Characterize The Mechanism Of Rhythmic Degradation Of GABA_A Receptor And Its Role Of Regulating Sleep

Sleep is an essential,evolutionarily conserved behavior.It is regulated by circadian rhythm and homeostatic mechanism.Circadian rhythm regulates timing of sleep,and homeostatic mechanism influences the amout of sleep.However,how the output of circadian clock determines the timing of sleep is still unknown.In vertebrates and invertebrates,sleep and wakefulness is modulated by the electrical activity of pacemaker neurons that are circadian-regulated and suppressed by inhibitory GABAergic inputs.Here,in Drosophila we found the protein level of RDL,homologous to mammalian GABA_A receptor,changed dynamically in the large ventral lateral clock neurons(l-LNvs).Through RNAi screen,we found GABA_A receptor is accumulated by knocking down Fbxl4 expression.Meanwhile,Fbxl4 mainly degrades GABA_A receptor at the daytime by ubiquitin dependent pathway and the process of degradation is dose dependent.Further findings are that the protein level of Fbxl4 alters across the 24 hr day and the transcription of fbxl4 in l-LNvs is CLOCK-dependent.Previously reported,the activity of l-LNvs was rhythmically regulated by GABAergic inputs.Then we performed whole-cell recordings of l-LNvs and showed that the spontaneous action potential rate in fbxl4 mutant is slower than wild type at dawn and fbxl4 mutant shows more sensitivity in the saturated-GABA puff experiment.Consequencely,fbxl4 mutant shows increased sleep and short latency.It indicated that Fbxl4 regulates sleep through promotion of rhythmic degradation of the GABA_A receptor.We propose that Fbxl4,as a clock output molecule,was expressed dynamically in the l-LNvs neuron.And Fbxl4 mediates RDL degradation,leading to increase arousal.It offers evidence to understand the relationship of circadian rhythm and homeostatic mechanism.