Peer review process
Revised: This Reviewed Preprint has been revised by the authors in response to the previous round of peer review; the eLife assessment and the public reviews have been updated where necessary by the editors and peer reviewers.
Read more about eLife’s peer review process.Editors
- Reviewing EditorHugo BellenBaylor College of Medicine, Houston, United States of America
- Senior EditorClaude DesplanNew York University, New York, United States of America
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, CaV5-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 α2are 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.