Figure 3. Axonal calcium imaging reveals that both mu and delta opioid receptors suppress presynaptic voltage-sensitive calcium channels.

(A) Two-photon image of a tdTomato-expressing basket cell filled with 30 µM Alexa 594 and 300 µM Fluo-5F in a brain slice taken from a PV-Cre; tdTom mouse. Scale bar: 50 μm. Inset shows the two axonal boutons where the line scan was carried out, with the orientation of the line scan indicated by the arrow. Scale bar: 5 μm. (B) Example of either a single action potential (AP) (left) or five APs (right) triggered in the cell body (top), and the resulting averaged, presynaptic Ca²⁺ transients, before and after application of DAMGO (top, blue, n = 8 cells, 16 boutons), SNC162 (red bottom, n = 7 cells, 14 boutons), and both drugs (top and bottom, purple). The transients are measured as the change in green signal (ΔG) , divided by G in saturating Ca²⁺ conditions (G_sat). Scale bars: top, 50 mV; bottom, x = 100 ms, y = 0.01 (left) or 0.02 (right) (ΔG/G_sat. (C) Summary of peak Ca²⁺ transients for DAMGO application in response to one AP (left) or five APs (right). One AP: BL 0.014 ± 0.001; DAMGO 0.011 ± 0.001; DAMGO+ SNC162 0.010 ± 0.001 (p = 0.042 and p = 0.0001, n = 10 pairs, Friedman test with Dunn’s multiple comparisons) five AP: BL 0.032 ± 0.004; DAMGO 0.025 ± 0.002, DAMGO+ SNC162 0.022 ± 0.003 (p = 0.076 and p = 0.0004, n = 10 pairs). (D) Summary of peak Ca²⁺ transients for SNC162 application in response to one AP (left) or five APs (right). One AP: BL 0.014 ± 0.002; SNC162 0.010 ± 0.002; SNC162+ DAMGO 0.008 ± 0.001 (p = 0.014 and p < 0.0001, n = 14 pairs, Friedman test with Dunn’s multiple comparisons). Five AP: BL 0.039 ± 0.004; SNC162 0.029 ± 0.003; SNC162+ DAMGO 0.023 ± 0.002 (p = 0.014 and p < 0.0001, n = 14 pairs).