Acetylcholine receptor at neuromuscular junctions by EM autoradiography using mask analysis and linear sources

Several methods of analyzing EM autoradiograms are now available. Two such procedures, the grain density distribution (or histogram) method and the mask method use the resolution of the EM autoradiographic technique to generate grain distributions expected from postulated sources, and compare these with the observed grains in the autoradiograms. These two methods are here compared in the analysis of label on linear sources: the distribution of labeled acetylcholine receptor (AChR) down the postjunctional folds of lizard and frog neuromuscular junctions. The receptors were labeled with I-25-α-bungarotoxin and the autoradiograms coated with the high resolution Kodak emulsion 129–01. We found that both methods gave similar results in confirming that the bulk of the AChR is concentrated on the thickened region of the membrane at the top ～2000 A of the junctional folds, and that there may be a gradient of receptor concentration down the folds. The grain density distribution method is simpler, but does not lend itself easily to quantifying the extent of deviation from simple models. Although computer graphics is not necessary for either method, its use allows the expected grains from linear sources to be generated quickly, making the mask analysis a feasible routine method for assigning the extent of label in different membrane regions.