长期低剂量氯胺酮对青春期大脑前额叶的损伤效应与机制研究
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摘要
1前言
氯胺酮(ketamine)是使用较为广泛的镇痛麻醉剂,但目前已成为主流娱乐性质的毒品,且滥用现象日趋严重。以英国为例,1999-2003年,娱乐性使用氯胺酮的人群增加了近2倍,且以青少年群体为主。研究提示,使用低于麻醉剂量的氯胺酮可出现难以言喻的愉快体验,如同从地狱跃入天堂。另有研究表明,正常成年人使用亚麻醉剂量的氯胺酮能够产生类似精神分裂症的阳性和阴性症状,包括知觉改变、幻视、幻觉、去人格化和现实感丧失等。滥用氯胺酮引起的个体和群体效应已经引起国际广泛关注。目前,氯胺酮在青少年亚群体中的普遍流行和滥用,原因之一是人们认为它不会导致躯体性依赖；其二,青少年群体具有追求新奇事物和新奇感觉的年龄特征。现氯胺酮在多数国家已成为限制性药物。
氯胺酮是N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMD A)受体的非竞争性拮抗剂,调节谷氨酸和天冬氨酸等兴奋性氨基酸(excitatory amino acids, EAAs)的功能,在突触可塑性和学习记忆中扮演着重要的角色。最近的研究发现,静脉给予成年人短期氯胺酮可损害认知功能和心理健康。但对长期低剂量氯胺酮是否对脑功能产生影响、其生理机制如何却鲜有研究。而长期低剂量使用方式与娱乐场所的氯胺酮滥用状态更为相似。
大多数成瘾药物是通过脑奖赏环路(VTA-NAcc-PFC神经环路)实现成瘾药物的奖赏效应。前额叶皮层(prefrontal cortex, PFC)接受来自腹侧被盖区(ventral tegmental area, VTA)的多巴胺能神经纤维支配,与情感行为、学习与记忆等诸多脑的高级功能密切相关。认知是指人们认识活动的过程,即个体对感觉信号接收、检测、转换、简约、合成、编码、储存、提取、重建、概念形成、判断和问题解决的信息加工处理过程。已经证实PFC是认知过程的关键脑区,构成了认知过程的中枢管理和工作记忆缓冲的基础结构。PFC内部合理的联系为合成不同范围的信息提供了完美的基础结构。有证据表明,PFC的多巴胺能系统对氯胺酮尤为敏感,对啮齿类动物研究发现,持续给予一定量氯胺酮(20mg/kg)可引起中枢神经系统(central nervous system, CNS)特定区域神经元细胞发生程序性细胞死亡,通过线粒体和胞质内质网通路凋亡通路,增加促凋亡因子的表达,加速细胞凋亡。且有证据表明,发育期大脑的这种损伤效应会对成年期的脑功能产生影响。
本研究假设长期娱乐剂量氯胺酮可能通过细胞凋亡途径引起大脑特定脑区,尤其是PFC脑区的细胞毒性作用,造成自发行为、认知功能的损伤。
2目的
2.1观察长期低剂量氯胺酮对青少年期ICR小鼠体重和神经肌肉强度、痛觉末梢和空间学习记忆的影响。
2.2观察长期低剂量氯胺酮对青少年期食蟹猴体重、自发行为的影响。
2.3探讨长期低剂量氯胺酮对青春期脑前额叶神经元凋亡表达的影响。
2.4揭示长期低剂量氯胺酮对青春期脑功能的影响和相关机制。
3材料和方法
3.1ICR小鼠实验动物模型制备
90只小鼠随机分为3组(每组30只),按给药时间长短分为一个月、三个月和六个月组。各组又随机分为对照组(10只)和氯胺酮组(20只),总实验周期为6个月。氯胺酮组每天腹腔注射给予盐酸氯胺酮30mg/kg,对照组给予生理盐水1mg/kg。
3.2ICR小鼠体重测量
实验开始前测量ICR小鼠基础体重,之后每周测量并记录体重变化情况,并随时调整给药剂量,观察长期给予氯胺酮是否会影响ICR小鼠体重增长。
3.3ICR小鼠行为学观察
在造模结束后进行连续3天的行为学测试,内容包括悬挂实验、热板实验和水迷宫实验。
3.4ICR小鼠前额叶神经元凋亡检测
采用核糖核酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, TUNEL)检测PFC中神经元细胞凋亡的情况。取PFC石蜡切片蛋白酶K (proteinase K, pK)孵育,平衡液处理,TUNEL反应混合液孵育,DAB显色,显微镜观察计数神经元凋亡的数量。
3.5ICR小鼠前额叶免疫印迹分析(Western Blotting)
总蛋白经抽提后蛋白定量,按照上样量为100μg的量计算上样体积,经加热变性处理后上样,电泳,5%浓缩胶80V,12%分离胶120V；电转后移至PVDF膜上,5%脱脂奶粉室温封闭1h,一抗4℃过夜,洗膜3次,二抗室温1h,洗膜3次,用ODYSSEY红外成像系统记录显影条带的光密度值。
3.6食蟹猴实验动物模型制备
24只食蟹猴随机分为3组(每组8只),按给药时间的长短分为一个月氯胺酮组,六个月氯胺酮组和六个月对照组。实验组每天静脉注射给予盐酸氯胺酮1mg/kg,对照组给予生理盐水1mg/kg。
3.7食蟹猴体重测量
实验开始前测量食蟹猴基础体重,之后每月测量并记录体重变化情况,观察长期氯胺酮是否会影响食蟹猴体重增长。
3.8食蟹猴行为学观察
在实验的第1、3、7、14、30、31、56、112、183、184、185天给药后录像观察食蟹猴自发行为(行为量表)15min。观察内容包括移动、行走、攀爬、跳跃四种行为。
3.9食蟹猴前额叶神经元凋亡检测
TUNEL染色来检测PFC神经元细胞凋亡情况。取PFC石蜡切片pK孵育,平衡液处理,TUNEL反应混合液孵育,DAB显色,显微镜观察计数神经元凋亡数目。
3.10食蟹猴前额叶免疫印迹分析Western Blotting
总蛋白经抽提后蛋白定量,按照上样量为30μg的量计算上样体积,经加热变性处理后上样,电泳,5%浓缩胶80V,12%分离胶120V；电转后移至PVDF膜上,5%脱脂奶粉室温封闭1h,一抗4℃过夜,洗膜3次,二抗室温1h,洗膜3次,暗室中显影定影,胶片经扫描后用Image J14.0软件分析目的条带的光密度值。
4实验结果
4.1长期低剂量氯胺酮对ICR小鼠体重增长的影响
实验过程中,对照组和氯胺酮组小鼠体重均有不同程度的增加,与初始体重比较均有统计学差异。与对照组相比,氯胺酮组体重增加速度缓慢。不同用药时间的三个组别中氯胺酮组与对照组的体重增长幅度无统计学差异。
4.2长期低剂量氯胺酮对ICR小鼠行为的影响
悬挂实验：一个月和三个月氯胺酮组小鼠掉落软垫所需的延迟时间与其对照组比较,差异无统计学意义,而六个月氯胺酮组在延迟时间上显著少于对照组,具有统计学差异。
热板实验：一个月和三个月氯胺酮组小鼠热板运动总和与其对照组比较,差异无统计学意义,而六个月氯胺酮组的热板运动总和显著少于对照组,具有统计学差异。
水迷宫实验：不同用药时间氯胺酮小鼠寻找逃避平台所需的潜伏期与对照组相比无统计学差异。
4.3长期低剂量氯胺酮对ICR小鼠大脑PFC神经元细胞凋亡的影响
氯胺酮组和对照组TUNEL阳性细胞计数无统计学差异。蛋白印迹分析结果显示,在一个月、三个月和六个月的氯胺酮组与对照组比较,Bax和caspase-3表达增高,Bax/Bcl-2比值增高,Bcl-2表达降低,但差异均无统计学意义。
4.4长期低剂量氯胺酮对青年期食蟹猴体重增加的影响
在6个月的实验过程中,各组食蟹猴体重均增加。氯胺酮组食蟹猴体重增长速度低于对照组,但差异无统计学意义。
4.5长期低剂量氯胺酮对青年期食蟹猴自发活动的影响
与对照组比较,氯胺酮组的自发运动有下降的趋势。给药一个月后,氯胺酮组跳跃显著低于对照组(F=7.439,P<0.01)；在其后的153天中,氯胺酮组移动、行走、跳跃和自发活动总量自身比较具有统计学差异(移动：F=4.048,P<0.05；行走：F=10.753,P<0.01；跳跃：F=4.180,P<0.05)。氯胺酮组移动、攀爬和自发行为总量显著少于对照组,差异有统计学意义(移动：F=10.798,P<0.001；攀爬：F=4.769,P<0.05；自发活动总量：F=5.793,P<0.05)。氯胺酮组自发活动总量随用药时间延长而减少(F=12.914,P<0.0001),且与药物处理存在交互作用(F=7.342,P<0.001)。
4.6长期低剂量氯胺酮对PFC细胞凋亡的影响
与对照组相比,一个月氯胺酮组前额叶TUNEL染色阳性细胞数、促凋亡蛋白(Bax和caspase-3)和Bcl-2表达水平均无显著性差异。六个月氯胺酮组PFC阳性细胞、促凋亡蛋白(Bax和caspase-3)表达水平明显增加,差异有统计学意义。
5结论
5.1长期低剂量氯胺酮可导致青春期小鼠和食蟹猴体重增长速度减慢；小鼠肌肉力量降低和伤害感受受损；抑制食蟹猴自发行为活动。
5.2长期低剂量氯胺酮可诱导食蟹猴前额叶发生明显地细胞凋亡现象,产生神经元细胞毒性作用,进而造成大脑功能受损。
5.3氯胺酮对中枢神经的损伤存在动物种属的差异,与啮齿类动物相比较,非人灵长类动物神经系统对氯胺酮更敏感。这为进一步研究慢性氯胺酮的生理心理机制和制定其临床靶向治疗策略提供了一定的理论基础。
1Introduction
Ketamine, a drug once used as an anesthetic for human and veterinary surgery, has increasingly become a mainstream'recreational' drug. In previous researches, ketamine at a dosage which was much smaller than anesthetic dosage could brought a pleasant sensation of incredible intensively to ketamine users, like'A K-hole can be anything from going to hell and meeting Satan to going to heaven and meeting God'. In the United Kingdom alone, the prevalence rates of ketamine has rapidly increased2-fold from1999to2003among the nightclub goers, especially among the adolescents. While, Ketamine at the subanesthetic dose produced a series of positive and negative schizophrenic-like symptoms including perceptual alternations, visions, delusions, depersonalization and derealization in healthy individuals, and those impairments of abuse ketamine have aroused increasing attention from the international community and various countries. Recently, ketamine has become a controlled (Schedule Ⅲ of The Controlled Substances Act) substance in the USA and other countries.
Ketamine is a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor, which regulates the action of excitatory aminoacids (EAAs) involving glutamate and asparte, and plays a crucial role in mechanisms of synaptic plasticity and neuronal learning. In the recent studies, many researches have validated the short-term effects of ketamine administration in healthy individuals, which induced impairments of cognitive function and psychological wellbeing, relatively little is known about the effects of long-term ketamine abuse. Obviously, the latter conforms to the status of sucking ketamine. Moreover, although rodents have been commonly used for drug administration studies, since the behavioral research of drug addiction and manifestation of working memory deficits, the monkey has emerged as a premier subject to model cognitive, behavioral and neuropsychopathological disorders, and to investigate novel drug treatments of the disease concerned cognitive dysfunction.
