摘要
研究发现,发育期大脑受到损伤可导致不可逆改变,且对药物滥用敏感。氯胺酮是近年迅速发展起来的滥用物质,作为非竞争性N-甲基-D-天门冬氨酸(N-methyl-D-aspartate, NMDA)受体拮抗剂,氯胺酮可导致幼龄大鼠和小鼠发生广泛的神经细胞凋亡,引起神经细胞死亡,导致脑结构发生不可逆改变。另外,有研究提示,青少年期是多巴胺(dopamine, DA)系统神经元重组、皮层突触重塑、受体消减的关键期,对药物滥用敏感。物质滥用可影响多巴胺(DA)神经系统的传递功能,导致生长发育和奖赏敏感性改变。
致幻作用是氯胺酮滥用的基本因素。有研究发现,长期滥用者出现社会功能受损、行为和人格改变、记忆力下降和身体多器官损害,其中,最明显的改变是中枢神经系统损伤。现有研究发现,氯胺酮滥用导致大脑皮质、脊髓、丘脑、海马和后扣带皮质等部位神经元调亡。
氯胺酮作为NMDA受体阻断剂,通过降低γ-氨基丁酸(gamma-Aminobutyric Acid, GABA)能神经元活性,导致γ-氨基丁酸(GABA)神经元抑制,引起广泛分布在皮层区域的谷氨酸能神经元脱抑制,引发谷氨酸过度释放和皮层兴奋,引起类似精神疾病的行为和认知功能异常。既往研究已经证实,急性氯胺酮应用主要作用于前额叶(prefrontal cortex, PFC)和周边皮层,在这些脑区可检测到谷氨酸和多巴胺(DA)水平升高。但慢性低剂量氯胺酮重复应用对前额叶(PFC)多巴胺(DA)神经元、工作记忆和功能执行的影响及分子机制尚不够明晰。
大多数成瘾药物是通过激活中脑-边缘多巴胺系统(mesolimbic dopaminesystem, MLDS)的多巴胺(DA)神经通路,实现成瘾药物的奖赏和强化作用。动物实验发现,在反复给予成瘾物质后,功能核磁共振成像(functional magnetic resonance imaging, fMRI)显示多巴胺(DA)分布区域包括纹状体、伏隔核及背侧丘脑出现明显激活。边缘叶多巴胺(DA)系统的激活,特别是纹状体多巴胺(DA)活性的增强,可破坏感觉运动控制功能。但随着新型成瘾物质的不断出现,不同成瘾物质对脑功能的作用部位不同,其作用方式具有区域选择性和作用靶点特异性。氯胺酮成瘾作用的靶点和神经通路尚不清楚。
麻醉剂量、急性氯胺酮使用可致成人工作记忆和执行功能的缺陷,使用者表现为面部情绪的处理加工、工作记忆、回忆、言语流畅和学习障碍。而慢性氯胺酮滥用研究报告较少,少有研究是关注长期低剂量氯胺酮滥用对青少年期相关脑区功能的影响及其生理机制。本实验将建立青少年期食蟹猴氯胺酮长期滥用模型,观察低剂量长期使用氯胺酮对动物体重生长、自主行为、脑功能和脑组织代谢的影响,并探讨可能的分子机制。
目的
1.观察长期低剂量氯胺酮滥用对青少年期食蟹猴体重、自发行为的影响。
2.探讨长期低剂量氯胺酮滥用对青少年期食蟹猴不同脑区功能的影响。
3.探讨长期低剂量氯胺酮滥用对额叶、纹状体脑组织代谢的影响。
4.观察长期低剂量氯胺酮滥用对豆状核(lentiform nucleus, LN)神经元凋亡的影响。
5.观察长期低剂量氯胺酮滥用对前额叶(PFC)皮质酪氨酸羟化酶(tyrosine hydroxylase, TH)的表达的影响。
材料和方法
1.实验模型制备
12只食蟹猴随机分为两组,8只实验组,4只对照组,实验时间为6个月。实验组每天给予盐酸氯胺酮1mg/kg静脉注射,对照组给予生理盐水1mg/kg。
2.体重测量
实验开始之前测量食蟹猴基础体重,之后每个月末测量并记录体重变化情况,观察长期氯胺酮滥用是否会影响食蟹猴体重增长。
3.行为学观察
在实验的第1、3、7、14、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。
4.功能磁共振成像(fMRI)检查
实验的第186天开始做放射影像学检查。功能磁共振成像(fMRI)扫描过程中外部闭路检测仪监控呼吸状态,由麻醉师全程检测麻醉状态和生命体征,采用轴位扫描。功能磁共振成像(fMRI)中,抬高食蟹猴右后肢(60°)频率1次/秒,诱发相应脑区激活,扫描时间12min48s。
5.核磁共振波谱检查(Magnetic Resonance Spectroscopy, MRS)
核磁共振波谱(MRS)扫描采用single voxel PROBE PRESS点解析波谱成像(point resolved spectroscopy),以轴位扫描,分别选取额叶、纹状体两个脑区域,尽量避开颅骨和脑脊液,扫描时间3mmin,每个部位扫描3次。
6.神经元凋亡检测
用核糖核酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, TUNEL)来检测豆状核(LN)中神经元凋亡情况。取LN石蜡切片蛋白酶K孵育,平衡液处理,TUNEL反应混合液孵育,DAB显色液,观察神经元凋亡数目。
7.酪氨酸羟化酶(TH)检测
影像学检查结束后,随机取4只食蟹猴(实验组3只对照组1只)留取脑组织豆状核(LN)及前额皮质(PFC),制成石蜡切片备用。
用免疫组织化学方法检测前额叶(PFC)酪氨酸羟化酶(TH)的表达。前额叶(PFC)石蜡切片抗原修复,1%的牛血清白蛋白和10%兔血清处理1h,多克隆羊抗人酪氨酸羟化酶过夜,辣根过氧化物酶结合的兔抗羊IgG室温下抚育,DAB试剂盒显像,观察酪氨酸羟化酶(TH)阳性细胞数目。
