To investigate the relationship between VGCC levels and neurotransmitter release properties at functionally distinct synapses, we took advantage of the two motor neuron subtypes with low and high release probabilities that innervate most Drosophila muscles (Aponte-Santiago and Littleton, 2020; Kurdyak et al., 1994). These glutamatergic neuromuscular junctions (NMJs) contain hundreds of individual synapses that are accessible to single AZ functional imaging using genetically encoded Ca²⁺ indicators.

In Drosophila, Cacophony (Cac) is the sole Ca_v2 pore-forming subunit and is the VGCC responsible for triggering synaptic transmission (Kawasaki et al., 2000; Macleod et al., 2006; Peng and Wu, 2007; Smith et al., 1996). To simultaneously monitor neurotransmitter release and VGCC levels, we swapped the N-terminal sfGFP tag in our well-characterized cac^sfGFP-N line for a Td-Tomato tag (cac^Td-Tomato-N) and confirmed that the tag does not impair synaptic function (Figure 1—figure supplement 1; Figure 1A–F; Ghelani et al., 2023; Gratz et al., 2019). We then expressed postsynaptically targeted GCaMP6f (SynapGCaMP6f; Newman et al., 2017), which reports Ca²⁺ influx through glutamate receptors in response to neurotransmitter release, in cac^Td-Tomato-N animals for a plus/minus readout. We and others have previously shown that Cac levels are highly predictive of P_r at individual type Ib AZs (Akbergenova et al., 2018; Gratz et al., 2019). To determine if VGCC levels are similarly predictive at high-P_r type Is AZs, we measured Cac^Td-Tomato-N fluorescence intensity and monitored neurotransmitter release in response to 0.2 Hz stimulus at individual synapses. To enable direct comparisons between the two inputs, we simultaneously imaged type Ib and Is synapses at NMJ 6/7 (Figure 1A). We quantified the number of times a vesicle was released over 120 stimuli to determine single-synapse P_r. As has been previously reported, we found that type Is synapses exhibited significant heterogeneity and higher average P_r than type Ib synapses (Figure 1B and C; Lu et al., 2016; Newman et al., 2022). Consistent with their higher P_r, type Is connections contain relatively fewer low-P_r and more high-P_r AZs (Figure 1D). We next investigated the correlation between P_r and VGCC levels and found that at type Is inputs, single-AZ Cac intensity positively correlates with P_r (Figure 1E). We also observe a strong positive correlation between VGCC levels and P_r at type Ib inputs to the same muscles (Figure 1F), consistent with our and others’ prior findings (Akbergenova et al., 2018; Gratz et al., 2019; Newman et al., 2022).

A simple prediction of the observation that VGCC levels correlate highly with P_r at individual AZs of both low- and high-P_r inputs is that Cac levels will be higher at synapses of type Is inputs than type Ib. We analyzed Cac^sfGFP-N levels at individual type Ib and Is synapses and found that average Cac levels are the same at type Ib and Is AZs (Figure 1G and H). Cac levels are also similarly distributed across AZs of the two inputs (Figure 1I). Together, these findings indicate that the relationship between VGCC levels and P_r differs between the two inputs. Consistently, when we directly compare the best-fit lines for the relationship between Cac levels and P_r at type Ib and Is inputs from our correlative functional imaging data (Figure 1E and F), we find that the slopes are significantly different (Figure 1J). Across type Is AZs, a similar range of VGCC levels supports a higher range of release probabilities. Thus, VGCCs can predict P_r within synaptic subtypes, but not between AZs of different synaptic subtypes, providing a framework for understanding seemingly contradictory findings on the role of VGCCs in determining P_r.