'Cognition' in nature, that is, the mental capacity to override or augment reflexive and habitual reactions in order to orchestrated behavior in accord with our intentions, is essential for what we recognize as intelligent behavior. In another word, the primary function of cognitive control is to extract the goal-relevant features of long-term memory programs for use in future circumstances. It has been validated that the prefrontal cortex (PFC) is centrally involved in this process, which requires two essential integrated components:a central executive and working memory buffers. Moreover, the proper interconnection of PFC provides a perfect infrastructure for synthesizing the various range of information needed for complex and extended behavior. Some evidences have showed that the dopaminergic system of PFC is particularly vulnerable to ketamine administration, but the effect of prolonged ketamine administration on PFC is still largely unknown. Currently, some studies performed on rodents have indicated that sustained exposure to ketamine produces programmed cell death in areas of the central nervous system (CNS) and increases the brain expression of pro-apoptosis factors belonging to the mitochondrial and the extrinsic apoptotic pathways. These studies open up the possibility that chronic exposure to ketamine in the CNS would interfere with learning and memory through a neurotoxic effect related to activation of apoptotic pathways. Because of the exacerbation of ketamine abuse in the world and the significance of PFC in cognitive control, we established the hypothesis:chronic ketamine administration of recreational dosage might produce permanent and irreversible deficits in brain functions due to neurotoxic effects involving the activation of apoptotic pathway in the prefrontal cortex.
2Objectives
2.1To observe body weight and neuromuscular strength, the sensitivity to a painful stimulus and spatial navigation learning and memory in ICR mice following chronic low dose of ketamine exposure.
2.2To observe body weight and behavioral changes in adolescent cynomolgus monkeys following chronic low dose of ketamine exposure.
2.3To investigate the effects of chronic low dose of ketamine on cell apoptosis of prefrontal cortex (PFC) in adolescence.
2.4To investigate the effects of chronic low dose of ketamine on the brain functions in adolescence and its underlying mechanism.
3Materials and methods
3.1Animal model of ICR mice established
Ninety male ICR mice were randomly divided into3groups receiving1-,3-or6-month of daily intra-peritoneal injection of ketamine of30mg/kg or saline. In each group of30mice,20were injected with ketamine and10with saline as controls.
3.2Body weight of ICR mice measurement
The weights of mice were recorded every week for adjustment of ketamine injection and monitoring of the well-being of the animals.
3.3Behavioral studies
Behavioral tests were made for3days after ketamine or saline injection. Behavior tests including wire hang test, hot plate test and water maze test.
3.4Apoptosis assay in ICR mice
After behavioral tests, ICR mice were randomly chosen to be sacrificed. Brains were dissected and embedded in paraffin wax. Sections of PFC were cut coronally from paraffin blocks at4-μm thickness.
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) was used to assay apoptosis. Brain tissues sections were incubated with proteinase K, and TUNEL reaction mixture. Finally, the sections were developed with DAB kit. Apoptotic cells were assayed.
3.5Western blotting analysis in ICR mice
PFC tissues were homogenized in cold lysis buffer. The sample was centrifuged at14,000rpm for30min at4℃, then the supernatant was collected and protein content was assayed colorimetrically.100μg total proteins were loaded onto a12% SDS-PAGE gel, electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with primary antibodies at4℃overnight following incubation of secondary antibodies for1h. The bound antibodies were then visualized and recorded using the ODYSSEY Infrared Imaging System (LI-COR Biosciences)
3.6Animal model of cynomolgus monkeys established
Twenty-four adolescent male cynomolgus monkeys were randomly divided into3groups of8monkeys:one group was given daily intravenous injection through the inner side of the lower limbs of1mg/kg body weight of ketamine in saline for1month, another group for6months and the last group were given daily saline injection as the control group also for6months.
3.7Body weight of cynomolgus monkeys measurement
Each monkey was weighed at the beginning of each week, so that the correct ketamine dose could be administered.
3.8Behavioral observation in cynomolgus monkeys
15-min video recordings were made for each monkey after ketamine or saline injection at1st,3rd,7th,14th,30th,31st,56th,112th,183rd,184th and185th day. Behavior observation including Move, Walk, Climb and Jump was conducted.
3.9Apoptosis assay in cynomolgus monkeys
TUNEL was also used to assay apoptosis. The protocol was same to the ICR mice's.
3.10Western blotting analysis in cynomolgus monkeys
PFC tissues were homogenized in cold lysis buffer. The sample was centrifuged at14,000rpm for30min at4℃, then the supernatant was collected and protein content was assayed colorimetrically.30μg total proteins were loaded onto a12%SDS-PAGE gel, electrophoretically transferred to PVDF membrane. The membranes were incubated with primary antibodies at4℃overnight following incubation of secondary antibodies for1h. The membranes were developed using an enhanced ECL detection system. The intensity of bands was determined using the Image J14.0software.
4Results
4.1Chronic low dose of ketamine administration inhibited the body weight increase in ICR mice following chronic ketamine exposure
The percentage increases in body weight for the ketamine and control groups at1,3and6months have increased significantly. The percentage increases were less in all ketamine groups than the percentage increases of the respective control groups. However, the differences in the percentage increases between ketamine groups and their respective control groups were not statistically significant.
