Finally, a third approach attempts to mirror the phosphoramidite-based protocols for in situ synthesis of high-density DNA arrays in order to produce in situ synthesized RNA microarrays. In this mini-review, we describe the chemistry and the engineering behind the fabrications methods, underlining the advantages and shortcomings of each, and illustrate how versatile these platforms can be by presenting some of their applications.DNA microarrays have become commonplace in the last two decades, but the synthesis of other nucleic acids biochips, most importantly RNA, has only recently been developed to a similar extent. RNA microarrays can be seen as organized surfaces displaying a potentially very large number of unique sequences and are of invaluable help in understanding the complexity of RNA structure and function as they allow the probing and treatment of each of the many different sequences simultaneously. Three approaches have emerged for the fabrication of RNA microarrays. The earliest examples used a direct, manual or mechanical, deposition of pre-synthesized, purified RNA oligonucleotides onto the surface in a process called spotting. In a second approach, pre-spotted or  Not oen route towards DNA-based digital information storage solutions ..en route22.1ex situ on solid support using standard phosphoramidite and 2′-O-TBDMS chemistries then purified, or purchased from a commercial source directly in pure form. Pre-synthesis also allows for the introduction of chemical modifications at the base, sugar or internucleotidic levels. Alternatively, RNA may be obtained by transcription of the template DNA sequence. In essence, spotting grants control over RNA sequence and purity, a probable necessity in the conception of the first RNA microchips, and confines all chemical steps to a single reaction: attaching the RNA molecule to the surface (O-(3-thiotriphosphate) guanosine ribonucleotide, and the resulting thio-containing RNA was immobilized onto a gold surface [via the formation of a Schiff base [Spotting is perhaps the simplest and most straightforward approach for the fabrication of RNA microarrays. It is indeed through spotting that the first DNA microarrays were produced, and it therefore comes as no surprise that the earliest attempts at arraying RNA onto a surface were also conducted using pre-synthesized RNA oligonucleotides. In so doing, the RNA is synthesized  surface . The ext surface . In one  surface . Multipl surface . Spottin surface . Finally surface . An alteiff base . These “2.2in situ [via hybridization [An entirely different approach to RNA array fabrication takes advantage of the rapidity and high-fidelity of enzymes to polymerize RNA from a DNA template, an approach that minimizes RNA handling and instead relies on a starting deoxyribose array, which is easier to manipulate, cheaper to synthesize, and already available in high-density. Indeed, the history of DNA microarray fabrication is at least a decade older than that of RNA and similarly started with spotting pre-synthesized DNA oligonucleotides, but the technology gained momentum as the phosphoramidite chemistry was successful ported to the glass surface, allowing for hundreds, thousands, then hundreds of thousands of different DNA sequences to be synthesized in parallel, and in situ . DNA arrdization . Alternadization . Up to e7 RNA sequences on the flow cell. An adaptation of this procedure uses the Tus protein, a known replication terminator, to halt RNA transcription once the RNA polymerase reaches this block. Due to incomplete polymerization, the RNA remains bound to the transcription complex [Diffusion and capture being an additional step in the process of RNA array fabrication and governed by its own set of physical and chemical rules, forcing the RNA to remain where it was originally transcribed on the surface appears convenient. However, in this approach, where template and transcript do not completely dissociate, the fate of the template DNA needs to be addressed. One solution is to leave the dsDNA template intact, all the while keeping the transcribed RNA loosely bound to its template. This was recently shown to be a viable method for RNA array fabrication on a sequencing flow cell as a platform for “on-array” transcription ,36. A li complex . Flow cein situ using a photolithographic approach, which uses UV light to trigger the removal of the photosensitive NPPOC group at the 5′-end of the growing oligonucleotide strand (vide infra) [in situ synthesis being a prime example of a chip fabrication process compatible with high density and high complexity, “on-array” transcription of in situ-synthesized DNA array can be seen as a real route towards high-density RNA microarrays.Template degradation is perhaps the closest step to an RNA-only microchip, but in order for this step to successfully proceed, the transcribed RNA must be bound to the surface independently from its DNA template, all the while avoiding diffusion into the solution phase so as to retain the uniformity and the spatial organization of the template DNA array. This challenge can be solved using proximity and primer elongation. Single-stranded DNA microarrays are first synthesized e infra) . After se infra) ,39. With2.3via phosphoramidite chemistry, an extremely robust, fast and efficient coupling reaction that can proceed just as efficiently on RNA phosphoramidites, albeit with longer coupling times. However, the synthetic protocols for RNA oligonucleotide elongation on solid-phase are not directly translatable to microarrays and the issue simply boils down to the choice of protecting group for the 2′ hydroxyl. In solid-phase synthesis, a bulky silyl group protects the 2′-OH and is post-synthetically removed with fluoride-containing organic salts or acids, an incompatible approach for microarray fabrication on glass as it would corrode the surface and wash away the synthesized RNA. The silyl protection strategy could be suitable for in situ RNA synthesis on non‑silicon based substrates, such as glassy carbon, PTFE or other polymers, but has not been pursued yet. Alternative 2′-protecting groups exist, but they are neither as universal as the standard TBDMS moiety nor are the corresponding phosphoramidites necessarily compatible with array synthesis. One of those alternatives relies on the use of the levulinyl (lev) group as a hydroxyl protection strategy, transforming the OH into a ketoester that can easily be removed under mild conditions with hydrazine hydrate: hydrazine first reacts with the ketone function to form a hydrazone, and the second amine can then react with the nearby ester function, releasing the hydroxyl group and a hydropyridazine as a byproduct [in situ on a silanized glass slide by maskless photolithography, where an array of small micromirrors, electronically controlled by a computer, can be tilted into a position so as to reflect the incoming UV light onto the surface , and with the layout of tilted mirrors being imaged 1:1 onto the substrate, the microscope slide can therefore contain as many features as there are mirrors and within the same area. Therefore, photolithography can be regarded as a high-density array fabrication method. We first reported on the synthesis of rU and rA homopolymers [The protocols for solid-phase synthesis of DNA oligonucleotides have long been adapted to the parallel synthesis of thousands of different DNA molecules on flat substrates, with photolithography, electrochemistry and inkjet printing three means of addressing precise and well-defined locations on a substrate. And technological improvements in the miniaturization of mirrors, electrodes and printing heads further increases the sequence complexity that a single flat surface can hold. Those improvements are made over the rock-solid foundation of DNA synthesis yproduct . A set oyproduct , and theyproduct . RNA oli surface . Oligonupolymers , but we polymers .Fig. 3Le3In many instances, a simple hybridization to the RNA with a fluorescently-labeled complementary strand is necessary and sufficient to indicate correct spotting/transcription/synthesis. But the usefulness of RNA microarrays goes beyond that of traditional DNA microarrays, extending for instance, to the study of biologically-relevant interactions such as ribozymes and enzymes, aptamer and RNA-binding proteins.3.1in situ synthesized microarrays, an RNase A assay on sequences containing a single rU monomer flanked by a varying number of dA nucleotides revealed kinetics and sequence preferences comparable to solution-phase data, i.e. substrates with a greater number of pyrimidine nucleobases around the rU insert were better recognized and cleaved by the enzyme [4 permutations of a 5-nt long region). RNase HII-mediated enzymatic degradation performed significantly better for substrates containing the 5′-dC-rC-3′ motif, but appeared to be independent of the nature of the DNA base 3′ to the RNA incorporation, which is the site of 2′-deoxy sensing by the enzyme. RNase H also recognizes and cleaves RNA oligos synthesized in situ, while leaving the corresponding DNA sequences intact [RNases are a class of enzymes that have been heavily investigated on RNA microarrays. RNA oligonucleotides ligated onto spotted DNA gold slides were exposed to RNase S and were shown to be correctly degraded using Surface Plasmon Resonance Imaging (SPRI) . When the enzyme . With RNs intact .32P radioactivity. Taken together, these assays not only serve to confirm the integrity and identity of the RNA, regardless of the microarray fabrication approach, they also verify the functionality and the accessibility of immobilized RNAs to small molecules, nucleic acids and enzymes.Enzymatic properties have been observed and described in very specific DNA and RNA sequences. The well-known RNA-cleaving 10–23 DNAzyme was used to degrade RNA oligonucleotides transcribed from high-density DNA arrays, while DNA sequences remained untouched. Similarly, RNase A degraded transcribed RNA but not the template DNA . Ribozym3.2via transcription of spotted DNA arrays and assayed in presence of the triarylmethane dye malachite green [A fair number of arrayed RNA oligonucleotides have been used in non-destructive binding experiments, most notably to proteins, in order to highlight the ability of the microchip to act as a detection platform. RNA arrays were successfully used to detect thrombin ,33, vascte green . The fluKd values comparable to earlier literature measurements [RNA microarrays transcribed from immobilized DNA on Illumina flow cells generated a colossal amount of data, owing to the high density and complexity of the available sequence space, from which RNA-protein dissociation constants could be extracted. Binding of a wild-type RNA aptamer to either the coat protein of the MS2 bacteriophage, the GFP protein or the negative elongation factor E (NELFe), or the Vts1 protein afforded urements , an impo4i.e. a single experiment) of sequence variations in presence of a given target, with each nucleic acids construct fixed and immobilized in a known and well-defined location that no other construct occupies, and the assay provides a complete overview of the reaction landscape, from poor substrates and poor binders to excellent substrates and strong aptamers. RNA microarrays certainly find their raison d'être in being interrogation platforms for a multitude of RNA·ligand interactions, but in order to perform and deliver a comprehensive understanding of binding/reaction mechanisms, RNA arrays need to be high-density. Here, the three approaches described in this review can be reduced down to two categories: one where the surface is a receptacle for pre-synthesized RNA oligonucleotides, and one where the surface actively participates in RNA synthesis either directly or through DNA. The first category refers to spotting and is an excellent approach to verify the functionality and usefulness of RNA molecules covalently attached to a slide, but is low-density due to the need for a pre-synthesized oligonucleotide for each spot on the surface. Conversely, in situ RNA synthesis, chemical or enzymatic, is a high-density fabrication method as it yields hundreds of thousands and up to several millions of variants and, de facto, appears to be the only way to probe a large enough permutation space. Synthetic and technical improvements may yet increase this capacity by a few orders of magnitude. However, high-density RNA arrays may still be insufficiently complex when it comes to finding and selecting entirely novel RNA structures that bind a chosen target, as SELEX experiments typically start with combinatorial libraries containing >1015 sequences. Microarrays also currently suffer from a dearth of modified nucleosides available for array fabrication —and microarrays are ideal tools to perform systematic substitutions between natural and synthetic analogs— [Herein lies a real advantage of microarrays over other high-throughput methods; they allow for the parallel study  and the Swiss National Science Foundation (PBBEP2_146174) are gratefully acknowledged for the support and the funding of the work on RNA photolithography presented in this review.The


