The overall goal of this work is to delineate the roles of neurotrophic factors in regulating development, differention, and function of the nervous system. During the past few years, we and others have generated targeted mutations that result in deficiencies in neurotrophins or neurotrophin receptors. Our laboratory has also generated mouse strains where athe coding region of a neurotrophin gene is replaced by lacZ, providing a convenient and sensitive assay for neurotrophin expression within individual cells. In addition, we have prepared function-inhibitory antibodies to some of these molecules which have been invaluable for immunocytochemical studies of their distributions and tests of their functions. In one project, the roles of the neurotrophins will be examined using mice with targeted mutations in and mice expressing ectopically individual neurotrophins. The distributions on the single cell level of neurotrophins in sensory ganglia and the hippocampus will be examined using the lacZ replacement strains of mice and these distributions will be compared to those of the neurotrophin receptors trkA, trkB, and trkC. Consequences of neurotrophin deficiency on neuronal precursor cell proliferation, neuronal fate determination, target innervation pattern, and acquisition of differentiated phenotype will be examined in sensory ganglia and the hippocampus. To avoid pleitrophic effects of neurotrophin deficiency, methods designed to target deficiencies spatially and temporally will be attempted. In the first, injection of Fab fragments of function-inhibitory anti-trkA, B, or C IgG will be used to assess effects of receptor inhibition at key developmental times. In a second approach, Adenovirus vectors will be used to express dominant-negative trk mutants in a few neurons. In a third, efforts will be made to generate conditional mutations by introducing loxP sites flanking essential trk receptor exons. Introduction of cre recombinase by virus or mating to a transgenic mouse strain will be used to eliminate neurotrophin or neurotrophin receptor in defined subsets of neurons at controlled times. This should make it possible to examine consequences of deficiency in a few cells on differentiation and/or function in an otherwise normal environment. Finally, we will extend initial observations indicating that hippocampal neurons express several integrin subunits, expression of some of which are strongly regulated by glutamate receptor activation. We will try to completely characterize athe repertory of integrin subunits expressed by hippocampal neurons. We will examine regulation of mRNA and protein expression by the neurotrophic factors in vitro and in response to neurotrophin deficiency in genetically altered mouse strains in vivo. Effects on expression of agents that affect glutamate receptor activation or LTP will be examined. Investigation of these molecules is potentially important because they are strong candidates to help mediate longer term responses to synaptic activity, involving sprouting, retraction of processes, and other structural changes.
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