A new brain dopamine-deficient Drosophila and its pharmacological and genetic rescue.
A new brain dopamine-deficient Drosophila and its pharmacological and genetic rescue.
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DOI：
10.1111/gbb.12353
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发表时间：
2017-03
期刊：
影响因子：
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通讯作者：
Hirsh J
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文献类型：
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作者：
Cichewicz K;Garren EJ;Adiele C;Aso Y;Wang Z;Wu M;Birman S;Rubin GM;Hirsh J
多巴胺(DA)是一种在无脊椎动物和脊椎动物之间具有保守行为作用的神经递质。除了它的神经功能，在昆虫中，DA是角质层着色和硬化的关键底物。果蝇酪氨酸羟化酶(DTH)是DA生物合成的限速酶。先前使用组织选择性GAL4-UAS二元表达挽救DTH零突变来产生可存活的脑DA缺陷果蝇，这些果蝇表现出特定的行为损伤。为了克服通过二元表达进行救援的局限性，我们利用基因组整合的突变体DTH实现了救援。正如预期的那样，我们的DA缺乏的果蝇在大脑中没有检测到DTH或DA，并表现出运动活动减少。这种缺陷可以通过L-多巴/卡比多巴喂养来挽救，类似于人类帕金森氏症的治疗。通过GAL4/UAS-DTH的遗传挽救也是成功的，尽管这需要产生一个新的UAS-DTH1转基因，没有大多数未翻译的区域，因为现有的UAS-DTH转基因在没有Gal4驱动的情况下通过内源性调节元件在大脑中表达。我们新构建的UAS-DTH1m的一个令人惊讶的发现是，即使在完全拯救DA的情况下，它也在多巴胺能GAL4驱动程序的调控下以不可检测的水平表达DTH，表明DTH免疫染色不一定是DA表达的有效标记。这一发现需要优化DA免疫组织化学，揭示DA对蘑菇体和中央复合体的神经支配的细节。当DA救援仅限于特定的DA神经元时，DA不会扩散到DTH表达终末之外，因此DA信号可以被限制在非常特定的脑区。
Dopamine (DA) is a neurotransmitter with conserved behavioral roles between invertebrate and vertebrate animals. In addition to its neural functions, in insects DA is a critical substrate for cuticle pigmentation and hardening. Drosophila tyrosine hydroxylase (DTH) is the rate limiting enzyme for DA biosynthesis. Viable brain DA deficient flies were previously generated using tissue selective GAL4-UAS binary expression rescue of a DTH null mutation and these flies show specific behavioral impairments. To circumvent the limitations of rescue via binary expression, here we achieve rescue utilizing genomically integrated mutant DTH. As expected, our DA deficient flies have no detectable DTH or DA in the brain, and show reduced locomotor activity. This deficit can be rescued by L-DOPA/carbidopa feeding, similar to human Parkinson’s disease treatment. Genetic rescue via GAL4/UAS-DTH was also successful, although this required the generation of a new UAS-DTH1 transgene devoid of most untranslated regions, since existing UAS-DTH transgenes express in the brain without a Gal4 driver via endogenous regulatory elements. A surprising finding of our newly constructed UAS-DTH1m is that it expresses DTH at an undetectable level when regulated by dopaminergic GAL4 drivers even when fully rescuing DA, indicating that DTH immunostaining is not necessarily a valid marker for DA expression. This finding necessitated optimizing DA immunohistochemistry, revealing details of DA innervation to the mushroom body and the central complex. When DA rescue is limited to specific DA neurons, DA does not diffuse beyond the DTH-expressing terminals, such that DA signaling can be limited to very specific brain regions.