Modular organization of axial microcircuits in zebrafish

Abstract

Locomotion requires precise control of spinal networks. In tetrapods and bipeds, dynamic regulation of locomotion is simplified by the modular organization of spinal limb circuits, but it is not known whether their predecessors, fish axial circuits, are similarly organized. Here, we demonstrate that the larval zebrafish spinal cord contains distinct, parallel microcircuits for independent control of dorsal and ventral musculature on each side of the body. During normal swimming, dorsal and ventral microcircuits are equally active, but, during postural correction, fish differentially engage these microcircuits to generate torque for self-righting. These findings reveal greater complexity in the axial spinal networks responsible for swimming than previously recognized and suggest an early template of modular organization for more-complex locomotor circuits in later vertebrates.

Fig. 1. Distinct excitatory microcircuits govern dorsal and ventral musculature

A) Top, recording set-up with schematic of example primary motor neuron (MN) axon arbors in the musculature. Bottom, dual whole-cell recording of EPSCs during swimming from MNs projecting to mid-dorsal and ventral territory. Traces vertically offset for clarity. Simultaneous ventral root recording (grey) shows that excitatory barrages occur in phase with the ipsilateral fictive bend. B) Expanded time scale, EPSCs recorded during swimming from example in-quadrant (mid-ventral and ventral) MNs (Fig. 1H). Asterisks indicate synchronous EPSCs in both neurons (Δt < 150 μs). Scale as in (D). C) 100 consecutive EPSCs recorded in the mid-ventral MN (grey, top; average in light brown) and simultaneously recorded signal in the ventral MN (grey, bottom) reveals a significant EPSC-triggered average (dark brown). D – E) As for B – C, for an example out of quadrant (mid-ventral and dorsal) MN pair, revealing few synchronous EPSCs and small EPSC-triggered average (dark blue). F) Summary EPSC-triggered averages, means ± SEM, for in quadrant (n = 15) and out of quadrant (n = 19) MN pairs. ***, p < 0.001, unpaired t-test of peak EPSC amplitude. Gray bar indicates period of baseline normalization. G) Cross-correlogram of EPSC timing, averages from all MN pairs. Means ± SEM; bin = 50 μs, N as in (F). ***, p < 0.001, unpaired t-test at 0 ms. H) Schematic of proposed premotor excitatory circuitry. An individual excitatory premotor neuron (open circles) preferentially synapses either on dorsal- or ventral-projecting MNs (solid lines), but not both.

Fig. 2. Distinct inhibitory microcircuits govern dorsal and ventral musculature

All panels as in Fig. 1, for IPSCs. A) Example dual recording of IPSCs from MNs projecting to mid-dorsal and ventral territory. B) Expanded time scale, IPSCs recorded during swimming from example in-quadrant (mid-dorsal and dorsal) MN pair. Scale as in (D). C) 100 consecutive IPSCs recorded in mid-dorsal MN (top) and simultaneously recorded signal in the ventral MN (bottom) reveals a significant IPSC-triggered average (dark blue). Scale as in (E). D – E) As for B – C, for an example out of quadrant (mid-ventral and dorsal) MN pair, revealing few synchronous IPSCs and small IPSC-triggered average (dark blue). F) Summary IPSC-triggered averages, means ± SEM, for in quadrant (n = 13) and out of quadrant (n = 17) MN pairs. ***, p < 0.001, unpaired t-test of peak IPSC amplitude. G) Cross-correlogram of IPSC timing, averages from all MN pairs. Means ± SEM; bin = 50 μs, N as in (F). **, p < 0.01, unpaired t-test at 0 ms. H) Schematic of proposed premotor inhibitory circuitry. An individual inhibitory premotor MN preferentially synapses either on D or V MNs (solid lines), but rarely on both (dotted lines).

Fig. 3. Intraspinal premotor inputs are segregated into dorsal and ventral microcircuits

A) Sagittal view of 4 dpf larva expressing alx:Gal4::UAS:CatCh in two premotor neurons with typical V2a morphology (ipsilateral axon, primarily descending). Rostral is to the left; white bars mark muscle segments. B) Example of synchronous EPSCs evoked optogenetically in a dorsal/mid-dorsal MN pair. Green bar indicates light exposure. C) Asynchronous EPSCs evoked in a dorsal/mid-ventral MN pair in another fish. D) Summary of EPSC-triggered averages for in quadrant and out of quadrant MN pairs (n = 5 pairs each). *, p < 0.05, unpaired t-test of peak amplitudes. Varying baseline period is due to frequent barrages of light-elicited EPSCs (as in B). E) IPSC-triggered average responses in phase with ipsilateral motor activity, as measured by ventral root recordings (schematic, bottom), from in quadrant (n = 7) and out of quadrant (n = 14) pairs. ***, p < 0.001, unpaired t-test of peak amplitudes. F) As in (E), but for recordings during mid-cycle, out of phase with ipsilateral motor activity.

Fig. 4. Descending vestibular information differentially recruits dorsal and ventral microcircuits

A) Time-lapse images of a 3 dpf larva engaging in self-righting from right ear up to dorsal-up orientation. Pictures were taken under normal illumination for display purposes only; behavioral data were gathered with infrared illumination. Scale, 1 mm. B) Example Ca²⁺ imaging data from primary MNs in one segment of a side-lying fish. Arrow indicates time of brief electrical stimulus applied to tail to induce swimming. Scale bars: 20% Δ F/F, 5 s. C) Ventral-projecting MNs exhibit larger Ca²⁺ signals than dorsal-projecting MNs on ear-up side (left; n = 13 MN pairs from 9 fish); pattern is reversed on ear-down flank (right; 7 pairs from 7 fish). *, p < 0.05, paired t-test. D) Schematic of asymmetric drive to the four quadrants of musculature, producing torque for self-righting. E) Ca²⁺ imaging summary as in (D), for rock solo mutant animals (ear-up: n = 7 pairs from 7 fish; ear-down: n = 8 pairs from 8 fish). n.s., not significant, paired t-test. F) Physiological recordings from MN out of quadrant pairs on the ear-up side of WT animals during fictive swimming reveals that EPSCs are more frequent in ventral-projecting MNs than dorsal (p < 0.001, paired t-test, n = 19 pairs), whereas IPSCs are more frequent in dorsal-projecting MNs than ventral (p < 0.001, 17 pairs). Each dot represents either EPSC or IPSC data from a single out of quadrant pair. Unity line is shown in dashed grey. G) As in (F), for vestibular-deficient animals. No differences are seen in the number of EPSCs (p = 0.11, n = 10 pairs) or IPSCs (p = 0.50, n = 9 pairs) between dorsal- and ventral-projecting MNs. H) Higher in quadrant than out of quadrant synchrony persists in rock solo mutants and otolith ablated animals for both EPSCs and IPSCs. **, p < 0.01, unpaired t-test of peak amplitude (EPSCs: n = 6 in quadrant, 10 out of quadrant pairs; IPSCs: n = 6 in quadrant, 9 out of quadrant pairs). Scale bars: 4 pA, 0.5 ms; 10 pA, 1.5 ms. I) Schematic summary of results: prior models of a single premotor circuit for dorsal and ventral control in fish (left); current model supporting parallel, modular microcircuits (middle); and proposed homology to limb control in tetrapods (right).