Specific presynaptic functions require distinct Drosophila Cav2 splice isoforms

  1. Christopher Bell
  2. Lukas Kilo
  3. Daniel Gottschalk
  4. Hanna Kern
  5. Jashar Arian
  6. Lea Deneke
  7. Oliver Kobler
  8. Christof Rickert
  9. Julia Strauß
  10. Martin Heine
  11. Carsten Duch
  12. Stefanie Ryglewski

  • Curated by eLife

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    Cav2 voltage-gated calcium channels play key roles in regulating synaptic strength and plasticity. In contrast to mammals, invertebrates like Drosophila encode a single Cav2 channel, raising questions on how diversity in Cav2 is achieved from a single gene. Here, the authors present convincing evidence that two alternatively spliced isoforms of the Cac gene (cacophony, also known as Dmca1A and nightblindA) enable diverse changes in Cav2 expression, localization, and function in synaptic transmission and plasticity. These valuable findings will be of interest to a variety of researchers.

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Abstract

The multiplicity of neural circuits that accommodate the sheer infinite number of computations conducted by brains requires diverse synapse and neuron types. At the vertebrate presynaptic active zone functional diversity can be achieved by the expression of different voltage gated calcium channels of the Ca v 2 family. In fact, release probability and other aspects of presynaptic function are tuned by different combinations of Ca v 2.1, Ca v 2.2, and Ca v 2.3 channels. By contrast, most invertebrate genomes contain only one Ca v 2 gene. The one Drosophila Ca v 2 homolog, cacophony, localizes to presynaptic active zones to induce synaptic vesicle release. We hypothesize that Drosophila Ca v 2 functional diversity is enhanced by two specific exon pairs that are mutually exclusively spliced and not conserved in vertebrates, one in the voltage sensor and one in the intracellular loop containing the binding site(s) for Caβ and G-protein βγ subunits. We test our hypothesis by combining opto- and electrophysiological with neuroanatomical approaches at a fast glutamatergic model synapse, the Drosophila larval neuromuscular junction. We find that alternative splicing in the voltage sensor affects channel activation voltage and is imperative for normal synapse function. Only the isoform with the higher activation voltage localizes to the presynaptic active zone and mediates evoked release. Removal of this Ca v 2 splice isoforms renders fast glutamatergic synapses non-functional. The By contrast, alternative splicing at the other alternative exon does not affect Ca v 2 presynaptic expression, but it tunes multiple aspects of presynaptic function. While expression of one exon yields normal transmission, expression of the other exon reduces channel number in the active zone and thus release probability. It also affects short term plasticity and abolishes presynaptic homeostatic plasticity. Thus, in Drosophila alternative splicing provides a mechanism to regulate different aspects of presynaptic functions with only one Ca v 2 gene.

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  1. Version published to 10.7554/elife.100394.1 on eLife
  2. Version published to 10.7554/elife.100394 on eLife
  3. eLife

    eLife assessment

    Cav2 voltage-gated calcium channels play key roles in regulating synaptic strength and plasticity. In contrast to mammals, invertebrates like Drosophila encode a single Cav2 channel, raising questions on how diversity in Cav2 is achieved from a single gene. Here, the authors present convincing evidence that two alternatively spliced isoforms of the Cac gene (cacophony, also known as Dmca1A and nightblindA) enable diverse changes in Cav2 expression, localization, and function in synaptic transmission and plasticity. These valuable findings will be of interest to a variety of researchers.

  4. eLife

    Reviewer #1 (Public Review):

    Summary:

    The manuscript by Bell et. al. describes an analysis of the effects of removing one of two mutually exclusive splice exons at two distinct sites in the Drosophila CaV2 calcium channel Cacophony (Cac). The authors perform imaging and electrophysiology, along with some behavioral analysis of larval locomotion, to determine whether these alternatively spliced variants have the potential to diversify Cac function in presynaptic output at larval neuromuscular junctions. The author provided valuable insights into how alternative splicing at two sites in the calcium channel alters its function.

    Strengths:

    The authors find that both of the second alternatively spliced exons (I-IIA and I-IIB) that are found in the intracellular loop between the 1st and 2nd set of transmembrane domains can support Cac function. However, loss of the I-IIB isoform (predicted to alter potential beta subunit interactions) results in 50% fewer channels at active zones and a decrease in neurotransmitter release and the ability to support presynaptic homeostatic potentiation. Overall, the study provides new insights into Cac diversity at two alternatively spliced sites within the protein, adding to our understanding of how regulation of presynaptic calcium channel function can be regulated by splicing.

