(A) Timeline of experiment in a chronic window animal. (B–D) Control studies of excitatory neuronal calcium activity before and after isoflurane induction. (B) Representative ∆F/F traces of somatic calcium activity from layer II/III CaMKIIa neurons in one animal. (C) A heatmap of CaMKIIa somatic calcium activity from 60 representative somatic ROIs in one animal under awake and isoflurane conditions. (D) Baseline-normalized neuronal calcium activity under isoflurane (Paired t-test; dots: one animal) showing an average 91% drop in calcium signaling in the first 10 min of isoflurane maintenance (1.5–2%). (E–H) Microglial calcium responses to isoflurane from a cohort of chronic window GCaMP6s;Cx3Cr1CreER-eYFP mice (see also: Figure 3—Video 1). (E) Representative ∆F/F traces under awake and isoflurane conditions from the depicted microglial cell using threshold-based segmentation. (F) A heat map of microglial calcium activity from 60 threshold-segmented process territories surveyed across multiple microglia in a single field of view from a representative animal. (G) Microdomains active and their signal areas from baseline through anesthesia (two-way ANOVA; *Dunnett’s post-hoc vs. baseline; #somata vs. processes) using the threshold-segmentation approach for quantification. Dashed lines represent the averaged data from each of five animals, while solid lines represent the mean ± SEM from the N = 5 animals. (H) Microglial process territory calcium activity was binned by minute across all territories studied (311 to 379 territories detected in different imaging periods). The mean minute-by-minute signal area during the baseline period (0.385 ∆F/F∙s, dashed line) was compared to minute-by-minute calcium signal areas during isoflurane exposure (one-sample t-test comparison to the baseline average), indicating significant calcium changes in microglial processes are detectable 6 min after exposure . (I) After masking the soma, uniform thresholding of the average intensity image by period shows increasing total process area during isoflurane exposure (dashed lines: individual animal; solid lines: group mean ± SEM; one-way ANOVA with Dunnett’s post-hoc vs. baseline). (J) Microglial process calcium activity under isoflurane was studied by motility/morphology characteristics. As displayed in the cartoon, microglial motility/morphology was categorized as processes that retracted under isoflurane, processes that remained stable, or processes that extended, showing new areas of outgrowth. Exact criteria and methodology are provided in the methods and displayed further in Figure 3—figure supplement 1. After subdividing processes based on motility characteristics, the percentage of processes exhibiting calcium activity is displayed (horizontal bars, Fisher’s exact test) along with the signal magnitude (one-way ANOVA with Tukey’s post-hoc test; the number of sampled regions fitting each criterion is provided near the bar; mean ± SEM). Scale bars: 50 µm (B), 10 µm (E). N = 4 AAV-CaMKIIa-GCaMP6s mice (A–C), N = 5 GCaMP6s;Cx3Cr1CreER-eYFP mice (D–G). *p<0.05, **, ##p<0.01, ***p<0.001, ****, ####p<0.0001.