An Automated Assay System to Study Novel Tank Induced Anxiety

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.

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“…A novel tank task and mirror test are used to measure anxiety ( Figure 1 ). Zebrafish that exhibit high anxiety levels spend more time at the edge of a novel tank than at the center (thigmotaxis) and the bottom of the tank than on the top ( Haghani et al, 2019 ). Aberrant swimming behaviors, such as increased freezing bouts and reduced inter-distance between fishes in a shoal also correspond to the anxiety level ( Speedie and Gerlai, 2008 ; Blaser et al, 2010 ).…”

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“…Normally, when put in a new environment, Danio rerio initially spend most of the time in the “protection zone” (i.e., in a lower part of the tank), and after they have acclimatized to the new place, they begin exploring it. The most important parameters recorded in the NTT that describe the anxiety-/depression-like behavior of tested subjects are the time spent in the upper and lower parts of the tank, number of entries to the upper part, delay in entering the upper part, freezing time, irregular movements, immobility, swimming speed, and travelled distance [ 33 , 51 , 52 ]. Many researchers underline an additional advantage of this test carried out in zebrafish, that is not attributed to experiments in rodents, i.e., the possibility to analyze the animal’s behavior in three dimensions, which provides more relevant data [ 53 , 54 ].…”

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“…The behavior of the fish was monitored for the next 5 min using the Noldus EthoVision XT system (Wageningen, Netherlands), with the camera placed approximately 1 m from the test tank. The tanks were uniformly illuminated from above at approximately 200 lx, reported to be the optimal illumination condition for NTT [ 23 ]. Background noise was kept at a minimum during the test.…”