夺(OGD)组和头孢曲松钠(CTX)预处理组。OGD组和CTX预处理组建立OGD模型。并检测各组的细胞活性、细胞谷氨酸转运体-1(GLT-1)mRNA表达、脑源性神经营养因子(BDNF)和白介素(IL)-6含量。结果与正常对照组比较,OGD组及CTX预处理组神经细胞活性减低,GLT-1 mRNA表达显著下调,细胞培养上清液中BDNF及IL-6含量显著增加(P<0.05～0.01);与OGD组比较,CTX预处理组神经细胞活性明显增加,GLT-1 mRNA表达上调,细胞培养上清液中BDNF含量增加(均P<0.01),IL-6含量无明显改变。结论 CTX预处理通过上调GLT-1和BDNF表达保护皮质细胞OGD损伤。     

左旋氨氯地平对成年大鼠神经干细胞氧/糖剥夺损伤的保护作用

目的 研究左旋氨氯地乎对成年大鼠神经干细胞氧/糖剥夺(OGD)损伤后细胞生长活力、增殖和凋亡情况的影响,探讨左旋氨氯地平对OGD后成年大鼠神经干细胞的保护作用.方法 制备成年大鼠海马神经干细胞OGD模型,按干预的左旋氨氯地平浓度分为4组:0mol/L组(即无干预对照组)、0.5 μmol/L组、1.0 μmol/L组、5.0 mol/L组.CCK-8法检测不同浓度左旋氨氯地平对OGD后成年大鼠神经干细胞生长活力的影响:Edu荧光染色法观察不同浓度左旋氨氯地平对OGD后成年大鼠神经干细胞增殖的影响:Annexin V-FITC/PI双染流式细胞仪检测OGD后成年大鼠神经干细胞的凋亡情况. 结果 OGD6 h后成功制备成年大鼠海马神经干细胞OGD模型.与无干预对照组比较,不同浓度左旋氨氯地平干预后成年大鼠海马神经干细胞的生长活力均明显增加,差异有统计学意义(P＜0.05); 5.0 μmol/L左旋氨氯地平组OGD后神经干细胞增殖率明显增加,差异有统计学意义(P＜0.05);1.0 μmol/L和5.0 μmol/L左旋氨氯地平组OGD后神经干细胞中凋亡细胞比例明显减少,差异有统计学意义(P＜0.05). 结论 成年大鼠神经干细胞OGD模型中,左旋氨氯地平具有一定的抗OGD损伤的保护作用.     

母爱剥夺对大鼠行为及纹状体前列腺凋亡反应蛋白基因表达的影响

目的 研究早期母爱剥夺对大鼠成年后的抑郁水平及纹状体前列腺凋亡反应蛋白(par-4)表达的影响,以及甲基化是否参与par-4基因表达的调控.方法 按照随机数字表法,将新生幼鼠按窝分为母爱剥夺组和对照组,每组17只;母爱剥夺组仔鼠在出生后的第1～14天,每天接受6 h母子分离,对照组不接受任何实验处理;13周龄时,采用强迫游泳实验和糖水偏爱实验测定大鼠的抑郁水平,采用实时定量聚合酶链反应检测纹状体par-4及多巴胺受体2(DRD2)信使RNA(mRNA)表达水平,采用亚硫酸盐测序法检测par-4基因启动子区DNA甲基化水平.结果 母爱剥夺组大鼠漂浮时间[(152.80±74.21)s]较对照组[(63.80±80.55)s]延长,糖水偏爱率(0.45±0.23)较对照组(0.69±0.25)降低,差异均有统计学意义(t=2.79,P<0.05;t=-2.57,P<0.05);母爱剥夺组大鼠纹状体内par-4(0.02±0.02)及DRD2 mRNA表达量(0.11±0.09)分别为对照组[(0.04±0.02)、(0.32±0.21)]的0.53倍和0.31倍;母爱剥夺组大鼠par-4基因启动子区DNA甲基化水平与对照组相比差异无统计学意义(t=-0.748,P>0.05).结论 母爱剥夺能引发大鼠抑郁样行为表现,并能抑制纹状体par-4的表达,基因甲基化可能不是其调控机制.

Abstract：

Objective To study the effect of maternal deprivation on the depressive behaviors and the expression of prostate apoptosis response-4(par-4)in adult rats'striatum,and to explore whether DNA methylaion be involved in the regulatory mechanism of par-4 expression.Methods Newborn rats were randomly divided into two groups,the maternal deprivation group rats(n=17)were deprived from their mother 6 hours per day from postnatal day 1 to 14,while the control group rats(n=17)received no experimental handle.When they grew up to thirteen weeks,their depressive level was assessed with forced swimming test and sucrose consumption test,the mRNA expression of par-4 and DRD2 in rats'striatum was detected by real-time Polymerase Chain Reaction(real-time PCR),and the DNA methylation level of par-4 was determined by bisulfated DNA sequencing.Results The float time of maternal deprivation rats [(152.80±74.21)s]was longer than the control rats[(63.80±80.55)s](t=2.79,P<0.05),and the sucrose preference rate of maternal deprivation rats (0.45±0.23)was reduced compared with the control rats(0.69 ±0.25)(t=-2.57,P<0.05).The fold changes of par-4(0.02±0.02)and DRD2(0.11±0.09)mRNA expression in maternal deprivation rats compared to control rats[(0.04±0.02),(0.32±0.21)]were 0.53 and 0.31 respectively.However,there was no significant difference between two groups in methylation level of par-4 promoter region(t=-0.748,P>0.05).Conclusion Maternal deprivation could induce the depressive behavior of rats and influence the expression of par-4,but DNA methylation may not be involved in the regulatory mechanism.     

