(a) Schematic time-line of the experiment. Three Thy1-GCamp3 mice with implanted cranial windows were imaged for calcium responses of MCs to eight monomolecular odors, both at baseline (day 0) and 30 days later, following microglia depletion induced by PLX5622 treatment. (b) In vivo two-photon micrograph of a representative field of MCs at the first imaging session (day 0). Scale bar = 10 μm. (c) Examples of calcium transients from the five neurons marked on the left image in response to eight odors during baseline. Odor stimulation was applied for 2 s, denoted as a black line under each trace. Black traces represent the three single trials and the red trace is the average. Asterisks denote a statistically significant response to that odor. Scale - 100% ∆F/F. Iso-a = Isoamyl acetate, Eth-b = Ethyl butyrate, Am-a = Amyl Acetate, 2-Phen = 2 phenylethanol, Eth-tig = Ethyl tiglate, α-pin = Alpha Pinen, Geran = Geraniol, Eucal = Eucalypyol. (d) Same as in panel b but for data collected from a different mouse, following 30 days of PLX5622 administration. (e) Examples of calcium transients from five neurons marked on the left image in response to eight odors. (f) Cumulative distribution of the proportion of MCs responding to 0–8 odors, before (Day 0; n = 622 MCs) and after (Day 30; n = 575 MCs) microglia depletion. The distributions in the two groups were not significantly different (Dn,n’=0.0649, p=0.155, two-sample Kolmogorov-Smirnov test). (g) Histograms showing the response magnitudes of all the significant cell odor pair responses. Data is based on imaging 622 neurons for a total of n = 1641 odor pairs on Day 0 (black bars), and 575 neurons for a total of n = 1492 odor pairs on Day 30 (red bars). Distributions are significantly different (Dn,n’=0.1114, ***p=6.30e-09, two-sample Kolmogorov–Smirnov test). (h) Box plots for the distributions shown in (g) (t(3131) = −4.9771, ***p=6.8e-07, two sample t-test). (i) In vivo two-photon micrographs of the same field of MCs before and after microglia depletion. Scale bar: 10 µm. (j) Representative examples of the mean calcium transients evoked by eight odors before (black) and after (red) microglia depletion. Data is from the four neurons marked on the image in F. Scale - 100% ∆F/F. Odors are the same as in A. (k) Scatter plot of the individual cell-odor pair responses from the neurons at day 0 (x-axis) and at day 30 (y-axis). n = 180 neurons with a total of n = 654 cell-odor pairs. (l) Box plots of the repeated neuronal responses before and after microglia depletion (t(653)=6.3525, ***p=3.97e-10, paired t-test).