Developmental ethanol exposure and its impact on behaviour and HPI axis activity of zebrafish

Ethanol exposure during pregnancy is one of the leading causes of preventable birth defects, leading to a range of symptoms collectively known as fetal alcohol spectrum disorder (FASD). More moderate levels of prenatal ethanol exposure (PNE) lead to a range of behavioural deficits including aggression, poor social interaction, poor cognitive performance and increased likelihood of addiction in later life. Current theories suggest that adaptation in the hypothalamic-pituitaryadrenal (HPA) axis and neuroendocrine systems contributes to mood alterations underlying behavioural deficits and vulnerability to addiction. This has led to the suggestion that corticotrophin-releasing factor (CRF) antagonists and glucocorticoid (steroid) inhibitors may be potential therapeutics to address the deficits of PNE and for the treatment of addiction. The zebrafish (Danio rerio) has several advantages over mammalian models, such as low cost of maintenance, short life cycle, easy embryological manipulation and the possibility of large-scale genetic screening. By using this model, our aim is to determine whether developmental ethanol exposure provokes changes in the HPA axis (HPI axis in fish), as it does in mammalian models, therefore opening the possibilities of using zebrafish to elucidate the mechanisms involved, and to test novel therapeutics to alleviate deleterious symptoms. Thus this thesis focuses solely on the effect of developmental ethanol exposure on the functioning of the HPI axis in zebrafish. Stress-reactivity in zebrafish larvae ethanol-treated 1-9 days post 4 fertilisation (dpf) was assessed using thigmotaxis and thigmotaxis following airstress. In both tests, lower stress-related responses were obtained with ethanol treated animals, in that they spent less time at the edges of the apparatus (P<0.01, n=3). They also showed lower total body cortisol (P=0.04, n=14). Larvae also showed the same behaviour pattern two weeks after ethanol exposure, (23dpf) (P=0.04, n=3), again with reduced total cortisol (P=0.03, n=4). HPI-related gene transcription was also assessed in 9dpf ethanol treated zebrafish larvae, by qRT-PCR. Revealing up-regulation of CRH, CRHBP and CRHR2, normalized against β-Actin, Elav1 and Gap43 housekeeping genes. In situ hybridization revealed no spatial changes in CRH, CRH-BP and POMC with animals at the same stage. Behavioural stress-reactivity differences in 6-months old adults that had been exposed developmentally to ethanol were assessed using novel tank diving and thigmotaxis. Both assays indicated a decrease in stress-like behaviour due to early ethanol exposure compared to controls (P<0.05, n=5 both). Finally, cortisol levels were assayed from 9dpf larvae and 6-month-old adults that had been treated with ethanol during early development showed a significant reduction in cortisol output when air-exposed stressed compared to controls (P=0.04, n=5). Conclusion: Early ethanol exposure produced significant changes in cortisol, HPI gene mRNA expression and stress-reactive behaviour in 9dpf animals. Changes in cortisol and behaviour were still detected in 6-months old adults, developmentally treated with ethanol, indicating that early ethanol exposure has permanent effects on the HPI axis. 5 As our data contradicts the findings in mammalian literature where early ethanol exposure increases stress-like behaviour in later life, it is also possible that more permanent effects of PNE in mammals may arise through maternal-offspring interactions, during and post gestation, such as breastfeeding and maternal grooming of the offspring, which are absent in the zebrafish model.BBSRC (grant: BB/F016913/1

Sustained Action of Developmental Ethanol Exposure on the Cortisol Response to Stress in Zebrafish Larvae and Adults

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedThis study was supported by the National Centre for the replacement, refinement and reduction of animals in research

Stress-reactivity in 9dpf larvae measured by whole-body cortisol concentration.

<p>Experimental animals were developmentally treated with ethanol from 1dpf-9dpf with 20mM and 50mM ethanol concentrations. At 9dpf animals were either flash frozen immediately or air exposed then frozen. Air stressed animals showed decreased tissue cortisol concentrations with increasing ethanol concentration exposure during development. Means ± SE, 6 batches of animals per group, *<i>P</i>< 0.05, **<i>P</i><0.01, by ANOVA and “t” test.</p

Effects of ethanol exposure from 1-9dpf on larval development A.

<p>size at 9dpf measured using eLaborant software. <b>B</b> dry weight at 9dpf. <b>C</b> body weight at 6 months. There were no differences between groups. Values are means ± SE, 3 samples were used per group, with 20 (A) or 25 (B) animals per sample.</p

Stress-reactivity measured by whole-body cortisol concentration of zebrafish 6-month-old adults.

<p>Treated animals were developmentally exposed to ethanol from 1dpf-9dpf with 20mM or 50mM ethanol concentrations. At 6 months animals were either flash frozen immediately or air exposed and frozen 6 minutes later. Zebrafish adults showed decreased cortisol content with increasing ethanol concentration exposure during development. Means ± SE, 9 batches of animals per group. *<i>P</i><0.05 **<i>P</i><0.01 “t” test and ANOVA.</p

Stress-reactivity measured by whole-body cortisol concentration of zebrafish 6-month-old adults.

<p>Animals were either flash frozen or stressed, either by air exposure at a specific time or acute exposure to ethanol (1%), followed by freezing 6 minutes later. Zebrafish adults showed increased cortisol concentration when air exposed for 30s, but not after 60s exposure. Means ± SE, 3 batches (on different days) of 3 samples of 10 animals were used. <i>P</i><0.01, ANOVA <0.01).</p

Assessment of embryonic tissue ethanol concentration compared to waterbath ethanol concentration following acute 24-hour ethanol exposure of zebrafish embryos.

<p><b>A</b> Alcohol was assayed in whole embryos developmentally exposed to an ethanol waterbath concentration of 100mM and 20mM for a period of 24 hours, using 1-24hpf, 24-48hpf and 48-72hpf periods. <b>B</b> Tissue ethanol content after exposure to 100mM ethanol during the whole period 1-9dpf. Values are means ± SE, 3 samples collected on different days were used for each time period, 25 animals per sample. Values for tissue ethanol in the absence of added ambient ethanol were less than 1% of those illustrated, and not significantly different from zero.</p

Stress-reactivity measured by novel tank diving in 6-month old adult zebrafish.

<p><b>A)</b> Time course of average time spent per minute at the bottom of the tank, <b>B)</b> overall average time spent per minute at the bottom of the tank each minute, <b>C)</b> mean distance travelled per minute during novel tank diving. <b>D)</b> Effect of diazepam on zebrafish stress-reactivity assessed by novel tank diving. A,B. Zebrafish that had been developmentally exposed to ethanol showed reduced bottom dwelling (<i>F</i> 2, 682 = 3.47, <i>P</i><0.05). There were no significant differences in distance travelled (C). D. Diazepam also significantly reduced time spent by adults at the bottom of the tanks compared to controls (<i>F</i> 1, 408 = 5.45, <i>P</i><0.001). Post hoc t-test, *** <i>P</i><0.001.</p