Getting more out of the zebrafish light dark transition test

Haigis, Ann-Cathrin; Ottermanns, Richard; Schiwy, Andreas; Hollert, Henner; Legradi, Jessica

doi:10.1016/j.chemosphere.2022.133863

Cited by 21 publications

(23 citation statements)

References 57 publications

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“…7 and 10). Darting swimming is an important swim factor to analyse because it is an indicator of the initial dark escape/light-seeking response as well as a sign of anxiety-like responses (Haigis et al, 2022). Slow swimming responses highlight more clearly any reduced swim response whilst zero movements show the innate freezing common with zebrafish as well as complete loss of movement because of sedation, paralysis or death (Kalueff et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

“…Previous publications have lacked consistency with respect to the length and number of light/dark transition periods for assays using 4 dpf zebrafish larvae (Table 1). Therefore, we assessed the optimal length of light to dark transition periods by replicating the protocols employed by Acevedo-Canabal (2019), Bachour (2020) and Haigis (2022), as well as assessing our own protocol. There were reproducible and robust dark phase locomotor responses with all transition periods.…”

Section: Five-minute Light/dark Protocol Produces a Robust Response I...mentioning

confidence: 99%

“…There is also the potential for the model to be used for toxicology and ecotoxicology research (Haigis et al, 2022). Previous research has identified the use of the light/dark assay for assessing anxiogenic and anxiolytic substances (Facciol et al, 2019).…”

Section: Potential Applicationsmentioning

confidence: 99%

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A Unified Approach to Investigating 4 dpf Zebrafish Larval Behaviour through a Standardised Light/Dark Assay

Hillman,

Kearn,

Parker

2023

Preprint

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Zebrafish have emerged as a dynamic research model in the domains of psychiatry, neuroscience and behaviour. Working with larvae ≤ 4 days post-fertilisation (dpf) offers an avenue for high-throughput investigation whilst aligning with the 3Rs principles of animal research. The light/dark assay, which is the most used behavioural assay for larval research, lacks experimental reliability and standardisation. This study aimed to formulate a robust, reproducible and standardised light/dark behavioural assay using 4 dpf zebrafish larvae. Considerable between-batch and inter-individual variability was found which we rectified with a normalisation approach to ensure a reliable foundation for analysis. We then identified that 5-minute light/dark transition periods are optimal for locomotor activity. No discernible impact of acclimation (length or lighting conditions) on larval performance was observed. Overall locomotion was found to be significantly greater in the early afternoon compared to morning and evening, but time of day of testing had no impact on light/dark transitions. Next, we confirmed the pharmacological predictivity of the standardised assay using ethanol which, as predicted, caused hyperlocomotion at low concentrations and hypolocomotion at high concentrations. Finally, the assay was validated by assessing the behavioural phenotype of hyperactive transgenic (adgrl3.1-/-) larvae, which was rescued with psychostimulant medications. Our standardised assay not only provides a clear experimental and analytical framework to work with 4 dpf larvae, but also facilitates between-laboratory collaboration using our normalisation approach.

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Section: Discussionmentioning

confidence: 99%

Section: Five-minute Light/dark Protocol Produces a Robust Response I...mentioning

confidence: 99%

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A Unified Approach to Investigating 4 dpf Zebrafish Larval Behaviour through a Standardised Light/Dark Assay

Hillman,

Kearn,

Parker

2023

Preprint

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“…In the present analyses, only two endpoints (total locomotor activity in either the Light or Dark period) are used to assess the Light/Dark locomotor response data. As the full 100 min, light/dark behavioral profiles are quite complex ( Supplementary Figure S2 ), there are many other behavioral endpoints to be captured and analyzed (e.g., [ 84 , 85 , 86 ]). Perhaps the larval zebrafish behavioral assessment community can capture those other behavioral characteristics in an organized and consistent manner so that “behavioral barcodes” linked to modes of action can be developed, much like how the acute effects of neuroactive chemicals have been indexed to unique behavioral patterns (e.g., [ 87 , 88 ]).…”

Section: Discussionmentioning

confidence: 99%

Developmental Neurotoxicity and Behavioral Screening in Larval Zebrafish with a Comparison to Other Published Results

