Article Figures & Data
Figures
- Figure 1.
Isoflurane impairs neuroexocytosis at cholinergic synapses in the adult Drosophila brain. A, Isoflurane anesthesia, equivalent to a 1.5% concentration, was induced in female Drosophila flies in an air-tight chamber for 15 min. The control condition consisted of only air. Brains were then dissected and mounted and imaged using a standard protocol of 10 s baseline, 2 min stimulation, and 5 min recovery. B, Schematic of synaptopHluorin activity in response to calcium-triggered SNARE-mediated release via CsChrimson activation. C, Neural skeleton of MB543B-Gal4 derived from FIB-SEM Drosophila brain volume (left) with a close-up of the synapses (right). Green arrow matches synaptopHluorin expression in D. D, MB543B-Gal4 expression pattern in the Drosophila brain immunostained for the presynaptic marker bruchpilot (nc82), image taken from Janelia FlyLight split-Gal4 collection. Confocal images of UAS-synaptopHluorin (top), UAS-CsChrimson-mCherry (middle), and their overlap (bottom) expressed in the cholinergic split-Gal4 (MB543B-Gal4). SynaptopHluorin expression is enriched in synapses (green arrow) but not in cell bodies or extrasynaptic compartments (red arrows). E, Standard deviation Z-projection of the synaptopHluorin fluorescence change observed during CsChrimson activation (left). An ROI selection is applied to individual release sites for analysis (right). F, Comparison of synaptopHluorin fluorescence changes under control (left) or isoflurane (right) during CsChrimson activation. Isoflurane decreases the density of release areas and peak fluorescence from the baseline. G, The average total neuron area between control and isoflurane conditions was not significantly different (n = 9 control; n = 8 isoflurane; p = 0.277; rank biserial correlation, r = −0.33; Mann–Whitney U test; ±SEM). H, Activity traces of synaptopHluorin release in control (black) and isoflurane (red) conditions during and after CsChrimson activation, normalized to the baseline and averaged over 10 s segments. Each trace is from a different fly. I, Total relative activity (average change in fluorescence per µm2) was significantly decreased under isoflurane anesthesia (p = 0.022; rank biserial correlation, r = −0.68; Mann–Whitney U test, ±SEM). J, In addition to decreased release, the density of release sites (number of ROIs relative to total neuron area in µm2) was significantly decreased under isoflurane anesthesia (p = 0.0206; rank biserial correlation, r = −0.667; Mann–Whitney U test; ±SEM).
- Figure 2.
Isoflurane affects release from smaller but not larger active zones. A, Top, Baseline fluorescence of synaptopHluorin in cholinergic synapses with (middle) the standard deviation of release during CsChrimson activation. Bottom, Classification of release sites into small (<0.08 µm2), medium (0.09–0.42 µm2), and large (>0.43 µm2). Classifications based on k-means clustering of individual release areas (scale bar, 5 µm). B, Average fluorescence traces normalized to the baseline for the control (left) and isoflurane (right) for each of the different release area groups. No significant difference in the activity between the control and isoflurane for each group. C, Frequency of each release group between control and isoflurane showed no significant difference in the absolute number of groups within each condition (absolute, small, F(1,18) = 1.89; p = 0.19; medium, F(1,18) = 0.41; p = 0.53; large, F(1,18) = 0.53; p = 0.48) or the relative frequency (relative, small, F(1,18) = 0.07; p = 0.80; medium, F(1,18) = 0.01; p = 0.94; large, F(1,18) = 0.15; p = 0.70; ANOVA). All data ± SEM. D, Relative activity for small (left), medium (middle), and large (right) release groups. Only the small and medium release groups showed a significant decrease in activity between control and isoflurane conditions (small, t(14) = 2.48; p = 0.0339; medium, t(14) = 2.38; p = 0.039). The large release group did not show a significant difference in activity (large, t(14) = 0.085; p = 0.934; ANOVA). All data ± SEM.
- Figure 3.
