Author response: Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans

Field: Neuroscience (Life Sciences)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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 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. https://doi.org/10.7554/eLife.45905.001 Introduction 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 (Nelson and Valakh, 2015; Vecchia and Pietrobon, 2012). Several studies have proposed that impaired inhibitory function may drive a shift in E/I balance toward excitation, and underlie the phenotypic changes observed in these disorders (Selten et al., 2018; Mainero and Louapre, 2014). While animal model studies provide support for this hypothesis, our understanding of the molecular and cellular mechanisms that lead to E/I imbalance remains limited. Mutations in the CACNA1A 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 (Catterall, 2000; Bidaud et al., 2006). CACNA1A mutations can cause episodic ataxia type 2 (EA2), epileptic seizures and familial hemiplegic migraine type 1 (FHM1) (Pietrobon, 2010). Episodic ataxia type 2 (EA2), whose clinical features include the lack of voluntary coordination of muscle movements and epileptic seizures, is associated with a range of missense, nonsense-, and splice site mutations throughout the CACNA1A gene. 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 (Adams and Snutch, 2007). Electrophysiological analyses suggest that EA2 mutations lead to diminished channel functions, whereas both gain- and loss-of-channel function phenotypes have been reported for FHM1-associated mutations (Cao et al., 2004; Tottene et al., 2002). Although these disorders have been conventionally distinguished, they exhibit considerable overlap in clinical presentations, leaving a precise correlation between genotype and phenotype unresolved. Animal model studies can provide mechanistic insights into the pathology of CACNA1A 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 (Tottene et al., 2009; Zhou et al., 2017; van den Maagdenberg et al., 2010). In FHM1 knock-in mice, glutamatergic neurotransmission in cortical pyramidal cells is enhanced, while GABAergic neurotransmission is unaltered. These findings suggest that FHM1 mutations cause a dysregulation of cortical E/I balance (Vecchia and Pietrobon, 2012). The genome of the nematode Caenorhabditis elegans encodes a single CaV2α subunit gene: unc-2 (Schafer and Kenyon, 1995). UNC-2/CaV2α is exclusively expressed in the nervous system (Mathews et al., 2003) and localizes to presynaptic zones, at synaptic vesicle release sites (Saheki and Bargmann, 2009), as well as at the plasma membrane of neural somas (Gao et al., 2018). Behaviorally, unc-2 loss-of-function (lf) mutants are sluggish and uncoordinated (Mathews et al., 2003). Furthermore, unc-2(lf) mutants have a reduced frequency of spontaneous excitatory postsynaptic currents (EPSCs) (Richmond et al., 2001), and a reduced intrinsic neuronal calcium oscillations of C. elegans motor neurons (Gao et al., 2018). In this study, we characterize a novel 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. Results zf35 mutants are hyperactive 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 (reversal frequency: zf35: 43.1 ± 2.0/3 min, n = 59; wild type: 6.8 ± 0.4/3 min, n = 59) (Figure 1; Video 1). This clonic seizure-like phenotype of zf35 mutants was accompanied by an increased locomotion rate during bouts of forward or backward locomotion. On average, zf35 mutants moved approximately 1.5 fold faster than wild-type animals (Figure 1A and B). Animals heterozygous for the zf35 mutation also displayed increased velocity and reversal frequency (Figure 1A–C), albeit to a lesser extent when compared to homozygous mutants. This indicates that the zf35 mutation is semi-dominant. zf35 mutant animals were slightly smaller than wild-type animals (0.82 ± 0.03 mm, n = 75 vs 1.00 ± 0.04 mm, n = 88) (Figure 1D) and had a reduced brood size (wild type: 207 ± 11, n = 5, zf35: 150 ± 16, n = 5). Furthermore, zf35 adults retained a reduced number of eggs in the uterus (zf35: 3.6 ± 0.2, n = 86; wild type: 14.1 ± 0.6, n = 80) (Figure 1E). zf35 mutants laid eggs that are at an earlier developmental stage than wild-type animals, indicating that the time between fertilization and egg laying was reduced (Figure 1F). Therefore, zf35 mutants are hyperactive in both locomotion and egg-laying behaviors. Figure 1 Download asset Open asset zf35 animals are hyperactive in both locomotion and egg-laying behaviors. (a) Representative traces from single worm tracking showing instantaneous velocity of indicated genotypes on OP50 thin lawn plates (see Materials and methods). Positive and negative values indicate forward and backward locomotion, respectively. Transition from positive to negative values indicates reversal events. (b) Shown is the average velocity for the wild-type (0.118 ± 0.01 worm lengths/s, n = 9), zf35 (0.156 ± 0.01 worm lengths/sec, n = 10), zf35 /+ (0.155 ± 0.01 worm lengths/s, n = 10) animals (c) Quantification of the reversal frequency in 3 min on regular OP50 plates: average reversal numbers made by wild type (6.8 ± 0.4 reversals, n = 59), zf35 (43.1 ± 2.0 reversals, n = 59) and zf35/+ (33.2 ± 1.6 reversals, n = 23). Error bars represent SEM for at least three trials. Statistical difference from wild type *p<0.05, ****p<0.0001, one-way ANOVA with Dunnett's multiple comparisons test. Statistical difference between zf35 and zf35/+ **p<0.01, unpaired t-test. (d) Representative images of wild type and zf35 animals. Average of midline lengths of the wild type: 1.00 ± 0.04 mm, n = 88 and zf35: 0.82 ± 0.03 mm, n = 75. Scale bar is 200 µm. (e) Representative Nomarski images of unlaid eggs in adult wild-type and zf35 animals. Arrowheads indicate eggs; asterisk denotes the position of the vulva. The average numbers of eggs in the uterus: wild type (14.1 ± 0.6 eggs, n = 80), zf35 (3.6 ± 0.2 egg, n = 86) animals. Scale bar, 50 µm. (f) Embryonic stages of freshly laid eggs of the wild type and zf35 mutants. 43% of the laid eggs from zf35 animals are at 1–16 cell stage, while only 5% from the wild type laid eggs are at 1–16 cell stage. Five independent trials with 75 animals for each genotype. Statistical difference from wild type ****p<0.0001, Chi-squared test. https://doi.org/10.7554/eLife.45905.002 Figure 1—source data 1 Source data for Figure 1. https://doi.org/10.7554/eLife.45905.003 Download elife-45905-fig1-data1-v3.xlsx Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg unc-2(zf35) mutants have an increased reversal frequency. Videos of locomotor behavior of the wild-type and unc-2(zf35) animals on NGM agar plates with seeded OP50. https://doi.org/10.7554/eLife.45905.004 zf35 mutant's hyperactivity is caused by a missense mutation in the unc-2/CaV2α gene We mapped the zf35 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 (Figure 2A). UNC-2/CaV2α consists of four homologous domains (I-IV) each containing six hydrophobic membrane-spanning segments (S1–S6, Figure 2—figure supplement 1). The zf35 mutation results in a glycine to arginine substitution (G1132R) in the highly conserved intracellular linker between III-S6 and IV-S1 (Figure 2B and C). To determine if UNC-2(G1132R) in zf35 mutant animals is sufficient to confer the hyperactive phenotype, we generated an unc-2(zf35) 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 (Figure 2E). 