摘要
研究背景：内源性大麻素系统包括大麻素受体(cannabinoid receptor, CB receptor)、内源性大麻素(endogenous cannabinoids, eCB)和与eCB合成、降解相关的酶类。目前已被证实的内源性大麻素主要有两种：N-花生四烯酸氨基乙醇(Arachidonoylethanolamine, AEA)和2-花生四烯酸甘油(2-AG),它们均具有与天然大麻素四氢大麻酚(THC)极为相似的三维结构。
外源性和内源性大麻素都是通过与大麻素受体结合发挥其生物学作用。目前证实体内存在两种大麻素受体：CB1受体和CB2受体。大麻素受体是G蛋白耦联受体,基因克隆研究发现这两种受体有44%的氨基酸序列同源。
CB2受体主要分布于外周免疫细胞,如脾脏边缘区、免疫细胞、扁桃体等,CB2功能主要与免疫调节有关。
CB1受体主要分布于脑、脊髓和外周神经系统中,脑内CB1受体主要分布于基底神经节(黑质、苍白球、外侧纹状体)、海马CA1区锥体细胞层,小脑和大脑皮层。CB1受体的这种分布可能与大麻素对记忆、认知、运动控制的调节功能有关：此外,在延髓、中脑导水管周围、脊髓后角等疼痛传导径路也有大量CB1受体分布。外周神经系统比中枢神经系统表达的CB1受体量少,它们分散地表达于诸如神经末梢等处,介导神经递质的释放。最近大量的研究数据提示,中枢神经系统内源性大麻素系统信号转导功能的失调有可能是抑郁症产生的原因。
伏隔核也称伏核,是基底前脑的一个较大的核团,位于基底核与边缘系统交界处,隔区的外下方,尾壳核的内下方,前方与嗅前核相连,后连接纹状核。伏隔核于周边核团投射关系比较复杂,其纤维联系与边缘系统较为密切,主要接受前脑的传入纤维,包括内侧前额皮质、杏仁核、海马和下丘脑；也接受脑桥区的投射纤维,包括腹侧被盖区、背侧中缝、脑桥网状结构腹侧为腹侧苍白球和嗅结节,是奖赏环路中一个重要的组成部分。最近研究发现,伏核功能的异常可能与兴趣缺失以及抑郁症其他症状的产生相关。研究目的：应用慢性非预期性应激(chronic unpredictable stress,CUS)的小鼠抑郁模型进行研究,探讨伏核区域内源性大麻素系统信号传递的改变是否是引起抑郁行为学改变的基础。
研究方法：
(1)经过6-8周的慢性非预期性应激(CUS)建立小鼠抑郁模型,同时观察1周的次慢性非预期性应激(sub-CUS)对实验动物的影响。
(2)抑郁模型完成后,进行与抑郁症相关的动物行为学实验检测,包括蔗糖水摄入实验、旷场实验、新环境抑制摄食实验和强迫游泳实验。
(3)行为学实验完成后,处死动物分离胸腺和肾上腺并称重,评估慢性非预期性应激对免疫系统和下丘脑-垂体-肾上腺(HPA)轴功能的影响；取血,应用酶联免疫吸附方法进行血清皮质酮含量测定；取脑并分离纹状体,应用高效液相色谱(HPLC)法对脑组织内AEA和2-AG含量进行测定。
(4)动物处死后制备脑片(brain slice),使用电生理设备记录CB1受体介导的神经突触可塑性变化的电生理指标,包括细胞内记录Depolarization-induced suppression of excitation (DSE),细胞外记录长时程抑制(LTD)和Ⅰ型代谢型谷氨酸受体(groupⅠmGluR)激动剂DHPG对场-兴奋性突触后电位(fEPSP)的抑制。
(5)在脑片上应用CB1受体激动剂WIN 55,212-2,细胞外记录CB1受体激动剂对场-兴奋性突触后电位(fEPSP)的抑制作用,比较应激组与非应激组之间CB1受体功能是否发生改变。
结果：
(1)给予慢性非预期性应激6周后,小鼠表现出抑郁样行为学改变,包括摄入蔗糖水绝对量和相对量减少,旷场实验自主活动性降低和中央停留时间缩短,新环境摄食潜伏期延长,强迫游泳实验不动时间延长。而次慢性非预期性应激(给予应激1周)后的动物没有出现明显的行为学改变。
(2)在脑片的伏核核心区(Nac core)记录兴奋性突触后电流(EPSCs),在单一使用去极化刺激条件下,无论是通过提高去极化程度(从-80mV到10或20mV)还是延长去极化时间(1s,5s和10s)都无法成功诱导DSE。在细胞外液中加入Ⅰ型mGluR的激动剂DHPG(5μM)后,从-80mV去极化到0mV持续5s可以成功诱导DSE。
(3)使用以上方法在伏核核心区域诱导DSE,慢性非预期性应激与对照组相比诱导出DSE的幅度降低,差异有统计学意义,而DSE的持续时间无统计学差异。而次慢性非预期性应激组诱导DSE无论在幅度还是持续时间上与对照组相比都无统计学差异。
