The rate of endogenous IPSCs, but not EPSCs, is reduced in the absence of MEC-15.

<p>(<b>A</b>) Recordings of endogenous inhibitory postsynaptic currents (IPSCs) were done on dissected adult <i>C. elegans</i>. IPSCs were recorded at 0 mV holding potential in the presence of 1 mM extracellular Ca²⁺. Left panels show representative traces recorded from wild-type animals, <i>mec-15</i> mutants and from <i>mec-15</i> mutants expressing <i>mec-15</i> in GABAergic neurons (rescue). Right panels show mean endogenous IPSC rates and amplitudes +/− SEM (n = 20, 13, and 12 for wild-type, <i>mec-15</i> and rescue, respectively). <b>*</b>indicates p  = 0.002, Student’s t-test. (<b>B</b>) Acetylcholine release is not changed in <i>mec-15</i> mutants. Endogenous excitatory postsynaptic currents (EPSCs) were measured as in (A) but at −60 mV holding potential. Left panels show representative traces, and right panels show mean endogenous EPSC rates and amplitudes +/− SEM of adult wild-type (n = 15), <i>mec-15</i> mutants (n = 12), and <i>mec-15</i> mutants expressing <i>mec-15</i> in GABAergic neurons (rescue) (n = 6).</p

SNB-1-GFP is reduced at GABAergic synapses and accumulates in cell bodies in <i>mec-15</i> mutants.

<p>(<b>A</b>) Representative fluorescence images of SNB-1-GFP in neurites of GABAergic motor neurons in the posterior dorsal nerve cord from wild-type animals and <i>mec-15</i> mutants. <i>mec-15</i> mutants have reduced fluorescence intensity of SNB-1-GFP puncta compared to wild-type animals. Expression of <i>mec-15</i> in GABAergic neurons rescues this phenotype (rescue 2). Right panels show quantifications of puncta fluorescence and densities. Reduced puncta fluorescence of <i>mec-15</i> mutants is rescued by expressing <i>mec-15</i> in GABAergic motor neurons (two rescuing lines are shown). (<b>B</b>) Representative fluorescence images (left) and summary data (right) are shown for GFP-tagged SNB-1 in posterior dorsal cord axons of cholinergic motor neurons (expressed with the <i>unc-129</i> promoter) in wild-type and mec-15 animals. Right panels show quantifications. No significant differences were observed. (<b>C</b>) Representative fluorescence images of the GABAergic cell bodies DD5 and VD10 from wild-type animals and <i>mec-15</i> mutants expressing the synaptic vesicle protein SNB-1-GFP in GABAergic motor neurons (arrows and arrowheads point to DD5 and VD10, respectively). The right panels show quantifications. <i>mec-15</i> mutants have increased fluorescence intensity of SNB-1-GFP in cell bodies compared to wild-type animals. (<b>D</b>) Representative fluorescence images of RAB-3-GFP in neurites of GABAergic motor neurons in the posterior dorsal nerve cord from wild-type animals and <i>mec-15</i> mutants. Right panels show quantifications of puncta fluorescence and densities. Puncta fluorescence and synapse density are normal in <i>mec-15</i> mutants. (<b>E</b>) As in (D) except that GFP-tagged SYD-2 (liprin-α) was expressed. Neither puncta fluorescence nor synapse density is changed in <i>mec-15</i> mutants. All data are means +/− SEM from 20–30 images (A, B, D, E) or 15–20 cell bodies (C). **p<0.001, *p<0.01, Student’s t-test. Scale bars  = 10 µm (A, B, D, E) and 5 µm (C).</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

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

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

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

Well-Defined Poly(α-amino-δ-valerolactone) via Living Ring-Opening Polymerization

This article demonstrates the synthesis of a new kind of cationic poly­(δ-valerolactone) with primary amino groups at α-positions (poly­(α-NH₂-VL)) via ring-opening polymerization (ROP) of α-NHBoc-valerolactone (α-NHB-VL) followed by a simple deprotection reaction. The ROP of α-NHB-VL using benzyl alcohol as an initiator and DBU/TU (1,8-diazabicyclo[5.4.0]­undec-7-ene/thiourea) as a catalytic system in THF at room temperature afforded poly­(α-NHB-VL) with narrow molecular weight distribution. The ¹H NMR and MALDI-TOF MS analysis of poly­(α-NHB-VL) indicated that each polymeric chain was capped by the initiator. Kinetic experiments confirmed the living nature of the DBU/TU-catalyzed ROP of α-NHB-VL in THF. The copolymerization result indicated that the polymerization activity of α-NHB-VL is comparable to that of ε-caprolactone (CL) and VL. In addition, block copolymers containing poly­(α-NHB-VL) were successfully synthesized regardless of whether hydrophilic PEG or hydrophobic PCL was used as the macroinitiator. Moreover, water-soluble poly­(α-NH₂-VL) was obtained by treatment with trifluoroacetic acid (TFA). It was found that poly­(α-NH₂-VL) degraded more slowly at pH 5.5 than at pH 7.4 through a hydrolysis kinetics study

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