Substance P mRNA expression during zebrafish development: influence of mu opioid receptor and cocaine

Zebrafish has emerged as an important vertebrate animal model for the study of human diseases and for developmental studies in mammals. Since there are few studies of the tachykinin 1 gene (TAC1), precursor of substance P (SP), in relation to embryonic development, we aimed to study the expression of SP transcript (mRNA) and determine the influence of cocaine and opioid receptors on the expression of this neuropeptide. In order to analyse the spatial and temporal SP mRNA expression in zebrafish, we cloned - based on human TAC1 sequence - the sequence that originates SP. Phylogenetic analyses of the precursor of SP, revealed an alignment in the fish cluster, with a clear distinction from other species (amphibians, birds and mammals). Real time PCR (qPCR) results showed that SP mRNA was expressed in several stages of embryonic development, where it increased progressively from gastrula-8hpf (hour post-fertilisation) to the end of the embryogenesis-72hpf. SP mRNA was expressed mainly in the spinal cord in embryos at 20-30hpf, whereas at 36, 42 and 48hpf embryos SP mRNA was expressed mainly in the CNS telencephalon, diencephalon, hypothalamus, rhombomeres, epiphysis and in peripheral areas (heart and somites). Exposure of embryos to 1.5μM cocaine altered the SP mRNA expression at 24 (increasing) and 48hpf (decreasing). We also report that knockdown of μ-opioid receptor induced an increase of SP mRNA expression while the knockdown of the two delta opioid receptors did not produce changes in SP mRNA expression. In conclusion, SP mRNA in zebrafish is expressed during embryonic development in the CNS and peripherally, suggesting that SP would play a critical role during embryogenesis. Furthermore, cocaine exposure and the knockdown of μ-opioid receptor affect the SP mRNA expression. These observations can be important in the pain and addiction field where SP is involved.