Ethanol affects behavior and HPA axis activity during development in zebrafish larvae

Recent studies have shown that long-term alcohol intake from food can lead to numerous mental disorders in humans, and the social and health effects of excessive intake of alcohol currently represent serious problems for governments and families worldwide. However, to date, it has not been determined how alcohol affects the hypothalamic–pituitary–adrenal (HPA) axis. The zebrafish offers a good model for studying the toxicology of food-grade ethanol. In the present study, using zebrafish larvae exposed to 1% ethanol, we performed zebrafish behavioral analysis. Samples were collected for enzyme-linked immunosorbent assay (ELISA) and quantitative real time-polymerase chain reaction (qRT-PCR) experiments, and statistical analysis was performed. We found that ethanol decreased the locomotor activity of zebrafish larvae, which showed a more intense reaction to external stimuli. Ethanol also increased the level of HPA axis hormones in zebrafish larvae, influenced the level of neurotransmitters, and altered the expression of key genes in neurotransmitter metabolism. Ethanol exposure affects zebrafish behavior, increases the level of HPA axis hormones in zebrafish larvae, affects the level of neurotransmitters, and affects the expression of key genes in dopamine and serotonin metabolism. These findings may help to elucidate the effects of ethanol on HPA axis activity.

Ethanol is a substance consumed daily by humans that can cause considerable harm to human health, especially in minors 1 . Indeed, the social and health effects of alcoholism are among the most serious problems faced by governments and families every year 2 . Recent studies have found that long-term consumption of ethanol can lead to numerous physical 2 , behavioral 3 , and mental disorders 4 . Furthermore, drinking during pregnancy can have a serious impact on fetal development, leading to fetal alcohol syndrome 5 . The main manifestations of this syndrome are skull deformity, cardiovascular dysplasia, and permanent nervous system damage 6 . Long-term drinking can also increase the risk of stroke, dementia, and hypertension, increase blood lipid concentration, and lead to a higher incidence of various cardiovascular and cerebrovascular diseases 7 . Because the liver and kidney are the primary organs of alcohol metabolism, acute alcoholism can lead to alcoholic liver and kidney diseases 5 . The most common pathological changes in alcoholic liver are a decrease in the rough endoplasmic reticulum, swelling and deformation of the mitochondria, and the occurrence of liver cirrhosis 8 . Epidemiological studies have also demonstrated that alcoholics are more likely to develop liver cancer. More recent studies have revealed that long-term drinking is associated with major mental disorders, such as depression 9 .
Numerous studies on alcohol toxicity have been based on the zebrafish model 10 . The zebrafish (Danio rerio), as a vertebrate, has a genome with a high degree of homology with that of humans, which makes it a valuable model for developmental, neurological, and toxicological research 11 . For example, Lockwood et al. 12 found that embryonic exposure to ethanol increased the susceptibility of larval zebrafish to chemically induced seizures 11 . Similarly, Matsui 13 found that high concentrations of ethanol can lead to abnormal development of the visual system, while even low concentrations of ethanol can lead to behavioral disturbances 12 . Carva et al. 14 found that the learning ability of adult and juvenile zebrafish treated with ethanol decreased significantly 14 . Accordingly, zebrafish is a good model for studying the toxicology of ethanol. While ethanol has been observed to cause depressive phenotypes in behavioral experiments, the underlying mechanism of this effect has not been elucidated.
The hypothalamic-pituitary-adrenal (HPA) axis is a bodily system that involves the hypothalamus, pituitary gland, and adrenal gland and is activated in response to environmental stress 15 . Specifically, the hypothalamus secretes corticotropin releasing-hormone (CRH) which, in turn, acts on the pituitary gland to release adrenocortico-tropic-hormone (ACTH). ACTH then acts on the adrenal gland to secrete glucocorticoids (GCs), which Scientific Reports | (2020) 10:21402 | https://doi.org/10.1038/s41598-020-78573-y www.nature.com/scientificreports/ regulate HPA axis activity through glucocorticoid receptors (GR or nr3c1) 16 . Cortisol is the main glucocorticoid in zebrafish, while corticosterone is the main glucocorticoid in rodents 17 . The HPA axis is an important aspect of the neuroendocrine system, as it controls the stress response and regulates numerous physical activities 16 . Numerous neurotransmitters are involved in regulating hypothalamic activity which, in turn, regulates the activity of the entire HPA axis, including specific changes in mood 18 . Clinical studies have shown that many patients with depression exhibit decreased GR expression and increased HPA axis activity 19 . On day 4 postfertilization (dpf), zebrafish show a fully functional HPA axis, and the HPA axis of zebrafish has remarkably similar functions. Indeed, zebrafish have been successfully utilized in studies on the HPA axis. Thus, zebrafish offer a good model for studying the function of the HPA axis 20 .
To determine whether the HPA axis is a target of ethanol, in this study, we exposed zebrafish larvae to 1% ethanol and examined their subsequent behavior, hormone levels, neurotransmitter levels, and gene expression levels. We attempted to determine the effects of ethanol as a stress factor on zebrafish behavior related to the HPA axis.

