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慢性可卡因暴露而受损。纹状体VMAT 2结合位点，VMAT 2 IR，多巴胺浓度，以及中脑黑色素化神经元和DAT mRNA的减少，我们已经检查了死后的人类样本。可卡因相关的人类神经毒性可能会导致奖励体验紊乱，以及特定症状，如渴望和抑郁。目标#1是证实人类可卡因使用者中脑中的多巴胺神经元丢失，这是由于纹状体多巴胺能纤维丢失。具体目标#1是测试假设，多巴胺神经元丢失的前背层的人类可卡因用户的pars延髓。将使用体视学方法和天然存在的黑色素内含物和酪氨酸羟化酶（TH）免疫组织化学染色，在可用标本的4个AP水平下定量测定主要多巴胺核中的多巴胺细胞数量，以识别多巴胺细胞。将评价GABA神经元以评估可卡因作用的特异性。VMAT 2的减少可能是由于可卡因诱导的VMAT 2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神经递质囊泡水平的多巴胺特异性调节。具体目标#2是测试的假设，囊泡相关蛋白，synaptotagmin 1，减少，而突触相关蛋白，syntaxin，没有，特别是在多巴胺神经元。将在中脑区域定量VMAT 2、突触融合蛋白和突触结合蛋白1 mRNA水平，而将在纹状体中评估突触结合蛋白亚型和其他突触蛋白水平的进一步表征。多巴胺神经元具有特定的、拓扑学上有组织的投射，这些投射有助于不同的运动功能与边缘功能。目标#3是更好地了解可卡因使用者多巴胺功能受影响的解剖程度，并确定是否存在局部模式。与多巴胺标记物不同，纹状体多巴胺转运体（DAT）功能增加，可能与长期接触可卡因的毒性作用有关。具体目标#3将检验可卡因使用者以共变模式显示纹状体VMAT 2结合位点减少和DAT结合增加的假设，其中在前腹内侧纹状体中最强烈。还将检查杏仁核中促进奖励/情感功能的DATNMAT 2表达。小鼠中的甲基苯丙胺毒性似乎涉及过量的钠流入多巴胺神经元，诱导能量（COX-1）和钠交换（NHE-1）酶的增加，这两者在6名可卡因使用者中均增加。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。具体目标#4是检验考克斯-1、NHE-1和相关mRNA在多巴胺神经元中增加的假设。

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.