TA modulates ASI neuropeptide release.

<p>(A) Wild-type animals expressing <i>Pgpa-4</i>::<i>gfp</i> were incubated with either TA or OA and analyzed for GFP fluorescence. (B) Quantitative PCR of several ASI neuropeptide-encoding genes and <i>gpa-4</i> in wild-type animals. (C-D) Animals expressing ASI::<i>nlp-14</i>::<i>gfp</i> or ASI::<i>nlp-18</i>::<i>gfp</i> transgenes in their respective null backgrounds were incubated with either TA or OA and analyzed for GFP fluorescence. (C) ASI::<i>nlp-14</i>::<i>gfp</i> in <i>nlp-14</i> or <i>nlp-14;tyra-3</i> null animals. (D) ASI::<i>nlp-18</i>::<i>gfp</i> in <i>nlp-18</i> null animals. The <i>gpa-4</i> promoter was used to drive ASI expression. All monoamine concentrations were 10 mM. * Denotes significantly different from control (<i>p ≤</i> 0.05). Data are presented as a mean ± SE (<i>n</i>) and were analyzed by One-way ANOVA (4A <i>n</i> = 31–33. 4B <i>n</i> = 9. 4C <i>n</i> = 22–28 One-way ANOVA: Total = <i>p</i> 0.020, Dunnett <i>t</i> (2-sided) = <i>p</i> 0.037, Axon = <i>p</i> 0.006, Dunnett T3 = <i>p</i> 0.011, Synaptic = <i>p</i> 0.006, Dunnett T3 = <i>p</i> 0.012. 4D <i>n</i> = 17–45 One-way ANOVA: Total = <i>p</i> 0.045, Dunnett <i>t</i> (2-sided) = <i>p</i> 0.025, Soma = <i>p</i> 0.035, Dunnett <i>t</i> (2-sided) = <i>p</i> 0.019).</p

Model of monoamine-specific ASI neuropeptide release.

<p>Neuropeptides encoded by <i>nlp-9</i>, <i>-14</i> and <i>-18</i> are expressed in the soma and predicted synaptic/perisynaptic and extra-synaptic sites in both ASI neurons. TA selectively stimulates the release of NLP-18::GFP from ASI soma and NLP-14::GFP from synaptic/presynaptic regions of both the right and left ASI axons. In contrast, OA selectively stimulates the asymmetric release of NLP-9::GFP from only synaptic/perisynaptic regions in the right ASI axon.</p

TA- and OA-dependent neuropeptides co-localize at ASI synapses.

<p>(A) Schematic representation of an ASI neuron highlighting the predicted synaptic/perisynaptic and extra-synaptic sites. (B-D) Translational neuropeptide fusions (ASI::<i>nlp-14</i>::<i>gfp</i>, ASI::<i>nlp-18</i>::<i>gfp</i> or ASI::<i>nlp-9</i>::<i>gfp)</i> expressed in their respective null backgrounds in the left ASI sensory neurons driven by the ASI-specific promoter <i>gpa-4</i>. Anterior is to the left and dorsal is up. (E) Straightened axons of <i>nlp-9</i> null animals co-expressing <i>gpa-4</i>::<i>nlp-9</i>::<i>gfp</i> and <i>srg-47</i>::<i>mCherry</i>::<i>rab-3</i> transgenes. Circles indicate the sites of predicted ASI synapses based on electron microscopy. Arrowheads denote potential additional extra-synaptic sites of release. (F) Straightened axons of an animal co-expressing <i>gpa-4</i>::<i>nlp-14</i>::<i>gfp</i> and <i>srg-47</i>::<i>nlp-9</i>::<i>mCherry</i> transgenes. (G) Extra-synaptic/synaptic ratios of ASI::<i>nlp-14</i>::<i>gfp</i> or ASI::<i>nlp-9</i>::<i>gfp</i> expressed in their respective null backgrounds. * Denotes significantly different to <i>nlp-14</i> (<i>p</i> = 0.0001). Data are presented as a mean ±SE (<i>n</i>) and were analyzed by two-tailed Student’s <i>t</i> test (<i>n</i> = 28–55).</p

