The sexually dimorphic cholinergic transmission is controlled by the sex identities of the entire nervous system. (A) Schematic for construction of the tra-2 (C15F1.3) conditional deletion lines. Two loxP sites were inserted into the 20th intron and the second loxP downstream the 3′ UTR using CRISPR/Cas9, the minigene encoding Cre recombinase under rgef-1, unc-4, and unc-25 promoters were used to knock out tra-2 gene in pan-neurons, cholinergic motor neurons, and GABAergic motor neurons. (B) Time course analysis of 1.4 mM aldicarb-induced paralysis in tra-2 conditional knockout hermaphrodites (Herm.) and males (Male). Knock out tra-2 gene in all neurons, cholinergic neurons, and GABAergic neurons to get pan-neuronal masculinized (Pan-neuronal masc.), Cholinergic neuronal masculinized (Cholinergic masc.), and GABAergic neuronal masculinized (GABAergic masc.) hermaphrodites. Transgenic animals with two loxP sites insertion but without Cre recombinase expression were used as controls (Control 1). (C) Locomotion behavior analysis of a single adult tra-2 conditional knockout hermaphrodite (Herm.) and a male (Male). The averaged and individual crawling locomotion velocities were plotted. (D and E) Endogenous acetylcholine transmission was assessed by recording mEPSCs from body-wall muscles of tra-2 conditional knockout hermaphrodites. Representative mEPSC traces (D), the mean mEPSC frequencies (E, top panel), and the mean mEPSC amplitudes (E, bottom panel) are shown. Transgenic animals with two loxP sites insertion but without Cre recombinase expression were used as control 1, and transgenic animals with pan-neuronal Cre recombinase expression, but without two loxP sites insertion were used as control 2. The data for WT are the same as in Fig. 1, H and I. (F) The puncta fluorescence intensities and densities, marked by the cholinergic synaptic UNC-43::split GFP in dorsal nerve cord axons in tra-2 conditional knockout hermaphrodites. Representative images (top panel, scale bar, 10 μm), mean puncta intensity (middle panel, and density (bottom panel) are shown. In B, for each of the group, n = 3 biologically independent samples; each sample contains ≥25 animals. Data are presented as mean values ± SEM. Two-way ANOVA comparing all of the time points. In C, E, and F, data are presented as mean values ± SEM. Ns represent the number of animals tested. One-way ANOVA with post-hoc Bonferroni’s multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.
Figure 8.

The sexually dimorphic cholinergic transmission is controlled by the sex identities of the entire nervous system. (A) Schematic for construction of the tra-2 (C15F1.3) conditional deletion lines. Two loxP sites were inserted into the 20th intron and the second loxP downstream the 3′ UTR using CRISPR/Cas9, the minigene encoding Cre recombinase under rgef-1, unc-4, and unc-25 promoters were used to knock out tra-2 gene in pan-neurons, cholinergic motor neurons, and GABAergic motor neurons. (B) Time course analysis of 1.4 mM aldicarb-induced paralysis in tra-2 conditional knockout hermaphrodites (Herm.) and males (Male). Knock out tra-2 gene in all neurons, cholinergic neurons, and GABAergic neurons to get pan-neuronal masculinized (Pan-neuronal masc.), Cholinergic neuronal masculinized (Cholinergic masc.), and GABAergic neuronal masculinized (GABAergic masc.) hermaphrodites. Transgenic animals with two loxP sites insertion but without Cre recombinase expression were used as controls (Control 1). (C) Locomotion behavior analysis of a single adult tra-2 conditional knockout hermaphrodite (Herm.) and a male (Male). The averaged and individual crawling locomotion velocities were plotted. (D and E) Endogenous acetylcholine transmission was assessed by recording mEPSCs from body-wall muscles of tra-2 conditional knockout hermaphrodites. Representative mEPSC traces (D), the mean mEPSC frequencies (E, top panel), and the mean mEPSC amplitudes (E, bottom panel) are shown. Transgenic animals with two loxP sites insertion but without Cre recombinase expression were used as control 1, and transgenic animals with pan-neuronal Cre recombinase expression, but without two loxP sites insertion were used as control 2. The data for WT are the same as in Fig. 1, H and I. (F) The puncta fluorescence intensities and densities, marked by the cholinergic synaptic UNC-43::split GFP in dorsal nerve cord axons in tra-2 conditional knockout hermaphrodites. Representative images (top panel, scale bar, 10 μm), mean puncta intensity (middle panel, and density (bottom panel) are shown. In B, for each of the group, n = 3 biologically independent samples; each sample contains ≥25 animals. Data are presented as mean values ± SEM. Two-way ANOVA comparing all of the time points. In C, E, and F, data are presented as mean values ± SEM. Ns represent the number of animals tested. One-way ANOVA with post-hoc Bonferroni’s multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant.

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