Neuropeptide processing mutations and

<p> The paralytic response to aldicarb treatment was analyzed in strains containing mutations that inactivate pro-neuropeptide processing enzymes (<i>egl-3</i> PC2 and <i>egl-21</i> CPE), or those inactivating RIC-7. The number of trials (∼20 animals/trial) is shown for each genotype. Values that differ significantly from wild type (*, p<0.01; **, p<0.001, Students t-test) are indicated. Error bars indicate SEM. Values that are not significantly different are indicated (ns).</p

Analysis of GABA transmission in <i>ric-7</i> mutants.

<p>(A) Intestinal muscle contractions during the defecation motor program (quantified as expulsions/pBoc) were analyzed in the indicated genotypes. (B) Endogenous IPSCs were recorded from adult body wall muscles of the indicated genotypes. Representative traces (left), and summary data (right) are shown. The number of animals analyzed is indicated for each genotype. (C) Representative images (left) and summary data (right) for GFP::SNB-1 (expressed by the <i>unc-25</i> promoter) in dorsal cord axons of the indicated genotypes. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type (**, p<0.001, ***, p<0.0001 Students t-test) and from <i>ric-7</i> mutants (#, p<0.05, ##, p<0.001, ###, p<0.0001 Students t-test) are indicated. Error bars indicate SEM. For rescue experiments, <i>ric-7</i> transgenes are as follows: ACh (<i>unc-17</i> promoter), GABA (<i>unc-47</i> promoter), pan-neuron (<i>snb-1</i> promoter), intestine (<i>vha-6</i> promoter).</p

Expression pattern of RIC-7.

<p>(A) The <i>ric-7</i> promoter expresses nuclear localized Cherry (HIS-24::wCherry) primarily in the nervous system of an adult worm. Anterior is left; ventral is up. The asterisk indicates fluorescence encoded by a co-injection marker. (B) The <i>ric-7</i> promoter is expressed in cholinergic (top panel) and GABAergic (bottom panel) motor neurons in the ventral cord. Cell bodies of cholinergic (<i>unc-17</i> promoter) and GABAergic (<i>unc-30</i> promoter) neurons were identified by expression of the indicated GFP reporter constructs. (C) Distribution of RIC-7, a synaptic vesicle marker (UNC-57 Endophilin) (top panel), and a DCV marker (NLP-21) (bottom panel) are compared in the dorsal cord axons of cholinergic motor neurons. (D) Distribution of RIC-7::GFP in cholinergic motor neurons of <i>unc-104</i> KIF1A mutants. Cell bodies (arrow) and ventral cord processes (arrow heads) are indicated.</p

Baseline ACh release is unaltered in <i>ric-7</i> mutants.

<p>Endogenous EPSCs (A) and stimulus-evoked EPSCs (B) were recorded from body wall muscles of wild type and <i>ric-7(nu447)</i> adults. Representative traces of endogenous EPSCs (A), averaged traces of stimulus-evoked responses (B), and summary data for both are shown. The number of animals analyzed is indicated for each genotype. No significant differences were observed. (C) Representative images (left) and summary data (right) are shown for GFP-tagged SNB-1 in dorsal cord axons of cholinergic motor neurons (expressed with the <i>unc-129</i> promoter) in wild type and <i>ric-7</i> adults. The number of animals analyzed is indicated for each genotype. No significant differences were observed. Error bars indicate SEM.</p

RIC-7 promotes neuropeptide release.

<p>YFP-tagged NLP-21 and INS-22 were expressed in cholinergic motor neurons using the <i>unc-129</i> promoter. Representative images (A) and summary data (B) are shown for NLP-21 (top) and INS-22 (bottom) fluorescence in dorsal cord axons of the indicated genotypes. The number of animals analyzed is indicated for each genotype. (C–D) Representative images (C) and summary data (D) are shown for NLP-21 and INS-22 fluorescence in coelomocytes of the indicated genotypes. The number of animals analyzed is indicated for each genotype. Values that differ significantly from wild type (**, p<0.001, ***, p<0.0001 Students t-test) and from <i>ric-7</i> mutants (#, p<0.01, ##, p<0.001, Students t-test) are indicated. Error bars indicate SEM. For rescue experiments, <i>ric-7</i> transgenes are as follows: pan-neuron (<i>snb-1</i> promoter), DA neuron (<i>unc-129</i> promoter).</p

Body muscle responses to ACh and GABA are unaltered in <i>ric-7</i> mutants.

