The dopaminergic neurons of the mesencephalon, innervating the telencephalon, have been the focus of intense investigation over the past three decades, and the information derived from such studies has revolutionized our understanding of CNS function. The development and normal maintenance of the dopaminergic projections has been studied in our laboratory using an in vitro dissociated cell-reaggregation system in which single cell suspensions prepared from specific areas of the embryonic mouse brain are allowed to reaggregate either by themselves or with cells from other areas of the brain to which they normally project axons and form synaptic connections. The primary advantage of this system is that it permits a selective reconstruction of neuronal pathways of the embryonic brain in which the neurons and glia can continue their normal development to the adult stage in a pattern remarkably similar to that observed in vivo. The reaggregate system will be used to examine the role of target and nontarget cells in dopaminergic cell survival and the size and specificity of the dopaminergic projections as well as the role of convergent mesencephalic and cortical inputs in regulating DARRP-32 phosphorylation in striatal medium spiny neurons. In addition, we have developed immortalized hybrid dopaminergic cell lines which produce and release the transmitter. Cell lines have also been generated from primary neurons of the corpus striatum which express an array of functional dopaminergic receptors and in some cases a cholinergic phenotype. The transmitter phenotype of both the mesencephalic dopaminergic and striatal cholinergic immortalized cells is markedly reduced in the presence of nontarget cells (cells which do not receive synaptic connections from the primary neurons from which these cells are derived). The down-regulation of the transmitter phenotype of immortalized cells by nontargets may be of functional significance in brain development and an attempt will be made to identify the recognition molecules involved in this process. New approaches involving introduction of the temperature-regulated SV40 large T antigen followed by somatic cell hybridization will be used for the immortalization of central dopaminergic neurons with temperature-regulated growth characteristics permitting the transplantation of these cells into the brain. Studies will be undertaken on the use of immortalized clonal dopaminergic cell lines, as well as mature dopamine neurons maintained in reaggregate culture, as a source of cells for neuronal replacement in lesion models of denervation and in the naturally occurring degeneration of dopaminergic neurons in the weaver mutant mouse. Such studies will be directed at examining the utility of immortalized dopaminergic cell lines, as well as the usefulness of the reaggregate culture system, as a means for maintaining dopaminergic neurons in culture for extended periods of time, which would then be available on demand for transplantation purposes in either experimental animals or in human disease states.

R01MH028942

1977-09-01

2000-07-31