Cystoviridae family rely on specific RNA–protein interactions to select appropriate +RNAs for packaging, viruses of the Reoviridae instead rely on specific inter-molecular interactions between +RNAs that guide multi-segmented genome assembly. While these families use distinct mechanisms to direct +RNA packaging, both yield progeny particles with a complete set of genomic dsRNAs.Segmented double-stranded (ds)RNA viruses share remarkable similarities in their replication strategy and capsid structure. During virus replication, positive-sense single-stranded (+)RNAs are packaged into procapsids, where they serve as templates for dsRNA synthesis, forming progeny particles containing a complete equimolar set of genome segments. How the +RNAs are recognized and stoichiometrically packaged remains uncertain. Whereas bacteriophages of the  Current Opinion in Virology 2018, 33:106–112Multicomponent viral systemsThis review comes from a themed issue on Stéphane Blanc and Yannis MichalakisEdited by Issue and the EditorialFor a complete overview see the Available online 23rd August 2018https://doi.org/10.1016/j.coviro.2018.08.001http://creativecommons.org/licenses/by/4.0/).1879-6257/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license RNA genomes, genome encapsidation requires recognition and packaging of a set of positive-sense (+) single-stranded (ss)RNAs in an environment rich in heterologous RNAs. Packaging may proceed by the sequential translocation of multiple +RNAs into pre-assembled procapsids ••,3,4••,Cystoviridae share structural similarities with those of the Reoviridae [T = 1 inner core that contains multiple copies of a viral RNA-dependent RNA polymerase (RdRP) [Cystoviridae are the only dsRNA viruses known to package +RNAs by translocation into pre-assembled cores [Cystoviridae is Pseudomonas virus φ6, whose assembly mechanisms have been examined using in vitro and in vivo systems [Members of the oviridae . Virusese (RdRP) ,7. Virale (RdRP) , 9, 10. ed cores . The bes systems ••,13••.  systems ••,14. Th systems , 16, 17. systems . Likewis systems . Remarka systems .Figure 1pac) signals present at the 5′ ends of φ6 +RNAs. Each pac signal resolves into two regions: a 5′ stretch of 18 nucleotides conserved among all three +RNAs and an adjoining downstream stretch of ∼200 nucleotides that folds to form a unique secondary structure [pac signal of the s+ RNA to a recognition site located on the vertices of the procapsid [pac signal [pac signal, leading to insertion of the l+ RNA  are packaged sequentially into procapsids in order of size, beginning with the smallest. Critical to the process are segment-specific packaging  layer. The cores of BTV and rotavirus are nearly indistinguishable, with copies of the viral RdRP and RNA capping enzyme (CAP) anchored to the interior core face near 5-fold vertices. By contrast, while MRV and CPV cores have similarly positioned RdRP molecules, their CAP components form turrets that extend through the core protein layer at 5-fold vertices.Unlike actories , 29, 30.ed cores ,32. IndeReoviridae packaging mechanism was established for BTV [prior to incorporation into core structures. In these experiments, efficient core assembly required all 10 BTV + RNAs and the presence of both, viral RdRP and CAP [The only cell-free reconstitution system available to study the  for BTV ••. Resul for BTV  and a co and CAP ••. Regio and CAP . Recent  and CAP •.Reoviridae +RNAs are important for RNA packaging.Studies performed using MRV reverse genetics systems indicate that the 5′-terminal 125–200 nucleotides and 3′-terminal 180–285 nucleotides of MRV + RNAs are essential for RNA assortment and packaging ••. Becauin vitro packaging system and, until recently, the lack of a robust reverse genetics system. Nevertheless, substantial progress has been made in understanding various aspects of rotavirus RNA replication and particle assembly through structural and biochemical studies. Rotaviruses are triple-layered particles (TLPs), consisting of core, intermediate, and outer protein layers [Studies on the rotavirus packaging mechanism have been challenging due to the lack of an n layers ,38,39. Dn layers ,41. Analn layers . Consistn layers •.in situ organization of CPV genome segments. Its 10 segments are organized within the core in a non-symmetrical, non-spooled manner, with each interacting with a polymerase complex anchored on the inner surface near a 5-fold vertex. Two of the 12 vertices of the CPV core lack polymerase complexes, but are occupied with dsRNA emanating from genomic segments linked to polymerases bound to neighboring vertices [Single-molecule imaging of transcribing rotavirus DLPs has revealed that each genome segment is associated with a dedicated polymerase complex, responsible for synthesis and extrusion of a single type of +RNA from 5-fold channels of the particle ••. A simvertices •,47•.in vitro [For rotaviruses, specific intermolecular interactions between distinct types of viral + RNAs may guide the assembly of an RNA assortment complex, prior to core encapsidation and RNA replication  10,48,4,448,49••in vitro , the rotin vitro ,50. Morein vitro ,16. As aThe rotavirus nonstructural proteins NSP2 and NSP5 are non-specific RNA binding proteins that accumulate to high levels in viroplasms , 55, 56.in vitro studies and reverse genetics experiments strongly support such a model [in vitro. Using two-color fluorescence correlation spectroscopy (FCCS)-based RNA–RNA interaction assays, NSP2 was found to promote de novo formation of sequence-specific inter-molecular contacts among the 11 rotavirus + RNAs [Current models favor the idea that rotavirus assortment is driven by intermolecular interactions occurring between the 11 species of viral + RNAs ••,57••.  a model •,58. Fors + RNAs •• RNAs may have the global features enabling packaging within a viral core •,63. Bin of NSP2 ••. Accor of NSP2 •,69,70. Reoviridae family employ mechanisms of RNA selection and packaging that are distinct from those utilized by members of the tri-segmented Cystoviridae. Such differences can be attributed to the different complexities of selecting and packaging 9–12 versus 3 +RNAs into progeny cores. In the case of Reoviridae, RNA selection and packaging take place in viroplasms, sites where +RNA and protein components are concentrated, including chaperone proteins that can act to facilitate and modulate such processes. The NSP2 octamer — an RNA-binding protein with a helix-destabilizing activity — is key for RNA selection and packaging in the case of the rotavirus. Notably, nonstructural proteins similar to NSP2 are produced by other members of the Reoviridae, suggesting a conserved essential role for NSP2-like proteins in the replication and packaging of all viruses in the family. Increasing evidence suggests that Reoviridae assortment is driven by interactions involving multiple regions of +RNAs, including those positioned within UTRs and even ORFs. Interestingly, RNA–RNA interactions that give rise to assortment are dependent on NSP2, and as a result appear likely to occur preferentially in viroplasms. Thus, free +RNAs in the cytosol may be precluded from interacting with each other, preventing the formation of RNA–RNA complexes that may interfere with translation or stimulate RNA-dependent innate immune responses. With the recent development of a completely plasmid-based rotavirus reverse genetics system it will become possible to identify sequences in +RNAs that direct the RNA–RNA interactions leading to complete assortment of all 11 viral + RNAs. From this should come information helping to explain how RNA assortment influences the maintenance of preferred genome constellations, a characteristic common for rotavirus strains isolated from different hosts and a factor influencing rotavirus evolution and genetic diversity.The multi-segmented dsRNA viruses of the • of special interest•• of outstanding interestPapers of particular interest, published within the period of review, have been highlighted as


Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(zf35gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(zf35gf) mutants. unc-2(zf35gf) mutants display increased cholinergic and decreased GABAergic transmission. Moreover, increased cholinergic transmission in unc-2(zf35gf) mutants leads to an increase of cholinergic synapses and a TAX-6/calcineurin-dependent reduction of GABA synapses. Our studies reveal mechanisms through which CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system.Mutations in pre-synaptic voltage-gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the  Maintenance of proper brain function requires the balance of excitatory and inhibitory synaptic transmission. There is an increasing amount of evidence that the disruption of E/I balance in neural circuits is associated with neurological disorders, including autism, epilepsy and migraine . SeveralCACNA1A gene, which encodes the pore-forming α subunit of the CaV2.1 (P/Q-type) voltage-gated calcium channel (VGCC), are associated with a broad spectrum of autosomal dominant neurological disorders. CaV2 VGCCs are the predominant channels in presynaptic nerve terminals, where they mediate the Ca2+ influx that triggers the fusion of synaptic vesicles with the presynaptic membrane (CACNA1A mutations can cause episodic ataxia type 2 (EA2), epileptic seizures and familial hemiplegic migraine type 1 (FHM1) , a severe variant of migraine that can co-occur with tonic-clonic seizures, has been found to be associated with missense mutations near the voltage sensors of the α1 subunit (Mutations in the membrane . CACNA1A1 (FHM1) . Episodi subunit . Electro subunit . AlthougCACNA1A mutations. Mice carrying FHM1 missense mutations R192Q or S218L in the cacna1a gene display gain-of-function CaV2 phenotypes with increased Ca2+ current density at lower voltages  mutants are sluggish and uncoordinated (unc-2(lf) mutants have a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs)  , and a r neurons .unc-2/CaV2α gain-of-function (gf) mutant, which, in sharp contrast to the loss-of-function mutant, exhibits hyperactive- as well as seizure-Iike motor behaviors. We show that the expression of an unc-2 gene carrying FHM1 mutations results in a similar hyperactive behavioral phenotype, while the intragenic suppressor alleles of unc-2(gf) resemble EA2 mutations and are lethargic. We reveal that the unc-2(gf) mutation shifts the E/I balance toward excitatory transmission, and that increased excitatory signaling leads to the destabilization of GABAergic synapses in a TAX-6/calcineurin-dependent manner.In this study, we characterize a novel C. elegans locomotion is biased toward sustained forward runs, interrupted by periodic brief reversals. From a forward genetic screen for animals with locomotion defects, we isolated a mutant, zf35, which failed to