marion murray - US grants

The first set of proposed experiments is designed to compare and contrast synaptogenesis occurring in a regenerating system, the goldfish retinotectal system, with lesion induced reorganization occurring by collateral sprouting in a non-regenerating system, the rat interpeduncular nucleus (IPN). The specific issues are related to a comparison of the factors regulating reestablishment of specific numbers and patterns of connections by regenerating goldfish retinal axons with the factors regulating collateral sprouting and reactive reinnervation by intact neurons after lesions in mammalian CNS. This information is essential in order to predict the functional consequences of lesion induced synaptogenesis in the adult CNS. A second goal is to determine those cellular processes which distinguish the response of the rat retinal ganglion cell, axotomized and induced to elongate its axon via implantation techniques, from the axotomized but non-regenerating rat retinal ganglion cell and from the regenerating goldfish retinal ganglion cell. These studies should contribute to an elucidation of the processes which account for the robust and directed growth of the axotomized goldfish optic axons and those which account for the normal failure of this growth in the axotomized mammalian central axons.

This Program Project focuses on mechanisms which account for recovery of locomotor function following spinal injury and on manipulations which can enhance the recovery Our studies carried out during previous granting periods have indicated that restoration of function may not require reestablishment of the original spinal circuitry limited regeneration together with other compensatory responses may suffice. Individual projects in the present Program Project will examine the correlates of motor recovery at the levels of specific molecules, synapses, circuits, and biomechanical systems. In a Project we will examine the mechanisms by which transplants of embryonic spinal cord improve motor function in rats with complete spinal cord transections. The physiologic basis of this recovery will be analyzed quantitatively and manipulated pharmacologically. These studies will allow us to distinguish the contributions to recovery made by supraspinal and propriospinal systems and those made by the reorganizing spinal cord caudal to the transection and transplant. In Tessler's Project we will determine whether identified neurotrophic factors delivered to the injured rat spinal cord by genetically modified cells can enhance the survival and regeneration of axotomized Clarke's nucleus, neurons, and whether transplants can allow these cells to maintain their normal circuitry. In rat vestibulospinal neurons to be studied in Tessler's Project and in goldfish Mauthner cells to be studied in Faber's Project we will examine the relationship between spontaneous regeneration or regeneration assisted by transplants and motor behaviors mediated by identified sets of brainstem neurons. In Fischer's Project parallel in vivo and in vitro experiments will examine the molecular mechanisms by which axotomized adult rat dorsal root ganglion neurons modify the expression and transport of cytoskeletal proteins as their axons change from stable structures to actively growing processes. Understanding these mechanisms will contribute to developing interventions that can enhance the regeneration of CNS axons. These projects together will provide quantitative information on the nature and extent of recovery of function in several different models. examine the extent and constraints on regeneration into the CNS and suggest therapeutic interventions that will enhance recovery.

DESCRIPTION (provided by applicant) The restoration of neurological function after injury to the spinal cord will certainly require a multifaceted genetic, cell biological and pharmacological approach accompanied by precise behavioral and physiological evaluation. Transplantation of fetal neural tissue permits some degree of motor competence to develop in rats and cats after spinal transection when the lesion and graft are made in neonates. We have also shown that grafts of genetically modified fibroblasts improve motor control after partial lesions in adults. Our hypothesis is that grafts of cells modified to secrete bioactive molecules will provide greater development or recovery of function after spinal transection in both neonates and adults because these grafts promote greater regeneration. Recovery after grafting, however, is unlikely to be complete. Pharmacological interventions, specifically with serotonergic agonists, have been shown to enhance recovery mediated by transplants. In these experiments we will continue investigation of pharmacological interventions to determine mechanisms by which these agonists act to improve function in spinal rats. We will also compare fetal transplants with transplants of genetically modified cells made in neonates and adults to determine whether pharmacological agents act synergistically with the transplant mediated effects to improve function further. Alternate strategies including co-grafting a cell line (RN46A-B14) that will secrete 5HT into lumbar cord may produce more long-lasting improvement in function. We will also extend our studies to include catecholaminergic agonists since we believe that the optimal pharmacological strategy may include stimulation of both serotonergic and noradrenergic receptors. Lesions made in neonates and in adults modify circuitry. Introduction of trophic factors into the spinal cord of neonates and adults is also likely to modify circuitry by stimulating growth of systems bearing appropriate receptors. The effects on lumbar circuitry of spinal injury and introduction of trophic factors are not known but must be important in understanding the limits on recovery of function and for developing principled therapeutic strategies. We will focus on changes in the descending monoaminergic systems and the peptidergic afferent systems and their receptor binding sites at the level of the transection/transplantation and in lumbar cord. Regeneration of descending and regeneration (or sprouting) of small caliber dorsal root axons will be studied immunocytochemically and changes in density of their receptor binding sites will be measured. Together these studies should provide significant information about mechanisms of pharmacological stimulation of function that may lead to development of therapies.