Identification of the Serum Complex Which Induces Cerebellar Granule Cell In Vitro Differentiation and Resistance to Excitatory Amino Acids


D. Mercanti and C. Galli, as above


The protein complex promoting in vitro terminal differentiation of cerebellar granule cells has been isolated from rabbit serum. We designate the complex the neurite outgrowth and adhesion complex (NOAC). The apparent molecular weight, evaluated by gel filtration, is 80–100 kDa. Rat cerebellar granule cells cultured in NOAC exhibit much lower glial cell contamination and survive, in their differentiated state, much longer than in 10% foetal calf serum. While they bind tetanus toxin, express specific antigens such as synapsin l, synaptophisin and A2B5, and release [3H]d-aspartate in a fashion similar to that shown by cells cultured in foetal calf serum, they show a 60% reduction in the total number of kainate binding sites. Excitatory amino acid (EAA)-triggered and depolarization-stimulated calcium influx, measured in the presence of different agonists, is 50–80% lower in NOAC-cultured cells. NOAC cells are resistant to excitotoxic stimuli carried by EAAs or by depolarizing treatments with 50 mM KCI or 6 μM veratridine. The marked resistance of NOAC-cultured neurons to EAAs can be attributed to decreased calcium entry through EAA-coupled and voltage-gated calcium channels and possibly to other, as yet unidentified, phenotypic properties of these cells. These findings demonstrate that rabbit serum contains one or more polypeptide(s) endowed with the properties of promoting in vitro survival and differentiation of rat cerebellar granule cells and of conferring an EAA-resistant phenotype.