4.2Effects on behavioral tests of Chronic low dose of ketamine abuse in ICR mice
According to the results of wire hang test and hot plate tests, after6months' ketamine administration, the latency time and the total number of movements of the6-month ketamine group was significantly less than the6-month control group. However, there were no significant differences and consistent trends of changes in ketamine treated mice for1and3months as compared to their respective controls.
According to the results of the water maze test, the escape latency time for the mice to locate and climb onto the platform showed no statistically significant differences between the1-,3-, and6-month ketamine groups as compared to their respective control groups. Although, the ketamine treated mice in different groups consistently used more time to climb onto the platform.
4.3Effects on PFC apoptosis of Chronic low dose of ketamine abuse in ICR mice
There were no statistically significant differences of the TUNEL positive cell counts in the prefrontal cortex between the ketamine groups of1-,3-,6-month and their respective control groups. Although Western blot results showed consistently in all1-,3-, and6-month ketamine groups had higher Bax, lower Bcl-2, higher Bax/Bcl-2, and higher caspase-3levels in PFC than their respective control groups, all differences were not statistically significant.
4.4Chronic low dose of ketamine administration inhibited the body weight increase in cynomolgus monkeys following chronic ketamine exposure
Both1-month and6-month ketamine and control groups increased their body weights about10%during the first4months. In the latter2months, the control group continued to gain about15%of body weight, while the ketamine group gained very little. However, there was no statistically significant difference between the groups.
4.5Chronic low dose of ketamine abuse in adolescent cynomolgus monkeys depressed motor behaviors
For behavior test, activities of Move, Walk, Climb and Jump of ketamine group exhibited obvious decreased tendency as time went by. With the exception of the results for Jump of1-and6-month ketamine groups versus controls (which were significantly different), results of other behavioral movements (Move, Climb, Walk and Total behavior) showed no significant differences between the ketamine and control groups. However, in the latter153days, results of Move, Walk, Jump and total behavior did show significant changes on different days. Furthermore, results of Total behavior movement showed significant differences both between different days and interactions between different days and control/ketamine groups. In addition, results of Move, Climb and Total behavior showed significant differences between the ketamine and control groups.
4.6Effects on PFC apoptosis of Chronic low dose of ketamine abuse in adolescent cynomolgus monkeys
TUNEL positive cells increased significantly (p<0.05) in the prefrontal cortex of the6-month ketamine group as compared with those of the control group; In Western blot assays, for the6-month ketamine group, Bax and caspase-3in the PFC both increased significantly (p<0.05) compared with the control group. Results of TUNEL positive cells, Bax, Bcl-2and caspase-3of the1-month ketamine group showed no significant differences as compared to the control group. Bcl-2decreased in the6-month ketamine group, but the difference as compared to the control group was not significant.
5Conclusion
Ketamine at the usual recreational dose after6months of use could produce stable and persistent damages to the brain's functions, decrease body weight gains. Mice showed significant deteriorations in the neuromuscular strength and nociception despite no significant apoptosis was found in prefrontal cortex. Monkeys showed locomotor activity decreased significantly, and ketamine treatment at recreational dose for6months might produce possibly permanent and irreversible deficits in brain function through the neurotoxic effect by activation of apoptotic pathway in PFC of adolescent monkeys. There might be regional differences in responses between different CNS regions and the detrimental effects of ketmaine on brains were not uniform across species, the primate animals were more sensitive to ketamine. These findings may have significant clinical implications in relation to chronic ketamine abuse and target therapeutic strategies.
氯胺酮(ketamine)是使用较为广泛的镇痛麻醉剂,但目前已成为主流娱乐性质的毒品,且滥用现象日趋严重。以英国为例,1999-2003年,娱乐性使用氯胺酮的人群增加了近2倍,且以青少年群体为主。研究提示,使用低于麻醉剂量的氯胺酮可出现难以言喻的愉快体验,如同从地狱跃入天堂。另有研究表明,正常成年人使用亚麻醉剂量的氯胺酮能够产生类似精神分裂症的阳性和阴性症状,包括知觉改变、幻视、幻觉、去人格化和现实感丧失等。滥用氯胺酮引起的个体和群体效应已经引起国际广泛关注。目前,氯胺酮在青少年亚群体中的普遍流行和滥用,原因之一是人们认为它不会导致躯体性依赖；其二,青少年群体具有追求新奇事物和新奇感觉的年龄特征。现氯胺酮在多数国家已成为限制性药物。
氯胺酮是N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMD A)受体的非竞争性拮抗剂,调节谷氨酸和天冬氨酸等兴奋性氨基酸(excitatory amino acids, EAAs)的功能,在突触可塑性和学习记忆中扮演着重要的角色。最近的研究发现,静脉给予成年人短期氯胺酮可损害认知功能和心理健康。但对长期低剂量氯胺酮是否对脑功能产生影响、其生理机制如何却鲜有研究。而长期低剂量使用方式与娱乐场所的氯胺酮滥用状态更为相似。
大多数成瘾药物是通过脑奖赏环路(VTA-NAcc-PFC神经环路)实现成瘾药物的奖赏效应。前额叶皮层(prefrontal cortex, PFC)接受来自腹侧被盖区(ventral tegmental area, VTA)的多巴胺能神经纤维支配,与情感行为、学习与记忆等诸多脑的高级功能密切相关。认知是指人们认识活动的过程,即个体对感觉信号接收、检测、转换、简约、合成、编码、储存、提取、重建、概念形成、判断和问题解决的信息加工处理过程。已经证实PFC是认知过程的关键脑区,构成了认知过程的中枢管理和工作记忆缓冲的基础结构。PFC内部合理的联系为合成不同范围的信息提供了完美的基础结构。有证据表明,PFC的多巴胺能系统对氯胺酮尤为敏感,对啮齿类动物研究发现,持续给予一定量氯胺酮(20mg/kg)可引起中枢神经系统(central nervous system, CNS)特定区域神经元细胞发生程序性细胞死亡,通过线粒体和胞质内质网通路凋亡通路,增加促凋亡因子的表达,加速细胞凋亡。且有证据表明,发育期大脑的这种损伤效应会对成年期的脑功能产生影响。
本研究假设长期娱乐剂量氯胺酮可能通过细胞凋亡途径引起大脑特定脑区,尤其是PFC脑区的细胞毒性作用,造成自发行为、认知功能的损伤。
2目的
2.1观察长期低剂量氯胺酮对青少年期ICR小鼠体重和神经肌肉强度、痛觉末梢和空间学习记忆的影响。
2.2观察长期低剂量氯胺酮对青少年期食蟹猴体重、自发行为的影响。
2.3探讨长期低剂量氯胺酮对青春期脑前额叶神经元凋亡表达的影响。
2.4揭示长期低剂量氯胺酮对青春期脑功能的影响和相关机制。
3材料和方法
3.1ICR小鼠实验动物模型制备
90只小鼠随机分为3组(每组30只),按给药时间长短分为一个月、三个月和六个月组。各组又随机分为对照组(10只)和氯胺酮组(20只),总实验周期为6个月。氯胺酮组每天腹腔注射给予盐酸氯胺酮30mg/kg,对照组给予生理盐水1mg/kg。
3.2ICR小鼠体重测量
实验开始前测量ICR小鼠基础体重,之后每周测量并记录体重变化情况,并随时调整给药剂量,观察长期给予氯胺酮是否会影响ICR小鼠体重增长。
3.3ICR小鼠行为学观察
在造模结束后进行连续3天的行为学测试,内容包括悬挂实验、热板实验和水迷宫实验。
3.4ICR小鼠前额叶神经元凋亡检测
采用核糖核酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, TUNEL)检测PFC中神经元细胞凋亡的情况。取PFC石蜡切片蛋白酶K (proteinase K, pK)孵育,平衡液处理,TUNEL反应混合液孵育,DAB显色,显微镜观察计数神经元凋亡的数量。
