Damien P. Kuffler - US grants

9353130 Zuazaga The proposal requests funds to establish an Imaging Center for the Institute of Neurobiology. Four instruments to perform major cell image analysis or tissue processing are requested: 1) a photometry/microscope system for quantitative fluorescence analysis, 2) a confocal microscope, 3) a ultramicrotome for serial sectioning, and 4) a three-dimensional reconstruction and morphometry system. The equipment will expand capabilities of eight research projects involving ten of the Institute's resident investigators, four of them Hispanic women, their students, and collaborators from mainland institutions. The development of cell imaging research will not only enhance current research at the Institute but will also further involve minority students in state- of-the-art neurobiological research. ***

Central nervous system injury normally results in permanent neuronal deficits, while neuronal connections lost due to peripheral nervous system injury are normally re-established. To understand how regeneration in the central nervous system can be made possible and to promote better re-innervation in the peripheral nervous system, we must understand the basic mechanisms underlying the reestablishment of synaptic connections: what induces neurons to extend processes (trophic influences), how is growth of axons directed towards their targets (tropic influences), and how do axons recognize their targets (targets recognition cues)? Favorable preparations for studying these problems at the cellular and molecular levels are provided by neurons, muscle fibers and macrophages isolated from adult frogs and maintained in tissue culture. This laboratory has established methods for isolating adult motor and sensory neurons which survive for more than four weeks and extend processes. Culture have been established of pure populations of Schwann cells, macrophages and isolated intact adult skeletal muscle fibers. Another preparation will be used is for an in vivo bioassay. This proposal examines 3 aspects of nerve regeneration: (1) A study of the trophic influences of denervated peripheral nerve and macrophages on neuronal process outgrowth. (2) A study of the tropic influence of denervated nerve and macrophages on neuronal process outgrowth (3) Determination of whether motoneuron synapses form with specificity at original synaptic sites on cultured adult muscle fibers (an indicator of selective target recognition). Sensory and motor neurons will be cultured with Schwann cells, macrophages and muscle fibers and in the presence of known and putative trophic factors and the interactions and influences observed using physiological, electrophysiological and morphological methods. Techniques to be used include tissue culture, immunocytochemistry, morphometric analysis of process outgrowth and electron microscopy of contacts between neurons, macrophages and muscle fibers. Electrophysiological studies of synaptic transmission between neurons and muscle fibers will be carried out in collaboration with Project 50. Electrophysiological and morphological studies of the interactions between sensory/motor neurons and co-culture glial cells will be carried out in collaborations with Project 51.

musculoskeletal regeneration; spinal cord; spine injury; clinical research;