Figure 2.

ArchT3.0 Activation in CA3-CA1 Axons Results in pH Transients that Are Restricted to Expressing Boutons

(A) Two-photon bouton pH imaging procedure. Upper: ArchT3.0-eYFP-expressing boutons (white arrows) were identified as boutons showing fluorescence at both 800 nm (green; 8-hydroxypyrene-1,3,6-trisulfonic acid [HPTS)] and 980 nm (red; eYFP). Lower left: calibration curve showing the measured HPTS fluorescence ratio (with a 525-nm filter) when exciting at 900 and 750 nm (two-photon) plotted at known pH values and fit with a sigmoidal function. Lower right: green (532-nm single-photon) light was delivered at 2 mW for 2 min. Fluorescence was measured with a 525-nm filter at 900 and 750 nm (two-photon) excitation immediately before light delivery (pre-light), immediately after light cessation (post-light), and 6 min after light cessation (post-recovery). The 900/750 fluorescence ratio was then used to calculate intra-bouton pH in each condition.

(B) Pre-light, post-light, and post-recovery pH measurements in ArchT3.0-eYFP-expressing CA3-CA1 boutons, neighboring non-expressing processes in the CA1 stratum radiatum, and ArchT3.0-eYFP-expressing CA3 somata.

(C) Plot showing correlation between estimated bouton surface-area-to-volume ratio and light-induced percentage change in bouton [H⁺] for ArchT3.0-eYFP-expressing boutons.

Error bars represent mean ± SEM.