However, when this procedure was repeated at 4—5 hpt hours post EDTA treatment , we saw that FM1—43 uptake was restored to qualitatively normal levels, suggesting that tip-links are restored by this time. We found similar effects on rheotaxis. Significantly fewer larvae performed rheotaxis at 30—90 min after EDTA treatment, but rheotaxis had fully recovered at 4—5 hpt Figure 3B. We do not believe that EDTA treatment affects the hair cells of inner ear sensory patches since no circling or balance behaviors were observed.
However, we were not able to confirm this by FM1—43 dye uptake since the inner ear hair cells are not directly exposed to the embryo medium. Given that EDTA treatment recapitulates the effect of neomycin on rheotaxis, we feel confident that lateral line hair cell activity is important for rheotaxis in the zebrafish larvae.
B,D Paired t-test.
We next tested whether differential damage to the lateral line system is reflected in systematic changes in rheotaxis. We find a dose-dependent change in rheotaxis, with decreasing alignment as the dose is increased Figure 3C. These experiments provide evidence that hair cell damage leads to a significant decrease in the ability to orient against flow in 5 dpf zebrafish larvae, therefore, indicating that lateral line input is important for flow detection as early as the initial stages of larval development. Since both neomycin and EDTA treatments implicate lateral line hair cells in rheotaxis, we wanted to know if this behavior is restored when hair cells regenerate.
Two days after neomycin-induced damage, the majority, but not all, of hair cells have regenerated and neurons have made contacts  ,  , . When we ablate hair cells using neomycin and assay rheotaxis at 4—5 hpt, we find that the larvae's ability to align against flow is significantly reduced Figure 3D. However, at 52—53 hpt the treated larvae display near-normal rheotaxis. We conclude that rheotaxis is restored following hair cell regeneration. Similar to previous observations  ,  , our results show that zebrafish larvae can align by positive rheotaxis i.
In their natural habitat, zebrafish spawn in areas of low flow  , but we do not know if zebrafish larvae perform rheotaxis under natural conditions. Nevertheless, the fact that larvae are capable of performing positive rheotaxis demonstrates that the sensory and motor systems responsible for this behavior are in place and functional at larval stages. Early experiments by Lyon  attributed rheotaxis of fish mainly to the visual system, suggesting that fish orient to movements across the visual field.
Orientation to movement of stimuli across the visual field results in optomotor behavior in zebrafish larvae, where fish swim in the direction of perceived motion . Montgomery and colleagues  showed that lateral line of adult fish is also critical for orientation. Likewise, we find that in 5 dpf zebrafish larvae, the visual system and the lateral line system are important for rheotaxis.
At an intermediate flow rate, we find that vision and lateral line can compensate for each other during rheotaxis when either input is inhibited. However, when inputs from both systems are removed rheotaxis is greatly reduced. Montgomery and colleagues  attribute the input from the superficial neuromasts and not canal neuromasts to be responsible for the lateral line dependent rheotaxis.
Evolution of the Mechanosensory and Electrosensory Lateral Line Systems | SpringerLink
In 5 dpf larval zebrafish only superficial neuromast are developed. We find that at flow rates up to 0. Despite the considerable reduction in rheotaxis, when both the lateral line and visual inputs are eliminated, the larvae still display significant alignment, suggesting that input from other sensory systems must feed into the rheotaxis circuitry. Lyon and Montgomery  ,  suggested that somatic inputs are also important for rheotaxis. We observed that larvae come in contact with the side or bottom of the chamber, also suggesting that they may use touch for orientation.
Whether larvae are more likely to use touch for orientation after lateral line damage would require additional monitors to track behavior in several dimensions. Fish can also use chemotaxis in combination with the lateral line system for additional guidance cues .
Performing the rheotaxis assay in an open system as opposed to the re-circulating system, showed no statistical difference between the two data not shown. Therefore, the potential release of odorants by the larvae in the re-circulating system was not sufficient to affect rheotaxis. It would be interesting to see if adding other odorants can increase the percentage of larvae aligning against current at any given time.
We found that regeneration of lateral line hair cells restores rheotaxis perturbed by neomycin treatment. Rheotaxis was restored to near normal levels by 48—52 hrs after neomycin exposure, even though the hair cell regeneration was incomplete at this time  , suggesting that perhaps there is a threshold of input needed from the lateral line above which the behavior does not change. Similar results were found by McHenry et al. A full complement of hair cells within lateral line organs may provide sensitivity to stimuli not measurable under either assay.
Our work has established a behavioral assay by which we can assess lateral line function. Because lateral line hair cells are situated on the body surface during larval development, zebrafish have proved to be an accessible model for investigating hair cell death, protection, regeneration, and synaptogenesis  ,  ,  ,  ,  ,  ,  ,  ,  ,  , .
Studying these mechanisms in such an accessible model could potentially shed light on similar processes of other types of hair cells, namely those found in the auditory and vestibular systems, which are not as amenable to perturbations and in vivo analyses. Finally, our assay could be beneficial in isolating functional mutants that affect not only the mechanosensory hair cells but their neuronal circuitry as well.
