Insulin signaling controls neurotransmission via the 4eBP-dependent modification of the exocytotic machinery
- University of Texas Health Sciences Center at San Antonio, United States
Figures
Figure 1
Effects of dietary protein concentrations on neurotransmission at the CM9 NMJ.
(A) Diagram of Drosophila head indicating the location of the Cibarial Muscle 9 (CM9). (B) 21-day feeding paradigm used for the analysis of dietary effects on neurotransmission. Animals were raised …
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Figure 1—source data 1
File contains the values represent the average value for the spontaneous release events per second determined in 5 s increments during the hypertonic stimulation of synaptic vesicle fusion at CM9 NMJs in animals raised on a 1X or 2X diet presented in Figure 1J.
Values for each animal (n = 7) are shown with time bins indicating time relative to the application of hypertonic recording solution. The results of Student’s t-tests for each 5-s time bin and the Kolmogorov-Smirnov test of the distributions are presented with data set.
- https://doi.org/10.7554/eLife.16807.004
Figure 2 with 2 supplements
Insulin/DILP signaling negatively regulates presynaptic release at the CM9 NMJ.
(A) Images from a proboscis extension reflex (PER) in response to tarsal stimulation with 0.5 M sucrose. Circle indicates location of sensory bristles tracked during the extension event resulting in …
Figure 2—figure supplement 1
Effects of diet and neuronal insulin signaling on SV exocytosis at larval NMJ.
(A) Representative evoked EPSP and spontaneous mEPSP traces from larval muscle 6 in larval of indicated genotype and diet conditions. In these experiments, larvae were on indicated diets for the …
Figure 2—figure supplement 2
Cycloheximide blocks the effects of diet switch on SV exocytosis.
(A) Model of the translational regulation of SV exocytosis by insulin signaling via 4eBP. (B) 21-day diet shift paradigm for testing the effects of cycloheximide on neurotransmission. Flies were …
Figure 3
Effects of diet on the release of neurotransmitter requires FOXO.
(A) Diagram depicts the regulation of 4eBP by either FOXO-dependent transcription or dTOR-dependent phosphorylation. (B) Relative mRNA expression levels of 4eBP in purified motor neurons from …
Figure 4
Effects of diet on the release of neurotransmitter is independent of dTOR.
(A) Diagram depicts the regulation of 4eBP by either FOXO-dependent transcription or dTOR-dependent phosphorylation indicating the effects of rapamycin. (B) To investigate the effect of rapamycin …
Figure 5
The role of Staufen during the regulation of neurotransmission by diet.
(A) Representative traces of EPSPs from CM9 NMJs from 21 day old staufenRNAi raised on a 1X diet or staufenRNAi and control flies subjected to a diet switch from 1X to 2X diet on day 20 and recorded …
Figure 6 with 1 supplement
Complexin levels regulate SV release in response to diet.
(A) Immunofluorescent images of CM9 NMJs from animals raised on a 1X (left panels) or 2X (right panels) diet co-stained for Complexin (red-upper panels and lower panels), Discs-large (Dlg, green …
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Figure 6—source data 1
File contains background-corrected values of max pixel intensity from complexin (Cpx) staining at the CM9 NMJ from indicated genotypes and diet conditions.
Included are data for quantification max pixel intensities for the Cpx staining presented in Figure 6C,D,J, and Figure 6—figure supplement 1. Data are presented in separate sheets as labeled. The results of statistical analyses (Student’s t-test, Kolmogorov-Smirnov test) are presented with each data set.
- https://doi.org/10.7554/eLife.16807.013
Figure 6—figure supplement 1
Diet effects on synaptic complexin levels at the lateral abdominal muscle NMJs.
(A) Diagram represents the area of the abdomen that is being analyzed (grey box). Image shows the NMJs on the lateral abdominal muscles (LAMs) used in these analyses. Staining for Dlg is shown and …
Tables
Table 1
Quantal analysis of neurotransmission at the CM9 NMJ.
