General morphology of the adult zebrafish Torus Longitudinalis (TL). (A) Schema of the brain in a lateral view representing the location of the TL (in red) along the rostro-caudal axis. Anterior to the left. The approximate levels of sections (B–D) and (K–M) are also shown. (B–E) Diagrams of the general anatomy of the TL at rostral, intermediate and caudal levels in transverse section. (F–I) Images of Nissl stained sections showing TL at approximately the same rostrocaudal levels as (B–E). (J) Glial processes along TL midline (arrowhead) after GFP immunostaining in Tg(gfap:GFP)^mi2001. (K) Neuropil in TL showing immunoreactivity against synaptic vesicles 2 (SV2). (L) Merged channels for SV2 (magenta) and Sytox green (green). Note that the non-immunoreactive areas in (I) correspond with location of cell bodies in green). (M–O) Horizontal sections [at levels indicated in (A)] showing the general anatomy and location of TL. Anterior to the left. For abbreviations, see list. Scale bars: 100 μm (A-L), 250 μm (M-O).

Cellular organization of zebrafish TL. (A–F) Micrographs of semithin transverse sections of zebrafish TL stained with toluidine blue-borax (A–E) and cryostat transverse sections immunostained against GAD67 (F). (A) Section through the rostral TL. Note that at this level, TL sits on top of the posterior commissure. Midline is indicated by dashed line. Area squared is shown in detail in (D). (B) Section through the intermediate region of the TL showing the peripheral band of cells and internal cell clusters. Black dashed line marks the approximate limit between both populations. Note also the presence of a group of large neurons (black thick arrow) in dorsal TL and the nuclei of ependymal cells (arrowheads). White dashed line marks the midline. (C) Detail of the peripheral band of cells below the ventricular surface showing small (Nu₂, black arrowheads) and medium size (Nu₁, white arrowheads) round nuclei, as well as more irregular nuclei with paler chromatin (Nu₃, white arrows). Midline is to the right. (D) Detail of the area squared in (A) showing small (Nu₂, black arrowheads) and medium size nuclei (Nu₁, white arrowheads), as well as slightly larger nuclei (Nu₃, arrows) with paler chromatin. Dashed line marks the OT border. (E) Section showing the location of large (C₃) cells in the dorsal region of TL (thick black arrows). Thin arrows: axons of the intertectal commissure. Midline is to the right. (F) Section through rostral TL showing GAD67-positive cell bodies (thick black arrows) in dorsal areas, as well as immunopositive fibers (white arrowheads). Asterisks, ventricle; Stars, blood vessels. For abbreviations, see the list. Scale bars: 50 μm (A–B, F); 20 μm (C,E); 10 μm (D).

Fine cell structure of the adult TL. (A–D) Electron micrographs showing the main three nucleus types found in TL. (A) Low magnification electron micrograph showing a cell cluster with medium-sized round nuclei with partially condensed chromatin (Nu₁, white star) and surrounded by a dense neuropil. (B) Detail of a medium-sized nucleus (Nu₁) surrounded by smaller nuclei (Nu₂ and Nu_2’). (C) Detail of a small-sized cell nucleus with non-homogeneously condensed chromatin (Nu_2’) showing the exit of the axon (black arrow) from the cell body. (D) Nucleus (Nu₃) with pale chromatin that belongs to the large-sized cells among other nuclei with more condensed chromatin (Nu₁). Note also the small compact bundle of non-myelinated axons (circle). m, mitochondria; black stars, blood vessels. Scale bars: 5 μm (A), 2 μm (B,D), 1 μm (C).