    Weaknesses:

    The authors find that one splice isoform (IS4B) in the first S4 voltage sensor is essential for the protein's function in promoting neurotransmitter release, while the other isoform (IS4A) is dispensable. The authors conclude that IS4B is required to localize Cac channels to active zones. However, I find it more likely that IS4B is required for channel stability and leads to the protein being degraded, rather than any effect on active zone localization. More analysis would be required to establish that as the mechanism for the unique requirement for IS4B.

  5. eLife

    Reviewer #2 (Public Review):

    This study by Bell et al. focuses on understanding the roles of two alternatively spliced exons in the single Drosophila Cav2 gene cac. The authors generate a series of cac alleles in which one or the other mutually exclusive exons are deleted to determine the functional consequences at the neuromuscular junction. They find alternative splicing at one exon encoding part of the voltage sensor impacts the activation voltage as well as localization to the active zone. In contrast, splicing at the second exon pair does not impact Cav2 channel localization, but it appears to determine the abundance of the channel at active zones. Together, the authors propose that alternative splicing at the Cac locus enables diversity in Cav2 function generated through isoform diversity generated at the single Cav2 alpha subunit gene encoded in Drosophila.

    Overall this is an excellent, rigorously validated study that defines unanticipated functions for alternative splicing in Cav2 channels. The authors have generated an important toolkit of mutually exclusive Cac splice isoforms that will be of broad utility for the field, and show convincing evidence for distinct consequences of alternative splicing of this single Cav2 channel at synapses. Importantly, the authors use electrophysiology and quantitative live sptPALM imaging to determine the impacts of Cac alternative splicing on synaptic function. There are some outstanding questions regarding the mechanisms underlying the changes in Cac localization and function, and some additional suggestions are listed below for the authors to consider in strengthening this study. Nonetheless, this is a compelling investigation of alternative splicing in Cav2 channels that should be of interest to many researchers.

  6. eLife

    Reviewer #3 (Public Review):

    Summary:

    Bell and colleagues studied how different splice isoforms of voltage-gated CaV2 calcium channels affect channel expression, localization, function, synaptic transmission, and locomotor behavior at the larval Drosophila neuromuscular junction. They reveal that one mutually exclusive exon located in the fourth transmembrane domain encoding the voltage sensor is essential for calcium channel expression, function, active zone localization, and synaptic transmission. Furthermore, a second mutually exclusive exon residing in an intracellular loop containing the binding sites for Caβ and G-protein βγ subunits promotes the expression and synaptic localization of around ~50% of CaV2 channels, thereby contributing to ~50% of synaptic transmission. This isoform enhances release probability, as evident from increased short-term depression, is vital for homeostatic potentiation of neurotransmitter release induced by glutamate receptor impairment, and promotes locomotion. The roles of the two other tested isoforms remain less clear.

    Strengths:

    The study is based on solid data that was obtained with a diverse set of approaches. Moreover, it generated valuable transgenic flies that will facilitate future research on the role of calcium channel splice isoforms in neural function.

    Weaknesses:

    (1) Based on the data shown in Figures 2A-C, and 2H, it is difficult to judge the localization of the cac isoforms. Could they analyze cac localization with regard to Brp localization (similar to Figure 3; the term "co-localization" should be avoided for confocal data), as well as cac and Brp fluorescence intensity in the different genotypes for the experiments shown in Figure 2 and 3 (Brp intensity appears lower in the dI-IIA example shown in Figure 3G)? Furthermore, heterozygous dIS4B imaging data (Figure 2C) should be quantified and compared to heterozygous cacsfGFP/+.

    (2) They conclude that I-II splicing is not required for cac localization (p. 13). However, cac channel number is reduced in dI-IIB. Could the channels be mis-localized (e.g., in the soma/axon)? What is their definition of localization? Could cac be also mis-localized in dIS4B? Furthermore, the Western Blots indicate a prominent decrease in cac levels in dIS4B/+ and dI-IIB (Figure 1D). How do the decreased protein levels seen in both genotypes fit to a "localization" defect? Could decreased cac expression levels explain the phenotypes alone?

    (3) Cac-IS4B is required for Cav2 expression, active zone localization, and synaptic transmission. Similarly, loss of cac-I-IIB reduces calcium channel expression and number. Hence, the major phenotype of the tested splice isoforms is the loss of/a reduction in Cav2 channel number. What is the physiological role of these isoforms? Is the idea that channel numbers can be regulated by splicing? Is there any data from other systems relating channel number regulation to splicing (vs. transcription or post-transcriptional regulation)?

