吗啡依赖小鼠海马神经元c-fos的表达

目的:探讨吗啡依赖小鼠海马不同亚区神经元c-fos表达的差异。方法:以剂量递增法皮下注射吗啡建立吗啡依赖小鼠模型,腹腔注射纳洛酮诱发戒断症状。根据小鼠戒断反应中出现的跳跃次数、体重下降等指标评定戒断反应强度。采用免疫组织化学法显示吗啡依赖小鼠和正常对照组小鼠海马神经元c-fos的表达。结果:吗啡依赖组海马CA1区和齿状回Fos阳性神经元数目明显增加(P<0.05),CA3区无明显改变(P>0.05)。纳洛酮催促戒断组海马CA1和CA3区Fos阳性神经元数目明显增加(P<0.05),齿状回Fos阳性神经元数目增加更为明显(P<0.01)。吗啡依赖组与纳洛酮催促戒断组CA3区阳性神经元数目有显著性差异(P<0.05)。结论:海马神经元c-fos的表达增强可能与吗啡依赖对神经元的损伤有关。     

CCK-8及其受体拮抗剂对吗啡戒断大鼠额叶皮质及海马CREB与pCREB表达的影响

 目的：观察八肽胆囊收缩素（CCK-8）及其受体拮抗剂对吗啡戒断大鼠额叶皮质和海马cAMP反应元件结合蛋白（CREB）表达及其磷酸化（pCREB）的影响,初步探讨CCK-8调节吗啡戒断大鼠的受体后机制。方法：建立大鼠吗啡慢性依赖及纳络酮催促戒断模型,并给予CCK-8、CCK1受体拮抗剂L-364718和CCK2受体拮抗剂LY-288513慢性干预,应用Western blotting和免疫组织化学技术观察额叶皮质和海马CREB与pCREB 表达的变化。结果：（1） 正常组大鼠额叶皮质神经元胞浆、胞核均表达CREB蛋白,pCREB蛋白则仅在胞核中高表达;海马CA1区锥体细胞层神经元中,CREB蛋白在胞浆中高表达,胞核低表达,pCREB蛋白则仅在胞核中表达。（2） 慢性吗啡作用后CREB无明显变化,pCREB增加;急性纳洛酮催促戒断后CREB仍无明显变化,pCREB进一步升高。（3） 与戒断组相比,CCK-8、L-364718和LY-288513慢性干预对吗啡依赖戒断大鼠额叶皮质CREB蛋白表达无明显影响,pCREB蛋白表达均明显降低;L-364718和LY-288513慢性干预后,海马CREB与pCREB表达均明显降低,而CCK-8慢性干预对CREB蛋白表达无明显影响,仅pCREB蛋白表达明显降低。结论：CCK-8及其受体拮抗剂可能通过调节核转录因子CREB减轻吗啡戒断症状,并具有脑区特异性。     

吗啡慢性处理及戒断后不同时期大鼠海马脑源性神经营养因子与trkB表达的变化

目的:观察吗啡慢性处理及戒断后不同时期大鼠海马内脑源性神经营养因子(BDNF)与trkB的表达.方法: 实验组雄性成年SD大鼠以剂量递增的方法连续腹腔注射吗啡10d,每天2次.动物在戒断后0、1、7、14d和21d处死.对照组动物注射等量生理盐水,按同样方法处理.用免疫组织化学方法(ABC法)检测海马CA1区BDNF与trkB的表达.用Motic 3.2图像分析系统测定免疫阳性产物的平均光密度值.结果: 实验组BDNF免疫阳性产物的平均光密度值在戒断后0、1、7、14d和21d分别低于相应对照组,差异具有统计学意义,以戒断后14d下降最为明显.实验组trkB免疫阳性产物的平均光密度值在戒断后0、1、7、14d和21d分别也低于相应对照组,差异具有统计学意义.结论: 大鼠慢性吗啡成瘾及戒断后不同时间点BDNF与trkB在海马CA1区的表达明显下降,这种下降可能与大鼠吗啡成瘾及戒断后不同时期的行为学变化有关.     