Results
Exposure to ethanol affects zebrafish behavior. To investigate whether ethanol affects the autonomous behavior of zebrafish, we selected 120 hpf control and ethanol-exposed zebrafish larvae for behavioral analysis. The results showed that under identical conditions, the swimming distance of zebrafish exposed to 1% ethanol was significantly shorter than that of the control fish (Fig. 1A), while the swimming speed per unit time was significantly lower (Fig. 1B). Furthermore, the average swimming time per unit time of the ethanol group was significantly lower than that of the control group (Fig. 1C). Taken together, these results suggest that exposure to ethanol decreases the locomotor activity of zebrafish larvae.
To test the exposed zebrafish's ability to respond to external stimuli, we observed their responsiveness to alternating stimulation of light and dark (30 min of light and 30 min of darkness). Compared to the control group, the ethanol group showed significantly greater activity under light conditions (Fig. 1D,E) but a significantly reduced swimming distance under dark conditions (Fig. 1D,E). These findings indicate that the ethanol-exposed zebrafish had a more intense reaction to external stimuli. Exposure to ethanol affects the level of HPA axis hormones. To investigate whether ethanol exposure affects zebrafish HPA axis activity, we used ELISA to detect the levels of related hormones. The results showed that the level of CRH was significantly elevated after exposure to ethanol at 3, 4, 5, and 6 dpf ( Fig. 2A), as was the level of ACTH (Fig. 2B) and the level of cortisol (Fig. 2C). Taken together, these results suggest that ethanol increases the level of HPA axis hormones in zebrafish larvae. affects HPA axis activity, we performed qRT-PCR analysis to investigate HPA axis gene expression. We found that the expression of crha exhibited no significant change at 3 and 4 dpf, but there was a significantly greater upregulation at 5 and 6 dpf in the ethanol group compared with the control group (Fig. 3A). Furthermore, the expression of the crhb gene showed a significant increase at 4, 5, and 6 dpf compared with the control group (Fig. 3B); there was no significant difference at 3 dpf (Fig. 3B). The expression of pomca and pomcb increased significantly with development time. Specifically, the expression of pomca showed clear upregulation at 4, 5, and 6 dpf, but there was no significant difference at 3 dpf (Fig. 3C). The expression of pomcb did not show a significant difference at 3 and 4 dpf, but it did show significant upregulation at 5 and 6 dpf in the ethanol group (Fig. 3D). The expression of nr3c1 increased with the development time, although there was a slight decrease in expression at 6 dpf, and the ethanol group did not show significant differences from the control group (Fig. 3E). Taken together, these results indicate that the HPA axis gene expression patterns of zebrafish larvae were notably affected by exposure to ethanol.  To investigate whether ethanol exposure influences the level of neurotransmitters, particularly dopamine and serotonin, we used ELISA. The results showed that the level of dopamine showed a significantly stronger increase at 3, 5, and 6 dpf in the ethanol group than in the control group, although it decreased at 4 dpf (Fig. 4A). Serotonin, on the other hand, showed significantly stronger increases at all four time points in the ethanol group compared to the control group (Fig. 4B). These results indicate that exposure to ethanol influences the level of neurotransmitters.
Exposure to ethanol affects the expression of key genes in dopamine and serotonin metabolism. Key genes act as rate-limiting enzymes in the synthesis and metabolism of certain neurotransmitters.
We selected three key genes that served as rate-limiting enzymes for the above two neurotransmitters and performed qRT-PCR analysis. The results showed that compared with the control group, the expression of monoamine oxidase-a(maoa) significantly increased at 4, 5, and 6 dpf in the ethanol group; however, there was no significant difference at 3 dpf (Fig. 5A). Furthermore, the expression of tyrosine hydroxylase(th) was significantly greater in the ethanol group at 5 and 6 dpf but showed no significant difference at 3 and 4 dpf (Fig. 5B). Finally, the expression of dopamine β-hydroxylase(dbh) was significantly downregulated at 5 dpf in the ethanol-treated group compared with the control group (Fig. 5C). Thus, exposure to ethanol affects the expression of key genes during neurotransmitter metabolism.

