Locomotory phenotypes associated with L-AChR<i>(gf)</i> expression require neuropeptide signaling.

<p>(<b>A</b>) Average body bends/min measured in liquid for the genotypes indicated. The strong locomotory defects of <i>egl-3</i> and <i>egl-21</i> mutants prevented analysis of L-AChR(<i>gf</i>) effects on agar (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584.s003" target="_blank">Fig. S3A, B</a>). Mutation of <i>pkc-1</i> normalized the locomotor effects of L-AChR(<i>gf</i>) in both liquid and on agar (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584.s003" target="_blank">Fig. S3C–E</a>). (<b>B</b>) Movement trajectories of wild type, L-AChR<i>(gf), nlp-12</i>;L-AChR<i>(gf)</i> animals as indicated. Each black line shows the trajectory of one animal monitored for 45 s on food. (<b>C</b>) Average body bend amplitude for the genotypes indicated. Each bar represents the mean (±SEM) of values calculated from recordings of at least 15 animals. For (A) and (C) ***, p<0.0001 by ANOVA with Sidak's post-hoc test. (<b>D</b>) Wide-field epifluorescent image of an adult animal expressing <i>nlp-12::SL2::mCherry</i>. The image is oriented with the head to the left. White rectangle: nerve ring. Arrow: DVA cell body. (<b>E</b>) Average body bend amplitude for the indicated genotypes and effects of DVA ablation (–DVA). Ablations were performed on L2 stage animals. Body bend amplitude was measured from recordings of young adult animals 2 days following laser ablation. Error bars indicate mean (±SEM) of at least 8 animals. ***, p<0.0001 student's t-test.</p

Modulation of body bend depth through NLP-12/CCK signaling is critical for local food searching.

<p>(<b>A</b>) Schematic representation of the neural circuit underlying NLP-12 modulation of local searching. Synaptic connections (triangles, brackets) are as described by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584-Garrison1" target="_blank">[42]</a>. DVA receives synaptic input from the dopaminergic neuron PDE and makes connections with both motor neurons and interneurons involved in locomotory control. DVA makes synaptic contacts onto all of the motor and interneurons indicated by brackets. In addition, DVA is connected to AVB and PVC by gap junctions. Assignments of interneurons into layers are as described by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584-Gray1" target="_blank">[13]</a>. Other neuron classes are as described in the text and references therein. DOP-1 modulation of DVA activity regulates NLP-12 release from DVA, altering the motor pattern during local food searching. (<b>B</b>) Average body bend amplitude for the genotypes as indicated measured during an initial five minute time interval (0–5) immediately following removal from food and a second five minute time interval 30 minutes after removal from food (30–35). Bars represent mean values (±SEM) calculated from 9–14 animals. (<b>C</b>) Representative tracks of wild type and <i>nlp-12(ok335)</i> mutants during an initial five minute period (0–5) following removal from food. Note the decreased number of reorientations and increased frequency of long forward runs for <i>nlp-12(ok335)</i> mutant as compared to the wild type. (<b>D</b>) Total directional reorientations measured during the 0–5 and 30–35 minute intervals following removal from food for wild type, transgenic wild type animals expressing Tetanus toxin in DVA <i>[DVA::Tetx]</i>, <i>nlp-12(ok335)</i> and <i>ckr-2(tm3082)</i> mutants. Bars represent mean (±SEM) for at least 12 animals. (<b>E</b>) Quantification of reversal coupled omega turns and reorientations in the absence of omega turns during the first 5 minutes following removal from food for the genotypes indicated. (WT and <i>nlp-12(ok335)</i>: n = 17). Rescue refers to the <i>nlp-12(ok335)</i> mutant expressing an extrachromosomal array carrying <i>Pnlp-12::nlp-12::SL2::mCherry</i> (n = 9). Bars represent mean (±SEM). ***, p<0.0001; **, p<0.001 by ANOVA with Sidak's post-hoc test.</p

The dopamine receptor DOP-1 is required in DVA for NLP-12 modulation of food searching.

<p>(<b>A</b>) Frequency of high angled reorientations for wild type and <i>nlp-12(ok335)</i> animals quantified for 5 minutes after transfer to food free plates in the presence (+) or absence (−) of dopamine (DA). Bars represent mean (±SEM) for at least 12 animals. Dopamine mechanosensory signaling is strongly enhanced at low osmotic strength <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584-Schafer1" target="_blank">[73]</a>. Therefore, these assays were conducted following transfer of the animals to low osmotic-strength assay plates as described previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584-Hills1" target="_blank">[14]</a>. We observed a modest increase in basal reorientation frequency across all genotypes under these conditions. (<b>B, C</b>) Representative images (B) and quantification (C) of NLP-12::VenusYFP fluorescence in the ventral cord region of the DVA process of wild type, <i>dop-1(vs100)</i>, and <i>dop-1(vs100) Ex DVA::dop-1</i> animals before (−) and after (+) 10 minutes dopamine (DA) treatment (wild type: n = 12 for (−) and (+) DA; <i>dop-1(vs100)</i>: n = 12 for (−) and 9 for (+) DA). <i>Ex DVA::dop-1</i> refers to specific rescue of <i>dop-1</i> expression in DVA using the <i>nlp-12</i> promoter (−DA, n = 12; +DA, n = 11). Bars represent mean ±SEM. ***, p<0.0005; *, p<0.05 student's t-test. (<b>D</b>) Single slice confocal images of the DVA neuron in a transgenic animal expressing <i>nlp-12::SL2::mCherry</i> (upper panel) together with <i>Pdop-1::GFP</i> (middle panel). White arrow denotes the DVA interneuron in all cases. Asterix denotes a ventral cord motor neuron expressing the <i>dop-1</i> reporter. Scale bars in B and D, 20 µm. (<b>E</b>) Total directional reorientations measured during 0–5 and 30–35 minute intervals following removal from food for the genotypes as indicated. WT: n = 10; <i>dop-1(vs101)</i>: n = 12, <i>dop-1(vs100)</i>: n = 14, <i>dop-1(vs100) Ex DVA::dop-1</i>: n = 12 and <i>dop-3(vs106)</i>: n = 8. Bars represent mean (±SEM). For (A) and (E) ***, p<0.0005, **, p<0.005 by ANOVA with Sidak's post-hoc test.</p