Circadian rhythm in cortical chloride homeostasis underpins variation in network excitability

The main inhibitory synaptic currents, gated by gamma-aminobutyric acid (GABA), are mediated by Cl--conducting channels1-3, and are therefore sensitive to changes in the chloride electrochemical gradient. GABAergic activity dictates the neuronal firing range4,5 and timing6-9, which in turn influences the rhythms of the brain, synaptic plasticity, and flow of information in neuronal networks7,10-12. The intracellular chloride concentration [Cl-]i 13is, therefore, ideally placed to be a regulator of neuronal activity. Chloride levels13 have been thought to be stable in adult cortical networks, except when associated with pathological activation14-17. Here, we used 2-photon LSSmClopHensor imaging14, in anaesthetized young adult mice, to show a large physiological circadian fluctuation of baseline [Cl-] inside pyramidal cells, equating to an ~15mV positive shift in ECl at times when mice are typically awake (midnight), relative to when they are usually asleep (midday). The change in pyramidal [Cl-]i alters the stability of cortical networks, as demonstrated by a greater than 3-fold longer latency to seizures induced by 4-aminopyridine at midday, compared to midnight. Importantly, both [Cl-]i and latency to seizure, in night-time experiments, were shifted in line with day-time measures, by inhibition of NKCC1. The redistribution of [Cl-]i reflects circadian changes in surface expression and phosphorylation states of the cation-chloride-co-transporters, KCC2 and NKCC1, leading to a greatly reduced chloride-extrusion capacity at night (awake period). Our data show how changes in the biochemical state of neurons may be transduced into altered brain states.