Patterns of free calcium in zebrafish embryos

Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged Ca2+ throughout the first day of development. During early cleavages, a high Ca2+ zone is seen in the cleavage furrows. The high Ca2+ zone during first cleavage spreads as a slow wave (0.5 mu m/second) and is preceded by three Ca2+ pulses within the animal pole region of the egg. When Ca2+ concentrations are clamped at the resting level by BAPTA buffer injection into the zygote, all signs of development are blocked. In later development, Ca2+ patterns are associated with cell movements during gastrulation, with neural induction, with brain regionalization, with formation of the somites and neural keel, with otic placode formation, with muscle movements and with formation of the heart. Particularly remarkable is a sharp boundary between high Ca2+ in the presumptive forebrain and midbrain versus low Ca2+ in the presumptive hindbrain starting at 10 hours of development. When Ca2+ changes are damped by injection of low concentrations of BAPTA, fish form with greatly reduced eyes and hearts. The present study provides a first overview of Ca2+ patterns during prolonged periods of vertebrate development and points to new roles of Ca2+ in cellular differentiation and pattern formation

Data_Sheet_1_Behavioral effects of visual stimuli in adult zebrafish using a novel eight-tank imaging system.ZIP

IntroductionAnimals respond to various environmental cues. Animal behavior is complex, and behavior analysis can greatly help to understand brain function. Most of the available behavioral imaging setups are expensive, provide limited options for customization, and allow for behavioral imaging of one animal at a time.MethodsThe current study takes advantage of adult zebrafish as a model organism to study behavior in a novel behavioral setup allowing one to concurrently image 8 adult zebrafish.ResultsOur results indicate that adult zebrafish show a unique behavioral profile in response to visual stimuli such as moving lines. In the presence of moving lines, the fish spent more time exploring the tank and spent more time toward the edges of the tanks. In addition, the fish moved and oriented themselves against the direction of the moving lines, indicating a negative optomotor response (OMR). With repeated exposure to moving lines, we observed a reduced optomotor response in adult zebrafish.DiscussionOur behavioral setup is relatively inexpensive, provides flexibility in the presentation of various animated visual stimuli, and offers improved throughput for analyzing behavior in adult zebrafish. This behavioral setup shows promising potential to quantify various behavioral measures and opens new avenues to understand complex behaviors.</p

The loss and recovery of vertebrate vision examined in microplates

<div><p>Regenerative medicine offers potentially ground-breaking treatments of blindness and low vision. However, as new methodologies are developed, a critical question will need to be addressed: how do we monitor <i>in vivo</i> for functional success? In the present study, we developed novel behavioral assays to examine vision in a vertebrate model system. In the assays, zebrafish larvae are imaged in multiwell or multilane plates while various red, green, blue, yellow or cyan objects are presented to the larvae on a computer screen. The assays were used to examine a loss of vision at 4 or 5 days post-fertilization and a gradual recovery of vision in subsequent days. The developed assays are the first to measure the loss and recovery of vertebrate vision in microplates and provide an efficient platform to evaluate novel treatments of visual impairment.</p></div

Visual stimuli in 6-well plates.

<p>a) View of a single well in a 6-well plate. b) Rotating cross assay using 5 larvae per well. c) Rotating red cross assay using 1 larva per well. In each color, the cross first rotates 10 minutes clockwise (cw) and then 10 minutes counter-clockwise (ccw). R, G, B, Y, C = red, green, blue, yellow, cyan. % CW = the percentage of measurements in which larvae display a clockwise orientation. Differences in the response to cw and ccw visual stimuli were tested for significance using a two-tailed t-test with unequal variance (n = 12 wells) and a Bonferroni correction for multiple comparisons when only pairwise comparisons were considered (a,b) A repeated measures ANOVA was performed to test the effect of stimulus on the % CW for 4x repeated two-bar stimulus study. When sphericity was violated, a Greenhouse-Geisser correction was used (c).</p