Whole-brain atlas of spinal-projecting neurons in larval zebrafish.

a. Schematic illustrating the principle of optical backfilling using photoactivatable GFP to label spinal projection neurons. b. Image processing workflow for anatomical registration and alignment to a standard brain reference. c. Three-dimensional distribution of segmented soma locations of spinal projection neurons in 6-day post-fertilization (dpf) larval zebrafish. The arrow indicates the site of optical backfilling in the spinal cord. Neurons are color-coded according to projection laterality: ipsilateral (magenta) and contralateral (purple). d. Quantitative analysis of spinal-projecting neuron quantification across brain regions. The red line indicates the mean value and each circle represents a larva (n = 8 larvae).

Regional distribution and laterality of spinal-projecting neurons in larval zebrafish.

a. Coronal sections showing the spatial distribution of descending spinal projection neurons throughout the brain. The top left panel displays a lateral view of the reference brain atlas with lines indicating the anatomical position and thickness of each coronal section plane. Magenta lines below each coronal section denote the spinal cord location where optical backfilling was performed. b. Spatial organization of spinal projection neurons across anatomical regions defined by the Zbrain atlas. c. Density of projection laterality (Mean + SD, n = 8 larvae) (left panel) and laterality index (right panel) for across the anatomical regions studied. LI = [Nipsi − Ncontra] ÷ [Nipsi + Ncontra], where LI = Laterality Index, Nipsi = Number of spinal projection neurons on the ipsilateral side and Ncontra = Number of spinal projection neurons on the contralateral side. Anatomical abbreviations as in Figure 1d.

Telencephalic and diencephalic spinal-projecting neurons.

a1.spinal-projecting neurons revealed using the ZExplorer larval zebrafish atlas. The web interface (https://zebrafish.cn/LM-Atlas/) was queried to identify neurons with somata in the telencephalon and axonal projections to the spinal cord. a2. Spinal projection neurons identified by optical backfilling in the olfactory bulb. White arrows indicate somata of ipsilateral and contralateral projecting neurons; black arrows trace the trajectory of a representative descending axon. b. Distribution of spinal projection neurons in the preoptic area (POA) and posterior tuberculum (PT). Neurons are displayed within their respective anatomical boundaries in horizontal (top) and coronal (bottom) orientations. The right panel shows localization and expression pattern of oxytocin-expressing neurons of the POA in the Tg(oxt:GAL4;UAS:mCherry) transgenic line. Scale bar, 10 µm. c. Spinal projection neurons in the hypothalamus, subdivided into rostral (rHyp) and intermediate (iHyp) regions. d. Spinal projection neurons in the thalamus (Th) and accessory optic system (AOS). e. Spinal projection neurons in the preganglionic complex (PGC) and pretectum (PrT).

Identification of pretectal interstitial nuclei homologs in larval zebrafish.

a. Schematic representation of the mammalian pretectal interstitial nuclei organization and composition. b. Spatial distribution of GnRH2-expressing neurons in the Tg(gnrh2:eGFP) transgenic line. c1. Pretectal Edinger-Westphal (EW) nucleus homolog gnrh2+ neurons are not labeled by classical retrograde spinal dye backfills. Lower panels show horizontal (top) and coronal (bottom) views of anatomical masks for the EW and the classically designated nucleus of the mediolateral fasciculus (nMLF) (putative Interstitial Nucleus of Cajal - nucleus of Darkschewitsch (INC-ND) homolog). The EW nucleus is positioned medially to the INC-ND nuclei labeled by spinal backfills. Scale bar 40 µm. c2. Anatomical comparison of pretectal GFP+ neurons in the Tg(gnrh2:GFP) line with the expression patterns of cart2 and uts1 mRNA from the mapZbrain atlas. d. Confocal image series through the pretectal interstitial region from ventral (left) to dorsal (right). Proposed homology assignments for zebrafish pretectal interstitial nuclei relative to mammalian counterparts are indicated. MLF, mediolateral fasciculus.

