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
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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.
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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
