Gap junctions synchronize action potentials and Ca2+ transients in Caenorhabditis elegans body wall muscle

Abstract

The sinusoidal locomotion of Caenorhabditis elegans requires synchronous activities of neighboring body wall muscle cells. However, it is unknown whether the synchrony results from muscle electrical coupling or neural inputs. We analyzed the effects of mutating gap junction proteins and blocking neuromuscular transmission on the synchrony of action potentials (APs) and Ca2+ transients among neighboring body wall muscle cells. In wild-type worms, the percentage of synchronous APs between two neighboring cells varied depending on the anatomical relationship and junctional conductance (Gj) between them, and Ca2+ transients were synchronous among neighboring muscle cells. Compared with the wild type, knock-out of the gap junction gene unc-9 resulted in greatly reduced coupling coefficient and asynchronous APs and Ca2+ transients. Inhibition of unc-9 expression specifically in muscle by RNAi also reduced the synchrony of APs and Ca2+ transients, whereas expression of wild-type UNC-9 specifically in muscle rescued the synchrony defect. Loss of the stomatin-like protein UNC-1, which is a regulator of UNC-9-based gap junctions, similarly impaired muscle synchrony as unc-9 mutant did. The blockade of muscle ionotropic acetylcholine receptors by (+)-tubocurarine decreased the frequencies of APs and Ca2+ transients, whereas blockade of muscle GABAA receptors by gabazine had opposite effects. However, both APs and Ca2+ transients remained synchronous after the application of (+)-tubocurarine and/or gabazine. These observations suggest that gap junctions in C. elegans body wall muscle cells are responsible for synchronizing muscle APs and Ca2+ transients.

FIGURE 1.

The percentage of synchronous APs corresponded to the level of junctional conductance (G_j). A, photo of a filleted wild-type worm showing the two ventral quadrants of body wall muscle cells and the ventral nerve cord (VNC) between them. Muscle cells in the right quadrant are designated as R1 and R2, whereas those in the left quadrant are designated as L1 and L2. B, representative traces of APs (left panel) and junctional currents (right panel) recorded from various pairs of body wall muscle cells. The red and blue traces show temporal correlation between APs from the two cells in each pair. The asterisks indicate synchronized APs (≤50 ms in peak time difference). C, distribution of peak time differences for consecutive APs recorded from the L1L2 and R1R2 pairs. D, percentages of synchronous AP and levels of G_j in different pairs of muscle cells. E, the mean peak time difference for synchronous APs in different pairs of muscle cells. Several columns (e.g. L1L2+R1R2) represent pooled data of two different pairs of muscle cells because the two cell pairs have analogous anatomical locations and are indistinguishable in functional properties. The asterisk indicates a significant difference compared with the other groups (p < 0.05, one-way ANOVA with Bonferroni post hoc test). In both D and E, the data are shown as the means ± S.E., and the number of cell pairs analyzed is indicated inside the column.

FIGURE 2.

Action potential synchrony and junctional conductance were compromised in unc-9 and unc-1 mutants. APs and junctional currents were recorded from the L1L2 and R1R2 pairs of muscle cells of unc-9(fc16), unc-1(e719), and the double mutant unc-9(fc16);unc-1(e719). The data were compared with the same WT data shown in Fig. 1. A, representative traces of APs (left panel) and junctional currents (right panel) from the WT and mutant worms. The red and blue traces show temporal correlation between APs from the two cells in each pair. B, percentages of synchronous APs and levels of junctional conductance (G_j) in different groups. The asterisk indicates a significant difference compared with the corresponding WT data (p < 0.01, one-way ANOVA with Bonferroni post hoc test). C, the peak time difference for synchronous APs among the different groups. In both B and C, the data are shown as the means ± S.E., and the number of cell pairs analyzed is indicated inside the column.

FIGURE 3.

Coupling coefficient was greatly decreased in unc-9 and unc-1 mutants compared with the wild type. The mutants analyzed were unc-9(fc16), unc-1(e719), and the double mutant unc-9(fc16);unc-1(e719). Negative currents were injected into one muscle cell, whereas membrane voltage changes in response to the current injections were measured in both the injected cell (Cell 1) and a contacting neighboring cell (Cell 2) of the L1L2 or R1R2 pair. A, diagram of the current injection steps (left panel) and representative membrane voltage traces (right panel). B, membrane voltage changes (ΔV_m) in response to the current injections in Cell 1 and Cell 2. C, the coupling coefficient (the ratio of ΔV_m of Cell 2/ΔV_m of Cell 1). In both B and C, the data are shown as the means ± S.E., and the asterisk indicates a significant difference compared with the WT (p < 0.01, one-way ANOVA with Bonferroni post hoc test). The number of cell pairs analyzed was 33 for WT, 13 for unc-9, 13 for unc-1, and 12 for the double mutant.

FIGURE 4.

