Cultures of brain cells from gilthead sea bream (Sparus aurata L.) as a new in vitro model for ecotoxicological studies.

Abstract Nowadays, there is a critical need to develop species-specific cell cultures from marine fish. Many attempts to obtain fish brain cells in primary cultures often yields heterogeneous cell populations that can hamper qualitative and comparative analyses. Out of nearly 300 cell lines have been obtained from fish worldwide, yet none from the brain of gilthead sea bream (Sparus aurata L.) with the only exception of the fibroblast cell line SAF-1 by Bejar, Borrego & Alvarez (1997) and the primary glial culture previously obtained in our laboratory. This is the first attempt to make a coculture of glial and neural cells from the brain of sea bream juveniles. Since the early 1970s, many comparative studies have been performed using immunocytochemical screening on the brains of several vertebrate taxa, including teleosts, in order to identify these cells, even if the data are sometimes contrasting. Due to several discrepancy, arose the need for improving differentiation and enrichment procedures able to generate highly pure populations of glia, neurons and ultimately neural stem cells (NSC) from the teleost brain. By the use of double immunofluorescence (IF) analysis, we unquestionably identified as “radial glia” those phase contrast leaf shaped cells, with spindle ends elongating in extended processes, previously mentioned in adult Anamnia as the only nonependymal astroglial cells in the nervous system. Fish oligodendrocytes, the myelinating cells of the CNS, were found morphologically similar to those in mammals. Their cell bodies showed a bipolar shape and after differentiating were found to myelinate different axons. Purified neurons were viable, expressed mature specific neuronal markers, and could replicate during long term culturing. Glia was found highly mitotic and could developed mature astrocytes. Furthermore, we demonstrated that NSC can self-renew and differentiate into neurons and glial cells during extended culturing. Once established the monolayer, cells were successfully tested for viability by the MTT cytotoxicity assay. Findings illustrate the efficacy of isolating highly pure populations of viable NSC, glia and neurons from sea bream brain, all characterized by cell phenotype markers. Results demonstrate that 6-weeks CNS cultures highly regulated expression of specialized nervous type-specific functions by IF and MTT, thus providing a useful tool as new fish model for neurotoxicity, viral diagnostics, ecotoxicological and neurofunctional studies.