Discussion
Zebrafish are a good model organism for studying neuroendocrine and behavioral effects 20 . There are numerous established behavioral and physiological tests and methods for generating genetic mutations in zebrafish, and numerous studies have utilized zebrafish to study ethanol toxicology 21 . In this study, we employed behavioral experiments, hormone measurements, and gene expression analyses to explore the effects of ethanol and  www.nature.com/scientificreports/ found that ethanol affected zebrafish larvae activity, increased HPA axis activity, and disrupted neurotransmitter metabolism. Exposure to 1% ethanol led to changes in the swimming behavior of zebrafish, including decreased swimming distance and speed. Furthermore, the light-dark alteration experiment showed that ethanol-exposed zebrafish showed significantly more activity in the light and lower activity under dark conditions than did the zebrafish in the control group. In the natural environment, sudden darkness is a dangerous environment for animals and can cause animals to increase their activity 22 . Thiago Acosta et al. reported that alcohol impairs predation risk response and communication in zebrafish 27 . This finding suggests that under light conditions, compared with the control zebrafish, 1% ethanol-treated zebrafish had significantly higher activity,while a significantly reduced swimming distance under dark conditions.
As noted above, the HPA axis is the primary system by which animals respond to changes in their external environment and stress, and the HPA axes of zebrafish and humans are highly similar 24 . Past studies have shown that the shallow water test leads to a clear increase in cortisol level and HPA activity 25 . Previous studies revealed that low concentrations of alcohol decrease cortisol production 26 , but our results show that 1% alcohol increases cortisol production 27 . In contrast to previous results, alcohol exposure reduces HPA axis activity [22][23][24] , and we think two reasons may explain this difference. First, for the exposure time, we exposed zebrafish larvae at 24 hpf; at this time, the HPA axis matured early, indicating that early-stage alcohol exposure can affect HPA axis activity. Second, in our experiment, ethanol exposure lasted for a long time, which was one of the reasons for the difference in results from the previous study.
GR is the core molecule in the HPA axis that regulates downstream gene expression. GR expression is usually downregulated in patients with depression 28 . In zebrafish, deletion of the gr gene can also lead to mood disorder 29 . In our study, we saw a trend to decrease in the expression of the gr gene. In addition, we found no difference in HPA axis activity at 3 and 4 dpf, possibly because the zebrafish larvae did not form a fully functional HPA axis before 4 dpf 30 . In addition, numerous compounds encountered during pregnancy can lead to epigenetic changes in gr 31 which, along with development, have an effect on adolescents' personality and mood 32 . Whether embryo or maternal alcohol exposure can lead to differences in the epigenetic regulation of zebrafish gr and mood changes in later developmental stages warrants further investigation.
The HPA axis is a neuroendocrine regulation axis linked to the level of neurotransmitters associated with the development of mental illness [33][34][35] , particularly dopamine 36 and serotonin 33,35 . We found significant increases in the levels of both neurotransmitters after exposure to ethanol along with increased expression of the maoa and th genes and decreased expression of the dbh gene. The th 37 is a rate-limiting enzyme in serotonin synthesis, dbh 38 converts dopamine into norepinephrine, and maoa 34,36 genes encode rate-limiting enzymes in dopamine metabolism. Thus, our results suggest that ethanol exposure increases the levels of dopamine and serotonin, perhaps partly due to decreased dbh. Previous studies have also found that in the hypothalamus, dopaminergic and serotonergic neurons can project to ACTH neurons to regulate the HPA axis 37 ; however, the detailed mechanism of this projection has not been elucidated. Whether ethanol exposure leads to changes in neurotransmitters that, in turn, cause behavioral changes or whether ethanol exposure directly affects behavioral abnormalities caused by HPA axis activity is a matter for further research.