UNC-9 deficiency in muscle was responsible for reduced junctional conductance and action potential synchrony in unc-9 mutant. A, unc-9 RNAi inhibited the expression of GFP-tagged UNC-9 in body wall muscle cells (the punctate expression in the top portion of the left panel). The broad fluorescent signal at the bottom of both panels resulted from autofluorescence of the gut. B, sample traces of APs and junctional currents from the WT, unc-9(fc16), unc-9 RNAi worms, and unc-9(fc16) with muscle specific rescue. The red and blue traces show temporal correlation between APs from the two cells in each pair. C, comparison of junctional conductance (G_j) and the percentage of synchronous APs. The asterisk indicates a significant difference compared with WT (p < 0.01), whereas the pound sign indicates a significant difference compared with unc-9 (p < 0.05 for AP and p < 0.01 for G_j, one-way ANOVA with Bonferroni post hoc test). In C, the data are shown as the means ± S.E., and the number of cell pairs analyzed is indicated inside the column. The WT and unc-9 data are the same as those shown in Fig. 2.

FIGURE 5.

Ca²⁺ transient synchrony was compromised in unc-9 mutant, unc-1 mutant, and unc-9 RNAi worms. A, representative Ca²⁺ transients of the WT, unc-9(fc16), unc-1(e719), worms with muscle unc-9 expression inhibited by RNAi, and unc-9(fc16) rescued by expressing wild-type UNC-9 specifically in muscle. Ca²⁺ transients (F/F₀) of three selected cells are plotted (red trace for Cell a, blue trace for Cell b, and green trace for Cell c). The arrowheads and numbers in the Ca²⁺ transient traces indicate the time points of the imagines shown below. Movies of these representative experiments may be found in the supplemental materials (supplemental Movies S1–S5). B, comparisons of Ca²⁺ transient properties. The data are shown as the means ± S.E. The asterisk indicates a significant difference compared with WT, whereas the pound sign indicates a significant difference compared with unc-9 (p < 0.01, one-way ANOVA with Bonferroni post hoc test). The number of worms analyzed was 21 WT, 17 unc-9, 15 unc-1, 13 unc-9 RNAi, and 11 unc-9 rescue. trans., transient; syn., synchronous; freq., frequency.

FIGURE 6.

Gabazine blocked muscle GABA_A receptors. A, a representative experiment showing the effect of gabazine (0.5 mm) on spontaneous postsynaptic currents (“minis”) mediated by GABA_A receptors (upward events). B, a representative experiment showing the effect of gabazine (0.5 mm) on photo-evoked IPSCs in a worm expressing channelrhodopsin-2 in GABAergic neurons. The traces from top to bottom are light pulses, postsynaptic currents including IPSCs at a longer time scale, and the IPSCs at a shorter time scale. C, comparisons of the effects of gabazine on the frequency of “minis” and amplitude of IPSCs. The asterisk indicates a significant difference (p < 0.01, paired t test) compared with the control (before gabazine treatment). The data are shown as the means ± S.E. Minis and IPSCs were recorded from six and eight muscle cells, respectively.

FIGURE 7.

Action potentials remained synchronous after blocking acetylcholine and/or GABA_A receptors. APs were recorded from the L1L2 and R1R2 pairs. A, representative AP traces showing the effects of (+)-tubocurarine (TBC, 0.5 mm) and/or gabazine (0.5 mm). The red and blue traces show temporal correlation between APs from the two cells in each pair. B, effects the various treatments on the resting membrane potential, AP frequency, and AP synchrony. Comparisons were made between the control period (before the treatment) and during the treatment period with the asterisk indicating a significant difference (p < 0.05, paired t test). AP frequency and synchrony percentage were subsequently normalized by values of the control period and displayed in the bar graphs. The data are shown as the means ± S.E. The numbers of cell pairs analyzed were five for TBC, six for gabazine, and five for TBC plus gabazine. The control in the graphs represents pooled data.

FIGURE 8.

Ca²⁺ transients remained synchronous after blocking acetylcholine and/or GABA_A receptors. APs were recorded from the L1L2 and R1R2 pairs. A, representative traces of Ca²⁺ transients (F/F₀) from a control worm and worms treated with TBC (0.5 mm) and/or gabazine (0.5 mm). The colored traces show temporal correlation of Ca²⁺ transients in three randomly selected cells within the camera imaging field. Movies matching these experiments are provided in the supplemental materials (supplemental Movies S6–S9). B, the frequency, mean amplitude, and area (F/F₀ × time) of Ca²⁺ transients are shown as ratios of the values (during/before the treatment). The asterisk indicates a significant difference compared with the control group (p < 0.05, one-way ANOVA with Bonferroni post hoc tests). The data are shown as the means ± S.E. The sample number was 8 for Control, 10 for TBC, 12 for gabazine, and 9 for TBC plus gabazine.