(4)在伏核核心区域诱导内源性大麻素系统介导的LTD,次慢性非预期性应激组与对照组相比LTD幅度无统计学差异,而慢性非预期性应激影响LTD的形成。
(5)应用抗抑郁药氟西汀(Fluoxetine)治疗对于慢性非预期性应激造成的行为学异常和内源性大麻素系统信号的传递受损都有改善作用。
结论：研究结果表明,慢性非预期性应激可以导致实验动物抑郁样行为改变和伏核区域eCB/CB1受体介导的信号转导受损,次慢性应激则没有以上作用；应用抗抑郁药物氟西汀可以逆转慢性应激造成的改变。以上结果提示慢性非预期型应激抑郁模型动物伏核区域的内源性大麻素系统信号转导功能受损。
意义：研究慢性应激导致的内源性大麻素系统功能异常可以加深我们对抑郁症发病机制的认识,促进抗抑郁治方案的优化,同时为抗抑郁药物的研发提供了新的靶点。
研究背景：人类吸食大麻(Cannabinoids,CB)的历史已经有一千多年,大麻中的主要有效成分是四氢大麻酚(THC)。人类应用大麻类物质可以产生精神兴奋、改善抑郁症状的作用,在动物实验中应用大麻类物质以及大麻素受体1(CB1 receptor)激动剂可以产生抗抑郁样作用,而治疗肥胖症的药物CB1受体拮抗剂利莫那班(Rimonabant)在临床实验中则增加患者焦虑和抑郁的发生率。因此,调节内源性大麻素系统(eCB system)的功能有可能是治疗抑郁症的一种新的有效方法。直接的CB1受体激动剂可以增强内源性大麻素系统的功能,但其缺陷是CB1受体激动剂无差别的广泛作用于全部CB1受体。通过选择性的阻断内源性大麻素物质降解而达到增强内源性大麻素系统的功能则可以克服以上缺点,在增强eCB功能的同时保证在正常生理状态下CB1受体激动的部位和持续时间。脑内最主要的两种eCB配体AEA和2-AG分别通过脂肪酰胺水解酶(FAAH)和单酰基甘油脂酶(MAGL)降解。有研究表明FAAH和AEA运输的抑制剂可以对啮齿类动物产生抗焦虑和抗抑郁的作用,然而对MAGL的抑制是否能产生相似的作用仍然未知。研究目的：JZL184是新合成的一种高选择性MAGL抑制剂,可以选择性的抑制脑内MAGL,增加2-AG含量。通过对慢性非预期性应激小鼠长期应用JZL184,探讨对中枢神经系统内MAGL的抑制是否可以产生抗抑郁作用。
研究方法：
(1)经过6-8周的慢性非预期性刺激(CUS)建立小鼠抑郁模型。
(2)抑郁模型完成后,进行相关的动物行为学实验检测,包括旷场实验,新鲜环境抑制摄食实验和强迫游泳实验。
(3)用免疫组织化学方法测定各组海马齿状回(DG) BrdU阳性和DCX阳性新生神经元数量。
(4)记录DG区域神经突触的基本电生理特征如paired-pluse ratio(PPR), input-output(I/O)relationships,诱导新生神经元依赖性LTP。
(5)对小鼠进行条件性位置偏爱(conditioned place preference,CPP)实验,评价治疗剂量下(8mg/kg)小鼠有无对JZL184产生依赖。
结果：
(1)慢性非预期性应激后小鼠产生抑郁样行为学改变,在体内应用MAGL抑制剂JZL184后产生抗抑郁样行为学效应。
(2)慢性非预期性应激后DG区域的BrdU阳性和DCX阳性细胞与对照组相比明显减少,应用JZL184治疗后,新生神经元的数量增加。
(3)慢性非预期性应激和JZL治疗对PPR无显著影响。
(4)慢性非预期性应激降低I/O曲线的斜率,JZL184治疗后I/O曲线斜率恢复。
(5)慢性非预期性应激后,DG新生神经元依赖性LTP受损,经JZL184治疗后LTP得到改善。CUS和JZL184治疗都未影响非新生神经元依赖性LTP。
(6)CPP实验表明使用JZL184 8mg/kg的剂量对小鼠无成瘾效应。
结论：体内应用MAGL抑制剂JZL184治疗慢性非预期性应激抑郁小鼠产生抗抑郁样效应,并且增加了海马新生神经元数量,增强海马新生神经元依赖的LTP,表明MAGL抑制剂可以通过增加海马神经发生产生抗抑郁作用。
Background Endocannabinoid system include cannabinoid receptors, endocannabinoids and the enzymes that involve in the synthesise and the hydrolyse the endocannabinoids.
