Figure 1. Increased microglial motility in the hippocampus of awake mice.

Experimental paradigm of repetitive microglia imaging in dorsal CA1 (stratum radiatum [SR]) in the same mice under varying conditions. (b) Schematic of the accessible hippocampal layers in CA1 through a chronic hippocampal cranial window. CX3CR1-GFP::thy-1-YFP-H transgenic mice allowed for simultaneous visualization of microglia (green) and neurons (magenta). (c) Representative image of a microglia cell and its processes in an awake mouse imaged in CA1, SR. Subsequent time points (0, 5 min) were superimposed (Δt=5 min) to measure gained (green arrow), lost (red arrow), and stable (yellow area) processes. (d) Turnover rate of microglia fine processes (i.e. motility) at varying conditions (n=6 mice ‘anesthesia’, ‘awake’; n=4 ‘awake+tetrodotoxin [TTX]’); one-way ANOVA with Bonferroni’s multiple comparison test, F(2,13) = 10.22; p=0.0291 (anesthesia vs. awake), p=0.0014 (awake vs. awake+TTX). Error bars: SEM. Scale bars: (b) 100 μm, (c) 10 μm, 2 μm; *p<0.05, **p<0.01.

Figure 1—figure supplement 1. Isoflurane anesthesia decreases neuronal activity in the hippocampus.

(a) Exemplary image of Ca²⁺ imaging (GCaMP6m) in CamKII+ cells of the CA1 region in the dorsal CA1 of the hippocampus in an awake mouse averaged over 60 s at 30 Hz sampling rate. (b) Ca²⁺ imaging under isoflurane anesthesia (1% in O₂). (c) Exemplary traces of changes in somatic GCaMP6m fluorescence in 10 cells over 60 s in an awake mouse. (d) Fluorescence changes under anesthesia. (e) Comparison of average Ca²⁺ responses in both conditions. Data shown for three mice and 50–100 cells per recording in two different fields of views, respectively. Paired t-test, p=0.0435; *p<0.05. Error bars: SEM.

Figure 1—figure supplement 2. Examples of microglia in CA1 under anesthesia, awake, and tetrodotoxin (TTX)-treated conditions.

(a–c) Exemplary in vivo images of microglia from stratum radiatum (SR) of dorsal CA1 under (a) isoflurane anesthesia, (b) awake, and (c) awake but after topical TTX treatment. We did not observe deramification under conditions (b) and (c). Scale bar: 5 µm.

Figure 1—video 1. Microglial motility in an anesthetized mouse.

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Microglia fine process motility recorded in the hippocampus of a mouse under isoflurane anesthesia (1% in O₂) using two-photon microscopy over 45 min. Z-stacks were acquired 5 min apart each spanning 100 µm starting under the CA1 cell layer. For analysis, a maximum intensity projection of 25 µm was created for each time point. Scale bar: 10 μm.

Figure 1—video 2. Microglial motility in an awake mouse.

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Microglia fine process motility recorded in the hippocampus of an awake mouse head-fixed on a circular treadmill using two-photon microscopy. Z-stacks were taken 5 min apart each spanning 100 µm starting under the CA1 cell layer. For analysis a maximum intensity projection of 25 µm was created for each time point. Scale bar: 10 μm.

Figure 1—video 3. Calcium imaging of CA1 pyramidal neurons under isoflurane anesthesia.

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Exemplary video of Ca²⁺ imaging (GCaMP6m) in CamKII+ cells in the CA1 region of the hippocampus in an anesthetized mouse (isoflurane, 1% in O₂) over 60 s. Scale bar: 50 µm.

Figure 1—video 4. Calcium imaging of CA1 pyramidal cells in an awake mouse.

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Exemplary video of Ca²⁺ imaging (GCaMP6m) in CamKII+ cells in the CA1 region of the hippocampus of an awake mouse over 60 s. Scale bar: 50 µm.