3.5ICR小鼠前额叶免疫印迹分析(Western Blotting)
总蛋白经抽提后蛋白定量,按照上样量为100μg的量计算上样体积,经加热变性处理后上样,电泳,5%浓缩胶80V,12%分离胶120V；电转后移至PVDF膜上,5%脱脂奶粉室温封闭1h,一抗4℃过夜,洗膜3次,二抗室温1h,洗膜3次,用ODYSSEY红外成像系统记录显影条带的光密度值。
3.6食蟹猴实验动物模型制备
24只食蟹猴随机分为3组(每组8只),按给药时间的长短分为一个月氯胺酮组,六个月氯胺酮组和六个月对照组。实验组每天静脉注射给予盐酸氯胺酮1mg/kg,对照组给予生理盐水1mg/kg。
3.7食蟹猴体重测量
实验开始前测量食蟹猴基础体重,之后每月测量并记录体重变化情况,观察长期氯胺酮是否会影响食蟹猴体重增长。
3.8食蟹猴行为学观察
在实验的第1、3、7、14、30、31、56、112、183、184、185天给药后录像观察食蟹猴自发行为(行为量表)15min。观察内容包括移动、行走、攀爬、跳跃四种行为。
3.9食蟹猴前额叶神经元凋亡检测
TUNEL染色来检测PFC神经元细胞凋亡情况。取PFC石蜡切片pK孵育,平衡液处理,TUNEL反应混合液孵育,DAB显色,显微镜观察计数神经元凋亡数目。
3.10食蟹猴前额叶免疫印迹分析Western Blotting
总蛋白经抽提后蛋白定量,按照上样量为30μg的量计算上样体积,经加热变性处理后上样,电泳,5%浓缩胶80V,12%分离胶120V；电转后移至PVDF膜上,5%脱脂奶粉室温封闭1h,一抗4℃过夜,洗膜3次,二抗室温1h,洗膜3次,暗室中显影定影,胶片经扫描后用Image J14.0软件分析目的条带的光密度值。
4实验结果
4.1长期低剂量氯胺酮对ICR小鼠体重增长的影响
实验过程中,对照组和氯胺酮组小鼠体重均有不同程度的增加,与初始体重比较均有统计学差异。与对照组相比,氯胺酮组体重增加速度缓慢。不同用药时间的三个组别中氯胺酮组与对照组的体重增长幅度无统计学差异。
4.2长期低剂量氯胺酮对ICR小鼠行为的影响
悬挂实验：一个月和三个月氯胺酮组小鼠掉落软垫所需的延迟时间与其对照组比较,差异无统计学意义,而六个月氯胺酮组在延迟时间上显著少于对照组,具有统计学差异。
热板实验：一个月和三个月氯胺酮组小鼠热板运动总和与其对照组比较,差异无统计学意义,而六个月氯胺酮组的热板运动总和显著少于对照组,具有统计学差异。
水迷宫实验：不同用药时间氯胺酮小鼠寻找逃避平台所需的潜伏期与对照组相比无统计学差异。
4.3长期低剂量氯胺酮对ICR小鼠大脑PFC神经元细胞凋亡的影响
氯胺酮组和对照组TUNEL阳性细胞计数无统计学差异。蛋白印迹分析结果显示,在一个月、三个月和六个月的氯胺酮组与对照组比较,Bax和caspase-3表达增高,Bax/Bcl-2比值增高,Bcl-2表达降低,但差异均无统计学意义。
4.4长期低剂量氯胺酮对青年期食蟹猴体重增加的影响
在6个月的实验过程中,各组食蟹猴体重均增加。氯胺酮组食蟹猴体重增长速度低于对照组,但差异无统计学意义。
4.5长期低剂量氯胺酮对青年期食蟹猴自发活动的影响
与对照组比较,氯胺酮组的自发运动有下降的趋势。给药一个月后,氯胺酮组跳跃显著低于对照组(F=7.439,P<0.01)；在其后的153天中,氯胺酮组移动、行走、跳跃和自发活动总量自身比较具有统计学差异(移动：F=4.048,P<0.05；行走：F=10.753,P<0.01；跳跃：F=4.180,P<0.05)。氯胺酮组移动、攀爬和自发行为总量显著少于对照组,差异有统计学意义(移动：F=10.798,P<0.001；攀爬：F=4.769,P<0.05；自发活动总量：F=5.793,P<0.05)。氯胺酮组自发活动总量随用药时间延长而减少(F=12.914,P<0.0001),且与药物处理存在交互作用(F=7.342,P<0.001)。
4.6长期低剂量氯胺酮对PFC细胞凋亡的影响
与对照组相比,一个月氯胺酮组前额叶TUNEL染色阳性细胞数、促凋亡蛋白(Bax和caspase-3)和Bcl-2表达水平均无显著性差异。六个月氯胺酮组PFC阳性细胞、促凋亡蛋白(Bax和caspase-3)表达水平明显增加,差异有统计学意义。
5结论
5.1长期低剂量氯胺酮可导致青春期小鼠和食蟹猴体重增长速度减慢；小鼠肌肉力量降低和伤害感受受损；抑制食蟹猴自发行为活动。
5.2长期低剂量氯胺酮可诱导食蟹猴前额叶发生明显地细胞凋亡现象,产生神经元细胞毒性作用,进而造成大脑功能受损。
5.3氯胺酮对中枢神经的损伤存在动物种属的差异,与啮齿类动物相比较,非人灵长类动物神经系统对氯胺酮更敏感。这为进一步研究慢性氯胺酮的生理心理机制和制定其临床靶向治疗策略提供了一定的理论基础。
1Introduction
Ketamine, a drug once used as an anesthetic for human and veterinary surgery, has increasingly become a mainstream'recreational' drug. In previous researches, ketamine at a dosage which was much smaller than anesthetic dosage could brought a pleasant sensation of incredible intensively to ketamine users, like'A K-hole can be anything from going to hell and meeting Satan to going to heaven and meeting God'. In the United Kingdom alone, the prevalence rates of ketamine has rapidly increased2-fold from1999to2003among the nightclub goers, especially among the adolescents. While, Ketamine at the subanesthetic dose produced a series of positive and negative schizophrenic-like symptoms including perceptual alternations, visions, delusions, depersonalization and derealization in healthy individuals, and those impairments of abuse ketamine have aroused increasing attention from the international community and various countries. Recently, ketamine has become a controlled (Schedule Ⅲ of The Controlled Substances Act) substance in the USA and other countries.
Ketamine is a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptor, which regulates the action of excitatory aminoacids (EAAs) involving glutamate and asparte, and plays a crucial role in mechanisms of synaptic plasticity and neuronal learning. In the recent studies, many researches have validated the short-term effects of ketamine administration in healthy individuals, which induced impairments of cognitive function and psychological wellbeing, relatively little is known about the effects of long-term ketamine abuse. Obviously, the latter conforms to the status of sucking ketamine. Moreover, although rodents have been commonly used for drug administration studies, since the behavioral research of drug addiction and manifestation of working memory deficits, the monkey has emerged as a premier subject to model cognitive, behavioral and neuropsychopathological disorders, and to investigate novel drug treatments of the disease concerned cognitive dysfunction.
'Cognition' in nature, that is, the mental capacity to override or augment reflexive and habitual reactions in order to orchestrated behavior in accord with our intentions, is essential for what we recognize as intelligent behavior. In another word, the primary function of cognitive control is to extract the goal-relevant features of long-term memory programs for use in future circumstances. It has been validated that the prefrontal cortex (PFC) is centrally involved in this process, which requires two essential integrated components:a central executive and working memory buffers. Moreover, the proper interconnection of PFC provides a perfect infrastructure for synthesizing the various range of information needed for complex and extended behavior. Some evidences have showed that the dopaminergic system of PFC is particularly vulnerable to ketamine administration, but the effect of prolonged ketamine administration on PFC is still largely unknown. Currently, some studies performed on rodents have indicated that sustained exposure to ketamine produces programmed cell death in areas of the central nervous system (CNS) and increases the brain expression of pro-apoptosis factors belonging to the mitochondrial and the extrinsic apoptotic pathways. These studies open up the possibility that chronic exposure to ketamine in the CNS would interfere with learning and memory through a neurotoxic effect related to activation of apoptotic pathways. Because of the exacerbation of ketamine abuse in the world and the significance of PFC in cognitive control, we established the hypothesis:chronic ketamine administration of recreational dosage might produce permanent and irreversible deficits in brain functions due to neurotoxic effects involving the activation of apoptotic pathway in the prefrontal cortex.
2Objectives
2.1To observe body weight and neuromuscular strength, the sensitivity to a painful stimulus and spatial navigation learning and memory in ICR mice following chronic low dose of ketamine exposure.
