Proportional Downscaling of Glutamatergic Release Sites by the General Anesthetic Propofol at Drosophila Motor Nerve Terminals

Propofol decreases glutamate release similarly from different synapse types. A, Left, Schema of Drosophila larval NMJ preparation. The black structure is the brain, rectangles are muscle segments. Middle, Muscle 6 motor nerve terminals labeled with HRP to identify neurons and nc82 to identify active zones. 1s and 1b boutons are indicated. Scale bar, 4 μm. Right, Diagram illustrating the configuration for focal macropatch recording from single 1s and 1b boutons at the fly larval NMJ. B, Example traces of EJCs and mEJCs (inset) recorded from 1s and 1b boutons in the CS strain. C, Chemical structures of propofol (1,6-diisopropylphenol) and its nonanesthetic analog, 2,4-diisopropylphenol. D, Normalized QC in drug-treated preparations (3 μm) for both bouton types. Parameters were normalized to their respective averages in non-drug-treated controls (dashed line). Raw data are presented in Extended Data Figure 1-1. *p < 0.05; Student’s t test. Graphs show individual data points (animals), and error bars show the average ± SEM. E, Cumulative plot of quanta released per stimulus, for both bouton types and both drugs.

The upper limit for n is set by the number of T-bars. A, Schema of release sites at a synapse. Active release sites (*) contribute to the average release p. B, Electron micrographs (EMs) of a 1s (top) and a 1b (bottom) bouton, with T-bars (synapses) indicated with arrowheads. C, Average number (±SEM) of release sites for 1s and 1b boutons, determined from the T-bar counts from the EM data. See Materials and Methods, and Extended Data Figure 2-1 for other examples. D, [Ca²⁺]_e was raised from 1.0 to 1.5 mm in focal macropatch recordings in CS larvae. Right, Sample recordings from single 1s and 1b boutons revealed increases in the amplitudes of EJCs and mEJCs at both bouton types under higher calcium concentrations. E, Average EJC (±SEM) for 1s and 1b recordings. F, Average mEJC (±SEM) for 1s and 1b recordings. G, As [Ca²⁺]_e was raised, calculations of QCs revealed no increase in 1s QC, whereas there was an increase in 1b QC. H, I, There were no increases in p (H) or in n (I) at 1s boutons, whereas there were increases in both p (H) and n (I) at 1b boutons, contributing to the increase in 1b QC (G). J, We observed that at 1s boutons, all the release sites were already activated at 1.0 mm [Ca²⁺]_e, with no additional increase being observed when [Ca²⁺]_e was further raised. At 1b boutons, not all of the release sites were activated at 1.0 mm [Ca²⁺]_e, whereas they were all activated when [Ca²⁺]_e was further raised. K, We generated a graph to test whether values of n approached the number of T-bars per bouton as [Ca²⁺]_e was raised, by normalizing n (Fig. 2I) to the number of T-bars per bouton (Fig. 2C). The dashed line at 1.0 is representative of when n equates to the number of T-bars per bouton. These results indicate that n is limited by the number of T-bars per bouton type. *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t test. 1.0 mm [Ca²⁺]_e data are the same as in Figure 1, shown here for comparison. Graphs show individual data points (animals), and all error bars are ±SEM.

Propofol decreases the number of active release sites at synapses. A, Estimated number of active release sites (n; see Materials and Methods) in 1s and 1b boutons, for control and drug-treated conditions, in CS animals. B, Estimated release probability (p, see “Materials and Methods”) in 1s and 1b boutons, for control and drug-treated conditions, in CS animals. C, Data from A and B, normalized to non-drug-treated controls (dashed line). Graphs in A–C are averages ± SEM; individual data points (animals) are shown. *p < 0.05; **p < 0.01; ***p < 0.05, Student’s t test. D, Quantal content plotted against n, from all 1s and 1b recordings in control and drug conditions. E, Rank-ordered plot of n for drug versus control conditions. Dashed line indicates no effect of drug on n. F, Rank-ordered plot of p for drug versus control conditions. Dashed line indicates no effect of drug on p.