“THERMAL NOCICEPTION STUDIES IN ZEBRAFISH”

Background Nociception was defined by Sherrington as the mechanism used to sense painful events. Despite the value of current animal models for nociception and pain, we have not yet understood in full how these mechanisms operate. Aim Here we propose a novel approach using zebrafish (Danio rerio) larvae in order to determine if the fish is suitable for the study of pain stimuli. Taking advantage of the fact that, in response to noxious stimuli, the expression of several genes in mammals is induced in neurons of the spinal cord and the dorsal root ganglia (DRG), we investigated if such mechanism is evolutionary conserved in the Zebrafish. Materials and Methods We cloned homologues of mammals pain marker genes (PMGs) in the fish and we tested their expression after painful stimuli. Thus, we determined an easy and ubiquitous method of noxious stimuli induction in 5dpf larvae by heat shock. We quantitatively assayed these larvae for over-expression of the PMGs by real-time PCRs, to determine if any of these genes is up regulated in specific time points. In another set of experiments we tested, by in situ hybridization, the localization of the PGMs over-expression in the larvae. To confirm the up-regulation of these genes is pain related we looked for the possible heat receptor that could mediate such sensation. Thus, we cloned Zebrafish trpv1, analyzed the expression pattern, tested its function with loss of function experiment and by analysis of changes in intracellular calcium levels after various painful stimuli in HEK293T cell line. Results and Conclusions These experiments show peculiar over-expression of all the PMGs in time and space, in particular they are expressed in what are probably the spinal cord and the DGR. We cloned and studied trpv1 as a good candidate for heat sensation mediation. However, zebrafish trpv1 responds only to low pH when tested in human cell line. Thus, we need further in vivo analysis to determine if trpv1 cooperates with other factors to mediate noxious heat in zebrafish.