Kaneko M, Park J H, Cheng Y, Hardin P E, Hall J C
Department of Biology, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
J Neurobiol. 2000 Jun 5;43(3):207-33. doi: 10.1002/(sici)1097-4695(20000605)43:3<207::aid-neu1>3.0.co;2-0.
To study the function of clock-gene-expressing neurons, the tetanus-toxin light chain (TeTxLC), which blocks chemical synaptic transmission, was expressed under the control of promoters of the clock genes period (per) and timeless (tim), each fused to GAL4-encoding sequences. Although TeTxLC did not affect cycling of a clock-gene product at the gross level, it disrupted the rhythmic behavior of adult Drosophila. In constant darkness, the proportion of rhythmic flies was reduced in flies expressing active TeTxLC compared to controls, including those expressing inactive toxin. The behavior of TeTxLC-expressing flies was less synchronized to light:dark cycles than that of controls. To determine which neurons are responsible for these effects on behavior, the toxin was also expressed in restricted subsets of per/tim-expressing, laterally located pacemaker neurons by expressing TeTxLC under the control of a driver in which GAL4-encoding sequences are fused to the promoter of the pigment dispersing factor (pdf) gene. pdf-gal4-driven TeTxLC expression had relatively little effect on behavioral rhythms, implying that per/tim neurons other than pdf-expressing lateral neurons participate in the generation of rhythmic behavior. In another set of experiments, period gene products were expressed under the control of per-gal4 or tim-gal4. This resulted in an increased level of PER protein in many brain cells and reduction of bioluminescence cycling reported by a per-luciferase transgene, especially in the case of per expression affected by tim-gal4. This indicates a disruption of the transcriptional feedback loop that is a part of the oscillatory mechanism underlying Drosophila's circadian rhythms. Consistent with this molecular defect, the proportion of rhythmic individuals in constant darkness was subnormal in flies expressing PER under the control of tim-gal4, and their behavior in light:dark cycles was abnormal.
为了研究表达生物钟基因的神经元的功能，破伤风毒素轻链（TeTxLC），它能阻断化学突触传递，在生物钟基因周期（per）和无时间性（tim）的启动子控制下表达，每个启动子都与编码GAL4的序列融合。尽管TeTxLC在总体水平上不影响生物钟基因产物的循环，但它扰乱了成年果蝇的节律行为。在持续黑暗中，与对照组相比，包括那些表达无活性毒素的果蝇，表达活性TeTxLC的果蝇中节律性果蝇的比例降低。表达TeTxLC的果蝇的行为与明暗周期的同步性比对照组差。为了确定哪些神经元对这些行为效应负责，通过在一种驱动子的控制下表达TeTxLC，使毒素也在表达per/tim的、位于侧面的起搏器神经元的特定亚群中表达，在该驱动子中，编码GAL4的序列与色素分散因子（pdf）基因的启动子融合。pdf-gal4驱动的TeTxLC表达对行为节律的影响相对较小，这意味着除了表达pdf的侧面神经元之外的per/tim神经元参与了节律行为的产生。在另一组实验中，周期基因产物在per-gal4或tim-gal4的控制下表达。这导致许多脑细胞中PER蛋白水平升高，并且per-荧光素酶转基因报告的生物发光循环减少，特别是在tim-gal4影响per表达的情况下。这表明转录反馈环受到破坏，而转录反馈环是果蝇昼夜节律振荡机制的一部分。与这种分子缺陷一致，在tim-gal4控制下表达PER的果蝇在持续黑暗中节律性个体的比例低于正常水平，并且它们在明暗周期中的行为也不正常。