结果
1.长期低剂量氯胺酮滥用抑制青少年期食蟹猴体重的增加速度
实验过程中两组食蟹猴体重均增加,差异无统计学意义。但与对照组比较,氯胺酮组体重增加缓慢,对照组在第2个月末时体重增加10%,在后来的4个月里体重增加10%,而氯胺酮组在第4个月末时体重增加10%,6个月末时两组体重平均差为10%。
2.长期低剂量氯胺酮滥用抑制食蟹猴自发运动
与对照组比较,氯胺酮组的自发运动有下降趋势。与对照组比较,氯胺酮组在第183、184、185天自发行为包括移动、行走、攀爬下降明显,有统计学意义(p<0.05)。
3.长期低剂量氯胺酮滥用导致食蟹猴广泛性脑功能失调
功能磁共振成像(fMRI)检查结果中,氯胺酮组右侧大脑半球豆状核(LN)、梭状回(fusiform gyrus, FG)和内嗅球(entorhinal cortex, Ent)可观察到明显活跃脑区,其中豆状核(LN)激活最明显,梭状回(FG)明显激活,内嗅球(Ent)的激活次之。
对照组左侧大脑半球的黑质(substantial nigra in midbrain, SN)、腹侧被盖核(VTA)、视皮质(visual cortex, VC)及右侧大脑半球的后扣带皮质(posterior cingulate cortex, CGp)及躯体感觉区(somatosensory cortex, SC)明显活跃。其中,视皮质(VC)、躯体感觉区(SC)激活最明显,黑质(SN)与腹侧被盖核(VTA)有明显激活,后扣带皮质(CGp)的激活次之。
4.长期低剂量氯胺酮滥用导致食蟹猴脑组织代谢异常
与对照组比较,氯胺酮组食蟹猴额叶、纹状体中N-天门冬氨酸(NAA)、肌酸(Cr)、NAA/Cr数值降低,但差异无统计学意义；与对照组比较,氯胺酮组食蟹猴额叶、纹状体中胆碱(Cho)的水平明显降低(p<0.05)。
5.未观察到明显神经元凋亡
与对照组比较,氯胺酮组豆状核(LN)神经元凋亡数目未见显著性增加。
6.长期低剂量氯胺酮滥用降低食蟹猴前额叶(PFC)酪氨酸羟化酶(TH)表达
酪氨酸羟化酶(TH)检测结果发现,氯胺酮组酪氨酸羟化酶(TH)阳性细胞数目明显少于对照组(p<0.01)。
结论
1.长期低剂量氯胺酮滥用可抑制生长发育过程中体重增加。
2.长期低剂量氯胺酮滥用产生可导致运动抑制,白发行为减少,提示产生行为耐受性。
3.长期低剂量氯胺酮滥用明显抑制左侧大脑半球黑质(SN)与腹侧被盖核(VTA),可能为实验动物未出现觅药行为与成瘾性的主要原因。主要通过激活纹状体中豆状核(LN)而导致感觉运动控制障碍,导致运动失调,可能是自发行为减少、运动抑制的原因；激活梭状回(FG)可引起视幻觉,而致幻作用为氯胺酮滥用最基本因素；激活内嗅球(Ent),使内嗅球(Ent)-纹状体通路活化,从而在精神分裂样症状的出现及物质滥用过程中起重要作用。除了上述脑区外,与以往的研究不同,我们还发现躯体感觉区(SC)活性降低,提示长期低剂量氯胺酮滥用损害躯体感觉功能,首次明确了氯胺酮滥用对躯体感觉区(SC)的损害作用,补充了中枢作用机制,为研究青少年物质滥用及戒毒提供了实验依据。
4.长期低剂量氯胺酮滥用主要通过影响神经胶质细胞膜的合成、周转和代谢,影响青少年期食蟹猴额叶、纹状体代谢。
5.豆状核(LN)未见明显的神经元凋亡。可能引起不同于经典细胞凋亡的其他形式的细胞死亡,虽然最终发生以胞核变化为特征的细胞凋亡,但凋亡进程会明显延迟,提示随着用药时间的延长可能导致细胞凋亡
6.长期低剂量氯胺酮滥用可损害前额叶(PFC)多巴胺(DA)系统功能,进而导致食蟹猴认识功能和执行功能的损害,自发运动减少。
Studies indicate that developing brain is sensitive to drug abuse, in which damage may induce inreversible changes. Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that was abused rapidly recent years, with profound socioeconomic and health impacts. It was reported that even exposed little dose of ketamine to infant or adolescence can trigger widespread apoptotic neurodegeneration in brain tissue. Moreover, adolescence is a biologically critical period in neurodevelopment where reorganization, synaptic remodeling and receptor pruning occur in the dopaminergic system. Drug abuse can influence transmission of dopaminergic neurons, and then induce changes of growth and sensibility of reward system.
