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. 2020 Nov 11;10:19569. doi: 10.1038/s41598-020-76287-9

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Treatment timeline and treatment groups during the fear conditioning. (A) Schematic of treatment timeline during the fear conditioning. On Day-2, the fish was transferred to either the preferred compartment or non-preferred compartment (at random), and control fish were treated with 5-min of water in preferred and non-preferred compartments (Non-AS, Group 1). Treatment groups (Groups 2–4) were exposed to alarm substance (AS) or water, respectively for 5-min. After 5-min treatment, the fish was transferred to the other side of compartment (preferred to non-preferred or vice versa) and exposed with AS or water for 5-min followed by transferring to the acclimatisation tank (AS-treated group, Group 2). For morphine-treated group (Groups 3 and 4), after one hour of recovery from fear conditioning, the fish were then treated with morphine (2 mg/l) or vehicle (water) for 30-min and transferring to the acclimatisation (housing) tank. On Day-3, the fish were placed in the centre compartment of experimental tank with the separators. After familiarisation period, the fish was again treated with morphine before the post-conditioning test. For Kiss1-treated group (Group 4), morphine-treated fish were centrally administered with 1 µl of Kiss1 (zebrafish kisspeptin1-15) at the dose of 10^–21 mol/fish or 1 µl of water (control) before transferred to the acclimatisation (housing) tank. Four different treatment groups were summarized in (B).