(A) Confocal image of the posterior ventral nerve cord of an adult animal co-expressing a GABA-specific marker (Punc-47∷mCherry) and an ACR-2 specific marker (Pacr-2∷GFP). No overlap is observed between GFP and mCherry expressing neurons. The animal is oriented with the posterior (tail) on the right. (B) Quantification of movement on a food-free agar plate. Average number of body bends per minute for wild type, acr-2(ok1887) mutants and acr-2(ok1887) mutants expressing full-length ACR-2∷GFP (ufIs42) over a five-minute period are shown. Data represents mean ± SEM of at least 10 trials. ** denotes significance of p<0.01 compared to wild type. (C) Time course of paralysis in the presence of the cholinesterase inhibitor aldicarb (1 mM) for wild type, acr-2(ok1887) mutants and acr-2(ok1887) mutants expressing full-length ACR-2∷GFP (ufIs42). The percentage of immobilized animals calculated every 15 minutes over a time course of 2 hours is shown. Each data point represents the mean ± SEM of at least 4 trials. * denotes significance of p<0.001 (2-way ANOVA).

(A and B) Still image of a wild type animal (A) and a transgenic animal expressing the ACR-2(L/S) transgene (B). Note the coiled posture and reduced size that occurs as a result of ACR-2(L/S) expression. (C) Quantification of movement on a food-free agar plate. Average number of body bends per minute for wild type, acr-2(ok1887) mutants and transgenic animals expressing full-length ACR-2(L/S) (ufIs25) counted over a five-minute period are shown. Data represents mean ± SEM of at least 10 trials. * and ** denote significance of p<0.02 and p<0.0001 respectively.

(A and A₁) DIC images of an adult wild type animal (A) and an adult transgenic animal expressing ACR-2(L/S) (A₁). Asterisks denote lesions observed along the ventral nerve cord transgenic animals expressing ACR-2(L/S). Images show a region directly posterior of the vulva and are oriented with the ventral surface facing up and the anterior of the animal to the left. (B and B₁) Wide-field epifluorescent images of a transgenic animal expressing the cholinergic neuron marker Punc-17∷GFP (vsIs48) (B) and a transgenic animal coexpressing ACR-2(L/S) with Punc-17∷GFP (B₁). The few Punc-17∷GFP labeled motor neurons that remain in transgenic ACR-2(L/S) animals include the 6 VCs (indicated) and a more variable group of approximately 10–12 neurons (arrowheads). (C and C₁) Wide-field epifluorescent images of a transgenic animal expressing Punc-47∷mCherry (C) and a transgenic animal coexpressing Punc-47∷mCherry with ACR-2(L/S) (C₁). The full complement of Punc-47∷mCherry labeled motor neurons remains in transgenic ACR-2(L/S) animals and are indicated. (E) Quantification of the total number of motor neurons in wild type (gray) and ACR-2(L/S) animals (black). (***P<.0001). For all images, animals are positioned with the head on the left side of the image.

(A) Wide-field epifluorescent images of wild type and transgenic ACR-2(L/S) animals expressing Punc-17∷GFP imaged 16, 28, 38 and 48 hours after bleach synchronization of embryos. (B) Confocal images of first larval stage (L1) wild type and transgenic ACR-2(L/S) animals expressing Punc-17∷GFP. Swollen or dying neuronal cell bodies are indicated (arrowhead). (C) Quantification of the average number of cell bodies at the time points indicated for wild type (black bars) and transgenic ACR-2(L/S) animals (gray bars). Bars represent the mean ± SEM for 5–8 animals at each time point.

(A) Quantification of the average number of body bends/minute for wild type animals, acr-12(ok367), unc-63(ok1075), unc-38(e264), unc-74(e883) and unc-50(e306) mutants in the absence (gray) or presence (black bars) of the ACR-2(L/S) transgene. Animals were placed on a food-free agar plate and the average number of body bends/minute was quantified over a five-minute period. Data represent the mean ± SEM of at least 10 animals for each genotype. (B) Schematic of the membrane topology of ACR-12 with approximate location, allele names and molecular nature of loss-of-function mutations that suppress ACR-2(L/S) toxicity indicated. (C) Quantification of the average number of body bends/minute for the following genotypes: wild type, acr-12(ok367), transgenic ACR-2(L/S), acr-12 mutants expressing ACR-2(L/S), acr-12 mutants expressing ACR-2(L/S) together with an extrachromosomal array containing Punc-47∷ACR-12, and acr-12 mutants expressing ACR-2(L/S) together with an extrachromosomal array containing the Pacr-2∷ACR-12 cDNA. Data represent the mean ± SEM for 5–10 animals. (D–H) Still images of adult animals on NGM plates without food for the genotypes indicated. “GABA” and “ACh” refer to specific expression of the acr-12 cDNA in GABAergic or cholinergic neurons using the unc-47 or acr-2 promoters, respectively.

(A–F) Representative wide-field images of Punc-17∷GFP fluorescence in the ventral nerve cord of adult animals for the genotypes indicated. For each image, the head is oriented to the left. The alleles used were ced-3(ok2734), cnx-1(ok2234) and crt-1(ok948). (G) Quantification of the average number of Punc-17∷GFP labeled cell bodies present in the ventral nerve cord for the genotypes indicated. Data represent the mean ± SEM for at least 10 animals/genotype. * and ** denote significance of at least p<.01 compared to transgenic ACR-2(L/S) animals.

(A and B) Frames showing the movement of L1 transgenic ACR-2(L/S) (A) or cnx-1;crt-1;ACR-2(L/S) (B) animals 30s (t=0), 60s (+30), or 90s (+60) after transfer to an agar plate. The black arrow marks the starting positions of the worms in each frame. The transgenic ACR-2(L/S) animal does not move from where it was placed on the plate. The dashed line shows the movement path of the L1 cnx-1;crt-1;ACR-2(L/S) animal over the course of 60s. The additional tracks on the plate show the path of the animal in an approximately 30s period before imaging began. (C and D) Confocal images of a first larval stage (L1) (C) or adult cnx-1;crt-1;ACR-2(L/S) animals expressing an integrated Punc-17∷GFP (D). Images show Z-projections of 15 confocal planes (0.5μm/slice) (C) or 16 confocal planes (0.5 μm/slice)(D). Arrows (D) indicate positions of commissures. Dashed box (D) indicates area with multiple neuronal defects. (E–G) Confocal images of cnx-1;crt-1;ACR-2(L/S) animals taken at hatch (E), 24 hours post hatch (F) and adulthood (G). In each case, a region immediately posterior of the vulva was imaged and the ventral nerve cord is positioned at the top. Arrows indicate commissural processes, arrowheads indicate the ventral nerve cord. Images show Z-projections of 12 confocal planes (E), 23 confocal planes (F) or 33 confocal planes (G)(0.5 μm/slice). Scale bars represent 10 μm. (H) Quantification of percent of animals moving at hatch in ACR-2(L/S) and cnx-1;crt-1;ACR-2(L/S) animals. Data represent the mean number of animals ± SEM making more than two consecutive body bends during a three minute period. (I) Quantification of the percent of cnx-1;crt-1 and cnx-1;crt-1;ACR-2(L/S) animals with defective neuronal process morphology. Observed defects include defasciculation of the ventral nerve cord, wandering commissural processes or ectopic branching.

(A–C) Quantification of the average number of Punc-17∷GFP labeled cell bodies present in the ventral nerve cord in adult animals treated with dantrolene (A), L1 animals treated with EGTA (B) and adult unc-68(e540) mutant animals (C). Data represent the mean ± SEM for at least eight animals. ** and *** denote significance of at least p<0.01 compared to untreated ACR-2(L/S) animals. (D and E) Confocal images of adult wild type (D) or cnx-1;crt-1 (E) animals expressing ACR-2∷GFP. Images are taken immediately posterior to the vulva and show Z-projections of seven confocal planes (0.5 μm/slice). (F and G) Quantification of fluorescence levels in cell bodies (F) and ventral nerve cord processes (G) of wild type and ACR-2(L/S) animals transgenically expressing ACR-2∷GFP. Data represent the mean ± SEM for at least 10 animals. *** denotes significance of p<0.01 compared to ACR-2(L/S) animals.