复方瑞康欣可逆转吗啡戒断焦虑大鼠海马、杏仁核和伏核突触形态结构的可塑性

目的 观察复方瑞康欣(CR)对吗啡戒断焦虑大鼠海马、杏仁核和伏核突触形态结构可塑性的影响.方法 雄性SD大鼠84只,随机分为生理盐水对照组、吗啡戒断焦虑模型组、CR 高、中、低剂量组和丁螺环酮组.以剂量递增方式皮下注射吗啡,10 d后自然戒断,于戒断1～3 d CR灌胃治疗后行高架十字迷宫实验.采用透射电镜技术比较各组...     

八肽胆囊收缩素在吗啡依赖及戒断大鼠相关脑区的表达变化

目的:探究慢性吗啡依赖及戒断大鼠相关脑区八肽胆囊收缩素(CCK-8)表达的变化。方法:利用Wistar大鼠建立慢性吗啡依赖及戒断模型,分为对照组(control)、吗啡依赖组(MOR)、纳洛酮催促戒断组(NAL)。Gellert-Holtzman评分评价吗啡成瘾及戒断程度,采用免疫组织化学和原位杂交技术分别检测相关脑区CCK-8蛋白及CCK mRNA表达的变化。结果:戒断组Gellert-Holtzman评分与对照组和吗啡依赖组比较显著增加;前额叶皮质(PFC)、黑质致密部(SNc)、中脑腹侧被盖区(VTA)CCK-8及CCK mRNA表达依赖组较对组明显升高,戒断后表达下降;杏仁核、蓝斑(LC)吗啡依赖后CCK-8及CCK mRNA表达较对照组显著增高,海马齿状回(PoDG)、杏仁核、LC戒断后较依赖组表达进一步升高;尾壳核(CPU)、伏隔核(NAc)、中脑导水管周围灰质(PAG)未检测到CCK-8及CCK mRNA的表达。结论:CCK-8参与了大鼠吗啡依赖与戒断过程并具有脑区特异性。     

Akt/mTOR信号通路在海人酸损伤大鼠海马组织中的激活

目的 研究Akt/mTOR信号通路在海人酸诱导的大鼠海马神经元损伤中的激活和HIF1α的表达情况。方法 成年SD大鼠随机分为3组,其中2组接受脑室注射,一组为假手术组。大鼠麻醉后颅骨钻孔,侧脑室注射KA 1.0 ?g（KA组）或等体积生理盐水（NS组）或相应位置颅骨钻孔,不予注射（假手术组）。8 和16 h及1 、3 和5 d后,用Western blot观察Akt和mTOR的表达及其磷酸化水平,用免疫组化观察HIF1α的表达情况。结果 尼氏染色显示KA组大鼠海马CA3区1 d时即开始有死亡,5 d时累及CA1区。Akt在KA损伤16 h后就开始激活并持续至第5 d,mTOR于第1 d开始激活,第3 d时达峰值。CA3区的HIF1α于第1 d明显升高,CA1区的HIF1α表达则于3 d时明显升高。NS组和假手术组上述诸指标变化均不明显。结论 KA诱导大鼠海马中Akt/mTOR通路有激活,可能调节神经元的存活。     

吗啡成瘾戒断对大鼠海马CA1区P-CREB表达的影响

目的探讨吗啡成瘾戒断后大鼠海马CA1区P-CREB的表达变化。方法48只健康雄性成年SD大鼠,随机分为实验组和对照组:实验组腹腔注射吗啡起始剂量5mg/kg,1d2次,逐日递增,连续10d;对照组:以生理盐水代替吗啡。停药后7d、14d和21d处死。用免疫组织化学方法(ABC法)检测海马CA1区P-CREB的表达,图像分析系统测定阳性反应产物的平均灰度值。结果P-CREB表达在7d、14d实验组与对照组相比表达上调(P<0.05),21d与对照组比较无显著差异(P>0.05)。结论海马CA1区CREB磷酸化在吗啡依赖的形成中发挥作用。     