<p>(A) Time course of levamisole (200 µM) induced paralysis is shown for wild type and <i>ric-7(nu447)</i> adults. Three trials (∼20 animals/trial) were performed for each genotype. (B) ACh (top) and Muscimol (bottom)-activated currents were recorded from body wall muscles of <i>ric-7</i> and wild type adults. Representative responses (left) and summary data (right) are shown. Wild type and <i>ric-7</i> mutant responses were not significantly different. (C–D) Representative images (above) and summary data (below) are shown for ACR-16::GFP (C) and UNC-49::GFP (D) expressed in body muscles of wild type and <i>ric-7</i> adults (using the <i>myo-3</i> promoter). No significant differences were observed. The number of animals analyzed is indicated for each genotype (panels B–D). Error bars indicate SEM.</p

ASH activity is associated with locomotion arousal.

<p>Locomotion behavior during the L4/A lethargus (A-C) and in adults (D) of single worms whose ASH neurons were ablated by transgenic overexpression of CED-3 in ASH neurons (<i>sra-6</i> promoter) was analyzed in the indicated genotypes. Animals were analyzed by fluorescence microscopy after locomotion recordings to determine if ASH neurons were ablated (1–2 ASH: animals with 1 or 2 ASH intact neurons; 0 ASH: animals lacking viable ASH neurons). Instantaneous locomotion velocity (A), average motile fraction (B), and average locomotion velocity (C-D) are plotted. The <i>npr-1</i> locomotion defect during the L4/A lethargus, but not in adults, was partially suppressed in the transgenic animals in which both of ASH neurons were ablated (0 ASH). (E-H) Copper-evoked calcium transients in ASH were analyzed in L4, L4/A, and adults of the indicated genotypes using cameleon as a calcium indicator. Averaged responses (E, G), and the amplitudes of individual trials (F, H) are shown for each genotype. Each trace represents the average percentage change in YFP/CFP fluorescence ratio. The light tan rectangle indicates the duration for which 10 mM copper was applied. Dark gray shading of each trace indicates SEM of the mean response. (E-F) Copper-evoked calcium transients in ASH neurons were significantly reduced during L4/A lethargus, and this effect was abolished in <i>npr-1</i> mutants. (G-H) This defect during L4/A lethargus was rescued by transgenes expressing NPR-1 in the RMG circuit (RMG rescue, <i>flp-21 promoter</i>) or in ASH neurons (ASH rescue, <i>sra-6 promoter</i>). (I-J) Forced depolarization of ASH neurons increased adult locomotion velocity (I) and aldicarb sensitivity (J). Rat TRPV1 was ectopically expressed in ASH neurons (using the <i>sra-6</i> promoter). (I) Locomotion behavior of adult transgenic worms was analyzed with or without capsaicin treatment (5 hours). Average locomotion velocity (I) is plotted. Capsaicin treatment increased adult locomotion velocity in transgenic animals expressing TRPV1 in ASH neurons, but not in wild type controls. The number of animals analyzed is indicated for each genotype. (J) The percentage of animals paralyzed on 1 mM aldicarb at 80 min with or without capsaicin treatment (2–3 hours pretreatment) were plotted for the indicated genotypes. The number of trials is indicated for each genotype. Full time courses for aldicarb-induced paralysis are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005359#pgen.1005359.s002" target="_blank">S2G Fig</a>. Capsaicin treatment increased aldicarb sensitivity in transgenic animals expressing TRPV1 in ASH neurons, but not in wild type controls. Error bars indicate SEM. Values that differ significantly are indicated (***, <i>p</i> <0.001; ns, not significant).</p

Cholinergic transmission at NMJs is enhanced by increased sensory activity in <i>npr-1</i> adults.