Many differences between low-P_r type Ib and high-P_r type Is AZs have been described (Aponte-Santiago and Littleton, 2020; Aponte-Santiago et al., 2020; Atwood et al., 1993; He et al., 2023; Jetti et al., 2023; Kurdyak et al., 1994; Lu et al., 2016; Medeiros and O’Connor-Giles, 2023). Perhaps most notably, type Is AZs experience ~twofold greater Ca²⁺ influx than type Ib (He et al., 2023; Lu et al., 2016). While this alone could explain the estimated 3-fold greater P_r at type Is AZs and is certainly a key factor, several lines of evidence argue for additional contributors. A recent study using a botulinum transgene to isolate type Ib and Is synapses for electrophysiological analysis found that increasing external [Ca²⁺] from physiological levels (1.8 mM) to 3 mM or even 6 mM does not result in a 3-fold increase in EPSCs or quantal content at type Ib synapses and type Ib synapses continue to facilitate at 3 mM external [Ca²⁺] (He et al., 2023). Using this approach, they further found that type Ib synapses are more sensitive to the slow Ca²⁺ chelator EGTA, indicating looser VGCC-SV coupling.

We investigated the spatial distribution of VGCCs at type Ib and Is AZs using 3D dSTORM single-molecule localization microscopy (SMLM). An individual VGCC complex is estimated to be ~10 nm in diameter with the most common immunolabeling techniques adding significantly to their size and creating a linkage error of ~20 nm between the target molecule and fluorescent reporter (Früh et al., 2021; Liu et al., 2022; Thomas, 2000). For following VGCC dynamics using single-particle tracking via photoactivation localization microscopy (sptPALM), we recently incorporated mEOS4b (Paez-Segala et al., 2015) at the same N-terminal site we previously used to endogenously tag Cac, achieving a linkage error of less of than 5 nm (Ghelani et al., 2023; Gratz et al., 2019). To gain more flexibility in labeling Cac without adding to the linkage error, we swapped the mEOS tag for a similarly sized HaloTag (cac^HaloTag-N). cac^HaloTag-N flies are fully viable, do not display significant defects in synaptic function, and exhibit normal Cac localization at AZs as observed in super-resolution optical reassignment images, where Brp is arranged in rings surrounding puncta of VGCCs (Figure 1—figure supplement 1; Figure 2A–C, Ghelani et al., 2023). HaloTag, which covalently binds synthetic ligands, is 3.3 nm in diameter (Los et al., 2008; Yazaki et al., 2020), yielding a linkage error well under 5 nm. cac^HaloTag-N larvae were stained with JaneliaFluor646 HaloTag ligand (Grimm et al., 2015) and horseradish peroxidase (HRP) to distinguish between type Ib and Is branches and enable simultaneous imaging of the two inputs at a single NMJ. We then used density-based spatial clustering of applications with noise (DBSCAN) analysis to identify Cac clusters at type Ib and Is AZs (Figure 2D and E; Ehmann et al., 2014). We find that the average size of Cac^HaloTag-N clusters is similar at low- and high-P_r AZs (Figure 2F), with mean diameters of approximately 102 nm and 105 nm, respectively. This is similar to the Cac^mEOS4b-N type Ib cluster size observed by sptPALM imaging (Ghelani et al., 2023). In agreement with our confocal level data, the number of localizations per cluster was similar at low- and high-P_r AZs (Figure 2G). We then calculated the average Cac density per AZ and found that VGCCs are significantly more densely organized at high-P_r type Is AZs than low-P_r type Ib AZs (Figure 2H, I). Greater VGCC AZ density at type Is AZs is consistent with a recent SMLM study using antibodies to label Cac, indicating these results are robust to different labeling approaches (Newman et al., 2022). Together, these findings suggest that more compact organization of VGCCs increases their coupling to SVs and contributes to the steeper relationship between VGCC levels and P_r at high-P_r type Is AZs.

