Figure S5. TNFα controls GABA A R... | Rockefeller University Press

$TNFα controls GABAAR plasticity. (a) Conditional deletion of TNFα from CX3CR1-expressing cells in organotypic slices and in vivo assessed by ELISA. Graphs show TNFα concentration (pg/ml) as mean ± SEM. Left: Complete lack of LPS-induced TNFα production by organotypic slices upon 4-OHT-induced recombination of microglial TNFα in a CX3CR1CreERT2/+:TNFf/f background but not control slices (TNFf/f). n = 2 replicates from two individual experiments. Middle: Following feeding with tamoxifen-containing food, LPS- or BzATP-induced TNFα release by adult mouse primary microglia observed in transgenic controls (tCTL, CX3CR1GFP/+:TNFf/f) but not on microglia-TNFα depleted mice (micTNFα-KO, CX3CR1CreERT2/+:TNFf/f). n = two to six mice per group. Right: On spleen cultures from tamoxifen-treated mice, LPS-induced TNFα release is not affected in both tCTL and micTNF-KO, demonstrating microglia-specific TNFα deletion in vivo. n = 3 mice per condition. (b) Mean intensity of GABAARα1 clusters at gephyrin+ clusters upon blockade of TNFα cleavage (TAPI), neutralization of TNFR1 (n-N1), TNFR2 (n-N2), and control IgGs (IgG). n = 62 to 89 FOVs from five to nine independent experiments. *P < 0.05, nested one-way ANOVA followed by Sidak’s multiple comparison test. (c) Representative images showing increase of GABAARα1 (cyan) at gephyrin+ clusters (arrowhead, green) after 20 min treatment with recombinant TNFα (rTNFα). Scale bars, 1 μm. (d) Effect of rTNFα and TNFR1 activating antibodies (TNFRA1), but not TNFR2 activating antibodies (TNFRA2), on GABAARα1 at L1 synapses. Left: Mean intensity of GABAARα1 clusters at gephyrin+ clusters. ***P < 0.001, nested two-tailed t test (bsl compared to rTNFα); and no statistical significance by nested one-way ANOVA followed by Sidak’s multiple comparison test (for bsl, TNFR1A and TNFR2A). Right: Cumulative fraction of the intensity of GABAARα1 clusters at gephyrin+ cluster. ****P < 0,0001, Kolmogorov–Smirnoff. (e) Mean intensity of GABAARα1 clusters at extrasynaptic sites. (f) Fraction of gephyrin+ clusters colocalized to GABAARα1. (e and f) No statistical significance by nested two-tailed t test (bsl compared to rTNFα) and nested one-way ANOVA followed by Sidak’s multiple comparison test (for bsl, TNFR1A, and TNFR2A). (d–f)n = 65 to 77 FOVs from eight independent experiments. (b and d–f) Results are presented as a minimum to maximum box plots.$

Figure S5.

TNFα controls GABA _A R plasticity. (a) Conditional deletion of TNFα from CX3CR1-expressing cells in organotypic slices and in vivo assessed by ELISA. Graphs show TNFα concentration (pg/ml) as mean ± SEM. Left: Complete lack of LPS-induced TNFα production by organotypic slices upon 4-OHT-induced recombination of microglial TNFα in a CX3CR1^CreERT2/+:TNF^f/f background but not control slices (TNF^f/f). n = 2 replicates from two individual experiments. Middle: Following feeding with tamoxifen-containing food, LPS- or BzATP-induced TNFα release by adult mouse primary microglia observed in transgenic controls (tCTL, CX3CR1^GFP/+:TNF^f/f) but not on microglia-TNFα depleted mice (micTNFα-KO, CX3CR1^CreERT2/+:TNF^f/f). n = two to six mice per group. Right: On spleen cultures from tamoxifen-treated mice, LPS-induced TNFα release is not affected in both tCTL and micTNF-KO, demonstrating microglia-specific TNFα deletion in vivo. n = 3 mice per condition. (b) Mean intensity of GABA_ARα1 clusters at gephyrin⁺ clusters upon blockade of TNFα cleavage (TAPI), neutralization of TNFR1 (n-N1), TNFR2 (n-N2), and control IgGs (IgG). n = 62 to 89 FOVs from five to nine independent experiments. *P < 0.05, nested one-way ANOVA followed by Sidak’s multiple comparison test. (c) Representative images showing increase of GABA_ARα1 (cyan) at gephyrin⁺ clusters (arrowhead, green) after 20 min treatment with recombinant TNFα (rTNFα). Scale bars, 1 μm. (d) Effect of rTNFα and TNFR1 activating antibodies (TNFRA1), but not TNFR2 activating antibodies (TNFRA2), on GABA_ARα1 at L1 synapses. Left: Mean intensity of GABA_ARα1 clusters at gephyrin⁺ clusters. ***P < 0.001, nested two-tailed t test (bsl compared to rTNFα); and no statistical significance by nested one-way ANOVA followed by Sidak’s multiple comparison test (for bsl, TNFR1A and TNFR2A). Right: Cumulative fraction of the intensity of GABA_ARα1 clusters at gephyrin⁺ cluster. ****P < 0,0001, Kolmogorov–Smirnoff. (e) Mean intensity of GABA_ARα1 clusters at extrasynaptic sites. (f) Fraction of gephyrin⁺ clusters colocalized to GABA_ARα1. (e and f) No statistical significance by nested two-tailed t test (bsl compared to rTNFα) and nested one-way ANOVA followed by Sidak’s multiple comparison test (for bsl, TNFR1A, and TNFR2A). (d–f)n = 65 to 77 FOVs from eight independent experiments. (b and d–f) Results are presented as a minimum to maximum box plots.

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