Use of central neuronal cultures for the detection of neuronotrophic agents

Neuronotrophic factors, a class of macromolecules thought to be present within the neuronal environment are required to support the survival in vitro of peripheral neurons. In the present study we have established bioassay culture systems suitable for the identification of similar agents for intrinsic neurons of the central nervous system. The striatum, hippocampus and septum of 18 day fetal rats were dissociated and plated in a serum-free medium on a neurite conducive substratum which allows an easy recognition of neurons under phase contrast microscopy. These cultures contain predominantly neurons as assessed by tetanus toxin labelling, a well recognized neuronal marker. Seeding the cell suspensions at decreasing densities yields after 24 h a density dependent survival of the neuronal population. Thus a low seeding density could be chosen where survival of these neurons required an exogenous source of trophic factors. Survival of central neurons was promoted by several conditioned media derived from rodent glial cell cultures, both primary (astroglia, Schwann) and clonal (C6 glioma, Schwannoma). Serial dilutions of these media allowed the titration of their respective neuronotrophic activities. In addition, conditioned media derived from the central neuronal cultures themselves, when seeded at a high density, were also able to support the survival of low density seeded central neurons.