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 (Figure 2D). The fluorescence expression pattern of UNC-2(GF/G1132R)::GFP animals displayed no obvious difference with that of a UNC-2(WT)::GFP transgene (Figure 2D) (Saheki and Bargmann, 2009). This indicates that UNC-2(G1132R) is properly processed and trafficked to presynaptic sites. Figure 2 with 1 supplement see all Download asset Open asset zf35 is a novel allele of the CaV2α subunit gene unc-2. (a) The genetic map and gene structure of unc-2. Coding sequences are represented as black boxes. The zf35 allele is a single nucleotide transition (GGA to AGA) resulting in a glycine to arginine (G to R) amino acid substitution at position 1132. (b) Diagram of the secondary structure of UNC-2/CaV2α. UNC-2/CaV2α consists of four domains (I–IV) each containing six alpha-helix transmembrane (TM) segments (S1 – S6). The UNC-2 (G1132R) mutation localizes in the intracellular loop between TM domain III and IV, indicated by the blue circle. Purple circles indicate positions of intragenic unc-2(zf35) suppressors, red circles indicate the location of human FHM1 mutations. (c) The G1132R mutation occurs in a highly conserved region of the CaV2α subunit. Amino acid alignment of C-terminus region of the transmembrane III alpha-helix segment 6 (III S6) and the beginning of the third intracellular loop of CaV2α subunits from human (Homo sapiens, CACNA1A), rainbow fish (Poecilia reticulata, cacna1a), fly (Drosophila melanogaster, Cacophony) and nematode (C. elegans. UNC-2). Identities are shaded in dark gray, similarities in light gray. Location of the G1132R mutation is indicated. (d) Representative images of GFP tagged UNC-2(WT) and UNC-2(GF/G1132) in the ventral nerve cord. Asterisks point the cell bodies of the motor neurons and arrows indicate the presynaptic sites. Both constructs are expressed under pan-neuronal promoter tag-168. Scale bar, 10 μm. (e) Quantification of the reversal frequency: wild type (6.6 ± 0.4, n = 70), unc-2(zf35) (43.3 ± 1.9, n = 65), unc-2(e55lf) (2.4 ± 0.2, n = 59), wild-type animals expressing unc-2(wt) transgene (7.5 ± 0.6, n = 10) and unc-2(zf35) transgene (33 ± 2.1, n = 22), and unc-2(e55lf) rescued with unc-2(wt) transgene (3.8 ± 0.7, n = 12) and unc-2(zf35) transgene (41.3 ± 3.6, n = 21). Error bars represent SEM for at least three trials with indicated totaling animals number. Statistical difference from wild type ****p<0.0001, one-way ANOVA with Dunnett's multiple comparisons test. (f) Intragenic unc-2(lf) mutations suppress unc-2(zf35) hyperactive locomotion. Shown are numbers of thrashes in 30 s in M9 for the wild type (107.0 ± 14.0, n = 60), unc-2(zf35) (128.1 ± 13.5, n = 60), unc-2(lf) (4.8 ± 2.1, n = 57), unc-2(zf35 zf109) (6.9 ± 4.3, n = 53); unc-2(zf35 zf113) (5.6 ± 3.7 thrashes, n = 57); unc-2(zf35 zf114) (80.2 ± 9.9, n = 60); unc-2(zf35 zf115) (6.9 ± 3.8, n = 56); unc-2(zf35 zf124) (5.3 ± 3.1, n = 57); unc-2(zf35 zf130) (67.1 ± 22.5, n = 58); unc-2(zf35 zf134) (31.2 ± 17.9, n = 50). Error bars represent SEM. Statistical difference from unc-2(zf35) mutants unless otherwise indicated, ****p<0.0001, one-way ANOVA with Tukey's multiple comparisons test. https://doi.org/10.7554/eLife.45905.005 Figure 2—source data 1 Source data for Figure 2. https://doi.org/10.7554/eLife.45905.007 Download elife-45905-fig2-data1-v3.xlsx Intragenic mutations suppress the unc-2(zf35) hyperactivity phenotype unc-2 loss-of-function (lf) mutants are sluggish and exhibit reduced motor activities (Mathews et al., 2003). 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 (Figure 2B). Four suppressors, zf109, zf113, zf115 and zf124 reverted the zf35 hyperactivity phenotype to sluggish locomotion, similar to the canonical loss-of-function unc-2(e55) allele (Figure 2F). The 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). A G to R substitution in CaV2α intracellular III-IV linker leads to increased CaV2 channel activity To 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 (Figure 2C). CaV2.1α expression constructs were transfected into a HEK 293 cell line that stably expresses the auxiliary β1c and α2δ subunits (Piedras-Renteria et al., 2001). Whole-cell patch clamp experiments (Figure 3A) showed that the CACNA1A(G1518R) CaV2.1α channel exhibited a −10 mV shift in activation potential when compared to the wild-type CaV2.1α channel (Figure 3B and C). The maximal current density was 1.7-fold larger for G1518R channels (80.6 ± 5.7 pA/pF, n = 11) compared to wild type (47.5 ± 4.3 pA/pF, n = 13) (Figure 3B). Figure 3 Download asset Open asset The UNC-2(G1132R) corresponding mutation in human CACNA1A, CaV2.1α, subunit results in increased channel activity. (a) Representative macro-currents of wild type and G1518R CaV2.1 channels. Currents were generated by stepping membrane potential to voltages between −55 and 40 mV in 5 mV increments for 200 ms from a holding potential of −120 mV. (b) Voltage dependence of whole-cell current density for wild type and G1518R CaV2.1 channels. Current density values were obtained by dividing current amplitudes and cell capacitance. (Wild type, n = 13; G1518R, n = 11). (c) Voltage dependence of Ba2+ current activation. The activation curve of G1518R exhibits a significant shift of the V0.5 value towards more negative membrane potentials. (d) Steady-State inactivation curves. The G1518R mutation causes a slight positive shift in the midpoint voltage in the steady-state inactivation curves (V0.5inact= -55.0 ± 1.0 and −47.3 ± 1.0 for wild type and G1518R, respectively). 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 the Boltzmann equation: (Imax=(1+exp[(V-V0.5)/kin]) - 1). All recordings were carried out in Ba2+ solution to exclude the effects from calcium-dependent inactivation. https://doi.org/10.7554/eLife.45905.008 Figure 3—source data 1 Source data for Figure 3. https://doi.org/10.7554/eLife.45905.009 Download elife-45905-fig3-data1-v3.xlsx The slope of the activation curve was not significantly affected in the CACNA1A(G1518R) channel (KaWT = 3.8 ± 0.2 mV; Ka G1518R = 4.1 ± 0.1 mV, Figure 3C). Both wild-type and G1518R CaV2.1 channels decayed with similar mono-exponential time courses (Tinac 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 (Figure 3D). The membrane potential at which half of the current was inactivated in the G1518R channels exhibited a mV shift to more positive compared to wild type (V0.5inact= -55.0 ± 1.0 mV and −47.3 ± 1.0 mV for the wild-type and G1518R respectively). This indicates that the of channels is increased for CACNA1A(G1518R) channels at a membrane potential. the G1518R mutation leads to channels that are at lower membrane and inactivated at membrane potentials. these properties lead to increased current density by The in the linker between TM III and TM between C. elegans and suggests that UNC-2(G1132R) exhibits similar gain-of-function effects in activation and inactivation of CaV2α channel. these experiments were with the human CACNA1A channel in HEK we cannot exclude the that the corresponding UNC-2/CaV2α G1132R mutation may have effects on channel function in C. elegans. mutations in UNC-2/CaV2α lead to behavioral hyperactivity Several missense mutations in the human gene result in familial hemiplegic migraine type 1 (FHM1) (Pietrobon, 2010). Electrophysiological analyses of the effects on CaV2.1 channel of FHM1 mutations in expression and can be while that the R192Q mutation CaV2.1 calcium (Cao et al., 2004; Tottene et al., that the mutation results in an increased calcium influx at lower membrane et al., van den Maagdenberg et al., In knock-in the R192Q and FHM1 mutations increased Ca2+ current density indicating a gain-of-function den Maagdenberg et al., 2004; Tottene et al., 2009; van den Maagdenberg et al., 2010). To determine the effects of FHM1 mutations in C. we introduced R192Q and S218L mutations into unc-2 (Figure Figure 2—figure supplement 1). Pan-neuronal expression of the or transgene in C. elegans in phenotypes similar to unc-2(zf35gf) mutants. both and animals exhibited increased reversal n = and n = when compared to wild-type animals n = (Figure also displayed hyperactive egg-laying behavior (Figure transgenic animals laid eggs that are at an earlier developmental stage and retained eggs in the uterus 5.7 ± 0.4, n = ± 0.6, n = when compared to wild-type animals ± n = 23). These experiments provide genetic evidence similar to the FHM1 mutations are gain-of-function mutations that lead to