Preparation-dependent distribution of intramembranous particles in freeze-fracture replicas of the neuromuscular junction of the locust Schistocerca gregaria

Summary Freeze-fracture replicas of the neuromuscular junction were prepared from untreated retractor unguis muscles of the locust Schistocerca gregaria that were rapidly frozen by contact with a copper block cooled by liquid helium. These replicas were compared with others prepared from tissue following fixation with glutaraldehyde and cryoprotection in glycerol. Freeze-fracture of rapidly frozen tissue produced replicas of high quality with little evidence of tissue damage by ice crystals in the superficial layers. The gross fracturing characteristics of the neuromuscular junction were consistent with replicas from fixed and cryoprotected tissue; all of the membrane specializations were recognisable but with some alterations in infrastructure. In tissue replicas prepared using either method intramembranous particles in the presynaptic membrane were arranged in a bar-like array. The intramembranous particles of this presynaptic bar array of the rapidly frozen material were large and found on the E-face of the cleaved membrane. This contrasts with the P-face distribution of the comparable particles in muscles fixed in glutaraldehyde and cryoprotected in glycerol, in which they are also smaller and more numerous. This difference in partitioning between rapidly frozen, and fixed, cryoprotected nerve terminals is not found at cholinergic synapses and thus may reflect functional differences between the two types of junction.Indentations of the nerve-terminal membrane occur in replicas from rapidly frozen muscle as well as fixed and cryoprotected muscle suggesting they are not fixation or glycerol-induced artifacts. It is suggested from their position and size that these indentations are more likely to be part of a membrane retrieval system than exocytotic figures.This work was supported by an S.E.R.C. project grant to I.R.D.