Figure S2. GABA A Rs plasticity across... | Rockefeller University Press

$GABAARs plasticity across the light/dark cycle. (a) Left: Confocal images of Iba1+ cells revealing partial depletion in the brain, but not in the spleen, by 2-wk feeding with PLX3397-containing food (PLX). Scale bars, 200 μm. Right: Density of Iba1+ cells normalized to CTL. n = 5 mice for CTL and PLX. **P < 0.01, unpaired two-tailed Mann–Whitney test. Data are mean ± SEM. (b) Left: Cumulative fraction of GABAARγ2 clusters’ intensities at gephyrin+VGAT+ synapses in cortical L1. ****P < 0.0001, Kolmogorov–Smirnoff. (c) Representative confocal images showing increase of GABAARα1 (cyan) at cortical L1 inhibitory synapses (arrowhead: gephyrin+VGAT+) at ZT6 in comparison to ZT18. Scale bars, 5 and 1 μm. Dashed box corresponds to enlarged detail in ZT18. (d) Left: Mean intensity of GABAARα1 clusters at gephyrin+VGAT+ synapses (synaptic) and at extrasynaptic sites. *P < 0.05, nested two-tailed t test. Right: Cumulative fraction of GABAARα1 clusters’ intensities at gephyrin+VGAT+ synapses. ****P < 0.0001, Kolmogorov–Smirnoff test. (e) Fraction of gephyrin+VGAT+ synapses colocalized to GABAARs clusters. *P < 0.05 and **P < 0.01, nested two-tailed t test. (f) Fraction of somatic VGAT+ cluster colocalized to GABAARγ2 clusters in cortical L5. No statistical significance by nested two-tailed t test. (g) Mean intensity of total GABAARγ2 (left) and GABAARα1 signal (right). No statistical significance by nested two-tailed t test. (b–g)n = 53-60 FOVs from 5 mice per group. Results are presented as minimum to maximum box plots. (h–j) Amounts of vigilance states over 24 h in controls CTL (black) and PLX3397 treated (green) mice in Wake (h), NREMS (i), and REMS (j). Left panels: Amounts were reported by 2 h segments. Two-way rANOVA; Wake: treatment, F(1,14) = 4.128, P = 0.062; time: F(4.253,59.54) = 46.97, P < 0.0001; interaction F(11,154) = 1.751, P = 0.067; NREMS, treatment, F(1,14) = 3.596, P = 0.079; time: F(11,154) = 15.32, P < 0.0001; interaction F(11,154) = 1.452, P = 0.155, REMS, treatment, F(1,14) = 1.578, P = 0.2296; time: F(3.267,45.73) = 22.47, P < 0.0001; interaction F(11,154) = 0.650, P = 0.7832. Right panels: amounts were reported by 12 h segments. Mann–Whitney tests, **P < 0.01 CTL versus PLX3397. CTL, seven mice; PLX3397 treated, nine mice. Data are represented as mean ± SEM. In agreement with previous work (Corsi et al., 2022; Liu et al., 2021) microglia depletion affects time in wake and NREM sleep only in the dark phase.$

Figure S2.

GABA _A Rs plasticity across the light/dark cycle. (a) Left: Confocal images of Iba1⁺ cells revealing partial depletion in the brain, but not in the spleen, by 2-wk feeding with PLX3397-containing food (PLX). Scale bars, 200 μm. Right: Density of Iba1⁺ cells normalized to CTL. n = 5 mice for CTL and PLX. **P < 0.01, unpaired two-tailed Mann–Whitney test. Data are mean ± SEM. (b) Left: Cumulative fraction of GABA_ARγ2 clusters’ intensities at gephyrin⁺VGAT⁺ synapses in cortical L1. ****P < 0.0001, Kolmogorov–Smirnoff. (c) Representative confocal images showing increase of GABA_ARα1 (cyan) at cortical L1 inhibitory synapses (arrowhead: gephyrin⁺VGAT⁺) at ZT6 in comparison to ZT18. Scale bars, 5 and 1 μm. Dashed box corresponds to enlarged detail in ZT18. (d) Left: Mean intensity of GABA_ARα1 clusters at gephyrin⁺VGAT⁺ synapses (synaptic) and at extrasynaptic sites. *P < 0.05, nested two-tailed t test. Right: Cumulative fraction of GABA_ARα1 clusters’ intensities at gephyrin⁺VGAT⁺ synapses. ****P < 0.0001, Kolmogorov–Smirnoff test. (e) Fraction of gephyrin⁺VGAT⁺ synapses colocalized to GABA_ARs clusters. *P < 0.05 and **P < 0.01, nested two-tailed t test. (f) Fraction of somatic VGAT⁺ cluster colocalized to GABA_ARγ2 clusters in cortical L5. No statistical significance by nested two-tailed t test. (g) Mean intensity of total GABA_ARγ2 (left) and GABA_ARα1 signal (right). No statistical significance by nested two-tailed t test. (b–g)n = 53-60 FOVs from 5 mice per group. Results are presented as minimum to maximum box plots. (h–j) Amounts of vigilance states over 24 h in controls CTL (black) and PLX3397 treated (green) mice in Wake (h), NREMS (i), and REMS (j). Left panels: Amounts were reported by 2 h segments. Two-way rANOVA; Wake: treatment, F(1,14) = 4.128, P = 0.062; time: F(4.253,59.54) = 46.97, P < 0.0001; interaction F(11,154) = 1.751, P = 0.067; NREMS, treatment, F(1,14) = 3.596, P = 0.079; time: F(11,154) = 15.32, P < 0.0001; interaction F(11,154) = 1.452, P = 0.155, REMS, treatment, F(1,14) = 1.578, P = 0.2296; time: F(3.267,45.73) = 22.47, P < 0.0001; interaction F(11,154) = 0.650, P = 0.7832. Right panels: amounts were reported by 12 h segments. Mann–Whitney tests, **P < 0.01 CTL versus PLX3397. CTL, seven mice; PLX3397 treated, nine mice. Data are represented as mean ± SEM. In agreement with previous work (Corsi et al., 2022; Liu et al., 2021) microglia depletion affects time in wake and NREM sleep only in the dark phase.

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