ELECTRON MICROSCOPE RADIOAUTOGRAPHY AS A QUANTITATIVE TOOL IN ENZYME CYTOCHEMISTRY

The distribution of diisopropylfluorophosphate (DFP)-sensitive enzyme sites at the neuromuscular junction was determined quantitatively by electron microscope radioautography after incubation of muscle fragments in DFP-3H. Most of the sensitive sites were located in the subneural apparatus at a concentration of 90,000 sites per µ3 of cleft tissue or 12,000 sites per µ2 of postjunctional membrane surface area. A considerable concentration is also present in the teloglial cap. It has previously been demonstrated (Rogers et al., 1966) that one-third of the DFP-sensitive sites at the endplate can be reactivated by pyridine-2-aldoxime methiodide (2-PAM)—a compound which selectively reactivates phosphorylated acetylcholinesterase. In the present study, it was found that this ratio of 1:2 holds also on a fine-structural level. Muscle mast cells were found to have a heavy concentration of bound DFP.

CHOLINESTERASES AND MOTOR END-PLATES IN DEVELOPING DUCK SKELETAL MUSCLE

End-plates in the thigh muscles of duck embryos were first recognized with myristoylcholine as substrate at the 19th day of incubation. Each appeared as a cholinesterase-positive dot surrounded by a small halo which rapidly increased in size during the 20th and 21st days. The endplates were usually oval in shape, averaging 33 µ x 25 µ with a subneural apparatus 5-12µ wide. The latter contained refringent lamellas arranged transversely in a palisade fashion. From the 21st day to the day of hatching (27-29 days) the number of end-plates progressively increased. After hatching, the myristoylcholine-reacting end-plates were difficult to locate. With acetylthiocholine as substrate, the embryonal end-plates were not demonstrable; however, the posthatched tissues showed numerous end-plates. The nerve trunks and nerve fibers gave a faintly positive myristoylcholine reactions in all stages after the 19th day of incubation. On the basis of the effects of eserine and diisopropyl fluorophosphate, the structures reacting with myristoylcholine and acetylthiocholine contained specific chohinesterase. The end-plates containing nonspecific cholinesterase also appeared on the 19th day of incubation and appeared to increase gradually in number until the 15th postembryonic day; thereafter they seemed to decrease.

SUBMICROSCOPIC ORGANIZATION OF THE POSTSYNAPTIC MEMBRANE IN THE MYONEURAL JUNCTION

Cross-striated muscles of frogs and rats were fixed in 3.3 per cent lead nitrate solution. Frozen sections 30 micra thick were mounted in different media and observed by polarization microscopy. The subneural apparatus of myoneural junctions exhibits a strong birefringence in these sections. Birefringence is exerted by a highly organized lipoprotein framework (postsynaptic material) which builds up the "organites" (junctional folds) of the postsynaptic membrane. Synaptic cholinesterase is closely associated with this material. Freezing and/or formalin fixation results in a destruction of the molecular organization of the postsynaptic material, but does not influence the synaptic enzyme activity. It is hypothesized from this study that the junctional folds (postsynaptic "organites") consist of regularly arranged, sheet-like lamellar micellae in the frog and of less regular, mainly radially arranged submicroscopic units in the rat. The micellar organization as revealed by polarization analysis is in good agreement with the electron microscopic findings reported in the literature. Intramicellar protein molecules of the resting postsynaptic membrane are arranged longitudinally, lipids transversely. Supramaximal stimulation or treatment with acetylcholine + eserine results in a disorganization of proteins and a rearrangement of lipids. Denervation results in a rearrangement of lipids without any significant alterations of proteins. All these functional stresses influence only the molecular and not the micellar structure of the membrane. The function of the organized lipoprotein framework as an acetylcholine receptor is suggested.

OBSERVATIONS ON THE FINE STRUCTURE AND CYTOCHEMISTRY OF MOUSE AND HUMAN INTERCOSTAL NEUROMUSCULAR JUNCTIONS

The fine structure of the mouse and human intercostal muscle neuromuscular junction was studied after brief fixation in a new formol-sucrose fixative. This primary formalin fixation was followed by brief postosmication in buffered 1 per cent osmium tetroxide. Muscle blocks were embedded in methacrylate or Epon 812 epoxy resin. Marked similarities between mouse and human motor end-plates were observed. Neuromuscular junctions from both mouse and human intercostal muscle showed synaptic vesicles, primary and secondary synaptic clefts, and layered differentiation of the amorphous surface material (ASM) present on the surface of the Schwann cell plasma membrane and on the muscle surface membrane in the region of the neuromuscular junction. An attempt to stain the ASM with lead was unsuccessful. Observations on thick and thin plastic-embedded sections stained by PAS after diastase digestion showed that the ASM within the subneural apparatus is PAS positive. Alcian blue stained the endoneurium and perineurium of peripheral nerve bundles and portions of the end-plates. The similarity of the PAS-positive ASM to other basement membranes described in other sites is discussed and its possible physiologic significance within the subsynaptic apparatus is considered.

Electron Microscope Study of the Human Neuromuscular Junction

A preliminary electron microscope study of human neuromuscular junction is presented. The biopsy material was taken from the palmarus longus, and fixed routinely in osmium tetroxide and embedded in methacrylate. The structure of the motor endings and the relationship of the synaptic vesicles to the axolemmal membrane are described. The synaptic clefts are filled with an homogeneous material in continuity with the basement membrane covering the muscle fiber. The subneural apparatus is described, and special attention is paid to a vesicular component present in the sarcoplasm of the junctional area, which differs from synaptic vesicles and is presumed to be a derivate of the sarcoplasmic reticulum.