increased CaV2 activity. Figure Download asset Open asset FHM1 mutations in unc-2 gene result in a hyperactive (a) The amino acid alignment of the conserved region of transmembrane domain I membrane-spanning segments I and the following linker region from human and worm CaV2α Identities are dark and similarities are light gray. are the human FHM1 R192Q and (b) Shown is the average number of in s on thin lawn OP50 plates: wild type ± n = unc-2(zf35gf) ± n = ± n = and ± n = (c) Average numbers of eggs in the adult uterus: wild type ± eggs, n = unc-2(zf35gf) ± 0.2, n = ± 0.4, n = and ± 0.6, n = bar the ± SEM for at least three trials with indicated totaling animals number. Statistical difference from ****p<0.0001, one-way ANOVA with Dunnett's multiple comparisons test. (d) Quantification of on 1 data point the ± SEM of the of animals of the wild-type animals were at unc-2(lf) animals were to the effects of and at unc-2(zf35gf) mutants were to of the unc-2(zf35gf) mutants were at of heterozygous unc-2(zf35gf) mutants at 40 independent trials with at least 50 animals for each **p<0.01, ****p<0.0001, ANOVA with Tukey's multiple comparisons test. (e) Quantification of on 1 at the min time ± of wild type, ± of and ± of expressed in wild-type animals, ± of unc-2(lf) animals, ± of of and of and in unc-2(lf) **p<0.01, one-way ANOVA with Dunnett's multiple comparisons test. Figure data 1 Source data for Figure Download unc-2/CaV2α gain-of-function mutations increase to Our recordings suggested the channel may increase Ca2+ resulting in To if unc-2(zf35gf) mutants have synaptic transmission, we to the C. elegans from excitatory cholinergic motor neurons et al., and causes the of muscle and et al., of wild-type animals to 1 1 with findings et al., et al., while than unc-2(lf) mutants were 1 (Figure In sharp of unc-2(zf35gf) mutants 30 min (Figure mutants also more than the that the unc-2(zf35gf) mutation is semi-dominant. Pan-neuronal expression of or also induced to (Figure This is not due to overexpression of the unc-2 transgene because expression of a wild-type unc-2 transgene, which restored the locomotion defects in unc-2(lf) mutants, to wild-type to (Figure Therefore, animals with gain-of-function mutations in unc-2/CaV2α are to which may increased release at the unc-2(zf35gf) mutants exhibit increased cholinergic and decreased GABAergic spontaneous postsynaptic currents at the To the of the unc-2(zf35gf) mutation on synaptic we the frequency of spontaneous release in recordings of postsynaptic currents from C. elegans C. elegans are by both excitatory and inhibitory motor neurons et al., et al., et al., To the spontaneous we recordings under where both cholinergic and GABAergic as currents mV holding see and methods). unc-2(zf35gf) mutants showed an increase in the frequency of spontaneous when compared to wild-type animals (Figure and with no significant changes in the (Figure and C). Figure 5 Download asset Open asset The unc-2(zf35gf) mutation leads to increased spontaneous and decreased spontaneous (a) Representative traces of spontaneous postsynaptic currents from ventral in wild-type and unc-2(zf35gf) mutants. and spontaneous frequency and of wild-type and unc-2(zf35gf) mutants. (d) Representative traces of spontaneous cholinergic in and unc-2(zf35gf) mutants. and spontaneous frequency and and unc-2(zf35gf) mutants. Representative traces of spontaneous GABAergic in wild-type and unc-2(zf35gf) mutants. and frequency and of wild-type animals and unc-2(zf35gf) mutants. Error bars SEM. *p<0.05, **p<0.01, test. Figure data 1 Source data for Figure Download excitatory and inhibitory to be affected in FHM1 (Tottene et al., 2009; Vecchia et al., Vecchia et al., we the of the unc-2(zf35gf) mutation on cholinergic and GABAergic transmission. To cholinergic we recordings at a holding potential of mV in the GABA mutant The frequency of spontaneous excitatory postsynaptic currents (EPSCs) was increased by approximately in mutants compared