FIGURE SUMMARY
Title

Gaze-stabilizing central vestibular neurons project asymmetrically to extraocular motoneuron pools

Authors
Schoppik, D., Bianco, I.H., Prober, D.A., Douglass, A.D., Robson, D.N., Li, J.M.B., Greenwood, J.S.F., Soucy, E., Engert, F., Schier, A.F.
Source
Full text @ J. Neurosci.

Vestibular nucleus neurons labeled in Tg(6.7FRhcrtR:gal4VP16). A, The expression pattern of Tg(6.7FRhcrtR:gal4VP16); Tg(UAS-E1b:Kaede)s1999t (purple) is shown as a horizontal MIP, with one vestibular neuron, colabeled by focal electroporation of gap43-EGFP (white). Arrows point to the tangential (TVN) and medial vestibular nuclei (MVN) and the MLF. Inset, Schematic of a dorsal view of a larval zebrafish. Magenta rectangle represents the location of the image. Scale bar, 50 μm. Horizontal (B) and sagittal (C) MIP of vestibular neurons in Tg(6.7FRhcrtR:gal4VP16);Tg(UAS-KillerRed) (purple);Tg(isl1:GFP) (green, image γ = 0.5) showing cranial motoneuron somata from nIII/nIV, nV, and nVII (green text). Arrows indicate neurons in the vestibular nuclei (VN) and the MLF. Scale bar, 50 μm. D–F, Close-up of white boxed region in C, showing major branch patterns of vestibular neuron axon fascicle (purple) relative to extraocular motoneurons (green). D, Motoneurons from Tg(isl1:GFP) (green) in nIV (magenta arrow), superior rectus motoneurons of nIII (cyan arrow), and the midbrain/hindbrain boundary (white dotted line). E, Branches of the vestibular neuron axon fascicle (purple), emerging from the MLF (white arrow) in Tg(6.7FRhcrtR:gal4VP16);Tg(UAS-KillerRed), projecting to nIV (magenta arrow) and nIII (cyan arrow). F, Merge of D and E. Scale bar, 20 μm. G–I, Broad and close-up views of vestibular neuron axonal projection (purple) to nIII cell bodies (green), taken at the horizontal plane delineated by the cyan dotted line in F, SR motoneurons (nIII) encircled in cyan. G, Cyan arrows localize close-ups in H and I. Scale bar, 10 μm. J–L, Broad and close-up view of vestibular neuron axonal projection (purple) to nIV cell bodies (green), taken at the horizontal plane delineated by the magenta dotted line in F, SO motoneurons (nIV, green) encircled in magenta. J, Arrows point to close-up in K and L. Scale bar, 10 μm.

Projections from singly labeled vestibular nucleus neurons. A, Horizontal MIP of a single vestibular neuron labeled with UAS-ChR2(H134R)-EYFP (purple) in Tg(6.7FRhcrtR:gal4VP16);Tg(isl:GFP) (green). γ = 0.5 to highlight the sparse label. Scale bar, 100 μm. Pink triangle represents the data in Figure 7D. Twenty-three of 27 neurons studied projected similarly. B, Sagittal MIP of the neuron in A highlighting nIII (cyan arrow), nIV (magenta arrow), and projection to nIV (white arrow). Scale bar, 20 μm. C, Horizontal MIP of nIV (green cell bodies in dotted magenta outline) from A. Vestibular neuron projection (purple, white arrow). Scale bar, 10 μm. D, Horizontal MIP of nIII (green cell bodies in dotted cyan outline) with no proximal vestibular neuron projection (purple). E, Sagittal MIP of a single axon expressing 14×UAS-E1b:hChR2(H134R)-EYFP (purple) in Tg(6.7FRhcrtR:gal4VP16);Tg(isl1:GFP) (green); Tg(atoh7:gap43-RFP) (cyan) fish. Expression of bright GFP bleeds into the purple channel, making the cell bodies white. nIV (magenta arrow), nIII (cyan arrow), and the vestibular neuron projection to SR motoneurons in nIII (white arrow). Scale bar, 20 μm. Four of 27 neurons projected similarly, exclusively to nIII. F, Horizontal MIP of nIII (cells in blue outline) from E, purple projections from vestibular neuron (white arrow). Scale bar, 10 μm. G, Horizontal MIP of nIV (cells in magenta outline) from E with no purple vestibular neuron projection. Scale bar, 10 μm.

