Ca²⁺ channel and active zone protein abundance intersects with input-specific synapse organization to shape functional synaptic diversity

Reviewer #1 (Public Review):

Calcium channels are key regulators of synaptic strength and plasticity, yet how these channels are differentially utilized to enable synaptic diversity is not clear. In this manuscript, the authors use new endogenous tagging of the Drosophila CaV2 channel Cac and three auxiliary subunits to investigate distinct calcium channel functions at two motor neuron subtypes at the fly NMJ, Is and Ib. Although it is clear from previous studies that Pr is higher at Is over Ib, it is not clear why. The authors confirm these differences using postsynaptic calcium imaging combined with post-hoc Cac-TdTomato imaging. Then, through a series of confocal and super resolution imaging studies, the authors describe differences in calcium channel and active zone structure between Is and Ib motor neuron terminals, and the role of Brp and homeostatic plasticity in regulating channel abundance. Finally, the authors show that while the CaBeta subunit is present at similar levels at Is and Ib active zones, there is an interesting reduction in Stj at Is active zones. The authors conclude that these differences in active zone structure and architecture contribute to the generation of the observed heterogeneity in synaptic strength.

Overall the manuscript is well written, and the successful generation of the new endogenous Cac tags (Td-Tomato, Halo) and CaBeta, stj, and stolid genes with V5 tags will be powerful reagents for the field to enable new studies on calcium channels in synaptic structure, function, and plasticity. There are also some interesting, though not entirely unexpected, findings regarding how Brp and homeostatic plasticity modulate calcium channel abundance. The key factors generating diversity in synaptic strength beyond simple Ca2+ influx are well articulated in framing this study. Beyond the particularly useful new reagents for the field presented, the new data demonstrating a concerted and coupled increase in Cac, Stj, and CaB together after plasticity provides an interesting new dimension to the study and a foundation for new work moving forward.

Reviewer #2 (Public Review):

The authors aim to investigate how voltage-gated calcium channel number, organization, and subunit composition lead to changes in synaptic activity at tonic and phasic motor neuron terminals, or type Is and Ib motor neurons in Drosophila. These neuron subtypes generate widely different physiological outputs, and many investigations have sought to understand the molecular underpinnings responsible for these differences. Additionally, these authors explore not only static differences that exist during the third-instar larval stage of development but also use a pharmacological approach to induce homeostatic plasticity to explore how these neuronal subtypes dynamically change the structural composition and organization of key synaptic proteins contributing to physiological plasticity. The Drosophila neuromuscular junction (NMJ) is glutamatergic, the main excitatory neurotransmitter in the human brain, so these findings not only expand our understanding of the molecular and physiological mechanisms responsible for differences in motor neuron subtype activity, but also contribute to our understanding of how the human brain and nervous system functions.

The authors employ state-of-the-art tools and techniques such as single-molecule localization microscopy 3D STORM and create several novel transgenic animals using CRISPR to expand the molecular tools available for exploration of synaptic biology that will be of wide interest to the field. Additionally, the authors use a robust set of experimental approaches from active zone level resolution functional imaging from live preparations to electrophysiology and immunohistochemical analyses to explore and test their hypotheses. All data appear to be robustly acquired and analyzed using appropriate methodology. The authors make important advancements to our understanding of how the different motor neuron subtypes, phasic and tonic-like, exhibit widely varying electrical output despite the neuromuscular junctions having similar ultrastructural composition in the proteins of interest, voltage gated calcium channel cacophony (cac) and the scaffold protein Bruchpilot (brp). The authors reveal the ratio of brp:cac appears to be a critical determinant of release probability (Pr), and in particular, the packing density of VGCCs and availability of brp. Importantly, the authors demonstrate a brp-dependent increase in VGCC density following acute philanthotoxin perfusion (glutamate receptor inhibitor). This VGCC increase appears to be largely responsible for the presynaptic homeostatic plasticity (PHP) observable at the Drosophila NMJ. Lastly, the authors created several novel CRISPR-tagged transgenic lines to visualize the spatial localization of VGCC subunits in Drosophila. Two of these lines, CaV5-C and stjV5-N, express in motor neurons and in the nervous system, localize at the NMJ, and most strikingly, strongly correlate with Pr at tonic and phasic-like terminals.

