中脑腹侧被盖区多巴胺能神经元参与抑郁发生的机制研究

发布时间：2018-01-12 19:00

本文关键词：中脑腹侧被盖区多巴胺能神经元参与抑郁发生的机制研究　出处：《中国人民解放军军事医学科学院》2016年硕士论文　论文类型：学位论文

【摘要】：抑郁症(Depression)是一种慢性长期的精神类疾病,全球范围内约1.2亿人口深受其害。虽然目前关于抑郁症发生的病因和神经生物学机制的研究已经取得了突破性进展,但是关于抑郁症发生的电生理和分子机制仍需要待进一步阐明。VTA多巴胺能神经元在奖励、动机、药物成瘾等动物行为中起重要作用。最近有文章报道,实验证明中脑腹侧被盖区(Ventral tegmental area,VTA)多巴胺能神经元可能参与了抑郁症的发生。特异性激活或者抑制中脑腹侧被盖区多巴胺能神经元的自发放电能明显影响抑郁动物的行为学表现,但其具体的分子机制还需要进一步的探究。慢性疼痛、慢性应激和抑郁症的发生存在重要的联系,临床上许多慢性疼痛和长期遭受应激的患者常伴随抑郁症状,但其机制确不清楚。研究目的:本研究拟模拟临床上慢性疼痛和长期遭受应激的患者,建立慢性神经痛和慢性温和不可预知性刺激(CUMS)两种抑郁大鼠模型;在此基础上,运用在体多通道神经电生理记录系统(Multichannel Acquisition Processor(MAP)Neurophysiology System)记录抑郁发生后的VTA多巴胺能神经元自发电活动;并在药物治疗恢复后,记录分析VTA多巴胺能神经元的电活动的变化情况;并应用免疫组化的方法,分析在此病理过程中超极化激活的环核苷酸门控的离子通道(HCN)通道表达改变的情况,从而初步探讨VTA多巴胺能神经元参与抑郁症发生的电生理分子机制。研究方法:1.抑郁大鼠模型的建立:(1)慢性神经痛诱发抑郁症:我们采用坐骨神经分支选择性损伤模型(spared nerve injury,SNI),采用机械性刺激缩足阈值、糖水偏好、强迫游泳以及旷场实验作为行为学评定指标。实验开始前,48只SPF成年健康大鼠在隔离屏障环境适应一周后,进行随机分组,分为假手术组大鼠12只和SNI组大鼠36只,并测得机械痛阈值基值。只暴露假手术组大鼠神经而不进行结扎,结扎并剪断SNI组大鼠的腓总神经和胫神经,并且需要完整保留,不能牵拉腓肠神经。在SNI手术后的第3、14、28、56天,进行机械性刺激缩足阈值测得,第14、28、56天进行矿场实验、糖水偏好、强迫游泳实验进行抑郁行为学评定。将SNI组大鼠随机分为两组,分别腹腔注射一种临床上广泛使用的神经病理性疼痛镇痛药加巴喷丁(100 mg/kg)和生理盐水14天,进行行为学观察。(2)建立另一种大鼠抑郁症模型:即采用慢性不可预知轻度应激(chronic unpredictable mild stress,CUMS)刺激的方法对大鼠进行长期应激压力刺激造模。采用百分比糖水偏好、旷场下活动距离以及体重变化作为抑郁发生与否的行为学评定指标。实验前,大鼠在实验动物中心屏障隔离环境饲养一周,使其适应CUMS刺激环境。并按照随机分组的原则将实验动物随机分为对照组和CUMS组。然后进行CUMS造模,在造模过程中,对照组大鼠3-4只合笼正常饲养,实验组大鼠单笼饲养并进行CUMS造模6-8周,随后根据行为学结果,经统计分析后得到具有抑郁行为大鼠。把经CUMS刺激后具有抑郁表型的大鼠进行氟西汀治疗恢复抑郁表型:其中一部分大鼠腹腔注射21天生理盐水,另一部分大鼠连续腹腔注射21天的氟西汀,并给药结束后,进行行为学评价,得到抑郁恢复的大鼠以及抑郁大鼠,所有实验动物均需要进行行为学评价。2.多通道电生理技术记录分析:我们采用Plexon公司的多通道神经电生理记录分析系统对实验动物进行在体电生理记录,并分析中脑腹侧被盖区VTA多巴胺能神经元的自发放电特征。在CUMS造模后筛选抑郁症状大鼠,随后进行氟西汀注射进行治疗,记录分析氟西汀治疗后大鼠、抑郁症大鼠、以及对照组大鼠的中脑腹侧被盖区多巴胺能神经元自发电活动,分析自发放电频率与爆发式放电活动。在慢性神经痛诱发抑郁症实验中,通过筛选出慢性神经痛诱导的抑郁症状的大鼠进行加巴喷丁注射,记录分析加巴喷丁治疗组大鼠、神经损伤组大鼠,以及假手术组大鼠的VTA多巴胺能神经元自发放电特征。3.大鼠中脑腹侧被盖区免疫荧光分析:在慢性神经痛诱发抑郁症大鼠的VTA脑区,免疫荧光双标记酪氨酸羟化酶(Tyrosine hydroxylase,TH)与超极化激活的环核苷酸离子通道(Hyperpolarization-activated cyclic nucleotide-gated channel,HCN)蛋白,观察并统计分析HCN通道蛋白在中脑腹侧被盖区多巴胺能神经元上的表达变化。研究结果:1.在慢性神经痛诱发抑郁症实验中,相对比与假手术组大鼠,我们发现SNI大鼠的机械刺激缩足阈值下降,表现为明显的统计学差异(n=6,P0.01),糖水偏好(n=6,P0.01),旷场行走总路程(n=8,P0.01)均有所下降,强迫游泳静止时间增加(n=10,P0.01)。在经过14天加巴喷丁治疗后,SNI组大鼠的抑郁行为得到缓解,相比较于SNI注射生理盐水组的大鼠,加巴喷丁注射组大鼠的机械性刺激缩足反射阈值增加,(n=10,P0.001),在旷场下活动的总路程增加(n=6,P0.001),具有明显的统计学意义。在强迫游泳中的静止时间减少(n=8,P0.01),在糖水偏好方面增加(n=8,P0.01),表现为明显的统计学差异。SNI组大鼠VTA多巴胺能神经元自发放电频率较假手术组大鼠增加,出现明显的统计学差异(P0.001),簇状放电活动显著增加(P0.001);经过加巴喷丁治疗后,加巴喷丁治疗组大鼠VTA多巴胺能神经元自发放电频率较SNI组大鼠有所降低,存在统计学差异(P0.05),但簇状放电活动显著增加,表现出明显的统计学差异(P0.01);但相比较于Sham组大鼠,加巴喷丁治疗组大鼠的VTA多巴胺能神经元自发放电频率仍然增加(P0.01),簇状放电活动出现高度统计学差异(P0.001,n=132(SNI+Saline),n=81(SNI+GBP),n=71(Sham))。同时HCN1蛋白在加巴喷丁治疗组大鼠与盐水注射组大鼠和Sham组大鼠的VTA脑区表达没有差异(P0.05);相比较于Sham组大鼠,HCN2在加巴喷丁治疗组大鼠(P0.05)以及盐水组大鼠中均高调(P0.01),但是二者之间没有差异(P0.05),我们没有发现HCN3在VTA的表达,同时没有找到HCN4和TH在VTA共表达的细胞。2.在CUMS抑郁症模型实验中,在第一次8周CUMS造模后,与对照组大鼠相比较,CUMS造模组大鼠的糖水偏好比率下降,出现高度统计学差异(n=12,P0.001),体重增长缓慢,出现统计学差异(n=12,P0.05),旷场下的活动总路程没有差异(n=12,P0.05),经过21天的氟西汀治疗后,氟西汀注射组大鼠的糖水偏好比率较盐水注射组大鼠增加,表现为明显统计学差异(n=12,P0.01),而体重变化与旷场活动总路程没有变化(n=12,P0.05)。CUMS大鼠VTA多巴胺能神经元自发放电频率较对照组大鼠明显增加,有显著的统计学差异(P0.01),簇状放电增加,表现为高度的统计学差异(P0.001);经过21天氟西汀治疗后,氟西汀治疗组大鼠的VTA多巴胺能神经元自发放电频率较CUMS组大鼠降低,但是没有统计学差异(P0.05),但较对照组大鼠表现出统计学差异(P0.05)。氟西汀治疗组大鼠VTA多巴胺能神经元簇状放电活动中动作电位的数量相比较CUMS降低,表现出统计学差异;较对照组大鼠仍然增加,表现为统计学差异(P0.05);而簇状放电的持续时间同CUMS组大鼠比较没有差异,同对照组大鼠比较表现为高度的统计学差异(P0.001)(n=61(control);n=58(CUMS);n=83(CUMS+fluoxetine))。研究结论:1.在慢性疼痛和慢性应激(CUMS)诱发的两种抑郁症模型大鼠中,中脑腹侧被盖区(VTA)多巴胺能神经元电信号都发生改变。该研究提示,慢性应激(疼痛或者刺激)导致的VTA脑区多巴胺能神经元兴奋性改变,与抑郁症的发生具有很强的相关性。2.慢性疼痛诱发抑郁症状的模型大鼠中,超极化激活的环核苷酸离子通道蛋白(HCN)的表达高调。由于HCN在神经元的兴奋性调节中发挥重要的作用,本研究提示,长期的疼痛刺激诱发的抑郁症,可能与VTA多巴胺能神经元HCN表达高调,从而引发的神经元兴奋性改变相关。
