RIC-7 Promotes Neuropeptide Secretion

Secretion of neurotransmitters and neuropeptides is mediated by exocytosis of distinct secretory organelles, synaptic vesicles (SVs) and dense core vesicles (DCVs) respectively. Relatively little is known about factors that differentially regulate SV and DCV secretion. Here we identify a novel protein RIC-7 that is required for neuropeptide secretion in Caenorhabditis elegans. The RIC-7 protein is expressed in all neurons and is localized to presynaptic terminals. Imaging, electrophysiology, and behavioral analysis of ric-7 mutants indicates that acetylcholine release occurs normally, while neuropeptide release is significantly decreased. These results suggest that RIC-7 promotes DCV–mediated secretion

An Aversive Response to Osmotic Upshift in Caenorhabditis elegans

Abstract Environmental osmolarity presents a common type of sensory stimulus to animals. While behavioral responses to osmotic changes are important for maintaining a stable intracellular osmolarity, the underlying mechanisms are not fully understood. In the natural habitat of Caenorhabditis elegans, changes in environmental osmolarity are commonplace. It is known that the nematode acutely avoids shocks of extremely high osmolarity. Here, we show that C. elegans also generates gradually increased aversion of mild upshifts in environmental osmolarity. Different from an acute avoidance of osmotic shocks that depends on the function of a transient receptor potential vanilloid channel, the slow aversion to osmotic upshifts requires the cGMP-gated sensory channel subunit TAX-2. TAX-2 acts in several sensory neurons that are exposed to body fluid to generate the aversive response through a motor network that underlies navigation. Osmotic upshifts activate the body cavity sensory neuron URX, which is known to induce aversion upon activation. Together, our results characterize the molecular and cellular mechanisms underlying a novel sensorimotor response to osmotic stimuli and reveal that C. elegans engages different behaviors and the underlying mechanisms to regulate responses to extracellular osmolarity

Supplemental Material for Zhao, Hao, and Kaplan, 2018

Supplemental Figure

Confidence-based iterative efficient large-scale stereo matching

In this study, we integrate confidence into efficient large-scale stereo (ELAS) matching to produce a more accurate approach to binocular stereo for high-resolution image matching. Elas ensures good performance in the presence of poorly textured and slanted surfaces, but one of its deficiencies is its unsatisfactory ability to capture disparity discontinuities. Our formulation explicitly models the effects of confidence as a likelihood term in a principled manner using the Bayes rule. Because it is an iterative method, we associate each point with a variable confidence value and update this value based on a given confidence updating rule. Meanwhile, complementary support points are selected from stable points whose confidence value exceeds a predefined threshold, which differs from ELAS, whose support points are matched in advance and kept unchanged in the subsequent process. Confidence also plays a vital role in avoiding expensive computation, and the adjustment of support points makes disparity estimation more flexible. Quantitative evaluation demonstrates the effectiveness and efficiency of the proposed formulation in improving the accuracy of disparity estimation

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

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

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