MESENCEPHALIC TYPE-I ASTROCYTES MEDIATE THE SURVIVAL OF SUBSTANTIA-NIGRA DOPAMINERGIC-NEURONS IN CULTURE

We previously demonstrated that substantia nigra (SN) support cells selectively increase SN dopamine (DA) neuron survival in dissociated primary culture. Increased survival was elicited specifically by nigral support cells; glia from other brain regions exerted lesser effects. We now report that Type I astrocytes, the principal component of SN support cell monolayers, mediate the enhanced DA cell survival. Initially, the predominant glial subtypes in SN support cell cultures were identified. Postnatal day 1 rat SN was dissociated and cells were grown to confluence (7-9 days in vitro; DIV). Monolayers were immunostained with antibodies against glial fibrillary acidic protein (GFAP; an astrocyte-specific marker), myelin basic protein (MBP; an oligodendrocyte marker), or A2B5 (recognizes 0-2A progenitors and Type II astrocytes). The number of GFAP+ cells far exceeded MBP+ and A2B5+ cells, suggesting that astrocytes constituted the predominant subpopulation. Further, direct comparison of GFAP+ (Type I and Type II astrocytes) and A2B5+ (Type II astrocytes) cells indicated that the vast majority were Type I astrocytes. Greater than 98% of cells reacted with glial antibodies. To definitively characterize the cellular subtype that augments survival of DA neurons, glial subcultures were established. At 2 DIV, enriched populations of Type I or Type II astrocytes, or oligodendrocytes, were tested for the ability to elicit DA neuron survival. Embryonic day 16 rat SN dissociates were added and DA cell number was assessed with antibody against tyrosine hydroxylase (TH), the DA biosynthetic enzyme. After 2 days in coculture, the Type I astrocyte group exhibited greater than a 2-fold increase in TH cell number, while oligodendrocyte and Type II astrocyte cocultures did not differ from control. Our observations indicate that Type I astrocytes alone enhanced DA neuron survival.