Gene expression changes during zebrafish development induced by morphine exposure

I Rodriguez-Martin1, MJ Herrero-Turrion2,3, RE Rodriguez2,3. 1European University of Madrid, Department of Basic Biomedical Sciences, 28670, Spain, 2University of Salamanca, Institute of Neuroscience of Castilla y León (INCYL), 37007, Spain, 3University of Salamanca, Department of Biochemistry and Molecular Biology, 37007, Spain

MicroRNAs (e.g. miRNAs), endogenously expressed small non-coding RNAs, control gene expression by regulating target messenger RNA at post-transcriptional level. Recent studies have identified a specific role for microRNAs as key regulators of neuronal differentiation and/or plasticity. For example, miR-133b, regulates the differentiation, maturation and function of dopaminergic neurons. Our research is focused in the molecular mechanisms underlying addiction that develops from the gradual adaptation of the brain to chronic drug exposure, and involves genetic reprogramming of neuronal function. In this sense, we have recently demonstrated that morphine, the most used analgesic to treat chronic pain, although its long-term administration produces tolerance and dependence through neuronal plasticity, decreases miR-133b expression, hence increasing the expression of its target, Pitx3, a transcription factor that activates tyrosine hydroxylase (TH) and dopamine transporter (DAT). We have used zebrafish embryos as a model to analyze in vivo molecular mechanisms related to the activity and function of the opioid system that cannot be fully established in other models. For instance, in contrast to mammalian embryos, which develop in the uterus and are influenced by the maternal biochemical processes, zebrafish embryos develop externally, avoiding the maternal effect on these embryos. This is essential when dealing with drug exposure, as the effects observed in mammalian embryos might be due to the susceptibility of the mother and not the embryo per se.

Based in our previous results and with the goal of looking for new insights into the transcriptional changes induced by morphine exposure, we present here the analysis of zebrafish transcripts using the Affymetrix GeneChip® Zebrafish Genome Array. We have studied the differential expression of over 14900 transcripts in zebrafish embryos after exposure to morphine and also in combination with the over-expression of the previously named miR-133b. We will present the computational analysis of same genes whose expression changes in the different conditions studied. Also, the expression changes will be corroborated by qRT-PCR and the confirmed data will be used to establish hypotheses about the molecular adaptations underlying morphine effects. In particular, we have studied Wntless (Wnl), a known gene encoding for mu-opioid receptor (MOR) interacting protein, is significantly up-regulated in zebrafish embryos after morphine treatment, injection of miRNA-133b, and exposure to morphine plus miRNA-133b. Our results indicate that not only the application of morphine appears to cause (1) a redistribution of Wnl protein from cytosol to the cell surface, (2) enhanced MOR/Wnl complex formation at the plasma membrane and (3) an inhibition of Wnt secretion, but also alters their gene expression. The study of the morphine direct effects in the embryos will provide a better understanding on the molecular mechanisms that underlie the physical and neurobehavioural defects shown in fetuses and offsprings after maternal morphine consumption.