To understand how these nanoscale differences in VGCC organization might be established, we investigated the AZ scaffolding protein Brp. Brp/CAST/ELKS family proteins function as central organizers of both VGCCs and SV release sites at developing synapses (Dai et al., 2006; Dong et al., 2018; Hallermann et al., 2010; Held et al., 2016; Kittel et al., 2006; Liu et al., 2014; McDonald et al., 2020; Radulovic et al., 2020). Like Cac, Brp is more densely arranged at type Is AZs as measured through SMLM and stimulated emission depletion (STED) imaging studies (He et al., 2023; Jetti et al., 2023; Mrestani et al., 2021). We simultaneously imaged type Ib and Is inputs and found lower Brp levels at type Is AZs (Figure 3A and B). Since Cac levels are similar at AZs of the two inputs, lower Brp levels result in a significantly higher Cac:Brp ratio at type Is synapses, which we hypothesize promotes compact organization of VGCCs (Figure 3C). In contrast, we and others have previously shown that Brp levels positively correlate with P_r among AZs of low-P_r type Ib inputs (Gratz et al., 2019; Muhammad et al., 2015; Newman et al., 2017; Peled et al., 2014; Reddy-Alla et al., 2017). Consistently, Brp and Cac levels strongly correlate at type Ib AZs (Gratz et al., 2019) and we observe a similarly strong correlation across individual type Is AZs (Figure 3D). Thus, like VGCCs, Brp levels contribute in distinct ways to synaptic heterogeneity within vs. between low- and high-P_r inputs, likely due to differences in AZ organization between the two synaptic subtypes.

We next investigated the requirement for Brp in promoting VGCC accumulation at low- and high-P_r inputs by analyzing Cac^sfGFP-N levels in brp null mutants (brp^-/-; Figure 3E and F). Cac^sfGFP-N levels are diminished at both type Ib and Is AZs, demonstrating a conserved role for Brp in promoting Cac accumulation at both inputs (Figure 3G). The relative decrease in Cac levels at type Ib AZs is significantly greater than at type Is AZs, indicating a greater requirement for Brp in regulating VGCC levels at low-P_r type Ib synapses (Figure 3H). This suggests that an additional factor or factors function with or upstream of Brp to establish differences in Brp dependence at low- and high-P_r AZs.

In response to acute or chronic inhibition of glutamate receptors at NMJs, Drosophila motor neurons homeostatically increase neurotransmitter release to maintain synaptic communication (Davis and Müller, 2015; Frank, 2014; James et al., 2019). Pharmacological inhibition of glutamate receptors with the wasp toxin Philanthotoxin-433 (PhTx) induces acute presynaptic homeostatic potentiation of release (PHP) within minutes (Frank et al., 2006). We and others have demonstrated that acute PHP involves rapid changes in VGCC and other AZ protein levels at type Ib AZs (Böhme et al., 2019; Gratz et al., 2019; Weyhersmüller et al., 2011). Recent studies have revealed significant differences in the induction of PHP at low- and high-P_r synaptic inputs under different conditions (Genç and Davis, 2019; Newman et al., 2017; Sauvola et al., 2021). PhTx induces acute PHP at both type Ib and Is synapses (Genç and Davis, 2019), but the molecular changes underlying PHP at high-P_r type Is AZs remain unknown. To compare the dynamic modulation of VGCCs at low- and high-P_r AZs, we treated cac^sfGFP-N larvae with non-saturating concentrations of PhTx for 10 min, then quantified Cac and Brp levels at type Ib and Is AZs (Figure 4A and B). We observe a significant PhTx-induced increase in Brp and Cac^sfGFP-N levels at type Is AZs similar to type Ib (Figure 4C and D; Gratz et al., 2019). Thus, despite their distinct baseline transmission and organizational properties, PhTx-induced potentiation of neurotransmitter release involves the rapid accumulation of VGCCs at both low- and high-P_r AZs.

