Abstract.A scanning electron microscope (SEM) autoradiographic technique was calibrated and used to determine the site density of acetylcholine receptors within 250 ~tm of the neuromuscular junction in innervated as well as 3-and 10-d denervated sternomastoid muscle of the mouse. In all these groups sharp gradients of receptor site density are seen around the endplates in the first 2-7 ~n, continuing less sharply to between 25 and 50 Ixm. Beyond 50 ~tm (to 250 ~tm) a spatial density gradient is present 3 d after denervation, but none exist by 10 d. These results suggest that the postdenervation steady-state extrajunctional receptor site density is reached sooner near the junction than away from the junction.The usefulness of SEM autoradiography to study the expression and distribution of membrane molecules at high resolution is demonstrated.I NFORMATION on the quantitative distribution of cell surface receptors is usually obtained using light microscope or transmission electron microscope (TEM), ~ autoradiography, biochemical-binding studies, and physiological tests. These procedures are limited either by resolution or by an inability to assess variations in receptor distribution over large areas in a morphologically intact cell. In the present study we demonstrate the value of using scanning electron microscope (SEM) autoradiography for that purpose. A SEM technique, modified from that of Junger and Bachmann (1980) is described. The procedure had already been illustrated in a nonquantitative manner to study acetylcholine receptors (AChRs) on striated muscle cells in culture (Neugebauer et al., 1985;Salpeter, 1986). In the present study, we calibrated the procedure for sensitivity and chemography and used it to demonstrate gradients of AChRs around the neuromuscular junction (nmj) on innervated and denervated mouse sternomastoid muscle. The results are consistent with, and extend those previously derived using other techniques.We conclude that SEM autoradiography can be a reproducible quantitative procedure, with sensitivities comparable to those of TEM and light microscope autoradiography and with the potential of answering questions regarding the distribution of surface receptors on a fine structural scale.1. Abbreviations used in this paper: AChR, acetylcholine receptor; nmj, neuromuscular junction; SEM, scanning electron microscope; TEM, transmission electron microscope.
Materials and Methods
Labeling the ReceptorsThe acetylcholine receptors in the mouse sternomastoid muscle were saturated in vivo by topically applying ~25I-¢t-bungarotoxin onto the surgically exposed muscle in anesthetized mice (10% Nembutal in 10% ethanol, 0.1 cc/10 g body wt) as previously described (Fertuck et al., 1975). Innervated muscles, or muscles denervated either 3 or 10 d previously from adult female mice (25-35 g) were used. Once the receptors on a muscle were saturated, the wound was sutured and the animal allowed to recover for ,o3 h. (We have found that nonspecific binding is more effectively removed in active animals than by ...