Preparation of fetal rat hindbrain neuronal cultures and effects of an astrocyte-derived trophic factor

To facilitate developmental studies of the central nervous system (CNS), a procedure for the preparation of fetal rat hindbrain neuronal cell cultures was established which achieves an optimal balance between neuronal purity and dispersion, and neuronal survival. This method employs embryonic day 17 hindbrain tissue, dissociation by mechanical sieving, a plating density of 3.7 ;This trophic factor for CNS neurons is different from other reported astrocyte-derived factors. Its possible role in normal development is discussed.;There is little known about trophic interactions in the CNS. While establishing neuronal culture methods, it became evident that neuronal survival decreased in the absence of glia. It was possible to use mixed glial cultures from rat cerebral cortex in several ways to promote long-term survival of isolated neurons without direct contamination. Since astrocytes were the major cell type in the glial cultures, virtually pure neonatal rat astrocyte cultures were subsequently used. Astrocyte-conditioned medium (ACM) promoted long-term survival of the neuronal cultures. The effect was not reproduced by conditioned medium from rat fibroblast, rat skeletal muscle cell, or mouse L1210 cultures. By dialysis and ultrafiltration of ACM, activity was found to be associated with an apparent molecular weight between 20,000 and 30,000 Daltons. Though 2.5S-NGF falls within this range, 2.5S-NGF did not reproduce the trophic effect and antiserum to 2.5S-NGF did not prevent it. Initial evidence suggests that this factor or one of similar size also stimulates the growth of immature glia.