At low-P_r type Ib AZs, Brp is a critical regulator of PHP-induced accumulation of proteins associated with SV priming and release, specifically Unc13A and Syntaxin-1A (Böhme et al., 2019). At type Ib AZs, PhTx also induces a Brp-dependent increase in Cac density and decrease in channel mobility (Ghelani et al., 2023). Notably, Brp itself becomes more densely organized during PHP (Ghelani et al., 2023), consistent with its denser organization at high-P_r type Is AZs (Mrestani et al., 2021). Since baseline accumulation of VGCCs depends less on Brp at high-P_r type Is AZs, we investigated the role of Brp in promoting dynamic increases in VGCC levels at type Ib and Is AZs by treating cac^sfGFP-N; brp^-/- larvae with PhTx followed by quantification of Cac^sfGFP-N levels (Figure 4E and F). We find that PhTx failed to induce accumulation of Cac at either type Ib or Is AZs in brp^-/- mutants, demonstrating a shared requirement for Brp in regulating VGCC dynamics at both inputs (Figure 4G). In contrast to no change at type Ib AZs, Cac^sfGFP-N levels are significantly decreased at type Is AZs (Figure 4G), revealing an input-specific role for Brp in maintaining VGCC levels during the dynamic reorganization of AZs. Consistently, Ghelani et al., 2023 found that whereas PhTx induces a decrease in Cac mobility at wild-type type Ib AZs, in brp^-/- mutants Cac mobility increases (Ghelani et al., 2023). Together, these findings suggest potentiating synapses must coordinate the accumulation of new VGCCs with the stabilization of existing channels, and that meeting this challenge is more dependent upon Brp at high-P_r AZs.

In addition to the pore-forming α subunits, VGCCs comprise auxiliary α2δ and β subunits that regulate forward channel trafficking, membrane insertion, and function (Figure 5A; Campiglio and Flucher, 2015; Dolphin and Lee, 2020; Weiss and Zamponi, 2017). β subunits interact with pore-forming α subunits intracellularly, whereas GPI-anchored α2δ subunits are largely extracellular. Beyond their interaction with α subunits, α2δs have been shown to interact with a growing number of extracellular proteins to promote synaptogenesis (Bauer et al., 2010; Dolphin, 2018; Risher et al., 2018). The Drosophila genome encodes one synaptic Ca_v2 α subunit (Cac), one β subunit, and three α2δ subunits (Littleton and Ganetzky, 2000). Auxiliary subunits are both spatially and temporally regulated and broadly able to interact with α subunits. Thus, the subunit composition of channel complexes is a potential source of significant diversity in both the spatial and functional regulation of VGCCs.

Ca-β encodes the sole Drosophila β subunit and has been shown to enhance Ca²⁺ transients in sensory neurons (Kanamori et al., 2013). Drosophila α2δ–3, also known as Straightjacket (Stj), has well-characterized roles at the NMJ in promoting Ca²⁺ channel clustering, homeostatic plasticity, and, independently of Cac, synapse formation and organization (Dickman et al., 2008; Hoover et al., 2019; Kurshan et al., 2009; Ly et al., 2008; Schöpf et al., 2021; Wang et al., 2016). While low sequence homology between α2δ subunits within and across species makes 1:1 mapping difficult, the remaining two Drosophila α2δs map more closely to mammalian α2δ–3 and –4 than α2δ–1 and –2. Stolid was recently shown to promote dendritic Cac expression in motor neurons, whereas Ma2d is known to function in muscle where it is broadly expressed (Heinrich and Ryglewski, 2020; Reuveny et al., 2018). The synaptic localization of endogenous auxiliary subunits with VGCCs remains unknown in Drosophila.

To explore potential differences in VGCC subunit composition at type Ib and Is synapses, we used CRISPR gene editing to incorporate endogenous V5 tags in sequence common to all isoforms of stj, stolid, and Ca-β (Bruckner et al., 2017; Gratz et al., 2014). We inserted V5 after the N-terminal signal peptides of Stj and Stolid and near the C-terminus of Ca-β (see Materials and methods for details), and confirmed that the incorporation of the peptide tags did not impair neurotransmission (Figure 5B–E). We investigated the expression of each endogenously tagged subunit in the larval ventral ganglion and found that all subunits are expressed in the synaptic neuropil in a pattern similar to the α subunit Cac (Figure 5F–H; Gratz et al., 2019). Similar to Cac, Ca-β^V5-C is highly enriched in the mushroom bodies of the larval brain. We next investigated expression at the larval NMJ where Cac localizes in a single punctum at each AZ and found that only Ca-β^V5-C and Stj^V5-N are present (Figure 5F–H). This aligns with the recent finding that Stolid does not play a role in regulating Ca²⁺ transients at the larval NMJ (Heinrich and Ryglewski, 2020). We also observe Ca-β^V5-C expression in muscle as expected for the sole Drosophila β subunit (Figure 5F and see Figure 6C).