Conclusion
Ethanol exposure was observed to reduce zebrafish locomotor activity, increase HPA axis activity, and lead to significant changes in the levels of dopamine and serotonin. These results provide a new understanding of the effects of ethanol on the HPA axis.

Methods and materials
All experimental procedures were approved by the Yantai University Animal Care and Use Committee and were in accordance with governmental regulations of China.
Fish maintenance. Adult zebrafish of the AB line were raised in a recirculating water system under a 14/10-h light/dark (L/D) cycle at 28 °C. The zebrafish were fed three times per day. To produce embryos, two male and two female adult zebrafish were paired in one 2-L tank in the evening, and spawning occurred the next day within 1 h after the lights were switched on. The embryos were placed in 10-cm Petri dishes with egg water containing methylene blue (0.3 ppm) and were raised in a light-controlled (14/10-h L/D) incubator at 28 °C.
Ethanol treatment procedure. According to data obtained in a previous experiment 27 , 1% ethanol significantly alters zebrafish larvae behavior and gene expression; therefore, at 24 h postfertilization (hpf), 50 larvae were exposed to 25 ml 1% ethanol (directly dissolved in E3 solution) in each petri dish. A total of 500 embryos were treated. A control group consisting of sibling larvae raised in E3 solution (34.8 g NaCl/L; 1.6 g KCl/L; 5.8 g CaCl 2 ·2H 2 O/L; 9.78 g MgCl 2 ·6H 2 O/L) was also applied. Each treatment was repeated three times, and three independent experiments were performed. For each group, the ethanol solution was refreshed daily until the sampling stage was reached. At 3, 4, 5, and 6 dpf, 60 larval fish were collected for the enzyme-linked immunosorbent assay (ELISA), and 30 larval fish were collected for quantitative real time-polymerase chain reaction (qRT-PCR) experiments.

Zebrafish behavioral analysis.
Locomotor activity analysis was performed as described in a previous study with several modifications 38 . At 5 dpf, a single larva was placed in each well of a 48-well plate (24 control and 24 ethanol-exposed larvae), thereby enabling simultaneous tracking of the larvae. The locomotor activities of the larvae were monitored for 30 min using an automated video-tracking system (Videotrack, ViewPoint . At least three independent samples were examined in this analysis, with each being examined in triplicate. The mRNA level of the genes of interest was calculated using the 2−∆CT method and presented as relative (-fold) values of the control group after normalization to the β-actin mRNA levels.
The target gene levels were normalized to β-actin, and triplicates were run for each sample. In each reaction with the target gene primers, 1 µl of undiluted cDNA was used, whereas the cDNA used for β-actin amplification was diluted 1:10. The relative expression of the target genes was calculated using the 2(−ΔΔCT) method. The relative primers are listed in Table 1.
Enzyme-linked immunosorbent assay (ELISA). Samples from the ethanol and control groups in the 3, 4, 5 and 6 dpf zebrafish were extracted. We analyzed HPA axis hormones and neurotransmitters using an ELISA kit obtained from Fanke Biotech (Shanghai, China) according to the manufacturer's protocol.
Statistical analyses. The data are presented as the mean ± SD. Statistical differences between two or more groups were determined using Student's t-test and one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test, respectively. All experiments were repeated three times, and the representative results are described in "Discussion". A p-value < 0.05 was considered to indicate significance.  ACT CTC ACC GAC AA  CAG AGC TCC AGA CGG AGA GT qRT-PCR   crhb  CTC GCC ACT TTT TGA CAT GA  GCT GCT CTC GAT GGC TCT AC  qRT-PCR   pomca  GCT CAG TGT TGG GAA AAT GC  GGT AGA CGG GGG TTT CAT CT  qRT-PCR   pomcb  GTG CAG ATC GGA CCA AGA AT  GCA AAC CCA AGC TCA GAC TC qRT-PCR   nr3c1  GGC CAG TTT ATG CTT TTC CA  TTG TGT GTG CCA GTC TTT CC  qRT-PCR   maoa  GAA TCC TGT GGT CCT GGA AG  GAA TGC GGT TTT GAG TTG GT  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://creat iveco mmons .org/licen ses/by/4.0/.