Ethanol in Food affects behaviour and HPA axis activity during development in zebrafish larvae Subhead: Ethanol in Food affects Nerves during development in zebrafish larvae

Long-term alcohol intake from food can lead to numerous mental disorders in humans, and cause serious problems for governments and families worldwide. However, currently, it is unclear how alcohol affects the hypothalamic–pituitary–adrenal (HPA) axis. In the present study, using zebrafish larvae exposed to 1% ethanol, we made Zebrafish behavioural analysis, samples were collected for the enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) experiments,and made statistical analyses at last. we found that ethanol decreases the locomotor activity of zebrafish larvae,showed a more intense reaction to external stimuli,increases the secretion of HPA axis hormones in zebrafish larvae,influences the secretion of neurotransmitters,alters key gene expression during neurotransmitter metabolism. Ethanol exposure reduced zebrafish locomotor activity, increased their HPA axis activity, and led to significant changes in the secretion of dopamine and serotonin. These findings provide us with a new understanding of the effects of ethanol on the HPA axis.

development, leading to foetal alcohol syndrome [6]. The main manifestations of this syndrome are skull deformity, cardiovascular dysplasia, and permanent nervous system damage [7]. 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 [8]. Because the liver and kidney are the main organs of alcohol metabolism, acute alcoholism can lead to alcoholic liver and kidney diseases [5]. The most common pathological changes of alcoholic liver are a decrease in the rough endoplasmic reticulum, swelling and deformation of the mitochondria, and the occurrence of liver cirrhosis [9]. Epidemiological studies have also shown 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 [10].
Numerous studies on alcohol toxicity have been based on the zebrafish model [11]. The zebrafish (Danio rerio), as a vertebrate, has a genome with a high degree of homology with that of humans, this makes it a valuable model for developmental, neurological, and toxicological research [12]. Lockwood, for example, found that embryonic exposure to ethanol increases the susceptibility of larval zebrafish to chemically induced seizures [12].
Similarly matsui found that high concentrations of ethanol can lead to abnormal development of the visual system, while even low concentrations of ethanol can lead to behavioural disturbances [13]. Carva et al. found that the learning ability of adult and juvenile zebrafish treated with ethanol decreased significantly [14]. Accordingly, zebrafish are a good model for studying the toxicology of ethanol. While ethanol has been found to cause depressive phenotypes in behavioural experiments, the underlying mechanism of this effect remains unknown.
The hypothalamic-pituitary-adrenal (HPA) axis is a bodily system involving the hypothalamus, pituitary gland, and adrenal gland that is activated in response to environmental stress [15]. Specifically, the hypothalamus secretes adrenocorticotropinreleasing hormone (ACTH), which in turn acts on the pituitary gland to release adrenocorticotropin (CRH). CRH then acts on the adrenal gland to secrete glucocorticoids (GCs), which regulate HPA axis activity through the glucocorticoid receptors (gr or nr3c1) [16]. Cortisol is the main glucocorticoid in both humans and 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, such as digestion, the immune system, mood and emotion, sexual behaviour, and energy storage and consumption [16]. Upstream in the HPA axis, 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 a lot of patients with depression exhibit decreased GR expression and increased HPA axis activity [19]. At four days post-fertilization (dpf), zebrafish show a fully functional HPA axis, and the HPA axis of zebrafish has remarkably similar functions that of humans. Indeed, zebrafish have been successfully applied in studies on the HPA axis in humans. Thus, zebrafish offer a good model for studying the function of the HPA axis [20].
In this study, we exposed zebrafish larvae to 1% ethanol and examined their subsequent behaviour, hormones, neurotransmitters, and gene expression. The results showed that ethanol treatment reduced zebrafish activity and increased their response to environmental stimuli as well as their HPA axis activity. In addition, there were significant changes in the secretion of certain neurotransmitters. This study provides new understanding of the effects of ethanol on the HPA axis, and provide new evidence on whether or not alcohol-control.

Methods And Materials
Fish maintenance and ethanol treatment procedure All experimental procedures were approved by the Yantai University Animal Care and Use Committee and were in accordance with governmental regulations of China.
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. They were fed three times per day. To produce embryos, male and female zebrafish were paired 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 raised in a lightcontrolled (14/10-h L/D) incubator at 28 °C. According to the data of our previous experiment, 1% ethanol significantly alters zebrafish larvae behaviour and gene expression, so at 24 h post-fertilization (hpf), the larvae were exposed to 1% ethanol, A control group consisting of sibling larvae raised in E3 solution were applied too. For each group, the ethanol solution was refreshed daily until the sampling stage. At 3, 4, 5, and 6 dpf, samples were collected for the enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) experiments.