Ketamine abuse can induce hallucinations, which is regarded as the main reason of drug addicts. Lots of reports demonstrated that ketamine abuser showed up objection of social ability, behavior and personality changes, damage of memory and organs, and especial evident damage of central nervous system. Present studies indicate that ketamine abuse can induce neuron apoptosis in many encephalic regions, including cerebral cortex, spinal cord, thalamus, hippocampus cingulate gyrus cortex and so on.
Ketamine induces hypofunction of NMDAR including excessive glutamate release through decreased activity of GABAergic interneurons, and subsequent exciteds cortex which may induce psychosis-like behavior and cognitive anomalies. Previous studies have demonstrated that acute ketamine administration mainly influenced prefrontal lobe and periphery cortex. There are evidently increased levels of glutamic acid and dopamine in these different encephalic regions. However, the mechanism of chronic ketamine administration on prefrontal lobe, dopamine neurons is still unclear.
The dopaminergic neurons play an important part in the reward system, in which glutamatergic-dopaminergic pathway interaction within the mesolimbic dopamine system are thought to contribute to addiction-related behavior in ketamine abuse. Functional magnetic resonance imaging (fMRI) about animal experiments showed that drug abuse enhanced activity of dopamine system in the mesolimbic, especially in striatum, robustly disrupting sensorimotor gating. With the emerging of new abused drugs successively, the modes of action and target areas of them are specificity differently. At present, little is known about the target areas and brain circuits of chronic ketamine administration.
Anaesthetic dose and acute ketamine administration in adult lead to impaired working memory and executive function. Disorders of face emotion processing, working memory, language fluency and learning were observed. There is little report about chronic low dose of ketamine administration, especially the effects on brain function and physiological mechanism in adolescent. This study was designed to determine the consequences of chronic ketamine administration in adolescent cynomolgus monkeys following an exposure pattern that mimics adolescence drug abuse. The chronic drug effects on monkey's weight, behavior, brain functions and brain metabolism were investigated and the molecule mechanisms were explored.
1. Objectives
1.1. To observe body weight and behavioral changes in adolescent cynomolgus monkey following chronic low dose of ketamine exposure.
1.2. To investigate the effects of chronic low dose of ketamine on brain function in adolescent cynomolgus monkey.
1.3. To investigate the brain metabolism in striatum and frontal lobe following chronic low dose of ketamine exposure in adolescent cynomolgus monkey.
1.4. To assay apoptosis in lentiform nucleus (LN) in adolescent cynomolgus monkey following chronic low dose of ketamine exposure.
1.5. To examine expression of tyrosine hydroxylase (TH) in the prefrontal cortex (PFC) following chronic low dose of ketamine administration in adolescent cynomolgus monkey.
2. Materials and methods
2.1. Animal model established
Twelve adolescent male cynomolgus monkeys were randomly divided into2 groups:eight to be administered with ketamine and four with saline as controls. A ketamine dose of1mg/kg in an injection volume of1ml (in saline) freshly prepared on the day of injection was given daily intravenously via arm vein under mild physical constraints for6months. Control monkeys were given sterile saline (1ml) daily.
2.2. Body weight measurement
Animal body weights were recorded monthly to monitor the monkeys'well-being throughout the whole experiment.
2.3. Behavioral observation
15-min video recordings were made for each monkey after ketamine or saline injection at day1, day3, day7, day14, day56, day112, day183, day184and day185. Behavior observation including moving, walking, climbing and jumping was conducted.
2.4. fMRI
fMRI study was performed on186day. Respiration rate and body temperature were continuously monitored during the fMRI scan. For stimulation, the monkeys' right lower limb was moved (raised leg to60°) once per second. Average scanning time of fMRI for one monkey was about12min48s. Images were acquired in axial level in order to localization accurately.
2.5. MRS
MRS use single voxel PROBE PRESS point resolved spectroscopy. We examined15x15x10mm area in striatum and frontal lobe regions, avoiding skull and cerebrospinal fluid. Average scanning time of MRS for one monkey was about3min, and3times were performed.
2.6. Apoptosis assay
After fMRI scanning,1control and3ketamine-abuse monkeys were randomly chosen to be sacrificed. Brains were dissected and embedded in paraffin wax. Sections of left PFC and LN were cut coronally from paraffin blocks at4-um 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.
2.7. Tyrosine hydroxylase (TH)
For Immunohistochemistry TH positive cells in PFC was used to study morphological changes under light microscope. Brain tissues sections were treated with inactivate endogenous peroxidases. They were then blocked with1%bovine serum albuminand10%normal rabbit serum for1h, and then were incubated with horse radish peroxidase (HRP)-conjugated rabbit anti-goat IgG for1h. Finally, the sections were developed with a DAB kit. TH positive cells were assayed.
3. Results
3.1. Chronic low dose of ketamine administration inhibited the body weight increase in adolescent cynomolgus monkey following chronic ketamine exposure.
