Peripheral glia and neurons jointly regulate activity-induced synaptic remodeling at the Drosophila neuromuscular junction
- Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, United States
- Department of Physiology and Neuroscience, Keck School of Medicine, University of Southern California, United States
Figures
Figure 1
Endogenous Shv tagged with eGFP is detected in glial cells.
(A) Schematic of eGFP knock-in to the Shv gene generated using CRISPR/Cas9 system (top). Exons are in orange, signal peptide in red. Western blots using antibodies against GFP and Shv confirm the presence of eGFP in Shv. β-Tubulin is used as a loading control. (B) Low magnification images of the third-instar larval brain showing weak Shv-eGFP signal throughout the brain (left). Scale bar = 50 µm. Zoomed-in view of the brain hemisphere and ventral nerve cord with neurons and glia marked by Elav and Repo antibodies, respectively. Asterisks label glial cells with Shv expression. Scale bar for brain and VNC are 10 and 15 µm, respectively. (C) Shv-eGFP can be detected at the neuromuscular junction (NMJ). Glial membrane is marked by membrane targeted tdTomato (driven by glial specific repo-GAL4) and neuronal membrane labeled by HRP. Zoomed-in views show that Shv-eGFP colocalizes with glial membrane (magenta arrow) and synaptic boutons (yellow arrow). Shv-eGFP also weakly labels the muscle. Note that a single optical slice of the NMJ at muscle 6/7, abdominal segment 2 is shown, which highlights Shv-eGFP colocalization with glia and synaptic boutons in this permeabilized prep. The full glial stalk is not visible because it lies in a different focal plane from the branch of interest. Scale bar = 10 µm in the upper panels, and 2 µm in the lower panels.
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Figure 1—source data 1
PDF file containing original western blots for Figure 1A, showing the relevant bands and molecular weight marker.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot analysis are shown in Figure 1A.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig1-data2-v1.zip
Figure 2 with 1 supplement
Shv in glia is necessary for activity-induced synaptic remodeling.
(A) Tissue-specific knockdown of shv. Reducing Shv in neurons or glia blocks activity-induced synaptic remodeling, but not when shv is knocked down in muscles. Notably, glia-specific knockdown of shv increases the levels of GluRIIC, whereas neuronal Shv knockdown decreases basal GluRIIC. (B) Acute knockdown of shv in glia using the inducible repo-GeneSwitch-GAL4 driver affects basal GluR intensity and abolishes activity-induced synaptic changes. (A, B) Scale bar = 2 µm. All values are normalized to unstimulated control and presented as mean ± SEM. Control contains TRiP RNAi control vector driven by the indicated driver. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated neuromuscular junctions (NMJs) across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 when comparing stimulated to unstimulated NMJs.
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Figure 2—source data 1
Data for relative bouton size and GluR intensity.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Independent Shv-RNAi line and efficacy of shv knockdown by the GeneSwitch system.
(A) Glial-specific knockdown of shv by shv-RNAi37507 recapitulates the shv-RNAi phenotypes shown in Figure 2. All values are normalized to unstimulated control and presented as mean ± SEM. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated neuromuscular junctions (NMJs) across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05; ***p ≤ 0.001 when comparing stimulated to unstimulated NMJs. (B) Western blot against Shv to show the efficiency of the GeneSwitch system. Complex V is used as an internal control. The control contains TRiP RNAi control vector driven by the indicated driver. Student’s t-test was used to compare control to knockdown. *p ≤ 0.05; ***p ≤ 0.001.
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Figure 2—figure supplement 1—source data 1
Raw data for relative bouton size, GluR intensity, and protein levels.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig2-figsupp1-data1-v1.xlsx
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Figure 2—figure supplement 1—source data 2
PDF file containing original western blots, showing the relevant bands and molecular weight marker.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
Original files for western blots.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig2-figsupp1-data3-v1.zip
Figure 3
Expression of shv in perineurial (PG) and subperineurial glia (SPG) is required for activity-induced synaptic remodeling.
(A) Diagram of relative membrane position and extension of wrapping glia (WG), SPG, and PG at the neuromuscular junction (NMJ). Astrocyte-like glia can be detected in the central brain and ventral nerve chord. (B) Representative images and quantification of synaptic changes following knockdown of shv in glia subtypes. Knockdown of shv in SPG and PG recapitulates the phenotypes of pan-glial knockdown, as well as abolishes activity-induced synaptic remodeling. Scale bar = 2 µm. All values are normalized to unstimulated control and presented as mean ± SEM. Control contains TRiP RNAi control vector driven by the indicated driver. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated NMJs across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. #p ≤ 0.05; ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 when comparing stimulated to unstimulated NMJs.
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Figure 3—source data 1
Data for relative bouton size and GluR intensity shown in Figure 3B.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig3-data1-v1.xlsx
Figure 4
Glial Shv rescues defective synaptic plasticity observed in shv1 mutant.
