An analysis of grain distributions around a radioactive line source (consisting of polystyrene-1H) showed that the shape of the distribution was independent of the factors that influence resolution, i .en section and emulsion thickness, silver halide crystal, and developed grain size. These factors did effect the spread of the distribution, however, and thus the distance from the line source within which 50 % of the total developed grains fell . We called this distance "half distance" (HD) and determined it for a variety of specimens . When grain distributions were normalized in units of HD, one could plot universal grain distributions for specimens with radioactive sources of various shapes . The use of HD and the universal curves in interpreting radioautograms is discussed .

We recorded miniature endplate currents (mEPCs) using simultaneous voltage clamp and extracellular methods, allowing correction for time course measurement errors. We obtained a 20-80% rise time (tr) of "80 pis at 22°C, shorter than any previously reported values, and tr variability (SD) (12). Analog mEPCs were also used to select a low pass filter cutoff of 9 kHz

SUMMARY1. The distribution of acetylcholine receptors (AChR) at frog cutaneous pectoris neuromuscular junctions was studied quantitatively using [125I]a-bungarotoxin (a-BTX) labelling and EM autoradiography.2. We found that, as in mouse end-plates, the AChR is localized uniformly along the thickened post-junctional membrane. In the frog muscle this specialized membrane constitutes approximately the top 50 % of the junctional folds.3. The receptor site density is 26,000 + 6000 sites/,um2 on the thickened postjunctional membrane and falls sharply to 50 sites/fum2 within 15 Itm from the axon terminal.4. a-BTX site density on the presynaptic axonal membrane was directly determined to be at most 5 % of the value on the thickened post-junctional membrane.5. The high post junctional AChR site density leads us to conclude that: (a) each quantum of ACh needs to spread only over a very small post-junctional area (to be called the 'critical area') before it encounters as many AChR (plus AChE) sites as there are ACh molecules in the quantum (for a packet of 104 ACh molecules this critical area is-03 #um2), (b) the average concentration of ACh prevailing in

Exposed sternomastoid muscles of anaesthetized mice were bathed in 1251-labeled a-bungarotoxin until all neurally evoked muscle contractions were eliminated. The distribution of label was then determined by electron microscope autoradiography. It was found that the label was localized at the top of the junctional folds, i.e., at the postjunctional membrane nearest the axon. Since the a-bungarotoxin had fully eliminated the physiological muscle response, these results indicate that the active acetylcholine receptor occupies a limited area of the junctional folds and is not distributed uniformly throughout this membrane. Specialized membrane densities seem to coincide with the labeled regions. Fig. 2A).The present communication refutes this assumption. We found that the active AChR, as judged by '25I-labeled abungarotoxin binding, is concentrated in the region of the junctional folds adjacent to the axonal membrane. MATERIALS AND METHODSBiological System. Three mice were used for the results reported here. The exposed sternomastoid muscle of an anaesthetized mouse was bathed in 12I-labeled a-bungarotoxin, while the nerve was stimulated by a suction electrode. Muscle contractions were monitored with a delicate strain gauge and recorded on a two-channel polygraph. Stimulation conditions, chosen to give a maximal tetanic muscle response, were as follows:The stimulating frequency was 90-100 sec-1 (well above mechanical fusion frequencies), and for each animal the stimulating voltage was adjusted for maximum contraction.12'I-Labeled a-bungarotoxint (2,uM) at 135 Ci/mmol was then applied topically in Krebs' buffer and the nerve stimulation was repeated intermittently (once every 15 min) until the neurally evoked muscle response was eliminated. Autoradiographic Calibration. Although iodine-125 was one of the first isotopes used for electron microscope autoradiography (12), it had not been calibrated for quantitative interpretation of the autoradiograms. In an earlier study we established that the sensitivity with this isotope is higher than with tritium (13). For the present study we tested its resolution by a method similar to that used for tritium (14).We found that for 126I, 1000-A sections and Ilford L4 emulsion t Mr. Peter M. Ravdin of Cornell University purified and iodinated the bungarotoxin using lactoperoxidase (9) based on the procedure described by Eldefrawi and Fertuck (10). The specificity of the iodinated bungarotoxin was compared with noniodinated toxin by its lethal dose, by the concentration and time taken to inactivate the muscle response, by its localization at the endplate using light autoradiography, and by its competition for ACh receptor sites in torpedo electroplax membrane fractions (10).