[Abstract]:Depression (Depression) is a chronic mental illness, approximately 120 million of the population worldwide suffer. Although the current research on the etiology of depression and neurobiological mechanisms has made breakthrough progress, but about the depression and the electrophysiological and molecular mechanisms still need to be elucidated.VTA dopaminergic neurons in reward that motivation plays an important role in drug addiction and other animal behavior. The article reported recently, experiments show that the ventral tegmental area (Ventral tegmental, area, VTA) dopaminergic neurons may be involved in the occurrence of depression. The specificity of activation or inhibition of midbrain VTA dopamine neurons from firing could significantly affect the animal depression the behavioral performance, but its molecular mechanism needs further study. Chronic pain, chronic stress and depression There is an important relationship, many clinical chronic pain and long suffering stress patients often associated with depressive symptoms, but the mechanism is not clear. Objective: This study intends to simulate clinical chronic pain and long suffering stress in patients with established chronic neuropathic pain and chronic unpredictable mild stimulation (CUMS) two models rats with depression; on this basis, using the in vivo multi-channel electrophysiological recording system (Multichannel Acquisition Processor (MAP) Neurophysiology System) recorded depression VTA dopaminergic neurons spontaneous electrical activity; and in the drug treatment after recovery, changes in recording electrical activity analysis of VTA dopaminergic neurons; and immunohistochemical analysis of ion channels, this pathological process of hyperpolarization activated cyclic nucleotide gated (HCN) channel expression changes, and from the preliminary study of VTA DOPA Dopaminergic neuronal electrophysiological mechanisms involved in depression. Methods: a rat model of depression: 1. (1) chronic neuropathic pain induced depression: we use the spared nerve injury model (spared nerve injury, SNI), the mechanical withdrawal threshold, sucrose preference, forced swimming and open field the experiment for the behavioral assessment indicators. Before the start of the experiment, 48 SPF adult rats adapted for one week in the isolation barrier environment, were randomly divided into sham operation group, 12 rats in group SNI and 36 rats, and measured the mechanical pain threshold base value. Only exposed rats in sham operation group without nerve ligation, ligation of the common peroneal nerve and tibial nerve cut SNI rats, and the need to retain the integrity, not to pull the sural nerve. After SNI surgery at 3,14,28,56 days, were measured the mechanical withdrawal threshold of the 14,28,56 day For field test, sucrose preference, forced swimming test for depression behavior evaluation. Rats in group SNI were randomly divided into two groups, neuropathic pain and analgesic gabapentin were injected a clinically widely used (100 mg/kg) and saline for 14 days, behavioral observation. (2) the establishment of another a rat model of depression: the chronic unpredictable mild stress (chronic unpredictable mild stress, CUMS) stimulation method for long-term stress on rat model. The percentage of stimulation of sucrose preference, open field activity distance and weight change as the Depression Behavior and whether or not the evaluation index. Before the experiment, rats in the experimental animal center of environmental barrier