Establishing the R-OptIoN approach.

<p><b>A)</b> Schematic of the Ca²⁺ imaging approach showing single animal and bulk measurement conditions (lower inset, left and right, respectively, showing RCaMP fluorescence in BWMs). The 470 and 590 nm LEDs were used to stimulate ChR2 expressed in neurons, and for exciting RCaMP expressed in BWMs, respectively. The micro-wells (upper inset) were prepared in a thick 10% agar pad, pierced with a heated LED cooler element. <b>B)</b> Schematic of the epifluorescence microscope used for Ca²⁺ imaging. Two high power LEDs are coupled into the excitation light train with a dichroic mirror; light passes through a 470/593 nm double band-pass excitation filter and a 605 nm beam splitter onto the animals in the micro-wells. Emission light passes a 647 nm filter before reaching the camera. <b>C)</b> Comparison of changes in body length of animals expressing ChR2 in cholinergic motoneurons in wild type background (black trace), <i>unc-49(e407)</i> (GABA_AR) mutants (red trace), or <i>unc-26(s1710)</i> (synaptojanin) mutants (orange trace). In the absence of ATR, rendering ChR2 non-functional, no contraction is observed (grey trace). Shown is the mean normalized body length (± SEM; n = 8–21). Blue bar marks period of illumination. <b>D)</b> Light-induced activation of cholinergic neurons expressing ChR2 causes essentially identical changes in body length (contraction) with and without RCaMP expression in BWMs (red and black trace, respectively). In the absence of ATR, no contraction is observed (grey trace). Shown is the mean normalized body length (± SEM; n = 11–27 animals). <b>E)</b> Ca²⁺ response in BWMs of single animals fixed on polystyrene beads during photostimulation of ChR2 expressed in cholinergic motoneurons. Displayed are mean ΔF/F₀ values (± SEM) of wild type control (black trace), <i>unc-47(e307)</i> mutants (green trace), and <i>unc-49(e407)</i> mutants (red trace) of n = 5–15 animals. <b>F)</b> Ca²⁺ response in BWMs during photostimulation of ChR2 expressed in cholinergic motoneurons in bulk measurements. Displayed are mean ΔF/F₀ values (± SEM) of wild type control (black trace), <i>unc-47(e307)</i> mutants (green trace), and <i>unc-49(e407)</i> mutants (red trace). Shown is the mean of n = 3 measurements of ~ 1000 animals each. <b>G)</b> Ca²⁺ response in BWMs of single animals fixed on polystyrene beads during photostimulation of ChR2 expressed in GABAergic motoneurons. Displayed are mean ΔF/F₀ values (± SEM) of wild type control (black trace), <i>unc-47(e307)</i> mutants (green trace) and no-ATR control (grey trace), of n = 6–9 animals for each genotype/condition. <b>H)</b> Ca²⁺ response in BWMs during photostimulation of ChR2 expressed in GABAergic motoneurons in bulk measurements. Displayed are mean ΔF/F₀ values (± SEM) of wild type control (black trace), <i>snt-1(md290)</i> mutants (orange trace), <i>unc-47(e307)</i> mutants (green trace), and <i>unc-25(n2569)</i> mutants (magenta trace); means ± SEM of n = 3 measurements of ~1000 animals each.</p

Electrophysiology verifies SV loading, release and recycling phenotypes.

<p>Photoevoked excitatory post-synaptic currents were measured in patch-clamped muscle, during repeated photostimulation of cholinergic neurons expressing ChR2, at 0.5 Hz. <b>A)</b> Representative original current traces of wild type, compared to mutants affecting SV loading (<i>unc-17(e113)</i> vAChT), SV recycling (<i>unc-26(s1710)</i> synaptojanin, or Ca²⁺ influx (<i>unc-2(ra612)</i> VGCC). <b>B)</b> Group data and statistical analysis, corresponding to the experiments in A. Wild type data is shown in black. Left panel: Mean inward currents (pA) ± SEM, in response to repeated stimulation with 10 ms blue light at 0.5 Hz. Right panel: Data was normalized to the first peak inward current [%]. Statistically significant differences were assessed by one-way ANOVA compared to wild type (* P < 0.05, ** P < 0.01, *** P < 0.001). For analysis of miniature PSC currents and frequencies of these strains, see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135584#pone.0135584.s005" target="_blank">S5 Fig</a>.</b></p

Further analysis of selected genes from the RNAi screen cluster #1, by Ca²⁺ imaging (A, B) and by electrophysiology (C-G).