<p>Locomotion behavior of single adult worms was analyzed for the indicated genotypes. Instantaneous locomotion velocity (A) and average locomotion velocity (B-C) are plotted. (A-C) The <i>npr-1</i> adult locomotion defect was rescued by transgenes expressing NPR-1 in the RMG circuit (RMG rescue, <i>flp-21</i> promoter), and suppressed in double mutants lacking TAX-4/CNG channels. (D-F) The percentage of animals paralyzed on 1 mM aldicarb at 80 min were plotted for the indicated genotypes. The number of trials is indicated for each genotype. Full time courses of aldicarb-induced paralysis are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005359#pgen.1005359.s002" target="_blank">S2A–S2C Fig</a>. (D) The <i>npr-1</i> aldicarb hypersensitivity was rescued by transgenes expressing NPR-1 in the RMG circuit (RMG rescue, <i>flp-21</i> promoter) but not by those expressed in GABAergic neurons (GABA rescue, <i>unc-25</i> and <i>unc-30</i> promoters). (E-F) The <i>npr-1</i> aldicarb hypersensitivity was blocked by mutations inactivating TAX-4/CNG channels or OCR-2/TRPV channels. (G-L) mEPSCs were recorded from body wall muscles of adult worms for the indicated genotypes. Representative traces of mEPSCs (G and J) and summary data are shown (H, I, K, and L). (G-I) The <i>npr-1</i> cholinergic transmission defect was rescued by transgenes expressing NPR-1 in the RMG circuit (RMG rescue, <i>flp-21</i> promoter) but not by those expressed in GABAergic neurons (GABA rescue, <i>unc-30</i> promoter). (J-L) The <i>npr-1</i> cholinergic transmission defect was abolished by mutations inactivating TAX-4 or OCR-2. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly are indicated (*, <i>p</i> <0.05; **, <i>p</i> <0.01; ***, <i>p</i> <0.001; ns, not significant).</p

PDF-1 secretion is not altered in <i>glr-2</i> mutants.

<p>PDF-1 secretion was analyzed in the indicated genotypes. YFP-tagged PDF-1 was expressed with the <i>pdf-1</i> promoter. Representative images (A) and summary data (cumulative fraction) (B-C) are shown for coelomocyte fluorescence in L4/A lethargus and 1-day old adults of the indicated genotypes. PDF-1::YFP coelomocyte fluorescence was dramatically increased in <i>npr-1</i> mutants during the L4/A lethargus and in adults as previously reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005359#pgen.1005359.ref011" target="_blank">11</a>]. Mutations inactivating GLR-2 did not alter PDF-1::YFP coelomocyte fluorescence during L4/A lethargus (B) and in adults (C) in either wild type or <i>npr-1</i> mutants. Scale bar indicates 10 μm. <i>p</i> values are indicated for each comparison (Kolmogorov-Smirnov test).</p

Glutamate released by sensory neurons is required for the <i>npr-1</i> locomotion and the cholinergic transmission defects.

<p>Locomotion behavior of single worms during the L4/A lethargus (A-C) and in adults (D-E) was analyzed in the indicated genotypes. Instantaneous locomotion velocity (A, D), average motile fraction (B), and average locomotion velocity (C, E) are plotted. The <i>npr-1</i> locomotion defect was suppressed by mutations inactivating EAT-4/VGLUT, and partially reinstated by transgenes expressing EAT-4 in ASH neurons (<i>sra-6</i> promoter) and touch neurons (<i>mec-4</i> promoter) in <i>eat-4;npr-1</i> double mutants using the indicated promoters. An EAT-4 transgene expressed in ASK neurons (<i>sra-9</i> promoter) lacked rescuing activity. (F) The <i>npr-1</i> aldicarb hypersensitivity was suppressed by mutations inactivating EAT-4/VGLUT. The percentage of animals paralyzed on 1 mM aldicarb at 80 min were plotted for the indicated genotypes. The number of trials is indicated for each genotype. Full time courses of aldicarb-inuced paralysis are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005359#pgen.1005359.s002" target="_blank">S2F Fig</a>. (G-I) The <i>npr-1</i> cholinergic transmission defect was abolished by mutations inactivating EAT-4/VGLUT. mEPSCs were recorded from body wall muscles of adult worms for the indicated genotypes. Representative traces of mEPSCs (G) and summary data are shown (H-I). The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly are indicated (*, <i>p</i> <0.05; **, <i>p</i> <0.01; ***, <i>p</i> <0.001; ns, not significant).</p