Alteration of neurotransmitters in the zebrafish brain
To investigate the effects of different treatments on neurotransmitter secretion in the zebrafish head, the levels of neural hyperactivity-related neurotransmitters in each group were determined (Fig. 1B). On day 1, the concentration of DA in the caffeine, melatonin and probiotic groups was significantly increased, while the concentrations of γ-GABA decreased, and the concentration of 5-HT did not change significantly compared with those in the control group. The imbalance indicated that caffeine had successfully induced neurotransmitter secretion disorders in the zebrafish brain. After 14 days of equal treatment in different experimental groups, the level of the neurotransmitters in each group changed. In the caffeine group, the contents of DA, γ-GABA, and 5-HT returned to almost normal levels. While the level of DA gradually returned to normal in the melatonin group. The γ-GABA levels in the melatonin and probiotic groups were significantly higher than those in the control group (145.98%, p=0.0456; 154.66%, p=0.0414), and the concentration of 5-HT in the melatonin group was markedly higher than that in the remaining three groups, which rose 212.2% compared with the average level (p=0.0162).
Effects of different treatments on altered zebrafish intestinal microbiota composition
The distance based on weighted and unweighted UniFrac in Fig. 2A and Fig. S1 indicated that the distance between control and treatment groups narrowed gradually with the extension of the treatment time. The distance between the control and melatonin group on day 14 was the smallest, followed by the probiotic group, and then the caffeine group. PCA presented in Fig. S2 also demonstrated that the points representing caffeine, melatonin, and probiotic groups were highly disordered but different from the control group on day 1, this indicated that the intestinal microbiota of zebrafish was disturbed by caffeine. After 14 days of corresponding treatment, the microbial community structure in each treatment group was similar to that in the control group after 14 days of treatment, the melatonin group had the highest similarity with the control group, followed by the probiotic and caffeine groups. Taken together, these data suggested that caffeine induction results in some destabilization in the zebrafish microbial community, while melatonin and probiotic supplementation regulated this effect.
Comparison of the core microbiota and different microbes among the different treatment groups in each timepoint
Based on the high-throughput 16S rRNA sequencing data, the effects of different treatments on the gut microbiota of zebrafish were evaluated, and the alpha and beta diversity of the zebrafish intestinal microbiota in different groups were compared on day 1and on day 14 (Table S1-S2). After 14 days of equal treatment, the Chao1, ACE, Shannon, and Simpson indices in the caffeine group were significantly higher than those in the control group. Moreover, supplementation with melatonin decreased intestinal microbial diversity compared with that in the caffeine group, while that in the probiotic group remained at a high level. Since different treatments had specific effects on the intestinal microbiota of zebrafish, we next selected the core genera accounting for more than 1% in each group after 14 days of treatment for comparative analysis (Fig. 2B), and further screened the differential genus at each time point to observe the variation of specific genus with time in each group (Fig. 3A and Fig. S3). Based on determining the differences in microbial composition, we further refined the differential genera among the groups on day 1 and day 14 (Table 1, Table S3 and Table S4). After two-day caffeine exposure, the intestinal microbial community structure of zebrafish was disturbed. Many of the differentially abundant bacterial genera showed a tendency to decrease compared with those in the control group (p<0.05). After 14 days of different treatments, the intestinal microbiota composition of zebrafish in each experimental group changed in different ways (Table S5). There was no significant difference compared with those in the control group, indicating that melatonin supplementation gradually reversed caffeine-induced intestinal microbiota dysbiosis. For the probiotic group, we observed that probiotics introduced differentially abundant genera, and there were 6 differentially abundant genera compared with those in the caffeine group.
The construction of the calm index of microbiota (CIM)
Based on the above results, we found that both melatonin and probiotics regulate the intestinal microbiota. Then, we selected the differentially abundant genera to draw a ternary diagram (Fig. S4). As shown in Figure 3B-6D, most of the bacterial genera showed a downward trend compared with those in the control group at baseline after caffeine induction, while a few specific bacteria were enriched in the gut of the zebrafish induced by caffeine (Fig. 3B). On day 14, the microbial community structure of each group showed some recovery, with the different bacteria decreasing (Fig. 3C).
To more directly observe the disturbance degree of caffeine interference, melatonin and probiotics supplement on intestinal microbiota, a “calm index of microbiota” was derived based on the distinguishing bacteria between control and treatment groups. 27 differentially abundant bacteria between the control and caffeine-induced zebrafish on day 1 were chosen as markers to construct the CIM day 1, and the CIM day 1 of each sample was calculated. The results indicated that a significant difference existed in the CIM day 1 between the control and the other three groups (Fig. 3D), and after recalculation on day 14, the CIM day 1 was improved (Fig. S5). Meanwhile, 11 differentially abundant genera among groups on day 14 were selected to build the CIM day 14. Based on the CIM day 14, we observed that the gaps between the melatonin group, probiotic group, and control group were smaller than that in the caffeine group (Fig. 3D). This indicated that melatonin played a further role in promoting the recovery of the intestinal microbiome relative to that in the caffeine group. The formulas were concluded as follow:
[Due to technical limitations, the formulas could not be displayed here. Please see the supplementary files section to access the formulas.]