    (4) Although not supported by statistics, and as appreciated by the authors (p. 14), there is a slight increase in PSC amplitude in dIS4A mutants (Figure 2). Similarly, PSC amplitudes appear slightly larger (Figure 3J), and cac fluorescence intensity is slightly higher (Figure 3H) in dI-IIA mutants. Furthermore, cac intensity and PSC amplitude distributions appear larger in dI-IIA mutants (Figures 3H, J), suggesting a correlation between cac levels and release. Can they exclude that IS4A and/or I-IIA negatively regulate release? I suggest increasing the sample size for Canton S to assess whether dIS4A mutant PSCs differ from controls (Figure 2E). Experiments at lower extracellular calcium may help reveal potential increases in PSC amplitude in the two genotypes (but are not required). A potential increase in PSC amplitude in either isoform would be very interesting because it would suggest that cac splicing could negatively regulate release.

    (5) They provide compelling evidence that IS4A is required for the amplitude of somatic sustained HVA calcium currents. However, the evidence for effects on biophysical properties and activation voltage (p. 13) is less convincing. Is the phenotype confined to the sustained phase, or are other aspects of the current also affected (Figure 2J)? Could they also show the quantification of further parameters, such as CaV2 peak current density, charge density, as well as inactivation kinetics for the two genotypes? I also suggest plotting peak-normalized HVA current density and conductance (G/Gmax) as a function of Vm. Could a decrease in current density due to decreased channel expression be the only phenotype? How would changes in the sustained phase translate into altered synaptic transmission in response to AP stimulation?

    (6) Why was the STED data analysis confined to the same optical section, and not to max. intensity z-projections? How many and which optical sections were considered for each active zone? What were the criteria for choosing the optical sections? Was synapse orientation considered for the nearest neighbor Cac - Brp cluster distance analysis? How do the nearest-neighbor distances compare between "planar" and "side-view" Brp puncta?

    (7) Cac clusters localize to the Brp center (e.g., Liu et al., 2011). They conclude that Cav2 localization within Brp is not affected in the cac variants (p. 8). However, their analysis is not informative regarding a potential offset between the central cac cluster and the Brp "ring". Did they/could they analyze cac localization with regard to Brp ring center localization of planar synapses, as well as Brp-ring dimensions?

    (8) Given the accelerated PSC decay/ decreased half width in dI-IIA (Fig. 5Q), I recommend reporting PSC charge in Figure 3, and PPR charge in Figures 5A-D. The charge-based PPRs of dI-IIA mutants likely resemble WT more closely than the amplitude-based PPR. In addition, miniature PSC decay kinetics should be reported, as they may contribute to altered decay kinetics. How could faster cac inactivation kinetics in response to single AP stimulation result in a decreased PSC half-width? Is there any evidence for an effect of calcium current inactivation on PSC kinetics? On a similar note, is there any evidence that AP waveform changes accelerate PSC kinetics? PSC decay kinetics are mainly determined by GluR decay kinetics/desensitization. The arguments supporting the role of cac splice isoforms in PSC kinetics outlined in the discussion section are not convincing and should be revised.

    (9) Paired-pulse ratios (PPRs): On how many sweeps are the PPRs based? In which sequence were the intervals applied? Are PPR values based on the average of the second over the first PSC amplitudes of all sweeps, or on the PPRs of each sweep and then averaged? The latter calculation may result in spurious facilitation, and thus to the large PPRs seen in dI-IIB mutants (Kim & Alger, 2001; doi: 10.1523/JNEUROSCI.21-24-09608.2001).

    (10) Could the dI-IIB phenotype be simply explained by a decrease in channel number/ release probability? To test this, I propose investigating PPRs and short-term dynamics during train stimulation at lower extracellular Ca2+ concentration in WT. The Ca2+ concentration could be titrated such that the first PSC amplitude is similar between WT and dI-IIB mutants. This experiment would test if the increased PPR/depression variability is a secondary consequence of a decrease in Ca2+ influx, or specific to the splice isoform.

    (11) How were the depression kinetics analyzed? How many trains were used for each cell, and how do the tau values depend on the first PSC amplitude? Time constants in the range of a few (5-10) milliseconds are not informative for train stimulations with a frequency of 1 or 10 Hz (the unit is missing in Figure 5H). Also, the data shown in Figures 5E-K suggest slower time constants than 5-10 ms. Together, are the data indeed consistent with the idea that dI-IIB does not only affect cac channel number, but also PPR/depression variability (p. 9)?

