Behavioural phenotyping of casper mutant and 1-pheny-2-thiourea treated adult zebrafish

Abstract

The ability to visualise neural circuits in zebrafish in vivo is one of the most useful aspects of this model organism in neuroscience. To maintain the transparency of embryos, however, drugs, such as 1-pheyl-2-thiourea (PTU) must be added, or researchers can use mutants that do not develop pigment (e.g., the casper). The behavioural characteristics of such strains, however, have not been documented. Here, we tested adult zebrafish from the casper line, as well as wild-type (Tübingen, TU) and wild-types treated as embryos with PTU on three commonly used behavioural endpoints in neuroscience: novel tank test (similar to open-field in rodents), conditioned place preference for nicotine, and social cohesion (using a new method of cluster analysis). We found no differences between the casper and the TU, but the adult TU treated with PTU as embryos showed a marked increase in anxiety during the novel tank test. These data suggest that where possible, labs interested in analysis of developmental processes involved in adult phenotypes should avoid the use of PTU in favour of transparent mutants, such as casper.

FIG. 1. Cluster analysis.

Groups of n = 5 fish were observed in a rectangular tank (W × L × H: 35 × 50 × 15 cm) over a 10 min period. The tanks were split into eight equal segments (locations) for analysis. Cluster scores (Clus) were calculated for each time point (T) once every 30-s to ascertain shoaling by dividing the maximum number of fish in one location of the tank (Max_T) by the total number of segments occupied (Total_T).

FIG. 2. Tank used in novel tank test.

Fish were placed in the novel tank for 5-min and the duration (s) spent at the bottom of the tank was recorded via an automated video tracking system (Ethovision).

FIG. 3. Tank used to measure conditioned place preference (CPP).

After assessment of baseline preference for spots or stripes, fish were conditioned for 2 days to their least-preferred stimulus with 5 μM nicotine solution. Preference was then reassessed in a drug-free probe trial.

FIG. 4. Novel tank test.

Time (s) spent in the bottom third of a novel tank over a 5-min exposure for TU w/t, TU w/t treated with PTU (TU w/t + PTU) and casper mutants. Error bars represent standard error.

FIG. 5. Conditioned place preference for nicotine.

Time spent in the proximity of the conditioned cue before and after conditioning with 5 μM nicotine for 2 days for TU w/t, TU w/t treated with PTU (TU w/t + PTU) and casper mutants. Error bars represent standard error. **p < 0.01.

FIG. 6. Cluster analysis of zebrafish shoals over 10-min.

Individual group dynamics for shoals from casper, TU w/t, and TU w/t + PTU, ordered by intercept (bottom-left–top-right panels). Group cohesion (clustering) was measured every 30-s (instantaneous sampling) for a 10-min undisturbed period. Lines represent first order least-squares regression (see text for details).