| Genotype (condition) | Diet | N | mEPSP (mV) | EPSP (mV) | QC | RMP (mV) | IR (MΩ) |
|---|---|---|---|---|---|---|---|
| w1118 | 1X | 8 | 0.94 ± 0.04 | 3.46 ± 0.30 | 3.66 ± 0.28 | −40.89 ± 1.37 | 7.56 ± 0.80 |
| w1118 | 2X | 8 | 0.83 ± 0.04 | 1.65 ± 0.08 | 2.01 ± 0.12 | −39.67 ± 0.57 | 7.00 ± 0.80 |
| w1118 (12 hr shift) | 1-2X | 8 | 0.88 ± 0.02 | 2.74 ± 0.21 | 3.12 ± 0.24 | −38.40 ± 2.17 | 8.13 ± 1.01 |
| w1118 (24-hr shift) | 1-2X | 8 | 0.89 ± 0.03 | 2.25 ± 0.27 | 2.58 ± 0.27 | −35.65 ± 1.53 | 8.75 ± 0.62 |
| E49-Gal4/+ | 1X | 8 | 0.96 ± 0.03 | 3.40 ± 0.16 | 3.55 ± 0.16 | −35.53 ± 3.24 | 7.48 ± 0.55 |
| E49-Gal4/+ | 2X | 8 | 0.92 ± 0.02 | 2.06 ± 0.09 | 2.23 ± 0.07 | −32.24 ± 0.83 | 7.12 ± 0.58 |
| UAS-4eBPRNAi/+ | 1X | 8 | 0.94 ± 0.04 | 3.31 ± 0.33 | 3.50 ± 0.30 | −41.02 ± 1.40 | 7.88 ± 0.79 |
| UAS-4eBPRNAi/+ | 2X | 8 | 0.83 ± 0.02 | 1.62 ± 0.12 | 1.98 ± 0.17 | −39.67 ± 0.57 | 8.25 ± 0.82 |
| UAS-4eBPRNAi/+ | 1-2X | 8 | 0.93 ± 0.01 | 1.90 ± 0.05 | 2.05 ± 0.07 | −37.96 ± 0.54 | 8.25 ± 0.62 |
| E49-Gal4/+; UAS-4eBPRNAi/+ | 1X | 7 | 0.92 ± 0.02 | 1.50 ± 0.06 | 1.64 ± 0.07 | −38.88 ± 0.68 | 9.14 ± 0.77 |
| E49-Gal4/+; UAS-4eBPRNAi/+ | 2X | 8 | 0.91 ± 0.03 | 1.67 ± 0.13 | 1.86 ± 0.19 | −38.13 ± 0.50 | 8.00 ± 1.00 |
| E49-Gal4/+; UAS-4eBPRNAi/+ | 1-2X | 8 | 0.94 ± 0.02 | 1.79 ± 0.10 | 1.90 ± 0.11 | −38.68 ± 0.67 | 8.75 ± 0.62 |
| UAS-chicoRNAi/+ | 1X | 8 | 0.90 ± 0.04 | 3.56 ± 0.33 | 3.96 ± 0.30 | −37.31 ± 1.49 | 8.63 ± 0.30 |
| UAS-chicoRNAi/+ | 2X | 8 | 0.83 ± 0.02 | 1.74 ± 0.10 | 2.10 ± 0.15 | −39.45 ± 0.47 | 8.25 ± 0.73 |
| E49-Gal4/UAS-chicoRNAi | 1X | 8 | 0.92 ± 0.03 | 4.60 ± 0.30 | 5.09 ± 0.43 | −34.75 ± 1.07 | 8.31 ± 0.47 |
| E49-Gal4/UAS-chicoRNAi | 2X | 8 | 0.95 ± 0.05 | 4.09 ± 0.28 | 4.34 ± 0.24 | −38.52 ± 4.49 | 8.40 ± 0.77 |
| E49-Gal4/UAS-chicoRNAi | 1-2X | 8 | 0.91 ± 0.04 | 4.02 ± 0.23 | 4.48 ± 0.28 | −35.47 ± 2.82 | 8.75 ± 0.68 |
| E49-Gal4/UAS-chicoRNAi; UAS-4eBPRNAi/+ | 2X | 8 | 0.84 ± 0.05 | 2.18 ± 0.12 | 2.60 ± 0.13 | −39.51 ± 1.96 | 7.75 ± 0.85 |
| UAS-InRDN/+ | 2X | 8 | 0.86 ± 0.01 | 2.07 ± 0.17 | 2.42 ± 0.21 | −32.19 ± 1.55 | 8.06 ± 0.79 |
| E49-Gal4/UAS-InRDN | 2X | 8 | 0.85 ± 0.03 | 2.87 ± 0.15 | 3.43 ± 0.24 | −35.92 ± 2.20 | 8.69 ± 0.54 |
| w1118 | 1X | 8 | 0.92 ± 0.02 | 3.25 ± 0.25 | 3.53 ± 0.26 | −34.61 ± 1.77 | 7.88 ± 0.69 |
| w1118 | 2X | 8 | 0.84 ± 0.03 | 1.82 ± 0.10 | 2.18 ± 0.14 | −36.62 ± 1.14 | 8.50 ± 0.80 |
| w1118 (+CXM) | 1-2X | 8 | 0.99 ± 0.04 | 4.31 ± 0.20 | 4.39 ± 0.26 | −40.58 ± 1.84 | 7.88 ± 0.69 |
| w1118 (+Veh (CMX)) | 1-2X | 8 | 0.95 ± 0.02 | 2.50 ± 0.11 | 2.63 ± 0.11 | −40.01 ± 2.56 | 8.00 ± 0.68 |
| w1118 (+CXM) | 1X | 8 | 1.04 ± 0.03 | 4.23 ± 0.23 | 4.09 ± 0.25 | −39.09 ± 0.89 | 9.00 ± 0.82 |
| w1118 (+rapamycin) | 1-2X | 8 | 0.86 ± 0.03 | 2.11 ± 0.13 | 2.48 ± 0.22 | −41.29 ± 1.19 | 7.63 ± 0.78 |