    (12) The GFP-tagged I-IIA and mEOS4b-tagged I-IIB cac puncta shown in Figure 6N appear larger than the Brp puncta. Endogenously tagged cac puncta are typically smaller than Brp puncta (Gratz et al., 2019). Also, the I-IIA and I-IIB fluorescence sometimes appear to be partially non-overlapping. First, I suggest adding panels that show all three channels merged. Second, could they analyze the area and area overlap of I-IIA and I-IIB with regard to each other and to Brp, and compare it to cac-GFP? Any speculation as to how the different tags could affect localization? Finally, I recommend moving the dI-IIA and dI-IIB localization data shown in Figure 6N to an earlier figure (Figure 1 or Figure 3).

  7. eLife

    Author response:

    eLife assessment

    Cav2 voltage-gated calcium channels play key roles in regulating synaptic strength and plasticity. In contrast to mammals, invertebrates like Drosophila encode a single Cav2 channel, raising questions on how diversity in Cav2 is achieved from a single gene. Here, the authors present convincing evidence that two alternatively spliced isoforms of the Cac gene (cacophony, also known as Dmca1A and nightblindA) enable diverse changes in Cav2 expression, localization, and function in synaptic transmission and plasticity. These valuable findings will be of interest to a variety of researchers.

    We suggest replacing “two alternatively spliced isoforms of the Cac gene” by “two alternatively spliced mutually exclusive exon pairs of the Cac gene”.

    Public Reviews:

    Reviewer #1 (Public Review):

    Summary:

    The manuscript by Bell et. al. describes an analysis of the effects of removing one of two mutually exclusive splice exons at two distinct sites in the Drosophila CaV2 calcium channel Cacophony (Cac). The authors perform imaging and electrophysiology, along with some behavioral analysis of larval locomotion, to determine whether these alternatively spliced variants have the potential to diversify Cac function in presynaptic output at larval neuromuscular junctions. The author provided valuable insights into how alternative splicing at two sites in the calcium channel alters its function.

    Strengths:

    The authors find that both of the second alternatively spliced exons (I-IIA and I-IIB) that are found in the intracellular loop between the 1st and 2nd set of transmembrane domains can support Cac function. However, loss of the I-IIB isoform (predicted to alter potential beta subunit interactions) results in 50% fewer channels at active zones and a decrease in neurotransmitter release and the ability to support presynaptic homeostatic potentiation. Overall, the study provides new insights into Cac diversity at two alternatively spliced sites within the protein, adding to our understanding of how regulation of presynaptic calcium channel function can be regulated by splicing.

    Weaknesses:

    The authors find that one splice isoform (IS4B) in the first S4 voltage sensor is essential for the protein's function in promoting neurotransmitter release, while the other isoform (IS4A) is dispensable. The authors conclude that IS4B is required to localize Cac channels to active zones. However, I find it more likely that IS4B is required for channel stability and leads to the protein being degraded, rather than any effect on active zone localization. More analysis would be required to establish that as the mechanism for the unique requirement for IS4B.

    We agree that we need to explain more clearly why IS4B is unlikely required for channel stability, but instead, likely has a unique function at the presynaptic active zone of fast synapses. We will address this by revising text and by providing additional data. If IS4B was required for evoked release because it supported channel protein stability, then the removal of IS4B should cause protein degradation throughout all sub-neuronal compartments and throughout the CNS, but this is not the case. First, upon removal of IS4B in adult motoneurons (which use cac channels at the presynapse and somatodendritically, Ryglewski et al., 2012) evoked release from axon terminals is abolished (as at the larval NMJ), but somatodendritic cac inward current is present. If IS4B was required for cac channel stability, somatodendritic current should also be abolished. We will add these data to the ms. Second, immunohistochemistry for tagged IS4B channels reveals that these are present not only at presynaptic active zones at the NMJ but also throughout the VNC motor neuropils. Excision of IS4B causes the absence of cac channels from the presynaptic active zones at the NMJ and throughout the VNC neuropils (and accordingly this is lethal). By contrast, tagged IS4A channels (with IS4B excised) are not found at the presynaptic terminals of fast synapses, but instead, in other distinct parts of the CNS. We will also provide data to show this. Together these data are in line with a unique requirement of IS4B at presynaptic active zones (not excluding additional functions of IS4B), whereas IS4A containing cac isoforms mediate different functions.

