Over a three year period we will examine an in vitro cell culture system for the neurotoxic and neurodevelopmental effects caused by inorganic and organic lead (Pb) compounds commonly found in the environment. We intend to study the influence of this heavy metal on an in vitro experimental system that accurately reflects in vivo CNS function and development. This cell culture system offers the advantage of exposing neurons and glial cells to known concentrations of potential toxins at diffferent developmental stages and permits direct assessment of neural damage and recovery as well as exploration of the underlying molecular mechanisms and possible intervention methods. Cells from 16 day old embryonic mouse brains shall be dissociated into single cell suspensions and placed into rotary culture where they resume their growth and differentiation by reaggregating, developing axonal networks, myelinating, and exhibiting neurotransmitter synthesis, storage, release and uptake. At four stages of in vitro development (equivalent to the prenate, neonate, adolescent and young adult CNS), the effects of inorganic and organic lead compounds on subsequent developmental events and the organization of neuronal and glial cells shall be evaluated by exposing the reaggregates to four different concentrations of lead compounds for 24 hours. At the time of harvesting, aliquots of reaggregates shall be taken to determine the influence of these lead compounds on 1) the cellular organizaiton of the reaggregates as determined by conventional and electron microscopy, 2) protein and phosphoprotein composition 3) myelin membrane synthesis, and 4) signal tranduction mechanisms involving second messenger systems. Control cells shall be taken from flasks treated identically, but in which no lead compound has been added. Definition of the specific CNS anatomical, physiological and biochemical changes caused by lead at various developmental stages using this in vitro experimental system will provide information for preventing and possibly therapeutically intervening in the molecular processes responsible for lead's neurotoxic effects.
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