GCaMP indicators targeted to pre- and post-synaptic compartments.

(A) Schematic of the presynaptic ratiometric Syt::mScarlet::GCaMP (Scar8f/Scar8m) Ca2+ indicators showing localization to synaptic vesicles via fusion to the Ca2+ sensor Synaptotagmin (SYT). (B) Representative images of NMJs expressing the indicated reporter driven in motor neurons with the OK319-GAL4 driver (w1118; OK319-GAL4/UAS-Scar8f) immunostained with anti-GFP (GCaMP) and anti-SYT. Note that endogenous mScarlet signals were obtained without antibody labeling. (C) Schematic of the BRP::mScarlet::GCaMP8f (Bar8f) ratiometric Ca2+ indicator, which targets GCaMP to active zones via fusion to the BRP-short protein (Schmid et al., 2008). (D) Representative images of NMJs expressing the indicated reporter driven in motor neurons (w;OK319-GAL4/Bar8f) immunostained with anti-GFP (GCaMP) and anti-BRP. Note that native mCherry or mScarlet signals were obtained without antibody labeling. (E) Schematic of the SynapGCaMP indicator, which targets GCaMP to postsynaptic compartments via a Shaker PDZ domain.(Newman et al., 2017) (F) Representative NMJs expressing the indicated reporter (w;MHC-CD8-GCaMP6f-Sh/+;+, w;;MHC-CD8-GCaMP8f-Sh/+, w;;MHC-CD8-GCaMP8m-Sh/+) immunostained with anti-GFP (GCaMP), - GluRIIC (glutamate receptors), and -DLG (postsynaptic density).

“CaFire” - a Python-based analysis program for quantifying synaptic Ca2+ imaging data.

(A) Workflow showing how Ca2+ imaging data and downstream analysis is performed. Raw timelapse movies are processed with SVI Huygens software to correct and deconvolve image artifacts. ROIs are then selected in ImageJ Fiji software and intensities are extracted. Data are exported to Excel for manual inspection or directly analyzed using CaFire software. (B) Screenshot of the CaFire user interface. Users can load fluorescence intensity data, detect Ca2+ events, partition evoked events, calculate peak amplitude, and rise and decay time constants. Detected events are automatically marked on the raw traces and associated values are displayed in the data table below. (C) Examples of two distinct analysis pipelines implemented in CaFire. Evoked event analysis: (1) Thresholds are set for automatic peak detection; (2) Events are automatically partitioned based on stimulus intervals (e.g., 1 Hz and 5 Hz); (3) Parameters such as peak amplitude, rise time constant (τrise), and decay time constant (τdecay) are calculated using exponential fits. Mini event analysis: (1) Users define amplitude thresholds for event detection; (2) CaFire automatically identifies candidate events; (3) Missed or misidentified events can be manually corrected; (4) Event parameters are exported for each validated event.

Scar8m is an optimal presynaptic Ca2+ indicator.

(A) Schematic of the UAS-Syt::mScarlet3::GCaMP8m (Scar8m) ratiometric Ca2+ indicator consisting of mScarlet3 and GCaMP8m targeted to synaptic vesicles via fusion to Synaptotagmin (SYT). (B) Representative images of a MN-Ib bouton expressing Scar8m (w;OK319-GAL4/+;Scar8m/+) resonant scanned at ∼115 fps. Fluorescence from GCaMP8m (green) and mScarlet3 (magenta) is shown at baseline, peak, decay, and recovery to baseline. (C) Normalized Ca2+ signals following single action potential (AP) stimulation. Averaged traces compare the kinetics of GCaMP6s, GCaMP8f, and GCaMP8m. (D) Representative GCaMP and mScarlet signals recorded from Syt::GCaMP6s, Scar8f, and Scar8m in response to single AP stimuli. The mScarlet reference signal remains stable throughout the recording. (E) Quantification of average peak amplitude (ΔR/R, GCaMP/mScarlet ratios), rise time constant (τrise), and decay time constant (τdecay) from the indicated sensors. Scar8m yields significantly higher peak ΔR/R signals, similar rise time kinetics, and a modestly slower decay compared to Scar8f. (F) Presynaptic Ca2+ responses of the indicated sensors to 5 Hz and 10 Hz stimulation trains. All comparisons in bar graphs are statistically significant unless explicitly noted otherwise. Error bars represent ±SEM. Unless indicated by an “ns” label, all values are significantly different; detailed statistics including p-values are presented in Table S1.

Scar8m captures differences in Ca2+ levels between motor neuron subtypes and after plasticity.

(A,B) Representative images of Scar8m expressed at both MN-Ib (A) and MN-Is (B) motor neuron subtypes immunostained with anti-GFP. (C) ΔF/F traces of GCaMP8m and mScarlet3 responses from single AP stimulation at MN-Ib and MN-Is, with ∼2x higher responses observed at MN-Is over -Ib, as expected. (D) Quantification of ΔR/R responses from the two inputs. (E) ΔF/F traces of GCaMP8m and mScarlet3 responses from single AP stimulation at MN-Ib in wild type (w;OK319-GAL4/+;Scar8m/+) and GluRIIA mutants (w;OK319-GAL4,GluRIIAPV3/GluRIIAPV3;Scar8m/+), which express presynaptic homeostatic plasticity (PHP). Note the enhanced presynaptic Ca2+ levels induced after PHP plasticity. (F) Quantification of ΔR/R responses from the indicated genotypes. Error bars represent ±SEM. Detailed statistics including p-values are provided in Table S1.

Evaluating the ability of Bar8f to capture active zone-specific Ca2+ dynamics.