The few limitations in this study could be addressed with some commentary, a few minor follow-up analyses, or experiments. The authors use a postsynaptically expressed calcium indicator (mhc-Gal4>UAS -GCaMP) to calculate Pr, yet do not explore the contribution that glutamate receptors, or other postsynaptic contributors (e.g. components of the postsynaptic density, PSD) may contribute. A previous publication exploring tonic vs phasic-like activity at the drosophila NMJ revealed a dynamic role for GluRII (Aponte-Santiago et al, 2020). Could the speed of GluR accumulation account for differences between neuron subtypes?

The observation that calcium channel density and brp:cac ratio as a critical determinant of Pr is an important one. However, it is surprising that this was not observed in previous investigations of cac intensity (of which there are many). Is this purely a technical limitation of other investigations, or are other possibilities feasible? Additionally, regarding VGCC-SV coupling, the authors conclude that this packing density increases their proximity to SVs and contributes to the steeper relationship between VGCCs and Pr at phasic type Is. Is it possible that brp or other AZ components could account for these differences. The authors possess the tools to address this directly by labeling vesicles with JanellaFluor646; a stronger signal should be present at Is boutons. Additionally, many different studies have used transmission electron microscopy to explore SVs location to AZs (t-bars) at the Drosophila NMJ.

In reference to the contradictory observations that VGCC intensity does not always correlate with, or determine Pr. Previous investigations have also observed other AZ proteins or interactors (e.g. synaptotagmin mutants) critically control release, even when the correlation between cac and release remains constant while Pr dramatically precipitates.

To confirm the observations that lower brp levels results in a significantly higher cac:brp ratio at phasic-like synapses by organizing VGCCs; this argument could be made stronger by analyzing their existing data. By selecting a population of AZs in Ib boutons that endogenously express normal cac and lower brp levels, the Pr from these should be higher than those from within that population, but comparable to Is Pr. I believe the authors should also be able to correlate the cac:brp ratio with Pr from their data set generally; to determine if a strong correlation exists beyond their observation for cac correlation.

For the philanthotoxin induced changes in cac and brp localization underlying PHP, why do the authors not show cac accumulation after PhTx on live dissected preparations (i.e. in real time)? This also be an excellent opportunity to validate their brp:cac theory. Do the authors observe a dynamic change in brp:cac after 1, or 5 minutes; do Is boutons potentiate stronger due to proportional increases in cac and brp? Also regarding PhTx-induced PHP, their observations that stj and α2are more abundant at Is synapses, suggests that they may also play a role in PhTx induced changes in cac. If either/both are overexpressed during PhTx, brp should increase while cac remains constant. These accessory proteins may determine cac incorporation at AZs.
Taken together this study generates important data-driven, conceptional, and theoretical advancements in our understanding of the molecular underpinnings of different motor neurons, and our understanding of synaptic biology generally. The data are robust, thoroughly analyzed, appropriately depicted. This study not only generates novel findings, but also generated novel molecular tools which will aid future investigations and investigators progress in this field.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For The Authors):

Major points:

(1) A central question regarding VGCC differences at Is vs Ib active zones is why is calcium influx higher at Is active zones compared to Ib. Ideally, the authors would have started this study by showing correlations between Cac abundance, presynaptic calcium influx, and Pr at Is vs Ib active zones. If they had, they would likely find that Cac abundance scales with calcium influx and Pr within Is vs Ib, but that calcium influx is over two-fold enhanced at Is over Ib when normalized to the same Cac abundance. This is more than sufficient to explain the Pr differences, so the rest of the study should have focused on revealing why influx is different at Is over Ib despite an apparently similar level of Cac abundance. Then the examination of CaBeta, Stj, etc could have been used to help explain this conundrum.

