McDonald Paul W, Hardie Shannon L, Jessen Tammy N, Carvelli Lucia, Matthies Dawn Signor, Blakely Randy D
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-8548, USA.
J Neurosci. 2007 Dec 19;27(51):14216-27. doi: 10.1523/JNEUROSCI.2992-07.2007.
The catecholamine dopamine (DA) functions as a powerful modulatory neurotransmitter in both invertebrates and vertebrates. As in man, DA neurons in the nematode Caenorhabditis elegans express a cocaine-sensitive transporter (DAT-1), presumably to regulate synaptic DA signaling and limit DA spillover to extrasynaptic sites, although evidence supporting this is currently lacking. In this report, we describe and validate a novel and readily quantifiable phenotype, swimming-induced paralysis (SWIP) that emerges in DAT-1-deficient nematodes when animals exert maximal physical activity in water. We verify the dependence of SWIP on DA biosynthesis, vesicular packaging, synaptic release, and on the DA receptor DOP-3. Using DAT-1 specific antibodies and GFP::DAT-1 fusions, we demonstrate a synaptic enrichment of DAT-1 that is achieved independently of synaptic targeting of the vesicular monoamine transporter (VMAT). Importantly, dat-1 deletions and point mutations that disrupt DA uptake in cultured C. elegans neurons and/or impact DAT-1 synaptic localization in vivo generate SWIP. SWIP assays, along with in vivo imaging of wild-type and mutant GFP::DAT-1 fusions identify a distal COOH terminal segment of the transporter as essential for efficient somatic export, synaptic localization and in vivo DA clearance. Our studies provide the first description of behavioral perturbations arising from altered trafficking of DATs in vivo in any organism and support a model whereby endogenous DA actions in C. elegans are tightly regulated by synaptic DAT-1.
儿茶酚胺多巴胺（DA）在无脊椎动物和脊椎动物中均作为一种强大的调节性神经递质发挥作用。与人类一样，线虫秀丽隐杆线虫中的DA神经元表达一种对可卡因敏感的转运体（DAT-1），推测其作用是调节突触DA信号传导并限制DA扩散到突触外位点，不过目前尚缺乏支持这一观点的证据。在本报告中，我们描述并验证了一种新的且易于量化的表型——游泳诱导性麻痹（SWIP），该表型出现在缺乏DAT-1的线虫中，当动物在水中进行最大程度的体力活动时就会出现。我们证实了SWIP对DA生物合成、囊泡包装、突触释放以及DA受体DOP-3的依赖性。使用DAT-1特异性抗体和GFP::DAT-1融合蛋白，我们证明了DAT-1在突触处的富集，这一过程独立于囊泡单胺转运体（VMAT）的突触靶向作用而实现。重要的是，破坏培养的秀丽隐杆线虫神经元中DA摄取和/或影响体内DAT-1突触定位的dat-1缺失和点突变会产生SWIP。SWIP分析以及野生型和突变型GFP::DAT-1融合蛋白的体内成像表明，转运体的一个远端COOH末端片段对于有效的体细胞输出、突触定位和体内DA清除至关重要。我们的研究首次描述了任何生物体中DATs体内转运改变所引起的行为扰动，并支持了一种模型，即秀丽隐杆线虫中内源性DA的作用受到突触DAT-1的严格调控。