Sx1a mobility dynamics are altered by neuronal Ca2+ stimulation and isoflurane anesthesia in cholinergic synapses. A, Schematic of the SNARE complex proteins with an mEos3.2 photoconvertible tag attached to the extracellular C-terminus of Sx1a (Sx1a-mEos3.2). B, Low-resolution unphotoconverted Sx1a-mEos3.2 in cholinergic synapses (left), super-resolution photoconverted Sx1a-mEos3.2 (middle, highlighted inset of a single molecule), and trajectories from analyzed Sx1a-mEos3.2 molecules. C, CsChrimson activation of cholinergic synapses increases the MSD and the AUC of Sx1a-mEos3.2 molecules (n = 11 baseline; n = 13 stimulated; p = 0.026; rank biserial correlation, r = 0.538; Mann–Whitney U test; ±SEM). D, Sx1a-mEos3.2 were relieved from clusters under CsChrimson activation (left, p = 0.02; rank biserial correlation, r = −0.574; Mann–Whitney U test) in line with an increase of mobility but did not significantly alter the time spent in clusters (middle, p = 0.208; rank biserial correlation, r = −0.32; Mann–Whitney U test) or the number of trajectories per cluster (right, p = 0.203; rank biserial correlation, r = −0.323; Mann–Whitney U test). All data ± SEM. E, Isoflurane anesthesia significantly decreased the mobility of Sx1a-mEos3.2 in cholinergic synapses in response to CsChrimson activation (n = 11 control; n = 10 isoflurane; p = 0.0008; rank biserial correlation, r = −0.818; Mann–Whitney U test; ±SEM). F, Clustering phenotype of Sx1a-mEos3.2 under isoflurane anesthesia was significantly altered. The cluster area (left, p = 0.022; rank biserial correlation, r = 0.6; Mann–Whitney U test), lifetime (middle, p = 0.0079; rank biserial correlation, r = 0.673; Mann–Whitney U test), and number of trajectories per cluster (right, p = 0.0051; rank biserial correlation, r = 0.70; Mann–Whitney U test) all significantly increased during CsChrimson activation and isoflurane anesthesia. All data ± SEM. G, Segment overlap plots generated by segNASTIC highlighting increased clustering in the isoflurane condition around boutons (indicated by white arrows).
- Figure 4.
TeTx-LC disruption of cholinergic synapse development impairs Sx1a-mEos3.2 mobility. A, Schematic of the SNARE complex with Sx1a-mEos3.2 and TeTx-LC cleavage of VAMP2. B, Expression patterns of Sx1a-mEos3.2 within the synapses of the MB543B-Gal4 cholinergic MBON in low resolution (top) and super resolved (bottom) in 3–5-d-old female fruit fly brains (scale bar, 5 µm). C, Expression pattern of Sx1a-mEos3.2 as in B but with developmental TeTx-LC expression (scale bar, 5 µm). D, MSD analysis of Sx1a-mEos3.2 for unclustered trajectories reveals a significant decrease in mobility with TeTx-LC cleavage of VAMP2, resulting from disordered synaptic organization (control, n = 12; TeTx-LC, n = 11). E, The average instantaneous diffusion of both clustered and unclustered Sx1a-mEos3.2 trajectories reveals a significant decrease in diffusion of the unclustered population without affecting the mobility within clusters (clustered control, ns p = 0.2351; rank biserial correlation, r = 0.3; Mann–Whitney U test; unclustered control, p = 0.00025; rank biserial correlation, r = −0.91; Mann–Whitney U test). F, The number of clustered to unclustered trajectories significantly increased with TeTx-LC (p = 0.00025; rank biserial correlation, r = 0.91; Mann–Whitney U test). G–I, Alongside an increase in cluster number, TeTx-LC also significantly increased the cluster area (p = 0.009; rank biserial correlation, r = 0.636), radius (p = 0.009; rank biserial correlation, r = 0.636), and density of Sx1a-mEos3.2 of trajectories in cholinergic synapses (p = 0.0006; rank biserial correlation, r = 0.848; Mann–Whitney U test). All data ± SEM.
- Figure 5.
