Isoflurane decreases transmitter release. (A) A schematic of the Drosophila larval neuromuscular junction (NMJ) recording preparation is shown. Ground for stimulation wire (gray) and recording ground wire shielded with nail polish (black) plus perfusion set up (blue) with the vacuum (orange) is shown. The recording electrode (red) is impaled into muscle 6 of segment 3 of dissected third-instar larvae. A stimulating electrode (green) encases the nerve, which innervates the muscle. Dashed lines represent a more detailed schematic of muscle 6 synaptic boutons (purple arrowheads) which release glutamate onto the muscle. Not all muscles or nerves are shown for simplicity, and the brain is shown in this schematic for illustration purposes only (brain is removed when recording). (B) i: Time course of NMJ experiment, showing duration of the recording (in min) with red arrow denoting when isoflurane perfusion begins, and green arrow denoting time window when isoflurane perfusion data was sampled. ii: Example recording trace, showing evoked excitatory junctional potentials (EJPs, black arrowhead) which are recorded when the stimulus is presented (stimulus trace shown below recording). Spontaneous miniature EJPs (mEJPs, gray arrowhead) are also shown. (C) Example recording traces from wild-type (Canton-S, [CS]) NMJs showing EJPs before isoflurane perfusion (i) and during isoflurane perfusion (ii), with stimulus traces shown. (D) Two example recording traces from wild-type (CS) NMJs showing mEJPs before isoflurane perfusion (i) and during isoflurane perfusion (ii). (E) Normalized EJP amplitude in wild-type (CS) before (solid bar) and after 6 min of isoflurane perfusion (shaded bar). *P < 0.05, one-way ANOVA with Dunnett multiple comparisons test. (F) Normalized mEJP amplitude in wild-type (CS) before (solid bar) and after 6 min of isoflurane perfusion (shaded bar). Data represent >700 individual measures. ns = not significant. (G) Normalized quantal content before (solid bars) and after 6 min of isoflurane perfusion (shaded bars) in CS (red), isoCJ1 (blue), and syxH3-C (black). Quantal content is calculated by dividing the mean EJP amplitude by the mean mEJP amplitude. *P < 0.05, one-way ANOVA with Dunnett multiple comparisons test. Quantal content with isoflurane perfusion is significantly higher in syxH3-C compared with isoCJ1. *P < 0.05, two-way ANOVA with Sidak multiple comparison test. Data in E to G represent the mean ± SEM from: CS (n = 12), isoCJ1 (n = 8), and syxH3-C (n = 8).