To investigate Ca-β^V5-C and Stj^V5-N localization at type Ib and Is AZs, we used super-resolution optical reassignment microscopy. Both subunits localize to AZs labeled with Cac or the CAST/ELKS AZ cytomatrix protein Brp (Figure 6A and B). We observe Brp rings surrounding puncta of VGCCs including Ca-β^V5-C (Figure 6A). The tight localization of both subunits to central AZ puncta suggests they are associated with α subunits and predicts that Ca-β^V5-C and Stj^V5-N levels, like Cac, will be similar at the low- and high-P_r synapses. To test this, we imaged Ca-β^V5-C and Stj^V5-N levels at both inputs simultaneously using confocal microscopy and measured fluorescence intensity (Figure 6C and D). As predicted, we found that Ca-β^V5-C levels are similar at type Ib and Is AZs (Figure 6E). In contrast, Stj^V5-N levels are significantly lower at high-P_r type Is AZs (Figure 6F). Thus, while Cac and Ca-β are present in similar ratios at AZs of both inputs, surprisingly, the same is not true of Stj/α2δ–3 with high-P_r type Is AZs exhibiting lower levels of Stj. This unexpected finding indicates that α:α2δ–3 stoichiometry is not always 1:1 in vivo and differs at low- and high-P_r synapses. This is consistent with studies of mammalian subunits indicating that in contrast to β subunits, α2δ interactions with α subunits may be transient, leading to a pool of VGCCs lacking α2δ (Müller et al., 2010; Voigt et al., 2016). Our results indicate this pool may be present in vivo and larger at high-P_r type Is inputs.

To further investigate the contribution of Stj to synaptic heterogeneity, we analyzed the relationship between Cac and Stj levels at individual AZs of type Is inputs. Stj^V5-N and Cac^sfGFP-N levels are highly positively correlated at type Is AZs (Figure 6G). We observe the same relationship between Stj^V5-N and Cac^sfGFP-N levels at type Ib AZs (Figure 6H). Because P_r is highly positively correlated with Cac levels within synaptic subtypes, this indicates that Stj levels are also positively correlated with P_r within, but not between, inputs.

α2δ subunits are critical regulators of α subunit forward trafficking. In flies and mammals, overexpression of α2δ subunits increases α subunit abundance, whereas overexpression of the α subunit alone does not (Cao et al., 2004; Cunningham et al., 2022; Hoppa et al., 2012). These findings suggest that α2δ may be dynamically regulated together with Cac during PHP, a prediction we can now test with our endogenously tagged line. Following PhTx exposure, we find that Stj^V5-N is recruited on a rapid timescale to both low- and high-P_r AZs, increasing by a similar percentage at both type Ib and Is AZs (27% and 26%, respectively) as predicted (Figure 7A-C). Cac levels are similarly increased (33% at type Ib and 30% at type Is; See Figure 4D), suggesting coordinated regulation. We have previously shown that Cac abundance is also increased in chronic PHP, which is induced by genetic loss of the GluRIIA receptor subunit (Gratz et al., 2019; Li et al., 2018; Petersen et al., 1997). We investigated Stj dynamics in GluRIIA^-/- null animals and observed elevated Stj^V5-N levels at AZs of both type Ib and Is inputs (18% and 17%, respectively; Figure 7D–F), indicating similar dynamics during chronic PHP. These data are consistent with the recent finding that in addition to its previously uncovered role in promoting an increase in the readily releasable pool of SVs (Wang et al., 2016), Stj is required for the accumulation of Cac at type Ib AZs during both acute and chronic PHP (Zhang et al., 2023). Thus, the abundance of multiple key AZ proteins distinguishes low- and high-P_r synapses within, but not between, inputs at baseline and during homeostatic plasticity.

