Whole brain mapping of spinal-projecting neurons in larval zebrafish

Key results

1. ShownPan-neuronal PAGFP backfills reveal many more spinal-projecting neurons than earlier dye tracing in larvae. The cells span hindbrain, midbrain and diencephalon, with the largest population in the hindbrain and an overall ipsilateral bias.

   Unilateral photoactivation labelled 654.5 ± 53 (mean ± SD) neurons per larva (n = 8 larvae), from which the whole population is estimated at 1,309 ± 106; earlier larval tracing labelled fewer than 200. Region counts against Zbrain masks put the largest population in the hindbrain, most of it in the medulla oblongata. · Brainwide distribution of spinal descending…; Introduction; Discussion; Figure legends

2. SuggestedHindbrain spinal-projecting neurons are assigned to putative homologs of mammalian reticulospinal (e.g. gigantocellular), vestibulospinal and raphe-spinal groups. Across six vertebrates, reticulospinal neurons share a Y-shaped layout in dorsal view, and zebrafish fits it.

   Assignments follow Watson et al. 2019 brainstem subdivisions and use soma size, laterality and Tg(pet1:GFP) atlas expression (two medullary columns flank the inferior raphe). Figure 9 plots published maps for lamprey larvae, ray, lizard, snake and mouse with zebrafish, binned into Hoevell's (1911) superior, middle and inferior reticular nuclei; the comparison is qualitative, with no statistics. · Rhombencephalic projections; Reticulospinal neurons: conserved core of motor…; Figure legends

3. SuggestedA GnRH2-expressing cluster in the interstitial tegmental region is proposed to be the Edinger-Westphal (EW) nucleus. This recasts the classical nucleus of the medial longitudinal fasciculus (nMLF) as the interstitial nucleus of Cajal and the nucleus of Darkschewitsch, both lying lateral to it.

   In Tg(GnRH2:EGFP) larvae, biocytin Alexa Fluor 594 spinal backfills labelled the nMLF but not the GFP+ cluster, which lies medial to it. cart2 and uts1 mRNA from mapZbrain (homologs of the mammalian EW peptides CART and Ucn) overlap the GnRH2+ cluster. The support is atlas overlap, not co-labelling in the same larvae. · Diencephalic projections; Diencephalic descending projections; Figure legends

4. SuggestedDiencephalic spinal-projecting neurons include the rostral oxytocinergic preoptic cluster, which projects only ipsilaterally, and two posterior tuberculum clusters at the positions of dopaminergic groups DC2 and DC4. Hypothalamic, thalamic, accessory optic system and pretectal groups also project to the cord.

   Preoptic backfilled cells coincide with Tg(oxt:GAL4;UAS:mCherry) neurons whose axons descend only ipsilaterally; the tuberculum clusters sit anterior and posterior to the caudal oxytocin cluster. A per-region laterality index (n = 8 larvae) shows the accessory optic system projecting bilaterally with a strong contralateral bias and the rostral hypothalamus only ipsilaterally. · Diencephalic projections; Diencephalic descending projections; Figure legends

5. SuggestedA few olfactory bulb neurons project to the spinal cord, and atlas tracings add ipsilaterally projecting subpallial neurons. The authors take this as evidence that telencephalospinal projections are not unique to mammals.

   Olfactory bulb neurons were backfilled in 4 of 8 larvae and matched single-neuron tracings in the ZExplorer atlas. The atlas also showed subpallial spinal-projecting neurons that were not detected optically; their atlas morphologies extend only to the rostral-most spinal cord. · Telencephalic projections to spinal cord; Telencephalic descending projections