Larvae orient when placed in a flow of 0. Larvae are engaged in several behaviors, including rheotaxis, turning and directed swimming. Perturbation of lateral line hair cells decreases the ability of 5 day-old zebrafish larvae to perform rheotaxis. We thank Eric Martinson for assistance in building the rheotaxis apparatus, Fritz Reitz for constructing the IR illumination platform, Glen MacDonald and Brandon Warren for writing the image analysis macro, and Harold Burgess for helpful comments on the manuscript.
Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract The lateral line sensory system, found in fish and amphibians, is used in prey detection, predator avoidance and schooling behavior. Introduction Mechanosensory hair cells are specialized cells with protruding apical stereocilia whose deflection leads to voltage changes that are transmitted to the central nervous system.
Rheotaxis apparatus and assay A 1. Download: PPT. Results Five day-old zebrafish larvae were tested for rheotaxis in continuous, non-turbulent flow. Figure 2. Zebrafish larvae use the lateral line system to align in flow. Figure 3. Rheotaxis depends on level of hair cell impairment or damage and is restored during regeneration. Supporting Information. Movie S1. Movie S2.
The Mechanosensory Lateral Line
Acknowledgments We thank Eric Martinson for assistance in building the rheotaxis apparatus, Fritz Reitz for constructing the IR illumination platform, Glen MacDonald and Brandon Warren for writing the image analysis macro, and Harold Burgess for helpful comments on the manuscript. References 1. Genes Dev — View Article Google Scholar 2. J Comp Neurol — The caudal box outlines the region shown at higher power in panels C, D; the rostral box outlines the region shown at higher power in panels E—G.
B—G In situ hybridization shown in skin-mounts in panels C—G: white dotted lines indicating the approximate position of lateral line canals, within which neuromasts are buried reveals expression of hair cell ribbon synapse-associated genes in ampullary organs, as well as neuromasts, for B Cacna1d , encoding the pore-forming subunit of Ca v 1. Abbreviations: ao, ampullary organs; e, eye; io, infraorbital neuromast line; nm, neuromasts; ol, otic neuromast line; so, supraorbital neuromast line. A color version of this figure is available online.
The gene encoding otoferlin, Otof , was present in the paddlefish lateral line-enriched gene-set and expressed in ampullary organs as well as neuromasts Fig. Taken together, these gene expression studies, albeit descriptive, suggest that the synaptic transmission mechanisms at the electroreceptor ribbon synapse are conserved with those of hair cells. Both Sox2 and Atoh1 were also present in the paddlefish lateral line-enriched gene-set and proved to be expressed in developing ampullary organs, as well as neuromasts Fig.
Transcription factor genes important for hair cell development are expressed in developing electrosensory organs in the paddlefish. A—E In situ hybridization in paddlefish embryos reveals expression in both developing neuromasts and ampullary organs of A Six1 and B Eya1 expression of both is also seen in the posterior lateral line primordium migrating along the trunk ; C Sox2 , D Atoh1 the inset shows neuromast lines at an earlier stage of development and E Pou4f3 Brn3c.
The dotted white lines in panel G show the approximate position of the neuromast lines. H—H 2 show a transverse section through the head near the eye. Panel H shows Neurod4 expression in an ampullary organ.
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Abbreviations: ao, ampullary organ; e, eye; nm, neuromasts; pll, posterior lateral line primordium. The expression of all these transcription factor genes in developing ampullary organs suggests that they are likely to be important for electroreceptor differentiation, not just hair cell differentiation. Another bHLH transcription factor gene present in the paddlefish lateral line-enriched gene-set, Neurod4 , may be important specifically for electroreceptor development, as its expression was restricted to developing ampullary organs, and never seen in neuromasts Fig.
Indeed, putative hair cell homologs have been reported in tunicates the closest invertebrate chordate relatives of the vertebrates; Fig. Thus, ribbon-less cellular homologs of retinal and, most likely, pineal photoreceptors were already present in separate locations within the central nervous system of the ancestral chordate. Instead, the recruitment of Ribeye to the presynaptic active zone machinery, and thus the formation of presynaptic ribbons, must represent independent evolutionary events during the evolution of vertebrate retinal cells, pineal cells, and hair cells.
The gene expression data from paddlefish suggest that ribbon synapses in electroreceptors use hair cell-specific transmission mechanisms i. Similarly, the fact that so many transcription factor genes essential for hair cell differentiation, including Atoh1 and Pou4f3 , are also expressed in developing ampullary organs Fig. Taking all these lines of evidence together, and with the caveat that nothing is yet known at the molecular level about electroreceptors in lampreys, it seems less likely that electroreceptors and hair cells evolved independently from a ciliated secondary sensory cell in the vertebrate ancestor, as this would also require electroreceptors to have recruited Ribeye to their presynaptic active zones, and formed presynaptic ribbons, independently of hair cells.
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Evolution of the Mechanosensory and Electrosensory Lateral Line Systems
Close mobile search navigation Article Navigation. Volume Article Contents. The mechanosensory division of the lateral line system.