The following fly lines used in this study were obtained from the Bloomington Drosophila Stock Center (BDSC, NIH P40OD018537): w¹¹¹⁸ (RRID:BDSC_5905), vasa-Cas9 (RRID:BDSC_51324), piggyBac transposase (RRID:BDSC_8283), and Df(2 R)brp^6.1 (Gratz et al., 2014; Horn et al., 2003). brp⁶⁹ and GluRIIA^sp16 (GluRIIA^-/-) alleles were generously provided by Stephan Sigrist (Freie Universität Berlin; Fouquet et al., 2009; Kittel et al., 2006) and Aaron DiAntonio (Petersen et al., 1997) respectively. brp loss-of-function experiments were performed in brp⁶⁹/Df(2 R)brp^6.1. Drosophila melanogaster stocks were raised on molasses food (Lab Express, Type R) in a 25 °C incubator with controlled humidity and 12 hr light/dark cycle. Endogenously tagged cac, Ca-β, stolid, and straightjacket (stj) alleles were generated using our piggyBac-based CRISPR approach as previously detailed (https://flycrispr.org/; Bruckner et al., 2017; Gratz et al., 2019). We used FlyBase (release FB2024_01) to obtain genomic sequences (doi.org/10.1093/genetics/iyad211). Td-Tomato and Halo tags were incorporated at the N-terminus of Cac, where we have previously incorporated fluorescent tags (Ghelani et al., 2023; Gratz et al., 2019). V5 tags were incorporated at the N-terminus of Stj and Stolid after their signal peptide sequences, immediately after Asparagine 34 of Stj and after Isoleucine 34 of Stolid. For Ca-β, V5 was inserted immediately after Proline 446 of isoform Ca-β-PN. All endogenously tagged lines are fully viable in homozygous males and females and were molecularly confirmed by Sanger sequencing. All genetic reagents generated in this study are available upon request. gRNAs sequences used:

All antibodies used, associated fixation methods, and incubation times can be found in Supplementary file 1b. Male wandering third-instar larvae were dissected in ice-cold saline and fixed either for 6 min at room temperature with Bouin’s fixative, 5 min on ice with 100% methanol, or 30 min at room temperature (RT) in 4% PFA. Dissections were permeabilized with PTX (PBS with 0.1% Triton-X 100) and blocked for 1 hr at RT using 5% goat serum and 1% bovine serum albumin. Stained larvae were mounted in Vectashield (Vector Laboratories, #H-1000) under Fisherbrand coverglass (Fisher Scientific, #12541B) for confocal microscopy, with Prolong glass mounting medium (Thermo Fisher Scientific, #P36980) under Zeiss High Performance Coverglass (Zeiss, #474030-9000-000) for super-resolution optical reassignment microscopy, or buffer (see STORM imaging and analysis section) under Zeiss coverglass with edges sealed using vacuum grease for STORM microscopy.

Functional imaging was performed on a Nikon A1R resonant scanning confocal mounted on a FN1 microscope using a Nikon Apo LWD 25x1.1 NA objective and a Mad City Labs piezo drive nosepiece. Dissections and data collection were performed as previously described in Gratz et al., 2019. Briefly, cac^Td-Tomato-N; Mhc-GCaMP6f male 3rd instar larvae were dissected in HL3 containing 0.2 mM Ca²⁺ and 25 mM Mg²⁺ with motor axons severed and the larval brain removed. Larval filets were placed in HL3 containing 1.5 mM Ca²⁺ and 25 mM Mg²⁺ for recording. Nerves were suctioned into 1.5 mm pipettes and stimulus amplitude was adjusted to recruit both type Ib and Is inputs. Motor terminals from segments A2-4 at NMJ 6/7 were imaged for Cac^Td-Tomato-N levels first using a galvanometer scanner, then a resonant scanner to collect GCaMP6f events in a single focal plane continuously for 120 stimulations at a 0.2 Hz stimulation frequency.