NCX1在吗啡依赖大鼠前额皮质和海马中的表达变化

目的:观察吗啡条件性位置偏好(CPP)大鼠的前额皮质(PFC)和海马(Hip)中钠钙交换体亚型1(NCX1)的变化。方法:40只健康雄性SD大鼠随机分为:对照组(control)和吗啡组(morphine)。吗啡以起始剂量10 mg/kg皮下注射到大鼠颈背部,每日递增,连续注射10 d,第10 d剂量为100 mg/kg。每次注射20 min后,将大鼠放置黑、白箱(morphine)中进行训练。在最后一次训练48 h后,进行CPP实验以确认大鼠吗啡依赖模型建立成功,并且立即将两组大鼠处死后取脑。分离PFC和Hip脑区,通过Western Blot和real time RT-PCR技术分别检测两个脑区中NCX1的蛋白和mRNA的表达水平。结果:CPP结果显示,吗啡依赖大鼠在吗啡伴药箱中停留时间较对照组明显延长(P 0. 05);训练后吗啡组的大鼠在吗啡伴药箱中停留的时间明显长于对照组(P 0. 05)。与对照组相比,依赖组大鼠PFC和Hip中NCX1的m RNA和蛋白表达水平显著升高(P 0. 05)。结论:NCX1在吗啡依赖大鼠PFC和Hip部位表达增加,提示PFC和Hip部位的NCX1可能与吗啡依赖产生机制有关。     

大鼠触液核中TRPC6的分布及其在吗啡依赖与戒断条件下的表达

目的:研究TRPC6在大鼠触液核(cerebrospinal fluid-contacting nucleus,CSF-CN)中的分布及其在吗啡依赖与戒断条件下的表达变化。方法:雄性SD大鼠随机分为4组:正常大鼠组、生理盐水组、吗啡依赖组和吗啡戒断组。每组大鼠进行戒断总评分(total withdrawal scores,TWS);采用侧脑室注射CB-HRP,并结合CB-HRPP/TRPC6免疫荧光双标技术,观察大鼠触液核内TRPC6的表达情况。结果:上述4组大鼠的戒断总评分分别为2.0±0.6、2.1±0.7、2.6±0.7和41.0±4.6,其中吗啡戒断组与其他三组相比,差异具有显著性(P<0.01);在侧脑室注射CB-HRP后,上述4组大鼠触液核内可观察到大量的CB-HRP标记神经元;同时在触液核内也可观察到部分神经元表达TRPC6免疫阳性。经计数,CB-HRP/TRPC6双标神经元的数量分别为81.78±4.93、79.44±7.09、254.61±15.36和260.00±12.04,其中吗啡依赖组和吗啡戒断组分别与其他两组相比,差异具有显著性(P<0.01);但吗啡依赖组和吗啡戒断组相比,差异无显著性(P>0.05)。结论:正常大鼠触液核内表达TRPC6;触液核可能通过上调TRPC6的表达参与吗啡依赖和戒断的形成。     

苯丙胺对大鼠行为、学习能力及海马CA3区突触素表达的影响

目的观察苯丙胺对大鼠行为、空间辨别性学习能力和海马CA3区突触素表达的影响。方法将45只健康雄性SD大鼠随机分为:正常对照组、生理盐水组和苯丙胺组。①苯丙胺组每天肌注0.5mg/kg苯丙胺1次;②生理盐水组即注射等体积生理盐水;③正常对照组不予任何处理。每组大鼠分14d、28d和42d三个时间段进行一般行为学、学习记忆能力及海马CA3区突触素表达的检测。结果①苯丙胺组大鼠在注射苯丙胺10d后出现直立、点头、狂躁等行为改变。正常对照组、生理盐水组均无此现象出现;②苯丙胺组大鼠空间辨别性学习记忆能力的影响,在第1、2个时间段与对照组的比未见组间差异,在第3时间段的平均运行时间和正确率比对照组的延长和降低(P<0.05)。③苯丙胺组大鼠海马CA3区突触素表达在第一阶段比对照组的减少,并随着用药时间的延长减少的更明显(P<0.05)。结论苯丙胺对空间辨别性学习记忆时大鼠行为及海马CA3区突触素表达有影响。     

