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Fig. 4 Figure 4. Mosaic Labeling of th2+ Neurons Reveals Long-Range Projections from the PON and Reciprocal Connectivity between Hc, Hi, and PT (A) Experimental workflow. Fish were injected at the one-cell stage with a low concentration of the th2:gfp-aequorin plasmid to achieve sparse labeling. At 6 dpf, fish were fixed and stained for GFP and total ERK. Confocal stacks were transformed for registration to the z-brain atlas using the total ERK channel, and the same transformation was then applied to the GFP channel. Neurons were manually traced, and the anatomical locations of the soma and axonal termini were identified using the z-brain viewer in MATLAB [37]. Cyan boundaries mark annotated brain regions from the z-brain reference atlas. (B–E) Maximum intensity projection of confocal stacks showing th2+ neurons (green) in Hc (B), Hi (C), PT (D), and PON (E) morphologically registered to the z-brain reference atlas (magenta). IO, inferior olive; nMLF, nucleus of the medial longitudinal fasciculus; TS, torus semicircularis. (F and G) Example ascending (F) and locally projecting (G) th2+ neurons in Hc. (H and I) Example ascending (H) and locally projecting (I) th2+ neurons in Hi. (J and K) Example descending (J) and locally projecting (K) th2+ neurons in PT. (L–O) Example th2+ neurons in PON. All scale bars, 20 μm.