isolation feed for a week, make it adapt to the stimulation of CUMS environment. And in accordance with the principles of randomized experimental animal were randomly divided into control group and CUMS group. Then in the CUMS model, the In the process of modeling, the rats in the control group 3-4 only mated with normal feeding, experimental rats reared in single cage and CUMS model for 6-8 weeks, then according to the results of behavior, after statistical analysis with depressive behavior of rats after CUMS stimulation. The phenotype of rats with depression recovery of fluoxetine in the treatment of depression phenotype: some of the rats by intraperitoneal injection of normal saline for 21 days, another part of the rats by intraperitoneal injection for 21 days and fluoxetine, after medication, behavioral assessment, get recovery from depression rats and rats with depression, all experimental animal are required to the evaluation of behavior of.2. multi channel electrophysiology Technology of recording and analysis: we use multi channel neural Plexon, electrophysiological recording and analysis system of experimental animal in vivo electrophysiological recording, and analyze the characteristics of spontaneous discharges of VTA dopaminergic neurons in the VTA in CUMS. After modeling the screening of depressive symptoms in rats, followed by injection of fluoxetine treatment, recording and analysis of fluoxetine in the treatment of rats after depression rats and control group rats midbrain VTA dopaminergic neurons and spontaneous activity, analysis of spontaneous discharge frequency and the bursting activity. In chronic neuropathic pain induced depression in the experiment for injection by screening out, gabapentin chronic neuropathic pain induced depression rats, recording and analysis of gabapentin in the treatment group rats, nerve injury rats, and the rats in the sham operation group VTA spontaneous discharge characteristics of.3. dopaminergic neurons in ventral tegmental area: immunofluorescence analysis in VTA brain regions of chronic nerve the pain induced depression rats, immunofluorescence double labeling of tyrosine hydroxylase (Tyrosine hydroxylase, TH) and hyperpolarization activated cyclic nucleotide ion channels (Hyperpolarization-act Ivated cyclic nucleotide-gated channel, HCN) protein, observation and statistical analysis of HCN channel protein in VTA dopaminergic neurons. Expression of the results: 1. in chronic neuropathic pain induced depression in the experiment, compared with the sham operation group rats, we found that SNI rats mechanical withdrawal threshold decline, showed statistically significant difference (n=6, P0.01), sugar preference (n=6, P0.01), open field walking distance (n=8, P0.01) were decreased, forced swimming immobility time increased (n=10, P0.01). After 14 days of gabapentin treatment, the depressive behaviors of rats in group SNI were alleviated SNI, compared with the injection of saline group rats, rats injected with gabapentin mechanical stimulus withdrawal threshold increased (n=10, P0.001), the total distance in open field under the increase of (n=6, P0.001), which was statistically significant in the forced. A reduction in the swimming time (n=8, P0.01), the increase in sucrose preference on (n=8, P0.01, VTA) showed significant difference in the.SNI group rats dopamine neurons firing frequency than those in the sham operation group rats increased, there was significant differences (P0.001), cluster discharge activity increased significantly (P0.001); after