There are two main endocannabinoids that have been identified clearly, arachidonoylethanolamine (anandemide or AEA) and 2-AG, they are similar in molecular structure with terahydrocannabinol(THC), the natural component of Marijuanan.
There are currently two known subtypes, termed CB1and CB2. The CB1 receptor is expressed mainly in the brain (central nervous system, CNS), but also in the lungs, liver and kidneys. The CB2 receptor is mainly expressed in the immune system and in hematopoietic cells. The protein sequences of CB1and CB2 receptors are about 44% similar. When only the transmembrane regions of the receptors are considered, amino acid similarity between the two receptor subtypes is approximately 68%.
The nucleus accumbens (NAc) is a critical component of the reward circuitry and dysfunction of the NAc may account for anhedonia and other symptoms of depression. The NAc is an important anatomical substrate for motivation and reward and dysfunction of the NAc could account for anhedonia, decreased sex drive, social withdrawal, and other symptoms of depression. The present study has identified that alterations of eCB signaling might occur in the NAc in depression.
Objective To investigated whether alterations in endocannabinoid (eCB) signaling in the NAc contribute to depression-like behaviors induced by chronic unpredictable stress (CUS) in mice.
Method
1. Male C57BL/6J mice (8-10 weeks of age) were subjected to chronic mild unpredictable stress (CUS) for a totle of 8-10 week to establish the depression model.
2. After the CUS procedure, mice were subject to a batter of behavior test to evaluate the state of depression. The behavior test include sucrose-intake test open field test (OPT), novelty suppressed feeding (NSF) and forced swimming test.
3. Control and CUS-exposed mice were anesthetized by isoflurane inhalation and decapitated and the brains were removed immediately and frozen by dry ice, the striatum were disseccted. Trunk blood was collected, and adrenals and thymus were excised and weighed immediately. Serum corticosterone concentrations were determined by enzyme-linked immunosorbent assay kit. Brain AEA and 2-AG tissue contents were determined using isotope-dilution liquid chromatography electrospray ionization tandem mass spectrometry.
4. Striatal slices containing the NAc (250μm thick) were prepared and three types of eCB/CB1 receptor-mediated synaptic plasticity were compared, including depolarization-induced suppression of excitation (DSE), long-term depression (LTD), and the depression of field excitatory postsynaptic potentials (fEPSPs) induced by group I metabotropic glutamate receptor agonist DHPG and the effect of CB1 receptor agonist WIN 55,212-2.
Result CUS (5-6 week exposure to stressors), but not sub-CUS (1 week exposure to stressors), induces depression-like behaviors and impairs these forms of eCB/CB1 receptor-mediated plasticity examined in the NAc core. Neither sub-CUS nor CUS altered the tissue contents of the eCBs anandamide and 2-arachidonoylglycerol in the striatum. However, exposure to CUS, but not sub-CUS, attenuated the depression of fEPSPs induced by the CB1 receptor agonist WIN 55,212-2. CUS exposure reduced the maximal effect without affecting the EC50 of WIN 55,212-2 to induce fEPSP depression. Thus, impaired CB1 receptor function could account for CUS-induced deficiency in eCB signaling in the NAc. Both CUS-induced deficiency in eCB signaling and depression-like behaviors were reversed by in vivo administration of antidepressant fluoxetine.
Conclusion These results suggest that down-regulation of the eCB signaling in the NAc occurs following CUS and contributes to the pathophysiology of depression.
Marijuana (cannabis) and its active constituentΔ9-tetrahydrocannabinol (THC) elevate mood and ease anxiety in humans and CB1 receptor agonists exert antidepressant-like behavioral effects in animals. Modulation of the endocannabinoid (eCB) system may represent a useful strategy for the treatment of depression. Inhibitors of eCB degradation amplify endogenous cannabinoid activity in a temporal- and spatial-specific manner and could be superior to direct-acting CB1 agonists as therapeutic agents. The eCB ligand 2-arachidonoylglycerol (2-AG) is inactivated by monoacylglycerol lipase (MAGL). However, whether MAGL inhibition produces antidepressant-like behavioral effects remains unknown. JZL184 is a recently developed selective MAGL inhibitor that increases 2-AG levels in the brain. We report that chronic in vivo administration of JZL184 produced antidepressant-like behavioral effects in a chronic unpredictable stress (CUS) model of depression in mice, and these effects were blocked by the CB1 antagonist rimonabant. Chronic JZL184 reversed CUS-induced decreases in the number of bromodeoxyuridine (BrdU)-labeled neural progenitor cells and doublecortin-positive immature neurons in the in the dentate gyrus of the hippocampus and facilitated a form of long-term potentiation (LTP) that is dependent on hippocampal neurogenesis. These results suggest that MAGL inhibition produces antidepressant-like effects by prompting hippocampal neurogenesis and that MAGL is a potential target for pharmacotherapy of depression.
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