Optogenetic dissection of a behavioural module in the vertebrate spinal cord

Abstract

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer-Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer-Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer-Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer-Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

Figure 1. Optical stimulation of specific spinal neurons leads to distinct locomotor behaviors

a, Spontaneous swim (superimposed frames). b, Optical stimulation (circle) of Gal4^s1020t/UAS:LiGluR evokes a “spontaneous swim”-like behavior. c, Comparison of deflection angle traces corresponding to a) (top, black) and b) (bottom, magenta, bar for stimulation) (inset: 94% of responses were a swim (n=18)). No difference in angle (p > 0.51; n=9) (d), or frequency (f), but more oscillations (e), in light-induced swims. g, Escape elicited by a water jet (trapeze) consists of sharp C-turn away from stimulus followed by a forward swim. h, Light-induced escape induced by stimulation of RB cells in Gal4^s1102t/UAS:LiGluR larvae. i, Tail deflection traces corresponding to g) (top) and h) (bottom) (inset: 79% of responses were an escape (n= 11)). j-l, No difference in j) deflection angle (p > 0.13; n=7); k, frequency (p > 0.42; n=7); l, number of oscillations ((p > 0.4101;n=7).

Figure 2. The Gal4^s1020t line drives expression in motoneurons and KA neurons

a, Expression in ventral cells including motoneurons projecting out of cord (arrows) (lateral view). b-e, Random labeling in Gal4^s1020t/BGUG identifies solely two cell types: b, primary (top) and secondary (bottom) motoneurons. Dorsal (c) and lateral (d-f) views of neuron with a central ipsilateral ascending axon. Note contact feet (red stars in c-d) and a “toothbrush” morphology (e) (cilia, yellow arrows) characteristic of KAs. In c), larva was slightly tilted to show enlarged contacts on axon (midline, red line and segment, white lines). e, Dense BGUG pattern with multiple KAs shows the alignment of the brush of cilia (arrows) with central canal. f, The ascending axon runs near the ventral edge of the spinal cord before aiming dorsally. g,h, KAs at 5dpf in Gal4^s1020t/BGUG (green) are GABAergic neurons (anti-GAD (g) and anti-GABA (h) immunostaining in red). Scale bars = 25μm.

Figure 3. Optical stimulation of KAs of Gal4^s1003t line induces a forward swim

a, Expression in spinal cord is confined to cells close to the midline (dorsal view). b, Lateral view with BGUG shows ventral neurons with a central axon forming contact feet (red stars), characteristic “toothbrush” morphology (arrow) of KAs. c, d, These cells (green) are GABAergic (anti-GAD and anti-GABA staining in red). e-f, Optical stimulation induces a swim-like response. e, Superimposed images. f, Deflection angle trace. g-i, No difference in deflection angle (g), frequency (h) and number of oscillations (i) between Gal4^s1003t (blue) to Gal4^s1020t (magenta) (respectively p>0.85; p>0.98, p>0.36, n=9). j, Side-to-side comparison of number of oscillations evoked by a 100ms pulse of light shows that only lines expressing in KAs show a swim-like response while line with motoneurons (MNs) and no KAs (Gal4^s1041t, black; Hb9:Gal4, orange), do not. k, Reduction of the spontaneous swimming frequency in Gal4^s1003t/UAS:TeTxLC-CFP (p> 0.0075; n=10) but no change in the probability of touch-response (p> 0.45; n=12).

Figure 4. Dissection of the light-evoked responses in Gal4^s1020t and Gal4^s1102t by unilateral stimulation and lesion studies

a-c, Patterned illumination for stimulation. a, semi-restrained Gal4^s1020t larva aimed bilaterally (cartoon and fluorescence image of Kaede expression in three segments, top) and on left (L) or right (R) side (bottom panels, Scale bar = 25μm). b-c, Deflection angle traces and mean values induced by L (green) and R (red) stimulation; b) L and R activations induce similar symmetric oscillations of tail in Gal4^s1020t line (n=5). c, L and R activations induce large and opposite directed C-bends in Gal4^s1102t (n=9). d-f, Effect induced by isolation of the spine. d, Pattern in Gal4^s1020t pre (top) and post (below) lesion. e-f, No reduction of the light-induced swim behavior in Gal4^s1020t ((e), n=7) but abolition of the light-induced escape behavior in Gal4^s1102t ((f), n=4) (pre and post-lesion, top and bottom).