Monoamines differentially modulate neuropeptide release from distinct sites within a single neuron pair

<div><p>Monoamines and neuropeptides often modulate the same behavior, but monoaminergic-peptidergic crosstalk remains poorly understood. In <i>Caenorhabditis elegans</i>, the adrenergic-like ligands, tyramine (TA) and octopamine (OA) require distinct subsets of neuropeptides in the two ASI sensory neurons to inhibit nociception. TA selectively increases the release of ASI neuropeptides encoded by <i>nlp-14</i> or <i>nlp-18</i> from either synaptic/perisynaptic regions of ASI axons or the ASI soma, respectively, and OA selectively increases the release of ASI neuropeptides encoded by <i>nlp-9</i> asymmetrically, from only the synaptic/perisynaptic region of the right ASI axon. The predicted amino acid preprosequences of genes encoding either TA- or OA-dependent neuropeptides differed markedly. However, these distinct preprosequences were not sufficient to confer monoamine-specificity and additional N-terminal peptide-encoding sequence was required. Collectively, our results demonstrate that TA and OA specifically and differentially modulate the release of distinct subsets of neuropeptides from different subcellular sites within the ASIs, highlighting the complexity of monoaminergic/peptidergic modulation, even in animals with a relatively simple nervous system.</p></div

<i>C</i>. <i>elegans</i> mutants with increased cuticular permeability are hypersensitive to 5-HT-dependent paralysis.

<p><b>A-B.</b> Paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on NGM agar plates. <b>A.</b> Wild type animals examined for 5-HT-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3). <b>B.</b> Dose-response curves for 5-HT-dependent paralysis on NGM plates at 10 min exposure for wild type and 5-HT <i>quint</i> animals. <b>C-D.</b> Paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on non-NGM agar (hypotonic) plates. <b>C.</b> Wild type animals were examined for 5-HT-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3). <b>D.</b> Dose-response curves for 5-HT-dependent paralysis in hypotonic conditions at 15 min exposure for wild type and 5-HT <i>quint</i> animals. <b>E-F.</b> 5-HT-dependent paralysis of wild type and mutant <i>C</i>. <i>elegans</i> on NGM agar plates. <b>E.</b> 5-HT (0.25 mM)-dependent paralysis of wild-type, <i>bus-8</i> (<i>e2968</i>), <i>bus-16</i> (<i>e2802</i>) and <i>bus-17</i> (<i>e2800</i>) mutants. Data are presented as mean ± SE (n = 3). <b>F.</b> Dose-response curves for 5-HT-dependent paralysis at 10 min exposure for wild type and <i>bus</i> mutants.</p

PAPP paralyzes <i>C</i>. <i>elegans</i> via SER-4 and DOP-3.

<p><b>A-C.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic non-NGM agar plates. <b>A.</b> PAPP (0.5 mM)-dependent paralysis of wild-type, 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing SER-4 in the cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>B.</b> Dose-response curves for PAPP-dependent paralysis at 15 min exposure for wild type, 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing SER-4 in the cholinergic motor neurons (P<i>unc-17β</i>). <b>C.</b> PAPP (0.5 mM)-dependent paralysis of 5-HT <i>quint</i> and 5-HT <i>quint</i> animals expressing P<i>dop-3</i>::<i>dop-3</i> RNAi. Data are presented as mean ± SE (n = 3). ‘*’ p≤0.001, significantly different from 5-HT <i>quint</i> animals assayed under identical conditions.</p

5-HT and 5-HT receptor agonists selectively paralyze <i>C</i>. <i>elegans</i> 5-HT receptor mutant animals expressing nematode, insect or human 5-HT₁-like receptors in the cholinergic motor neurons.