Sleep deprivation attenuates experimental stroke severity in rats

Indirect epidemiological and experimental evidence suggest that the severity of injury during stroke is influenced by prior sleep history. The aim of our study was to test the effect of acute sleep deprivation on early outcome following experimental stroke. Young male Sprague-Dawley rats (n = 20) were subjected to focal cerebral ischemia by reversible right middle cerebral artery occlusion (MCAO) for 90 min. In 10 rats, MCAO was performed just after 6-h of total sleep deprivation (TSD) by “gentle handling”, whereas the other rats served as controls. Neurological function during the first week after stroke was monitored using a battery of behavioral tests investigating the asymmetry of sensorimotor deficit (tape removal test and cylinder test), bilateral sensorimotor coordination (rotor-rod and Inclined plane) and memory (T-maze and radial maze). Following MCAO, control rats had impaired behavioral performance in all tests. The largest impairment was noted in the tape test where the tape removal time from the left forelimb (contralateral to MCAO) was increased by ∼ 10 fold (p < 0.01). In contrast, rats subjected to TSD had complete recovery of sensorimotor performance consistent with a 2.5 fold smaller infarct volume and reduced morphological signs of neuronal injury at day 7 after MCAO. Our data suggest that brief TSD induces a neuroprotective response that limits the severity of a subsequent stroke, similar to rapid ischemic preconditioning.     

Increase in the threshold of a nociceptive test induced by naloxone in morphine-tolerant rats

The effects of various doses of naloxone (3–1000 μg/kg i.v.) on the vocalization threshold elicited by pressure on the paw were evaluated in rats chronically treated with high doses of morphine. In addition to the well known precipitation of withdrawal induced by naloxone, an unexpected dose-related increase in the vocalization threshold was observed.     

Severe deficit in brain reward function associated with fentanyl withdrawal in rats.

BACKGROUND: During the last decade, there has been a strong increase in the use of the mu-opioid receptor agonist fentanyl. The aim of these studies was to investigate the effects of fentanyl withdrawal on brain reward function and somatic withdrawal signs. METHODS: Fentanyl and saline were chronically administered via minipumps. An intracranial self-stimulation procedure was used to provide a measure of brain reward function. Somatic signs were recorded from a checklist of opioid abstinence signs. RESULTS: The opioid receptor antagonist naloxone induced a dose-dependent elevation in brain reward thresholds and somatic withdrawal signs in fentanyl-treated rats. Discontinuation of fentanyl administration resulted in a time-dependent elevation of brain reward thresholds and somatic withdrawal signs. CONCLUSIONS: These findings indicate that fentanyl withdrawal is associated with affective and somatic withdrawal signs. The severity of the deficit in brain reward function in this animal model suggests that affective fentanyl withdrawal symptoms may be a strong deterrent to abstinence.     

Accumbens activity during a multiple schedule for water and sucrose reinforcement in rats

Electrophysiological recording procedures were used to examine nucleus accumbens (Acb) cell firing during operant responding for water reinforcement vs. a highly palatable sweet solution (0.6 M sucrose). Rats (n = 8) were trained on a multiple schedule to press one lever for water (fixed ratio 1, FR1; 15 min) followed by a 20-sec timeout period (chamber dark, levers retracted), and extension of a second spatially distinct lever that the animals pressed for sucrose reinforcement (FR1; 15 min). Of 84 cells, 55 neurons (65%) displayed one of three types of patterned discharges (increases or decreases in firing rate) immediately before or following the sucrose- or water-reinforced response. The major finding of this report was that the majority of these neurons (36/55 cells; 65%) showed similar types of neuronal firing patterns across the two reinforcer conditions. The remaining cells (19/55; 35%) exhibited patterned activity specific to responding for one reinforcer only (water or sucrose). These findings are discussed with respect to how Acb neurons encode goal-directed behaviors for "natural" reinforcers including food, water, and a palatable sweet solution.     

Preoptic Area Infusions of Morphine Disrupt-and Naloxone Restores-Parental-Like Behavior in Juvenile Rats

As in the adult lactating female, opioids disrupt (and naloxone restores), parental behavior in juvenile rats (25 days of age). Because the preoptic area regulates the display of parental behavior in lactating females, we examined its parental behavior role in the juvenile rat. At 21 days of age, juvenile rats were implanted with bilateral cannulae aimed at the preoptic area using a modified Kopf stereotaxic and extrapolating from a developing-rat brain atlas [58], and divided into two groups: initiation and maintenance. On day 25, the Initiation group received bilateral infusions of either morphine (0.50 μg), saline (0.25 μl), or morphine plus naloxone (0.25 μg). Thirty minutes later, they were exposed to three 1–6-day-old pups; the maintenance group was exposed to pups until they displayed 2 consecutive days of parental behavior, then infused. Morphine disrupted parental behavior in both the Initiation and Maintenance groups, and naloxone restored the behavior to control/saline levels. Parental behavior in the juvenile animal of both sexes, therefore, is under opioid regulation that parallels the adult female.     

