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. 2020 Jul 27;9:e56502. doi: 10.7554/eLife.56502

Figure 6. DREADD-based modulation of excitatory neuronal activity is sufficient to induce microglial calcium signaling.

(A) AAVs injected into the somatosensory cortex of WT mice in order to study DREADD-based changes in neuronal calcium activity (top). Experiment outline (bottom). (B–C) Representative ∆F/F traces of layer 2/3 CaMKIIa neuronal calcium activity at baseline and after CNO injection in the Gi (B) and Gq (C) systems. (D) Saline or CNO dose-dependent effects on neuronal calcium activity in the Gi and Gq systems over 15 min imaging periods (one-way ANOVA with Dunnett’s post-hoc comparison to saline). (E) Summary of DREADD-based effects on neuronal calcium activity (at the time of peak effect: 30–45 min post injection; two-way ANOVA with Dunnett’s post-hoc comparison to baseline). (F) Method to study DREADD-based changes in neuronal activity on microglial calcium signaling. mCherry expression served as a positive control for successful DREADD transfection. (G–H) Representative ∆F/F traces of microglial soma and process territory calcium activity at baseline and after CNO injection in the Gi (G) and Gq (H) systems. The threshold-based ROI approach was used to detect process regions. (I) Microglial process territory calcium activity in the Gi and Gq systems over 15 min imaging periods (one-way ANOVA with Dunnett’s post-hoc vs. saline). (J) Summary of DREADD-based effects on microglial process territory calcium activity (at the time of peak effect: 45–60 min post injection; two-way ANOVA with Dunnett’s post-hoc comparison to baseline). (K) The mean ± SEM calcium signal area is plotted for microglial process territories that remained stable, demonstrated new process outgrowth, or exhibited retraction 45–60 min after 5 mg/kg CNO injection in the Gi DREADD system (one-way ANOVA with Tukey’s post-hoc test; process sample size given in the bar). The percentage of processes exhibiting calcium activity by motility/structural characteristic is displayed in the horizontal bars (Fisher’s exact test). (L) The same analyses as performed in (K) for the Gq DREADD system (see Figure 6—figure supplement 1 for more detail). Scale bars: 50 µm (B, C, and F), 10 µm (G-H). (A-E) N = 8 WT mice, N = 4/8 receiving AAV-CaMKIIa-hM4D(Gi) and N = 4/8 receiving AAV-CaMKIIa-hM3D(Gq). (F-J) N = 6 GCaMP6s;Cx3Cr1CreER-eYFP mice, half receiving Gq or Gi DREADD. Grouped data represent mean ± SEM; dots represent individual animals (E, J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. See also: Figure 6—Video 1.

Figure 6—source data 1. Microglial calcium activity during Gi and Gq DREADD activation in CaMKIIa neurons.

Figure 6.

Figure 6—figure supplement 1. CaMKIIa Gi and Gq activation results in microglial process extension and coordinated process calcium activity.

Figure 6—figure supplement 1.

(A) A field of view with multiple microglia under awake baseline (red) and 45-60 min following Gi DREADD activation (green). (B) Quantification of the microglial process area across imaging periods (mean and SEM). (C) Examples of stable, extending, or retracting processes and their calcium activity 45-60 min after CNO injection. For extending and retracting processes, the time of extension/retraction start and end is overlaid on the ∆F/F calcium trace. (D) A field of view with multiple microglia under awake baseline (red) and 45-60 min following Gq DREADD activation (green). (E) Quantification of the microglial process area across imaging periods (mean and SEM). (F) Examples of stable, extending, or retracting processes and their calcium activity 45-60 min after CNO injection. For extending and retracting processes, the time of extension/retraction start and end is overlaid on the ∆F/F calcium trace. *p<0.05, **p<0.01, ***p<0.001; one-way ANOVA with Dunnett's post-hoc comparison to baseline.
Figure 6—video 1. Microglial calcium activity at baseline and following CNO administration in Gi and Gq systems.
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