We have established a novel, primary culture of E14 rat ventral mesencephalic dopaminergic neurons, in which, at 12 hr after plating, 95% of the cells are neurons (NSE+), 20% are tyrosine hydroxylase positive (TH+), 5% are glioblasts~neuroblasts (vimentin~positive) and glial fibrillary acidic protein (GFAP) is ex~pressed in <1% of the cells. Dopaminergic neurons in culture exhibit distinct glia independent (at DIV0~DIV9) and glia~dependent (after DIV9) phases of development, even when grown in a medium supple~mented with 10% serum. Serum~deprivation caused the selective death of TH+ neurons, while the per~centage of GABA~IR neurons increased. These findings, combined with a microisland culturing method, have been used to develop a reliable, sensitive bioassay for dopaminergic neurotrophic factors(DNTFs), which will be crucially important in our purification studies. The lysed extracts from type~1 astrocytes and O~2A progenitor cells, prepared from the ventral mesencephalon, and their conditioned media protect dopaminergic neurons from death. The comparative potency of the protective effect of type~1 astrocytes and O~2A progenitor cells is 1:5. This demonstration of the potent dopaminergic neurotrophic effect of O~2A progenitor cells is a major new finding. We are growing ventral, mesencephalic type~1 astrocytes and O~2A progenitors on a large scale, and will identify, purify and achieve the molecular character~ization of at least one DNTF. Preliminary results, based on gel filtration studies using a Sephadex G~75, column indicate that 8 major peaks, all of molecular mass <50 KD, are present in the type~1 astrocytes lysate~conditioned medium, and that significant dopaminergic, neurotrophic activity is retained in peak #7 (fraction #71). Our functional studies demonstrate that GpIa muscle spindle afferents are tightly coupled to their homonymous alpha motoneurons (alpha~MNs) in spinalized, neonatal, PN7 rats that recover rhythmic stepping in their hindlimbs, but not in spinalized, PN14 rats, which do not recover. Trans~plantation of a suspension of dopaminergic neurons into the lumbar region of the spinal cord of the PN14 rat, previously spinalized at the middle thoracic level, was associated with functional recovery in the hindlimbs, similar to that seen in the spinalized, PN7 rat. A dense, neodopaminergic innervation of the lumbosacral spinal cord was also observed. We are now uniquely placed to investigate the neuro~physiological basis of functional recovery in the mammalian spinal cord that is associated with a dense, dopaminergic reinnervation.