Vestibular nucleus neurons show synaptophysin-positive puncta on their motoneuron targets. A, Sagittal MIP of a labeled SO motoneuron (magenta arrow) in green and the purple synaptic puncta labeled in Tg(−6.7FRhcrtR:gal4VP16); Tg(5×UAS:sypb-GCaMP3). Dotted lines indicate the planes in B, C. Scale bar, 20 μm. B, C, Close-up slice of the motoneuron somata in A with puncta (magenta arrow). Scale bar, 10 μm. D, Close-up of a retrogradely labeled SR motoneuron soma (green) with visible purple puncta (cyan arrow). Scale bar, 10 μm. E, Close-up of the dendrites of SR motoneurons (green) with visible purple puncta (cyan arrow). Scale bar, 10 μm.

Vestibular nucleus neurons labeled in Tg(6.7FRhcrtR:gal4VP16) are necessary for both nose-up and nose-down gaze stabilization. A, Horizontal MIP of vestibular and control neurons (nVII) in rhombomeres 4–8 in Tg(6.7FRhcrtR:gal4VP16); Tg(UAS-E1b:Kaede)s1999t; Tg(isl1:GFP) fish before and after targeted photo-ablation of vestibular neuron cell bodies. γ = 0.5 highlights dim signal. Colors represent depth over ∼150 μm. White arrows indicate the general region of targeted cell bodies in either the vestibular nuclei (top row) or the facial nucleus (nVII). Scale bar, 150 μm. For anatomical localization, compare with the right side of Figure 1B. B, Vestibulo-ocular reflex gain preablation and postablation of vestibular neurons. C, Vestibulo-ocular reflex gain preablation and postablation of facial nucleus neurons. D, Vestibulo-ocular reflex gain wild-type siblings (WT) and fish with pharmacogenetic (nitroreductase, “nfsb”) and optogenetic ablation (Killer-Red [KR]) of neurons in Tg(−6.7FRhcrtR:gal4VP16).

The simplified neural circuit underlying the ocular response to pitch and roll tilts. Cyan represents nose-down. Magenta represents nose-up channels. A, Wiring diagram of one hemisphere of the excitatory vestibulo-ocular circuit showing utricular hair cells (cyan/magenta), stato-acoustic ganglion (SAG), central vestibular neurons (VN, cyan and magenta), extraocular motoneuron pools in nIII (SR, IR, IO) and nIV (SO). B, During a roll tilt to the fish's left, the left utricle hair cells (cyan/magenta) are activated, causing cocontraction of superior (SO/SR) eye muscles ipsilateral to the activated utricle, and inferior (IO/IR) muscles contralateral to the activated utricle. C, Utricular hair cells sensitive to nose-up pitch tilts (magenta) ultimately activate only vestibular neurons that project to both nIII and nIV, activating SO (contralateral) and IR (ipsilateral). D, Utricular hair cells sensitive to nose-down pitch tilts (cyan) ultimately activate vestibular neurons that project to exclusively to nIII, activating SR (contralateral) and IO (ipsilateral).

Early ablations of vestibular neurons leave fish unable to inflate their swim bladders. A, Tg(−6.7FRhcrtR:gal4VP16); Tg(14×UAS-E1b:hChR2(H134R)-EYFP); mitfa−/− fish swimming in a cuvette in the dark at 144 hpf. Red arrows point to swim bladders. B, Sibling fish where the vestibular neurons in these fish were photoablated at 72 hpf, before swim bladder inflation. Note the absence of a swim bladder, evaluated here at 144 hpf. Images are taken from Figures 9-1 and 9-2.

Acknowledgments
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