Fig. 2: Differential role of Cl- co-transporter in baseline Cl- regulation.
a Representative fields imaged at ZT5 and ZT17 before, and 40 min after, topical application of the NKCC1 inhibitor bumetanide (bume, 55 µM): [Cl-]i decreased in all neurons in the field. Calibration bar 40 μm. The colour bar indicates [Cl-]i in mM. b Change of [Cl-]i measured in the same neurons before and after bumetanide treatment. Data from 4 mice (188 neurons) imaged at ZT5, and 5 mice (218 neurons) imaged at ZT17. Box plots show the interquartile range, median (central line) and mean (small symbol). Whiskers extend down to the 5th centile and up to the 95th centile. (p = 2.2*10−16, Mann–Whitney, two-sided). c Cumulative frequency distributions of the effects of bumetanide treatment on [Cl-]i at ZT17. The thin lines are the distributions from individual mice and the thick lines are the average distributions (Mann–Whitney, two-sided, p = 7.5*10−6). d Two representative fields imaged at ZT5, before and after topical application of the KCC2 inhibitor VU0463271 (VU, 10 µM). Calibration bar 40 μm. e Effects of the VU0463271 treatment on all imaged neurons at ZT5 (control 4 mice, 176 neurons; VU0463271 3 mice, 163 neurons, Mann–Whitney, two-sided, p = 4.7*10−5). The thin lines are the distributions from individual mice and the thick lines are the average distributions. f Schematic representation of the suggested mechanism for the daily change in [Cl-]i. During the day (rest phase) the baseline [Cl-]i is mainly determined by the extrusion mediated by KCC2 and the inhibition of NKCC1 does not produce a discernible effect. In contrast, at night, the opposite picture is true, as the higher activity of NKCC1 raises the [Cl-]i baseline level. Source data are provided in the Source Data file.