Z-stacks and movies were loaded into Nikon Elements Software (NIS) where movies were motion corrected, background subtracted, and denoised. Change in fluorescence (ΔF) movies were then created by subtracting the average of the previous 10 frames from each frame. A substack of only stimulation frames was further processed using a gaussian filter followed by the Bright Spots detection module in the Nikon GA3 software to identify the location of each postsynaptic event. Cac^Td-Tomato-N fluorescence intensity levels and coordinate locations were measured for 531 AZs for type Ib and 365 AZs for type Is terminals across six animals. X-Y coordinate positions of fluorescent signals from GCaMP6f postsynaptic events were aligned to Cac^Td-Tomato-N puncta locations and each post synaptic event assigned to a Cac punctum using nearest neighbor analysis. Postsynaptic events that did not map within 960 nm of a Cac^Td-Tomato-N punctum were discarded from the analysis. Pearson’s correlation was used to determine the correlation between P_r and Cac levels normalized to average to account for variability between imaging sessions. Cac intensity-P_r heat maps were generated using Python matplotlib and seaborn plotting packages.

STORM imaging was performed on a Nikon Eclipse Ti2 3D NSTORM with an Andor iXon Ultra camera, Nikon LUN-F 405/488/640 nm lasers, and a Nikon 100x1.49 NA objective. STORM buffer (10 mM MEA (pH 8.0), 3 U/mL pyranose oxidase, and 90 U/mL catalase, 10% (w/v) glucose, 10 mM sodium chloride, and 50 mM Tris hydrochloride) was made fresh each imaging day and pH adjusted to between 7.0–8.0 using acetic acid. cac^HaloTag-N NMJs were labeled as detailed in Supplementary file 1b and immediately imaged for HRP in 488 channel to identify type Ib and Is terminals. Cac^HaloTag-N was then imaged using the 640 nm laser line at 33 Hz for 5000 frames. 405 nm laser power was gradually increased over the course of imaging to compensate for run-down of blinking rates. A back aperture camera was used to ensure beam focus and position for each imaging session to ensure high signal to noise. Data were binned with a CCD minimum threshold of 100 and drift correction was applied using the NIS Software STORM package. ROIs of single boutons were drawn in NIS using HRP in the 488 nm channel followed by a DBSCAN analysis with criteria of 10 molecules within 50 nm to determine clusters. Positional coordinates of localizations within clusters from DBSCAN were exported from NIS and run through a Python script published with this manuscript. Using the implementation developed in Mrestani et al., 2021 as a starting point, we wrote custom code to use the alpha shapes component of the CGAL package (https://www.cgal.org), via a python wrapper (https://anaconda.org/conda-forge/cgal), to measure the area of Ca²⁺ channel clusters, the number of localizations, and calculate cluster density. To achieve an average lateral localization accuracy of ~30 nm, all localizations with >50 nm localization accuracy were removed prior to analysis. Using this custom code, Cac^HaloTag-N area was analyzed using an alpha value of 0.015, which controls the complexity of cluster boundaries (not restricted to be convex).

For quantitative AZ analysis of larval NMJs, dissections stained in the same dish were imaged on a Nikon Eclipse Ni A1R+confocal microscope using an Apo TIRF 60x1.49 NA oil-immersion objective for larval NMJs. NMJs containing both type Is and type Ib branches from muscles 6/7 in segments A2-4 were collected. ROIs were drawn using HRP staining to differentiate between type Ib and Is branches. To analyze individual AZs, Nikon Elements GA3 Software was used to process images with Gaussian and rolling ball filters and measure fluorescence intensity levels at individual puncta identified by the Bright Spots module. When experimental design allowed, Brp fluorescence signal was used to create a binary mask to aid in the identification of AZ ROIs for analysis. Otherwise, binary masks were created based on the fluorescence signal of the channel analyzed. Quantifications were conducted masked to genotype and/or treatment. Confocal fluorescence intensity level data are reported as the sum fluorescence intensity per AZ averaged over individual NMJs. For Figure 5, larvae were stained separately and imaged using a Nikon Plan-Apo 20x0.75 NA objective (ventral ganglia) or Apo TIRF 60x1.49 NA oil-immersion objective (NMJs). Super-resolution optical reassignment images were obtained on a Nikon CSU-W1 SoRa (Spinning Disk Super Resolution by Optical Pixel Reassignment) with a Photometrics Prime BSI sCMOS camera and a 60x1.49 NA oil-immersion objective. Images were acquired using Nikon NIS and deconvolved using Richardson-Lucy deconvolution with 15–20 iterations.