Zebrafish behavioural analysis
Locomotor activity analysis was performed as described in a previous study, with some modifications. At 5 dpf, a single larva was placed in each the wells of a 48-well plate (24 control and 24 ethanol-exposed larvae), thereby allowing for simultaneous tracking of the larvae. The locomotor activities of the larvae were monitored for 30 min using an automated video-tracking system (Videotrack, ViewPoint LifeSciences), while their movement was recorded and analysed using Zebralab 3.10 software (ViewPoint Life Sciences). The 48-well plates were placed inside the Zebrabox Observation Chamber, wherein they were exposed to continuous infrared light or 10 min light ON and 10 min light OFF. Instruments were placed in the chamber to maintain a constant temperature of 28.5 °C. The Videotrack quantization parameters were set as in the previous study. The test was performed 3 times. Data were further analysed using Microsoft Excel.

RNA extraction and qRT-PCR
For the qRT-PCR analysis, total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Following DNase treatment, RNA (1 µg) was reverse-transcribed with oligo (dT) 18 primers and M-MLV reverse transcriptase. The qRT-PCR analysis was then carried out in the StepOne Real-Time PCR System (ABI) using SYBR® Premix Ex TaqTM (Takara Bio Inc., Dalian, Liaoning). At least three independent samples were examined in this analysis, each in triplicate. The mRNA level of the interested genes was calculated using 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 for each sample triplicates were run. 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. Table 1 The relative primers of genes Gene name Forward primer Reverse primer Samples from the ethanol and control groups in the 3, 4, 5 and 6 dpf zebrafish were extracted. We analysed the 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 below 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 Sect. 3. A p-value of < 0.05 was considered to indicate statistical significance.

Exposure to ethanol affects zebrafish behaviour
To investigate whether ethanol affects the autonomous behaviour of zebrafish, we selected the 120 hpf control and ethanol-exposed zebrafish larvae for behavioural analysis. The results showed that under the same 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 faster (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 tests the exposed zebrafish's ability to respond to external stimuli, we observed their responsiveness in alternating stimulation of light and dark (30 minutes of light and 30 minutes of darkness). Compared to the control group, the ethanol group showed significantly reduced activity in the light condition ( Fig. 1D-E), but significantly greater swimming distance in the dark condition ( Fig. 1D-E). These findings indicate that the ethanol-exposed zebrafish showed a more intense reaction to external stimuli.
Exposure to ethanol affects the secretion of HPA axis hormones To investigate whether ethanol exposure affects the zebrafish HPA axis activity, we used the ELISA method to detect the secretion of related hormones. The results showed that the secretion of CRH was significantly elevated after exposure to ethanol at 3, 4, 5, and 6 dpf ( Fig. 2A), as was the secretion of ACTH (Fig. 2B) and the secretion of cortisol (Fig. 2C).
Taken together, the results suggest that ethanol increases the secretion of HPA axis hormones in zebrafish larvae.
Exposure to ethanol affects the expression of HPA axis genes Further to investigate how ethanol affects HPA axis activity, we performed a qRT-PCR analysis to investigate HPA axis gene expression. We found that the expression of crha showed 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 crhb gene showed a statistically significant increase at 4, 5, and 6 dpf compared with the control group ( Figure.3B); there was no significant difference at 3 dpf (Fig. 3B). The expression of pomca and pomcb increased significantly with the development time. Specifically, the expression of pomca showed clear upregulation at 4, 5, and 6 dpf, but there was not a statistically significant difference at 3 dpf (Fig. 3C). The expression of pomcb did not show a significant differences at 3 and 4 dpf, but it did show significant up-regulation 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). Altogether, the HPA axis genes expression patterns of zebrafish larvae showed clear changes after exposure to ethanol.

Exposure to ethanol affects the secretion of neurotransmitters
Numerous monoamine neurotransmitters play an important role in the regulation of the HPA axis, particularly the regulation of individual behaviour and mood. To investigate whether ethanol exposure influences the secretion of neurotransmitters, particularly dopamine and serotonin, we used the ELISA method. The results showed that the secretion of dopamine showed a significantly greater 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 greater 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 secretion 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 further qRT-PCR analysis. The results showed that compared with the control group, the expression of maoa significantly increased at 4, 5, and 6 dpf in the ethanol group; however, there was no statistically significant difference at 3 dpf (Fig. 5A). Furthermore, the expression of 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 dbh was significantly downregulated at 5 dpf in the ethanol treated group compared with the control group (Fig. 5C).
Thus, the exposure to ethanol alters key gene expression during neurotransmitter metabolism.