There was no statistically significant difference between control and ketamine groups. But body weights of ketamine group increased more slowly compared with control group. Body weights increased for about10%in the control monkeys during the first2months, while the ketamine abuse monkeys took4months to gain the same10%increase in body weights. The mean difference at the end of6month was10%between the ketamine and control groups.
3.2. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey depressed motor behaviors
For behavior test, activities of moving, walking, climbing and jumping of ketamine group exhibited obvious decreased tendency as time went by. There were significant reduced activities of moving, walking and climbing of ketamine group in day183,184and185compared to control group (p<0.05).
3.3. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey caused disturbance in the central nervous system
First, significantly higher levels of neural activities were observed in the right hemisphere of LN, fusiform gyrus (FG) and entorhinal cortex (Ent) in ketamine-challenged monkeys compared with controls. Second, monkeys in the control group had significantly higher neural activities in the substantia nigra (SN), ventral tegmental area (VTA), posterior cingulate cortex (CGp), visual cortex, and somatosensory cortex (SC) compared with ketamine-challenged monkeys.
3.4. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey caused disturbance in brain metabolism
In MRS, Cho in striatum and frontal lobe in ketamine group are lower than those of control (p<0.05). Chronic ketamine abuse might affect cell metabolism such as membrane breakdown, turn over, synthesis, myelination and lipid metabolism in striatum and frontal lobe.
3.5. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey did not cause obvious apoptosis
There were not significant differences of apoptotic cells between two groups in lentiform nucleus.
3.6. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey decreased expression of TH protein in the prefrontal cortex
Tyrosine hydroxylase (TH) pilot experiment showed there was a qualitative reduction in the positively stained projective axon of dopaminergic neuron in the prefrontal cortex of monkeys with chronic ketamine abuse (p<0.01).
4. Conclusion
4.1. Chronic low dose of ketamine abuse inhibits the body weight increase in adolescent cynomolgus monkey following chronic ketamine exposure.
4.2. Chronic low dose of ketamine abuse decreases locomotor activity, which suggesting induced tolerance after chronic ketamine administration in adolescent cynomolgus monkey.
4.3. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey leads to depression in VTA and SN, which is correlated no drug seeking and behavior addiction. Significant brain activation in LN implicates a dysfunction of sensorimotor gating, which plausibly leading to motor inhibition. Activation in FG causes visual hallucination, which is regarded as the main reason of drug addicts. Activation in Ent-striatum may play an important role in schizophrenia and substance abuse. We identify the damage of SC firstly, which implicates injury function of somatosensory. This compensates the central action of ketamine, providing experiment proof and abstinence of ketamine abuse in adolescence.
4.4. Chronic low dose of ketamine abuse in adolescent cynomolgus monkey might affect neuroglial cell metabolism such as membrane turn over, synthesis and lipid metabolism in striatum and frontal lobe.
4.5. There is no significant apoptosis. Perhaps chronic low dose of ketamine abuse in adolescent cynomolgus monkey induces other paths of apoptosis. Although apoptosis will take place at last, the procee of apoptosis was delayed.
4.6. Chronic low dose of ketamine abuse impaires the function of prefrontal dopaminergic system, causing damage of cognitive function and decreased locomotor activity.
引文
[1]Bubenikova-Valesova V, Horacek J, Vrajova M, Hoschl C. Models of schizophrenia in humans and animals based on inhibition of NMD A receptors [J]. Neuroscience and Biobehavioral Reviews,2008,32(5):1014-1023.
[2]Morgan CJ, Riccelli M, Maitland CH, Curran HV. Long-term effects of ketamine:evidence for a persisting impairment of source memory in recreational users[J]. Drug Alcohol Depend,2004,75(3):301-308.
[3]胡绚丽,卞士中.氯胺酮滥用对中枢神经系统的毒性作用[J].中国司法鉴定,2008,2：31-33.
[4]Homayoun H, Moghaddam B. NMDA receptor hypofunction produces opposite effects on prefrontal cortex interneurons and pyramidal neurons[J]. J Neurosci, 2007,27:11496-500.
[5]Rujescu D, Bender A, Keck M, Hartmann AM, Ohl F, Raeder H, Giegling I, Genius J, McCarley RW, Moller HJ, Grunze H. A pharmacological model for psychosis based on N-methyl-D-aspartate receptor hypofunction:molecular, cellular, functional and behavioral abnormalities[J]. Biol Psychiatry,2006, 59:721-9.
[6]Young C, Jevtovic-Todorovic V, Qin YQ, Tenkova T, Wang H, Labruyere J, Olney JW. Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain[J]. Br J Pharmacol, 2005,146(2):189-197.
[7]Doyle KM, Feerick S, Kirkby DL, et al. Comparison of various NMDA receptor antognists in a modle of short-term memory and on overt behavior[J]. Behav Pharmacol,1998,9(8):671-681.
[8]Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vockler J, Dikranian K, Tenkova T, Stefovska V, Turski L, Olney JW. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain[J]. Science,1999,283:70-74.
[9]Ikonomidou C, Bittigau P, Ishimaru MJ, Koch C, Genz K, Price MT, Stefovska V, Horster F, Tenkova T, Dikranian K, Olney JW. Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome[J]. Science,2000,287(5455): 1056-1060.
