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 channels[1][1]-[3][2], and are therefore sensitive to changes in the chloride electrochemical gradient. GABAergic activity dictates the neuronal firing range[4][3],[5][4] and timing[6][5]-[9][6], which in turn influences the rhythms of the brain, synaptic plasticity, and flow of information in neuronal networks[7][7],[10][8]-[12][9]. The intracellular chloride concentration [Cl-]i [13][10]is, therefore, ideally placed to be a regulator of neuronal activity. Chloride levels[13][10] have been thought to be stable in adult cortical networks, except when associated with pathological activation[14][11]-[17][12]. Here, we used 2-photon LSSmClopHensor imaging[14][11], in anaesthetized young adult mice, to show a large physiological circadian fluctuation of baseline [Cl-] inside pyramidal cells, equating to a ∼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. (235 words) ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-3 [3]: #ref-4 [4]: #ref-5 [5]: #ref-6 [6]: #ref-9 [7]: #ref-7 [8]: #ref-10 [9]: #ref-12 [10]: #ref-13 [11]: #ref-14 [12]: #ref-17