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Acetylcholine (ACh), a vital neurotransmitter for both the peripheral (PNS) and central nervous systems (CNS), signals through nicotinic ACh receptors (nAChRs) and muscarinic ACh receptors (mAChR). Here, we explore the expression patterns of three nAChR subunits, chrna3, chrnb4, and chrna5, which are located in an evolutionary conserved cluster. This close genomic positioning, in a range of vertebrates, may indicate co-functionality and/or co-expression. Through novel transgenic zebrafish lines, we observe widespread expression within both the PNS and CNS. In the PNS, we observed expression of chrna3tdTomato, chrnb4eGFP, and chrna5tdTomato in the intestinal enteric nervous system; chrna5tdTomato and chrnb4eGFP in sensory ganglia of the lateral line; and chrnb4eGFP in the ear. In the CNS, the expression of chrnb4eGFP and chrna5tdTomato was found in the retina, all three expressed in diverse regions of the brain, where a portion of chrna3tdTomato and chrnb4eGFP cells were found to be inhibitory efferent neurons projecting to the lateral line. Within the spinal cord, we identify distinct populations of chrna3tdTomato-, chrnb4eGFP-, and chrna5tdTomato-expressing neurons within the locomotor network, including dmrt3a-expressing interneurons and mnx1-expressing motor neurons. Notably, three to four primary motor neurons per hemisegment were labeled by both chrna3tdTomato and chrnb4eGFP. Interestingly, we identified an sl-type secondary motor neuron per hemisegement that strongly expressed chrna5tdTomato and co-expressed chrnb4eGFP. These transgenic lines provide insights into the potential roles of nAChRs within the locomotor network and open avenues for exploring their role in nicotine exposure and addiction in a range of tissues throughout the nervous system.

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.

The zebrafish lateral line is a frequently used model to study the mechanisms behind peripheral neuronal innervation of sensory organs and the regeneration thereof. The lateral line system consists of neuromasts, a cluster of protruding hair cells, which are innervated by sensory afferent and modulatory efferent neurons. These flow-sensing hair cells are similar to the hair cells in the mammalian ear. Though, while hair cell loss in humans is irreversible, the zebrafish neuromasts are regarded as the fastest regenerating structure in vertebrates, making them an ideal model to study regeneration. However, one component of the lateral line system, the efferent projections, has largely been omitted in regenerative studies. Here, for the first time, we bring insights into the fate of efferent axons during ablation and regeneration of the hair cells in the zebrafish lateral line. Our behavioral analysis showed functional recovery of hair cells and sensory transmission within 48 h and their regeneration were in line with previous studies. Analysis of the inhibitory efferent projections revealed that in approximately half the cases the inhibitory efferent axons degenerated, which was never observed for the sensory afferent axons. Quantification of hair cells following ablation suggests that the presence of mature hair cells in the neuromast may prevent axon degeneration. Within 120 h, degenerated efferent axons regenerated along the axonal tract of the lateral line. Reanalysis of published single cell neuromast data hinted to a role for Bdnf in the survival of efferent axons. However, sequestering Bdnf, blocking the Trk-receptors, and inhibiting the down-stream ERK-signaling, did not induce axon degeneration, indicating that efferent survival is not mediated through neurotrophic factors. To further explore the relation between hair cells and efferent projections, we generated atoh1a mutants, where mature hair cells never form. In larvae lacking hair cells, inhibitory efferent projections were still present, following the tract of the sensory afferent without displaying any innervation. Our study reveal the fate of efferent innervation following hair cell ablation and provide insights into the inherent differences in regeneration between neurons in the peripheral and central nervous system.

The wiring of neuronal networks is far from understood. One outstanding question is how neurons of different types link up to form subnetworks within the greater context. Cadherins have been suggested to create an inclusion code where interconnected neurons express the same subtypes. Here, we have used a CRISPR/Cas9 knock-in approach to generate a transgenic zebrafish reporter line for protocadherin 9 (pcdh9), which is predominantly expressed within the central nervous system. Expression of eGFP was detected in subsets of neurons in the cerebellum, retina and spinal cord, in both larvae and juveniles. A closer characterization of the spinal locomotor network revealed that a portion of distinct classes of both excitatory and inhibitory interneurons, as well as motor neurons, expressed pcdh9. This transgenic line could thus be used to test the cadherin network hypothesis, through electrophysiological characterization of eGFP positive cells, to show if these are synaptically connected and form a discrete network within the spinal cord.

Despite growing knowledge, much remains unknown regarding how signaling within neural networks translate into specific behaviors. To pursue this quest, we need better understanding of the behavioral output under different experimental conditions. Zebrafish is a key model to study the relationship between network and behavior and illumination is a factor known to influence behavioral output. By only assessing behavior under dark or light conditions, one might miss behavioral phenotypes exclusive to the neglected illumination setting. Here, we identified locomotor behavior, using different rearing regimes and experimental illumination settings, to showcase the need to assess behavior under both light and dark conditions. Characterization of free-swimming zebrafish larvae, housed under continuous darkness or a day/night cycle, did not reveal behavioral differences; larvae were most active during light conditions. However, larvae housed under a day/night cycle moved a shorter distance, had lower maximum velocity and maximum acceleration during the startle response under light conditions. Next, we explored if we could assess behavior under both dark and light conditions by presenting these conditions in sequence, using the same batch of larvae. Our experiments yielded similar results as observed for naive larvae: higher activity during light conditions, regardless of order of illumination (i.e. dark-light or light-dark). Finally, we conducted these sequenced illumination conditions in an experimental setting by characterizing behavioral phenotypes in larvae following neuromast ablation. Depending on the illumination during testing, the behavioral phenotype following ablation was characterized differently. In addition, the results indicate that the order in which the light and dark conditions are presented has to be considered, as habituation may occur. Our study adds to existing literature on illumination-related differences in zebrafish behavior and emphasize the need to explore behavioral phenotypes under both light and dark condition to maximize our understanding of how experimental permutations affect behavior.

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.

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.

Optical projection tomography (OPT) is a 3D imaging alternative to conventional microscopy which allows imaging of millimeter-sized object with isotropic micrometer resolution. The zebrafish is an established model organism and an important tool used in genetic and chemical screening. The size and optical transparency of the embryo and larva makes them well suited for imaging using OPT. Here, we present an open-source implementation of an OPT platform, built around a customized sample stage, 3D-printed parts and open source algorithms optimized for the system. We developed a versatile automated workflow including a two-step image processing approach for correcting the center of rotation and generating accurate 3D reconstructions. Our results demonstrate high-quality 3D reconstruction using synthetic data as well as real data of live and fixed zebrafish. The presented 3D-printable OPT platform represents a fully open design, low-cost and rapid loading and unloading of samples. Our system offers the opportunity for researchers with different backgrounds to setup and run OPT for large scale experiments, particularly in studies using zebrafish larvae as their key model organism.