PV阳性神经元在孤独症模型大鼠情绪与认知相关脑区内的表达

为显示小白蛋白(Parvalbumin,PV)中间神经元在孤独症脑内的形态变化,更好地理解PV中间神经元在孤独症发病过程中的作用,本研究用免疫组化法观察了孕鼠腹腔注射丙戊酸钠(VPA)和反复冷冻刺激(RCS)法建立的两种孤独症动物模型杏仁体、前额叶皮层和海马区的PV阳性神经元的表达特点。结果显示:两种孤独症模型鼠杏仁体、前额叶皮层和海马区内的PV阳性神经元的胞体形态、大小、突起的长度和密度等与正常对照组(CTL)相比,都发生了不同程度的变化。这些结果提示,PV中间神经元在孤独症的发病过程中发挥重要的作用,它们的改变可能削弱了对孤独症相关的神经环路中锥体神经元的抑制作用。     

Expression of cocaine sensitization: regulation by the medial prefrontal cortex.

Extracellular levels of dopamine are increased in response to systemic administration of cocaine in several brain areas including the nucleus accumbens and medial prefrontal cortex. While the cocaine-induced increase in extracellular dopamine levels in the nucleus accumbens is augmented after repeated daily cocaine, the response of extracellular dopamine levels in the medial prefrontal cortex is attenuated. Since dopamine in the medial prefrontal cortex has an inhibitory effect on nucleus accumbens dopamine levels and locomotor activity, the role of medial prefrontal cortex dopamine tolerance in the expression of sensitized locomotor behavior was further examined by injection of D-amphetamine sulfate into the prelimbic portion of the medial prefrontal cortex just prior to cocaine challenge in cocaine-sensitized rats. Male Sprague-Dawley rats were non-handled (naive) or injected with either saline (1 ml/kg, i.p.) or cocaine (15 mg/kg, i.p.) for five consecutive days. After a seven to 12 day withdrawal period, rats were microinjected with either saline or various doses of amphetamine into primarily the prelimbic region of the medial prefrontal cortex followed by systemic injection of saline or cocaine. In naive rats, intramedial prefrontal cortex amphetamine produced a trend toward decreased locomotor responding to cocaine challenge while no effect of amphetamine was evident in daily saline pretreated rats. Daily cocaine pretreated rats that received saline in the medial prefrontal cortex demonstrated a sensitized locomotor response compared to their daily saline pretreated counterparts. This sensitization was blocked by a low dose of amphetamine (0.175 microg/side) in the medial prefrontal cortex, an effect which disappeared in animals administered higher amphetamine doses. The results suggest that in rats sensitized to cocaine, decreased medial prefrontal cortex dopamine levels in response to cocaine challenge may contribute to behavioral sensitization. Furthermore, the data indicate the possibility that there is an optimal range at which medial prefrontal cortex amphetamine exerts maximal behavioral inhibition. These findings implicate a role for decreased cortical control in producing sensitized behavioral responding to cocaine.     

Up-regulation of murine double minute clone 2 (MDM2) gene expression in rat brain after morphine, heroin, and cocaine administrations

Repeated administration of addictive drugs induces neuronal apoptosis and the underlying mechanisms are not clear. Our present study investigated the effects of treatments with different addictive drugs on gene expression of murine double minute clone 2 (MDM2), a key negative regulator of p53 and an important mediator in cell apoptosis. The level of MDM2 gene expression in rat brain was assessed using in situ hybridization histochemistry. In normal adult rat brain, MDM2 expression was at a very low level but MDM2 mRNA-positive cells were detected in various regions including cortex, hippocampus, thalamus, amygdala, periaqueductal gray and locus ceruleus. After a single morphine injection, MDM2 gene expression increased significantly in hippocampus, amygdala and cortex; however, such up-regulation of MDM2 gene expression was significantly reduced after repeated morphine administration. Moreover, 24 h after cessation of chronic morphine exposure, MDM2 mRNA increased again to a level comparable to that of the acute morphine group. Acute heroin or cocaine administration also significantly increased MDM2 gene expression in hippocampus, but not in cortex. In thalamus, no change was detected after acute or chronic treatment with morphine, heroin, or cocaine. Thus we demonstrated for the first time that the administration of addictive drugs regulate MDM2 gene expression in distinct rat brain regions and these data suggest that MDM2 may play an important role in the development of drug addiction.     