2.2To observe body weight and behavioral changes in adolescent cynomolgus monkeys following chronic low dose of ketamine exposure.
2.3To investigate the effects of chronic low dose of ketamine on cell apoptosis of prefrontal cortex (PFC) in adolescence.
2.4To investigate the effects of chronic low dose of ketamine on the brain functions in adolescence and its underlying mechanism.
3Materials and methods
3.1Animal model of ICR mice established
Ninety male ICR mice were randomly divided into3groups receiving1-,3-or6-month of daily intra-peritoneal injection of ketamine of30mg/kg or saline. In each group of30mice,20were injected with ketamine and10with saline as controls.
3.2Body weight of ICR mice measurement
The weights of mice were recorded every week for adjustment of ketamine injection and monitoring of the well-being of the animals.
3.3Behavioral studies
Behavioral tests were made for3days after ketamine or saline injection. Behavior tests including wire hang test, hot plate test and water maze test.
3.4Apoptosis assay in ICR mice
After behavioral tests, ICR mice were randomly chosen to be sacrificed. Brains were dissected and embedded in paraffin wax. Sections of PFC were cut coronally from paraffin blocks at4-μm thickness.
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) was used to assay apoptosis. Brain tissues sections were incubated with proteinase K, and TUNEL reaction mixture. Finally, the sections were developed with DAB kit. Apoptotic cells were assayed.
3.5Western blotting analysis in ICR mice
PFC tissues were homogenized in cold lysis buffer. The sample was centrifuged at14,000rpm for30min at4℃, then the supernatant was collected and protein content was assayed colorimetrically.100μg total proteins were loaded onto a12% SDS-PAGE gel, electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were incubated with primary antibodies at4℃overnight following incubation of secondary antibodies for1h. The bound antibodies were then visualized and recorded using the ODYSSEY Infrared Imaging System (LI-COR Biosciences)
3.6Animal model of cynomolgus monkeys established
Twenty-four adolescent male cynomolgus monkeys were randomly divided into3groups of8monkeys:one group was given daily intravenous injection through the inner side of the lower limbs of1mg/kg body weight of ketamine in saline for1month, another group for6months and the last group were given daily saline injection as the control group also for6months.
3.7Body weight of cynomolgus monkeys measurement
Each monkey was weighed at the beginning of each week, so that the correct ketamine dose could be administered.
3.8Behavioral observation in cynomolgus monkeys
15-min video recordings were made for each monkey after ketamine or saline injection at1st,3rd,7th,14th,30th,31st,56th,112th,183rd,184th and185th day. Behavior observation including Move, Walk, Climb and Jump was conducted.
3.9Apoptosis assay in cynomolgus monkeys
TUNEL was also used to assay apoptosis. The protocol was same to the ICR mice's.
3.10Western blotting analysis in cynomolgus monkeys
PFC tissues were homogenized in cold lysis buffer. The sample was centrifuged at14,000rpm for30min at4℃, then the supernatant was collected and protein content was assayed colorimetrically.30μg total proteins were loaded onto a12%SDS-PAGE gel, electrophoretically transferred to PVDF membrane. The membranes were incubated with primary antibodies at4℃overnight following incubation of secondary antibodies for1h. The membranes were developed using an enhanced ECL detection system. The intensity of bands was determined using the Image J14.0software.
4Results
4.1Chronic low dose of ketamine administration inhibited the body weight increase in ICR mice following chronic ketamine exposure
The percentage increases in body weight for the ketamine and control groups at1,3and6months have increased significantly. The percentage increases were less in all ketamine groups than the percentage increases of the respective control groups. However, the differences in the percentage increases between ketamine groups and their respective control groups were not statistically significant.
4.2Effects on behavioral tests of Chronic low dose of ketamine abuse in ICR mice
According to the results of wire hang test and hot plate tests, after6months' ketamine administration, the latency time and the total number of movements of the6-month ketamine group was significantly less than the6-month control group. However, there were no significant differences and consistent trends of changes in ketamine treated mice for1and3months as compared to their respective controls.
According to the results of the water maze test, the escape latency time for the mice to locate and climb onto the platform showed no statistically significant differences between the1-,3-, and6-month ketamine groups as compared to their respective control groups. Although, the ketamine treated mice in different groups consistently used more time to climb onto the platform.
4.3Effects on PFC apoptosis of Chronic low dose of ketamine abuse in ICR mice
There were no statistically significant differences of the TUNEL positive cell counts in the prefrontal cortex between the ketamine groups of1-,3-,6-month and their respective control groups. Although Western blot results showed consistently in all1-,3-, and6-month ketamine groups had higher Bax, lower Bcl-2, higher Bax/Bcl-2, and higher caspase-3levels in PFC than their respective control groups, all differences were not statistically significant.
4.4Chronic low dose of ketamine administration inhibited the body weight increase in cynomolgus monkeys following chronic ketamine exposure
Both1-month and6-month ketamine and control groups increased their body weights about10%during the first4months. In the latter2months, the control group continued to gain about15%of body weight, while the ketamine group gained very little. However, there was no statistically significant difference between the groups.
4.5Chronic low dose of ketamine abuse in adolescent cynomolgus monkeys depressed motor behaviors
For behavior test, activities of Move, Walk, Climb and Jump of ketamine group exhibited obvious decreased tendency as time went by. With the exception of the results for Jump of1-and6-month ketamine groups versus controls (which were significantly different), results of other behavioral movements (Move, Climb, Walk and Total behavior) showed no significant differences between the ketamine and control groups. However, in the latter153days, results of Move, Walk, Jump and total behavior did show significant changes on different days. Furthermore, results of Total behavior movement showed significant differences both between different days and interactions between different days and control/ketamine groups. In addition, results of Move, Climb and Total behavior showed significant differences between the ketamine and control groups.
4.6Effects on PFC apoptosis of Chronic low dose of ketamine abuse in adolescent cynomolgus monkeys
TUNEL positive cells increased significantly (p<0.05) in the prefrontal cortex of the6-month ketamine group as compared with those of the control group; In Western blot assays, for the6-month ketamine group, Bax and caspase-3in the PFC both increased significantly (p<0.05) compared with the control group. Results of TUNEL positive cells, Bax, Bcl-2and caspase-3of the1-month ketamine group showed no significant differences as compared to the control group. Bcl-2decreased in the6-month ketamine group, but the difference as compared to the control group was not significant.
5Conclusion
Ketamine at the usual recreational dose after6months of use could produce stable and persistent damages to the brain's functions, decrease body weight gains. Mice showed significant deteriorations in the neuromuscular strength and nociception despite no significant apoptosis was found in prefrontal cortex. Monkeys showed locomotor activity decreased significantly, and ketamine treatment at recreational dose for6months might produce possibly permanent and irreversible deficits in brain function through the neurotoxic effect by activation of apoptotic pathway in PFC of adolescent monkeys. There might be regional differences in responses between different CNS regions and the detrimental effects of ketmaine on brains were not uniform across species, the primate animals were more sensitive to ketamine. These findings may have significant clinical implications in relation to chronic ketamine abuse and target therapeutic strategies.
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6. Lankenau, S.E., B. Sanders, J.J. Bloom, D. Hathazi, E. Alarcon, S. Tortu, and M.C. Clatts. First injection of ketamine among young injection drug users (IDUs) in three U.S. cities. Drug Alcohol Depend,2007.87(2-3):p.183-93.
7. Morgan, C.J., A. Mofeez, B. Brandner, L. Bromley, and H.V. Curran. Ketamine impairs response inhibition and is positively reinforcing in healthy volunteers:a dose-response study. Psychopharmacology (Berl),2004.172(3): p.298-308.
8. Lankenau, S.E. and M.C. Clatts. Ketamine Injection among High Risk Youth: Preliminary Findings from New York City. J Drug Issues,2002.32(3):p. 893-905.
9. Lankenau, S.E. and M.C. Clatts. Drug injection practices among high-risk youths:the first shot of ketamine. J Urban Health,2004.81(2):p.232-48.
10. Group, C. Hightlights and Executive Summary. Epidemiologic Trends in Drug Abuse1,2005.
11. Crews, F., J. He, and C. Hodge. Adolescent cortical development:a critical period of vulnerability for addiction. Pharmacol Biochem Behav,2007.86(2): p.189-99.
