Experimental design and the effects of neural hyperactivity on the levels of neurotransmitters (DA, γ-GABA, and 5-HT) in the zebrafish brain. (A) Experimental design. In the main experiment, the fish were divided into four groups. The control group was treated with normal feed and regular filtered water throughout the experiment. The caffeine, melatonin, and probiotic groups were subjected to two consecutive days of caffeine exposure and then maintained under the corresponding conditions (caffeine group: normal feed + regular filtered water, melatonin group: normal feed + filtered water with 1 μM melatonin, probiotic group: feed containing 8.0 log10 Lactobacillus plantarum HNU082/g of feed + regular filtered water). On days 1 and 14 after caffeine induction, the brain and intestine of each zebrafish were collected for neurotransmitter level determination and high-throughput sequencing, respectively. For the verification experiment, the study added the GF zebrafish groups, and the experimental design and feed conditions were the same as the main experiment. (B) Effects of neural hyperactivity on the levels of neurotransmitters (DA, γ-GABA, 5-HT) in the zebrafish brain; the variation in the secretion levels of neurotransmitters in response to melatonin and probiotic supplementation were determined by ELISA. Mann-Whitney test was used to assess the differences in neurotransmitter levels in the zebrafish brain on days 1 and 14. *p < 0.05 compared with the control group. #p < 0.05 compared with the caffeine group.
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