The long-term objective of this proposal is to understand the process by which environmental chemicals can destroy distinct populations of neurons in the brain-especially during development. Organotins such as trimethyl tin (TMT) induce preferential toxicity in the hippocampus- a region of the brain critical for development of strategies for certain types of learning and memory. This effect occurs prominently during development. TMT induces a variety of cellular effects in vitro, but as yet the mechanism(s) by which TMT induces cell loss, or the reason for the predilection for hippocampus remains unclear. Among the myriad of effects which TMT has in the hippocampus are alterations in the levels of proteins which interact with the neuronal cytoskeleton, and changes in ionized [Ca2+], a critical regulator of growth cone elongation. Mechanical tension on neurons is an important regulator of neuron process development. Simple exertion of tension on brain neurons with force-calibrated glass needles can cause them to begin to develop their characteristic branching pattern, extend their major process, the axon, and cause them to retract from other cells. Further, growth and spreading of the neuron from the growth cone is also the result of mechanical tension produced by pulling growth cones. As a first step in attempting to examine the effects of TMT on hippocampal neuron growth, differentiation and survival, we propose to examine the effect which TMT has on generation of neuronal tension in developing hippocampal pyramidal neurons in primary culture, or transformed neuronal cells- rat pheochromocytoma (PC12) cells- a commonly-used model for neuronal growth, and a cell type which exhibits sensitivity to TMT. The proposal has five discrete aims. They are: 1) Does exposure to TMT inhibit initiation of axonal outgrowth from cultured rat hippocampal pyramidal cells? 2) Does TMT exposure compromise the normal capacity of the axonal growth cone to exert tension? 3) Does TMT exposure alter the quantitative relationship between axonal elongation rate and tension magnitude? 4) Does TMT exposure alter the active contraction of axons in response to slackening? 5) Does TMT exposure alter the elastic-like behavior of axons in response to tension? Glass needles of known bending constant will be used to initiate axonal outgrowth from (untreated) control and TMT-poisoned neurons. The two groups will be compared for the magnitudes of force required for and the frequency of success at initiating neurites by experimentally applied tension, or that exerted by the advancing growth cones of individual neurons in response to sub-lethal doses of TMT. The final specific aim focuses on the elastic, non-growth responses of cultured pyramidal neurons to experimentally-applied tension. Calibrated glass needles will be used to apply rapid """"""""plucks"""""""" to the axons of individual neurons to determine whether TMT causes a change in neurite stiffness and/or rest tension. The proposed studies will begin to provide information regarding the ability of organotins to disrupt neural development by interfering with the sensitivity of the neuron to mechanical tension.