(A) Selective expression of shv in glia or neurons of shv1 mutants is sufficient to rescue activity-dependent synaptic changes, but glial shv expression did not restore basal bouton size. Scale bar = 2 µm. (B) Representative mEPSP and eEPSP recordings conducted using HL3 solution containing 0.5 mM Ca2+. Average eEPSP amplitude is plotted after nonlinear summation correction. (C) Normalized eEPSP before and following tetanus (10 Hz for 2 min) at the indicated time points. Recordings were done using HL3 solution containing 0.25 mM Ca2+. shv1 showed significantly diminished post-tetanic potentiation (PTP) following stimulation. Expression of shv in glial cells of shv1 rescued PTP. The number of neuromuscular junctions (NMJs) examined is shown in parentheses. Student’s t-test was used to compare between control and the indicated genotypes. * shows that shv1 displays PTP that is significantly lower than the control (p ≤ 0.05), starting from the indicated time and onwards. All values are mean ± SEM. For (A) and (B), one-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated NMJs across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 when comparing stimulated to unstimulated NMJs.
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Figure 4—source data 1
Data for relative bouton size and GluR intensity, and electrophysiology data.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig4-data1-v1.xlsx
Figure 5 with 2 supplements
Glial Shv does not activate integrin signaling but modulates neuronal release of Shv.
(A) Representative images showing that Shv can be detected extracellularly when expressed using glial-specific driver. Extracellular Shv-HA (Shvextra) is monitored using an antibody against HA under detergent-free staining condition. (B) Expression of Shv using the glial-specific driver does not rescue pFAK levels during unstimulated and stimulated conditions, revealing that glial Shv does not activate integrin. (C) Knockdown of shv in glia upregulated pFAK, whereas upregulation of shv in glia blocked the activity-dependent increase normally seen in control. (D) Expression of shv in shv1 mutants shows activity-dependent release of Shv by neurons, but not by glia. HAextra indicates extracellular Shv stained under non-permeabilizing (detergent-free) conditions. Permeabilized staining protocol washes away extracellular staining and thus mainly detects intracellular Shv. Intracellular levels of Shv in neurons or glia did not change following stimulation. Asterisk indicates glial membrane overlay with neurite at the neuromuscular junction (NMJ). (E) Knockdown of endogenous Shv-eGFP in neurons or glia did not diminish extracellular presence of Shv-eGFP at the NMJ, suggesting homeostatic regulation of Shv protein level. Right panels show control NMJ (nsyb-GAL4/+) stained using non-permeabilizing (Non-perm) and permeabilizing (Perm) conditions. CSP, an intracellular protein, is only observed when the sample is stained under permeabilizing conditions, suggesting that our non-permeabilizing protocol is selective for extracellular proteins. Scale bar = 2 µm for all panels. All values are normalized to unstimulated control and presented as mean ± SEM. One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated NMJs across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. #p ≤ 0.05; ##p ≤ 0.01; ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 when comparing stimulated to unstimulated NMJs.
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Figure 5—source data 1
Relative staining intensities for the indicated antibodies and conditions.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Neuronal and glial expression of Shv and efficiency of shv knockdown by RNAi in neurons and glia.
(A) Western blots using antibodies against HA show no detectable post-translational modification between glial and neuronal OE Shv. β-Tubulin is used as a loading control. (B) Staining of the larval brain confirms that the RNAi approach effectively reduces Shv-eGFP in the selective cell types. Yellow arrows highlight neurons (Elav positive), magenta arrows point to glial cells (Repo positive). Scale bar = 10 µm.
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Figure 5—figure supplement 1—source data 1
PDF file containing original western blots, showing the relevant bands and molecular weight marker.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original files for western blots.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Integrity of peripheral glial cell membranes.
The top panels show representative images of the segmental nerves in the XY axis (left) and the orthogonal YZ axis view (right). The dashed line indicates the orthogonal projection plane. Knockdown of shv in glia does not affect the overall glial membrane integrity. Scale bar = 5 μm. Middle panels show that knockdown of shv in glia does not alter the general morphology of peripheral glia at the neuromuscular junction (NMJ). Scale bar = 10 μm. Lower panels show a magnified view of glia closely associated with proximal synaptic boutons (boxed area in the upper panels). Scale bar = 2 μm.
Figure 6 with 2 supplements
Glial Shv regulates ambient extracellular glutamate concentration.