Mesencephalic spinal-projecting neurons.

a. Distribution of spinal projection neurons in the optic tectum (OT). b. Distribution of spinal projection neurons in the torus semicircularis (TS). c. Distribution of spinal projection neurons in the tegmentum (Teg). d. Confocal image series through the mediolateral tegmentum (boxed region in inset) from ventral (bottom) to dorsal (top). Proposed homology assignments for zebrafish midbrain reticular formation (mRF) and nucleus of the lateral lemniscus (nLL) are indicated. e. Confocal images of the mid-tegmentum (boxed region in inset) showing the contralaterally projecting red nucleus (RN). Lower panels display ventral and dorsal aspects of the RN with constituent neurons delineated by white outlines. Right panels show coronal (top), sagittal (middle), and schematic (bottom) representations of RN neuronal organization. f. Single-cell projection patterns of cerebello-rubral neurons identified using the mapZbrain single-cell atlas (https://mapzebrain.org). The RN region was used as a seed to query neurons with axonal projections to this target area.

Rhombencephalic spinal-projecting neurons: prepontine region.

a. Distribution of spinal projection neurons in the prepontine region. b. Confocal image series through the prepontine region from ventral (bottom) to dorsal (top). Each image represents a maximal projection stack of 20 µm. Proposed homology assignments for zebrafish are indicated in magenta dash lines. PnO, pontis oralis; LC, locus coeruleus; PB, parabrachial nucleus; PTg; LDT, Laterodorsal tegmentum; SR, superior raphe nucleus; mRT, midbrain reticular formation; CuF, cuneiform nucleus. c. Confocal images of the prepontine region showing the projecting neurons in the caudal SR after bilateral optical spinal backfills.

Rhombencephalic spinal-projecting neurons in the pontine and retropontine regions.

a. Distribution of spinal projection neurons in the pontine region. b. Distribution of spinal projection neurons in the retropontine region. c. Confocal image series through the pontine-retropontine region from ventral (bottom) to dorsal (top). Each image represents a maximal projection stack of 20 µm. Proposed homology assignments for zebrafish are indicated in magenta dash lines. PnO, pontis oralis; PnC, pontis caudalis. ION, intermediate octavomotor nuclei; PON, posterior octavomotor nuclei; s5, sensory trigeminal nuclei. d. Confocal images of the pontine-retropontine region showing the distribution of the projecting neurons from the vestibular system in larval zebrafish. Each image represents a maximal projection stack of 10 µm. M, Mauthner cell.

Rhombencephalic spinal-projecting neurons: Medullary region.

a. Distribution of spinal projection neurons in the medullary region. b. Confocal image series in the coronal plane showing the spatial distribution of descending spinal projection neurons throughout the medulla from rostral (med1) to caudal (med3). c. Confocal image series through the medulla region from ventral (top-left) to dorsal (bottom-right). Each image represents a maximal projection stack of 20 µm. ci. Anatomical comparison of medullary GFP+ neurons in the Tg(pet1:GFP) line from the mapZbrain atlas with the location of the SPNs. Proposed homology assignments for zebrafish are indicated in magenta dash lines. GiA, gigantocellular reticular nucleus pars alpha; Gi, gigantocellular reticular nucleus; lRt, intermediate reticular nucleus; PCRt, parvocellular reticular nucleus; LPGi, lateral paragigantocellular nucleus; IR, inferior Raphe nucleus. MdV, ventral medullary reticular nucleus; MdD, dorsal medullary reticular nucleus. Sp5, spinal trigeminal tract.

Comparative distribution of reticulospinal neurons across vertebrates.

Dorsal view schematic illustrating the distribution of reticulospinal neurons in six vertebrate species: lamprey larvae, ray, larval zebrafish, lizard, snake, and mouse. Each dot represents a single retrogradely labeled reticulospinal neurons. Neurons are color-coded according to their assignment to one of three reticular subdivisions following the parcellation of van Hoevell (1911): the superior reticular nucleus (SRN, orange), spanning from the isthmus to rhombomere 2; the middle reticular nucleus (MRN, green), extending from rhombomere 3 to rhombomere 6; and the inferior reticular nucleus (IRN, purple), running from rhombomere 7 to the obex. Dashed lines indicate rhombomere boundaries. Rostral is up. Data for each species were compiled from: lamprey larvae (Swain et al., 1993), ray (Smeets and Timerick, 1981b), lizard (Wolters et al., 1982), snake (Ten Donkelaar, 1982a), and mouse (Wang et al., 2022). Vestibulospinal and raphespinal neurons were excluded from this comparison (see text).