A lesson might be gleaned in how to structure this narrative from the Rebola 2019 study, which the authors cite and discuss at length. Similar to the current study, that paper started with two synapses ("strong" vs "weak") and sought to explain why they were so different in synaptic strength. First, they examined presynaptic calcium influx, and surprisingly found that the strong synapse had reduced calcium influx compared to the weak. Then the rest of the paper sought to explain why synaptic strength (Pr) was higher at the strong synapse despite reduced calcium influx. The authors do not use this logical flow and narrative in the present study, despite the focus being on how Cav2 channels contribute to strong vs weak synapses - and the primary function of Cav2 channels is to pass calcium at active zones to drive vesicle fusion.

Although the authors did not show that presynaptic calcium influx is higher at Is vs Ib active zones in the current manuscript, other studies have previously established that calcium influx is two-fold higher at Is active zones vs Ib (as the authors cite). Rather than focusing so much on Pr at Is vs Ib active zones, which as the authors know can be influenced by myriad differences, it seems the more relevant parameter to study is simply to address presynaptic calcium influx at Is vs Ib, which is the primary function of Cac. Put more simply, if Cac levels are the same at Is vs Ib active zones, why is calcium influx at least two-fold higher at Is?

It would therefore seem crucial for the authors to determine presynaptic calcium influx levels (ideally at individual AZs) to really understand how Cac intensity levels correlate with calcium influx. The authors instead map Pr at individual AZs, but as the authors know there are many variables that influence whether a SV releases in addition to calcium influx. There are a number of options for this kind of imaging in Drosophila, including genetically encoded calcium indicators targeted to active zones. But since several studies have previously established that influx is higher at Is active zones over Ib, this may not be necessary. That being said, there is a lot of value in quantitatively analyzing Cac/Stj/CaBeta abundance, calcium influx, and Pr together at individual active zones.

We appreciate the perspective that we could have focused on why Ca2+ influx is 2x greater at type Is active zones, which we agree is an important and interesting question. However, growing evidence indicates that Ca2+ influx alone, like Ca2+ channel abundance, does not reliably predict synaptic strength between inputs. So, here we focused instead on how other differences between synapses influence Pr and contribute to synaptic heterogeneity between and/or among synapses formed by strong and weak inputs. We have changed our title and framing to better reflect this focus.

As Reviewer 1 notes, Rebola et al. (2019) found that lower Pr granule synapses exhibit higher Ca2+ influx (and Ca2+ channel abundance). In another example, Aldahabi et al. (2022) demonstrated that even when Ca2+ influx is greater at high-Pr synapses, it does not necessarily explain differences in synaptic strength as raising Ca2+ entry at low-Pr synapses to high-Pr synapse levels was not sufficient to increase synaptic strength to high-Pr input levels. Similar findings have been reported at tonic and phasic synapses of the Crayfish NMJ (Msghina, 1999).

Several lines of evidence argue that factors beyond Ca2+ influx also play important roles in establishing distinct release properties at the Drosophila NMJ. A recent study using 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 despite the prediction that the increase would be even greater given the power dependence of release on between Ca2+ concentration (He et al., 2023). The authors further found that type Ib synapses are more sensitive than type Is synapses to the slow Ca2+ chelator EGTA, indicating looser Ca2+ channel-SV coupling.

Consistently, we find that although VGCC levels are similar at the two inputs, their density is greater at type Is active zones (Figs. 1 and 2). Our findings also reveal additional molecular differences that may contribute to the observed differences in neurotransmitter release properties between the two inputs, including lower levels of the active zone protein Brp (Fig 3) and the auxiliary subunit α2δ-3/Stj (Fig. 6) at high Pr type Is inputs. In contrast, levels of each of these proteins positively correlate with synaptic strength among active zones of a single input, whether low- or high-Pr (Figs. 1, 3, 6). Similarly, levels of each of these proteins increase during homeostatic potentiation of neurotransmitter release (Figs. 4 and 7). Thus, we propose that two broad mechanisms contribute to synaptic diversity in the nervous system: (1) spatial organization and relative molecular content establish distinct average basal release probabilities that differ between inputs and (2) among individual synapses of distinct inputs, coordinated modulation of Ca2+ channel and active zone protein abundance independently tunes Pr. These intersecting mechanisms provide a framework for understanding the extensive and dynamic synaptic diversity observed across nervous systems.