(A) Shv expression in glia maintains ambient glutamate concentration at the neuromuscular junction (NMJ). The left panel shows a schematic of the tripartite synapse at the NMJ. iGluSnFR expression in neurons via the GAL4/UAS system can detect extracellular ambient glutamate concentration at the synapse, while glia-specific knockdown or upregulation of Shv is achieved using the LexA/LexAop system. iGluSnFR signals are seen in green and mtdTomato marks neuronal membrane. In addition, overexpression of postsynaptic GluR using mhc-GluRIIA does not affect iGluSnFR signals. HRP-Cy3 was added after live imaging of iGluSnFR. Lower graph shows quantitation of the relative ambient glutamate concentration at the NMJ. Only Shv from glia affects ambient glutamate concentration, Shv from neurons does not. (B) Incubating the NMJ with 2 mM glutamate rescues synaptic remodeling in case of glial Shv knockdown. Vehicle controls represent NMJs dissected in parallel and incubated with HL3 without 2 mM glutamate for the same length of time. Scale bar = 2 µm. All values are normalized to unstimulated control and presented as mean ± SEM. Control contains TRiP RNAi control vector driven by the indicated driver. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated NMJs across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 when comparing stimulated to unstimulated NMJs.
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Figure 6—source data 1
Data for relative iGluSnFR sensor intensity, bouton size, and GluR levels.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Validation of GluR intensity in MhcGluRIIA.
MhcGluRIIA line shows higher basal GluRIIA and GluRIIC levels that failed to further increase upon stimulation. All values are normalized to unstimulated control and presented as mean ± SEM. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated neuromuscular junctions (NMJs) across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05; **p ≤ 0.001 when comparing stimulated to unstimulated NMJs.
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Figure 6—figure supplement 1—source data 1
Data for relative levels of GluRIIA and GluRIIC subunits.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Defective activity-induced synaptic remodeling caused by the loss of neuronal Shv is not rescued by incubation with 2 mM glutamate.
Vehicle controls represent neuromuscular junctions (NMJs) dissected in parallel and incubated with HL3 without 2 mM glutamate for the same length of time. Scale bar = 2 µm. All values are normalized to unstimulated control and presented as mean ± SEM. Control contains TRiP RNAi control vector driven by the indicated driver. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated NMJs across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. ##p ≤ 0.01; ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05 when comparing stimulated to unstimulated NMJs.
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Figure 6—figure supplement 2—source data 1
Data for relative bouton size and GluR intensity for the indicated conditions.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig6-figsupp2-data1-v1.xlsx
Figure 7
Acute ablation of glia elevated basal GluR levels and disrupted activity-induced synaptic remodeling, but extracellular glutamate incubation is sufficient to compensate for the loss of glia.
(A) Transient rpr expression is sufficient to trigger death of glial cells. The diagram shows the RU486 feeding protocol used to induce glial cell death in third-instar larvae. Lower panels show images of the segmental nerves (left) and the neuromuscular junction (NMJ, middle). The zoomed-in region of the NMJ (yellow box) is magnified on the right. Glial membrane is marked by CD4-tdGFP, which appears fragmented in the presence of rpr expression, indicating glial cell death. Scale bars indicate size in 50, 10, and 2 µm from left to right. (B) Images and (C) quantitation of synaptic bouton size and GluR abundance. Incubating the NMJ for 10 min with 2 mM glutamate is sufficient to overcome loss of glial cells and restore basal GluR level and activity-induced synaptic remodeling, suggesting a main function of peripheral glia in synaptic plasticity regulation is to maintain a high ambient glutamate concentration. Scale bar = 2 µm in (B). All values are normalized to unstimulated control and presented as mean ± SEM. Control contains TRiP RNAi control vector driven by the indicated driver. Statistics: One-way ANOVA followed by Tukey’s multiple comparison test was used to compare between unstimulated control and unstimulated NMJs across genotypes. Student’s t-test was used to compare between unstimulated and stimulated NMJs of the same genotype. ###p ≤ 0.001 when comparing unstimulated samples to unstimulated control. *p ≤ 0.05; ***p ≤ 0.001 when comparing stimulated to unstimulated NMJs.
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Figure 7—source data 1
Data for relative bouton size and GluR intensity for the indicated conditions.
