To optimize CNS graft survival, fetal donor tissue is essential. Given the ethical and logistical constraints of obtaining human fetal tissue to replace damaged CNS neurons, one alternative strategy is the development of neuronal cell lines whose mitotic activity and neuronal differentiation in vivo can be regulated. The goal of this research is to functionally restore the damaged spinal cord with novel neuronal cell lines. Immortal, neurotransmitter-specific, cell lines will be developed from specific regions of the embryonic CNS which can be induced to stop dividing both in vitro and in vivo and differentiate with neuronal properties. These cell lines will be transplanted into spinal cord in an attempt to functionally restore experimentally depleted endogenous neurotransmitter. To develop these cell lines, a retrovirus construct containing DNA sequences encoding the temperature-sensitive (ts) mutant of the SV40 large T antigen will be used as the transforming vector. The rationale for this strategy is that cells transformed with this construct are mitotically active at """"""""permissive"""""""" temperatures (33C), where the T antigen transforming protein is functional. At """"""""non-permissive"""""""" temperature (39 C), the thermolabile oncogenic protein is inactive and the cells stop dividing and """"""""resume differentiation"""""""", presumably in the direction that was interrupted at the time of viral infection. Ts-cell lines from locus coeruleus (tsLC) and medullary raphe nucleus (tsRN) will be grown in vitro at 39 C and characterized morphologically for differentiation toward a neuronal phenotype and immunohistochemically for the expression of glialor neuronal-specific antigens. tsLC and tsRN cell lines which differentiate neuronally will be further characterized immunohistochemically and biochemically for the ability to synthesize norepinephrine (NE) and serotonin (5HT) respectively. tsLC ad tsRN cell lines which synthesize the appropriate neurotransmitter will be transplanted into 6-OHDA and 5,7-DHT-lesioned rodent lumbar spinal cord to determine whether these ts-cell lines can replace the denervated monoaminergic fibers. One month post- transplantation, lower limb flexor and extensor reflexes, which are modulated by NE and 5HT respectively, will be assessed adn the grafted area examined histologically, immunohistochemically and biochemically for the extent of graft survival, differentiation and possible re- innervation.
