Morphologycal analysses of the neural components of the input and output pathways involved in the Drosophila circadian system, with special reference to the cholinergic neurons.

在德拉索菲拉，各种证据表明，负责日常运动节律的昼夜节律起搏器驻留在位于延髓前皮的细胞中，称为“外侧神经元”(LNS)。然而，人们对将这些起搏细胞与行为节律联系起来的神经通路知之甚少。为了确定果蝇昼夜节律时钟系统的输入和输出通路的神经成分，我们使用免疫细胞化学技术观察了大脑，特别是参照胆碱能神经元。一种抗果蝇胆碱乙酰转移酶(ChAT)的单抗对复眼后缘的一组视网膜外光感受器进行了染色，这组光感受器被称为“小孔”。从小孔发出的轴突终止于前髓区域，可能是LNS的树突区所在。LN对抗果蝇nAChR抗体呈免疫反应，提示胆碱能传入LNS。这一间接证据表明…更多地在小孔处参与了有助于昼夜节律夹带的输入路径。携带含有1.2kb顺式调控序列的ChAT基因的转基因果蝇，在特定的胆碱能神经元亚群，如终末位于延髓远端的神经元中，ChAT的表达明显降低，LNS的输出纤维的曲张位于该区域。一项形态计量学研究显示，在转基因果蝇中，延髓中的LN静脉曲张明显大于野生型果蝇。转化子被带入光明：黑暗周期，并表现出早晚高峰的双峰运动模式，与对照苍蝇(白色)一样。当光照水平较低时(即0.24勒克斯)，转化子表现出不寻常的单相运动活动，仅发生在整个光期，与对照的双峰活动形成对比。这些观察结果表明，ChAT表达的缺陷导致了转化子中异常的运动节律。较少

In Drasophila, various lines of evidence suggest that the circadian pacemakers for daily locomotor rhythms reside in the cells called "lateral neurons" (LNs) lying in the anterior rind of the medulla. However, little is known about the neural pathways linking these pacemaker cells to behavioral rhythms. To identify the neural components of the input and output pathways of the Drosophila circadian clock system, we have surveyed the brain using immunocytochemical techniques, especially with reference to cholinergic neurons. A monoclonal antibody against Drosophila choline acetyltransferase (ChAT) stained a group of extraretinal photoreceptors at the posterior margin of the compound eye, which is called the "eyelet". The axons emerging from the eyelet terminated in the region of the anterior medulla, where the possible dendritic field of LNs resides. LNs were immunoreactive to an antibody against Drosophila nAChR, implying cholinergic input to LNs. This circumstantial evidence suggests th … More at the eyelet is involved in an input pathway contributing to circadian entrainment. Transformed flies carrying the transgene composed of ChAT cDNA with 1.2 kb of the cis-regulatory sequence showed obviously reduced ChAT expression in specific subsets of cholinergic neurons, such as the neurons terminating in the distal region of the medulla, where the varicosity of the output fibers of LNs resides. A morphometric study revealed that LN varicosity in the medulla was -significantly larger in the transformants than in the wild-type flies. The transformants entrained to light : dark cycles, and exhibited a double-peaked locomotor activity pattern with morning and evening peaks, as did control flies (white). When the illumination level was low (I.e., 0.24 lux), the transformants showed unusual monophagic locomotor activity exclusively occurring during the entire light phase, in contrast to double-peaked activity in the controls. These observations suggest that defects in ChAT expression resulted in the extraordinary locomotor rhythms in the transformants. Less