[10]Belforte JE, Zsiros V, Sklar ER, Jiang Z, Yu G, Li Y, Quinlan EM, Nakazawa K. Postnatal NMDA receptor ablation in corticolimbic interneurons confers schizophrenia-like phenotypes[J]. Nat Neurosci,2010,13:76-83.
[11]Rujescu D, Bender A, Keck M, Hartmann AM, Ohl F, Raeder H, Giegling I, Genius J, McCarley RW, Moller HJ, Grunze H. A pharmacological model for psychosis based on N-methyl-D-aspartate receptor hypofunction:molecular, cellular, functional and behavioral abnormalities[J]. Biol Psychiatry,2006, 59:721-9.
[12]Narendran R, Frankle WG, Keefe R, Gil R, Martinez D, Slifstein M, Kegeles LS, Talbot PS, Huang Y, Hwang DR, Khenissi L, Cooper TB, Laruelle M, Abi-Dargham A. Altered prefrontal dopaminergic function in chronic recreational ketamine users[J]. Am J Psychiatry,2005,162:2352-9.
[13]Li Q, Cheung C, Wei R, Cheung V, Hui ES, You Y, Wong P, Chua SE, McAlonan GM, Wu EX. Voxel-based analysis of postnatal white matter microstructure in mice exposed to immune challenge in early or late pregnancy[J]. Neuroimage,2010,52:1-8.
[14]Liao Y, Tang J, Ma M, Wu Z, Yang M, Wang X, Liu T, Chen X, Fletcher PC, Hao W. Frontal white matter abnormalities following chronic ketamine use:a diffusion tensor imaging study[J]. Brain,2010,133:2115-22.
[15]Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits [J]. J Neurosci,2003,23(3):876-882.
[16]Farber NB, Wozniak DF, Price MT, Labruyere J, Huss J, St Peter H, Olney JW. Age-specific neurotoxicity in the rat associated with NMDA receptor blockade: potential relevance to schizophrenia?[J]. Biol Psychiatry,1995,38(12):788-796.
[17]Fu CH, Abel KM, Allin MP, Gasston D, Costafreda SG, Suckling J, Williams SC, McGuire PK. Effects of ketamine on prefrontal and striatal regions in an overt verbal fluency task:a functional magnetic resonance imaging study[J]. Psychopharmacology (Berl),2005,183:92-102.
[18]Stoet G, Snyder LH. Effects of the NMDA antagonist ketamine on task-switching performance:evidence for specific impairments of executive control[J]. Neuropsychopharmacology,2006,31:1675-1681.
[19]Abel KM, Allin MP, Kucharska-Pietura K, Andrew C, Williams S, David AS, Phillips ML. Ketamine and fMRI BOLD signal:distinguishing between effects mediated by change in blood flow versus change in cognitive state[J]. Hum Brain Mapp,2003,18:135-45.
[20]Marota JJ, Mandeville JB, Weisskoff RM, Moskowitz MA, Rosen BR, Kosofsky BE. Cocaine activation discriminates dopaminergic projections by temporal response:an fMRI study in rat[J]. Neuroimage,2000,11(1):13-23.
[21]Koch M, Schnitzler HU. The acoustic startle response in rats--circuits mediating evocation, inhibition and potentiation[J]. Behav Brain Res,1997, 89:35-49.
[22]Chambers RA, Taylor JR, Potenza MN. Developmental neurocircuitry of motivation in adolescence:a critical period of addiction vulnerability[J]. Am J Psychiatry,2003,160:1041-52.
[23]Slikker W Jr, Zou X, Hotchkiss CE, Divine RL, Sadovova N, Twaddle NC, Doerge DR, Scallet AC, Patterson TA, Hanig JP, Paule MG, Wang C. Ketamine-induced neuronal cell death in perinnata Rhesus monkey [J]. Toxicol Sci, 2007,98(1):145-158.
[24]Young C, Jevtovic-Todorovic V, Qin YQ, Tenkova T, Wang H, Labruyere J, Olney JW. Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain[J]. British J of Pharmaco,2005,146:189-197.
[25]Ikonomidou C, Bittigau P, Koch C, Genz K, Hoerster F, Felderhoff-Mueser U, Tenkova T, Dikranian K, Olney JW.Neurotransmitters and apoptosis in the developing brain[J].Biochemical Pharmacology,2001,62:401-405.
[26]Bubenikova-Valesova V, Horacek J, Vrajova M, Hoschl C.Models of schizophrenia in humans and animals based on inhibition of NMDA receptors[J].Neurosci Biobehav Rev,2008,32(5):1014-23.
[27]Crews F, He J, Hodge C. Adolescent cortical development:a critical period of vulnerability for addiction[J]. Pharmacol Biochem Behav,2007,86:189-99.
[28]Stoet G, Snyder LH. Effects of the NMDA antagonist ketamine on task-switching performance:evidence for specific impairments of executive control[J]. Neuropsychopharmacology,2006,31:1675-81.
[29]Bubenikova-Valesova V, Horacek J, Vrajova M, Hoschl C. Models of schizophrenia in humans and animals based on inhibition of NMDA receptors[J]. Neurosci Biobehav Rev,2008,32:1014-23.
[30]Fang M, Li J, Lu G, Gong X, Yew DT. A fMRI study of age-related differential cortical patterns during cued motor movement[J]. Brain Topogr,2005,17:127-37.