    We appreciate the additional reviewer suggestions to the authors that we will address point by point when revising the ms.

    Reviewer #2 (Public Review):

    This study by Bell et al. focuses on understanding the roles of two alternatively spliced exons in the single Drosophila Cav2 gene cac. The authors generate a series of cac alleles in which one or the other mutually exclusive exons are deleted to determine the functional consequences at the neuromuscular junction. They find alternative splicing at one exon encoding part of the voltage sensor impacts the activation voltage as well as localization to the active zone. In contrast, splicing at the second exon pair does not impact Cav2 channel localization, but it appears to determine the abundance of the channel at active zones. Together, the authors propose that alternative splicing at the Cac locus enables diversity in Cav2 function generated through isoform diversity generated at the single Cav2 alpha subunit gene encoded in Drosophila.

    Overall this is an excellent, rigorously validated study that defines unanticipated functions for alternative splicing in Cav2 channels. The authors have generated an important toolkit of mutually exclusive Cac splice isoforms that will be of broad utility for the field, and show convincing evidence for distinct consequences of alternative splicing of this single Cav2 channel at synapses. Importantly, the authors use electrophysiology and quantitative live sptPALM imaging to determine the impacts of Cac alternative splicing on synaptic function. There are some outstanding questions regarding the mechanisms underlying the changes in Cac localization and function, and some additional suggestions are listed below for the authors to consider in strengthening this study. Nonetheless, this is a compelling investigation of alternative splicing in Cav2 channels that should be of interest to many researchers.

    We agree that some additional information on cac isoform localization (in particular for splicing at the IS4 site) will strengthen the manuscript. We will address this by providing additional data and revising text (see responses to reviewers 1 and 3). We are also grateful for the additional reviewer suggestions which we will address point by point when revising the ms.

    Reviewer #3 (Public Review):

    Summary:

    Bell and colleagues studied how different splice isoforms of voltage-gated CaV2 calcium channels affect channel expression, localization, function, synaptic transmission, and locomotor behavior at the larval Drosophila neuromuscular junction. They reveal that one mutually exclusive exon located in the fourth transmembrane domain encoding the voltage sensor is essential for calcium channel expression, function, active zone localization, and synaptic transmission. Furthermore, a second mutually exclusive exon residing in an intracellular loop containing the binding sites for Caβ and G-protein βγ subunits promotes the expression and synaptic localization of around ~50% of CaV2 channels, thereby contributing to ~50% of synaptic transmission. This isoform enhances release probability, as evident from increased short-term depression, is vital for homeostatic potentiation of neurotransmitter release induced by glutamate receptor impairment, and promotes locomotion. The roles of the two other tested isoforms remain less clear.

    Strengths:

    The study is based on solid data that was obtained with a diverse set of approaches. Moreover, it generated valuable transgenic flies that will facilitate future research on the role of calcium channel splice isoforms in neural function.

    Weaknesses:

    (1) Based on the data shown in Figures 2A-C, and 2H, it is difficult to judge the localization of the cac isoforms. Could they analyze cac localization with regard to Brp localization (similar to Figure 3; the term "co-localization" should be avoided for confocal data), as well as cac and Brp fluorescence intensity in the different genotypes for the experiments shown in Figure 2 and 3 (Brp intensity appears lower in the dI-IIA example shown in Figure 3G)? Furthermore, heterozygous dIS4B imaging data (Figure 2C) should be quantified and compared to heterozygous cacsfGFP/+.

    We understand the reviewer’s comment and will do the following to convincingly demonstrate absence of cac from presynaptic active zones upon IS4B excision. First, we will show selective enlargements of IS4A and IS4B with Brp in presynaptic active zones to show distinct cac label in active zones following excision of IS4A but not following excision of IS4B. Second, we will provide Pearson’s co-localization coefficients of Brp with IS4B and with IS4A, respectively. Third, we will reduce the intensity of the green channels in figures 2C and 2H to the same levels as in 2A and B, and H control to allow a fair comparison of cac intensities following excision of IS4B versus excision of IS4A and control. We had increased intensity to show that following excision of IS4B, no distinct cac label is found in active zones, even at high exaggerated image brightness. However, we agree with the reviewer that the bright background hampers interpretation and thus will show the same intensity in all images that need to be compared.

