Computer-assisted quantification of axo-somatic boutons at the cell membrane of motoneurons

This paper presents a system for computer-assisted quantification of axe-so matic boutons at motoneuron cell-surface membranes, Different immunohistoch emical stains can be used to prepare tissue of the spinal cord. Based on mi crographs displaying single neurons, a finite element balloon model has bee n applied to determine the exact location of the cell membrane, A synaptic profile is extracted next to the cell membrane and normalized with referenc e to the intracellular brightness. Furthermore, a manually selected referen ce cell is used to normalize settings of the microscope as well as variatio ns in histochemical processing for each stain. Thereafter, staining, homoge neity, and allocation of boutons are determined automatically from the syna ptic profiles. The system is evaluated by applying the coefficient of varia tion (C-v) to repeated measurements of a quantity. Based on 1856 motoneuron al images acquired from four animals with three stains, 93% of the images a re analyzed correctly. The others were rejected, based on process protocols . Using only rabbit anti-synaptophysin as primary antibody, the correctness increases above 96%, C-v values are below 3%, 5%, and 6% for all measures with respect to stochastic optimization, cell positioning, and a large rang e of microscope settings, respectively, A sample size of about 100 is requi red to validate a significant reduction of staining in motoneurons below a hemi-section (Wilcoxon rank-sum test, alpha = 0.05, beta = 0.9), Our system yields statistically robust results from light micrographs. In future, it is hoped that this system will substitute for the expensive and time-consum ing analysis of spinal cord injury at the ultra-structural level, such as b y manual interpretation of nonoverlapping electron micrographs.