12. Stansfield, K.H. and C.L. Kirstein. Chronic cocaine or ethanol exposure during adolescence alters novelty-related behaviors in adulthood. Pharmacol Biochem Behav,2007.86(4):p.637-42.
13. Weber, F., H.Wulf, M.Gruber, Biallas R. S-ketamine and s-norketamine plasma concentrations after NAsal and i.v.administration in anesthetized children. Paediatr Anaesth,2004.14(12):p.983-988.
14. AM.Hughes, J.Rhodes, and G.Fisher. Assessment of the effect of dextromethorphan and ketamine on the acute nociceptive threshold and wind-up of the second pain response in healthy male volunteers. Br J Clin Pharmacol,2002.53(6):p.604-612.
15. 赵平,聂连之。氯胺酮用于学龄前儿童静脉滴注的药代动力学。 中华麻醉杂志,1996.16：p.150.
16. 王玉瑾,刘玲,贾娟,廖林川,王英元。氯胺酮的薄层层析和气相色谱-质谱分析。中国医院药学杂志,2005.25(6)：p.497-499.
17. 贾娟,曹洁,王玉瑾,贠克明。氯胺酮在大鼠体内的分布。中国医院药学杂志,2008.14：p.1146-1148.
18. Britt, G.C. and E.F. McCance-Katz. A brief overview of the clinical pharmacology of "club drugs". Subst Use Misuse,2005.40(9-10):p. 1189-201.
19. Jevtovic-Todorovic, V., R.E. Hartman, Y. Izumi, N.D. Benshoff, K. Dikranian, C.F. Zorumski, J.W. Olney, and D.F. Wozniak. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci,2003.23(3):p.876-82.
20. Lau, A. and M. Tymianski. Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers Arch,2010.460(2):p.525-42.
21. Collingridge, G.L. and T.V. Bliss. Memories of NMD A receptors and LTP. Trends Neurosci,1995.18(2):p.54-6.
22. Newcomer, J.W. and J.H. Krystal. NMDA receptor regulation of memory and behavior in humans. Hippocampus,2001.11(5):p.529-42.
23. JM.Fuster. The Prefrontal Cortex. Academic Press,2008.
24. Robbins, T.W. From arousal to cognition:the integrative position of the prefrontal cortex. Prog Brain Res,2000.126:p.469-83.
25. J.Olds. Pleasure centers in the brain. Scientific American 1956.195:p. 105-116.
26. Thompson-Schill, S.L., J. Jonides, C. Marshuetz, E.E. Smith, M. D'Esposito, I.P. Kan, R.T. Knight, and D. Swick. Effects of frontal lobe damage on interference effects in working memory. Cogn Affect Behav Neurosci,2002. 2(2):p.109-20.
27. Rowe, J.B., I. Toni, O. Josephs, R.S. Frackowiak, and R.E. Passingham. The prefrontal cortex:response selection or maintenance within working memory? Science,2000.288(5471):p.1656-60.
28. Arnsten, A.F. Stress signalling pathways that impair prefrontal cortex structure and function. Nat Rev Neurosci,2009.10(6):p.410-22.
29. Grafman, J. Handbook of Neuropsychology,2nd Edition:The Frontal Lobes. Elsevier Health Sciences,2002.
30. Vazquez-Borsetti, P., R. Cortes, and F. Artigas. Pyramidal neurons in rat prefrontal cortex projecting to ventral tegmental area and dorsal raphe nucleus express 5-HT2 A receptors. Cereb Cortex,2009.19(7):p.1678-86.
31. Spinella, M. Relationship between drug use and prefrontal-associated traits. Addict Biol,2003.8(1):p.67-74.
32. Lu, H., B. Lim, and M.M. Poo. Cocaine exposure in utero alters synaptic plasticity in the medial prefrontal cortex of postnatal rats. J Neurosci,2009. 29(40):p.12664-74.
33. Crawley, J.N. Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res,1999.835(1):p.18-26.
34. Miyakawa, T., M. Yamada, A. Duttaroy, and J. Wess. Hyperactivity and intact hippocampus-dependent learning in mice lacking the M1 muscarinic acetylcholine receptor. J Neurosci,2001.21(14):p.5239-50.
35. Pick, C.G., J. Cheng, D. Paul, and G.W. Pasternak. Genetic influences in opioid analgesic sensitivity in mice. Brain Res,1991.566(1-2):p.295-8.
36. Gulinello, M., M. Gertner, G Mendoza, B.P. Schoenfeld, S. Oddo, F. LaFerla, C.H. Choi, S.M. McBride, and D.S. Faber. Validation of a 2-day water maze protocol in mice. Behav Brain Res,2009.196(2):p.220-7.
37. RJ.Jaffard, B.Bontempi, and F.Menzaghi. Theoretical and Practical Considerations for the Evaluation of Learning and Memory in Mice. Methods of Behavior Analysis in Neuroscience,2001. FL(CRC LLC):p.301-329.
38. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol,1992.119(3):p.493-501.
39. Baba, N., T. Koji, M. Itoh, and A. Mizuno. Reciprocal changes in the expression of Bcl-2 and Bax in hypoglossal nucleus after axotomy in adult rats: possible involvement in the induction of neuronal cell death. Brain Res,1999. 827(1-2):p.122-9.
40. Morton, G.J., D.E. Cummings, D.G. Baskin, G.S. Barsh, and M.W. Schwartz. Central nervous system control of food intake and body weight. Nature,2006. 443(7109):p.289-95.
41. Coppari, R., G. Ramadori, and J.K. Elmquist. The role of transcriptional regulators in central control of appetite and body weight. Nat Clin Pract Endocrinol Metab,2009.5(3):p.160-6.
42. Hamann, M., M.H. Meisler, and A. Richter. Motor disturbances in mice with deficiency of the sodium channel gene Scn8a show features of human dystonia. Exp Neurol,2003.184(2):p.830-8.
43. Morris, R.. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods,1984.11(1):p.47-60.
44. Brody, D.L. and D.M. Holtzman. Morris water maze search strategy analysis in PDAPP mice before and after experimental traumatic brain injury. Exp Neurol,2006.197(2):p.330-40.
45. Iivonen, H., L. Nurminen, M. Harri, H. Tanila, and J. Puolivali. Hypothermia in mice tested in Morris water maze. Behav Brain Res,2003.141(2):p. 207-13.
46. Sinner, B. and B.M. Graf. Ketamine. Handb Exp Pharmacol,2008(182):p. 313-33.
47. Quirk, M.C., D.L. Sosulski, C.E. Feierstein, N. Uchida, and Z.F. Mainen. A defined network of fast-spiking interneurons in orbitofrontal cortex:responses to behavioral contingencies and ketamine administration. Front Syst Neurosci, 2009.3:p.13.
48. Verdejo-Garcia, A. and A. Bechara. A somatic marker theory of addiction. Neuropharmacology,2009.56 Suppl 1:p.48-62.
49. Yeung, L.Y., M.S. Wai, M. Fan, Y.T. Mak, W.P. Lam, Z. Li, G. Lu, and D.T. Yew. Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine. Toxicol Lett,2010.193(2):p.189-93.
50. H.Tsukada, S..Nishiyama, D.Fukumoto, K.Sato, T.Kakiuchi, and EF.Domino. Chronic NMDA antagonism impairs working memory, decreases extracellular dopamine, and increases D1 receptor binding in prefrontal cortex of conscious monkeys.. Neuropsychopharmacology 2005.30:p.1861-1869.
1. Shively, C.A., D.P. Friedman, H.D. Gage, M.C. Bounds, C. Brown-Proctor, J.B. Blair, J.A. Henderson, M.A. Smith, and N. Buchheimer. Behavioral depression and positron emission tomography-determined serotonin 1A receptor binding potential in cynomolgus monkeys. Arch Gen Psychiatry,2006. 63(4):p.396-403.
2. Shively, C.A., T.C. Register, D.P. Friedman, T.M. Morgan, J. Thompson, and T. Lanier. Social stress-associated depression in adult female cynomolgus monkeys (Macaca fascicularis). Biol Psychol,2005.69(1):p.67-84.
3. Jevtovic-Todorovic, V., R.E. Hartman, Y. Izumi, N.D. Benshoff, K. Dikranian, C.F. Zorumski, J.W. Olney, and D.F. Wozniak. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci,2003.23(3):p.876-82.