(2) In addition to key points made above, it seems the authors should at least consider (if not experimentally test) what other differences might contribute to the higher calcium influx at Is over Ib:

- Distinct splice isoforms of Cac (and/or Stj/Cabeta): The recent RNAseq analysis of gene expression at Is vs Ib motor neurons from Troy Littleton's group may inform this consideration?

- Stj reduction at Is: Do channel studies in heterologous systems give any insight into VGCC channel function with and without a2d-3? Do Cav2 channels without a2d pass more calcium? This would then offer an obvious solution to the key conundrum underlying this study.

These are excellent questions that we are actively pursuing. While there is no evidence of differentially expressed splice isoforms of Stj or Ca-β in the recent RNA-seq data from Jetti et al., 2023, subtle changes in Cac isoform usage were observed that may contribute to differences in Ca2+ influx. In heterologous systems, α2δ expression generally increases Ca2+ channel membrane insertion and Ca2+ currents. However, in vivo α2δ’s can also mediate extracellular interactions that may modulate channel function. We address these points in greater detail in the revised discussion.

(3) Assess Stj and CaBeta levels at AZs after PhTx: The successful generation of endogenously tagged Stj and CaBeta enables some relatively easy experiments that would be of interest, similar to what the authors present for Cac. Does Brp similarly control Stj and CaBeta at Is vs Ib compared to what they show for Cac? In addition, does homeostatic plasticity similarly change Stj and CaBeta at Is vs Ib compared to what the authors have shown for Cac? i.e., do they both similarly increase in intensity, by the same amount, as Cac?

We agree and have included an analysis of α2δ-3/Stj levels following PhTx exposure (Fig. 7A-C). We have also investigated the regulation of Stj during chronic presynaptic homeostatic potentiation (Fig. 7D-F). In both cases, StjV5-N levels significantly increase at type Ib and Is active zones, consistent with our finding that among AZs of either type Ib or Is inputs, Stj levels correlate with Cac abundance and, thus, Pr. Together with our and others’ findings, this suggests that coordinated increases Ca2+ channel, auxiliary subunit, and active zone protein abundance positively tunes synaptic strength at diverse synaptic subtypes.

Minor points:

(1) Including line numbers would make reviewing/commenting easier.

We apologize for this oversight and have added line numbers to the revised manuscript.

(2) Fig. 2I: It is not apparent what the mean cluster density is between Ib vs Is (as it is in Fig. 2F-H graphs). The mean and error bars should be included in 2I as it is in 2G. Same with Fig. 3C.

Thank you for pointing this out. We have added error bars to the paired analysis in 2I as well as in 3C and 1C.

(3) Fig. 4 - it might make more sense to normalize Brp and Cac intensity as a percentage of baseline (PhTx at Is or Ib) rather than normalizing everything to control Ib.

We have revised the graphs as suggested in Figure 4 and throughout.

(4) Page 5 bottom - REFS missing after Fig. 1E.

Thank you for catching this. We have fixed it.

Reviewer #2 (Recommendations For The Authors):

This reader found differentiating between low Pr sites (deep purple) and cac measurements (black) difficult in Fig 1B. You may consider depicting this differently.

Thank you for this feedback. We have changed the color scheme to improve readability.

I found it difficult to discern the difference between experiments Fig 1E and Fig 1J. Why are individual dots distributed differently?

The individual data points are the same as in 1E and 1F, but we have removed the individual NMJ dimensionality to combine all Is and Ib data points together along with best fit lines for comparison of their slopes. We have added text to the revised manuscript to clarify this.

Results section, second paragraph, add references, remove 'REF': We next investigated the correlation between Pr and VGCC levels and found that at type Is inputs, single-AZ Cac intensity positively correlates with Pr (Fig. 1E; REFS).

Thank you. We have corrected this error.