Maintenance of homeostatic plasticity at the Drosophila neuromuscular synapse requires continuous IP3-directed signaling
- University of Iowa Carver College of Medicine, United States
- University of Iowa, United States
Figures
Figure 1
Maintenance of presynaptic homeostatic potentiation requires PLCβ, but induction does not.
(A) GluRIII knockdown induces a significant decrease in quantal size for both driver control and Plc21C knockdown genetic backgrounds. (B) EPSP amplitudes are maintained with GluRIII knockdown alone …
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Figure 1—source data 1
Raw electrophysiology data for Figure 1.
Values include EPSP amplitude in mV, resting membrane potential (RMP) in mV, mEPSP amplitude in mV, mEPSP frequency (Hz), quantal content, and quantal content corrected for non-linear summation (NLS).
- https://doi.org/10.7554/eLife.39643.003
Figure 2
IP3 sequestration blocks PHP maintenance but not PHP induction.
(A) Screen data, plotting baseline quantal content (QC, x-axis, genetic manipulation alone) versus QC of the homeostatically challenged condition (y-axis, GluRIII RNAi or GluRIIA mutant). Blue = GluR…
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Figure 2—source data 1
Raw electrophysiology data for UAS-IP3-sponge experiements in Figure 2.
Values as in Figure 1—source data 1.
- https://doi.org/10.7554/eLife.39643.005
Figure 3
IP3 sequestration does not impair NMJ growth.
(A–E) NMJs were co-stained with anti-DLG (red) and anti-Synapsin antibodies (green) to visualize synaptic boutons, with anti-HRP (blue) to visualize presynaptic membranes. Genotypes or conditions as …
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Figure 3—source data 1
Raw synapse growth data for Figure 3.
These measures include bouton counts, muscle area (µm2), and boutons per µm2.
- https://doi.org/10.7554/eLife.39643.007
Figure 4
Xestospongin C blocks PHP maintenance but not PHP induction.
Xestospongin C acutely applied to NMJs to impair IP3R function. (A) The GluRIIASP16 deletion mutation diminishes quantal size for all experimental conditions. (B) EPSP amplitudes are somewhat …
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Figure 4—source data 1
Raw electrophysiology data for Figure 4.
Values as in Figure 1—source data 1.
- https://doi.org/10.7554/eLife.39643.009
Figure 5
2-APB blocks PHP maintenance but not PHP induction.
2-APB acutely applied to NMJs to impair IP3R function. Wild-type and GluRIIA control data sets are replotted from Figure 4 for visual comparison. (A) The GluRIIASP16 deletion mutation diminishes …
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Figure 5—source data 1
Raw electrophysiology data for Figure 5.
Values as in Figure 1—source data 1.
- https://doi.org/10.7554/eLife.39643.011
Figure 6
The maintenance of PHP requires continuous RyR function, but PHP induction does not.
Ryanodine or Dantrolene acutely applied to NMJs to impair RyR function. Wild-type and GluRIIA control data sets are replotted from Figures 4 and 5 for visual comparison. (A) The GluRIIASP16 deletion …
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Figure 6—source data 1
Raw electrophysiology data for Figure 6.
Values as in Figure 1—source data 1.
- https://doi.org/10.7554/eLife.39643.013
Figure 7
There are no additive effects of genetic IP3 signaling inhibition and pharmacological RyR inhibition.
(A) GluRIII knockdown diminishes quantal size for all experimental conditions. (B) When challenged with GluRIII knockdown, EPSP amplitudes are maintained for the GAL4 driver control background but …
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Figure 7—source data 1
Raw electrophysiology data for Figure 7.
Values as in Figure 1—source data 1.
- https://doi.org/10.7554/eLife.39643.015
Figure 8
Combined pre-and postsynaptic IP3 signaling maintains PHP.
UAS-IP3-sponge transgene expression in single tissue types impairs PHP maintenance, but does not block it. IP3-sponge either in neurons (A–C) or muscle (D–F). Wild-type and GluRIIA control data sets …
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Figure 8—source data 1
Raw electrophysiology data for Figure 8.
Values as in Figure 1—source data 1.
- https://doi.org/10.7554/eLife.39643.017
Figure 9
Model depicting PLCβ/IP3R/RyR signaling underling the maintenance of PHP in both muscle and neuron.