Rop experiences lateral trapping and clustering under isoflurane anesthesia in cholinergic neurons. A, Schematic showing the SNARE complex with Rop tagged with an mEos3.2 photoconvertible tag on its C-terminus. B, Low-resolution (left), super-resolution photoconverted (middle), and trajectories (right) of Munc18-mEos3.2 molecules in cholinergic synapses using the split MB543B-Gal4. C, The MSD and AUC for Munc18-mEos3.2 mobility during CsChrimson activation significantly decreased under isoflurane anesthesia (n = 11 control; n = 10 isoflurane; p = 0.0295; rank biserial correlation, r = −0.56; Mann–Whitney U test; ±SEM). D, In conjunction with a decrease in mobility, Munc18-mEos3.2 molecules experienced a significant increase in the cluster area consistent with Sx1a-mEos3.2 (p = 0.01; rank biserial correlation, r = 0.7; Mann–Whitney U test; ±SEM).
- Figure 6.
Isoflurane fails to cluster Sx1a in extrasynaptic compartments of cholinergic neurons. A, Neural skeleton of MB543B-Gal4 derived from FIB-SEM Drosophila brain volume (left) with a close-up of the extrasynapses expressing Sx1a-mEos3.2 (right). Red arrow indicates the area of the close-up. B, Isoflurane anesthesia is able to significantly restrict CsChrimson-activated Sx1a-mEos3.2 mobility in the absence of synaptic architecture by decreasing the MSD and AUC (n = 9 control; n = 8 isoflurane; p = 0.0006; rank biserial correlation, r = −0.92; Mann–Whitney U test; ±SEM). C, Compared to the synaptic compartment, isoflurane was unable to alter the clustering dynamics of Sx1a-mEos3.2 in the extrasynapse. No significant change in the cluster area (left, p = 0.37; rank biserial correlation, r = 0.28; Mann–Whitney U test), lifetime (middle, p > 0.999; rank biserial correlation, r = 0; Mann–Whitney U test), or number of trajectories per cluster (right, p = 0.541; rank biserial correlation, r = 0.194; Mann–Whitney U test) was observed. All data ± SEM. D, Comparison between the number of clusters in the presence of isoflurane between synaptic and extrasynaptic compartments was significantly lower for extrasynapses (p = 0.043; rank biserial correlation, r = −0.575; Mann–Whitney U test); however, a number of trajectories that were clustered between the two were not significantly different (p = 0.203; rank biserial correlation, r = 0.375; Mann–Whitney U test). All data ± SEM.
- Figure 7.
Sx1a dynamics are unaffected by isoflurane in GABAergic and glutamatergic MBONs. A, A GABAergic MBON driven by MB112C-Gal4, with imaging, is indicated (synapses). B, Sx1a-mEos3.2 trajectories for MB112C-Gal4 (GABA) inhibitory synapses. C, Sx1a-mEos3.2 MSD, AUC, and the cluster area are unaffected by isoflurane anesthesia in GABA synapses (n = 8 control; n = 7 isoflurane; AUC, p = 0.728; rank biserial correlation, r = 0.125; cluster area, p = 0.867; rank biserial correlation, r = 0.071; Mann–Whitney U test). All data ± SEM. D, A glutamatergic MBON driven by MB433B-Gal4, with imaging, is indicated (synapses). E, Sx1a-mEos3.2 trajectories for MB433B-Gal4 (Glut) inhibitory synapses. F, Sx1a-mEos3.2 MSD, AUC, and the cluster area are unaffected by isoflurane anesthesia in glutamatergic inhibitory synapses (n = 8 control; n = 7 isoflurane; AUC, p = 0.955; rank biserial correlation, r = 0.036; cluster area, p = 0.054; rank biserial correlation, r = −0.607; Mann–Whitney U test). All data ± SEM.
- Figure 8.
