Markers for Membrane Components to Study “Recycling” at Neuromuscular Junctions

Abstract

We recently applied an improved quick-freezing technique at frog nerve muscle preparations and were able to show that fusion of synaptic vesicles with the axolemma and quantal release of ACh are coincident. This result fulfills an important requirement of the vesicle hypothesis of transmitter release. The continued and intense secretion of quanta over long period of time requires some means of maintaining the population of synaptic vesicles. Over the short term this is done by the endocytosis of membrane from the axolemma. However, almost nothing is known about this important membrane conservation process. In particular, it is not known how rapidly endocytosis follows upon exocytosis, whether the sites of endocytosis are distant from the sites of exocytosis or what factors control the rate of endocytosis. Much of our ignorance concerning endocytosis stems from the difficulty in tracing the movements of vesicle membrane. Specific markers for vesicle membrane that could be localized at the E.M. level allow many of the controversial questions mentioned above to be settled. We have therefore developed immunohistochemistry techniques in order to visualize at frog neuromuscular junctions specific markers for the synaptic vesicle and nerve terminal membranes. Synapsin I has been shown by immunohistochemistry to be present in many mammalian terminals and in brain synaptosomes it is associated with the outer half of the synaptic vesicle membrane. Our work shows that Synapsin I is also present in frog nerve terminals. We have applied similar immunofluorescence techniques to study the binding of -latrotoxin, the active component of black widow spider venom, to nerve muscle preparations. The binding is highly localized and seems to be present only at the nerve endings. If these two proteins prove to be specific markers for the synaptic vesicle membrane and axolemma, respectively, it would be of immense value to study membrane turnover at neuromuscular junction.