Brain dopamine alterations in human cocaine users

描述(由申请人提供)：人类使用者的大脑多巴胺神经元似乎因长期接触可卡因而受到损害。在我们检测的身体标本中，纹状体VMAT2结合位点、VMAT2 IR和多巴胺浓度以及中脑黑化神经元和DAT mRNA都减少了。人类与可卡因相关的神经毒性可能会导致奖励体验紊乱，以及特定的症状，如渴望和抑郁。第一个目标是确认人类可卡因吸毒者中脑中多巴胺神经元的丢失，与纹状体多巴胺能纤维的丢失平行。第一个具体目标是测试人类可卡因吸毒者致密部前背层多巴胺神经元丢失的假说。将使用体视学方法和自然产生的黑色素包涵体和酪氨酸羟基酶(TH)免疫组织化学染色对现有标本中四个AP水平的主要多巴胺核中的多巴胺细胞数量进行定量，以识别多巴胺细胞。将对GABA神经元进行评估，以评估可卡因效应的特异性。VMAT2减少可能是可卡因诱导的VMAT2蛋白或神经递质囊泡的调节所致。第二个目标是阐明人类可卡因使用者是否经历了多巴胺对神经递质囊泡水平的特定调节。具体目的#2是测试假设，即囊泡相关蛋白synaptopagmin 1减少，而突触相关蛋白synaxin不减少，特别是在多巴胺神经元中。VMAT2、Synaxin和Synaptopagmin 1mRNA水平将在中脑区域定量，而纹状体中Synaptopagmin亚型和其他突触蛋白水平的进一步特征将被评估。多巴胺神经元有特定的、按地形组织的投射，这些投射服从于不同的运动和边缘功能。目标3是更好地了解可卡因吸毒者多巴胺功能受影响的解剖学范围，并确定是否存在区域模式。与多巴胺标志物不同的是，纹状体多巴胺转运体(DAT)功能增强，可能与长期接触可卡因的毒性作用有关。具体目标#3将测试这一假设，即可卡因使用者同时表现出纹状体VMAT2结合位点减少和DAT结合增加的共同变化模式，其中在前腹内侧纹状体最为强烈。DATNMAT2在杏仁核中的表达也将被检测到，这些核团服务于奖励/情绪功能。甲基苯丙胺对小鼠的毒性似乎涉及过量的钠流入多巴胺神经元，导致能量酶(COX-L)和钠交换酶(NHE-1)的增加，这两种酶在六名可卡因使用者中都有所增加。目标4是确认这些增加，并确定相关基因是否在人类可卡因使用者中发生诱导。具体目的#4是验证在多巴胺神经元中COX-1、NHE-1和相关的mRNA增加的假说。

DESCRIPTION (provided by applicant): Brain dopamine neurons appear to be injured by chronic cocaine exposure in human users. Striatal VMAT2 binding sites, VMAT2 IR, and dopamine concentrations, as well as midbrain melanized neurons and DAT mRNA are decreased in post mortem human samples that we have examined. Cocaine-related neurotoxicity in humans could contribute to disordered reward experience, and specific symptoms such as craving and depression. Goal #1 is to confirm that dopamine neurons are lost in midbrain, paralleled by loss of striatal dopaminergic fibers, in human cocaine users. Specific Aim #1 is to test the hypotheses that dopamine neurons are lost in the anterior dorsal tier of pars compacta of human cocaine users. Dopamine cell numbers will be quantitated in major dopamine nuclei at four AP levels in available specimens, using stereological methods and naturally occurring melanin inclusions and tyrosine hydroxylase (TH) immunohistochemical staining to identify dopamine cells. GABA neurons will be evaluated to assess the specificity of the cocaine effect. VMAT2 decreases could result from cocaine-induced regulation of VMAT2 protein, or neurotransmitter vesicles. Goal #2 is to clarify if human cocaine users experience dopamine-specific regulation of neurotransmitter vesicle levels. Specific Aim #2 is to test the hypotheses that the vesicle-associated protein, synaptotagmin 1, decreases, while the synapse-associated protein, syntaxin, does not, specifically in dopamine neurons. VMAT2, syntaxin, and synaptotagmin 1 mRNA levels will be quantitated in midbrain regions, while further characterization of synaptotagmin subtypes and other synaptic protein levels will be assessed in striatum. Dopamine neurons have specific, topographically organized projections that subserve distinct motor versus limbic functions. Goal #3 is to better understand the anatomical extent that dopamine function is affected in cocaine users and to determine if regional pattems exist in stdatum. Distinct among dopamine markers, striatal dopamine transporter (DAT) function is increased, and could be related to the toxic effects of long-term cocaine exposure. Specific Aim #3 will test the hypothesis that cocaine users display both decreased striatal VMAT2 binding sites and increased DAT binding in a co-varying pattern that is most intense in anterior ventromedial striatum. DATNMAT2 expression in amygdalar nuclei subserving reward/emotional functions will also be examined. Methamphetamine toxicity in mice appears to involve excessive sodium influx into dopamine neurons, inducing increases in energetic (COX-l) and sodium exchange (NHE-1) enzymes, which were both increased in six cocaine users. Goal #4 is to confirm these increases and determine if induction of related genes occurs in human cocaine users. Specific Aim # 4 is to test the hypotheses that COX-1, NHE-1 and related mRNA's are increased in dopamine neurons.