Presynaptic ryanodine receptors are required for normal quantal size at the Caenorhabditis elegans neuromuscular junction

Abstract

Analyses of the effect of ryanodine in vertebrate brain slices have led to the conclusion that presynaptic ryanodine receptors (RYRs) may have several functions in synaptic release, including causing large-amplitude miniature postsynaptic currents (mPSCs) by promoting concerted multivesicular release. However, the role of RYRs in synaptic release is controversial. To better understand the role of RYRs in synaptic release, we analyzed the effect of RYR mutation on mPSCs and evoked postsynaptic currents (ePSCs) at the Caenorhabditis elegans neuromuscular junction (NMJ). Amplitudes of mPSCs varied greatly at the C. elegans NMJ. Loss-of-function mutations of the RYR gene unc-68 (uncoordinated 68) essentially abolished large-amplitude mPSCs. The amplitude of ePSCs was also greatly suppressed. These defects were completely rescued by expressing wild-type UNC-68 specifically in neurons but not in muscle cells, suggesting that RYRs acted presynaptically. A combination of removing extracellular Ca2+ and UNC-68 function eliminated mPSCs, suggesting that influx and RYR-mediated release are likely the exclusive sources of Ca2+ for synaptic release. Large-amplitude mPSCs did not appear to be caused by multivesicular release, as has been suggested to occur at vertebrate central synapses, because the rise time of mPSCs was constant regardless of the amplitude but distinctive from that of ePSCs, and because large-amplitude mPSCs persisted under conditions that inhibit synchronized synaptic release, including elimination of extracellular Ca2+, and mutations of syntaxin and SNAP25 (soluble N-ethylmaleimide-sensitive factor attachment protein 25). These observations suggest that RYRs are essential to normal quantal size and are potential regulators of quantal size.

Figure 1.

Ryanodine receptor mutations inhibited mPSCs and ePSCs at the C. elegans neuromuscular junction. A, a-c, Representative traces of mPSCs of the wild type (WT) and the unc-68 mutants (r1162, r1162). In WT, mPSCs showed great variability in amplitudes. Many events have amplitudes >50 pA. In r1162 and r1161, the frequency and amplitudes of mPSCs were decreased compared with WT. d, mPSC amplitude histograms showing that large-amplitude mPSCs (operationally defined as >50 pA) were essentially absent in the mutants. e, Cumulative mPSC amplitude distribution showing that mPSCs had smaller amplitudes in the mutants. f, The frequency of mPSCs was significantly decreased in the mutants. g, The mean amplitude of mPSCs was significantly reduced in the mutants (WT, n = 25; r1162, n = 10; r1161, n = 7). B, a, Representative traces of ePSCs in WT and r1162. b, The amplitude of ePSCs was significantly reduced in r1162 (n = 9) compared with WT (n = 14). The asterisk indicates a statistically significant difference (p < 0.05).

Figure 2.

Ryanodine (Rya; 100 μm) inhibited mPSCs in wild-type but not unc-68 mutant preparations. A, In wild-type preparations, ryanodine inhibited the frequency and the mean amplitude of mPSCs (n = 5). B, In the unc-68(r1162) mutant, ryanodine showed no effect on either mPSC frequency or amplitude (n = 5). The preryanodine and postryanodine mPSC amplitude distributions overlap completely (b). The wild-type mPSC amplitude histogram shown in Figure 1 A is replotted here for comparison.

Figure 3.

RYRs play a similar role in various subsets of neuromuscular junctions. Neuromuscular transmissions through nicotinic (Nic) receptors, levamisole (Lev) receptors, and GABA receptors were analyzed independently using unc-49(e407);unc-29(e1072) mutant, unc-49(e407) mutant in the presence of dihydro-β-erythroidine (5 μm), and wild-type animals in the presence of 0.5 mm d-TBC, respectively. The function of RYRs in synaptic transmission was evaluated by comparing mPSCs and ePSCs during the control period (Ctr) and after the administration of 100 μm ryanodine (Rya). A, Representative current traces and cumulative amplitude plots showing that ryanodine reduced mPSC frequency and shifted mPSCs toward smaller amplitudes. B, Representative traces showing that ryanodine inhibited the amplitudes of ePSCs mediated by either nicotinic or levamisole receptors. The ePSCs mediated by the two receptors were kinetically very different. The ePSCs mediated by nicotinic receptors had a single decay time constant (τ = 7.2 ± 0.9 ms; n = 5), whereas those mediated by levamisole receptors had two decay time constants (τ₁ = 5.4 ± 1.2 ms; τ₂ = 43.5 ± 7.8 ms; n = 6). C, Bar graphs showing mPSC frequencies (Nic, n = 5; Lev, n = 6; GABA, n = 7) and ePSC amplitudes (Nic, n = 5; Lev, n = 6) before and after ryanodine. D, Sample traces showing the effect of d-TBC on ePSC amplitude in a wild-type preparation and a typical ePSC in unc-49(e407). The ePSCs in wild-type preparations were abolished by d-TBC (Ctr, 1.73 ± 0.36 nA; n = 5; d-TBC, 0.00 ± 0.00 nA; n = 5), which could be partially reversed by washing (wash, 0.69 ± 0.20; n = 3). The amplitude of ePSCs in unc-49(e407) was 2.02 ± 0.16 nA (n = 9).

Figure 4.

Body wall muscle sensitivity to acetylcholine was not different between the wild-type (WT) and the unc-68(r1162) mutant. A, Representative traces showing acetylcholine-induced inward currents in WT and r1162. B, Comparison of the amplitudes of acetylcholine-induced inward currents between WT and r1162 (WT, n = 8; r1162, n = 6).

