Figure 1.
Neuropeptide processing and release pathway regulate acr-2(gf) convulsions.
All mutations are loss of function alleles, except for acr-2(gf), which designates acr-2(n2420). Mean convulsion frequencies are shown. Error bars indicate SEM. Numbers in the graph indicate sample sizes. Statistics: ***: p<0.001, **: p<0.01, *: p<0.05 by ANOVA and Bonferroni post hoc test. (A) Loss of function in egl-3 and sbt-1 significantly enhances acr-2(gf) convulsions; and the increased convulsion caused by egl-3(lf) is dependent on unc-31. (B) egl-3 functions in the cholinergic motor neurons to suppress acr-2(gf) convulsions. The number of independent transgenic lines tested are the following: Prgef-1::egl-3; 4 lines, Punc-17β::egl-3; 3 lines, Pglr-1::egl-3; 3 lines, Pacr-2; 2 lines. Quantification data is shown for one representative line.
Figure 2.
Loss of both flp-1 and flp-18 enhances acr-2(gf) convulsions.
Null mutants of candidate neuropeptide genes were tested for effects on acr-2(gf) convulsions. flp-18(lf) indicates flp-18(tm2179); the allele number for other genes are listed in materials and methods. No significant effects were observed for selected FMRF-amide (flp) (A), neuropeptide like proteins (nls) (B), or insulins (ins) (C). (D) Double mutants of candidate peptide genes with flp-18. Loss of both flp-1 and flp-18 leads to a significant enhancement of acr-2(gf) convulsions. Numbers in the graph indicate sample sizes. Mean convulsion frequencies are shown. Error bars indicate SEM. Statistics: *: p<0.05 by ANOVA and Dunnett's post hoc test.
Figure 3.
flp-1 and flp-18 act as inhibitory neuropeptides in the acr-2(gf) background.
(A) Convulsion frequency of acr-2(gf) in combination with loss of function (lf) mutations in flp-1(yn4), flp-18(tm2179), or flp-18(db99). The enhanced convulsion frequency of flp-1(lf); flp-18(lf) acr-2(gf) animals is rescued with transgenic expression of flp-1 under the pan-neuronal promoter Prgef-1. Two independent transgenic lines were tested as indicated by line 1 and 2. Mean convulsion frequencies are shown. Error bars indicate SEM. Numbers in the graph indicate sample sizes. Statistics: ***: p<0.001, **: p<0.01 by ANOVA and Dunnett's post-hoc test. (B, C) Rate of paralysis on 150 µM aldicarb plates in acr-2(gf) background. flp-1(lf); flp-18(lf) mutants in the acr-2(gf) background showed enhanced aldicarb sensitivity compared to acr-2(gf) (B). Pan-neuronal expression of flp-1 rescues the increased aldicarb sensitivity of the flp-1(lf); flp-18(lf) acr-2(gf) mutants (C). n = 10 for one group per trial; and results of three to five independent trials are shown. Mean rate of paralysis are shown for each time point. Error bars indicate SEM. Two independent transgenic lines were tested, only one is shown in the graph. Statistics in B, C: **: p<0.01, *: p<0.05 by two-way ANOVA and Bonferroni post-hoc test.
Figure 4.
Neuropeptide modulation primarily affects GABAergic neuromuscular transmission.
(A) Shown are the electrophysiology recording data on the neuromuscular junctions. Top panels are representative traces of genotypes indicated. Middle panels are mean rates of endogenous EPSCs and IPSCs; and bottom panels are cumulative fractions of the animal number with endogenous EPSC rate or IPSC rate less than indicated values in X-axis of genotype indicated. (B) Mean amplitudes of endogenous EPSCs and IPSCs from genotypes shown in A. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Statistics, two-tailed t-test, ***, p<0.001; *, p<0.05.
Figure 5.
FLP-18 expression is selectively increased in the cholinergic motor neurons in the acr-2(gf) background.
(A–B) Ventral nerve cord expression of Pflp-18::flp-18::SL2::gfp in wild type (N2) and acr-2(gf) background, respectively. Increased fluorescence intensity and cell body expression is seen in the ventral cord in the acr-2(gf) background. Arrows point to the ventral nerve cord posterior to the vulva, and arrowheads point to cell bodies. Scale bar = 25 µm. Two Pflp-18::flp-18::SL2::gfp transgenic lines, juEx4062 and juEx4073, were tested. Images from juEx4073 are shown. (C) Quantification of average fluorescence intensity in the ventral nerve cord posterior to the vulva. Mean fluorescence intensities are shown. N = 37 (wild type), = 38 (acr-2(gf)). ***: p<0.001 by student's t-test. Error bars indicate SEM. (D) Quantification of the number of cell bodies in the ventral cord with visible GFP expression. ***: p<0.001, **: p<0.01 by Student's t-test. Each dot indicates quantification from one animal. Means are indicated by lines. Error bars indicate SEM. Two transgenic lines were tested. (E–H) Identification of the cells showing up-regulation of Pflp-18::flp-18::SL2::gfp in the acr-2(gf) background. (E) Co-expressing mCherry in GABAergic (Pttr-39) motor neurons did not show overlap with Pflp-18::flp-18::SL2::gfp. (F) Expression of mCherry in B-type (Pacr-5) cholinergic motor neurons overlapped extensively with Pflp-18::flp-18:SL2::gfp expression. (G) mCherry expression in A-type (Punc-4) cholinergic motor neurons mostly did not overlap with Pflp-18::flp-18::SL2::gfp expression. (H) Quantification of the number of cell bodies that showed overlapping expression of Pflp-18::flp-18::SL2::gfp and Pacr-5::mCherry or Punc-4::mCherry in F–G. Each dot indicates quantification from one animal. Means are indicated by lines. Error bars indicate SEM.
Figure 6.
Induced expression of FLP-18 in acr-2(gf) correlates with the onset of convulsions, and high levels of FLP-18 or FLP-1 suppress convulsions.
(A) Quantification of the number of cell bodies in the ventral cord that showed Pflp-18::flp-18::SL2::gfp expression in larval and adult stages. Each dot indicates quantification from one animal. Means are indicated by lines. Error bars indicate SEM. Two independent lines juEx4062 and juEx4073 were tested. Result from juEx4073 is shown. (B) Convulsion of acr-2(gf) was suppressed by expression of Pflp-18::flp-18::SL2::gfp or pan-neuronal expression of flp-1. The suppression by flp-18 overexpression was blocked by loss of both npr-1 and npr-5, or npr-4 and npr-5. The same set of npr mutations did not affect the suppression effect of flp-1 overexpression. Mean convulsion frequencies are shown. Error bars indicate SEM. Statistics: ***: p<0.001, *: p<0.05 by ANOVA and Dunnett's post-hoc test. (+/+) indicates strains with no mutations in any of the neuropeptide receptor genes.
Figure 7.
NPR-1, NPR-4, and NPR-5 act together to mediate the effects of neuropeptides on convulsions.
(A–B). Convulsion frequencies of acr-2(gf) combined with loss of function mutations in npr-1(ok1447), npr-4(tm1782), npr-5(ok1583) (A) and with flp-1(yn4) (B). (C) Convulsion frequency of animals with cell type-specific expression of npr-1 and npr-5. npr-5 expression in the muscle rescued the increased convulsion frequency of npr-5(lf); npr-1(lf) acr-2(gf) triple mutant; two independent lines were tested. npr-1 expression in GABAergic motor neurons did not significantly rescue the increased convulsion frequency; three lines were tested. All strains contain acr-2(gf). Mean convulsion frequencies are shown. Error bars indicate SEM. Statistics: ***: p<0.001, *: p<0.05 by ANOVA and Bonferroni post hoc test. (+/+) indicates strain with no mutations in any of the neuropeptide receptor genes.