- https://cdn.elifesciences.org/articles/104126/elife-104126-fig7-data1-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Drosophila melanogaster) | repo-GAL4 | BDSC | 7415, RRID:BDSC_7415 | Pan-glial driver |
| Strain, strain background (D. melanogaster) | 24B-GAL4 | BDSC | 1767, RRID:BDSC_1767 | Muscle driver |
| Strain, strain background (D. melanogaster) | elav-GAL4 | BDSC | 458, RRID:BDSC_458 | Pan-neuronal driver |
| Strain, strain background (D. melanogaster) | nSyb-GAL4 | BDSC | 51635, RRID:BDSC_51635 | Pan-neuronal driver |
| Strain, strain background (D. melanogaster) | Gliotactin-GAL4 | BDSC | 9030, RRID:BDSC_9030 | Subperineurial glial driver |
| Strain, strain background (D. melanogaster) | Nrv- GAL4 | BDSC | 6800, RRID:BDSC_6800 | Wrapping glial driver |
| Strain, strain background (D. melanogaster) | alrm-GAL4 | BDSC | 67032, RRID:BDSC_67032 | Astrocyte driver |
| Strain, strain background (D. melanogaster) | NP6293-GAL4 | Kyoto | 105188, RRID:DGGR_105188 | (PG-GAL4) perineurial glial driver |
| Strain, strain background (D. melanogaster) | TRiP-RNAi control | BDSC | 35788, RRID:BDSC_35788 | Non-specific RNAi control |
| Strain, strain background (D. melanogaster) | iGluSnFR | BDSC | 59611, RRID:BDSC_59611 | Glutamate concentration sensor |
| Strain, strain background (D. melanogaster) | UAS-CD4-tdGFP | BDSC | 35836, RRID:BDSC_35836 | Membrane form reporter |
| Strain, strain background (D. melanogaster) | UAS-IVS-myr::tdTomato | BDSC | 32221, RRID:BDSC_32221 | Membrane form reporter |
| Strain, strain background (D. melanogaster) | Elav-LexA | BDSC | 52676, RRID:BDSC_52676 | Pan-neuronal driver |
| Strain, strain background (D. melanogaster) | repo-LexA | Gift from Dr. Henry Y. Sun | Pan-neuronal driver | |
| Strain, strain background (D. melanogaster) | repo-GeneSwitch-GAL4 | Artiushin et al., 2018 | Drug inducible pan-glial driver | |
| Strain, strain background (D. melanogaster) | Mhc.GluRIIA.Myc | BDSC | 64258, RRID:BDSC_64258 | GluRIIA expression in muscles |
| Strain, strain background (D. melanogaster) | UAS-shv-RNAi | VDRC | 108576, RRID:Flybase_FBst0480386 | |
| Strain, strain background (D. melanogaster) | UAS-shv-RNAi37507 | BDSC | 37507, RRID:BDSC_37507 | |
| Strain, strain background (D. melanogaster) | UAS-Shv | Lee et al., 2017 | shv transgene for overexpression | |
| Strain, strain background (D. melanogaster) | shv1 | Lee et al., 2017 | shv mutant | |
| Strain, strain background (D. melanogaster) | Shv-eGFP | This study | eGFP insertion line | |
| Strain, strain background (D. melanogaster) | Cas9 | BDSC | 55821, RRID:BDSC_55821 | CRISPR Fly injection |
| Strain, strain background (D. melanogaster) | LexAop-Shv- RNAi | This study | shv-RNAi designed based on sequence from VDRC 108576 | |
| Strain, strain background (D. melanogaster) | LexAop-Shv | This study | shv transgene for overexpression | |
| Strain, strain background (D. melanogaster) | P{CaryP}attP18 | BDSC | 32107, RRID:BDSC_32107 | |
| Recombinant DNA reagent | pU6-BbsI-chiRNA vector | Addgene | 45946, RRID:Addgene_45946 | |
| Recombinant DNA reagent | pHD-DsRed | Addgene | 51434, RRID:Addgene_51434 | |
| Recombinant DNA reagent | pJFRC19-13XLexAop2-IVS-myr::GFP vector | Addgene | 26224, RRID:Addgene_26224 | |
| Antibody | Rabbit polyclonal anti-pFAK | Invitrogen | Catalog #44-624G, RRID:AB_2533701 | 1:250 |
| Antibody | Rabbit polyclonal anti-HA | Sigma | Catalog #H6908, RRID:AB_260070 | 1:1000 |
| Antibody | Rabbit polyclonal anti-GluRIIC | Chang et al., 2024 | 1:1000 | |
| Antibody | Rat monoclonal anti-Elav | DSHB | 7E8A10, RRID:AB_528218 | 1:500 |
| Antibody | Mouse monoclonal anti-Repo | DSHB | 8D12, RRID:AB_528448 | 1:50 |
| Antibody | Mouse monoclonal anti-GFP | DSHB | 4C94C9, RRID:AB_2617422 | 1:100 |
| Antibody | Cy3 conjugated anti-HRP | Jackson ImmunoResearch | RRID:AB_2338959 | 1:100 for non-permeabilized staining. 1:250 for permeabilized staining. |
| Antibody | Alexa-647 conjugated anti-HRP | Jackson ImmunoResearch | RRID:AB_2338967 | 1:100 for non-permeabilized staining. 1:250 for permeabilized staining. |
| Antibody | Alexa Fluor 488 | Invitrogen | RRID:AB_143165 | 1:250 |
| Antibody | Alexa Fluor 405 | Invitrogen | RRID:AB_221604 | 1:250 |
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Peripheral glia and neurons jointly regulate activity-induced synaptic remodeling at the Drosophila neuromuscular junction
eLife 14:RP104126.
https://doi.org/10.7554/eLife.104126.3
