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Using chronoamperometry to investigate serotonin and dopamine transporter function.

机译：使用计时电流法研究5-羟色胺和多巴胺转运蛋白的功能。

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摘要

The global hypothesis of this thesis is that modest but biologically important alterations in neurotransmission underlie phenotypic alterations in mice (and humans) with altered gene expression. Chronoamperometry with carbon fiber microelectrodes was utilized to investigate changes in serotonin and dopamine uptake in SERT knockout mice and in mutant human A53T alpha-synuclein transgenic mice. Four primary findings are described in detail. The first is that partial reductions in SERT expression lead to similar magnitude decreases in SERT function in mice, similar to what has been shown to occur in humans with differential SERT expression. Characterization of uptake by chronoamperometry in neurosynaptosomes demonstrated that transport of serotonin was enhanced 7-fold in the presence of oxygen and abolished when synaptosomes were stirred. The results indicated that 50% reductions in SERT expression in SERT +/- mice resulted in 60% decreases in serotonin uptake rates measured in brain stem, frontal cortex and striatum compared to SERT +/+ mice. Uptake of serotonin was not detected in SERT-/- mice in any of these brain regions. These findings contradict those from previous radiochemical assay experiments in which differential uptake was not detected between SERT+/+ and SERT+/- mice.; The second main finding addresses the lack of sensitivity of radiochemical methods for detecting differential changes in transporter function in mice with partial reductions in SERT expression. The data indicate that absence of oxygen in the incubation medium and most importantly, the disruption of synaptosomal plasma membranes during the filtration step used in radiochemical assays together contribute to low uptake rates and the poor resolution of this method. Specifically, uptake rates measured by radiochemical methods are 200-fold lower than those determined by chronoamperometry even in the presence of oxygen. In addition, differential uptake rates between SERT +/+ and SERT+/- mice were only observed in frontal cortex and striatum but not in brain stem synaptosomes after oxygenation of the incubation medium in radiochemical uptake experiments. Most striking, the results indicate that the filtration of synaptosomal suspensions results in a 75% loss of serotonin taken up contributing to the lower uptake rates calculated by this method and to its poor ability to detect differential uptake rates between SERT+/+ and SERT+/- mice.; The third major finding is that reductions in SERT expression and function do not lead to neuroadaptive changes in dopamine transporter expression and function. Furthermore, we did not find evidence for promiscuous uptake of dopamine by the SERT in wildtype mice. Thus, behavioral changes in SERT knockout mice appear to be a direct consequence of alterations in serotonergic neurotransmission in SERT knockout mice.; Finally, the results of experiments in alpha-synuclein transgenic mice demonstrate that expression of A53T mutant human alpha-synuclein leads to reduced dopamine uptake rates in striatum. These alterations in dopamine neurotransmission are associated with adult onset locomotor hyperactivity in A53T alpha-synuclein transgenic mice.
机译：该论文的全球假设是，神经传递的适度但生物学上重要的改变是基因表达改变的小鼠（和人类）的表型改变的基础。碳纤维微电极计时安培法用于研究SERT基因敲除小鼠和突变型人A53Tα-突触核蛋白转基因小鼠中5-羟色胺和多巴胺摄取的变化。详细描述了四个主要发现。首先是SERT表达的部分降低会导致小鼠SERT功能的下降幅度相似，这与具有差异SERT表达的人的情况相似。通过计时安培法测量神经突触小体的摄取特征表明，在有氧存在下，血清素的转运增强了7倍，而当搅拌突触小体时，5-羟色胺的转运被取消了。结果表明，与SERT + / +小鼠相比，SERT +/-小鼠中SERT表达的降低50％导致脑干，额叶皮层和纹状体中血清素摄取率降低了60％。在任何这些大脑区域的SERT-/-小鼠中均未检测到血清素的摄取。这些发现与先前的放射化学分析实验相反，在先前的放射化学分析实验中，在SERT + / +和SERT +/-小鼠之间未检测到差异摄取。第二项主要发现解决了放射化学方法缺乏灵敏性的问题，该方法无法检测SERT表达部分降低的小鼠转运蛋白功能的差异。数据表明温育培养基中没有氧气，最重要的是，放射化学分析所用的过滤步骤中突触体质膜的破坏共同导致了该方法的低吸收率和较差的分辨率。具体而言，即使在有氧存在的情况下，通过放射化学方法测得的摄取率也比通过计时安培法测定的摄取率低200倍。此外，在放射化学吸收实验中将培养液充氧后，仅在额叶皮层和纹状体中观察到SERT + / +和SERT +/-小鼠之间的差异摄取率，而在脑干突触体中未观察到。最惊人的是，结果表明突触体悬浮液的过滤导致所吸收的5-羟色胺损失了75％，这导致该方法计算出的摄取率降低，并且无法检测出SERT + / +和SERT +/-之间的差异摄取率。老鼠。;第三个主要发现是，SERT表达和功能的降低不会导致多巴胺转运蛋白表达和功能的神经适应性改变。此外，我们没有发现野生型小鼠中SERT混杂摄取多巴胺的证据。因此，SERT基因敲除小鼠的行为改变似乎是SERT基因敲除小鼠血清神经能神经传递改变的直接结果。最后，在α-突触核蛋白转基因小鼠中的实验结果表明，A53T突变型人α-突触核蛋白的表达导致纹状体中多巴胺摄取率降低。多巴胺神经传递的这些改变与A53Tα-突触核蛋白转基因小鼠中的成年发作的运动过度活跃有关。

著录项

  • 作者

    Perez, Xiomara A.;

  • 作者单位

    The Pennsylvania State University.;

  • 授予单位 The Pennsylvania State University.;
  • 学科 Chemistry Analytical.; Biology Neuroscience.; Health Sciences Pharmacology.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 233 p.
  • 总页数 233
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 化学;神经科学;药理学;
  • 关键词

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