The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days

Cation chloride cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl^-]_i) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA.¹ Na⁺ K⁺ Cl^- (NKCC) and K⁺ Cl^- (KCC) cotransporters transport Cl^- into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl^-]_i can lead to an excitatory and depolarizing response of the neuron upon GABA_A receptor opening, while KCC dominance has the opposite effect.¹ This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length,^2-4 and its dysregulation is associated with neurodevelopmental disorders such as epilepsy.^5-8 In Drosophila melanogaster, constant light normally disrupts circadian clock function and leads to arrhythmic behavior.⁹ Here, we demonstrate a function for CCCs in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of CCC expression in circadian clock neurons elicits rhythmic behavior in constant light. We observed the same effects after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl^-]_i-dependent manner. Patch-clamp recordings from the large LNv clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC and NKCC downregulation reduces or increases morning behavioral activity during long photoperiods, respectively. In summary, our results support a model in which the regulation of [Cl^-]_i by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions.