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. 2018 Mar 12;14(3):e1007263. doi: 10.1371/journal.pgen.1007263

Fig 4. casy-1 mutants show decreased GABAergic synaptic transmission at the NMJ.

Fig 4

(A) Expression of the CASY-1C specifically in DD and VD neuronal subtypes using GABAergic motor neuron specific promoter (Punc-30) establish that CASY-1 is explicitly functioning in GABAergic motor neurons to regulate synaptic transmission. Assays were done 3 times as indicated in Figures (~20 C. elegans/ assay). Data are represented as mean ± S.E.M. (***P<0.0001 using one-way ANOVA and Bonferroni's Multiple Comparison Test). “ns” indicates not significant in all Figures. (B) Optogenetic stimulation of GABAergic neurons using Channelrhodopsin (ChR2) [zxIs3 (unc-47p::ChR2(H134R)::YFP)] showed that the casy-1 mutant animals relax significantly less than WT C. elegans upon blue light exposure (percent change in body length before and after optogenetic stimulation). Expressing the CASY-1C isoform under a GABAergic neuron specific promoter (Punc-25) completely rescues the relaxation defect in casy-1 mutant animals. Data is shown for two independent rescue lines. The graph shows the percentage change in body length for both +ATR and–ATR controls. The numbers of animals analyzed for each genotype are indicated. Data are represented as mean ± S.E.M. (*p<0.01 using one-way ANOVA and Bonferroni's Multiple Comparison Test). “ns” indicates not significant in all Figures. (C) mIPSCs were recorded from body wall muscles of adult animals for the indicated genotypes. Representative traces of mIPSCs and summary data for frequency and amplitude are shown. The casy-1 mutants showed a significant decrease in mIPSCs rate compared to WT C. elegans, suggesting a decreased GABAergic neurotransmission at the NMJ. The mIPSC amplitude, however, remains unaltered suggesting normal muscle responsiveness in the mutant. The decreased mIPSCs rate of casy-1 mutants can be significantly rescued by expressing CASY-1C specifically in GABA motor neurons (Punc-25). (D) Depicts traces and quantified data for the mEPSCs recorded from the C. elegans body-wall muscles. There is a subtle but significant increase in mEPSC frequency in casy-1 mutants, and this defect was not rescued by expressing CASY-1C in GABA motor neurons. For both C and D, the number of animals analyzed for each genotype is indicated. Data are represented as mean ± S.E.M. *p<0.05 with respect to WT and # p<0.05 with respect to casy-1 mutants, using the one-way ANOVA with Dunnett’s post test).