Abstract
New environments are known to be anxiogenic initially for many animals including the zebrafish. In the zebrafish, a novel tank diving (NTD) assay for solitary fish has been used extensively to model anxiety and the effect of anxiolytics. However, studies can differ in the conditions used to perform this assay. Here, we report the development of an efficient, automated toolset and optimal conditions for effective use of this assay. Applying these tools, we found that two important variables in previous studies, the direction of illumination of the novel tank and the age of the subject fish, both influence endpoints commonly measured to assess anxiety. When tanks are illuminated from underneath, several parameters such as the time spent at the bottom of the tank, or the transitions to the top half of the tank become poor measures of acclimation to the novel environment. Older fish acclimate faster to the same settings. The size of the novel tank and the intensity of the illuminating light can also influence acclimation. Among the parameters measured, reduction in the frequency of erratic swimming (darting) is the most reliable indicator of anxiolysis. Open source pipeline for automated data acquisition and systematic analysis generated here and available to other researchers will improve accessibility and uniformity in measurements. They can also be directly applied to study other fish. As this assay is commonly used to model anxiety phenotype of neuropsychiatric ailments in zebrafish, we expect our tools will further aid comparative and meta-analyses.
Highlights
Unfamiliar surroundings elicit a response of cautious exploration among animals
We find that illumination direction changes time spent and distance traversed in the bottom of the tank and the frequency of transitions to the top—that is, it affects most measures used to ascertain the level of anxiety in zebrafish
We matched the conditions of the experiment described by Levin et al (2007), who introduced the test in the form used most commonly and examined the subjects for 10-min with the expectation that fish acclimate to the novel environment over this time
Summary
Unfamiliar surroundings elicit a response of cautious exploration among animals. Among these, the open field test, originally introduced in 1934, Hall (1934) examines the motivational drive. Tanks can be backlit while observing or video recording from the front (Bencan and Levin, 2008; Bencan et al, 2009; Pittman and Ichikawa, 2013), or lit from the top in a darkened room (Maximino et al, 2011, 2013a,b), or may be placed in ambient light (Egan et al, 2009; Cachat et al, 2010, 2013; Sackerman et al, 2010) with a light reflective surface at the bottom or at the tank’s back wall, or left undescribed (Grossman et al, 2010; Wong et al, 2010; Khor et al, 2011; Parker et al, 2013, 2014; Kulkarni et al, 2014; Mezzomo et al, 2016) Whether these differences influence the endpoints measured has not been systematically evaluated. We expect these will allow more reliable and consistent phenotyping in studies using innate anxiety tests to investigate the genetics of comorbid mental disorders (Blaser and Rosemberg, 2012; Stewart et al, 2012; Kim et al, 2014; Meshalkina et al, 2018)
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