Diurnal variation in neuronal chloride levels and seizure susceptibility, in neocortex, reflecting changes in activity of chloride-cation-cotransporters

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 is, therefore, ideally placed to be a regulator of neuronal activity. Chloride levels have been thought to be stable in adult cortical networks, except when associated with pathological activation13–16. Here, we used 2-photon LSSmClopHensor imaging, in anaesthetized young adult mice13, to show that [Cl-] inside pyramidal cells shows a physiological diurnal rhythm, with an approximately 1.8-fold range, equating to an ~15mV positive shift in ECl at times when mice are typically awake (midnight), relative to when they are usually asleep (midday). This change of [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 diurnal 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 demonstrate a means by which changes in the biochemical state of neurons are transduced into altered brain states.