<p><b>A-C.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic, non-NGM agar plates. <b>A.</b> 5-HT (1 mM)-dependent paralysis of 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>B.</b> 8-OH-DPAT (2 mM)-dependent paralysis of 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3). <b>C.</b> Sumatriptan (1 mM)-dependent paralysis of wild type, 5-HT <i>quint</i> animals expressing either <i>C</i>. <i>elegans</i> 5-HT₁-like (SER-4), <i>Drosophila</i> 5-HT₁-like, or human 5-HT_1A receptor in cholinergic motor neurons (P<i>unc-17β</i>). Data are presented as mean ± SE (n = 3).</p

The 5-HT/SER-4-dependent inhibition of either the AIB interneurons or cholinergic motor neurons causes locomotory paralysis.

<p><b>A.</b> Confocal images of 5-HT <i>quint</i> expressing SER-4::GFP in the AIB interneurons (P<i>npr-9</i>)(A1) or cholinergic motor neurons (P<i>unc-17β</i>)(A2). GFP fluorescence (A2) or GFP fluorescence overlaid on DIC image (A1). The red stain in A2 is coelomocyte-specific RFP screening marker. <b>B.</b> Paralysis of wild type, mutant and transgenic <i>C</i>. <i>elegans</i> on hypotonic, non-NGM agar plates. Wild type, quadruple 5-HT receptor null animals expressing only SER-4 (SER-4 <i>quad</i>) or 5-HT <i>quint</i> expressing the <i>C</i>. <i>elegans</i> 5-HT₁-like receptor, SER-4, in either the cholinergic motor neurons (P<i>unc-17β</i>) or the two AIB interneurons (P<i>npr-9</i>) were examined for 5-HT (1 mM)-dependent paralysis as outlined in Methods. Data are presented as mean ± SE (n = 3).</p

The two ASI sensory neurons integrate both monoaminergic and peptidergic signaling to modulate nociceptive behavior.

<p>The two ASI sensory neurons express Gα_q-coupled TA, OA and 5-HT receptors that differentially modulate neuropeptide release and, in turn, differentially modulate aversive responses mediated primarily by the two ASH sensory neurons. For example: 1) the ASI OA receptor, SER-6, and neuropeptides encoded by <i>nlp-6</i>, <i>-7</i> and <i>-9</i> are essential for the OA inhibition of aversive responses to 100% 1-octanol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196954#pone.0196954.ref006" target="_blank">6</a>], 2) the ASI TA receptor, TYRA-3, and neuropeptides encoded by <i>nlp-1</i>, <i>-14</i> and <i>-18</i> are essential to inhibit the 5-HT stimulation of aversive responses to 30% 1-octanol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196954#pone.0196954.ref011" target="_blank">11</a>], and 3) the ASI 5-HT receptor, SER-1, appears to stimulate the release of neuropeptides encoded by <i>nlp-24</i> that activate the Gα_o-coupled ASI opiate receptor, NPR-17, to interfere with TYRA-3 signaling in response to 30% 1-octanol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196954#pone.0196954.ref005" target="_blank">5</a>].</p

Truncation of the ASI::NLP-14 preproprotein does not alter TA and OA sensitivity.

<p>(A) Amino acid sequence for the full length NLP-14 preproprotein. (B) Amino acid sequence for the truncated NLP-14. Bold denotes a dibasic cleavage site, blue denotes putative neuropeptides, red denotes neuropeptides confirmed by either Edman degradation, Matrix Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry, or Quadrupole-Time of Flight Mass Spectrometry, and GFP sequence is underlined green. Amino acid sequences adapted from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196954#pone.0196954.ref021" target="_blank">21</a>]. (C) Animals expressing the truncated ASI::<i>nlp-14</i>_{<i>(1–95)</i>}::<i>gfp</i> transgene in an <i>nlp-14</i> null background were incubated with either TA or OA and analyzed for GFP fluorescence. The <i>gpa-4</i> promoter was used to drive ASI expression. * Denotes significantly different from control (<i>p ≤</i> 0.05). Data are presented as a mean ± SE (<i>n</i>) and were analyzed by One-way ANOVA (7C <i>n</i> = 22–42, One-way ANOVA: Native NLP-14: <i>p =</i> 0.012, Dunnett T3 = <i>p</i> 0.025, NLP-14_(1–95) <i>p =</i> 0.022, Dunnett T3 = <i>p</i> 0.012).</p