Figure 6

Figure 6

(A) Experimental design diagram. Fish from the stock were weight-graded and divided into control and overfeeding-induced obesity (OIO) groups. Animals were fed at 2 and 8%, respectively, for 8 weeks and sampled for morphological parameters every two weeks. Following the feeding protocol, the behavioural tests began with a novel tank diving test (NTDT). Animals were subsequently isolated in individual tanks for 24 h (n = 16 for each treatment). Fish were then tested for short-term memory (STM), and subsequently trained for 4 days for LTM tests. Finally, the fish were euthanised and sampled for morphological parameters. Eight fish were used for lipid quantification and the brains of the remaining eight fish were dissected for monoamine determination by HPLC. (B) Novel tank diving test setup. Fish were placed in trapezoidal tanks, and their trajectories were acquired for 10 min using Ethovision 16 XT. (C) Zantiks AD unit representation, used for aversive learning tests. (D) Short-term memory test protocol. Fish were habituated for 10 min to the experimental conditions in which the screen was half-divided into ‘check’ and ‘grey’ switching positions every 5 min. The potential preference for any background was recorded for 30 min (baseline). The same schedule used for the habituation period was then repeated 3 times. Throughout the conditioning phase, the conditioned stimulus (CS) was displayed for 1.5 s and paired to the unconditioned stimulus (US, 9 V DC shock for 750 ms) followed by 8.5 s of non-conditioned stimulus (‘check’ or ‘grey’). This protocol was repeated 12 times and learning and STM were tested for 2 min. (E) Long-term memory test protocol. Fish were initially trained for 4 consecutive days. The conditioning phase was followed by an inter-trial interval (ITI) of 15 min to allow memory consolidation, and the probe phase was performed 24 h after the last training session. The CS was displayed for 5 s and linked to the US (5 shocks of 9 V DC, 750 ms each). This protocol was repeated 12 times and subsequently non-CS was shown for 1 min followed by the ITI (absence of stimulus). Conditioning + ITI were repeated 6 times for each training session. Finally, 24 h after the last training session, the probe phase took place. In both STM and LTM tests, the preference for conditioned stimulus (PCS) was calculated as total time in CS/(time in CS + time in non-CS). Significant differences between baseline and probe PCSs were analysed. The significantly lower probe PCS, the memory enhancer, was achieved.