    (2) They conclude that I-II splicing is not required for cac localization (p. 13). However, cac channel number is reduced in dI-IIB. Could the channels be mis-localized (e.g., in the soma/axon)? What is their definition of localization? Could cac be also mis-localized in dIS4B? Furthermore, the Western Blots indicate a prominent decrease in cac levels in dIS4B/+ and dI-IIB (Figure 1D). How do the decreased protein levels seen in both genotypes fit to a "localization" defect? Could decreased cac expression levels explain the phenotypes alone?

    We will precisely define channel localization, and we will explain why it is highly unlikely that the absence of IS4B channels as well as the lower number of I-IIA channels are simply a consequence of reduced expression, but instead of splice variant specific channel function and localization. For example, upon excision of IS4B no cac channels are found at the presynaptic active zones and these synapses are thus non-functional. The isoforms containing the mutually exclusive IS4A exon are expressed and mediate other functions (see also response to reviewer 1) but cannot substitute IS4B containing isoforms at the presynapse. In fact, our Western blots are in line with reduced cac expression if all isoforms that mediate evoked release are missing, again indicating that the presynapse specific cac isoforms cannot be replaced by other cac isoforms (see also below, response to (3)). Feedback mechanisms that regulate cac expression in the absence of presynapse specific cac isoforms are beyond the scope of this study.

    (3) Cac-IS4B is required for Cav2 expression, active zone localization, and synaptic transmission. Similarly, loss of cac-I-IIB reduces calcium channel expression and number. Hence, the major phenotype of the tested splice isoforms is the loss of/a reduction in Cav2 channel number. What is the physiological role of these isoforms? Is the idea that channel numbers can be regulated by splicing? Is there any data from other systems relating channel number regulation to splicing (vs. transcription or post-transcriptional regulation)?

    We will provide additional evidence that mutually exclusive splicing at the IS4 site results in cac channels that localize to the presynaptic active zone (IS4B) versus cac channels that localize to other brain parts and/or other subneuronal compartments (see response to reviewer 1). In addition, we already show in figure 2J that IS4B is required for normal cac HVA current, and we can add data showing that IS4A is not essential for cac HVA current. Similarly, for I-II we find it unlikely that differential splicing regulates channel numbers, but rather splice variant specific functions in different brain parts and different sub-neuronal compartments. To substantiate this interpretation, we will add data from developing adult motoneurons showing that excision of I-IIA causes reduced activity induced calcium influx into dendrites (new data), but it does not reduce channel number at the larval NMJ (figure 4). In our opinion these data are not in line with the idea that splicing regulates cac expression levels, and this in turn, results in specific defects in distinct neuronal compartments. However, we agree that the lack of isoforms with specific functions results in altered overall cac expression levels as indicated by our Western data. If isoforms normally abundantly expressed throughout most neuropils are missing due to exon excision, we indeed find less cac protein in Westerns. By contrast, the lack of isoforms with little abundance has little effect on cac expression levels. This may be the results of unknown feedback mechanisms which are beyond the scope of this study.

    (4) Although not supported by statistics, and as appreciated by the authors (p. 14), there is a slight increase in PSC amplitude in dIS4A mutants (Figure 2). Similarly, PSC amplitudes appear slightly larger (Figure 3J), and cac fluorescence intensity is slightly higher (Figure 3H) in dI-IIA mutants. Furthermore, cac intensity and PSC amplitude distributions appear larger in dI-IIA mutants (Figures 3H, J), suggesting a correlation between cac levels and release. Can they exclude that IS4A and/or I-IIA negatively regulate release? I suggest increasing the sample size for Canton S to assess whether dIS4A mutant PSCs differ from controls (Figure 2E). Experiments at lower extracellular calcium may help reveal potential increases in PSC amplitude in the two genotypes (but are not required). A potential increase in PSC amplitude in either isoform would be very interesting because it would suggest that cac splicing could negatively regulate release.

    There are several possibilities to explain this, but as none of the effects are statistically significant, we prefer to not investigate this in depth. However, given that we cannot find IS4A at the presynaptic active zone, IS4A is unlikely to have a direct negative effect on release probability. Nonetheless, given that IS4A containing cac isoforms mediate functions in other neuronal compartments it may regulate release indirectly by affecting action potential shape. We will provide data in response to the more detailed suggestions to authors that will provide additional insight.