<p>The genomic mutants <i>C27B7</i>.<i>7(ok2978)</i>, <i>inx-10(ok2714)</i>, <i>inx-8(gk42)</i>, <i>spp-10(gk349)</i> and <i>erp-1(ok462)</i> were crossed either to the ChR2(C128S); RCaMP strain for Ca²⁺ imaging, or to the <i>zxIs6</i> ChR2(H134R) strain, for measuring photo-ePSCs. In A and B, normalized Ca²⁺ signal difference traces are shown as color coded, maximum normalized data, based on the mean ΔF/F₀ Ca²⁺ response (± SEM) from bulk measurements of RNAi or mutant and control (n = 3–5 experiments, ~1000 animals each). In E—G, the averaged photo-ePSC measurements of the respective wild type animals (black), <i>C27B7</i>.<i>7(ok2978)</i> (red), <i>inx-10(ok2714)</i> (yellow), <i>inx-8(gk42)</i> (green), <i>spp-10(gk349)</i> (blue), or <i>erp-1(ok462)</i> mutants (orange) are shown as inward currents in pA ± SEM (left panels). Statistically significant differences were calculated by one-way ANOVA for individual time points, always compared to wild type (* P < 0.05), or for the whole data set as two-way ANOVA (** P < 0.01, **P<0.001). Animals were stimulated for 10 ms with blue light at a frequency of 0.5 Hz (n = 7–12). On the right, the same data are represented, but normalized to the mean peak currents of the first stimulus. For representative original current records, as well as for mini ePSC current and frequency analysis, see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135584#pone.0135584.s005" target="_blank">S5 Fig</a>.</b></p

R-OptIoN analysis of mutants that had previously been described to affect the synaptic vesicle cycle [25, 26].

<p><b>A)</b> Schematic representation of the synaptic vesicle cycle, showing (anti-clockwise) loading of SV with transmitter, transport of SV to the plasma membrane, docking, priming into the fusion-competent state, SV-plasma membrane fusion upon depolarization and Ca²⁺ influx, endocytosis/recycling of SV membrane and proteins, biogenesis of SV from endosomal compartment. <b>B-G)</b> Ca²⁺ traces resulting from bulk measurements in animals expressing ChR2(C128S) in cholinergic motoneurons and RCaMP in BWMs (displayed are means ± SEM of n = 3 experiments, ~1000 animals each). The blue bar marks the period of illumination. Graphs on the left describe ΔF/F₀ Ca²⁺ dependent RCaMP fluorescence signals, graphs on the right side show maximum normalized ΔF/F₀ Ca²⁺ response. The analyzed mutants affect different steps in the SV cycle such as SV loading (<b>B</b>; <i>unc-17(e113)</i>), priming (<b>C;</b><i>unc-13(e1091)</i>, Ca²⁺ influx (<b>D;</b><i>unc-2(ra612)</i>, VGCC), SV fusion and recycling (<b>E;</b><i>snt-1(md290)</i>), postsynaptic defects in an AChR subunit (<b>F;</b><i>unc-38(x20)</i>) and SV recycling (<b>G;</b><i>unc-26(s1710)</i> synaptojanin and <i>unc-57(e406)</i> endophilin A).</p

Construction and characterization of a strain with specific RNAi sensitivity in cholinergic motoneurons.

<p><b>A)</b> The genetic background lacks <i>rde-1</i>, eliminating RNAi in all tissues. RDE-1 is selectively rescued in cholinergic neurons, by expression from the <i>unc-17</i> promoter. Furthermore, neurons are sensitized to RNAi by overexpression of the SID-1 dsRNA uptake facilitator (using the panneuronal <i>unc-119</i> promoter). <b>B)</b> Specific knockdown (feeding RNAi) of YFP in cholinergic neurons (using GFP-RNAi bacteria that also target YFP), in an animal expressing YFP panneuronally (punc-119::YFP). Left, mock-RNAi control; right, YFP knockdown, residual YFP fluorescence in ventral nerve cord mainly from GABAergic neurons. <b>C)</b> Group data and statistics of animals as shown in B. Mean ± SEM, background-corrected fluorescence values resulting from the whole nerve cord YFP fluorescence. Compared are animals treated with mock-RNAi vector (L4440) (n = 21) or with L4440::GFP RNAi (n = 15), showing statistically significant reduction of YFP fluorescence (t-test, *** P<0.001). <b>D)</b> Pan-neuronal YFP expression in the RNAi-sensitive strain ZX1800. Animals were treated with bacteria containing the mock-control (L4440) (upper images), or (lower images) the GFP RNAi vector. Reduced YFP expression in the ventral nerve cord can be recognized. In the right images, cholinergic (blue) and GABAergic (red) motoneurons are labeled, in maximum projections of confocal stacks; shown is an enlarged region of the ventral nerve cord surrounding the vulva. Anti-GFP RNAi renders most cholinergic neurons undetectable due to YFP mRNA knock-down.</p

Representation of Ca²⁺ imaging data for high throughput analyses and recapitulation of mutant phenotypes by cholinergic neuron RNAi.

<p><b>A)</b> Maximum-normalized ΔF/F₀ Ca²⁺ signals from bulk measurements, before and during 120 s cholinergic neuron ChR2 photostimulation, in wild type control (black trace), and <i>unc-26(s1710)</i> synaptojanin mutant (orange trace). The difference graph (wild type—mutant trace) is shown in green. Mean of n = 3 experiments with ~1000 animals each. The difference trace is further plotted in color-code below the graph, color scale is shown on the left. Blue bar marks the period of illumination. <b>B)</b> Comparing color-coded Ca²⁺ signal difference traces from bulk measurements in genomic mutants (left panel) and respective RNAi animals (right panel); genotypes or mRNAs targeted, as indicated. Data averaged from n = 3–18 experiments with ~1000 animals each.</p