Intrinsic cell-class-specific modulation of intracellular chloride levels and inhibitory function, in cortical networks, between day and night.

Recent work showed unexpectedly large, daily modulation of intracellular chloride concentration (Cl - in ) in cortical pyramidal cells, with consequences for GABAergic function and network excitability (Alfonsa et al., 2023; Pracucci et al., 2023). One explanation for this Cl - in modulation is that it arises from variation in presynaptic drive. In that case, neuronal classes with similar synaptic inputs should show correlated changes in activity-dependent ionic redistribution. To examine this prediction, we performed in vivo, LSSm-ClopHensor imaging to measure Cl - in and pH in in populations of parvalbumin- (PV) and somatostatin-expressing (SST) interneurons in neocortical layer 2/3 of male and female adult mice. Imaging was performed at zeitgeber time (ZT) 5, and ZT17, when pyramidal cell Cl - in shows maximal divergence (Pracucci et al., 2023). Interestingly, PV interneurons also showed large physiological Cl - in modulation between these times, but out of phase with that in pyramidal cells, being raised at ZT5 and lower at ZT17, and with a far higher mean Cl - in . SST interneurons showed less modulation, with higher variance, and with a temporal dynamic resembling the pyramidal cell pattern. Notably, in vitro experimental assays of inhibition, involving these two classes of interneuron, differed markedly at ZT5 and ZT17. The persistence of these time-of-day effects in vitro, and the difference in Cl - in dynamics between pyramidal cells and PV interneurons in vivo, both point towards cell-intrinsic regulation being more important than activity-dependent effects in setting these slow, daily, physiological, ionic redistribution patterns. We discuss what other possible factors may influence variations in brain state through the day. Significance Statement We find that the three largest subclasses of supragranular neocortical neurons, pyramidal cells and parvalbumin- and somatostatin-expressing interneurons, show different patterns of daily modulation of Cl - in . Notably, the modulation in parvalbumin-interneurons is out-of-phase with the other two cell classes. We further observed differences in network inhibition in brain slices prepared at ZT5 and ZT17. We argue, based upon these various lines of evidence, that activity-dependent ionic redistribution is not the primary determinant of the slow daily Cl - in modulation. Instead, we discuss which cell-autonomous mechanisms may be involved, and what implications these findings have for our understanding of brain state differences.