execute sustained forward or backward runs and continually switched the direction of locomotion in a jerky manner   . Furthermore, zf35 adults retained a reduced number of eggs in the uterus   ; Video 1 animals . Animalsrequency , albeit  n = 88)  and had  n = 80) . zf35 mu reduced . Therefozf35 mutation to the left end of chromosome X between genetic markers lon-2 and dpy-3. This region contains a gene, unc-2, which encodes the α1 subunit of the C. elegans CaV2 voltage-gated calcium channel. Sequencing analysis of the zf35 allele revealed a single-base transition (GGAto AGA) in the 17th exon of unc-2 (zf35 mutation results in a glycine to arginine substitution (G1132R) in the highly conserved intracellular linker between III-S6 and IV-S1  cDNA clone, which encodes the UNC-2/CaV2α(G1132R) protein. Pan-neuronal expression of the unc-2(zf35) transgene, in both wild-type and unc-2 loss-of-function mutant (lf) backgrounds, induced hyperactive behavior similar to that of the zf35 mutant. Transgenic overexpression of the wild-type unc-2 cDNA rescued the uncoordinated and lethargic phenotype of unc-2(lf) mutants, but did not induce hyperactive behavior (unc-2(zf35) mutants did not display obvious defects in neural morphology (data not shown). To determine if the zf35 mutation affected UNC-2 localization, we generated transgenic animals carrying C-terminus GFP-tagged unc-2(zf35) cDNA. UNC-2(GF/G1132R)::GFP was observed in the cell soma, and in puncta along the neuronal processes (We mapped the of unc-2 . UNC-2/Cof unc-2 . The zf3nd IV-S1 . To detebehavior . unc-2(zrocesses . The flurocesses  (Saheki rocesses . This inunc-2 loss-of-function (lf) mutants are sluggish and exhibit reduced motor activities (unc-2(lf) mutants are also slightly longer than wild type animals, most likely due to reduced muscle contraction. The contrasting phenotypes between unc-2(zf35) and unc-2(lf) mutants suggested that the zf35 G1132R mutation is a rare gain-of-function mutation. If so, secondary, loss-of-function mutations in the unc-2 locus should function as intragenic suppressors of the hyperactivity phenotype of unc-2(zf35). From a screen of mutagenized unc-2(zf35) mutants, we identified seven intragenic suppressor alleles that harbor missense or non-sense secondary mutations in the unc-2 gene  allele (zf113(W550stop) and zf124(M1369stop) alleles result in premature stop codons and therefore likely represent null alleles of unc-2. The zf115(C341Y) and zf109(L1355F) missense mutations result in substitutions of conserved amino acids in the S5-S6 loop of domain I and IV, respectively. Two suppressors, zf134 and zf130, caused moderate locomotion defects. The zf134(I970T) mutation affects an amino acid in the conserved voltage sensor, and the zf130(G1475D) mutation affects the C-terminal region, between a conserved EF-hand and the IQ-like motif. One suppressor, zf114(D892N), which changes an amino acid in the domain III S2, restored locomotion behavior of unc-2(zf35) to approximately wild-type levels. These intragenic suppressors represent an allelic series of hypomorphic unc-2 mutations. Their ability to revert the hyperactive phenotype of zf35 mutants to that of the wild-type or unc-2(lf) mutants strongly suggest that the zf35 mutation is a gain-of-function allele of unc-2. Therefore, from here on the unc-2(zf35) allele will be referred to as unc-2(zf35gf).Four suppressors, ) allele . The zf1To investigate the functional consequences of the UNC-2/CaV2α G1132R gain-of-function mutation, we introduced the corresponding change (G1518R) into the human P/Q type CaV2.1 channel α1 subunit, CACNA1A . CaV2.1αaWT = 3.8 ± 0.2 mV; Ka G1518R = 4.1 ± 0.1 mV, inac CACNA1A(wt)=177 ± 45 ms and Tinac CACNA1A(G1518R)=196 ± 32 ms at a 0 mV pulse). This suggests that the transition from the open to the inactive states was not affected by the G1518R mutation. To determine if inactivation following closed states was altered, we compared steady-state inactivation properties of wild-type and G1518R channels . This displacement indicates that the proportion of activatable channels is increased for CACNA1A(G1518R) channels at a given membrane potential. Thus, the G1518R mutation leads to channels that are activated at lower membrane potentials, and inactivated at higher membrane potentials. Together, these properties lead to increased current density by CACNA1A(G1518R). The conservation in the linker between TM III and TM IV between C. elegans and mammals suggests that UNC-2(G1132R) exhibits similar gain-of-function effects in activation and inactivation kinetics of CaV2α channel. However, since these experiments were performed with the human CACNA1A channel in HEK cells, we cannot exclude the possibility that the corresponding UNC-2/CaV2α G1132R mutation may have different effects on channel function in C. elegans.The slope of the activation curve was not significantly affected in the CACNA1A(G1518R) channel   or unc-2(S218L) transgene in C. elegans resulted in phenotypes similar to unc-2(zf35gf) mutants. Specifically, both unc-2(R192Q) and unc-2(S218L) animals exhibited increased reversal frequencies  when compared to wild-type animals  (unc-2(FHM1) transgenic animals laid eggs that are at an earlier developmental stage and retained fewer eggs in the uterus (unc-2(R192Q): 5.7 ± 0.4, n = 37; unc-2(S218L): 8.4 ± 0.6, n = 32, respectively), when compared to wild-type animals . These experiments provide strong genetic evidence that, similar to unc-2(zf35), the FHM1 mutations are gain-of-function mutations that lead to increased CaV2 activity.Several missense mutations in the human 1 (FHM1) . Electro1 (FHM1) , others 1 (FHM1) . In knocn effect . To deteto unc-2 . Pan-neu n = 29) . They al n = 29) . unc-2 mutants have altered synaptic transmission, we analyzed their sensitivity to the acetylcholinesterase inhibitor, aldicarb. C. elegans body wall muscles receive input from excitatory cholinergic motor neurons (unc-2(lf) mutants were paralyzed within 1 hr (unc-2(zf35gf) mutants became paralyzed within 30 min (unc-2(zf35gf)/+ mutants also paralyzed more rapidly than the wild-type, confirming that the unc-2(zf35gf) mutation is semi-dominant.Our electrophysiological recordings suggested the UNC-2(GF/G1132R) channel may increase Ca neurons . Aldicar neurons . Approxi neurons , while lhin 1 hr . In sharn 30 min . Heterozunc-2(R192Q) or unc-2(S218L) also induced hypersensitivity to aldicarb  mutants, led to wild-type sensitivity to aldicarb .Pan-neuronal expression of aldicarb . This hyaldicarb . Therefounc-2(zf35gf) mutation on synaptic function, we measured the frequency of spontaneous neurotransmitter release events in recordings of postsynaptic currents (PSCs) from C. elegans body wall muscles. C. elegans body wall muscles are innervated by both excitatory (cholinergic) and inhibitory (GABAergic) motor neurons (unc-2(zf35gf) mutants showed an over two-fold increase in the overall frequency of spontaneous PSCs when compared to wild-type animals  mutation on cholinergic and GABAergic transmission. To isolate cholinergic currents, we performed recordings at a holding potential of −60 mV in the GABA receptor/unc-49 mutant background. The frequency of spontaneous excitatory postsynaptic currents (EPSCs) was increased by approximately 1.5-fold in unc-2(zf35gf); unc-49 double mutants compared with control unc-49 single mutants  mutation leads to increased cholinergic and decreased GABAergic transmission to body wall muscles. Thus, instead of causing a uniform increase of neural signaling, the UNC-2/CaV2α(GF) mutation differentially affects excitatory and inhibitory signaling, shifting the E/I balance toward excitatory transmission.Our data show that, despite being expressed by both cholinergic and GABAergic motor neurons, the Pacr-2::RAB-3::mCherry) and the postsynaptic nicotinic ACh receptor (AChR) UNC-29::GFP (Punc-29::UNC-29::GFP) (unc-2(zf35gf) mutants (unc-2(zf35gf) mutants to an AChR agonist, levamisole. Levamisole induces hyper-contraction and paralysis through the activation of a class of UNC-29-containing AChRs in body wall muscles (unc-2(zf35gf) mutants were hypersensitive to levamisole, consistent with an increased AChR expression on the muscle cell membrane  . RAB-3:: mutants , consist mutants . Importa mutants . To phar muscles . unc-2(zmembrane . These pPunc-25::RAB-3::mCherry) and the GABAA receptor UNC-49 (Punc-49::UNC-49::GFP). In unc-2(zf35gf) mutants, RAB-3::mCherry puncta were enlarged, to a level comparable to that observed for cholinergic NMJs (unc-2(zf35gf) mutants, RAB-3::mCherry puncta density was slightly increased, whereas UNC-49::GFP puncta density was reduced compared to wild type  mutants is in sharp contrast to the increased fluorescence of the UNC-29::GFP cholinergic receptor. RNA-seq experiments showed no obvious changes in the unc-49 and unc-29 expression level in wild-type vs unc-2(zf35gf) animals (unc-2(zf35gf) mutants’ response to the GABA receptor agonist muscimol. Muscimol induces hyperpolarization of body wall muscles through UNC-49/GABAA-mediated inward Cl- currents (unc-2(zf35gf) mutants exhibited reduced sensitivity to muscimol: most unc-2(zf35gf) mutants were able to generate backward locomotion upon the head touch (unc-2(zf35gf) mutants to muscimol-induced muscle relaxation is consistent with reduction of UNC-49/GABAA expression at the muscle cell surface.The reduced UNC-49::GFP fluorescence in  animals , suggestcurrents . Muscimocurrents . unc-2(zad touch . The parunc-2(zf35gf) mutants and the wild type. This suggests that the density of functional cholinergic and GABAergic receptors at individual synapses is unchanged in unc-2(zf35gf) mutants. As individual synapses can be difficult to resolve with confocal microscopy, single fluorescent puncta often represent multiple synapses. Therefore, the synaptic fluorescence changes we observe most likely indicate increases or decreases in the number of excitatory and inhibitory synaptic connections. An increased number of cholinergic synapses would account for the increased sEPSC frequency, the levamisole hypersensitivity, and increased UNC-29::GFP fluorescence intensity in the of nerve cord. Similarly, a reduced number of GABAergic synapses is consistent with a reduced spontaneous IPSC frequency, reduced sensitivity to muscimol, and reduced UNC-49::GFP fluorescence intensity in the nerve cord.Thus, consistent with the electrophysiological analyses, our pharmacological studies demonstrate that a gain-of-function mutation in UNC-2/CaV2α has distinct effects on cholinergic and GABAergic synapses. However, both spontaneous EPSC and IPSC amplitudes are not significantly different between unc-2(zf35gf)mutants is surprising since both cholinergic and GABAergic neurons express unc-2. Why does a gain-function-mutation in the presynaptic CaV2 channel lead to a reduction in the number of GABAergic synapses? The simplest explanation is that cholinergic and GABAergic synapses respond differently to increased presynaptic activity. For instance, while increased ACh release may result in the increase of cholinergic synapses, increased GABA release may result in a homeostatic reduction of GABAergic synapses. To test this possibility, we analyzed the GABAergic synaptic markers in animals that specifically express the unc-2(zf35gf) transgene in GABAergic motor neurons (Punc-47::UNC-2(GF)) in a wild-type background. Expression in GABAergic motor neurons alone resulted in an increase in both presynaptic RAB-3::mCherry and post-synaptic UNC-49::GFP fluorescence (Punc-47::UNC-2(GF) animals are partially resistant to aldicarb, indicative of increased GABAergic signaling onto the body wall muscles (unc-2(zf35gf) mutants is not a direct consequence of elevated GABAergic neuron activity. Instead, our results indicate that increased GABAergic motor neuron activity in principle leads to increases in both presynaptic and postsynaptic termini similar to that observed for cholinergic synapses.The striking difference in excitatory and inhibitory neuromuscular signaling in