The role of telomerase reverse transcriptase (TERT) has been widely investigated in the contexts of aging and age-related diseases. Interestingly, decreased telomerase activities (and accelerated telomere shortening) have also been reported in patients with emotion-related disorders, opening the possibility for subjective appraisal of stressful stimuli playing a key role in stress-driven telomere shortening. In fact, patients showing a pessimistic judgment bias have shorter telomeres. However, these evidences in humans are correlational and the causal inference of pessimism driving shortening of telomeres has not been established experimentally yet. We have developed and validated a judgment bias experimental paradigm to measure subjective evaluations of ambiguous stimuli in zebrafish. This behavioral assay allows classifying individuals in an optimistic-pessimistic dimension (i.e. from individuals that consistently evaluate ambiguous stimuli as negative to others that perceived them as positive). Using this behavioral paradigm we found that telomerase-deficient zebrafish (tert^−/−) were more pessimistic in response to ambiguous stimuli than WT zebrafish. The fact that individuals with constitutive shorter telomeres have pessimistic behaviors demonstrate for the first time in a vertebrate model a genetic basis of judgment bias.

Fish and Housing

Fish used for the development and validation of the judgment bias paradigm were 4 months old male wild-type (Tübingen strain) zebrafish (Danio rerio). The telomerase mutant line tert^AB/hu3430 was used to assess the role of telomerase in the modulation of judgment biases that produce subjective evaluations. All fish were bred and held at Instituto Gulbenkian de Ciência (IGC, Oeiras, Portugal). 

Ethical Statement

All procedures were performed in accordance with Institutional and National regulations and guidelines, reviewed by the Ethics Committee of the Instituto Gulbenkian de Ciência, and approved by the competent Portuguese authority (Direcção Geral de Alimentação e Veterinária).

Judgment bias test

In this study, we have developed and validated a judgment bias test for zebrafish that is based on the judgment bias experimental paradigm published by Harding et al. [4]. In this discrimination task, fish are trained to perform one response (Positive (P)) when one cue is presented (specific location/color cue) in order to experience a positive event. Furthermore, fish are also trained to perform a different response (Negative (N)) when presented with a different cue in order to avoid a negative event. For technical reasons, the responses used in this study were lower (P) or higher (N) latencies in entering the experimental arms of the behavioral apparatus. Once fish were trained on this discrimination task, they were then tested by exposing them to occasional unreinforced ambiguous cues.

Behavioral observations

The latency to enter in the target arm (60 s maximum) was recorded for each trial. Video recordings were analyzed by using computerized multi-event recorder software (The Observer XT, Noldus technology, version 9).

Statistical analyses

The validation of the judgment bias paradigm was analyzed by one-way analysis of variance (ANOVA) followed by the Holm–Sidak post hoc test. The effects of Genotype (tert^-/- mutants and WT) and Age (4 and 9 months old) on judgment bias were analyzed using two-way ANOVA followed by Holm–Sidak post hoc tests. Behavioral data of tert^-/- mutants and WT siblings were logarithmically transformed to meet the assumptions for parametric testing.