Functional features of the intestinal microbiota in different treatments
To investigate how caffeine interference and the supplementation of melatonin and probiotic affect metabolic pathways by regulating intestinal microbiota, we performed shotgun metagenomic sequencing for samples collected on day 14 (n=20). We annotated the assembled genes and compared with KEGG database to further annotate the microbial metabolic pathways. The Z score greater than 1.6 represent the significant differences. Analysis results after 14 days of equal treatment showed that different treatments significantly altered the metabolism of zebrafish intestinal microbiota (Fig. 4 and Fig. S6). Compared with normal zebrafish, arginine and proline metabolism (ko00330), glyoxylate and dicarboxylate metabolism (ko00630), propanoate metabolism (ko00640), butanoate metabolism (ko00650) and biotin metabolism (ko00780) were enriched in the caffeine group. Glycolysis / gluconeogenesis (ko00010), citrate cycle (ko00020), fructose and mannose metabolism (ko00051), lipopolysaccharide biosynthesis (ko00540), pyruvate metabolism (ko00620), thiamine metabolism (ko00730) and phosphotransferase system (ko02060) were enriched in the control group. However, the relative abundance of these microbial metabolic pathways in the zebrafish intestines of the melatonin and probiotic groups approached the normal zebrafish in the control group. Collectively, the caffeine induction and melatonin and probiotic supplementation markedly altered the metabolism of the zebrafish gut microbiota.
Verification experiment through a GF zebrafish model
To verify that the gut microbiota plays an essential role in the function of melatonin, a GF zebrafish model was used. Considering that the effects of neurotransmitter regulation were best in the melatonin group, we only retained four groups: the control, caffeine, GF, and melatonin groups. After caffeine interference, the zebrafish showed significant hyperactivity, and the rest time was significantly less than that in the control group (Fig. 5B). However, we did not observe any significant difference in the phenotype including body weight and body length among different groups (Fig. 5A, C). Moreover, after adding the same dose of melatonin, the rest time which could reflect brain activity was different between the GF and melatonin groups (Fig. 5B). Correspondingly, the neurotransmitter secretions γ-GABA, 5-HT, DA and their related synthetic genes were also found to be different (Fig.6A-C). This confirmed the primary mediate role of the intestinal microbiota in the efficacy of melatonin. Furthermore, six zebrafish genes related to the secretion of γ-GABA, 5-HT and DA were selected for gene expression analysis, among which there were four groups of genes (PINK1, corresponding to DA; trh, corresponding to γ-GABA; and trph2 and mao, corresponding 5-HT) showing high consistency with the results of zebrafish rest time, as shown in Fig. 6F-I.
To better explain the mechanism by which the intestine microbiota function in the process of melatonin regulation of neurotransmitter secretion disorders, we also determined the contents of metabolites in the gut of zebrafish. Since germ-free zebrafish had almost no intestinal microbiota, while the SCFAs were mainly produced by colonic anaerobic fermentation of undigested carbohydrates, SCFAs were not detected in the GF group here. Results showed that the contents of acetic acid and propionic acid in the intestine of zebrafish in the caffeine and melatonin groups decreased significantly after the treatment with caffeine, while the contents increased remarkably after melatonin treatment, resulting in levels that were obviously higher than those in the caffeine group (Fig. 6D, E).
Melatonin regulated neurotransmitter secretion disorders through the gut-brain axis
Through the verification experiment, we highlighted the importance of intestinal microbiota in the melatonin-induced neurotransmitter regulation effects. Based on the Spearman′s rank correlation coefficient, we constructed a network of the correlations among melatonin, genera, metabolic pathways, SCFAs, and neurotransmitters. As shown in Fig. 7A, supplementation with melatonin inhibited the growth of Shewanella, Deefgea and Enterobacteriaceae in the zebrafish gut, which were negatively correlated with propanoate metabolism, butanoate metabolism and phosphotransferase system, whereas a negative correlation was observed between Aeromonadaceae and Rhizobiales and melatonin. Additionally, it was found that melatonin supplementation elevated the levels of acetic acid and propionic acid, representing SCFAs, in the intestine, and the generated SCFAs further stimulated the secretion of neurotransmitters in the brain of zebrafish, accompanied by decreased levels of DA and elevated levels of γ-GABA and 5-HT, which revealed the potential mechanism by which melatonin regulates neurotransmitter secretion disorders through the gut-brain axis (Fig. 7B).