Oscillating PDF in termini of circadian pacemaker neurons and synchronous molecular clocks in downstream neurons are not sufficient for sustenance of activity rhythms in constant darkness

In Drosophila, neuropeptide Pigment Dispersing Factor (PDF) is expressed in small and large ventral Lateral Neurons (sLNv and lLNv), among which sLNv are critical for activity rhythms in constant darkness. Studies show that this is mediated by rhythmic accumulation and likely secretion of PDF from sLNv dorsal projections, which in turn synchronises molecular oscillations in downstream circadian neurons. Using targeted expression of a neurodegenerative protein Huntingtin in LNv, we evoke a selective loss of neuropeptide PDF and clock protein PERIOD from sLNv soma. However, PDF is not lost from sLNv dorsal projections and lLNv. These flies are behaviourally arrhythmic in constant darkness despite persistence of PDF oscillations in sLNv dorsal projections and synchronous PERIOD oscillations in downstream circadian neurons. We find that PDF oscillations in sLNv dorsal projections are not sufficient for sustenance of activity rhythms in constant darkness and this is suggestive of an additional component that is possibly dependent on sLNv molecular clock and PDF in sLNv soma. Additionally, despite loss of PERIOD in sLNv, their activity rhythms entrain to light/dark cycles indicating that sLNv molecular clocks are not necessary for entrainment. Under constant light, these flies lack PDF from both soma and dorsal projections of sLNv, and when subjected to light/dark cycles, show morning and evening anticipation and accurately phased morning and evening peaks. Thus, under light/dark cycles, PDF in sLNv is not necessary for morning anticipation.在果蝇（Drosophila）中，神经肽色素分散因子（Pigment Dispersing Factor，PDF）表达于小、大腹侧外侧神经元（small ventral Lateral Neurons，sLNv与large ventral Lateral Neurons，lLNv），其中小腹侧外侧神经元（sLNv）对于恒定黑暗条件下的活动节律至关重要。研究表明，该功能通过sLNv背侧投射中PDF的节律性积累与潜在分泌介导，进而同步下游昼夜节律神经元的分子振荡。本研究通过在LNv中靶向表达神经退行性蛋白亨廷顿蛋白（Huntingtin），诱导sLNv胞体中的神经肽PDF与时钟蛋白周期蛋白（PERIOD）发生选择性丢失，但sLNv背侧投射与lLNv中的PDF并未出现缺失。尽管sLNv背侧投射中仍保留PDF的节律振荡，且下游昼夜节律神经元的PERIOD振荡仍保持同步，这些果蝇在恒定黑暗条件下仍表现出行为性无节律。我们发现，sLNv背侧投射中的PDF节律振荡不足以维持恒定黑暗下的活动节律，这提示存在另一依赖于sLNv分子时钟与胞体PDF的额外调控组分。此外，尽管sLNv中的PERIOD发生丢失，这些果蝇的活动节律仍可被光暗周期同步，表明sLNv的分子时钟并非节律同步所必需。在恒定光照条件下，这些果蝇的sLNv胞体与背侧投射均缺失PDF；当被置于光暗周期中时，它们可表现出清晨与傍晚的活动预期，且清晨、傍晚的活动峰相位精准。因此，在光暗周期下，sLNv中的PDF并非活动预期所必需。