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Fig. 3
Cell populations projecting to the habenula identified by anterograde DiI labeling and/or transgene expression. (A-J) Schematic of a transverse section on the brain at the level of the habenulae (A). The pink box approximates the area shown in (B,D,F,I,J). Images are from transverse sections (B-F,I-J) or dorsal views with anterior to the top (G,H) of zebrafish brains. Dotted lines in (B,D,F,H-J) mark the limits of the habenula. Images are of adult brains unless otherwise stated. (B) Labeled fibers (arrow) dorsally in the right habenula after DiI application in the olfactory bulb. (C) GFP+ neurons (arrowheads) in preoptic areas of a Tg(isl1:GFP)rw0 fish. Dotted lines mark the approximate limit between preoptic areas (ventral) and pallium (dorsal). (D) Sparse GFP+ fibers in the right habenula (arrow) of an adult Tg(isl1:GFP)rw0 fish. (E) GFP+ neurons (arrowheads) in the subpallium of a Tg(1.4dlx5a-dlx6a:GFP)ot1 fish. Dotted line marks the approximate pallial-subpallial boundary. (F) Very few GFP+ fibers (arrow) in the habenula of a Tg(1.4dlx5a-dlx6a:GFP)ot1 fish. (G) GFP expression (green) in 1 month old (1mpf) Et(gata2:GFP)bi105 juvenile zebrafish counter stained against tubulin (magenta) showing GFP+ neurons in the pallium and parapineal (PP, arrowhead). Note that the parapineal sends axons that terminate in the left habenula (arrow). Image is a dorsal view, anterior to the top. (H) High magnification image of the left habenula shown in G (after subtracting tubulin channel) showing that GFP+ fibers in the habenula (arrow) originate from the parapineal (PP, arrowhead). (I) Confocal image of the habenula showing labeled fibers (arrows) in the habenula after DiI application to the rostral vENT. Although more intense labeling is observed in ventral regions of the habenula, precise habenula subnuclei could be delineated using this method. (J) Labeled fibers (arrows) in the ventral half of the habenula after DiI application to the posterior hypothalamic lobe. (K,L) Schematics showing a summary of the main afferent areas to the habenula (K: outline of the brain in lateral view, L: boxes diagram). (K) The brain nuclei that project to the habenula are shown as colored areas. (L) Boxes represent main nuclei projecting to the habenula, while arrows represent projections. Asymmetric projection from the parapineal (PP) to left habenula (lHB) is shown with a blue arrow. Asymmetric projection from mitral cells of the olfactory bulbs (OB) to right habenula (rHB) is shown with a pink arrow. All other afferent areas that seem to innervate both left and right habenula are represented with black arrows. Scale bars: 150 µm in (B-F,I-J); 100 µm in (G), 50 µm in (H).