Fig. 2: Astrocyte Ca²⁺ dynamics gate the effect of NE on synapses.

(A) Approach for astrocyte Ca²⁺ silencing with iβARK. (B) Representative IHC images of iβARK-mCherry expression in the hippocampal CA1, along with quantification of efficiency and specificity (n=5 slices). (C) Plot of the stimulation intensity/fEPSP slope relationship (left, unpaired Student’s t-test on slope/stim ratio) and summary bar graphs of PPF values (right) in RFP-control and iβARK slices at baseline. (D) Kymograph (each row shows the average fluorescence across ROAs of a single astrocyte) and 5 representative ΔF/F₀ traces (from individual ROAs) of spontaneous Ca²⁺ transients in RFP-control and iβARK slices. Horizontal time axis applies to the kymograph and representative traces. (E) Pots of the peak amplitude, frequency, and kinetics of spontaneous Ca²⁺ transients in RFP-control and iβARK slices. Each data point shows the average fluorescence across ROAs for an individual astrocyte. (F) Kymographs (each row shows the whole-cell fluorescence of a single astrocyte) and average ΔF/F₀ traces (± s.e.m.) across all astrocytes in response to 20μM NE application in RFP-control and iβARK slices. (G) Plot of the peak ΔF/F₀ response in RFP-control and iβARK conditions for experiments shown in (F). (H,I) Time-courses of the effect of 20μM NE on fEPSP slope and PPF, and representative traces, in RFP-control and iβARK slices. (J) Pair-wise quantifications of the effect of NE on fEPSP slope and PPF for the experiments shown in (H) and (I). (K) Plots summarizing the effect of NE on fEPSP slope in RFP-control and iβARK slices. (L) Correlation between the effect of 20μM NE on astrocyte peak Ca²⁺ responses and fEPSP slope across three methods of astrocyte silencing and RFP-controls (see SupFig.3, SupFig.4 and Table S1). Data are shown as mean ± s.e.m.