[31]Howard CVRM. Unbiased stereology. New York:Garland Science/BIOS Scientific Publishers,2005.
[32]Chou SY, Chen CY, Su HW, Hsu MI, Liang SR, Hsu CS. Ultrasonographic quantification of the endometrium during the menstrual cycle using computer-assisted analysis[J]. Taiwan J Obstet Gynecol,2011,50(3):297-300.
[33]Marco LA, Pendleton R, Reed TF, Brigham TE, Aldes LD, Chronister RB. Ketamine-induced tongue protrusions in rats[J]. Comp Biochem Physiol C,1989, 94(l):29-33.
[34]王红伟,任爱红,闫君宝,等.氯胺酮对小鼠离体十二指肠自主收缩活动的影响及受体途径[J].陕西医学杂志,2009,38(5)：525-528.
[35]Morton G J, Cummings D, Baskin DG, Barsh GS, Schwartz MW. Central nervous system control of food intake and body weight[J]. Nature,2006, 443(7109):289-95.
[36]Skibicka KP, Hansson C, Alvarez-Crespo M, Friberg PA, Dickson SL. Ghrelin directly targets the ventral tegmental area to increase food motivation[J]. Neuroscience,2011,180:129-37.
[37]Freese TE, Miotto K, Reback CJ. The effects and consequences of selected club drugs[J]. J Subst Abuse Treat,2002,23(2):151-156.
[38]Becker A, Peters B, Schroeder H, Mann T, Huether G, Grecksch G. Ketamine-induced changes in rat behaviour:A possible animal model of schizophrenia[J]. Prog Neuropsychopharmacol Biol Psychiatry,2003,27:687-700.
[39]Nelson CL, Burk JA, Bruno JP, Sarter M. Effects of acute and repeated systemic administration of ketamine on prefrontal acetylcholine release and sustained attention performance in rats[J]. Psychopharmacology (Berl),2002, 161:168-79.
[40]Cho HS, D'Souza DC, Gueorguieva R, Perry EB, Madonick S, Karper LP, Abi-Dargham A, Belger A, Abi-Saab W, Lipschitz D, Bennet A, Seibyl JP, Krystal JH. Absence of behavioral sensitization in healthy human subjects following repeated exposure to ketamine[J]. Psychopharmacology (Berl),2005,179(1): 136-143.
[41]Uchihashi Y, Kuribara H, Morita T, Fujita T. The repeated administration of ketamine induces an enhancement of its stimulant action in mice[J]. Jpn J Pharmacol,1993,61(2):149-151.
[42]Trujillo KA, Zamora JJ, Warmoth KP. Increased response to ketamine following treatment at long intervals:implications for intermittent use[J]. Biol Psychiatry,2008,63(2):178-183.
[43]Volkow ND, Wang GJ, Telang F, Fowler JS, Logan J, Childress AR, Jayne M, Ma Y, Wong C. Cocaine cues and dopamine in dorsal striatum:mechanism of craving in cocaine addiction[J]. J Neurosci,2006,26:6583-8.
[44]Caille S, Parsons LH. Intravenous heroin self-administration decreases GABA efflux in the ventral pallidum:an in vivo microdialysis study in rats[J]. Eur J Neurosci,2004,20(2):593-6.
[45]Hubner CB, Koob GF.The ventral pallidum plays a role in mediating cocaine and heroin self-administration in the rat[J]. Brain Res,1990,508(1):20-9.
[46]Everitt BJ, Robbins TW. Neural systems of reinforcement for drug addiction: from actions to habits to compulsion[J]. Nat Neurosci,2005,8:1481-9.
[47]Koch M, Schnitzler HU. The acoustic startle response in rats--circuits mediating evocation, inhibition and potentiation[J]. Behav Brain Res,1997, 89:35-49.
[48]Li Q, Cheung C, Wei R, Hui ES, Feldon J, Meyer U, Chung S, Chua SE, Sham PC, Wu EX, McAlonan GM. Prenatal immune challenge is an environmental risk factor for brain and behavior change relevant to schizophrenia:evidence from MRI in a mouse model[J]. PLoS One,2009,4(7):e6354.
[49]Vollenweider FX, Leenders KL, Scharfetter C, Antonini A, Maguire P, Missimer J, Angst J. Metabolic hyperfrontality and psychopathology in the ketamine model of psychosis using positron emission tomography (PET) and [18F] fluorodeoxyglucose (FDG)[J]. Eur Neuropsychopharmacol,1997,7:9-24.
[50]Akin A, Esmaoglu A, Guler G, Demircioglu R, Narin N, Boyaci A. Propofol and propofol-ketamine in pediatric patients undergoing cardiac catheterization[J]. Pediatr Cardiol,2005,26(5):553-7.
[51]Park MS, Sohn JH, Suk JA, Kim SH, Sohn S, Sparacio R. Brain substrates of craving to alcohol cues in subjects with alcohol use disorder[J]. Alcohol Alcohol, 2007,42:417-22.
[52]Braus DF, Wrase J, Grusser S, Hermann D, Ruf M, Flor H, Mann K, Heinz A. Alcohol-associated stimuli activate the ventral striatum in abstinent alcoholics[J]. J Neural Transm,2001,108(7):887-94.