Current-clamp recordings were performed as previously described (Bruckner et al., 2017). Male third-instar larvae were dissected in HL3 (70 mM NaCl, 5 mM KCl, 15 mM MgCl2, 10 mM NaHCO3, 115 mM sucrose, 5 mM trehalose, 5 mM HEPES, pH 7.2) with 0.25 mM Ca²⁺. Recordings were performed in HL3 at the external Ca²⁺ concentration indicated. Sharp borosilicate electrodes filled with 3 M KCl were used to record from muscle 6 of abdominal segments A3 and A4. Recordings were conducted on a Nikon FN1 microscope using a 40x0.80 NA water-dipping objective and acquired using an Axoclamp 900 A amplifier, Digidata 1550B acquisition system, and pClamp 11.0.3 software (Molecular Devices). For each cell with an initial resting potential between −60 and −80 mV and input resistance ≥5 MΩ, mean miniature excitatory junctional potentials (mEJPs) were collected for 1 min in the absence of stimulation and analyzed using Mini Analysis (Synaptosoft). EJPs were generated by applying a stimulus to severed segmental nerves at a frequency of 0.2 Hz using an isolated pulse stimulator 2100 (A-M Systems). Stimulus amplitude was adjusted to consistently elicit compound responses from both type Ib and Is motor neurons. At least 25 consecutive EJPs were recorded for each cell and analyzed in pClamp to obtain mean amplitude. Quantal content was calculated for each recording as mean EJP amplitude divided by mean mEJP amplitude.

Acute PHP was induced by incubating semi-intact preparations in 20 µM Philanthotoxin-433 (Figure 4: PhTx; Santa Cruz, sc-255421, Lot B1417 and Figure 7: PhTx; Sigma Aldrich, P207-2, Lot MKCK7405) diluted in HL3 containing 0.4 mM Ca²⁺ for 10 min at room temperature (Frank et al., 2006). Control preparations were given a mock treatment. Following control and experimental treatment, dissections were completed, fixed in 4% PFA for 30 min (cac^sfGFP-N) or 100% ice-cold methanol on ice for 5 min (stj^V5-N), and stained in the same dish. Analyses of fluorescent intensity levels were performed as previously described in the Confocal imaging and analysis section.

Statistical analyses were conducted in GraphPad Prism 9. Normality was determined by the D’Agostino–Pearson omnibus test. Comparisons of normally distributed data were conducted by Student’s t test (with Welch’s correction in the case of unequal variance) for single comparisons and ANOVA followed by Tukey’s test for multiple comparisons. For non-normally distributed data, the Mann–Whitney U test and Kruskal-Wallis test followed by Dunn’s multiple comparisons tests were used for single and multiple comparisons, respectively. Paired analysis of non-normally distributed data was conducted using Wilcoxon’s matched-pairs signed rank test. One-dimensional Pearson correlation coefficients (r) were used to compare intensity levels and neurotransmitter release probability. ANCOVA test was performed on all regression lines to determine if slopes were significantly different. Reported values are mean ± SEM. Sample size, statistical test, and p values for each comparison are reported in Supplementary file 1a. All source data, including statistical tests and raw images, can be found in the following Harvard Dataverse dataset https://doi.org/10.7910/DVN/GGP3UM.