Proton efflux from primary neuron–astrocyte cocultures labeled with WGA-pHRho. (A) Merged Airyscan image of the neuronal and astrocyte planes labeled with WGA-pHRho (red) and DAPI (blue). (B) Pseudocolor image showing the averaged ΔF/F₀ of the regions of interest (ROI) in A after glucose addition. Red shades indicate more activity over time. ROI are denoted by white boxes (A, astrocyte; N, neuron). Data were collected at 2 Hz. Scale bars represent 10 μm in A and B. (C) Magnified images of two astrocytes and neurons in B to show labeling coverage; 8-bit pseudo-color intensity scale bar is for panels in B and C. Scale bars represent 2 μm. (D) Cartoon depiction of the metabolic pathways expected to be affected by glucose starvation and rotenone mitochondrial poisoning. (E and F) Time courses of the (E) neuronal and (F) astrocytic pHRho signal during glucose (3 mM) and rotenone (100 µM) addition for the exemplar in B. (G) Averaged data from E and F (total number of cells in the plot: four neurons and four astrocytes from one single dish; this average is considered n = 1). Error bars represent SEM. (H) Variation of the neuronal and astrocytic responses to glucose and rotenone; 11–12 ROIs from dishes from three animals. (I) Maximal change in ΔF/F₀ (n = 4–6 dishes) for glucose and rotenone treatment, comprising a total of 19 neurons and 21 astrocytes in total; error bars represent SEM. A paired t test was used to determine significance (*, P < 0.05).