Fine cell processes structure of the adult TL. (A–H), Electron micrographs of the TL neuropil showing different axon terminals and dendrites. (A) Type I axon terminal (at₁), with a number of small vesicles and large mitochondria (m) besides a dendritic process (Den) with smaller mitochondria (asterisk). Arrows: wavy-shaped synaptic active zone. (B) Type I axon terminal (at₁) densely filled with vesicles in close contact with a dendrite (Den). Arrows: wavy-shaped synaptic active zone. Note that mitochondria in the post-synaptic dendrite (asterisk) are smaller and more abundant. (C) Axon terminal type I (at₁) making synapse (arrows) with two dendritic processes (Den1, Den2). (D) Axo-axonic contact between type I (at₁) and type II (at₂) terminals, as well as a type I axo-dendritic synapse (arrows). (E) An axo-dendritic synapse (arrow) for a type II axon terminal. Note a myelinated axons (ax_m) on the top-right and left. (F) Axo-axonic synapse (arrows) between type I (at₁) and II (at₂) terminals, as well as axo-dendritic synapses (arrows) with two dendritic processes (Den₁, Den₂). (G) Axon terminal type III (at₃) in the rostral TL intermingled with myelinated (ax_m) and unmyelinated (ax) axons and dendrites (Den). Note the thick and highly myelinated axons in the posterior commissure at the bottom of the image (star). (H) Detail of axon terminals type III (at₃), with small clear (black arrows) and large electron-dense vesicles (white arrows), next to type I (at₁) axon terminals. m, mitochondria. Scale bars: 1 μm (A–F,H); 2 μm (G).

(A–G), Schematic drawings of selected transverse sections of the brain of adult zebrafish from rostral to caudal at the levels indicated in the inset, showing the results of DiI application to the intermediate TL (star). Circles, cells labeled consistently; asterisks: cells labeled occasionally; lines and dots, labeled fibers. Dashed lines: areas of the brain. For abbreviations see the list. Scale bar for sections: 200 μm.

Labeled cells and fibers after direct DiI application to TL. (A–J) Photomicrographs of labeled structures observed in transverse sections at diencephalic and mesencephalic levels after DiI application to the TL. (A) Labeled cells in the central pretectal nucleus (PCe; arrowheads). Inset: detail of labeled cells in the PCe. (B,C) Retrogradely labeled cells (arrowheads) in the intercalated (Pi; in B and C) and paracommissural (PCo; in B) pretectal nuclei. (D) Confocal image showing retrogradely labeled cells in the ipsilateral PCo. Inset: contralateral PCo. (E) Faintly labeled cells (arrowheads) in the prethalamus and labeled terminals and fibers in the nucleus rostrolateralis and optic tract. (F) Retrogradely labeled tectal cells (black arrowheads) and anterogradely labeled bundles of marginal axons (arrows) through the optic tectum to reach the marginal layer (SM) where they spread giving beaded axon profiles. (G) Confocal image of the marginal layer showing a detail of beaded parallel fibers (arrows) spreading from one torofugal tract. (H) Retrogradely labeled cells (black arrowheads) and fibers (arrows) in the tectum. (I) Inverted image of the projection of a confocal stack with two fluorescent toropetal tectal cells showing dendritic arborization in the SGC (open arrowheads) and ventral SFGS (black arrowheads) and, their axon directed ventrally to the SAC (arrows). (I’) Tracing of the cells shown in (I). (J) Confocal image of a toropetal tectal cell. (J’) Tracing of cell shown in (J). Asterisk, ventricle. For abbreviations, see list. Scale bars: 200 μm (A); 100 μm (B,H); 50 μm (D,G,I–J,J’); 20 μm (inset in A,C,E,F,I’).

Labeled cells and fibers after direct DiI application to TL. Photomicrographs of transverse sections caudal to the tracer application point showing labeled structures observed after homolateral DiI application to intermediate TL. Ipsilateral side is to the left. (A–C) Sections showing the caudal course of the torovalvular tract (arrows) from the labeled TL (A,B) that enters and travels bilaterally through the cerebellar valvula. Note in (C) the (tecto-)isthmal tract (white arrow) also labeled from some DiI diffusion to the ipsilateral optic tectum. (D) Detailed view showing labeled cells ventrolaterally to the nucleus lateralis valvulae (arrowheads) squared in (C), which could represent the subvalvular nucleus in the zebrafish. Note a few labeled fibers from the torovalvular tract traversing the nucleus lateralis valvulae (arrows). (E) Nissl stained section showing larger cell bodies (putative subvalvular nucleus, arrowheads) ventrolaterally to the nucleus lateralis valvulae. (F) Labeled cells (arrowheads) and fibers (arrows) in the posterior mesencephalic lamina and the nucleus isthmi. (G) Nissl stained section showing the posterior mesencephalic lamina (arrowheads). (H) Section showing labeled cells and fibers in the optic tectum, as well as cells in the cerebellar body (arrowheads). (I) Labeled cells in the granule cell layer of the cerebellar body (arrowheads), detailed in the inset. Asterisk: ventricle. For abbreviations, see list. Scale bars: 200 μm (B,C,F–I); 100 μm (A,D,E).