4. Bruijn, L.I., T.M. Miller, and D.W. Cleveland. Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu Rev Neurosci,2004.27: p.723-49.
5. Newcomer, J.W. and J.H. Krystal. NMDA receptor regulation of memory and behavior in humans. Hippocampus,2001.11(5):p.529-42.
6. Yeung, L.Y., M.S. Wai, M. Fan, Y.T. Mak, W.P. Lam, Z. Li, G. Lu, and D.T. Yew. Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine. Toxicol Lett,2010.193(2):p.189-93.
7. Ghoneim, M.M., J.V. Hinrichs, S.P. Mewaldt, and R.C. Petersen. Ketamine: behavioral effects of subanesthetic doses. J Clin Psychopharmacol,1985.5(2): p.70-7.
8. Morgan, C.J., A. Mofeez, B. Brandner, L. Bromley, and H.V. Curran. Ketamine impairs response inhibition and is positively reinforcing in healthy volunteers:a dose-response study. Psychopharmacology (Berl),2004.172(3): p.298-308.
9. Zou, X., T.A. Patterson, R.L. Divine, N. Sadovova, X. Zhang, J.P. Hanig, M.G. Paule, W. Slikker, Jr., and C. Wang. Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain. Int J Dev Neurosci,2009. 27(7):p.727-31.
10. TH.Clutton-Brock and H. P. Primate social organization and ecology.. Nature, 1974:p.539-542.
11. IS.Bernstein, G. JPeter, E.Thomas, and Ruehlmann. Kinship, association, and social relationships in rhesus monkeys (Macaca mulatta). American Journal of Primatology,1993.31(41-53).
12. SV.Vellucci. Primate social behavior--anxiety or depression? Pharmacol Ther 1990.47:p.167-180.
13. C.Ehardt and IS.Bernstein. Patterns of Affiliation Among Immature Rhesus Monkeys (Macaca mulatta). American Journal of Primatology 1987.13:p. 255-269.
14. Spear, L.P. The adolescent brain and age-related behavioral manifestations. Neurosci Biobehav Rev,2000.24(4):p.417-63.
15. Sun, L., W.P. Lam, Y.W. Wong, L.H. Lam, H.C. Tang, M.S. Wai, Y.T. Mak, F. Pan, and D.T. Yew. Permanent deficits in brain functions caused by long-term ketamine treatment in mice. Hum Exp Toxicol,2010.30(9):p.1287-96.
16. Morton, G.J., D.E. Cummings, D.G. Baskin, G.S. Barsh, and M.W. Schwartz. Central nervous system control of food intake and body weight. Nature,2006. 443(7109):p.289-95.
17. Becker, A., B. Peters, H. Schroeder, T. Mann, G. Huether, and G Grecksch. Ketamine-induced changes in rat behaviour:A possible animal model of schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry,2003.27(4):p. 687-700.
18. Yilmaz, A., D. Schulz, A. Aksoy, and R. Canbeyli. Prolonged effect of an anesthetic dose of ketamine on behavioral despair. Pharmacol Biochem Behav, 2002.71(1-2):p.341-4.
19. Nelson, C.L., J.A. Burk, J.P. Bruno, and M. Sarter. Effects of acute and repeated systemic administration of ketamine on prefrontal acetylcholine release and sustained attention performance in rats. Psychopharmacology (Berl),2002.161(2):p.168-79.
20. Wise, R.A. and M.A. Bozarth. A psychomotor stimulant theory of addiction. Psychol Rev,1987.94(4):p.469-92.
21. Dumitriu, D., J. Hao, Y. Hara, J. Kaufmann, W.G. Janssen, W. Lou, P.R. Rapp, and J.H. Morrison. Selective changes in thin spine density and morphology in monkey prefrontal cortex correlate with aging-related cognitive impairment. J Neurosci,2010.30(22):p.7507-15.
22. Frances, R., S. MillerI, and A. Mack. Clinical Textbook of Addictive Disorders. 2011.
23. Fredriksson, A. and T. Archer. Neurobehavioural deficits associated with apoptotic neurodegeneration and vulnerability for ADHD. Neurotox Res,2004. 6(6):p.435-56.
24. Honey, GD., R.A. Honey, C. O'Loughlin, S.R. Sharar, D. Kumaran, J. Suckling, D.K. Menon, C. Sleator, E.T. Bullmore, and P.C. Fletcher. Ketamine disrupts frontal and hippocampal contribution to encoding and retrieval of episodic memory:an fMRI study. Cereb Cortex,2005.15(6):p.749-59.
25. JM.Fuster. The Prefrontal Cortex. Academic Press,2008.
2. Krystal, J.H., L.P. Karper, J.P. Seibyl, G.K. Freeman, R. Delaney, J.D. Bremner, G.R. Heninger, M.B. Bowers, Jr., and D.S. Charney. Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans. Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen Psychiatry, 1994.51(3):p.199-214.
3. Strayer, R.J. and L.S. Nelson. Adverse events associated with ketamine for procedural sedation in adults. Am J Emerg Med,2008.26(9):p.985-1028.
4. Gable, R.S. Acute toxic effects of club drugs. J Psychoactive Drugs,2004. 36(3):p.303-13.
5. Joe Laidler, K.A. The rise of club drugs in a heroin society:the case of Hong Kong. Subst Use Misuse,2005.40(9-10):p.1257-78.
6. Lankenau, S.E., B. Sanders, J.J. Bloom, D. Hathazi, E. Alarcon, S. Tortu, and M.C. Clatts. First injection of ketamine among young injection drug users (IDUs) in three U.S. cities. Drug Alcohol Depend,2007.87(2-3):p.183-93.
7. Morgan, C.J., A. Mofeez, B. Brandner, L. Bromley, and H.V. Curran. Ketamine impairs response inhibition and is positively reinforcing in healthy volunteers:a dose-response study. Psychopharmacology (Berl),2004.172(3): p.298-308.
8. Lankenau, S.E. and M.C. Clatts. Ketamine Injection among High Risk Youth: Preliminary Findings from New York City. J Drug Issues,2002.32(3):p. 893-905.
9. Lankenau, S.E. and M.C. Clatts. Drug injection practices among high-risk youths:the first shot of ketamine. J Urban Health,2004.81(2):p.232-48.
10. Group, C. Hightlights and Executive Summary. Epidemiologic Trends in Drug Abuse1,2005.
11. Crews, F., J. He, and C. Hodge. Adolescent cortical development:a critical period of vulnerability for addiction. Pharmacol Biochem Behav,2007.86(2): p.189-99.
12. Stansfield, K.H. and C.L. Kirstein. Chronic cocaine or ethanol exposure during adolescence alters novelty-related behaviors in adulthood. Pharmacol Biochem Behav,2007.86(4):p.637-42.
13. Weber, F., H.Wulf, M.Gruber, Biallas R. S-ketamine and s-norketamine plasma concentrations after NAsal and i.v.administration in anesthetized children. Paediatr Anaesth,2004.14(12):p.983-988.
14. AM.Hughes, J.Rhodes, and G.Fisher. Assessment of the effect of dextromethorphan and ketamine on the acute nociceptive threshold and wind-up of the second pain response in healthy male volunteers. Br J Clin Pharmacol,2002.53(6):p.604-612.
15. 赵平,聂连之。氯胺酮用于学龄前儿童静脉滴注的药代动力学。 中华麻醉杂志,1996.16：p.150.
16. 王玉瑾,刘玲,贾娟,廖林川,王英元。氯胺酮的薄层层析和气相色谱-质谱分析。中国医院药学杂志,2005.25(6)：p.497-499.
17. 贾娟,曹洁,王玉瑾,贠克明。氯胺酮在大鼠体内的分布。中国医院药学杂志,2008.14：p.1146-1148.
18. Britt, G.C. and E.F. McCance-Katz. A brief overview of the clinical pharmacology of "club drugs". Subst Use Misuse,2005.40(9-10):p. 1189-201.
19. Jevtovic-Todorovic, V., R.E. Hartman, Y. Izumi, N.D. Benshoff, K. Dikranian, C.F. Zorumski, J.W. Olney, and D.F. Wozniak. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci,2003.23(3):p.876-82.
20. Lau, A. and M. Tymianski. Glutamate receptors, neurotoxicity and neurodegeneration. Pflugers Arch,2010.460(2):p.525-42.