| w1118 (+Veh (rapa)) | 1-2X | 8 | 0.82 ± 0.03 | 2.05 ± 0.20 | 2.54 ± 0.29 | −39.87 ± 1.84 | 6.88 ± 0.61 |
| w1118 | 1X | 8 | 0.89 ± 0.03 | 3.37 ± 0.20 | 3.81 ± 0.27 | −31.25 ± 1.47 | 8.25 ± 0.75 |
| w1118 | 2X | 5 | 0.95 ± 0.03 | 1.91 ± 0.16 | 2.00 ± 0.13 | −34.05 ± 1.48 | 7.80 ± 0.97 |
| dFOXOdel94/dFOXO21 | 1X | 8 | 0.94 ± 0.02 | 1.98 ± 0.13 | 2.10 ± 0.13 | −34.60 ± 1.35 | 8.44 ± 0.48 |
| dFOXOdel94/dFOXO21 | 2X | 8 | 0.96 ± 0.03 | 1.69 ± 0.09 | 1.77 ± 0.13 | −38.21 ± 1.52 | 7.75 ± 0.75 |
| dFOXOdel94/dFOXO21 | 1-2X | 8 | 0.94 ± 0.01 | 1.58 ± 0.04 | 1.68 ± 0.03 | −34.54 ± 2.06 | 8.69 ± 0.74 |
| dFOXOdel94 / dFOXO21 , UAS-4eBP | 1X | 8 | 0.94 ± 0.04 | 1.95 ± 0.23 | 2.06 ± 0.19 | −31.36 ± 2.83 | 7.38 ± 0.74 |
| E49-Gal4/+; dFOXOdel94 / dFOXO21 , UAS-4eBP | 1X | 8 | 0.88 ± 0.04 | 3.28 ± 0.22 | 3.85 ± 0.43 | −31.83 ± 2.65 | 6.88 ± 0.75 |
| UAS-4eBP/+ | 1X | 8 | 0.96 ± 0.04 | 3.30 ± 0.16 | 3.47 ± 0.16 | −33.08 ± 1.03 | 7.79 ± 0.38 |
| E49-Gal4/+;UAS-4eBP/+ | 1X | 8 | 0.92 ± 0.03 | 4.79 ± 0.38 | 5.25 ± 0.46 | −34.77 ± 2.12 | 8.08 ± 0.58 |
| E49-Gal4/UAS-stauenRNAi | 1X | 8 | 0.89 ± 0.03 | 3.37 ± 0.20 | 3.81 ± 0.27 | −36.32 ± 1.22 | 8.32 ± 0.66 |
| E49-Gal4/UAS-staufenRNAi | 1-2X | 8 | 0.95 ± 0.04 | 3.45 ± 0.21 | 3.65 ± 0.16 | −39.64 ± 2.42 | 7.55 ± 0.32 |
| +/UAS-staufenRNAi | 1-2X | 9 | 0.90 ± 0.04 | 2.35 ± 0.13 | 2.68 ± 0.20 | −35.51 ± 1.21 | 8.02 ± 0.73 |
| W1118 | 1X | 8 | 0.93 ± 0.02 | 3.21 ± 0.19 | 3.47 ± 0.22 | −30.56 ± 1.49 | 8.31 ± 0.09 |
| W1118 | 2X | 8 | 0.95 ± 0.02 | 1.96 ± 0.15 | 2.07 ± 0.17 | −30.43 ± 1.20 | 8.06 ± 0.67 |
| +/+,cpxSH1/+ | 1X | 8 | 0.91 ± 0.01 | 4.23 ± 0.48 | 4.66 ± 0.51 | −32.32 ± 1.40 | 7.88 ± 0.74 |
| +/+,cpxSH1/+ | 2X | 8 | 0.96 ± 0.01 | 2.65 ± 0.30 | 2.75 ± 0.32 | −31.26 ± 2.87 | 7.94 ± 0.83 |
| UAS-Complexin/+ | 1X | 9 | 0.99 ± 0.05 | 4.15 ± 0.46 | 4.37 ± 0.56 | −36.12 ± 1.65 | 6.67 ± 0.67 |
| UAS-Complexin/+ | 2X | 9 | 0.95 ± 0.02 | 2.39 ± 0.19 | 2.54 ± 0.24 | −31.27 ± 1.99 | 7.22 ± 0.80 |
| E49-Gal4/UAS-Complexin | 1X | 9 | 0.90 ± 0.05 | 2.22 ± 0.28 | 2.48 ± 0.28 | −30.59 ± 1.97 | 6.79 ± 0.73 |
| E49-Gal4/UAS-Complexin | 2X | 9 | 0.98 ± 0.05 | 2.58 ± 0.21 | 2.63 ± 0.18 | −34.86 ± 2.42 | 7.72 ± 0.52 |
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Table contents ordered by order of appearance in body of text. All values represent the average value ± sem (N = animals, 1 recording per animal). For each recording, the EPSP value represents the average of 60 evoked responses and the value for mEPSP represents the average of 30 events. All stocks were backcrossed five generations and re-established in the w1118 background. Quantal content (QC) is determined for each NMJ by dividing the amplitude of the EPSP by the amplitude of the mEPSP for each recording. RMP = resting membrane potential of CM9 muscle fiber. IR = depolarizing input resistance of CM9 muscle fiber.