[53]Matochik JA, Eldreth DA, Cadet JL, Bolla KI. Altered brain tissue composition in heavy marijuana users[J]. Drug Alcohol Depend,2005, 77(1):23-30.
[54]Lee HW, Hong SB, Seo DW, Tae WS, Hong SC. Mapping of functional organization in human visual cortex:electrical cortical stimulation[J]. Neurology, 2000,54:849-54.
[55]Lee CU, Shenton ME, Salisbury DF, Kasai K, Onitsuka T, Dickey CC, Yurgelun-Todd D, Kikinis R, Jolesz FA, McCarley RW. Fusiform gyrus volume reduction in first-episode schizophrenia:a magnetic resonance imaging study[J]. Arch Gen Psychiatry,2002,59:775-81.
[56]Bellgowan PS, Buffalo EA, Bodurka J, Martin A. Lateralized spatial and object memory encoding in entorhinal and perirhinal cortices[J]. Learn Mem,2009, 16:433-8.
[57]Nordquist RE, Risterucci C, Moreau JL, von Kienlin M, Kunnecke B, Maco M, Freichel C, Riemer C, Spooren W. Effects of aripiprazole/OPC-14597 on motor activity, pharmacological models of psychosis, and brain activity in rats[J]. Neuropharmacology,2008,54:405-16.
[58]See RE, Elliott JC, Feltenstein MW. The role of dorsal vs ventral striatal pathways in cocaine seeking behavior after prolonged abstinence in rats[J]. Psychopharmacology (Berl),2007,194:321-31.
[59]Pearson JM, Hayden BY, Raghavachari S, Platt ML. Neurons in posterior cingulate cortex signal exploratory decisions in a dynamic multioption choice task[J]. Curr Biol,2009,19:1532-7.
[60]Haznedar MM, Buchsbaum MS, Hazlett EA, Shihabuddin L, New A, Siever LJ. Cingulate gyrus volume and metabolism in the schizophrenia spectrum[J]. Schizophr Res,2004,71:249-62.
[61]Newell KA, Zavitsanou K, Jew SK, Huang XF. Alterations of muscarinic and GAB A receptor binding in the posterior cingulate cortex in schizophrenia[J]. Prog Neuropsychopharmacol Biol Psychiatry,2007,31:225-33.
[62]胡文富,傅先明,钱若兵,季学兵,王昌新,魏祥品,牛朝诗,汪业汉.海洛因成瘾者后扣带回结构和功能连接的磁共振研究[J].中华行为医学与脑科学杂志.2011,20(7)：580-582.
[63]McCann UD, Szabo Z, Seckin E, Rosenblatt P, Mathews WB, Ravert HT, Dannals RF, Ricaurte GA. Quantitative PET studies of the serotonin transporter in MDMA users and controls using [11C]McN5652 and [11C]DASB[J]. Neuropsychopharmacology,2005,30:1741-50.
[64]Renshaw PF, Yurgelun-Todd DA, Cohen BM. Greater hemodynamic response to photic stimulation in schizophrenic patients:an echo planar MRI study[J]. Am J Psychiatry,1994,151:1493-5.
[65]Yoon JH, Maddock RJ, Rokem A, Silver MA, Minzenberg MJ, Ragland JD, Carter CS. GABA concentration is reduced in visual cortex in schizophrenia and correlates with orientation-specific surround suppression[J]. J Neurosci,2010, 30:3777-81.
[66]Cho RY, Konecky RO, Carter CS. Impairments in frontal cortical gamma synchrony and cognitive control in schizophrenia[J]. Proc Natl Acad Sci USA, 2006,103:19878-83.
[67]Wise RA Brain reward circuitry:insights from unsensed incentives [J]. Neuron, 2002,36(2):229-40.
[68]Tzschentke TM. Pharmacology and behavioral pharmacology of the mesocortical dopamine system[J]. Prog Neurobiol,2001,63(3):241-320.
[69]O'Brien CP, Gardner EL. Critical assessment of how to study addiction and its treatment:human and non-human animal models[J]. Pharmacol Ther,2005, 108(1):18-58.
[67]Licata SC, Renshaw PF. Neurochemistry of drug action:insights from proton magnetic resonance spectroscopic imaging and their relevance to addiction[J]. Ann N Y Acad Sci.2010,1187:148-71.
[68]lory GS, O'Malley J, Grant KA, Park B, Kroenke CD. Quantification of ethanol methyl (1)H magnetic resonance signal intensity following intravenous ethanol administration in primate brain[J]. Methods,2010,50:189-198.
[69]Moffett JR, Ross B, Arun P, Madhavarao CN, Namboodiri AM. N-Acetylaspartate in the CNS:From neurodiagnostics to neurobiology[J]. Progress in Neurobiology,2007,81:89-131.
[70]Scherk H, Backens M, Schneider-Axmann T, Kraft S, Kemmer C, Usher J, Reith W, Falkai P, Meyer J, Gruber O. Dopamine transporter genotype influences N-acetyl-aspartate in the left putamen[J]. World J. Biol. Psychiatry,2007,26:1-7.
[71]徐惠,李传福,厉保秋等.磁共振波谱成像在食蟹猴脑研究中的应用[J]. 山东大学学报(医学版).2010,48(10)：56-59.
