Transcriptional analysis of the effects of early life exposure to fluoxetine on telencephalon and hypothalamus in SR4G transgenic zebrafish larvae and adults

Our previous study suggested persistent transgenerational effects of fluoxetine (FLX) exposure, in both environmental and therapeutic concentrations, on stress response and behaviour up to 3 generation withdrawn from exposure in larvae and adult zebrafish. To lay the foundations of the mechanistic of such transgenerational effects, we studied the effect of FLX on stress response in brain tissues (telencephalon and hypothalamus) of larvae and adult zebrafish using differentially expressed genes at the transcriptome level. To monitor the stress response, we used a transgenic zebrafish line (SR4G) which are expressing green fluorescence protein (GFP) in response to endogenous and exogenous glucocorticoids. The transcriptome analysis of GFP RNA as an internal control, provided us with an invaluable tool to clearly visualize and differentiate between stressed and unstressed groups as per treatments. To stimulate stress in both larvae and adult zebrafish we used a modified net stressing technique consisting of consecutive sequence of air exposure and incubation intervals. A pool of 22 larvae heads and a pool of 5 telencephalon and 5 hypothalamus dissected tissues were used as the target tissues for RNA extraction. All experiments were replicated in n=6 per 4 different groups including Control-Unstressed (Cnt-U), Control-Stressed (Cnt-S), Fluoxetine-Unstressed (FLX-U), and Fluoxetine-Stressed (FLX-S) using pools of larvae heads, telencephalon, and hypothalamus. After total RNA extraction, using Illumina technology all samples were sequence and the list of differentially expressed genes (DEGs) were produced using DSEQ pipeline in R. Overall design: Three different target tissue, two from 6 months old adults (Telencephalon and Hypothalamus) and one from 6 days post fertilization larvae (dissected heads). One treatment, Fluoxetine, One control (ethanol as vehicle), two condition (Stressed, Unstressed)