Discussion
Zebrafish are a good model organism for neuroendocrine and behavioural research [20] ( Steenbergen et al.,2011). There are numerous established behavioural and physiological tests and methods of generating genetic mutations in zebrafish, and plenty of research using zebrafish to study ethanol toxicology [21]. In this study, we explored the effects of ethanol using behavioural experiments, hormone measurements, and gene expression, and found that ethanol led to decreased zebrafish locomotor activity, increased HPA axis activity, and neurotransmitter metabolism disorders.
Exposure to 1% ethanol led to changes in the swimming behaviour of zebrafish, including decreased swimming distance and increased speed. Furthermore, the light-dark experiment showed that that ethanol-exposed zebrafish were significantly more active in the dark condition. In the natural environment, darkness is a dangerous environment for animals [22]. This suggests that zebrafish exposed to ethanol showed a more anxious phenotype in response to danger. Previous studies have also found that after treated with ethanol, zebrafish adults and juveniles show anxiety-like behaviour, and this anxiety-like behaviour could be decreased using the antidepressant drug diazepam [23]. These results suggest that zebrafish juveniles exposed to ethanol exhibit a depression-like phenotype.
As noted above, the HPA axis is the main system by which animal response to changes in their external environment and stress, and the HPA axis of zebrafish and humans are exceedingly similar [24]. Past studies have shown that the shallow water test leads to a clear increase in cortisol secretion and HPA activity [25]. In the HPA axis, gr serves as a central molecule that shows decreased expression in depressed patients [26]. In zebrafish, deletion of the gr gene can also lead to mood disoder [27]. Based on these past findings, we examined the expression of key genes and secretion of hormones related to the HPA axis, and found that the expression of crh and pomca was significantly up-regulated at 5 and 6 dpf. Similarly, the secretion of hormones encoded by these two genes also increased significantly. Finally, ethanol exposure led to an increase in cortisol secretion.
These data together indicate that ethanol leads to a significant increase in HPA axis activity.
Notably, however, while there was a decrease in the expression of the gr gene, this decrease was not statistically significant. This might be because the exposure time was insufficient to cause down-regulation of gr. In addition, we found no difference in the HPA axis activity at 3 and 4 dpf, possibly because the zebrafish larvae did not form a fully functional HPA axis before 4 dpf [28]. In addition, numerous compounds during pregnancy can lead to epigenetic changes in gr [29].which, along with development, have an impact on adolescents' personality and mood [30]. Whether embryo or maternal alcohol exposure can lead to differences in epigenetic regulation of zebrafish gr and mood changes in later developmental stages remains to be investigated in future studies.
The HPA axis is a neuroendocrine regulation system linked to the secretion of neurotransmitters associated with the development of mental illness [31]particularly dopamine[32] and serotonin [33]. We found significant increases in the secretion 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 maoa [34], th [35] and dbh [36] genes are a rate-limiting enzymes of dopamine synthesis, serotonin synthesis, and dopamine metabolism, respectively. Thus, our results suggest that ethanol exposure increases the synthesis of dopamine and serotonin, while also reducing the metabolism of dopamine. 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 remains unclear. Whether ethanol exposure leads to changes in neurotransmitters than in turn produce behavioural changes, or whether ethanol exposure directly affects behavioural abnormalities caused by HPA axis activity, is a matter of further research.
Alcohol consumption is a major risk factor for road traffic crashes worldwide,and lead contributor to death from injuries. Drunk driving result significant economic costs, social costs (such as law enforcement,imprisonment, compensation payments, unemployment, disability in surance, and loss of productivity from injury or premature death).A lot of governments are adopting multipronged strategies to curb these costs, such as launched public service announcements,higher penalties for offenders, greater law enforcement presence on the roads, and so on [5].

Conflicts of interest
The authors have no competing interests to declare.
All presentations of case reports have consent to publish.
Availability of supporting data.
"Availability of Data and Materials" section in this manuscript detailing where the data supporting their findings can be found.
Wenxiaodu did this experiment and wrote some parts of the article.Other authors proofread and wrote part of the article.
All authors have read and understood Journal of Neuroinflammation's policies, and we believe that neither the manuscript nor the study violates any of these.All authors agree that this article can be published in the Journal of Neuroinflammation.This manuscript has not been published or presented elsewhere in part or in entirety and is not under consideration by another journal.   HPA axis gene expression patterns in zebrafish larvae after ethanol exposure.

Figure 4
Secretion of neurotransmitters in the zebrafish larvae after ethanol exposure.