kcc-2 mutants exhibit a decrease in the size of the synaptic vesicle population within HSN presynaptic termini. A, Confocal image of the HSNL neuron expressing the red fluorescent protein DsRed2 and the egg-laying muscles expressing the cyan fluorescent protein CFP. The HSN process forms presynaptic varicosities (arrowheads) that contact the egg-laying muscles near the vulva. Scale bar, 5 μm. Asterisk indicates the position of vulva. B, C, Expression of DsRed2 in wild-type (wt) (B) and kcc-2 mutant (C) HSNs. D, E, Expression of SNB-1::GFP in wild-type (D) and kcc-2 mutant (E) HSNs. In B and C, numbers indicate the DsRed2 volume for each varicosity, and, in D and E, numbers indicate the volume of the SNB-1::GFP-labeled structure within each varicosity. Images in B-E are two-dimensional representations of three-dimensional images in which each two-dimensional pixel is shown at the brightest individual voxel intensity from the stack of three-dimensional voxels that it represents. F, G, Merged images in which the SNB-1::GFP is represented as in D and E, but DsRed2 pixels above background that do not overlap with the SNB-1::GFP are false colored red, partially obscuring the green label. These images emphasize that the perimeters of the synaptic varicosities are labeled by DsRed2, and that the SNB-1::GFP-labeled structures are contained within the varicosities. The red-only perimeter is more prominent in kcc-2 than in the wild-type because of the decreased volume of SNB-1::GFP labeling in kcc-2. B–G, Brackets indicate synaptic varicosities, and arrows point to an artifact in the GFP channel caused by the vulval slit. Scale bars, 5 μm. H, No significant difference in total DsRed2 varicosity volume was observed between wild-type and kcc-2 mutant animals (p = 0.89). I, A significant difference in the total sum of SNB-1::GFP fluorescence in the HSN synaptic region was observed between the wild-type and kcc-2 mutant animals (p < 0.005). J, The average SNB-1::GFP fluorescence intensity within the GFP-labeled structures was not significantly different between the wild-type and kcc-2 mutant animals (p = 0.40), suggesting that the density of synaptic vesicles within these structures is unchanged. K, The total volume of SNB-1::GFP-labeled structures in the HSN synaptic region was significantly less in the kcc-2 mutant compared with the wild type (p < 0.005), indicating that the reduction in total SNB-1::GFP fluorescence is likely attributable to a smaller population of synaptic vesicles inside the HSN varicosities of the kcc-2 mutant. For H-K, error bars indicate SE; n = 29 varicosities from 11 animals for the wild type, and n = 36 varicosities from 12 animals for the kcc-2 mutant.