[72]Chang L, Ernst T, Speck O, Grob CS. Additive effects of HIV and chronic methamphetamine use on Brain metabolite abnormalities[J]. Am J Psychiatry,2005, 162(2):361-369.
[73]Haselhorst R, Dursteler-MacFarl and KM, Scheffler K, Ladewig D, Muller-Spahn F, Stohler R, Seelig J, Seifritz E. Frontocortical N-acetylaspartate reduction associated with long-term IV heroinuse[J]. Neurology,2002,58:305-7.
[74]Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V. Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways[J]. Neuroscience,2005,135(3):815-827.
[75]Davidson AJ. Anesthesia and neurotoxicity to the developing brain:the clinical relevance[J]. Paediatr Anaesth,2011,21(7):716-21.
[76]Mak YT, Lam WP, Lu L, Wong YW, Yew DT. The toxic effect of ketamine on SH-SY5Y neuroblastoma cell line and human neuron[J]. Microsc Res Tech,2010, 73(3):195-201.
[77]Suzuki M, Nelson AD, Eickstaedt JB, Wallace K, Wright LS, Svendsen CN. Glutamate enhances proliferation and neurogenesis in human neural progenitor cell cultures derived from the fetal cortex[J]. Eur J Neurosci,2006,24(3):645-653.
[78]Coyle J, Puttfareken P. Oxidative stress, glutamate and neurodegenerative disorders [J]. Science,1993,262(5134):689-694.
[79]Farber NB et al,1998-Farber NB, Newcomer JW, Olney JW. The glutamate synapse in neuropsychiatric disorders:focus on schizophrenia and Alzheimer's disease[J]. Prog Brain Res,1998,116:421-437.
[80]Martin LJ, Al-Abdulla NA, Brambrink AM, Kirsch JR, Sieber FE, Portera-Cailliau C. Neurodegeneration in excitotoxicity, global cerebral ischemia, and target deprivation:a perspective on the contributions of apoptosis and necrosis [J]. Brain Res Bull,1998,46(4):281-309.
[81]Kim SY, Lee H, Kim HJ, Bang E, Lee SH, Lee DW, Woo DC, Choi CB, Hong KS, Lee C, Choe BY. In vivo and ex vivo evidence for ketamine-induced hyperglutamatergic activity in the cerebral cortex of the rat:Potential relevance to schizophrenia[J]. NMR Biomed,2011,24(10):1235-42.
[82]Moghaddam B, Adams B, Verma A, Daly D. Activation of glutamatergic neurotransmission by ketamine:a novel step in the pathway from NMDA receptor blockade to dopaminergic and cognitive disruptions associated with the prefrontal cortex[J]. J Neurosci,1997,17:2921-2927.
[83]Lee SH, Kim SY, Woo DC, Choe BY, Ryu KN, Choi WS, Jahng GH, Yim SV, Kim HY, Choi CB. Differential neurochemical responses of the canine striatum with pentobarbital or ketamine anesthesia:A 3T proton MRS study[J]. J Vet Med Sci,2010,72(5):583-7.
[84]Rowland LM, Bustillo JR, Mullins PG, Jung RE, Lenroot R, Landgraf E, Barrow R, Yeo R, Lauriello J, Brooks WM. Effects of ketamine on anterior cingulated glutamate metabolism in Healthy humans:a 4T proton MRS study[J]. Am J Psychiatry,2005,162:394-396.
[85]Sun L, Lam WP, Wong YW, Lam LH, Tang HC, Wai MS, Mak YT, Pan F, Yew DT. Permanent deficits in brain functions caused by long-term ketamine treatment in mice[J]. Hum Exp Toxicol,2011,30(9):1287-96.
[86]王力,江虹,于洪鸾,孙琳,潘芳.低剂量氯胺酮对食蟹猴行为、海马和前额皮质超微结构的影响.山东大学学报(医学版)[J].2010,48(9)：64-67.
[87]Hokfelt T, Johansson O, Fuxe K, Goldstein M, Park D. Immunohistochemical studies on the localization and distribution of monoamine neuron systems in the rat brain Ⅱ[J]. Med Biol,1977,55:21-40.
[88]Duncko R, Kiss A, Skultetyova I, Rusnak M, Jezova D. Corticotropin-releasing hormone mRNA levels in response to chronic mild stress rise in male but not in female rats while tyrosine hydroxylase mRNA levels decrease in both sexes[J]. Psychoneuroendocrinology,2001,26:77-89.
[89]Wang LF, Xu CL, Xiong KR. Effects of electroacupuncture on expression of tyrosine hydroxylase and c-fos in hippocampal CA 1 area in ketamine-addiction rats[J]. Zhen Ci Yan Jiu,2011,36(1):23-7.
[90]Weinberger DR. Implications of the normal brain development for the pathogenesis of schizophrenia[J]. Arch Gen Psychiatry,1987,44:660-669.
[91]Tsai G, Coyle JT. Glutamatergic mechanisms in schizophrenia. Annu Rev Pharmacol Toxicol,2002,42:165-179.
[92]Guo N, Hwang DR, Lo ES, Huang YY, Laruelle M, Abi-Dargham A. Dopamine depletion and in vivo binding of PET D1 receptor radioligands: implications for imaging studies in schizophrenia[J]. Neuropsychopharmacology, 2003,28:1703-11.