rescence . Punc-47 muscles . Thus, tunc-2(zf35gf) mutants may negatively affect the formation of GABAergic synapses. Indeed, when we expressed unc-2(zf35gf) only in cholinergic neurons (Pacr-2::UNC-2(GF)), UNC-49::GFP fluorescence was reduced to a similar degree as in the unc-2(zf35gf) mutants (Pacr-2::UNC-2(GF) animals (unc-2(zf35gf) transgene in cholinergic motor neurons increased sensitivity to aldicarb, consistent with an expected increase in cholinergic signaling (unc-2(zf35gf) mutants is not only required but also causes the decrease in GABAergic synapses.Previous studies showed that cholinergic signaling affects the development and transmission of GABAergic neurons . Therefo mutants . Presyna animals , which mignaling . Togetheunc-2(zf35gf) mutants. ACR-12, expressed by cholinergic motor neurons, and UNC-29, expressed by body wall muscles, are subunits of ionotropic AChRs. The loss of ACR-12 reduces excitability of cholinergic motor neurons (unc-2(zf35gf); acr-12 and unc-2(zf35gf); unc-29 mutants, UNC-49::GFP fluorescence was restored to wild-type levels  transgenic animals exhibited normal presynaptic marker expression at GABAergic NMJs. However, similar to unc-2(zf35gf) mutants, postsynaptic UNC-49::GFP fluorescence was markedly reduced in L-AChR(gf) animals (Pmyo-3::L-AChR(wt)) did not affect UNC-49::GFP fluorescence. This indicates that increased cholinergic signaling onto muscles in unc-2(zf35gf) mutants negatively regulates GABAergic postsynapse formation.To directly test this possibility, we examined UNC-49::GFP expression in animals where we specifically increased cholinergic input to body wall muscles. Muscle-specific expression of the hyperactive levamisole-sensitive AChR (L-AChR(GF)) containing gain-of-function mutations in L-AChR subunits UNC-29 and UNC-38, leads to increased excitation of body wall muscles, but no obvious defects in muscle structure or cholinergic synapses . L-AChR mutants. UNC-49::GFP expression was not significantly different in tax-6(lf) mutants (tax-6(lf) mutation restored UNC-49::GFP fluorescence in unc-2(zf35gf) mutants. tax-6 is expressed in muscles and several neurons  mutants  mutants are hyperactive and exhibit seizure-like motor behaviors, in contrast to the sluggish behavior of unc-2(lf) mutants (unc-2(zf35gf) mutation results in a G-to-R substitution in a highly conserved region in the intracellular linker between TMIII and TMIV. Our electrophysiological analyses of the human CACNA1A channel in HEK cells indicate that this G-to-R substitution causes a shift to lower voltages of activation and reduced inactivation of the channel to increase Ca2+ influx. The increased current density of the CACNA1A(G1518R) channel could reflect increased channel conductance, and/or enhanced cell surface expression. We did not observe obvious differences in the expression and localization of the UNC-2(WT) and UNC-2(GF/G1132R) channel in C. elegans. This could suggest that this G-to-R substitution in the CaV2 channel may arise from an increased channel conductance. However, we cannot exclude that enhanced expression of the CACNA1A(G1518R) channel in HEK cells culture contributes to the increased current density. A similar G-to-R substitution in an intracellular linker of the human CaV1.2 channel results in similar defects in channel inactivation that underlies Timothy syndrome  are crucial regulators of neuronal excitability and synaptic transmission. Here, we report the isolation of a gain-of-function mutation in the  mutants . The uncsyndrome . The negsyndrome . While bsyndrome .unc-2(zf35gf) allele include both premature stop codons and missense mutations. Most intragenic suppressor mutations result in uncoordinated and lethargic phenotypes, indicating that they are hypomorphic alleles. Interestingly, some intragenic suppressor mutations resemble those found in CACNA1A in episodic ataxia type 2 (EA2) patients. The UNC-2(C341Y) mutation in the domain I S5-S6 loop is analogous to the CACNA1A(C287Y) mutation which was shown to alter channel trafficking and kinetics in whole-cell patch-clamp recordings of transfected COS-7 cells  mutants, whereas EA2-like CACNA1A(lf) mutations led to decreased motor activity. These studies provide strong genetic evidence that EA2 mutations are reduction-of-function mutations, while FHM1 mutations are gain-of-function mutations. C. elegans, which has a single CaV2α gene, thus provides an efficient in vivo system to determine the genetic nature of VGCC mutations associated with neurological disorders.We found that expression of an 2+ influx through CaV2 channels is tightly coupled to neurotransmitter release. unc-2 loss-of-function mutants are resistant to the acetylcholinesterase inhibitor aldicarb (unc-2(zf35gf) mutation increases Ca2+ influx, which would lead to an increase in neurotransmitter release probability. In accordance, unc-2(zf35gf) mutants are hypersensitive to aldicarb, and show a two-fold increase in spontaneous EPSC frequency. In contrast, spontaneous IPSC frequency is significantly reduced in unc-2(zf35gf) mutants. Therefore, even though UNC-2 is expressed by both cholinergic and GABAergic motor neurons, the UNC-2/CaV2α(GF) mutation differentially affects excitatory and inhibitory signaling, shifting the E/I balance toward excitatory transmission.Presynaptic Caaldicarb , and havaldicarb . The uncHuman studies indicate that cortical hyperexcitability in migraine patients , could rC. elegans neuromuscular system, where both excitatory (cholinergic) and inhibitory (GABAergic) motor neurons regulate muscle activity, provides a suitable and complementary model for mechanistic studies of E/I imbalance (unc-2(zf35gf) mutations led to a modest increase in RAB-3 expression in the neurites of both excitatory and inhibitory motor neurons, consistent with the notion that increased Ca2+ influx may potentiate the recruitment of synaptic vesicles (unc-2(zf35gf) mutation led to pronounced and opposite effects on the density of cholinergic and GABAergic receptors in the ventral nerve cord: an increase of AChR, but a marked decrease of GABAAR, which parallel the increased sEPSC frequency and reduced sIPSC frequency. Like the wild-type UNC-2/CaV2α, UNC-2/CaV2α(GF) channel proteins localize to presynapses to mediate Ca2+ influx and exocytosis of neurotransmitters so these effects are not attributable to channel mislocalization. Unchanged amplitudes of spontaneous EPSCs and IPSCs suggest the density of receptors at individual cholinergic and inhibitory synapse is not affected by unc-2(zf35gf). Therefore, the differential density of cholinergic and GABAergic receptors and the frequency of spontaneous EPSCs and IPSCs likely reflects the number of functional synapses from excitatory and inhibitory motor neurons to the body wall muscles.The mbalance . In our vesicles . Howeverunc-2(zf35gf) mutants is a consequence of increased cholinergic signaling onto the same muscle target. A possible explanation of this observation is a differential response of cholinergic and GABAergic synapses to increased stimulation: strengthening of excitatory and homeostatic compensation of inhibitory synapses  channel in principle should increase synaptic strength in both synapse types. Our results instead reveal that the reduction of GABAergic neuromuscular signaling is a consequence of increased cholinergic input to the muscle cells.Our results show that the reduced GABAergic neuromuscular signaling in synapses . HoweverA receptor clustering  mutants may similarly affect GABAA receptor stability to disassemble or prevent the assembly of GABAergic post-synapses. Increased excitatory signaling may thus lead to silencing of GABA synapses at the postsynaptic site, UNC-49/GABAA receptor localization to postsynaptic sites is restored by removing TAX-6/calcineurin, implicating a conserved activity-dependent mechanism for modulation of synaptic inhibition.In the mammalian brain, excessive neuronal excitation can induce long-term depression of GABAergic transmission . Long-teustering . NMDA recineurin . Sustaineceptors . GABAA r surface , utilizi surface . In C. eC. elegans neuromuscular system: increased excitatory signaling strengthens excitatory synapses, resulting in the destabilization of inhibitory synapses in a calcineurin-dependent manner We propose that UNC-2/CaV2 gain-of-function mutations change the E/I balance of the E. coli strain OP50 as a food source. Experiments were performed on young adult animals (24 hr post-L4 larva) at room temperature (22–24°C). The wild-type strain was Bristol N2. Transgenic strains were obtained by microinjection of plasmid DNA into the germline with coinjection marker lin-15 rescuing plasmid pL15EK both at 80 ng/µl into unc-2(e55); lin-15(n765ts) or lin-15(n765ts) animals unless stated otherwise. At least three independent transgenic lines were obtained for each injected construct. The data presented are from a single representative line. The following strains were utilized in this study:All strains were cultured at room temperature (22–24°C) on nematode growth media (NGM) agar plates with the unc-2(zf35gf) mutation was introduced in the Ptag-168::UNC-2(wt) clone (unc-2(zf35gf) transgene expression, cell-specific promoters for GABAergic (Punc-47) and cholinergic (Pacr-2) (Ptag-168::UNC-2(zf35gf) construct was digested with FseI and AscI to remove the Ptag-168 promoter and replaced with cell-specific promoters of interest. To generate the unc-2 transgenes carrying human FHM1 mutations (UNC-2(R192Q) and UNC-2(S218L)), the UNC-2 and human CACNA1A amino acid sequences were aligned to locate the corresponding amino acid substitutions in UNC-2/CaV2. The mutations were then introduced in the Ptag-168::UNC-2(WT) construct by site-directed mutagenesis. The wild-type human CaV2.1 cDNA used in the HEK cell recording was obtained from Y Cao and R Tsien (CACNA1A cDNA by site-directed mutagenesis.The t) clone  using si(Pacr-2)  motor ne R Tsien . To geneunc-2(zf35gf) allele was isolated in a screen for animals that were resistant to the immobilizing effects exogenous tyramine as previously described (unc-2(zf35gf) to LG X based on its hyperactive locomotion phenotype using SNP mapping (unc-2(zf35gf) to the left of lon-2 and close to dpy-3. DNA sequencing of the unc-2 gene was performed to identify the molecular change of unc-2(zf35gf) mutation.The escribed . We mapp mapping . Three-func-2(zf35gf) L4 animals (P0) were mutagenized with 0.5 mM N-ethyl-N-nitrosourea (ENU) for 4 hr. Approximately 10,000 F1 animals were bleached to obtain F2 eggs. F2 eggs were plated on NGM plates containing 0.25 mM aldicarb and examined for viable progeny after 7 and 14 days. Aldicarb resistant animals were individually transferred to fresh NGM plates, and their progeny were retested for aldicarb resistance. All suppressors isolated from the screen backcrossed with the wild-type N2. Suppressors that showed linkage to the X-chromosome were tested for complementation with unc-2(e55lf) mutants. Molecular changes of unc-2(zf35gf) intragenic suppressors were identified by DNA sequencing of the unc-2 gene.Spontaneous reversal frequency was scored on NGM plates with freshly seeded OP50. The animals were transferred from their culture plate to a new plate, and allowed to recover for 1 min. After the recovery period the number of reversals was counted for 3 min. To quantify the instantaneous velocity and average forward velocity, animals were transferred from their culture plate to a new NGM plate seeded with a thin bacterial lawn and allowed to recover for 1 min. After the recovery period, the animals were tracked for 90 s using a single worm tracker . Videos To examine movement defects, individual young adult worms were transferred into 96-well plates containing 50 μl M9 buffer in each well. After a 30 s recovery period, body bends were counted for 30 s. A body bend was defined as a change in direction of bending at the mid-body.Egg-laying assays were performed as described . Rates oTo quantify aldicarb and levamisole resistance, young adult