Brain dopamine alterations in human cocaine users

描述（由申请人提供）：人类使用者的脑多巴胺神经元似乎因长期暴露于可卡因而受损。纹状体VMAT2结合位点、VMAT2 IR和多巴胺浓度，以及中脑黑色素化神经元和DAT mRNA在我们研究的死后人类样本中都减少了。人类与可卡因相关的神经毒性可能导致奖赏体验紊乱，以及诸如渴望和抑郁等特定症状。目标1是确认可卡因使用者中脑多巴胺神经元的丢失，同时纹状体多巴胺能纤维的丢失。具体目标#1是测试多巴胺神经元在人类可卡因使用者的紧脑部前背层丢失的假设。利用体视学方法、天然黑色素包涵体和酪氨酸羟化酶（TH）免疫组化染色来鉴定多巴胺细胞，定量测定可用标本中主要多巴胺核中四个AP水平的多巴胺细胞数量。GABA神经元将被评估以评估可卡因效应的特异性。VMAT2的减少可能是由于可卡因诱导的VMAT2蛋白或神经递质囊泡的调节。目标2是澄清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异性调节。具体目标#2是测试假设，即囊泡相关蛋白突触塔蛋白1减少，而突触相关蛋白syntaxin没有，特别是在多巴胺神经元中。VMAT2、syntaxin和synaptotagmin 1 mRNA水平将在中脑区域被量化，而进一步表征synaptotagmin亚型和其他突触蛋白水平将在纹状体中被评估。多巴胺神经元具有特定的、有地形组织的投射，服务于不同的运动和边缘功能。目标3是更好地了解可卡因使用者多巴胺功能受到影响的解剖学程度，并确定在正常状态下是否存在区域模式。与多巴胺标志物不同，纹状体多巴胺转运体（DAT）功能增加，可能与长期可卡因暴露的毒性作用有关。特异性目标#3将验证可卡因使用者在纹状体VMAT2结合位点减少和DAT结合位点增加的共同变化模式下的假设，这种模式在前腹内侧纹状体中最为强烈。DATNMAT2在杏仁核中表达的奖励/情绪功能也将被检查。甲基苯丙胺对小鼠的毒性似乎与过量的钠流入多巴胺神经元有关，导致能量（cox -1）和钠交换（NHE-1）酶的增加，这在6名可卡因使用者中都有所增加。目标4是确认这些增加，并确定相关基因的诱导是否发生在人类可卡因使用者中。具体目的# 4是验证多巴胺神经元中COX-1、NHE-1和相关mRNA增加的假设。

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.