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Statistical Analysis of Zebrafish Locomotor Response.

Liu Y, Carmer R, Zhang G, Venkatraman P, Brown SA, Pang CP, Zhang M, Ma P, Leung YF - PLoS ONE (2015)

Bottom Line: The resulting time-series locomotor data have been used to reveal new insights into neurobiology and pharmacology.The results indicate that the larval activity was generally affected by stage, light stimulus, their interaction, and location in the plate.Intriguingly, this analysis also shows that biological and technical repeats had negligible effect on larval activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Statistics, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Zebrafish larvae display rich locomotor behaviour upon external stimulation. The movement can be simultaneously tracked from many larvae arranged in multi-well plates. The resulting time-series locomotor data have been used to reveal new insights into neurobiology and pharmacology. However, the data are of large scale, and the corresponding locomotor behavior is affected by multiple factors. These issues pose a statistical challenge for comparing larval activities. To address this gap, this study has analyzed a visually-driven locomotor behaviour named the visual motor response (VMR) by the Hotelling's T-squared test. This test is congruent with comparing locomotor profiles from a time period. Different wild-type (WT) strains were compared using the test, which shows that they responded differently to light change at different developmental stages. The performance of this test was evaluated by a power analysis, which shows that the test was sensitive for detecting differences between experimental groups with sample numbers that were commonly used in various studies. In addition, this study investigated the effects of various factors that might affect the VMR by multivariate analysis of variance (MANOVA). The results indicate that the larval activity was generally affected by stage, light stimulus, their interaction, and location in the plate. Nonetheless, different factors affected larval activity differently over time, as indicated by a dynamical analysis of the activity at each second. Intriguingly, this analysis also shows that biological and technical repeats had negligible effect on larval activity. This finding is consistent with that from the Hotelling's T-squared test, and suggests that experimental repeats can be combined to enhance statistical power. Together, these investigations have established a statistical framework for analyzing VMR data, a framework that should be generally applicable to other locomotor data with similar structure.

No MeSH data available.


Related in: MedlinePlus

VMR experimental scheme.The experimental scheme for the VMR assay was adopted from Emran and colleague (2008). In the scheme, the larvae arrayed in the 96-well plate were first dark adapted for 3.5 hrs (long black bar on the left). Then, they were subjected to three consecutive trials of light onset (Light-On; grey bars) and light offset (Light-Off; short black bars). Each Light-On or Light-Off session lasted for 30 mins. In this study, we extracted the data from 30 s before light change (red bars; not to scale) to 30 s after light change (blue bars; not to scale) for statistical analyses. In some cases, the analyses separately handled the extracted data around the Light-On stimulus (yellow boxes) and Light-Off stimulus (green boxes). Furthermore, in our MANOVA models, the effect of three consecutive trials was explicitly evaluated (purple boxes in different colour values), regardless of the nature of light change.
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pone.0139521.g001: VMR experimental scheme.The experimental scheme for the VMR assay was adopted from Emran and colleague (2008). In the scheme, the larvae arrayed in the 96-well plate were first dark adapted for 3.5 hrs (long black bar on the left). Then, they were subjected to three consecutive trials of light onset (Light-On; grey bars) and light offset (Light-Off; short black bars). Each Light-On or Light-Off session lasted for 30 mins. In this study, we extracted the data from 30 s before light change (red bars; not to scale) to 30 s after light change (blue bars; not to scale) for statistical analyses. In some cases, the analyses separately handled the extracted data around the Light-On stimulus (yellow boxes) and Light-Off stimulus (green boxes). Furthermore, in our MANOVA models, the effect of three consecutive trials was explicitly evaluated (purple boxes in different colour values), regardless of the nature of light change.

Mentions: The VMR assay was implemented based on the design by Emran and colleagues [5,12] as described below. The assay was conducted inside the ZebraBox system (ViewPoint Life Sciences, Lyon, France). In the system, the 96-well plate with the animals was isolated from environmental light, and stimulated by white light emitted by a light-controlling unit from the bottom of the plate. The animal movement was recorded by an infra-red camera at a rate of 30 frames per second under infra-red light illumination at 850 nm, which the animals could not perceive. Before the actual experiment, the 96-well plate with the animals was placed in the ZebraBox system for 3.5 hours of dark adaption to acclimatize the animals. The data collection was started at 0.5 hours before the first light onset. The actual test consisted of three consecutive trials of light onset (Light-On) and light offset (Light-Off) periods with each period lasted for 30 minutes (Fig 1). The light change (On or Off) was abrupt and was not fading. The Light-On stimulus was set at 100% of the output intensity, which was measured by a LX1010B light meter (Mastech, Taipei, Taiwan). The measurements were taken at nine evenly distributed locations across the surface of the light-controlling unit that would be covered by the 96-well plate. The mean illuminance of these nine locations was 1390.94 Lux and the standard deviation of the measurement was 155.05 Lux. Using this experimental scheme, the VMR was measured from the three WT strains. For each strain, two biological repeats were conducted. Each repeat was started with 96 embryos in the 96-well plate at 3 dpf. The VMR of the same animals in each biological repeat was measured from 3 to 9 dpf. In each day, the assay was started at around the same time at 2 p.m. The health of the animals was also inspected every day. During the inspection, half of the E3 medium was changed. Embryos or larvae that showed any signs of abnormality during the course of behavioural experiment were excluded from the final data analysis. The remaining healthy embryos were not fed during the experimental period to avoid any confounding errors introduced by feeding. All data are available at the Harvard Dataverse (http://dx.doi.org/10.7910/DVN/HTXXKW).


