Extended Data Figure 9. Microglia can suppress glutamate-induced cortical neuron activation in a CD39/ADO/A₁R-dependent fashion in vitro.

a-d, Experimental approaches for the assessment of adenosine-mediated regulation of cortical neuron activity in vitro. Embryonic cortical neurons were cultured on Axion microelectrode array (MEA) plates which allows for continuous electrical field recordings. a, A₁Rs modulate cortical neuronal activity at baseline and in response to glutamate. On day in vitro (DIV) 14, neuronal cultures were treated with vehicle, glutamate (10μM), A₁R agonist (CPA, 100nM), A₁R antagonist (DCPCX, 100nM), glutamate and A₁R agonist, or glutamate and A₁R antagonist. Dot plot shows the percentage change in mean firing rate of neurons 1 hour after treatment compared to their baseline prior to drug treatment. (n=7 wells, P<0.0001, One-way ANOVA with Tukey’s post hoc test). b, Adenosine suppresses neuronal activity via A₁R activation. On DIV14, cultures were treated with vehicle, adenosine (10μM), A₁R antagonist (DCPCX, 100nM), or co-treated with adenosine and A₁R antagonist. Dot plot shows percentage change in mean firing rate of neurons 1 hour after treatment compared to their baseline prior to drug treatment. (n=8 wells, P<0.0001, One-way ANOVA with Tukey’s post hoc test). c, Microglia suppress neuronal activity in response to glutamate-induced activation in an A₁R-dependent manner. Microglia were isolated from neonatal pups, plated onto the neuronal culture on DIV 14, and allowed to settle for 48 hrs. Mixed cultures were treated with vehicle and/or glutamate (10μM) and/or A1R antagonist (100nM) on DIV 16. Dot plot shows percentage change in mean firing rate of neurons 1 hour after treatment compared to their baseline prior to drug treatment. (left, n=12 wells, P<0.0001, right, n=4, 6, 9, and 7 wells, P=0.001, One-way ANOVA with Tukey’s post hoc test). d, Microglia suppress neuronal activity in a CD39-dependent manner in response to glutamate-induced activation. Microglia were isolated from neonatal pups, plated onto the neuronal culture on DIV 14, and allowed to settle for 48 hrs. Mixed cultures were pretreated with CD39 inhibitor (ARL67156, 200μM) or vehicle (30 min) and then treated with glutamate (10μM). Dot plot shows percentage change in mean firing rate of neurons 1 hour after treatment compared to the corresponding baseline neuronal activity levels prior to their baseline prior to drug treatment. (n=12, 12, 11, and 11 wells, P=0.0045, One-way ANOVA with Tukey’s post hoc test). Data shown as mean ± s.e.m. and representative of 2-3 independent experiments.