Jarema

Hunter

Hill

et al. 2022

Toxics

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With the abundance of chemicals in the environment that could potentially cause neurodevelopmental deficits, there is a need for rapid testing and chemical screening assays. This study evaluated the developmental toxicity and behavioral effects of 61 chemicals in zebrafish (Danio rerio) larvae using a behavioral Light/Dark assay. Larvae (n = 16–24 per concentration) were exposed to each chemical (0.0001–120 μM) during development and locomotor activity was assessed. Approximately half of the chemicals (n = 30) did not show any gross developmental toxicity (i.e., mortality, dysmorphology or non-hatching) at the highest concentration tested. Twelve of the 31 chemicals that did elicit developmental toxicity were toxic at the highest concentration only, and thirteen chemicals were developmentally toxic at concentrations of 10 µM or lower. Eleven chemicals caused behavioral effects; four chemicals (6-aminonicotinamide, cyclophosphamide, paraquat, phenobarbital) altered behavior in the absence of developmental toxicity. In addition to screening a library of chemicals for developmental neurotoxicity, we also compared our findings with previously published results for those chemicals. Our comparison revealed a general lack of standardized reporting of experimental details, and it also helped identify some chemicals that appear to be consistent positives and negatives across multiple laboratories.

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“…This includes the photomotor response that occurs in 1-2 dpf larvae, 44,45 phototaxis with a preference towards light, 24 and the light-to-dark transition. 46 Even within the light-dark transition space, there are variations in design. For example, we used a short exposure to white light on dark-acclimated larval fish preceding an extended dark phase.…”

Section: Discussionmentioning

confidence: 99%

The canonical HPA axis contributes to locomotion during photoadaptation but is not required

Lee

Thi

Boyum

et al. 2022

Preprint

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The hypothalamic-pituitary-adrenal (HPA) axis and its effector molecules—glucocorticoids—modulate diverse aspects of physiology in vertebrates ranging from metabolism to immune function, behavior, and circadian cycle. While the glucocorticoid receptor (nr3c1) is known to be involved in light adaptation (photoadaptation) of the retinal cells, the role of nr3c1 and other HPA axis signaling molecules in the behavioral phenotypes observed during photoadaptation have not been delineated. Therefore, we investigated locomotor adaptation to various light/dark durations using larval zebrafish that carry a mutated allele in key HPA axis receptors. First, we established baseline locomotion and compared WT and nr3c1 mutant larvae in constantly lit and dark conditions (for 12-hrs). WT larval zebrafish showed highest locomotor activity in the middle of the day and low activity during the early and later parts of the day, which was generally higher in the light. The baseline locomotion was depressed in nr3c1 mutants throughout the day in both environments with a more significant change noted in the light. Next, groups of larvae mutant in nr3c1, nr3c2 (mineralocorticoid receptor), or mc2r (melanocortin receptor type 2; adrenocorticotropic hormone (ACTH) receptor) along with their wildtype (WT) siblings were acclimated in the dark and underwent four cycles of dark-light illumination changes with different durations of illumination: 7.5, 6, 4, or 2 min. The nr3c1 and mc2r mutant fish showed significantly decreased locomotion during the dark phase when the illumination was provided for 4 or 2 min. However, with 4 min or longer illumination, if locomotor deficits were observed in mutant larvae, they demonstrated a “catch-up” phenotype with increasing locomotion during the later dark phases of the assay and ultimately reaching the swimming distances indistinguishable from their WT siblings in the assays with 7.5-min illumination. Finally, we looked at effects of light intensity, beginning with our short light exposure. A lower intensity of light failed to elicit any response even from WT fish after 1-min illumination. However, this dim light still evoked a robust response from nr3c1 mutants and their WT siblings after 7.5-min illumination. The nr3c2 mutant larvae consistently showed locomotor response similar to their WT siblings regardless of the length of the light phase. Thus, activation of the canonical HPA axis (i.e. nr3c1, mc2r) was necessary to induce a rapid locomotor adaptation following after light to dark transition with shorter light exposure times (~≤4 min), but it was not needed for locomotor responses observed after longer exposure to light (~> 4 min). Moreover, the locomotor response after transitioning to dark from a short (1 min) exposure was dependent on the intensity of light, whereas the longer exposure was not. Together, these findings suggest that locomotion after longer exposure to light is not dependent on the HPA axis, suggesting that either parallel independent pathway(s) are responsible for the locomotor response in these cases or that the HPA axis facilitates a primary pathway to increase sensitivity to light exposure.

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Getting more out of the zebrafish light dark transition test

Cited by 21 publications

References 57 publications

A Unified Approach to Investigating 4 dpf Zebrafish Larval Behaviour through a Standardised Light/Dark Assay

A Unified Approach to Investigating 4 dpf Zebrafish Larval Behaviour through a Standardised Light/Dark Assay

Developmental Neurotoxicity and Behavioral Screening in Larval Zebrafish with a Comparison to Other Published Results

The canonical HPA axis contributes to locomotion during photoadaptation but is not required

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