Neuroprotective effect of caffeine against early-life stress-induced anxiety and reproduction in the zebrafish

Early-life stress is a high risk to cause reproductive dysfunction and mood disorders during adult stage by altering hormonal regulation of hypothalamic-pituitary-adrenal and hypothalamic-pituitary-gonadal axes. Caffeine is known as one of the potential neuroprotective agents against stress. This study was designed to investigate the protective ability of caffeine from early-life stress-induced anxiety behaviour and reproductive dysfunction in the adult male zebrafish. This thesis contains 5 chapters; chapter 1 literature review, chapters 2-4 are experimental chapters and chapter 5 general discussion. We treated zebrafish larvae with caffeine at 3 days post fertilization (dpf) and a synthetic glucocorticoid, dexamethasone (DEX), which is widely used in clinical applications at 4 dpf, for 24 hours each to study the effect of the treatments on anxiety-like behaviour and reproductive activity during adulthood. In chapter 2 we observed that zebrafish exposed to DEX during early life exhibited anxiety-like behaviour during adulthood, but caffeine pre-treated zebrafish exhibited similar behaviour in comparison to the control. In chapter 3, we found that 2 mg/L DEX led to up-regulation of Kiss2 and GnRH3 which are key players in reproduction, and 200 mg/L DEX caused increased Kiss1 and GnRH2 mRNA level, which are believed to control fear and feeding behaviour, respectively. These changes were not observed in caffeine pre-treated zebrafish. Haematoxylin and eosin-stained histological analysis of the male gonads showed that both 2 mg/L and 200 mg/L DEX-treated zebrafish had lower number of spermatids in comparison to the control, and 2mg/L DEX caused five-fold decrease in the number of spermatids. This suggests that early-life DEX exposure has differential effects; 2 mg/L DEX has higher impact in reproduction as observed in Kiss2 and GnRH3 mRNA level in the brain and at gonadal level, while 200 mg/L DEX up-regulated Kiss1 and GnRH2. Hence, the decrease in gonad maturation by DEX induces over-compensation mechanism, which led to up-regulation of the GnRH and Kiss mRNA levels in the brain. We performed in silico analysis on the promoter regions of Kiss and GnRH genes to identify glucocorticoid response element (GRE) and putative binding sites of transcription factors involved in stress and early-life development to understand epigenetic regulation by DEX treatment in chapter 4. In silico analysis showed that there were stress, caffeine and development-related putative binding sites in all of the promoter regions. DNA methylation studies showed that there were no differences in methylation pattern of CpG residues along -1203 to -305 in Kiss1 promoter region in whole brain between control, DEX and caffeine pre-treated groups. Furthermore, we used laser-capture micro-dissection to capture regions which are known to express GnRH3 for -1757 to -1265 GnRH3 promoter for more specificity; CpG residues at position -1410, -1377 and -1355 were highly methylated in 2 mg/L DEX group when compared to control. Early-life DEX treatment increased methylation of CpG residues in the promoter of zebrafish GnRH3. Changes in behaviour and mRNA levels of Kiss and GnRH caused by early life DEX exposure were not observed in caffeine pre-treated zebrafish, suggesting that caffeine is protective from DEX effects on anxiety-like behaviour and reproduction. Early-life DEX treatment may change methylation pattern at the promoter region which regulates gene expression. It is possible that the up-regulation of GnRH3 observed in the second study is related to these methylation differences in the promoter.