Figure 1.

The serotonergic NSM neuron forms ENT-containing axon arbors in a specific neuroanatomical coordinate. A, B, Electron micrograph sequence of cross-sections through the pharynx of worm N2W (www.wormimage.org). NSM (outlined in purple) forms an axon arbor (vertical projection) at the edge of the pharynx (PX) near the pseudocoelom (clear space annotated as PC). In A, synaptic vesicle clusters (SV) are visible at the base of the arbor (line) and in the tip of the arbor (bracket). A dense projection (DP) onto the pseudocoelom is visible in the base of the arbor (line). In B, the arbor has turned posteriorly and appears discontinuous from the main axon shaft (AS). Synaptic vesicle clusters are still visible in the main NSM axon shaft and in the tip of the arbor. A dense projection (DP) is also visible in the tip of the branch (line), facing the pseudocoelom (PC). C, Simultaneous visualization of NSM and the nematode nerve ring. NSM was imaged by expressing cytosolic mCherry using the tph-1 promoter (Sze et al., 2002), and the nerve ring was imaged by expressing transgene rab-3p::RAB-3::GFP. Note that arbors form in regions adjacent to the nerve ring (brackets). D, Schematic diagram of NSM neuron (modified image adapted from www.wormatlas.org with permission). As shown in the schematic, arbors form in the isthmus region of the pharynx, proximal to the nerve ring. Dashed line indicates the approximate location of the EM cross-sections shown in A and B. E–G, NSM in the L1 nematode. E, Cytosolic mCherry expressed cell specifically in NSM. Note the absence of arbors in the nerve ring region (bracket). Arrow marks dorsal neurite, open arrowhead marks proprioceptive dendrite, and filled arrowhead marks ventral neurite. F, Distribution of serotonergic vesicle clusters. G, Merge. H–J, NSM in the 3-d-old adult nematode. H, As in E for adult. I, As in F, for adult. Note the presence of vesicle clusters in the terminal arbors (brackets). J, Merge. Arrows indicate mature arbors with synaptic vesicle clusters at their bases. Open arrowhead indicates mature arbor lacking synaptic vesicle cluster at the base. A total of 78% of axon arbors are associated with synaptic vesicle clusters at their base (n = 70 with a 95% confidence interval extending from 66.4 to 85.7%). K–M, Localization of CAT-1::GFP (L) and mCherry::RAB-3 (K) in NSM, with merge in M. N–P, Localization of SNB-1::YFP (O) and mCherry::RAB-3 (N) in NSM, with merge in P. Q–S, Localization of GFP::SYD-2 (R) and mCherry::RAB-3 (Q) in NSM, with merge in S. T, U, CAT-1:GFP localization in WT (T) or unc-104(e1265) mutant animals (U). In all images, anterior is oriented to the left and dorsal is oriented up, as in the diagram (D). Bracket represents the expected position of the nerve ring as it circumvents the pharyngeal isthmus, and asterisk denotes position of the NSM cell body. Scale bars (in E): E–J, 5 μm; (in K) C, K–U, 5 μm.