animals were transferred to NGM plates supplemented with 1 mM aldicarb or 0.5 mM levamisole. The percentage of paralyzed animals was scored at 15 min intervals. Animals were scored as paralyzed when they did not move when prodded with a platinum wire. To assay muscimol response, young adults were transferred onto NGM plates containing 1 mM muscimol for an hour. The rubberband phenotype was subsequently scored by analyzing the behavioral response upon touching the animal with an eyelash across its body, (posterior to the pharynx) . The rubA stable HEK 293 cell line expressing the calcium channel auxiliary subunits β1c and α2δ  was used2, 1 mM MgCl2, 10 mM HEPES, 40 mM TEACl, 10 mM glucose, and 87.5 mM CsCl, pH 7.4. Typically the pipettes exhibited resistances ranging from 2 to 4 MΩ and were filled with internal solution containing: 105 mM CsCl, 25 mM TEACl, 1 mM CaCl2, 11 mM EGTA, and 10 mM HEPES, pH 7.2.Whole-cell inward currents were recorded 24–36 hr after transfection with a HEKA EPC-9 patch clamp amplifier. Recordings were filtered at 2 kHz and acquired using Patchmaster software (HEKA). The extracellular recording solution contained 5 mM BaClrev) (1+exp (V0.5- V)/ka)−1 where G is membrane conductance, Erev is the reversal potential, V0.5 is the midpoint, and ka the slope of the voltage dependence. Current densities were obtained by dividing the current peak amplitude to the cell capacitance for each experiment.Cell capacitance  and series resistance  were measured from the current transient after a voltage pulse from −80 to −90 mV. Series resistance was typically compensated by 80–90%. Cells with large currents in which errors in voltage control might appear were discarded. I-V curves were generated by measuring the peak currents obtained after stepping the membrane potential from a holding potential of −120 mV to voltages between −55 and 40 mV in 5 mV increments for 200 ms. I-V curves were fitted with Equation 1.: I = G(G – Emax= (1 + exp[(V-V0.5)/kin]−1).To measure steady-state inactivation profiles, conditioning pre-pulses (10 s) from −90 to 20 mV in 10 mV steps were applied, and the membrane was then stepped to the peak of the I–V curve. Currents were normalized to the maximal value obtained at the test pulse and plotted as a function of the prepulse potential. Data were fitted with Boltzmann equations: I/IData analysis was performed using the IgorPro software ; figures, fitting and statistical analysis were done using the SigmaPlot software . Data are presented as mean ± SD. Significant differences were determined using Student’s t test with the significance value set at p<0.01.2, 0.1 mM; MgCl2, 5 mM; BAPTS, 1 mM; HEPES, 10 mM; Na2ATP, 5 mM; Na2GTP, 0.5 mM cAMP, 0.5 mM; cGMP, 0.5 mM. pH 7.2 with KOH,~320 mOsm. Extracellular solution: NaCl, 150 mM; KCl, 5 mM; CaCl2, 5 mM; MgCl2, 1 mM; glucose, 10 mM; sucrose, 5 mM; HEPES, 15 mM. pH 7.3 with NaOH,~330 mOsm, and the membrane potential was held at −60 mV. To isolate spontaneous excitatory postsynaptic currents, total spontaneous postsynaptic currents were recorded in Aunc-49/GABA receptor mutant background. To isolate spontaneous inhibitory postsynaptic currents, 0.5 mM d-tubocurarine (dTBC) was added to the extracellular solution to block acetylcholine receptors, and the membrane potential was held at −10 mV so IPSCs appeared as outward currents  plate and grown overnight at 37°C. Single colonies were picked from these plates and grown overnight at 37°C in LB Amp 100 μg/ml. Bacteria were concentrated to 4X and seeded on NGM RNAi plates containing 6 mM IPTG and 100 μg/ml Amp. Plates were dried overnight. Six L4 worms were transferred onto desired NGM RNAi plates for 24 hr to grow to adult. Adults were transfered to another NGM RNAi plate to set up limited egg laying time intervals to obtain age-synchronized animals for analyses. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.C. elegans" for consideration by eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Ronald L Calabrese (Reviewer #2).Thank you for submitting your work entitled "Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in As you will see from the reviews appended below, a major issue was the interpretation of the electrophysiology data. In particular, in the discussion following peer review, the concern was the conclusions based on the observed reduction in GABA synapse number without a concomitant change in mIPSC amplitude, especially since reduction in UNC-49 puncta intensity (using the same marker) has previously been shown to result in reduced mIPSC current amplitudes. Together with additional issues that were raised, the reviewers and editors concluded that this work would require extensive revisions and is better suited for a different journal.Reviewer #2:unc-2(gf), in the Caenorhabditis elegans CaV2 channel α1 subunit (unc-2 gene), which leads to increased calcium currents. Such mutants exhibit locomotor and egg-laying hyperactivity. Importantly expression of the unc-2 gene with substitutions associated with familial hemiplegic migraine lead to hyperactivity similar to that of unc-2(gf) mutants. unc-2(gf) mutants show increased cholinergic and decreased GABAergic-transmission associated with a reduction of GABAergic synapses and an increase in cholinergic synapses. The authors also present evidence that the unc-2(gf) mutants lead to reduction of GABA synapses in a TAX-6/calcineurin dependent manner. Thus, this work has great general interest because it provides a roadmap and an experimental system for exploring mechanistic links between Ca-channel function and changes in E/I balance in the nervous system.The molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance seen in disease such as familial hemiplegic migraine are poorly understood. This work identifies a gain-of-function mutation, The paper is clearly written, and the figures present the data needed and do so clearly.Reviewer #3:C. elegans to decipher the cellular and neuronal circuit effects of a novel gain of function Ca2+ channel mutation. Furthermore, the authors present data to indicate that this novel mutation phenotypically resembles Ca2+ channel mutations found in familial hemiplegic migraine type 1. The study is significative because while it is known the in humans these types of mutations lead to increased excitation and reduced inhibition, the molecular underpinnings of this effect are unknown.In this manuscript, Huang and colleagues, use The study is in general rigorously designed; I have the following reservations though:2+ currents was studied by introducing the mutation in the human homolog. The authors do not mention anywhere the percentage of identity and homology between the worm and the human unc-2 type channels. This is relevant because, while the loop where the mutation is located is highly conserved, other parts of the proteins may not share such a high degree of homology and they may influence the overall effect of this mutation on the function of the channel. The experiment should really be repeated by introducing the mutation in worm unc-2.1) The effect of the novel mutation the authors identified on Caunc-2(gf) of the unc-2/CaV2α(gf)? Especially in light of the fact that exact parallelism between the worm and the human mutant channels function has not been firmly established, this point is particularly relevant. The authors should name the mutations throughout the text and in the figures and not simply call them unc-2(gf) mutants.2) Throughout the text and in the figures, it is really hard to keep track of which gain of function mutation the authors are referring to. Is it the unc-2(wt)::GFP and unc-2(gf)::GFP to see if there is more mutant protein being expressed in vivo.3) It appears that the effect of the gain of function mutation is both on voltage dependence and either trafficking or single-channel conductance . Indeed, currents shown in Figure 3 were recorded under conditions of complete removal of inactivation (holding -120mV) yet the mutant currents are much larger than the wild type currents. The authors need to comment on this. The authors could also compare tax-6 acts in the muscle to decrease GABAergic synapses, rescue or RNAi in muscle should confirm the conclusion of the authors.4) While the results suggest that Reviewer #4:unc-2(zf35gf) mutant was isolated in a screen for altered locomotion, lacking persistent forward or reverse movements while retaining a high level of motility. Analogous mutations in mammalian CaV2 channels expressed in tissue culture cells shows that the zf35 mutation alters channel gating . Similar gf alleles were previously described in mammalian CaV2 and CaV1 channels. Extensive analysis of unc-2(zf35) mutant worms finds changes in spontaneous cholinergic and GABAergic synaptic currents, and changes in the abundance of pre-synaptic (RAB-3) and post-synaptic (UNC-49 and UNC-29) markers. Based on these studies, the authors suggest that unc-2(zf35gf) mutants have altered GABA synapse development and that this results from the increased excitatory transmission. These conclusions are not strongly supported by the data. I agree that transmission has changed in these mutants, but it could simply be what you expect from increased UNC-2 channel currents. The analysis of unc-2(gf) mutants is extensive and would likely be of interest to C. elegans labs; however, they do not provide a clear understanding of how nervous system development or function is altered in these mutants. For these reasons, I do not support publication in eLife.The authors analyze a novel gain of function (gf) allele of CaV2 N-type calcium channels. The Essential revisions:unc-2(gf) mutants but this was not accompanied by a corresponding decrease in the amplitude of the spontaneous IPSCs. Several prior papers found that mutations decreasing GABA receptor puncta intensities are closely matched by similar changes in IPSC current amplitudes . Given the absence of an effect on mIPSC amplitudes, these results suggest that the GABA receptors imaged here are non-synaptic. Altered non-synaptic receptors could be interesting but would require many new experiments to prove and document a functional consequence .1) Post-synaptic GABA receptor abundance was significantly reduced in unc-2(gf) mutants have decreased GABA synapse numbers, based on recordings indicating a decreased mIPSC rate. The mIPSC recordings were done in the presence of a cholinergic antagonist (dTBC), which by itself decreases mIPSC rate (since cholinergic neurons provide the excitatory input to GABA neurons). It seems possible that use of dTBC biases their results toward lower mIPSC rates. I recommend recording mIPSCs without dTBC .2) The authors conclude that unc-2(gf) mutants (as the authors propose), I would expect that synaptic puncta density (both RAB-3 and UNC-49) would be reduced. The authors should report density data for the synaptic markers.3) If lower synapse numbers occur in unc-2(gf) mIPSC rate defect cell autonomous to D neurons? If so, one would not need to propose altered cholinergic transmission in causing this phenotype (which the authors propose).4) If the 5) No controls are provided to determine if changes in synaptic puncta intensities are caused by transcriptional changes.6) The RAB-3 puncta intensity changes are hard to interpret without further experiments. Are other SV markers altered similarly? Is the SV pool size altered (either by electrophysiology or EM)? [Editors’ note: the authors’ appeal in response to the first round of peer review follows.]Reviewer #3:2+ channel mutation. Furthermore, the authors present data to indicate that this novel mutation phenotypically resembles Ca2+ channel mutations found in familial hemiplegic migraine type 1. The study is significative because while it is known the in humans these types of mutations lead to increased excitation and reduced inhibition, the molecular underpinnings of this effect are unknown.In this manuscript, Huang and colleagues, use C. elegans to decipher the cellular and neuronal circuit effects of a