    (5) They provide compelling evidence that IS4A is required for the amplitude of somatic sustained HVA calcium currents. However, the evidence for effects on biophysical properties and activation voltage (p. 13) is less convincing. Is the phenotype confined to the sustained phase, or are other aspects of the current also affected (Figure 2J)? Could they also show the quantification of further parameters, such as CaV2 peak current density, charge density, as well as inactivation kinetics for the two genotypes? I also suggest plotting peak-normalized HVA current density and conductance (G/Gmax) as a function of Vm. Could a decrease in current density due to decreased channel expression be the only phenotype? How would changes in the sustained phase translate into altered synaptic transmission in response to AP stimulation?

    Most importantly, HVA current is mostly abolished upon excision of IS4B (not IS4A, we think the reviewer accidentally mixed up the genotype). This indicates that the cac isoforms that mediate evoked release encode HVA channels. However, the somatodendritic current shown in figure 2J that remains upon excision of IS4B is mediated by IS4A containing cac isoforms. Please note that these never localize to the presynaptic active zone, thus the small inactivating HVA that remains in figure 2J does normally not mediate evoked release. Therefore, the interpretation is that specifically HVA current encoded by IS4B cac isoforms is required for synaptic transmission. Reduced cac current density is not the cause for this phenotype because a specific current component is absent.

    We agree with the reviewer that a deeper electrophysiological analysis of cac currents mediated by IS4B containing isoforms will be instructive. However, a precise analysis of activation and inactivation voltages and kinetics suffers form space clamp issues in recordings from the soma of such complex neurons (DLM motoneurons of the adult fly). Therefore, we will analyze the currents in a heterologous expression system and present these data to the scientific community as a separate study at a later time point.

    (6) Why was the STED data analysis confined to the same optical section, and not to max. intensity z-projections? How many and which optical sections were considered for each active zone? What were the criteria for choosing the optical sections? Was synapse orientation considered for the nearest neighbor Cac - Brp cluster distance analysis? How do the nearest-neighbor distances compare between "planar" and "side-view" Brp puncta?

    Max. z-projections would be imprecise because they can artificially suggest close proximity of label that is close in x and y but far away in z. Therefore, the analysis was executed in xy-direction of various planes of entire 3D image stacks. We considered active zones of different orientations (Fig. 4C, D). In fact, we searched the entire z-stacks until we found active zones of all orientations shown in figures 4C1-C6 within the same boutons. The same active zone orientations were analyzed for all exon-out mutants with cac localization in active zones. The distance between cac and brp did not change if viewed from the side.

    (7) Cac clusters localize to the Brp center (e.g., Liu et al., 2011). They conclude that Cav2 localization within Brp is not affected in the cac variants (p. 8). However, their analysis is not informative regarding a potential offset between the central cac cluster and the Brp "ring". Did they/could they analyze cac localization with regard to Brp ring center localization of planar synapses, as well as Brp-ring dimensions?

    In the top views (planar) we did not find any clear offset in cac orientation to brp between genotypes. This study focuses on cac splice isoform specific localization and function. Possible effects of different cac isoforms on Brp-ring dimensions or other aspects of scaffold structure are not central to our study, in particular given that Brp puncta are clearly present even if cac is absent from the synapse (Fig. 2H), indicating that cac is not instructive for the formation of the Brp scaffold.

    (8) Given the accelerated PSC decay/ decreased half width in dI-IIA (Fig. 5Q), I recommend reporting PSC charge in Figure 3, and PPR charge in Figures 5A-D. The charge-based PPRs of dI-IIA mutants likely resemble WT more closely than the amplitude-based PPR. In addition, miniature PSC decay kinetics should be reported, as they may contribute to altered decay kinetics. How could faster cac inactivation kinetics in response to single AP stimulation result in a decreased PSC half-width? Is there any evidence for an effect of calcium current inactivation on PSC kinetics? On a similar note, is there any evidence that AP waveform changes accelerate PSC kinetics? PSC decay kinetics are mainly determined by GluR decay kinetics/desensitization. The arguments supporting the role of cac splice isoforms in PSC kinetics outlined in the discussion section are not convincing and should be revised.

    We agree that reporting charge in figure 3 will be informative and will do so. We also understand the reviewer’s concern attributing altered PSC kinetics to presynaptic cac channel properties. We will tone down our interpretation in the discussion and list possible alterations in presynaptic AP shape or Cav2 channel kinetics as alternative explanations (not conclusions). Moreover, we will quantify postsynaptic GluRIIA abundance to test whether altered PSC kinetics are caused by altered GluRIIA expression. In our opinion, the latter is more instructive than mini decay kinetic analysis because this depends strongly on the distance of the recording electrode to the actual site of transmission in these large muscle cells.

