Ca2+ channel and active zone protein abundance intersects with input-specific synapse organization to shape functional synaptic diversity
- Neuroscience Graduate Training Program, Brown University, United States
- Department of Neuroscience, Brown University, United States
- Carney Institute for Brain Science, Brown University, United States
Figures
Figure 1 with 1 supplement
VGCC levels predict Pr within, but not between, inputs.
(A) Representative confocal Z-projection of CacTd-Tomato-N (magenta) with type Ib (blue) and type Is (red) terminals outlined. (B) AZ heat map of terminals in A with color indicating Pr and size representing sum Cac intensity levels in arbitrary units (AU). (C) Average single-AZ probability of release at type Ib and Is terminals. N=6 animals, 6 NMJs. (D) Quintile distribution of single-AZ Pr frequency at type Ib and Is inputs. (E, F) Correlation between normalized CacTd-Tomato-N intensity and Pr at type Is and Ib AZs of the same 6 NMJs. Each dot represents a single AZ and each color corresponds to an individual NMJ with linear regression lines indicated for each. (G) Top, representative confocal Z-projection of CacsfGFP-N. Bottom, CacsfGFP-N in green with HRP marking neuronal membranes in gray. Type Ib (blue) and type Is (red) terminals are outlined. (H) Quantification of CacsfGFP-N AZ intensity at type Ib and Is terminals. Each data point represents the average normalized single AZ sum intensity for an individual NMJ. (I) Distribution of normalized CacsfGFP-N intensity from single type Ib and Is AZs in H (X-axis cutoff at 5.0). (J) Comparison between normalized CacTd-Tomato-N and Pr of type Ib and Is AZs combined from E-F with linear regression lines (blue and red, respectively) and 95% confidence intervals (black lines) indicated. All scale bars = 5 µm, all error bars indicate S.E.M, ****p<0.0001; ns, not significant. N’s, absolute values, and statistical information is detailed in Supplementary file 1a.
Figure 1—figure supplement 1
Electrophysiological validation of endogenously tagged cacophony lines.
(A-C) Representative traces of EJPs (top) and mEJPs (bottom) in control, cacHaloTag-N, and cacTd-Tomato-N. (D-F) Quantification of EJPs, mEJPs, and quantal content (QC). All error bars indicate S.E.M., ns, not significant.
Figure 2
VGCC clusters are more compact at AZs of high-Pr type Is inputs.
(A–C) Representative SoRa Z-projection of CacHaloTag-N (green), Brp (magenta), and merge. (D, E) Representative boutons of STORM CacHaloTag-N clusters as identified by DBSCAN at type Ib and Is boutons as indicated. Each color represents an individual identified cluster with purple scattered dots identifying excluded background signal. (F–H) Analysis of STORM-acquired CacHaloTag-N clusters where each data point represents the respective single-cluster measurement averaged over individual boutons. (F) Quantification of CacHaloTag-N cluster area at type Ib and Is AZs. (G) Quantification of localizations per cluster at type Ib and Is boutons. (H) Calculated CacHaloTag-N cluster density at type Ib and Is AZs. (I) Paired analysis of calculated AZ cluster density averaged over individual type Ib and Is inputs to the same muscle. All scale bars = 1 µm, all error bars indicate S.E.M. **p<0.01; *p<0.05; ns, not significant. N’s, absolute values, and statistical information is detailed in Supplementary file 1a.
Figure 3
Differences in Bruchpilot (Brp) levels and function at low-and high-Pr inputs.
(A) Representative confocal Z-projection of Brp expression at type Ib (blue outline) and type Is (red outline) terminals. (B) Quantification of Brp AZ intensity at type Ib and Is terminals. (C) Ratio of normalized CacsfGFP-N:Brp levels at type Ib and Is inputs to the same muscles. (D) Correlation of CacsfGFP-N and Brp at type Ib and Is single AZs with linear regression lines (blue and red, respectively) and 95% confidence intervals (black dotted lines) indicated. (E, F) Representative confocal Z-projections of CacsfGFP-N (green), Brp (magenta), HRP (white), and merge at type Ib (blue outline) and Is (red outline) terminals of cacsfGFP-N (control) or cacsfGFP-N;brp-/- (brp-/-) animals. (G) Quantification of CacsfGFP-N normalized fluorescence intensity at type Ib and Is AZs of control vs brp-/- NMJs. (H) Ratio of CacsfGFP-N fluorescence intensity at type Ib and Is AZs between brp-/-and control NMJs. For B and G, each data point represents the average normalized single AZ sum intensity for an individual NMJ. All scale bars = 5 µm, all error bars indicate S.E.M., ****p<0.0001. N’s, absolute values, and statistical information is detailed in Supplementary file 1a.
Figure 4
Brp differentially regulates VGCC dynamics at low-and high-Pr inputs during presynaptic homeostatic potentiation.
