A method for detailed movement pattern analysis of tadpole startle response

Abstract

Prolonged space flight, specifically microgravity, presents a problem for space exploration. Animal models with altered connections of the vestibular ear, and thus altered gravity sensation, would allow the examination of the effects of microgravity and how various countermeasures can establish normal function. We describe an experimental apparatus to monitor the effects of ear manipulations to generate asymmetric gravity input on the tadpole escape response. To perform the movement pattern analysis, an imaging apparatus was developed that uses a high-speed camera to obtain time-resolved, high-resolution images of tadpole movements. Movements were recorded in a temperature-controlled test chamber following mechanical stimulation with a solenoid actuator, to elicit a C-start response. Temperature within the test cell was controlled with a recirculating water bath. Xenopus laevis embryos were obtained using a standard fertilization technique. Tadpole response to a controlled perturbation was recorded in unprecedented detail and the approach was validated by describing the distinct differences in response between normal and one-eared tadpoles. The experimental apparatus and methods form an important element of a rigorous investigation into the response of the tadpole vestibular system to mechanical and biochemical manipulations, and can ultimately contribute to improved understanding of the effects of altered gravity perception on humans.

Sonic hedgehog antagonists reduce size and alter patterning of the frog inner ear

“… Impact of Vismodegib treatement on tadpole swimming behavior. A, Schematic of imaging apparatus utilized for recording tadpole swimming behavior and the C‐start response following a controlled perturbation, as described by Zarei et al (). B and C, Images of the first 25 frames captured following initiation of the stimulus for both control animals (B) and animals treated with 6.25 µM Vismodegib (C).…”

“…In addition, we will investigate general phenotypic changes following treatment of Vismodegib, such as effects on olfactory, eye, hypaxial muscle, and brain development. Finally, using a newly developed imaging apparatus (Zarei et al, ), we report how mild phenotypic changes affect the swimming behavior of the tadpole, presumably through affecting the inner ear/Mauthner cell interactions mediating the fast escape response. These insights will guide follow up work assessing how disrupted ear development affects swimming behavior after grafting Vismodegib‐treated ears onto control Xenopus (Elliott et al, ,)…”

“…A total of 17 tadpoles (seven controls and ten animals treated with 6.25 µM Vismodegib) were used to study the effect of Shh inhibition on the escape behavior. Individual tadpoles were transferred to an experimental chamber to perform movement pattern analysis, as described (Zarei et al, ), and were allowed to adapt for approximately ten minutes before testing. The imaging apparatus uses a high‐speed camera to obtain real‐time, high‐resolution images of tadpole movements, recorded in a temperature‐controlled test chamber following mechanical stimulation with a solenoid actuator, to elicit a C‐start response.…”

“…Following the completion of each trial, each animal was allowed to recover for a minimum of 3–5 min prior to the start of the subsequent trial. Response time was determined as previously described (Zarei et al, ) and means were compared using a two‐sample t ‐test assuming unequal variances.…”

“…Following image acquisition, image montages for each trial were constructed consisting of the tadpole's movement in the first 2.5 ms following application of the stimulus for each of the four trials. C‐start response times for each behavioral sequence and flexion angles for each still frame were quantified using the same manual grading system defined in Zarei et al (). The maximum flexion angle, as measured from each animal's tail to the tip of the nose, was then identified for each trial.…”