Endogenous enkephalins, not endorphins, modulate basal hedonic state in mice

The aversive response to naloxone administration observed in human and animal studies suggests the presence of an endogenous opioid tone regulating hedonic state but the class(es) of opioid peptides mediating such opioid hedonic tone is uncertain. We sought to address this question using mice deficient in either beta-endorphin or pro-enkephalin in a naloxone-conditioned place aversion paradigm. Mice received saline in the morning in one chamber and either saline or naloxone (0.1, 1 or 10 mg/kg, s.c.) in the afternoon in another chamber, each day for 3 days. On the test day they were given free access to the testing chambers in the afternoon and the time spent in each chamber was recorded. Whereas wild-type and beta-endorphin-deficient mice exhibited a robust conditioned place aversion to naloxone, pro-enkephalin knockout mice failed to show aversion to naloxone at any dose tested. In contrast, these mice showed a normal conditioned aversion to the kappa opioid receptor agonist, U50,488 (5 mg/kg), and to LiCl (100 mg/kg) indicating that these mice are capable of associative learning. In a separate experiment, pro-enkephalin knockout mice, similar to wild-type and beta-endorphin-deficient mice, demonstrated a significant conditioned place preference to morphine (2.5, 5 and 10 mg/kg s.c.). These data suggest that enkephalins, but not endorphins, may mediate an endogenous opioid component of basal affective state and also indicate that release of neither endogenous enkephalins nor endorphins is critical for the acquisition or expression of the association between contextual cues and the rewarding effect of exogenously administered opiates.     

Opiate mechanism in reward-related neuronal responses during operant feeding behavior of the monkey

Reward-related neuronal activity and its modulation by morphine and naloxone was investigated by extracellular single neuron recording and electrophoretic application of drugs in the lateral hypothalamus, during operant feeding of the monkey. Morphine-sensitive neurons responded more often during bar press and ingestion-reward phases. Naloxone blocked only ingestion-reward responses, especially the inhibitory ones. The results suggest that the central opiate system can be involved in reward-related neuronal responses in the lateral hypothalamic area of the monkey.     

REM-sleep deprivation, stress and emotional behavior in rats

Eighty-eight adult white rats were divided into 9 groups. Groups 1 and 2 served as controls. The rats of Group 3 were repeatedly aroused during 4 days at the very onset of each REM-sleep period by direct midbrain reticular formation stimulation. This deprivation decreased the daily amount of REM-sleep by 70%, while slow-wave sleep was reduced by 10% only. In Group 4, the animals were given food and water for 1 h a day only. Groups 5 and 6 were subjected to immobilization and cold stress, respectively. Groups 7, 8 and 9 were deprived of REM-sleep on platforms of 15, 11 and 6.5 cm in diameter, respectively. Stress was estimated by the classical Selye's triad: weight of adrenals and thymus and gastric ulceration. Emotionality was measured in the open-field and also by self-stimulation of the lateral hypothalamus. Neither emotional behavior disturbances nor Selye's stress features were found after REM-deprivation in Group 3. Moreover, arousal deprivation induced a slight, though significant, reduction in adrenal weight. Also, no changes in emotional behavior were noted in stress-exposed groups (5 and 6). Only the interplay between REM-sleep deprivation and stress on the platforms (Groups 7, 8 and especially 9) led to a considerable shift in emotionality.     

Morphine reinforces post-discharge inhibition of α-motoneurons in man

Morphine chlorhydrate (0.25 mg/kg) produced a significant and constant reinforcement of post-discharge inhibition of α-motoneurons in both normal and paraplegic humans. This effect was reversed by naloxone hydrochloride (0.02 mg/kg). These data do not allow to differentiate the effects of morphine on recurrent inhibition and on A.H.P.     

Does naloxone suppress self-stimulation by decreasing reward or by increasing aversion?

Fifty-eight rats were implanted with electrodes in the ventrolateral midbrain central gray from which both self-stimulation reward and/or stimulation-produced analgesia can be obtained. Thirty-nine cases were positive for self-stimulation; of these, 24 also displayed significant stimulation-produced analgesia and 15 did not. Injections of the opiate receptor blocker, naloxone, suppressed self-stimulation by approximately 40% at both analgesic and non-analgesic reward sites. Since naloxone failed to act preferentially at analgesic reward sites, the hypothesis that naloxone suppresses self-stimulation primarily by antagonizing endorphin-mediated analgesia, and thereby increasing the aversive properties of the brain stimulation, was not supported. Rather, the data are consistent with the hypothesis that naloxone suppresses self-stimulation by antagonizing endorphin-mediated reward.