Statistical Analysis of Zebrafish Locomotor Response.

Liu Y, Carmer R, Zhang G, Venkatraman P, Brown SA, Pang CP, Zhang M, Ma P, Leung YF - PLoS ONE (2015)

VMR experimental scheme.The experimental scheme for the VMR assay was adopted from Emran and colleague (2008). In the scheme, the larvae arrayed in the 96-well plate were first dark adapted for 3.5 hrs (long black bar on the left). Then, they were subjected to three consecutive trials of light onset (Light-On; grey bars) and light offset (Light-Off; short black bars). Each Light-On or Light-Off session lasted for 30 mins. In this study, we extracted the data from 30 s before light change (red bars; not to scale) to 30 s after light change (blue bars; not to scale) for statistical analyses. In some cases, the analyses separately handled the extracted data around the Light-On stimulus (yellow boxes) and Light-Off stimulus (green boxes). Furthermore, in our MANOVA models, the effect of three consecutive trials was explicitly evaluated (purple boxes in different colour values), regardless of the nature of light change.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4593604&req=5

pone.0139521.g001: VMR experimental scheme.The experimental scheme for the VMR assay was adopted from Emran and colleague (2008). In the scheme, the larvae arrayed in the 96-well plate were first dark adapted for 3.5 hrs (long black bar on the left). Then, they were subjected to three consecutive trials of light onset (Light-On; grey bars) and light offset (Light-Off; short black bars). Each Light-On or Light-Off session lasted for 30 mins. In this study, we extracted the data from 30 s before light change (red bars; not to scale) to 30 s after light change (blue bars; not to scale) for statistical analyses. In some cases, the analyses separately handled the extracted data around the Light-On stimulus (yellow boxes) and Light-Off stimulus (green boxes). Furthermore, in our MANOVA models, the effect of three consecutive trials was explicitly evaluated (purple boxes in different colour values), regardless of the nature of light change.
Mentions: The VMR assay was implemented based on the design by Emran and colleagues [5,12] as described below. The assay was conducted inside the ZebraBox system (ViewPoint Life Sciences, Lyon, France). In the system, the 96-well plate with the animals was isolated from environmental light, and stimulated by white light emitted by a light-controlling unit from the bottom of the plate. The animal movement was recorded by an infra-red camera at a rate of 30 frames per second under infra-red light illumination at 850 nm, which the animals could not perceive. Before the actual experiment, the 96-well plate with the animals was placed in the ZebraBox system for 3.5 hours of dark adaption to acclimatize the animals. The data collection was started at 0.5 hours before the first light onset. The actual test consisted of three consecutive trials of light onset (Light-On) and light offset (Light-Off) periods with each period lasted for 30 minutes (Fig 1). The light change (On or Off) was abrupt and was not fading. The Light-On stimulus was set at 100% of the output intensity, which was measured by a LX1010B light meter (Mastech, Taipei, Taiwan). The measurements were taken at nine evenly distributed locations across the surface of the light-controlling unit that would be covered by the 96-well plate. The mean illuminance of these nine locations was 1390.94 Lux and the standard deviation of the measurement was 155.05 Lux. Using this experimental scheme, the VMR was measured from the three WT strains. For each strain, two biological repeats were conducted. Each repeat was started with 96 embryos in the 96-well plate at 3 dpf. The VMR of the same animals in each biological repeat was measured from 3 to 9 dpf. In each day, the assay was started at around the same time at 2 p.m. The health of the animals was also inspected every day. During the inspection, half of the E3 medium was changed. Embryos or larvae that showed any signs of abnormality during the course of behavioural experiment were excluded from the final data analysis. The remaining healthy embryos were not fed during the experimental period to avoid any confounding errors introduced by feeding. All data are available at the Harvard Dataverse (http://dx.doi.org/10.7910/DVN/HTXXKW).

Bottom Line: The resulting time-series locomotor data have been used to reveal new insights into neurobiology and pharmacology.The results indicate that the larval activity was generally affected by stage, light stimulus, their interaction, and location in the plate.Intriguingly, this analysis also shows that biological and technical repeats had negligible effect on larval activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Statistics, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
Zebrafish larvae display rich locomotor behaviour upon external stimulation. The movement can be simultaneously tracked from many larvae arranged in multi-well plates. The resulting time-series locomotor data have been used to reveal new insights into neurobiology and pharmacology. However, the data are of large scale, and the corresponding locomotor behavior is affected by multiple factors. These issues pose a statistical challenge for comparing larval activities. To address this gap, this study has analyzed a visually-driven locomotor behaviour named the visual motor response (VMR) by the Hotelling's T-squared test. This test is congruent with comparing locomotor profiles from a time period. Different wild-type (WT) strains were compared using the test, which shows that they responded differently to light change at different developmental stages. The performance of this test was evaluated by a power analysis, which shows that the test was sensitive for detecting differences between experimental groups with sample numbers that were commonly used in various studies. In addition, this study investigated the effects of various factors that might affect the VMR by multivariate analysis of variance (MANOVA). The results indicate that the larval activity was generally affected by stage, light stimulus, their interaction, and location in the plate. Nonetheless, different factors affected larval activity differently over time, as indicated by a dynamical analysis of the activity at each second. Intriguingly, this analysis also shows that biological and technical repeats had negligible effect on larval activity. This finding is consistent with that from the Hotelling's T-squared test, and suggests that experimental repeats can be combined to enhance statistical power. Together, these investigations have established a statistical framework for analyzing VMR data, a framework that should be generally applicable to other locomotor data with similar structure.

No MeSH data available.


Related in: MedlinePlus