(A) Schematic of the Bar8f indicator, consisting of GCaMP8f and mScarlet1 fused to the BRP-short domain, which traffics to individual active zones (AZs). (B) Live confocal image of a single MN-Ib bouton expressing Bar8f (w;OK319-GAL4/+;Bar8f/+). GCaMP8f (green) and mScarlet1 (magenta) localize to AZ puncta. Eight individual AZs are shown as ROIs. (C) Representative ΔR/R traces of single AP-evoked Ca2+ responses at the eight annotated AZs shown in (B). Numerical values indicate peak ΔR/R amplitude for each AZ. (D) Bar graph showing peak ΔR/R values from 29 individual AZs collected across multiple boutons from different NMJs. Note that only one AZ shows Ca2+ responses that are significantly different from the others (highlighted in red). (E) Scatter plots relating AZ size (measured by mScarlet1 fluorescence area) to ΔR/R. A weak negative correlation is observed between ΔR/R and AZ size, likely due to overestimation of ROI boundaries during manual AZ segmentation. (F) Scatter plot showing summed ΔF responses as a function of AZ size. A strong positive correlation is observed between summed ΔF and AZ size, reflecting the expected scaling of total GCaMP fluorescence signals with abundance at larger AZs. Error bars indicate ±SEM. Additional statistical details are shown in Table S1.

SynapGCaMP8m is an optimal postsynaptic Ca2+ indicator.

(A) Schematic of the SynapGCaMP8m reporter, with the GCaMP indicator targeted to postsynaptic compartments near glutamate receptors via a Shaker-PDZ motif. Super resolution image using STED microscopy showing the GCaMP8m reporter is localized outside of GluRs. (B) Live confocal images of muscle 6 NMJ boutons expressing SynapGCaMP8m were performed using resonant area scans. The indicated ROI shows representative frames at baseline and peak quantal Ca2+ transients acquired at ∼115 fps. (C) Averaged single miniature Ca2+ events recorded from SynapGCaMP6f, -8f, and -8m. Traces show ΔF/F responses with fitted rise and decay time constants (τ), along with amplitude values for each indicator. SynapGCaMP8m yields the highest peak signal and maintains rapid kinetics. The corresponding heatmaps below show the spatiotemporal fluorescence dynamics of each indicator. (D) Representative traces of quantal events imaged with the indicated SynapGCaMP sensor and quantal events (mEPSPs) recorded using electrophysiology. (E) Quantification of ΔF/F peak amplitude, and rise and decay time constants (τrise and τdecay) for each SynapGCaMP variant. All comparisons in bar graphs are statistically significant unless “ns” is shown. Error bars indicate ±SEM. Additional statistical details, including p-values, are presented in Table S1.

SynapGCaMP8m resolution approaches that of electrophysiology.

(A) Simultaneous recordings of quantal events at MN-Ib boutons using the indicated SynapGCaMP variant (green) and electrophysiology (black) after silencing MN-Is (w;Is-GAL4/+;UAS-BoNT-C/SynapGCaMP). Red circles indicate mEPSP events not captured by SynapGCaMP. SynapGCaMP8m captures quantal events with high sensitivity, comparable to electrophysiology. (B) Quantification of the proportion of quantal events captured by the indicated SynapGCaMP variant as a proportion of the total mEPSP events recorded by electrophysiology. SynapGCaMP6f detects only about half of electrophysiological events, while both SynapGCaMP8f and SynapGCaMP8m capture nearly all mEPSPs. (C) Scatter plot of paired miniature event amplitudes recorded simultaneously by SynapGCaMP6f and -8f and electrophysiology. (D) Scatter plot of paired miniature event amplitudes recorded simultaneously by SynapGCaMP8m and electrophysiology. A linear relationship with high correlation is observed for SynapGCaMP8m, indicating that optical signals scale proportionally with quantal amplitude. (E) Bar plots showing average mEPSP amplitudes (left) and ΔF/F amplitudes of quantal Ca2+ events (right) in the indicated genotypes (same as (A) above but with GluRIIA or GluRIIB mutant alleles included). SynapGCaMP8m accurately resolves quantal size differences with similar resolution as the electrophysiological data, with quantal amplitudes in both datasets exhibiting the expected differences (GluRIIA-/-<WT<GluRIIB-/-). (F) Cumulative frequency distributions of mEPSP amplitudes (left) and Ca2+ mini event amplitudes (ΔF/F) (right); each are significantly different using the Kolmogorov– Smirnov Test. See Table S1 for full statistical details including p-values.

GCaMP expression does not perturb synaptic transmission at the Drosophila NMJ.

(A) Representative electrophysiological traces in the indicated genotypes showing evoked excitatory postsynaptic potentials (EPSPs, top traces) and spontaneous miniature events (mEPSPs, bottom traces) from muscle 6. (B) Quantification of synaptic parameters: average mEPSP amplitude (left), evoked EPSP amplitude (middle), and quantal content (right). No significant differences in mEPSP, EPSP, or quantal content values are observed in genotypes expressing the indicated sensor with the exception of Scar8f, where a significant reduction in EPSP amplitude and quantal content was observed. Data are presented as mean ±SEM with individual data points shown. Error bars represent ±SEM. Detailed statistics including p-values are provided in Table S1.

Absolute values and statistical comparisons for Ca2+ imaging and electrophysiology data.

This table reports the full statistical details and properties for data presented in the indicated figures, including p-values, mean ± SEM, sample sizes (n), and genotypes for all conditions tested. Ca2+ imaging parameters include ΔF/F (or ΔR/R), rise time (τrise), and decay time (τdecay) constants. Electrophysiological parameters include mEPSP amplitude, EPSP amplitude, quantal content (QC), input resistance, and resting potential. p-values from one-way ANOVA with Tukey’s multiple comparison test are shown for key contrasts between genotypes and indicators. Data for outlier analysis, correlation analysis and linear regression analyses are included where applicable.