Fig. 6.

Differential effects of isoflurane on synaptic vesicle exocytosis in glutamatergic and GABAergic neurons is proportional to reduction Ca²⁺ influx. (A) Representative live-cell image of syn-pH-positive boutons (green, Left), GABAergic boutons immunolabeled using vGAT–Oyster 550 (red, Center), and merge shown as yellow (Right), with surrounding vGAT-positive boutons (red, Right) from untransfected neurons. (Scale bar: 20 μm.) (B Left) The effect of 0.70 ± 0.06 mM isoflurane on single AP exocytosis is greater in glutamatergic (vGAT-negative; 62 ± 3% block, n = 11) than in GABAergic (vGAT-positive; 38 ± 3% block, n = 5) boutons at 4 mM extracellular [Ca²⁺]_e, ***P < 0.001 by unpaired t test. (Insets) Exemplar single AP stimulus (arrow) syn-pH fluorescence traces before (black) and after (red) isoflurane in glutamatergic (Left) and GABAergic (Right) boutons. (Scale bars: 0.5% TRP, 100 ms.) (B, Right) Effect of isoflurane on presynaptic Ca²⁺ influx is greater in glutamatergic (26 ± 2% block, n = 7) than in GABAergic (9.2 ± 2% block, n = 7) boutons at 4 mM extracellular [Ca²⁺]_e, ***P < 0.001 by unpaired t test. (Insets) Exemplar single AP stimulus (arrow) syn-GCaMP3 fluorescence traces before (black) and after (red) isoflurane in glutamatergic and GABAergic boutons. (Scale bars: 30% ∆F/F₀, 200 ms.) (C) Effect of 0.70 ± 0.06 mM isoflurane on exocytosis as a function of Ca²⁺ influx in glutamatergic and GABAergic boutons. Exocytosis determined using syn-pH in response to single AP relative to TRP is plotted against relative increases in Ca²⁺ influx determined using syn-GCaMP3 over a range of [Ca²⁺]_e from 1.5 to 10 mM under similar conditions to yield the Ca²⁺ sensitivity of exocytosis (n = 26 for exocytosis, n = 24 for Ca²⁺ influx). The curve indicates the data from Fig. 5A. (D) Cumulative frequency distribution of 0.70 ± 0.06 mM isoflurane inhibition of peak [Ca²⁺]_i for GABAergic and glutamatergic boutons. Data from 272 boutons collected in 12 experiments.