At the Drosophila NMJ, PLCβ and effectors IP3R and RyR are required for the maintenance of HSP. Left: PLCβ signaling components depicted in both muscle and neuron at the Drosophila NMJ. We detected …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic Reagent (Drosophila melanogaster) | GluRIII[RNAi] or UAS-GluRIII[RNAi] | PMID: 25859184 | FlyBase ID:FlyBase_FBtp0110520 | UAS-pWiz transgene knocking downGluRIII gene function. This lab (CAF) is the source (Brusich et al., 2015). |
| Genetic Reagent (D. melanogaster) | Plc21C[RNAi] or UAS-Plc21C[RNAi] | Vienna Drosophila Resource Center (GD11359); PMID: 17625558 | RRID:FlyBase_FBst0456476 | UAS-RNAi transgene |
| Genetic Reagent (D. melanogaster) | Plc21C[RNAi] or UAS-Plc21C[RNAi] | Vienna Drosophila Resource Center (GD11359); PMID: 17625558 | RRID:FlyBase_FBst0456477 | UAS-RNAi transgene |
| Genetic Reagent (D. melanogaster) | UAS-IP3-sponge.m30 | PMID: 16540404 | FlyBase ID:FlyBase_FBtp0068098 | also referred to as UAS-IP3-sponge |
| Genetic Reagent (D. melanogaster) | UAS-IP3-sponge.m49 | PMID: 16540404 | FlyBase ID:FlyBase_FBtp0068099 | also referred to as UAS-IP3-sponge |
| Genetic Reagent (D. melanogaster) | GluRIIASP16 | PMID: 9427247 | RRID:BDSC_64202 | deletion allele; also referred to as GluRIIA |
| Genetic Reagent (D. melanogaster) | w1118 | PMID: 6319027 | RRID:BDSC_3605 | wild-type genetic background |
| Genetic Reagent (D. melanogaster) | elaV(C155)-Gal4 | PMID: 7917288 | RRID:BDSC_458 | also known as C155-Gal4 |
| Genetic Reagent (D. melanogaster) | Sca-Gal4 | PMID: 8893021 | FlyBase ID:FlyBase_FBtp0007534 | |
| Genetic Reagent (D. melanogaster) | BG57-Gal4 | PMID: 8893021 | FlyBase ID:FlyBase_FBti0016293 | also known as C57-Gal4 |
| Chemical Compound, Drug | Philanthotoxin-433; PhTox | Sigma-Aldrich (MilliporeSigma); Santa Cruz Biotechnology | CAS Number:(Sigma-Aldrich and Santa Cruz Biotechnology)_276684-27-6 | product P207 discontinued by Sigma-Aldrich |
| Chemical Compound, Drug | Xestospongin C | Abcam | CAS Number:Abcam_88903-69-9 | |
| Chemical Compound, Drug | 2-APB | Tocris | CAS Number:Tocris_524-95-8 | |
| Chemical Compound, Drug | Ryanodine | Tocris | CAS Number:Tocris_15662-33-6 | |
| Chemical Compound, Drug | Dantrolene | Tocris | CAS Number:Tocris_14663-23-1 | |
| Antibody | Monoclonal mouse anti-Synapsin | DSHB (3C11) | Cat#: DSHB_3C11; RRID:AB_2313867 | (1:50) |
| Antibody | Polyclonal rabbit anti-Dlg | PMID: 8893021 | (1:15,000) | |
| Antibody | Polyclonal goat anti-mouse 488 (DyLight) | Jackson ImmunoResearch | Cat #:Jackson_ 115-485-003; (no RRID) | (1:1000) discontinued; substitute with Cat# 115-485-068; RRID:AB_2338804 |
| Antibody | Polyclonal goat anti-rabbit 549 (DyLight) | Jackson ImmunoResearch | Cat#:Jackson_111-505-003; RRID:AB_2493180 | (1:2000) discontinued; substitute with Cat# 111-165-003; RRID:AB_2338000 |
| Antibody | Polyclonal goat anti-HRP (Alexa-647) | Jackson ImmunoResearch | Cat#:Jackson_123-605-021; RRID:AB_2338967 | (1:250) |
| Software, Algorithm | pClamp | Molecular Devices | RRID:SCR_011323 | |
| Software, Algorithm | MiniAnalysis Program | Synaptosoft | RRID:SCR_002184 | |
| Software, Algorithm | GraphPad Prism | GraphPad | RRID:SCR_002798 |
Additional files
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Supplementary file 1
Summary electrophysiological data for Figure 1 (Plc21C RNAi experiments).
