Journal: bioRxiv
Article Title: Astrocyte-to-microglia purinergic signaling mediates synaptic shielding and promotes neuronal activity
doi: 10.64898/2026.07.05.735345
Figure Lengend Snippet: (A) Experimental design for chemogenetic suppression of excitatory neurons during in vivo imaging of ATP dynamics and microglial morphology. AAV-CaMKII-Cre, AAV-hSyn-DIO-hM4D(Gi)-mCherry, and AAV-hSyn-GRAB-ATP1.0 were injected into the motor cortex of Cx3cr1 GFP/+ mice, followed by DCZ administration. (B) Representative time-lapse images showing ATP events and microglial morphology near mCherry-positive Gi-DREADD-expressing neurons after DCZ administration. Dashed lines indicate mCherry-positive neuronal somata. The arrowhead indicates an ATP hotspot, and the arrow indicates a newly formed BE. (C and D) Quantification of ATP hotspot frequency (C) and ATP hotspot size (D) before and after DCZ administration (one-way ANOVA with Dunnett’s multiple-comparisons test, n = 4 mice). (E) Nearest-neighbor distance between ATP hotspots and mCherry-positive Gi-DREADD-expressing neurons compared with a random spatial model (one-way ANOVA with Dunnett’s multiple-comparisons test, n = 4 mice). (F) Quantification of BEs per ATP hotspot before and after DCZ administration (one-way ANOVA with Dunnett’s multiple-comparisons test, n = 4 mice). (G) Correlation between BE number and ATP hotspot area after DCZ administration (simple linear regression; n = 30 ATP hotspots; data collected from 4 mice). (H) Experimental design for freely moving miniature two-photon imaging combined with EEG and EMG recordings to monitor ATP dynamics across sleep-wake states. (I) Representative EEG spectrograms, EMG traces, and vigilance-state classifications during dark and light phases. W, wake; N, NREM sleep; R, REM sleep. (J) Quantification of time spent in wake, NREM sleep, and REM sleep during dark and light phases (n = 6 mice). (K) Representative GRAB-ATP fluorescence images during dark and light phases. Arrowheads indicate ATP hotspots. (L) Quantification of ATP hotspot event frequency during dark and light phases (two-sided paired t-test, n = 6 mice). (M) Quantification of ATP hotspot area during dark and light phases (two-sided unpaired t-test; dark, n = 24 ATP hotspots; light, n = 57 ATP hotspots; data collected from 6 mice). (N) Representative images showing microglial BEs during awake and NREM sleep states. Insets show BE dynamics over time. (O) Quantification of BE number during awake and NREM sleep states (two-sided paired t-test, n = 5 mice). Data are shown as mean ± SEM. Individual points represent mice, ATP hotspots, or BEs as indicated. ns, not significant.
Article Snippet: For chemogenetic neuronal silencing experiments, deschloroclozapine (DCZ; #HY-42110, MedChemExpress) was administered intraperitoneally at 100 μg/kg during in vivo imaging.
Techniques: In Vivo Imaging, Injection, Expressing, Imaging, Fluorescence