gabapentin treatment, gabapentin treated rats VTA dopaminergic neurons firing frequency than the SNI rats decreased, there was significant difference (P0.05), but the cluster activity increased significantly, showing a statistically significant difference (P0.01); but compared to Sham rats the rats in the treatment group, gabapentin VTA dopaminergic neurons spontaneous discharge frequency (P0.01), there was still increased highly significant difference of cluster activity (P0.001, n=132 (SNI+Saline), n =81 (SNI+GBP), n=71 (Sham)). At the same time, HCN1 protein in gabah Gabapentin treated rats and saline injected group rats and Sham group rats VTA expression did not differ between brain regions (P0.05); compared with Sham rats, HCN2 rats in the treatment group of gabapentin (P0.05) and saline group rats were high (P0.01), but no difference between the two (P0.05), we found no HCN3 expression in VTA, but did not find the co expression of HCN4 and TH in VTA.2. cells in CUMS depression model experiment, in the first 8 weeks after CUMS, compared with the control group of rats, decreased sucrose preference ratio of CUMS model rats, appears highly statistically the difference (n=12, P0.001), a slow increase of body weight, there was significant difference (n=12, P0.05), no difference in open field under the total distance (n=12, P0.05), fluoxetine after 21 days of treatment, the sucrose preference ratio of fluoxetine in rats injected with saline injection group rats showed significant increase. No statistical difference (n=12, P0.01), and the changes of body weight and open field activity did not change the total distance (n=12, P0.05).CUMS VTA rat dopaminergic neurons firing frequency of rats significantly increased compared with the control group, there was a statistically significant difference (P0.01), cluster discharge is increased, the height difference the (P0.001); after 21 days after fluoxetine treatment, fluoxetine treatment group rats VTA dopamine neurons firing frequency than the CUMS rats decreased, but no significant difference (P0.05), but compared with the control group rats showed significant difference (P0.05). Fluoxetine in the treatment of VTA rats dopaminergic the number of clusters of neurons in action potential discharge activity compared to CUMS decreased, showing statistical differences; compared with the control group rats still showed statistically significant increase (P0.05); while the duration of burst discharge with the rats in the CUMS group compared with the no difference. According to comparison of performance group rats were statistically significant differences between the height (P0.001) (n=61 (control); n=58 (CUMS); n=83 (CUMS+fluoxetine)). Conclusions: 1. in chronic pain and chronic stress (CUMS) of two kinds of rat model of depression induced in the ventral tegmental area dopamine (VTA) these electric signals are changed. The research suggests that chronic stress (or pain stimulation) in VTA brain dopaminergic neuronal excitability changes in rat model has a strong correlation between.2. and chronic pain induced depression and depression in the camp hyperpolarization activated ion channel protein (HCN) expression high profile. Because HCN play an important role in regulating the excitability of neurons in this study suggest that chronic pain induced by depression, VTA and HCN high expression of dopaminergic neurons, causing changes in neuronal excitability Relevant.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.4

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