Synaptic release in GABAergic and glutamatergic MBONs is unaffected by isoflurane. A, Total relative fluorescence and activity from GABAergic synapses is unaffected by isoflurane (n = 7 control and isoflurane; p = 0.901; rank biserial correlation, r = 0.06; Mann–Whitney U test). All data ± SEM. B, The relative ROI density was unaffected by isoflurane in GABA synapses (p = 0.44; rank biserial correlation, r = 0.25; Mann–Whitney U test; ±SEM). C, Isoflurane anesthesia’s impact on the relative distribution of ROI sizes was assessed across small, medium, and large groups, revealing no significant differences (small group, F(1,12) = 0.011; p = 0.918; medium group, F(1,12) = 1.500; p = 0.244; large group, F(1,12) = 0.883; p = 0.366; ANOVA). All data ± SEM. D, Like GABA synapses, release from glutamatergic synapses is also unaffected by isoflurane anesthesia (n = 7 control and isoflurane; p = 0.71; rank biserial correlation, r = −0.14; Mann–Whitney U test). All data ± SEM. E, Relative ROI density unaffected by isoflurane in glutamatergic synapses (p = 0.53; rank biserial correlation, r = 0.224; Mann–Whitney U test; ±SEM). F, Small ROIs significantly decreased under isoflurane (F(1,14) = 5.515; p = 0.037; ANOVA), with no difference in release per region size across all sizes (medium, F(1,14) = 1.259; p = 0.284; large, F(1,14) = 4.665; p = 0.052; ANOVA). All data ± SEM. G, Comparison of total relative activities for the control condition between small, medium, and large for the three neurotransmitter circuits. MB543B-Gal4 had significantly higher activity from small and medium ROIs than MB112C-Gal4 and MB433B-Gal4 (F(2,27) = 14.35; p = 0.0018; and F(2,27) = 39.83; p = 2.4 × 10−7; ANOVA). In contrast, the large ROIs showed no significant difference (F(1,18) = 0.88; p = 0.366; ANOVA). H, Same comparison as G but for the isoflurane condition. Only the medium release group for MB543B-Gal4 was significantly higher than MB112C-Gal4 or MB433B-Gal4 (F(2,12) = 5.48; p = 0.017; F(2,12) = 4.34; p = 0.01; ANOVA).
- Figure 9.
Intracellular calcium activity is not significantly affected by isoflurane. A, Synaptic domain of cholinergic MB543B-Gal4, as revealed by CsChrimson-linked mCherry (scale bar, 5 μm). B, Separation of GCaMP6s activity in the same MB543-Gal4 neuron as in A into small (<0.08 μm2), medium (0.09–0.42 μm2), and large (>0.43 μm2) ROIs. Inset, an example region with three different ROI categories. C, Average GCaMP6s fluorescence traces (±SEM) normalized to the baseline for the control (black) and isoflurane (red) conditions. Inset, average total activity for the control and isoflurane (n = 14 control; n = 14 isoflurane; p = 0.66; rank biserial correlation, r = −0.1; Mann–Whitney U test). D, Average GCaMP6s fluorescence traces (±SEM) for small (left), medium (middle), and large (right) ROIs. Inset, average total activity for each ROI category (ns, not significant; Mann–Whitney U test). E–H, As with A–D, for the GABAergic MB112C-Gal4 driver line (n = 14 control; n = 14 isoflurane; p = 0.918; rank biserial correlation, r = −0.03; Mann–Whitney U test).
- Figure 10.
Schematic of the presynaptic effect of isoflurane on three MBONs. In the presence of isoflurane, excitatory cholinergic neurotransmitter release is impaired, while glutamatergic and GABAergic release is unaffected. Decreased excitation and sustained inhibition coupled to potentiation of postsynaptic GABA receptors would lead to successive bouts of inhibition in the brain.
Movies
- Movie 1.
(Associated with Fig. 1). Recording synaptopHluorin activity from cholinergic synapses in the Drosophila brain. Left, Example of the processed imaging experiments with synaptopHluorin, highlighting the true delta fluorescence of release activity during CsChrimson activation. Middle, A surface plot of the processed recordings, highlighting the restriction of activity to individual synaptopHluorin ROIs. Right, A trace curve of fluorescence activity being measured for all detected ROIs. [view online]
- Movie 2.
(Associated with Fig. 3). Imaging Sx1a-mEos3.2 single-molecule dynamics from cholinergic, GABAergic, and glutamatergic synapses. Top, Green Sx1a-mEos3.2 images of the three neurotransmitter circuits (acetylcholine, GABA, and glutamate). Bottom, Example recordings of photoconverted single-molecule Sx1a-mEos3.2 activity in the same circuits. [view online]
- Movie 3.
(Associated with Fig. 9). Imaging GCaMP6s in CsChrimson-activated cholinergic and GABAergic circuits. Left, Activated MB543B-Gal4 (cholinergic). Right, Activated MB112C-Gal4 (GABAergic). [view online]
