Perturbed development of calb2b expressing dI6 interneurons and motor neurons underlies locomotor defects observed in calretinin knock-down zebrafish larvae.

Calcium binding proteins are essential for neural development and cellular activity. Calretinin, encoded by calb2a and calb2b, plays a role during early zebrafish development and has been proposed as a marker for distinct neuronal populations within the locomotor network. We generated a calb2b:hs:eGFP transgenic reporter line to characterize calretinin expressing cells in the developing spinal cord and describe morphological and behavioral defects in calretinin knock-down larvae. eGFP was detected in primary and secondary motor neurons, as well as in dI6 and V0v interneurons. Knock-down of calretinin lead to disturbed development of motor neurons and dI6 interneurons, revealing a crucial role during early development of the locomotor network. Primary motor neurons showed delayed axon outgrowth and the distinct inhibitory CoLo neurons, originating from the dI6 lineage, were absent. These observations explain the locomotor defects we observed in calretinin knock-down animals where the velocity, acceleration and coordination were affected during escapes. Altogether, our analysis suggests an essential role for calretinin during the development of the circuits regulating escape responses and fast movements within the locomotor network.

A deep-dive into fictive locomotion - a strategy to probe cellular activity during speed transitions in fictively swimming zebrafish larvae.

Fictive locomotion is frequently used to study locomotor output in paralyzed animals. We have evaluated the character of swim episodes elicited by different strategies in zebrafish. Motor output was measured on both sides of a body segment using electrodes and a pipeline for synchronizing stimulation and recording, denoising data and peak-finding was developed. The optomotor response generated swims most equivalent to spontaneous activity, while electrical stimulation and NMDA application caused various artefacts. Our optimal settings, optomotor stimulation using 5-day-old larvae, were combined with calcium imaging and optogenetics to validate the setup's utility. Expression of GCaMP5G by the mnx1 promoter allowed correlation of calcium traces of dozens of motor neurons to the fictive locomotor output. Activation of motor neurons through channelrhodopsin produced aberrant locomotor episodes. This strategy can be used to investigate novel neuronal populations in a high-throughput manner to reveal their role in shaping motor output. This article has an associated First Person interview with the first author of the paper.

Characterization of Individual Projections Reveal That Neuromasts of the Zebrafish Lateral Line are Innervated by Multiple Inhibitory Efferent Cells.

The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprized of clusters of superficial hair cells called neuromasts. Modulation occurs via excitatory and inhibitory efferent neurons located in the brain. Using mosaic transgenic labeling we provide an anatomical overview of the lateral line projections made by individual inhibitory efferent neurons in 5-day old zebrafish larvae. For each hemisphere we estimate there to be six inhibitory efferent neurons located in two different nuclei. Three distinct cell types were classified based on their projections; to the anterior lateral line around the head, to the posterior lateral line along the body, or to both. Our analyses corroborate previous studies employing back-fills, but our transgenic labeling allowed a more thorough characterization of their morphology. We found that individual inhibitory efferent cells connect to multiple neuromasts and that a single neuromast is connected by multiple inhibitory efferent cells. The efferent axons project to the sensory ganglia and follow the sensory axon tract along the lateral line. Time-lapse imaging revealed that inhibitory efferent axons do not migrate with the primordium as the primary sensory afferent does, but follow with an 8-14 h lag. These data bring new insights into the formation of a sensory circuit and support the hypothesis that different classes of inhibitory efferent cells have different functions. Our findings provide a foundation for future studies focussed toward unraveling how and when sensory perception is modulated by different efferent cells.

Behavioral Characterization of dmrt3a Mutant Zebrafish Reveals Crucial Aspects of Vertebrate Locomotion through Phenotypes Related to Acceleration.

Vertebrate locomotion is orchestrated by spinal interneurons making up a central pattern generator. Proper coordination of activity, both within and between segments, is required to generate the desired locomotor output. This coordination is altered during acceleration to ensure the correct recruitment of muscles for the chosen speed. The transcription factor Dmrt3 has been proposed to shape the patterned output at different gaits in horses and mice. Here, we characterized dmrt3a mutant zebrafish, which showed a strong, transient, locomotor phenotype in developing larvae. During beat-and-glide swimming, mutant larvae showed fewer and shorter movements with decreased velocity and acceleration. Developmental compensation likely occurs as the analyzed behaviors did not differ from wild-type at older larval stages. However, analysis of maximum swim speed in juveniles suggests that some defects persist within the mature locomotor network of dmrt3a mutants. Our results reveal the pivotal role Dmrt3 neurons play in shaping the patterned output during acceleration in vertebrates.