Genotypes and/or conditions are denoted. For GAL4 drivers, ‘Pre +Post Gal4’ denotes a genetic combination of elaV(C155)-Gal4/Y; Sca-Gal4/+; BG57-Gal4/+. Average values ± SEM are presented for each electrophysiological parameter, with n = number of NMJs recorded. Values include miniature excitatory postsynaptic potential (mEPSP) amplitude, mEPSP frequency (Freq), excitatory postsynaptic potential (EPSP) amplitude, quantal content (QC), and QC corrected for non-linear summation (NLS). *p<0.05, **p<0.01, ***p<0.001 vs. unchallenged control.
- https://doi.org/10.7554/eLife.39643.019
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Supplementary file 2
Summary electrophysiological data for Figure 2 (screen and follow-up).
Genotypes and/or conditions are denoted. The data are split into two tables. The first table summarizes the screen data from Figure 2A. The second table summarizes the follow-up data examining the UAS-IP3-sponge reagent, including the homoeostatic block identified in the screen. Average values ± SEM are presented for each electrophysiological parameter, with n = number of NMJs recorded. Values include miniature excitatory postsynaptic potential (mEPSP) amplitude, mEPSP frequency (Freq), excitatory postsynaptic potential (EPSP) amplitude, quantal content (QC), and QC corrected for non-linear summation (NLS). *p<0.05, **p<0.01, ***p<0.001 vs. unchallenged control.
- https://doi.org/10.7554/eLife.39643.020
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Supplementary file 3
Summary electrophysiological data for Figure 4 (Xestospongin C application).
Genotypes and/or conditions are denoted. Average values ± SEM are presented for each electrophysiological parameter, with n = number of NMJs recorded. Values include miniature excitatory postsynaptic potential (mEPSP) amplitude, mEPSP frequency (Freq), excitatory postsynaptic potential (EPSP) amplitude, quantal content (QC), and QC corrected for non-linear summation (NLS). *p<0.05, **p<0.01, ***p<0.001 vs. unchallenged control.
- https://doi.org/10.7554/eLife.39643.021
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Supplementary file 4
Summary electrophysiological data for Figure 5 (2-APB application).
Genotypes and/or conditions are denoted. Average values ± SEM are presented for each electrophysiological parameter, with n = number of NMJs recorded. Values include miniature excitatory postsynaptic potential (mEPSP) amplitude, mEPSP frequency (Freq), excitatory postsynaptic potential (EPSP) amplitude, quantal content (QC), and QC corrected for non-linear summation (NLS). *p<0.05, **p<0.01, ***p<0.001 vs. unchallenged control.
- https://doi.org/10.7554/eLife.39643.022
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Supplementary file 5
Summary electrophysiological data for Figure 6 (Ryanodine and Dantrolene applications).
Genotypes and/or conditions are denoted. Average values ± SEM are presented for each electrophysiological parameter, with n = number of NMJs recorded. Values include miniature excitatory postsynaptic potential (mEPSP) amplitude, mEPSP frequency (Freq), excitatory postsynaptic potential (EPSP) amplitude, quantal content (QC), and QC corrected for non-linear summation (NLS). *p<0.05, **p<0.01, ***p<0.001 vs. unchallenged control.
- https://doi.org/10.7554/eLife.39643.023
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Supplementary file 6
Summary electrophysiological data for Figure 7 (manipulation interaction analyses).
Genotypes and/or conditions are denoted. Average values ± SEM are presented for each electrophysiological parameter, with n = number of NMJs recorded. Values include miniature excitatory postsynaptic potential (mEPSP) amplitude, mEPSP frequency (Freq), excitatory postsynaptic potential (EPSP) amplitude, quantal content (QC), and QC corrected for non-linear summation (NLS). *p<0.05, **p<0.01, ***p<0.001 vs. unchallenged control.
- https://doi.org/10.7554/eLife.39643.024
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Supplementary file 7
Summary electrophysiological data for Figure 8 (tissue specificity analyses).
Genotypes and/or conditions are denoted. Average values ± SEM are presented for each electrophysiological parameter, with n = number of NMJs recorded. Values include miniature excitatory postsynaptic potential (mEPSP) amplitude, mEPSP frequency (Freq), excitatory postsynaptic potential (EPSP) amplitude, quantal content (QC), and QC corrected for non-linear summation (NLS). *p<0.05, **p<0.01, ***p<0.001 vs. unchallenged control.
- https://doi.org/10.7554/eLife.39643.025
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Transparent reporting form
- https://doi.org/10.7554/eLife.39643.026
