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Measuring behavioral and endocrine responses to novelty stress in adult zebrafish

Abstract

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the 'novel tank' test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.

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Figure 1: Novel tank test for behavioral testing in adult zebrafish.
Figure 2: Behavioral effects of anxiolytic and anxiogenic manipulations in adult zebrafish tested in the 6-min novel tank test.
Figure 3: Typical representative trace images of zebrafish behavior in the 6-min novel tank test, generated by CleverSys or Noldus-based video-tracking systems.
Figure 4: Endocrine responses (whole-body cortisol levels, assessed by ELISA assay) to various experimental manipulations in adult zebrafish.
Figure 5: Temporal three-dimensional (3D) reconstructions of zebrafish traces.

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Acknowledgements

This study was supported by Tulane University's Gordon Fellowship and the Georges Lurcy Fellowship as well as Provost's Scholarly Enrichment Fund, Newcomb Fellows Grant, Louisiana Board of Regents' fund grant and National Alliance for Research on Schizophrenia and Depression (NARSAD) Young Investigator awards. The colors of the bar graphs were inspired by the New Orleans Saints.

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Contributions

A.S. organized the protocol, analyzed the data and drafted the paper. J.C. developed the methodology, analyzed the data and contributed to drafting and editing the paper. A.V.K. contributed to drafting and editing the paper, generating and analyzing the data and general oversight of the project. All other authors contributed to data collection, analysis, interpretation and paper preparation.

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Correspondence to Allan V Kalueff.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

Typical patterns of a Zebrafish novel tank test behavior (MOV 26088 kb)

Supplementary Information

TopScan is an alternative video-tracking program that allows the user to define the parameters and calibrate the system to analyze key locomotory and behavioral endpoints. This software can be used in lieu of Ethovision XT7 detailed in the protocol. (DOC 28 kb)

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Cachat, J., Stewart, A., Grossman, L. et al. Measuring behavioral and endocrine responses to novelty stress in adult zebrafish. Nat Protoc 5, 1786–1799 (2010). https://doi.org/10.1038/nprot.2010.140

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