novel gain of function CaThe study is in general, rigorously designed, I have the following reservations though:2+ currents was studied by introducing the mutation in the human homolog. The authors do not mention anywhere the percentage of identity and homology between the worm and the human unc-2 type channels. This is relevant because, while the loop where the mutation is located is highly conserved, other parts of the proteins may not share such a high degree of homology and they may influence the overall effect of this mutation on the function of the channel. The experiment should really be repeated by introducing the mutation in worm unc-2.1) The effect of the novel mutation the authors identified on CaWe thank the reviewer for their comments. UNC-2 and mammalian CaV2 α1 subunit classes share highly similar . Since the similarity was published we did not include in the manuscript. We agree that this is helpful to directly compare UNC-2 and associated mutation directly with CACNA1a. Therefore, we will include an alignment of UNC-2 and CACNA1a in the supplemental figures.unc-2(zf35) allele is a gain-of-function mutation of the CaV2α gene. We sought further evidence to demonstrate for the biophysical effect on channel function in cell transfection experiments. The HEK cell electrophysiology experiments show the effect of the G to R substitution on CaV2 channel function which is located in a highly conserved loop . The main point of these experiments is to show that the G to R mutation results in a negative shift in voltage dependence activation, and increased current density similar to those observed for CACNA1a channels that carry FHM1 mutations. The HEK cell experiments are consistent with our behavioral-, electrophysiological and genetic analyses, clearly demonstrating that UNC-2(zf35) generates a gain-of-function phenotype. Analysis of the biophysical effect of the zf35 mutation on the UNC-2/CaV2α channel would require the co-transfection of genes encoding the C. elegans ß (ccb-1) and α2∂ subunits (unc-36), and maybe other genes required for proper UNC-2 channel transport and localization . Unfortunately, no worm cell lines are available to perform these experiments. Mammalian cell lines stably transfected with ß and α2∂ subunits have been used extensively to study the effect of CaV2α mutations . This allowed us to make informative comparisons of the zf35 mutation with those of previously characterized FHM1 mutations. The reciprocal experiment, expression of the unc-2 transgene with corresponding human FHM1 mutations, provides strong behavioural and genetic evidence that FHM1 mutations are gain-of-function mutations.The genetic data, and suppressor screen demonstrate that the 2) Throughout the text and in the figures, it is really hard to keep track of which gain of function mutation the authors are referring to. Is it the unc-2(gf) of the unc-2/CaV2α(gf)? Especially in light of the fact that exact parallelism between the worm and the human mutant channels function has not been firmly established, this point is particularly relevant. The authors should name the mutations throughout the text and in the figures and not simply call them unc-2(gf) mutants.unc-2(gf) refers to the unc-2(zf35) allele. We will clarify this and refer to the gain-of-function allele as unc-2(zf35gf) throughout the manuscript to avoid confusion. For the protein we will refer to it as UNC-2/CaV2α(GF).The 3) It appears that the effect of the gain of function mutation is both on voltage dependence and either trafficking or single-channel conductance . Indeed, currents shown in Figure 3 were recorded under conditions of complete removal of inactivation (holding -120mV) yet the mutant currents are much larger than the wild type currents. The authors need to comment on this. The authors could also compare unc-2(wt)::GFP and unc-2(gf)::GFP to see if there is more mutant protein being expressed in vivo.We agree with the reviewer that the larger currents that we show for the CaV2α(GF) channel could reflect increased single channel conductance, enhanced expression or both. We observed an increase in current density in independent transfection experiments in HEK cells . We did compare expression level, and localization of the UNC-2(wt)::GFP and UNC-2(gf)::GFP in transgenic animals  and did not see any obvious differences. This suggests that the mutation, besides the change in the voltage dependence of activation, also affects conductance. We will explicitly mention these possibilities in the text.4) While the results suggest that tax-6 acts in the muscle to decrease GABAergic synapses, rescue or RNAi in muscle should confirm the conclusion of the authors.tax-6.We can perform this experiment to test the muscle requirement of Reviewer #4:The authors analyze a novel gain of function (gf) allele of CaV2 N-type calcium channels. The unc-2(zf35gf) mutant was isolated in a screen for altered locomotion, lacking persistent forward or reverse movements while retaining a high level of motility. Analogous mutations in mammalian CaV2 channels expressed in tissue culture cells shows that the zf35 mutation alters channel gating . Similar gf alleles were previously described in mammalian CaV2 and CaV1 channels. Extensive analysis of unc-2(zf35) mutant worms finds changes in spontaneous cholinergic and GABAergic synaptic currents, and changes in the abundance of pre-synaptic (RAB-3) and post-synaptic (UNC-49 and UNC-29) markers. Based on these studies, the authors suggest that unc-2(zf35gf) mutants have altered GABA synapse development and that this results from the increased excitatory transmission. These conclusions are not strongly supported by the data. I agree that transmission has changed in these mutants, but it could simply be what you expect from increased UNC-2 channel currents. The analysis of unc-2(gf) mutants is extensive and would likely be of interest to C. elegans labs; however, they do not provide a clear understanding of how nervous system development or function is altered in these mutants. For these reasons, I do not support publication in eLife.unc-2 is expressed in all neurons, this is not what would simply be expected from increased UNC-2 calcium currents. We present evidence that increased cholinergic transmission in unc-2/CaV2α(zf35gf) mutants leads to a reduction of GABA synapses in a TAX-6/calcineurin dependent manner. While additional detailed molecular and cellular mechanisms remain to be explored (as with any study), our data provide a deeper understanding of how alterations in Ca-channel function produce unexpected changes in E/I balance in the nervous system. Further, our analysis of FHM mutations using this model provides evidence that these mutations produce similar changes in E/I balance that have clear implications for human disease.We would like to respond to this reviewer’s comments and clarify these issues with the editors. As pointed out by the reviewer 2 and 3, the main message of the paper is that gain-of-function mutation in the presynaptic voltage gated calcium channel causes an imbalance in E/I transmission. Since the Essential revisions:1) Post-synaptic GABA receptor abundance was significantly reduced in unc-2(gf) mutants but this was not accompanied by a corresponding decrease in the amplitude of the spontaneous IPSCs. Several prior papers found that mutations decreasing GABA receptor puncta intensities are closely matched by similar changes in IPSC current amplitudes .We understand the reviewer’s point as there is currently some discrepancy in the field to what extent electrophysiological data correlate with fluorescence of synaptic markers.However, the papers cited by the reviewer do not show that decreased postsynaptic GABAR marker signals are closely matched by changes in IPSC amplitude. Instead, consistent with our findings, these studies showed that decreased GABAR intensity is closed matched by a reduction in IPSC frequency. The IPSC amplitudes, even of the same mutant, behaved differently in different preparations, and the reported changes do not strongly correlate with reported changes UNC-49::GFP fluorescence.To summarize the relevant data from these papers:madd-4, nlg-1 and nrx-1 mutants wereclosely matched by the reduction of the IPSC frequency. For example, nlg-1 mutants exhibited strong reduction of IPSC frequency and UNC-49 signals, but nrx-1 did not show defects in either. Here nlg-1 mutants exhibited a very small reduction of IPSC amplitude, which did not closely match the very strong reduction in UNC-49/GABAR clustering and fluorescence.Maro et al. (2015) showed that the degree of reduction of UNC-49 puncta signals in madd-4B(0) showed no significant change in UNC-49 intensity, but exhibited a significant reduction of IPSC frequency, and a milder reduction in amplitude.Tu et al. (2015) reported reduction of mIPSC frequency and amplitude in these three mutants, irrespective of their different degrees of reduction of the UNC-49/GABAR fluorescence. For example, nlg-1 mutants exhibited reduced UNC-49 fluorescence signals, reduced mIPSC frequency, and reduced mIPSC amplitude. The reduction in mIPSC amplitude reported in Tong et al. was larger than reported in the other two studies.Tong et al. (2015) also reported that the lin-2, frm-3) that exhibited a reduction in UNC-49/GABA receptor signals, reduced mIPSC amplitude reduction but no change in mIPSC frequency. Unlike unc-2 mutants, these two genes function strictly in muscles to regulate UNC49/GABA receptor levels. In another mutant, nrx-1, which was examined also in the other two papers, the authors found no overt fluorescent marker change, but an increase of mIPSC amplitude and no change in frequency. The reported mIPSC amplitude changes don’t follow an obvious trend with respect to changes in GABA receptor fluorescence intensity and are inconsistent across studies even for the same mutation.Tong et al. (2015) is the only paper that reported two other mutants (unc-2(gf) mutants show concomitant changes of UNC-49/GABA receptor intensity and IPSC frequency, which is consistent with the most salient findings reported in the cited papers.Our results for Given the absence of an effect on mIPSC amplitudes, these results suggest that the GABA receptors imaged here are non-synaptic. Altered non-synaptic receptors could be interesting but would require many new experiments to prove and document a functional consequence .unc-2(zf35), a decreased frequency of mIPSCs, and an unchanged amplitude of mIPSCs. A decrease in receptor number at existing postsynaptic sites would be predicted to change amplitude whereas a loss of postsynaptic sites would be most consistent with a change in frequency. Other mutations that have been described in the literature may produce both effects, accounting for the change in amplitude and frequency. In unc-2(gf) mutants there appears to be a decrease in the number of postsynaptic GABAR clusters: at some NMJs, we find presynaptic of RAB-3::mCherry puncta without punctate UNC-49::GFP apposition . This indicates the presence of orphan presynaptic sites and post-synaptic silencing of GABA synapses. A decrease in non-synaptic receptors could account for a reduction in UNC-49::GFP intensity without changed amplitude, but does not account for the observed reduction in mIPSC frequency. A more plausible explanation, as we outline in the text, is a decrease in the number of UNC-49-containing postsynaptic sites on the muscle arms in the ventral nerve cord. This accounts for the observed reduction in mIPSC frequency, the preserved mIPSC amplitude, and the reduced UNC49::GFP intensity in the ventral nerve cord.We report a decrease in UNC-49::GFP intensity in the ventral nerve cord of 2) The authors conclude that unc-2(gf) mutants have decreased GABA synapse numbers, based on recordings indicating a decreased mIPSC rate. The mIPSC recordings were done in the presence of a cholinergic antagonist (dTBC), which by itself decreases mIPSC rate (since cholinergic neurons provide the excitatory input to GABA neurons). It seems possible that use of dTBC biases their results toward lower mIPSC rates. I recommend recording mIPSCs without dTBC .We are measuring mIPSC frequency and amplitude specifically from GABAergic motor neuron (VD) synapses onto muscle. VDs are postsynaptic to cholinergic motor neurons. We include dTBC to eliminate cholinergic drive onto the VDs and isolate VD endogenous events. UNC-2(gf) is also predicted to promote cholinergic synaptic transmission onto the VDs. In the absence of dTBC, our e-phys recording would measure the compound effect of increased cholinergic drive onto VDs and VD’s endogenous IPSCs. Therefore, we believe the most clear experiment is to perform the analysis in the presence of dTBC as presented. We note that we have used this strategy to clearly distinguish mEPSCs and mIPSCs in numerous prior studies.