Characterization of blood glucose level regulation in mouse opioid withdrawal models

The regulation of blood glucose level in intracerebroventricular (i.c.v.) administration with opioid alone or opioid withdrawal model was studied in ICR mice. In the first group, we found that i.c.v. administered morphine or β-endorphin alone causes an elevation of blood glucose level. Blood glucose level induced by i.c.v. morphine or β-endorphin began to increase within 30 min and reached maximal level at 1 h, decreasing to the basal level after 2 h. In another group, we observed that intraperitoneal (i.p.) injection with naloxone (10 mg/kg) post-treated 3 h after either a single i.c.v. injection with morphine or β-endorphin did not affect the increased blood glucose level in either group. In the next study, we observed that multiple (1 time/day for 3 days) i.c.v. injection with morphine alone significantly increased the blood glucose level. However, i.p. injection with naloxone post-treated 3 h after the last i.c.v. injection with morphine caused a decrease of blood glucose level. We found that multiple (1 time/day for 3 days) i.c.v. injections with β-endorphin did not affect the blood glucose level. Furthermore, i.p. injection with naloxone did not affect the blood glucose level in the mice injected with multiply β-endorphin. Our results suggest that both morphine and β-endorphin administered i.c.v. acutely increases the blood glucose level. However, blood glucose levels in the groups of multiply administered morphine alone, β-endorphin alone, and naloxone-treated withdrawal model in multiply injected morphine and β-endorphin appear to be differentially regulated.     

大鼠主动逃避学习后pElk-1在脑内表达分布的时程变化

为探讨大鼠主动逃避学习后转录因子pElk1在脑内表达分布的时程变化,将55只成年SpragueDawley大鼠,分为正常对照组、Y迷宫训练组和假训练组,其中训练组与假训练组再各分为训练后0、1、3、6、24h组,每组动物各5只。训练组动物接受Y迷宫光-电结合训练,假训练组动物接受光电不结合假训练。应用免疫组织化学方法检测各组大鼠脑内各区pElk1分布及表达的变化。结果发现:pElk1免疫阳性神经元在全脑内分布广泛,在纹状体边缘区皮层大部、下丘脑、杏仁核、海马、尾壳核、边缘区、小脑均有较强表达;Y迷宫训练后0、1、3、6h,在海马、皮层大部、杏仁核、下丘脑、纹状体尾壳核及边缘区、小脑均有pElk1免疫阳性神经元表达的持续增强,训练后24hpElk1免疫阳性反应回归到正常组水平;假训练组在假训练后各时间点也有皮层大部、杏仁核等部位的表达增强,但在海马、尾壳核、纹状体边缘区等部位表达增强不明显,与训练组表达强度相比,差异有显著性。以上结果表明:pElk1在全脑分布广泛,Y迷宫学习增强海马、尾壳核、纹状体边缘区等区域的pElk1的表达,提示pElk1可能参与了Y迷宫相关的各脑区的学习记忆活动。     

Extremely low-frequency electromagnetic field exposure during chronic morphine treatment strengthens downregulation of dopamine D2 receptors in rat dorsal hippocampus after morphine withdrawal

The aim of this study was to investigate the effect of extremely low-frequency electromagnetic field (ELF-EMF) exposure during morphine treatment on dopamine D2 receptor (D2R) density in the rat dorsal hippocampus following withdrawal. Rats were exposed to ELF-EMF (20 Hz, 14 mT) or sham exposed for 1h per day before injection of morphine (10mg/kg, i.p.) once daily for 12 days. The saline control group was sham exposed for the same period. Immunohistochemistry was used to detect the density of D2Rs on the 1st, 3rd and 5th morphine withdrawal days. The results showed that the density of D2Rs in sham-exposed morphine-treated rats on the 1st and 3rd days of morphine withdrawal was significantly lower than that of the saline control group. The ELF-EMF-exposed morphine group also exhibited a significantly lower density of D2Rs on the 1st and 3rd withdrawal days relative to the sham-exposed morphine group. However, the D2R density in both groups tended to recover as morphine withdrawal days increased. The results suggest that dorsal hippocampal D2Rs are sensitive to morphine withdrawal and that this is potentiated by ELF-EMF pre-exposure during morphine treatment.