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RESEARCH INTERESTS
Neurobiology; fluorescent dye design
Most of my non-teaching time during the last several years has been devoted to designing, building, and managing the Department's computing and network facilities (see Biology Computing Facilities ). In the time left for research, I have been pursuing design of fluorescent dyes intended to identify particular neuron types in dissociated cell suspensions prepared for culture. Dissociated cell cultures permit many aspects of neuronal development and physiology to be investigated with a directness and rigor not presently achievable with intact nervous tissues, but full realization of the experimental potential in such cultures requires that it be possible to identify and control the neuron types present in them. And, a priori, such in-vitro identifications can't be based on cellular properties known to distinguish specific neuron types in vivo until it has been established that those properties are reliably retained, and restricted, after the cells are dissociated. Thus, some initial means of identification not subject to change in vitro must be employed, even to discover which cellular properties are, or are not, stable in culture.
The approach my lab has taken to this problem is to use fluorescent tracers to mark particular neuron types on the basis of properties that distinguish them while they are still in the organism. These fluorescent tags can then be used to identify the marked neurons following dissociation. One already successful implementation of this approach, employing 'retrograde transport' (from synaptic terminal to cell body) of a fluorescent molecule, marks neurons on the basis of their specific synaptic targets ("output" connections), for example, motor neurons on the basis of the muscles they innervate. A second, complementary strategy being explored would mark neurons activated by particular "inputs" e.g., particular chemical transmitters or sensory stimuli. The intent in this case is to label neurons active under particular experimental conditions, with a fluorescent tag that will remain with them for an extended subsequent period, permitting their later identification after dissociation. For this purpose, a new class of fluorescent dye has been designed, and one successfully synthesized example has been shown to accumulate selectively, and to be trapped, within neurons depolarized in its presence. Other work developing from this project involves design of trapped dyes that mark living cells, or cellular organelles, on the basis of internal pH or specific enzymatic activities.
In all these approaches, once the fluorescent tags have been attached, the marked neurons, or other cell types, can be readily identified in subsequently prepared dissociated cell suspensions or cultures, and they can be separated from other cells by fluorescence-activated cell sorting equipment. Populations of living neurons, purified by neuron type in this way, are useful not only for cell cultures as originally intended, but also for direct biochemical and immunological analyses and for neuron transplantation studies.
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