– has been used in several studies. We respectfully point out that this is not the only protocol used in the C. elegans field for recording IPSC events, and at least for us, we could not find data that demonstrated the specificity of recording conditions .The recommended recording – recording mIPSCs without dTBC, with a low chloride internal buffer and holding potential of 0mV Before applying our current electrophysiology protocol, as shown in Figure 2A-B in Maro et al. (2015), we tested the effect of holding membrane potentials and dTBCs to show that using our intracellular recording solution, at -10mV holding potential, and the in presence of 0.5mM dTBC, we blocked all EPSC events without blocking IPSC events in the muscle preparation.unc-49(lf) mutant background) in unc2(gf) made it more important to apply dTBC. The use of dTBC does not bias but isolate the mIPSC population from the GABAergic VD-class motor neurons in both wild-type and unc-2(gf) mutant for comparison.For the current study, a significant increase of EPSC events (recorded in 3) If lower synapse numbers occur in unc-2(gf) mutants (as the authors propose), I would expect that synaptic puncta density (both RAB-3 and UNC-49) would be reduced. The authors should report density data for the synaptic markers.UNC-49 abundance was quantified by calculating the overall intensity of UNC-49::GFP in the region of nerve cord imaged. This is a compound measurement, dependent on the density of clusters, intensity of each cluster, and any diffuse UNC-49::GFP that is not localized to a cluster. We also measured the cluster density, and found an increase in presynaptic RAB-3 density and decreased UNC-49 density. We can include these data in a revised manuscript. These measures parallel overall intensity measurements. We didn’t add the density measurements since they do not add much information to the overall intensity measurements. Our data are consistent with the interpretation that there are fewer GABAergic postsynapses, and orphan (silent) GABAergic presynapses.4) If the unc-2(gf) mIPSC rate defect cell autonomous to D neurons? If so, one would not need to propose altered cholinergic transmission in causing this phenotype (which the authors propose).unc-2(zf35) on ventral cord UNC-49::GFP intensity is the result of a dominant effect in cholinergic neurons. Expression of unc-2(gf) transgene in GABAergic neurons alone, in an otherwise wild-type background, resulted in a marked increase in UNC-49::GFP intensity, whereas specific transgene expression in cholinergic neurons decreased the intensity similar to unc-2(gf) mutants . Furthermore, cell specific expression of an unc-2(gf) transgene in GABAergic neurons increases aldicarb resistance, indicating an increase in GABAergic transmission. (Expression of unc-2(gf) in cholinergic neurons decreases aldicarb resistance). We can include these data in a revised version of the manuscript. Together, these experiments all indicate that the reduction of mIPSC rate in unc-2(gf) is not cell autonomous to the D-type motor neurons and further support our conclusion that altered cholinergic transmission is responsible for the transmission defects in unc-2(gf).We did not do this particular experiment, but we do show extensively that the effect of 5) No controls are provided to determine if changes in synaptic puncta intensities are caused by transcriptional changes.unc-2(gf) expression profiles showed no differences in unc-49 expression but did show an increase in unc-29 expression. We confirmed this data by qPCR. We can include these data in a revised manuscript.We have performed transcriptional analyses but did not include these data in this manuscript. RNA-seq experiments, in which we compared wild-type vs. 6) The RAB-3 puncta intensity changes are hard to interpret without further experiments. Are other SV markers altered similarly? Is the SV pool size altered (either by electrophysiology or EM)?unc-2(gf). We provide evidence that increased cholinergic signaling leads to a reduction GABAergic synapses and explain and support this with pharmacology, fluorescent imaging of pre- and postsynaptic components of excitatory and inhibitory NMJs, cell specific expression and in vitro electrophysiology, as well as the impact on circuit output using locomotory behavior as a readout.This is not the main point of the paper.As noted by reviewer 2 and 3, the main point of our study is to demonstrate that gain-of-function mutations, as found in FHM1 patients, result in E/I imbalance. E/I imbalance does not simply arise due to cell autonomous excitability increases in excitatory and inhibitory neurons, but arise primarily from increased excitatory signaling onto synaptic targets, in this case muscle cells. Our electrophysiology shows that there is increased excitatory input and decreased inhibitory input to ventral muscles in Reviewer #3:The study is in general, rigorously designed, I have the following reservations though:2+ currents was studied by introducing the mutation in the human homolog. The authors do not mention anywhere the percentage of identity and homology between the worm and the human unc-2 type channels. This is relevant because, while the loop where the mutation is located is highly conserved, other parts of the proteins may not share such a high degree of homology and they may influence the overall effect of this mutation on the function of the channel. The experiment should really be repeated by introducing the mutation in worm unc-2.1) The effect of the novel mutation the authors identified on CaWe thank the reviewer for this comment. UNC-2 and mammalian CaV2 α1 subunit classes share a high degree of similarity. Since this was published previously  we did not include in the initial version of the manuscript. We agree that this is helpful to directly compare UNC-2 and associated mutation directly with CACNA1A and now include an alignment of UNC-2 and CACNA1A in the supplemental figures .unc-2(zf35) allele is a gain-of-function mutation of the CaV2α gene. We sought further evidence to demonstrate the biophysical effects on channel function in cell transfection experiments. The HEK cell electrophysiology experiments show the effect of the G to R substitution on CaV2 channel function, which is located in a highly conserved loop . The main point of these experiments is to show that the G to R mutation results in a negative shift in the voltage dependence of activation, and increased current density, similar to those prior observations for CACNA1A channels that carry FHM1 mutations. The HEK cell experiments are consistent with our behavioral, electrophysiological and genetic analyses, demonstrating that UNC-2(G1132R) generates a gain-of-function phenotype. Analysis of the biophysical effect of the zf35 mutation on the UNC-2/CaV2α channel would require the co-transfection of genes encoding the C. elegans ß (ccb-1) and α2∂ subunits (unc-36), and maybe other genes required for proper UNC-2 channel transport and localization . Unfortunately, no worm cell lines are available to perform these experiments. Mammalian cell lines stably transfected with ß and α2∂ subunits have been used extensively to study the effect of CaV2α mutations . This allowed us to make informative comparisons of the zf35 mutation with those of previously characterized FHM1 mutations. The reciprocal experiment, expression of the unc-2 transgene with corresponding human FHM1 mutations, provides strong behavioral and genetic evidence that FHM1 mutations are gain-of-function mutations.The genetic data, and suppressor screen demonstrate that the 2) Throughout the text and in the figures, it is really hard to keep track of which gain of function mutation the authors are referring to. Is it the unc-2(gf) of the unc-2/CaV2α(gf)? Especially in light of the fact that exact parallelism between the worm and the human mutant channels function has not been firmly established, this point is particularly relevant. The authors should name the mutations throughout the text and in the figures and not simply call them unc-2(gf) mutants.unc allele as unc-2(zf35gf) throughout the manuscript. After characterization of the unc-2(zf35gf) allele in the manuscript we refer to the corresponding protein as UNC-2/CaV2α(GF). UNC-2(GF) is used in figures and figure legends for brevity. The FHM1 mutations in the UNC-2 are referred to by their FHM1 amino acid substitutions (UNC-2(R192Q) OR UNC-2(S218L). We hope this avoids confusion.Thank you for the suggestion. We now clarified this in the text and referred to the gain-of-function 3) It appears that the effect of the gain of function mutation is both on voltage dependence and either trafficking or single-channel conductance . Indeed, currents shown in Figure 3 were recorded under conditions of complete removal of inactivation (holding -120mV) yet the mutant currents are much larger than the wild type currents. The authors need to comment on this. The authors could also compare unc-2(wt)::GFP and unc-2(gf)::GFP to see if there is more mutant protein being expressed in vivo.We agree with the reviewer that the larger currents that we show for the CACNA1A(G1518R) channel could reflect increased single channel conductance, enhanced expression or both. We observed an increase in current density in independent transfection experiments in HEK cells . We did compare expression level, and localization of the UNC-2(WT)::GFP and UNC-2(GF)::GFP in transgenic animals  and did not see any obvious differences. This suggests that the mutation, besides the change in the voltage dependence of activation, also affects conductance. We comment on these possibilities in the Discussion section.4) While the results suggest that tax-6 acts in the muscle to decrease GABAergic synapses, rescue or RNAi in muscle should confirm the conclusion of the authors.tax-6 RNAi feeding experiments. Neurons are largely resistant to RNAi feeding in C. elegans , tax-6 RNAi feeding rescued UNC-49::GFP fluorescence in unc-2(zf35gf) mutants. These experiments are included in the revised manuscript .We thank the reviewer for the suggestion. We have performed a Reviewer #4:The authors analyze a novel gain of function (gf) allele of CaV2 N-type calcium channels. The unc-2(zf35gf) mutant was isolated in a screen for altered locomotion, lacking persistent forward or reverse movements while retaining a high level of motility. Analogous mutations in mammalian CaV2 channels expressed in tissue culture cells shows that the zf35 mutation alters channel gating . Similar gf alleles were previously described in mammalian CaV2 and CaV1 channels. Extensive analysis of unc-2(zf35) mutant worms finds changes in spontaneous cholinergic and GABAergic synaptic currents, and changes in the abundance of pre-synaptic (RAB-3) and post-synaptic (UNC-49 and UNC-29) markers. Based on these studies, the authors suggest that unc-2(zf35gf) mutants have altered GABA synapse development and that this results from the increased excitatory transmission. These conclusions are not strongly supported by the data. I agree that transmission has changed in these mutants, but it could simply be what you expect from increased UNC-2 channel