(A, B) Representative confocal Z-projections of CacsfGFP-N (top, green), Brp (middle, magenta), and both merged with HRP (bottom, gray) at untreated and PhTx-treated cacsfGFP-N NMJs showing type Ib (blue) and type Is (red) terminals. (C) Quantification of Brp fluorescence intensity at untreated and PhTx-treated type Ib and Is terminals. (D) Quantification of CacsfGFP-N fluorescence intensity at untreated and PhTx-treated type Ib and Is terminals. (E, F) Representative confocal Z-projections of CacsfGFP-N (top, green), Brp (middle, magenta), and both merged with HRP (bottom, gray) at untreated and PhTx-treated cacsfGFP-N;brp-/- NMJs showing type Ib (blue) and type Is (red) terminals. (G) Quantification of CacsfGFP-N fluorescence intensity at untreated and PhTx-treated cacsfGFP-N;brp-/- type Ib and Is terminals. For all quantifications, each data point represents the average normalized single AZ sum intensity for an individual NMJ. All scale bars = 5 µm, all error bars indicate S.E.M. ****p<0.0001; ***p<0.001; **p<0.01; ns, not significant. N’s, absolute values, and statistical information is detailed in Supplementary file 1a.
Figure 5
Endogenous tagging of VGCC auxiliary subunits reveals distinct synaptic expression patterns.
(A) Schematic of a Ca2+ channel complex with tagged auxiliary subunits (created with BioRender). (B) Schematic of Ca-β (isoform PL shown), Stj (isoform PC), and Stolid (isoform H/I) indicating endogenous tag locations. (C–E) Quantification of EJPs, mEJPs, and quantal content for each endogenously tagged line. (F–H) Representative confocal Z-projections of auxiliary subunit expression (green) at the larval ventral ganglion (VG, top, scale bars = 100 µm) and NMJs co-labeled with anti-HRP (magenta, bottom, scale bars = 5 µm). All error bars indicate S.E.M., ns, not significant. N’s, absolute values, and statistical information is detailed in Supplementary file 1a.
© 2024, BioRender Inc. Figure 5A was created using BioRender, and is published under a CC BY-NC-ND license. Further reproductions must adhere to the terms of this license.
Figure 6
Stj/α2δ–3 levels are lower at AZs of high-Pr type Is inputs.
(A) Representative SoRa Z-projections of Ca-βV5-C (green), Brp (magenta), and merge at a single bouton. (B) Representative SoRa Z-projections of CacsfGFP-N (green), StjV5-N (magenta), and merge at a single bouton. Scale bars for A and B=1 μm. (C, D) Representative confocal Z-projections of Ca-βV5-C expression and StjV5-N expression at type Ib (blue outline) and type Is (red outline) terminals. Scale bars = 5 μm. (E, F) Quantification of Ca-βV5-C and StjV5-N fluorescence intensity at type Ib and Is AZs. Each data point represents the average normalized single AZ sum intensity for an individual NMJ. (G, H) Correlation of CacsfGFP-N and StjV5-N fluorescence intensity levels at type Ib and Is single AZs with linear regression lines (blue or red line, respectively) and 95% confidence intervals (black lines). All error bars indicate S.E.M. ***p<0.001; ns, not significant. N’s, absolute values, and statistical information is detailed in Supplementary file 1a.
Figure 7
Stj/α2δ–3 levels are modulated at AZs of both low- and high-Pr inputs during presynaptic homeostatic potentiation.
(A, B) Representative confocal Z-projections of StjV5-N (top, green), HRP (middle, gray), and merge (bottom) at untreated and PhTx-treated stjV5-N NMJs showing type Ib (blue) and type Is (red) terminals. (C) Quantification of StjV5-N fluorescence intensity at untreated and PhTx-treated type Ib and Is terminals. (D–E) Representative confocal Z-projections of StjV5-N (top, green), HRP (middle, gray), and merge (bottom) at stjV5-N and stjV5-N;GluRIIA-/- NMJs showing type Ib (blue) and type Is (red) terminals. (F) Quantification of StjV5-N fluorescence intensity levels at stjV5-N and stjV5-N;GluRIIA-/- type Ib and Is terminals. For all quantifications, each data point represents the average normalized single AZ sum intensity for an individual NMJ. All scale bars = 5 µm, all error bars indicate S.E.M. ****p<0.0001. N’s, absolute values, and statistical information is detailed in Supplementary file 1a.
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/88412/elife-88412-transrepform1-v1.pdf
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Supplementary file 1
Tables detailing absolute values, statistics, and imaging conditions of endogenously-tagged genetic lines.
- https://cdn.elifesciences.org/articles/88412/elife-88412-supp1-v1.docx
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Ca2+ channel and active zone protein abundance intersects with input-specific synapse organization to shape functional synaptic diversity
eLife 12:RP88412.
https://doi.org/10.7554/eLife.88412.3
