The overall goal of these studies is to build an accurate three-dimensional model of striatal neurons that can be used in computer simulation studies of these cells, and to determine the extent to which both natural variation in cell morphology and variations due to experimental procedures may alter the integrative functions of the cell. In some animals, partial deafferentation will be performed by destruction of the nigro-striatal dopamine pathway, and changes in cell shape reassessed. These data will be integrated into an evolving neurophysiological model of the striatal projection neuron that includes realistic ion channels and passive cable properties derived from voltage clamp experiments on the same cell type. In the first part of the project, the effect of lesions of the dopaminergic input to neostriatal spiny neurons on the density, morphology, surface area and volume of dendritic spines will be determined. This lesion mimics the pattern of degeneration seen in Parkinson's disease. Cells from lesioned and unlesioned striata were selectively stained using Golgi impregnation. Tomographic reconstructions were performed using single axis tilt tomography from thick sections viewed with IVEM. Reconstructions were then visualized and processed using the program Analyze. We found that due to inhomogeneities in the Golgi stain and the small dimensions of the spine neck that the spiny dendrite could not be segmented automatically using morphological operations. Therefore, a software package, Xvoxtrace, was developed at the NCMIR for manual contouring of the datasets. Using this program, the contours of the dendritic shaft and dendritic spines can be traced on a series of 2D slices through the volume while the traced contours are displayed simultaneously on both a 3D volume of and the original tilt series. Conversion tools were also written so that the resulting contours could be read into Analyze as a volume in order to obtain surface area and volume measurements for each spine. The pilot portion of this study has been completed: spiny dendrites from a total of 4 animals, both lesioned and unlesioned sides, have been reconstructed and measured. The data has been collated and preliminary analysis has been performed. Although there were no statistical differences between any of the parameters measured (spine density, spine volume, surface area and length), there were trends in the data which indicated that additional reconstructions may be necessary. We are currently performing power analyses to determine how the next phase of the project should proceed.
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