currents. The analysis of unc-2(gf) mutants is extensive and would likely be of interest to C. elegans labs; however, they do not provide a clear understanding of how nervous system development or function is altered in these mutants. For these reasons, I do not support publication in eLife.unc-2 is expressed in all neurons, this is not what would simply be expected from increased UNC-2 calcium currents. We present evidence that increased cholinergic transmission in unc-2/CaV2α(zf35gf) mutants leads to a reduction of GABA synapses in a TAX-6/calcineurin dependent manner. While additional detailed molecular and cellular mechanisms remain to be explored (as with any study), our data provide a deeper understanding of how alterations in Ca-channel function produce unexpected changes in E/I balance in the nervous system. Further, our analysis of FHM mutations using this model provides evidence that these mutations produce similar changes in E/I balance that have clear implications for human disease.As pointed out by the reviewer 2 and 3, the main message of the paper is that gain-of-function mutation in the presynaptic voltage gated calcium channel causes an imbalance in E/I transmission. Since the Essential revisions:1) Post-synaptic GABA receptor abundance was significantly reduced in unc-2(gf) mutants but this was not accompanied by a corresponding decrease in the amplitude of the spontaneous IPSCs. Several prior papers found that mutations decreasing GABA receptor puncta intensities are closely matched by similar changes in IPSC current amplitudes .We understand the reviewer’s point as there is currently some discrepancy in the field to what extent electrophysiological data correlate with fluorescence intensity of synaptic markers. We do believe that the reviewer’s statement does not accurately represent the results from the cited papers.Specifically, the papers cited by the reviewer did not show that decreased postsynaptic GABAR marker signals are closely matched by changes in IPSC amplitude. Instead, and consistent with our findings, these studies showed that decreased GABA receptor marker intensity is closed matched by a reduction in IPSC frequency. The IPSC amplitudes, even of the same mutant, behaved differently in different preparations, and reported changes do not strongly correlate with changes in UNC-49::GFP fluorescence.To summarize the relevant data from these papers:madd-4, nlg-1 and nrx-1 mutants wereclosely matched by the reduction of the IPSC frequency. For example, nlg-1 mutants exhibited strong reduction of IPSC frequency and UNC-49 signals, but nrx-1 did not show defects in either. Here nlg-1 mutants exhibited a very small reduction of IPSC amplitude, which did not closely match the very strong reduction in UNC-49/GABAR clustering and fluorescence.Maro et al. (2015) showed that the degree of reduction of UNC-49 puncta signals in madd-4B(0) showed no significant change in UNC-49 intensity, but exhibited a significant reduction of IPSC frequency, and a milder reduction in amplitude.Tu et al. (2015) reported reduction of mIPSC frequency and amplitude in these three mutants, irrespective of their different degrees of reduction of the UNC-49/GABAR fluorescence. For example, nlg-1 mutants exhibited reduced UNC-49 fluorescence signals, reduced mIPSC frequency, and reduced mIPSC amplitude. The reduction in mIPSC amplitude reported in was larger than reported in the other two studies. Tong et al., (2015) reported two other mutants  that exhibited a reduction in UNC-49/GABA receptor signals, reduced mIPSC amplitude reduction but no change in mIPSC frequency. Unlike unc-2, these two genes function strictly in muscles to regulate UNC-49/GABA receptor levels. In another mutant, nrx-1, which was also examined in the other two papers, the authors found no overt fluorescent marker change, but an increase of mIPSC amplitude and no change in frequency. The reported mIPSC amplitude changes don’t follow an obvious trend with respect to changes in GABA receptor fluorescence intensity and are inconsistent across studies, even for the same mutation.Tong et al. (2015) reported that the unc-2(zf35gf) mutants show concomitant changes of UNC-49/GABA receptor intensity and IPSC frequency, which is consistent with the most salient findings reported in the cited papers.Our results for Given the absence of an effect on mIPSC amplitudes, these results suggest that the GABA receptors imaged here are non-synaptic. Altered non-synaptic receptors could be interesting but would require many new experiments to prove and document a functional consequence .unc-2(zf35), a decreased frequency of mIPSCs, and an unchanged amplitude of mIPSCs. A decrease in receptor number at existing postsynaptic sites would be predicted to change amplitude whereas a loss of postsynaptic sites would be most consistent with a change in frequency. Other mutations that have been described in the literature may produce both effects, accounting for the change in amplitude and frequency. In unc2(zf35gf) mutants there appears to be a decrease in the number of postsynaptic GABAR clusters: at some NMJs, we find presynaptic of RAB-3::mCherry puncta without punctate UNC-49::GFP apposition . This indicates the presence of orphan presynaptic sites and post-synaptic silencing of GABA synapses. A decrease in non-synaptic receptors could account for a reduction in UNC-49::GFP intensity without changed amplitude, but does not account for the observed reduction in mIPSC frequency. As we outline in the text, we believe that a decrease in the number of UNC-49containing postsynaptic sites on the muscle arms in the ventral nerve cord is a more plausible explanation. This accounts for the observed reduction in mIPSC frequency, the preserved mIPSC amplitude, and the reduced UNC-49::GFP intensity in the ventral nerve cord.We report a decrease in UNC-49::GFP intensity in the ventral nerve cord of 2) The authors conclude that unc-2(gf) mutants have decreased GABA synapse numbers, based on recordings indicating a decreased mIPSC rate. The mIPSC recordings were done in the presence of a cholinergic antagonist (dTBC), which by itself decreases mIPSC rate (since cholinergic neurons provide the excitatory input to GABA neurons). It seems possible that use of dTBC biases their results toward lower mIPSC rates. I recommend recording mIPSCs without dTBC .We are measuring mIPSC frequency and amplitude specifically from GABAergic motor neuron (VD) synapses onto muscle. VDs are postsynaptic to cholinergic motor neurons. We include dTBC to eliminate cholinergic drive onto the VDs and isolate VD endogenous events. The UNC-2(GF) channel is also predicted to promote cholinergic synaptic transmission onto the VDs. In the absence of dTBC, our electrophysiology recording would measure the compound effect of increased cholinergic drive onto VDs and VD’s endogenous IPSCs. Therefore, we believe the clearest experiment is to perform the analysis in the presence of dTBC as presented. We note that we have used this strategy to clearly distinguish mEPSCs and mIPSCs in prior published studies.– has been used in several studies. But we respectfully point out that this is not the only protocol used in the C. elegans field for recording IPSC events. At least for us, we could not find published data that demonstrated the specificity of stated recording conditions. The closest we found was “data not shown” in Madison et al., 2005.The recommended recording – recording mIPSCs without dTBC, with a low chloride internal buffer and holding potential of 0mV Before applying our electrophysiology protocol , we tested the effect of holding membrane potentials and dTBCs to show that using our intracellular recording solution, at -10mV holding potential, and the in presence of 0.5mM dTBC, we blocked all EPSC events without blocking IPSC events in the muscle preparation.unc-49(lf) mutant background) in unc-2(zf35gf) made it more important to apply dTBC. The use of dTBC does not bias but isolate the mIPSC population from the GABAergic VD-class motor neurons in both wild-type and unc-2(gf) mutant for comparison.For the current study, a significant increase of EPSC events (recorded in 3) If lower synapse numbers occur in unc-2(gf) mutants (as the authors propose), I would expect that synaptic puncta density (both RAB-3 and UNC-49) would be reduced. The authors should report density data for the synaptic markers.UNC-49 abundance was quantified by calculating the overall intensity of UNC-49::GFP in the region of nerve cord imaged. This is a compound measurement, dependent on the density of clusters, intensity of each cluster, and any diffuse UNC-49::GFP that is not localized to a cluster. We also measured the cluster density, and found an increase in presynaptic RAB-3 density and decreased UNC-49 density. We now include these data in the revised manuscript . These measures parallel overall intensity measurements. Our data are consistent with the interpretation that there are fewer GABAergic postsynapses, and orphan (silent) GABAergic presynapses.4) If the unc-2(gf) mIPSC rate defect cell autonomous to D neurons? If so, one would not need to propose altered cholinergic transmission in causing this phenotype (which the authors propose).unc-2(zf35gf) on ventral cord UNC-49::GFP intensity is the result of a dominant effect in cholinergic neurons. Expression of unc-2(zf35gf) transgene in GABAergic neurons alone, in an otherwise wild-type background, resulted in a marked increase in UNC-49::GFP intensity, whereas specific transgene expression in cholinergic neurons decreased the intensity similar to unc-2(zf35gf) mutants . Furthermore, cell specific expression of an unc-2(zf35gf) transgene in GABAergic neurons increases aldicarb resistance, indicating an increase in GABAergic transmission. (Expression of unc-2(zf35gf) in cholinergic neurons decreases aldicarb resistance). We now include these data in a revised version of the manuscript . Together, these experiments all indicate that the reduction of mIPSC rate in unc-2(zf35gf) is not cell autonomous to the D-type motor neurons and further support our conclusion that altered cholinergic transmission is responsible for the transmission defects in unc-2(zf35gf).We did not do this particular experiment, but we do show extensively that the effect of 5) No controls are provided to determine if changes in synaptic puncta intensities are caused by transcriptional changes.unc-2(zf35gf) animals showed no significant differences in unc-29, unc-49, myo-3, rab-3, unc-2 and rgef-1 expression. We include these data in a revised manuscript .We thank the reviewer for pointing this out. We did perform transcriptional analyses but did not include these data in this manuscript. RNA-seq experiments, in which we compared expression profiles of wild-type vs. 6) The RAB-3 puncta intensity changes are hard to interpret without further experiments. Are other SV markers altered similarly? Is the SV pool size altered (either by electrophysiology or EM)?unc-2(zf35gf). We provide evidence that increased cholinergic signalling leads to a reduction GABAergic synapses and explain and support this with pharmacology, fluorescent imaging of pre- and postsynaptic components of excitatory and inhibitory NMJs, cell specific expression and in vitro electrophysiology, as well as the impact on circuit output using locomotory behavior as a readout.We agree that future studies are needed to provide additional mechanistic insights. However, this is not the main point of the paper.As noted by reviewer 2 and 3, the main point of our study is to demonstrate that gain-of-function mutations, as found in FHM1 patients, result in E/I imbalance. E/I imbalance does not simply arise due to cell autonomous excitability increases in excitatory and inhibitory neurons, but arise primarily from increased excitatory signalling onto synaptic targets, in this case muscle cells. Our electrophysiology shows that there is increased excitatory input and decreased inhibitory input to ventral muscles in