Figure 5.

Expression of the wild-type RYR in neurons but not muscle cells rescued unc-68(r1162) synaptic transmission defects. A myc-tagged wild-type UNC-68 was expressed in neurons and body wall muscle cells independently. A, Animals expressing myc::UNC-68 under the control of the neuron-specific rab-3 promoter show myc immunoreactivity only in neurons and neuronal processes, such as the dorsal cord (a) and neurons in the head (d). The immunoreactivities of myc (a) and the active zone marker RIM (b) in the dorsal cord overlap (c), suggesting that UNC-68 exists at presynaptic release sites. e, Animals expressing myc::UNC-68 under the control of the muscle-specific myo-3 promoter show myc immunoreactivity only in muscle cells. No myc staining was observed in wild-type or r1162 animals that did not express the transgene (data not shown). Scale bars: c, d, 10 μm; e, 20 μm. B, Animals of wild type (WT), r1162, r1162 expressing myc::UNC-68 in neurons (Neuron), and r1162 expressing myc::UNC-68 in muscle cells (Muscle) were singly placed in the center of a circular field (7 mm) in culture plates to track their locomotion. a, Photographs taken 10 min after the animals were placed on the plates showing that the r1162 mutant produced much shorter tracks on the plate than the WT, which was rescued by expressing wild-type UNC-68 in neurons. b, The locomotion velocity of r1162 was significantly slower than that of WT, which was mostly rescued by expressing the wild-type UNC-68 in neurons. Expression of myc::UNC-68 in body wall muscle cells also showed a modest rescuing effect. WT, n = 11; r1162, n = 5; Neuron, n = 5; Muscle, n = 9. C, a, Representative traces of mPSCs from r1162 and rescued animals. b, c, Comparisons of mPSC frequency and amplitudes among WT (n = 25), r1162 (n = 10), and the rescued (Neuron, n = 8; Muscle, n = 7). D, a, Representative traces of ePSCs from r1162 and rescued animals. b, Comparison of ePSC amplitudes among WT (n = 14), r1162 (n = 9), and the rescued (Neuron, n = 5; Muscle, n = 7). The asterisk indicates a statistically significant difference compared with WT (p < 0.05).

Figure 6.

Ca²⁺ influx and ryanodine receptor-mediated release were the exclusive sources of Ca²⁺ for synaptic exocytosis, and the amplitude of mPSCs was independent of extracellular Ca²⁺. A, Representative traces showing the effect of removing extracellular Ca²⁺ on mPSCs. Removal of Ca²⁺ greatly reduced the frequency of mPSCs in the wild type but essentially abolished mPSCs in the unc-68(r1162) mutant. B, Amplitude distributions of mPSCs were identical in the presence and absence of extracellular Ca²⁺ in wild type (n = 5). C, Averaged mPSCs of wild type before and after Ca²⁺ removal overlap completely. The averages were obtained from the wild-type trace shown in A. D, Removal of extracellular Ca²⁺ did not change mPSC amplitudes in wild type (n = 5). E, Although removal of extracellular Ca²⁺ greatly reduced mPSC frequency in wild type (n = 5), it eliminated mPSCs in r1162 (n = 5). The asterisk indicates a statistically significant difference (p < 0.05). F, itr-1(sa73), a hypomorphic mutant of inositol 1,4,5-triphophate receptor, showed normal mPSCs (a, b) and ePSCs (c); mPSC frequency, 50.9 ± 4.8 Hz; mPSC amplitude, 26.7 ± 2.2 pA; ePSC amplitude, 1.43 ± 0.17 nA; n = 5.

Figure 7.

Large-amplitude mPSCs did not result from concerted multivesicular release. A, Scatterplots of mPSCs (a, b) and mPSCs grouped according to amplitudes (c, d) show that the rise time was constant regardless of the amplitude, whereas the rise slope was steeper for events with larger amplitudes; n = 6. e, f, Averaged mPSC and ePSC traces in one representative experiment showing that the rise time of ePSCs is much longer than that of mPSCs. B, Large-amplitude mPSCs still occurred in mutants defective in synchronized synaptic exocytosis. The syntaxin mutant unc-64(e246) and the SNAP25 mutant ric-4(ys7) showed desynchronized ePSCs (c). However, large-amplitude mPSCs still occurred in the mutants (a, b). In ys7, the frequency and amplitude of mPSCs (n = 9) were 40 ± 7 Hz and 25.5 ± 2.5 pA, respectively. ePSC amplitude was 67.6 ± 12.5 pA (n = 5). Statistics on mPSC and ePSC properties of e246 are described in a study by Wang et al. (2001).

Figure 8.

Synaptic vesicle size and number were similar between the wild type (WT) and unc-68 mutant. A, Representative electron micrographs of neuromuscular junctions in the WT and unc-68 mutant. Scale bar, 100 nm. Presynaptic densities are indicated by arrowheads. B, The vesicle diameters were 33.1 ± 0.6 nm in WT and 32.1 ± 0.6 nm in unc-68. The numbers of vesicles per synapse were 109.5 ± 14.1 in WT and 105.4 ± 14.0 in unc-68. The numbers of morphologically docked vesicles per synapse were 15.0 ± 1.8 in WT and 13.8 ± 1.1 in unc-68. No statistically significant differences were found between the WT and unc-68 mutant. The numbers of neuromuscular junctions analyzed were eight for WT and 10 for unc-68(r1161).