Microglial P2RX7-TNFα signaling drives GABAARs synaptic enrichment through CaMKIIα phosphorylation. (a) Representative confocal images showing increase of GABAARα1 (cyan) at gephyrin+ clusters (arrowheads) upon NMDA-induced inhibitory long-term potentiation (iLTP: 2 min 20 μM NMDA/10 μM CNQX plus 20 min recovery) in organotypic slices cortical L1 (CTL: control; bsl: baseline). Scale bars, 1 μm. (b–e) Mean intensity of GABAARα1 clusters at gephyrin+ cluster normalized to CTL at bsl. n = FOVs/independent experiments: (b) n = 44–69/5–6; (c) n = 47–53/5; (d) n = 66–102/5–7; (e) n = 49–68/7–9. *P < 0.05, **P < 0.01 and ***P < 0.001, nested one-way ANOVA followed by Sidak’s multiple comparison test. iLTP-induced synaptic GABAAR enrichment is abolished by: b, microglia depletion/inactivation (PLX: PLX3397; SAP: Mac1-saporin; minoc: minocycline); c, neutralization of TNFα (nTNFα); d, microglia-specific TNFα deletion through 4-hydroxy-tamoxifen (4-OHT)-induced recombination on CX3CR1CreERT2/+:TNFf/f but not on a TNFf/f background; e, ATP hydrolysis (apy), P2RX antagonist (PPADS) and P2RX7 antagonist (A74), but not P2Y12R inhibitor (PSB). (f) Left: Confocal images of GABAARα1 (cyan) at gephyrin+ clusters (arrowheads) in bsl and upon BzATP treatment. Scale bars, 1 μm. Right: Mean intensity of GABAARα1 clusters at gephyrin+ clusters normalized to CTL at bsl. n = 49–63/5–6. *P < 0.05, **P < 0.01, nested one-way ANOVA followed by Sidak’s multiple comparison test. (g) Left: Thr286-phosphorylated CaMKII is enhanced in L1 at the induction phase of plasticity (iLTP0’ or BzATP0’). Scale bars, 5 μm. Right: Mean intensity of Thr286-phosphorylated CaMKII puncta normalized to bsl. n = 25 to 33 FOVs from three independent experiments. *P < 0.05, one-way ANOVA followed by Sidak’s multiple comparison test. (h) Left: Western blot analysis showing iLTP0’-induced CaMKII Thr286-phosphorylation. Right: Ratio between Thr286-phosphorylated CaMKII and total CaMKII normalized to the respective iLTP-free control. n = 5–9 independent experiments. *P < 0.05 compared with respective control, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (b–h) Results are presented as minimum to maximum box plots. (i) Model. ATP released downstream NMDA-induced neuronal activity activates microglial P2RX7 which triggers the release of microglial TNFα. TNFα signaling gates CaMKIIα autophosphorylation which controls the enrichment of synaptic GABAARs in pyramidal neurons. Source data are available for this figure: SourceData F2.
Figure 2.

Microglial P2RX7-TNFα signaling drives GABA A Rs synaptic enrichment through CaMKIIα phosphorylation. (a) Representative confocal images showing increase of GABAARα1 (cyan) at gephyrin+ clusters (arrowheads) upon NMDA-induced inhibitory long-term potentiation (iLTP: 2 min 20 μM NMDA/10 μM CNQX plus 20 min recovery) in organotypic slices cortical L1 (CTL: control; bsl: baseline). Scale bars, 1 μm. (b–e) Mean intensity of GABAARα1 clusters at gephyrin+ cluster normalized to CTL at bsl. n = FOVs/independent experiments: (b) n = 44–69/5–6; (c) n = 47–53/5; (d) n = 66–102/5–7; (e) n = 49–68/7–9. *P < 0.05, **P < 0.01 and ***P < 0.001, nested one-way ANOVA followed by Sidak’s multiple comparison test. iLTP-induced synaptic GABAAR enrichment is abolished by: b, microglia depletion/inactivation (PLX: PLX3397; SAP: Mac1-saporin; minoc: minocycline); c, neutralization of TNFα (nTNFα); d, microglia-specific TNFα deletion through 4-hydroxy-tamoxifen (4-OHT)-induced recombination on CX3CR1CreERT2/+:TNFf/f but not on a TNFf/f background; e, ATP hydrolysis (apy), P2RX antagonist (PPADS) and P2RX7 antagonist (A74), but not P2Y12R inhibitor (PSB). (f) Left: Confocal images of GABAARα1 (cyan) at gephyrin+ clusters (arrowheads) in bsl and upon BzATP treatment. Scale bars, 1 μm. Right: Mean intensity of GABAARα1 clusters at gephyrin+ clusters normalized to CTL at bsl. n = 49–63/5–6. *P < 0.05, **P < 0.01, nested one-way ANOVA followed by Sidak’s multiple comparison test. (g) Left: Thr286-phosphorylated CaMKII is enhanced in L1 at the induction phase of plasticity (iLTP0’ or BzATP0’). Scale bars, 5 μm. Right: Mean intensity of Thr286-phosphorylated CaMKII puncta normalized to bsl. n = 25 to 33 FOVs from three independent experiments. *P < 0.05, one-way ANOVA followed by Sidak’s multiple comparison test. (h) Left: Western blot analysis showing iLTP0’-induced CaMKII Thr286-phosphorylation. Right: Ratio between Thr286-phosphorylated CaMKII and total CaMKII normalized to the respective iLTP-free control. n = 5–9 independent experiments. *P < 0.05 compared with respective control, Kruskal–Wallis test followed by Dunn’s multiple comparisons test. (b–h) Results are presented as minimum to maximum box plots. (i) Model. ATP released downstream NMDA-induced neuronal activity activates microglial P2RX7 which triggers the release of microglial TNFα. TNFα signaling gates CaMKIIα autophosphorylation which controls the enrichment of synaptic GABAARs in pyramidal neurons. Source data are available for this figure: SourceData F2.

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