Experimental dynamics of morphological parameters. Fish were sampled every two weeks in 1, 2, 3, and NT (NTDT testing day) and one week between NT and LTM (long-term memory test probe phase). (A) Weight, (B) length, and (C) biomass index (BMI). (D) Representative images of control and OIO females and males at the end of the study. Data are expressed by mean ± SEM and analysed by two-way ANOVA and Tukey’s multiple comparison test. Asterisks indicate significant differences between OIO and control within the same gender (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001), and # p = 0.051.

(A) The percentage of total lipids. (B) Triglyceride content (n = 8 per treatment, sex ratio 50:50). Data are expressed by mean ± SEM and analysed by an unpaired t-test (* p ≤ 0.05).

Novel tank diving test (NTDT). Anxiety-like behaviours were measured in three zones: bottom, middle, and top. (A) Distance travelled, (B) mean velocity per zone, (C) angular velocity, and (D) time spent in each zone. (E) Representative heatmaps of fish movements. The colour scale represents the cumulative time spent in each of the previously defined zones: bottom, middle, and top. Data are represented by mean ± SEM and analysed by two-way ANOVA. Asterisks indicate significant differences (* p ≤ 0.05, ** p ≤ 0.01), and the hashtag indicates an apparent significant difference (# p = 0.051–0.06).

Brain serotoninergic and dopaminergic metabolites. (A) 5-hydroxytryptophan (5HTP), serotonin (5HT), and 5HIAA/5HT ratio. (B) Levels of L-dihydroxyphenylalanine (L-dopa), dopamine (DA), and dihydroxyphenylacetic acid (DOPAC) and DOPAC/DA ratio (n = 8 each per treatment, sex ratio 50:50). Results are expressed in pg of metabolite per mg of total protein. Data are represented by mean ± SEM analysed by an unpaired t-test.

Effects of obesity on short- and long-term memory. (A) Short-term memory (STM) and (B) long-term memory (LTM). PCS, preference for conditioned stimulus. Data are represented by mean ± SEM. The dotted line indicates no stimulus preference (PCS = 0.5); low and high PCS mean less or more time spent in conditioned stimulus (CS), respectively. Results were analysed by repeated measures mixed-effect model and, when interaction was significant, uncorrected Fisher’s LSD test for multiple comparisons. Asterisks indicate significant differences between phases, baselines, and probes (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001), and letters indicate significant differences between control and OIO probes.

(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.