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).
Resolution for 125I-labeled specimens under electron microscope (EM) autoradiographic conditions was assessed experimentally. With this isotope the size of the silver halide crystal was the most important resolution-limiting factor. Heavy metal staining such as is routinely used in preparing animal tissues for EM autoradiography produced an improvement in resolution of -15-20%. For a 500-1,000-/~ biological tissue section fixed with OsO4 and stained with uranyl acetate, we obtained resolution (half distance, HD) values of -800 -120/~ using Ilford L4 emulsion and 500 -70 A using a Kodak NTE-type emulsion. General aspects of resolution-limiting factors and comparison with 3H and 14C values are discussed.On theoretical grounds, 125I is a highly favorable isotope (see Appendix A) for electron microscope (EM) autoradiography. This was early recognized by Kayes et al. (11) in 1962. However, although some calibration studies for both sensitivity and resolution have been performed using this isotope (6, 10), the resolution attainable with this isotope under varying experimental conditions has not yet been systematically assessed. In the present study we have adapted previous calibration procedures used for tritium and ~4C (2,7,20,22) to obtain resolution values for mzsI as a function of section thickness, photographic emulsions, developing procedures, and heayy metal staining as employed in Em autoradiography. The results allowed us to reassess the critical parameters affecting resolution for isotopes of different energy. MATERIALS AND METHODS Resolution SpecimensTwo resolution specimens were used. One was to test resolution in plastic specimens (density 1.1) as a function of section thickness and different emulsion-developer combinations; the second was to test the effect of increasing specimen density by the incorporation of heavy metals such as osmium and uranium, which are used in the fixation and staining of biological material for electron microscopy.RESOLUTION USING PLASTIC TEST SPECI-MENS: The calibration specimen was modeled after that described previously for tritium and 14C (2,20,22) in which a thin film of radioactive polystyrene was sandwiched between a polymerized Epon block and a layer of nonradioactive methacrylate. (The radioactive styrene was thus never exposed to the solvents normally involved in embedding for EM autoradiography, which would
Sensitivity in electron microscope autoradiography was determined for 125!. Values are given using Ilford L4 and Kodak NTE emulsions combined with different developers. The extent of self-absorption as a function of section thickness and heavy metal staining and the effect of radiation dose (dose dependence) were assessed. It was found that the overall efficiency for 125! was better than that for tritium and that, as is the case with tritium, there is a distinct "dose dependence" especially when Microdol X is the developer. Selfabsorption studies indicate that self-absorption is increased by about 15% when the specimen thickness is increased from 300 to 1000 A. An increase of under 15% is also introduced by heavy metal staining of sections in this thickness range.
Mouse sternomastoid muscles were incubated with diisopropylfluorophosphate (DFP) in vivo, and the time course of recovery was studied using histochemistry, EM autoradiography and physiology. We found that: (1) the ability of the muscle to sustain tetanus in response to nerve stimulation is eliminated when the esterases at the neuromuscular junctions are saturated with DFP. This ability is regained partially when less than 10% of the DFP-binding sites have recovered. (2) There is a positive correlation between the frequency of stimulation at which the tetanic response can be maintained and the extent of acetylcholinesterase (AChE) recovery. (3) Tetanic responses at fusion frequency (about 100 Hz) appear indistinguishable from controls with only about 25% of normal AChE. (4) Butyrylcholinesterase (BuChE) possibly of Schwann cell origin recovers more rapidly than does AChE. (5) The muscle shows fine structural changes involving Z band dissolution and the breakdown of sarcoplasmic reticulum within hours after esterase inactivation. (6) This myopathy reaches a peak at three days after esterase inactivation and is almost fully recovered by two weeks. (7) It can be eliminated if, at the time of esterase inactivation, the nerve is cut or the acetylcholine receptors at the endplate are inactivated by alpha-bungarotoxin. We suggest that the myopathy, seen after DFP, is mediated by Ca2+ fluxes due to prolonged action of acetylcholine (ACh) in the absence of esterases.
Acetylcholine receptors were inactivated in vivo at the mouse neuromuscular junction using alpha-bungarotoxin (alpha-BTX). It was found that neurally produced muscle contraction recovered within 4-8 days (halftime similar to 3 days). Actinomycin D interfered with this recovery, but did not affect normal nerve-stimulated muscle contraction. If the response was initially eliminated by [125-I]alpha-BTX and the end plates examined by EM autoradiography, no evidence of mass internalization of bound radioactivity during recovery was seen. The fine structure of the end plates and muscle was unaltered during the post-alpha-BTX recovery period.
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