Labeled cells and fibers in TL after DiI application to various regions and nuclei. (A–I), Labeled structures in the TL after unilateral DiI application to the optic tectum (A) pretectal nuclei (B,C), cerebellum (D-F), nucleus isthmi (G,H) and nucleus rostrolateralis (I). (A) Small labeled cells in the ipsilateral TL (arrows) after tracer application to the OT. (B) Very few labeled fibers (arrowheads) in the medial TL after DiI application to the APN and PCe region. (C) Labeled fibers (arrowheads) in the medial TL after tracer application to the paracommissural nucleus. (D–F) Bilaterally labeled fibers (arrowheads) in the ventrolateral TL after unilateral tracer application to the cerebellar corpus (D), cerebellar valvula (E), and granular eminence (F). (G,H) Labeled fibers (arrowheads) after unilateral DiI application to the nucleus isthmi region. Note labeled cells in the optic tectum (white arrowheads) and labeled fibers coursing the intertectal commissure dorsal to TL (thin arrows) in (G) and (H), so as a dorsal longitudinal fascicle (thick arrow) in (H). Note also some fibers in the ipsilateral torovalvular tract (arrowheads), likely labeled by DiI diffusion to nucleus isthmi neighboring areas (see Section “DISCUSSION”), and bilateral labeled fibers in the TL (H). (I), Ipsilateral faintly labeled cells (arrows) in the TL from the rostrolateral nucleus region Asterisk: ventricle. For abbreviations, see the list. Scale bars: Scale bars: 200 μm (G); 100 μm (A-F,H); 20 μm (I).

GFP + cells and fibers in the optic tectum of Tg(1.4dlx5a-dlx6a:GFP)^ot1. (A–D’) Labeled tectal cells (confocal images and tracings) in adult (A–B’) and 20dpf (E–E’) transgenic zebrafish Tg(1.4dlx5a-dlx6a:GFP)^ot1. (A-D), Images projection from confocal stacks. (A) Section through the optic tectum of an adult fish showing GFP + pyramidal or type I cells (arrowheads, labeled “I” in the photograph) with their characteristic dendritic arbors (arrows) and cell bodies in the periventricular cell layer that are likely to correspond to type XIV cells from Meek and Schellart (1978) (outlined arrowheads, labeled “XIV” in the photograph). (B,B’) Section showing 4 GFP + pyramidal cells (B) and their tracing (B’). Note the sparse distribution of the cell bodies (arrowheads). Cell marked with an asterisk in (B’) shows quite broad dendritic arbor, overlapping with adjacent ones. Area marked by dotted lines: SM. (C) Dorsal view of a whole mount brain of a 20dpf fish showing labeled structures in the optic tectum. Arrowheads point to the cell bodies of the cells traced in (D’). (D) Detail of the area squared in (C) showing the dendritic arbor (arrowheads) of a pyramidal cell. (D’) Tracing of two adjacent pyramidal cells observed in (D’), showing the area covered by their dendritic arbor (dashed line). Note that the dendritic arbor of the cells covers roughly circular and oval areas, and the slight overlap between them. Axis: D, dorsal; M, medial; R, rostral. For abbreviations, see list. Scale bars: 100 μm (A-C); 50 μm (D,D’).

GFP + pyramidal cells in Tg(1.4dlx5a-dlx6a:GFP)^ot1. (A–D,F–G) Images showing GFP + pyramidal cells in Tg(1.4dlx5a-dlx6a:GFP)^ot1 adult fish. All images are projections from confocal stacks. (E,I) Tracings of 13 pyramidal cells. Apical dendrites are represented with a thicker line than basal ones. Arrowheads in (I) point to axon. All cells are numbered 1-13. Scale bars: 20 μm.

GFP + cells and fibers in Tg(1.4dlx5a-dlx6a:GFP)^ot1.(A) GFP positive fibers in TL in Tg(1.4dlx5a-dlx6a:GFP)^ot1 adult fish. (B) GFP + cell bodies in the putative subvalvular nucleus of a Tg(1.4dlx5a-dlx6a:GFP)^ot1 adult fish. Scale bars: 100 μm.

Schematic representation of type I and type X cells in the optic tectum. The stratum marginale is marked in gray. Parallel fibers are represented with dotted lines. Limit between layers in the optic tectum are marked with dashed lines.