21. Collingridge, G.L. and T.V. Bliss. Memories of NMD A receptors and LTP. Trends Neurosci,1995.18(2):p.54-6.
22. Newcomer, J.W. and J.H. Krystal. NMDA receptor regulation of memory and behavior in humans. Hippocampus,2001.11(5):p.529-42.
23. JM.Fuster. The Prefrontal Cortex. Academic Press,2008.
24. Robbins, T.W. From arousal to cognition:the integrative position of the prefrontal cortex. Prog Brain Res,2000.126:p.469-83.
25. J.Olds. Pleasure centers in the brain. Scientific American 1956.195:p. 105-116.
26. Thompson-Schill, S.L., J. Jonides, C. Marshuetz, E.E. Smith, M. D'Esposito, I.P. Kan, R.T. Knight, and D. Swick. Effects of frontal lobe damage on interference effects in working memory. Cogn Affect Behav Neurosci,2002. 2(2):p.109-20.
27. Rowe, J.B., I. Toni, O. Josephs, R.S. Frackowiak, and R.E. Passingham. The prefrontal cortex:response selection or maintenance within working memory? Science,2000.288(5471):p.1656-60.
28. Arnsten, A.F. Stress signalling pathways that impair prefrontal cortex structure and function. Nat Rev Neurosci,2009.10(6):p.410-22.
29. Grafman, J. Handbook of Neuropsychology,2nd Edition:The Frontal Lobes. Elsevier Health Sciences,2002.
30. Vazquez-Borsetti, P., R. Cortes, and F. Artigas. Pyramidal neurons in rat prefrontal cortex projecting to ventral tegmental area and dorsal raphe nucleus express 5-HT2 A receptors. Cereb Cortex,2009.19(7):p.1678-86.
31. Spinella, M. Relationship between drug use and prefrontal-associated traits. Addict Biol,2003.8(1):p.67-74.
32. Lu, H., B. Lim, and M.M. Poo. Cocaine exposure in utero alters synaptic plasticity in the medial prefrontal cortex of postnatal rats. J Neurosci,2009. 29(40):p.12664-74.
33. Crawley, J.N. Behavioral phenotyping of transgenic and knockout mice: experimental design and evaluation of general health, sensory functions, motor abilities, and specific behavioral tests. Brain Res,1999.835(1):p.18-26.
34. Miyakawa, T., M. Yamada, A. Duttaroy, and J. Wess. Hyperactivity and intact hippocampus-dependent learning in mice lacking the M1 muscarinic acetylcholine receptor. J Neurosci,2001.21(14):p.5239-50.
35. Pick, C.G., J. Cheng, D. Paul, and G.W. Pasternak. Genetic influences in opioid analgesic sensitivity in mice. Brain Res,1991.566(1-2):p.295-8.
36. Gulinello, M., M. Gertner, G Mendoza, B.P. Schoenfeld, S. Oddo, F. LaFerla, C.H. Choi, S.M. McBride, and D.S. Faber. Validation of a 2-day water maze protocol in mice. Behav Brain Res,2009.196(2):p.220-7.
37. RJ.Jaffard, B.Bontempi, and F.Menzaghi. Theoretical and Practical Considerations for the Evaluation of Learning and Memory in Mice. Methods of Behavior Analysis in Neuroscience,2001. FL(CRC LLC):p.301-329.
38. Gavrieli, Y., Y. Sherman, and S.A. Ben-Sasson. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol,1992.119(3):p.493-501.
39. Baba, N., T. Koji, M. Itoh, and A. Mizuno. Reciprocal changes in the expression of Bcl-2 and Bax in hypoglossal nucleus after axotomy in adult rats: possible involvement in the induction of neuronal cell death. Brain Res,1999. 827(1-2):p.122-9.
40. Morton, G.J., D.E. Cummings, D.G. Baskin, G.S. Barsh, and M.W. Schwartz. Central nervous system control of food intake and body weight. Nature,2006. 443(7109):p.289-95.
41. Coppari, R., G. Ramadori, and J.K. Elmquist. The role of transcriptional regulators in central control of appetite and body weight. Nat Clin Pract Endocrinol Metab,2009.5(3):p.160-6.
42. Hamann, M., M.H. Meisler, and A. Richter. Motor disturbances in mice with deficiency of the sodium channel gene Scn8a show features of human dystonia. Exp Neurol,2003.184(2):p.830-8.
43. Morris, R.. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods,1984.11(1):p.47-60.
44. Brody, D.L. and D.M. Holtzman. Morris water maze search strategy analysis in PDAPP mice before and after experimental traumatic brain injury. Exp Neurol,2006.197(2):p.330-40.
45. Iivonen, H., L. Nurminen, M. Harri, H. Tanila, and J. Puolivali. Hypothermia in mice tested in Morris water maze. Behav Brain Res,2003.141(2):p. 207-13.
46. Sinner, B. and B.M. Graf. Ketamine. Handb Exp Pharmacol,2008(182):p. 313-33.
47. Quirk, M.C., D.L. Sosulski, C.E. Feierstein, N. Uchida, and Z.F. Mainen. A defined network of fast-spiking interneurons in orbitofrontal cortex:responses to behavioral contingencies and ketamine administration. Front Syst Neurosci, 2009.3:p.13.
48. Verdejo-Garcia, A. and A. Bechara. A somatic marker theory of addiction. Neuropharmacology,2009.56 Suppl 1:p.48-62.
49. Yeung, L.Y., M.S. Wai, M. Fan, Y.T. Mak, W.P. Lam, Z. Li, G. Lu, and D.T. Yew. Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine. Toxicol Lett,2010.193(2):p.189-93.
50. H.Tsukada, S..Nishiyama, D.Fukumoto, K.Sato, T.Kakiuchi, and EF.Domino. Chronic NMDA antagonism impairs working memory, decreases extracellular dopamine, and increases D1 receptor binding in prefrontal cortex of conscious monkeys.. Neuropsychopharmacology 2005.30:p.1861-1869.
1. Shively, C.A., D.P. Friedman, H.D. Gage, M.C. Bounds, C. Brown-Proctor, J.B. Blair, J.A. Henderson, M.A. Smith, and N. Buchheimer. Behavioral depression and positron emission tomography-determined serotonin 1A receptor binding potential in cynomolgus monkeys. Arch Gen Psychiatry,2006. 63(4):p.396-403.
2. Shively, C.A., T.C. Register, D.P. Friedman, T.M. Morgan, J. Thompson, and T. Lanier. Social stress-associated depression in adult female cynomolgus monkeys (Macaca fascicularis). Biol Psychol,2005.69(1):p.67-84.
3. Jevtovic-Todorovic, V., R.E. Hartman, Y. Izumi, N.D. Benshoff, K. Dikranian, C.F. Zorumski, J.W. Olney, and D.F. Wozniak. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci,2003.23(3):p.876-82.
4. Bruijn, L.I., T.M. Miller, and D.W. Cleveland. Unraveling the mechanisms involved in motor neuron degeneration in ALS. Annu Rev Neurosci,2004.27: p.723-49.
5. Newcomer, J.W. and J.H. Krystal. NMDA receptor regulation of memory and behavior in humans. Hippocampus,2001.11(5):p.529-42.
6. Yeung, L.Y., M.S. Wai, M. Fan, Y.T. Mak, W.P. Lam, Z. Li, G. Lu, and D.T. Yew. Hyperphosphorylated tau in the brains of mice and monkeys with long-term administration of ketamine. Toxicol Lett,2010.193(2):p.189-93.
7. Ghoneim, M.M., J.V. Hinrichs, S.P. Mewaldt, and R.C. Petersen. Ketamine: behavioral effects of subanesthetic doses. J Clin Psychopharmacol,1985.5(2): p.70-7.
8. Morgan, C.J., A. Mofeez, B. Brandner, L. Bromley, and H.V. Curran. Ketamine impairs response inhibition and is positively reinforcing in healthy volunteers:a dose-response study. Psychopharmacology (Berl),2004.172(3): p.298-308.
9. Zou, X., T.A. Patterson, R.L. Divine, N. Sadovova, X. Zhang, J.P. Hanig, M.G. Paule, W. Slikker, Jr., and C. Wang. Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain. Int J Dev Neurosci,2009. 27(7):p.727-31.
10. TH.Clutton-Brock and H. P. Primate social organization and ecology.. Nature, 1974:p.539-542.
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