    (9) Paired-pulse ratios (PPRs): On how many sweeps are the PPRs based? In which sequence were the intervals applied? Are PPR values based on the average of the second over the first PSC amplitudes of all sweeps, or on the PPRs of each sweep and then averaged? The latter calculation may result in spurious facilitation, and thus to the large PPRs seen in dI-IIB mutants (Kim & Alger, 2001; doi: 10.1523/JNEUROSCI.21-24-09608.2001).

    We agree that the PP protocol and analyses have to be described more precisely in the methods, and we will do so. PPR values are based on the PPRs of each sweep and then averaged. We are aware of the study of Kim and Alger 2001, but it does not affect our data interpretation because all genotypes were analyzed identically, but only the I-IIB excision resulted in the large data spread shown in figure 5.

    (10) Could the dI-IIB phenotype be simply explained by a decrease in channel number/ release probability? To test this, I propose investigating PPRs and short-term dynamics during train stimulation at lower extracellular Ca2+ concentration in WT. The Ca2+ concentration could be titrated such that the first PSC amplitude is similar between WT and dI-IIB mutants. This experiment would test if the increased PPR/depression variability is a secondary consequence of a decrease in Ca2+ influx, or specific to the splice isoform.

    In fact, the interpretation that decreased PSC amplitude upon I-IIB excision is caused mainly by reduced channel number is precisely our interpretation (see discussion page 14, last paragraph to page 15, first paragraph). In addition, we are grateful for the reviewer’s suggestion to triturate the external calcium such that the first PSC amplitude matches the one in ΔI-IIB to test whether altered short term plasticity is solely a function of altered channel number or whether additional causes, such as altered channel properties, also play into this. We will conduct these experiments and include them in the revised manuscript.

    (11) How were the depression kinetics analyzed? How many trains were used for each cell, and how do the tau values depend on the first PSC amplitude? Time constants in the range of a few (5-10) milliseconds are not informative for train stimulations with a frequency of 1 or 10 Hz (the unit is missing in Figure 5H). Also, the data shown in Figures 5E-K suggest slower time constants than 5-10 ms. Together, are the data indeed consistent with the idea that dI-IIB does not only affect cac channel number, but also PPR/depression variability (p. 9)?

    For each animal, the amplitudes of each PSC were plotted over time and fitted with a single exponential. For depression at 1 and 10 Hz, we used one train per animal, and 5-6 animals per genotype (as reflected in the data points in Figs 5H and 5L). Given that the tau values are highly similar between control and excision of I-IIA, but ΔI-IIA tends to have larger single PSC amplitudes, differences in first PSC amplitude do not seem to skew the data (but see also response to comment 10 above). We thank the reviewer for pointing out that tau values in the range of ms are not informative at 1 and 10 Hz stimulations (Figs 5H and 5L). We mis-labeled (or did not label) the axes. The label should read seconds, not milliseconds. We apologize, and this will be corrected accordingly.

    In sum, pending the outcome of additional important control experiments for GluRIIA abundance (see response to comment 8) and trituration of control PSC amplitude for the first pulse of paired pulses in ΔI-IIB (see response to comment 10) we will either modify or further support that interpretation.

    (12) The GFP-tagged I-IIA and mEOS4b-tagged I-IIB cac puncta shown in Figure 6N appear larger than the Brp puncta. Endogenously tagged cac puncta are typically smaller than Brp puncta (Gratz et al., 2019). Also, the I-IIA and I-IIB fluorescence sometimes appear to be partially non-overlapping. First, I suggest adding panels that show all three channels merged. Second, could they analyze the area and area overlap of I-IIA and I-IIB with regard to each other and to Brp, and compare it to cac-GFP? Any speculation as to how the different tags could affect localization? Finally, I recommend moving the dI-IIA and dI-IIB localization data shown in Figure 6N to an earlier figure (Figure 1 or Figure 3).

    We will show panels with all three labels matched as suggested by the reviewer. For the size of the puncta: this could be different numbers and types of fluorophores on the different antibodies used and thus different point spread, chromatic aberration, different laser and detector intensities etc. We will re-analyze the data to test whether there are systematic differences in size. We do not want to speculate whether the different tags have any effect on localization precision because of the abovementioned reasons as well as artificial differences in localization precision that can be suggested by different antibodies. We prefer to not move the figure because we believe it is informative to show our finding that active zones usually contain both splice variants together with the finding that only one splice variant is required for PHP.

  8. Version published to 10.1101/2024.06.14.598978v1 on bioRxiv