ON THE ISOLATION OF NERVE ENDINGS AND SYNAPTIC VESICLES

Since the first description of the "synaptic vesicles" (1, 2) the suggestion was made that they could be associated with acetylcholine and other chemical synaptic mediators. Later work gave further information about the possible composition of synaptic vesicles and their role in synaptic functions. It was shown that they degenerate very early in the nerve terminal after section of the axon (3), a finding that can be correlated with the decrease in transmitter content of the synapse (4, 5). It was also found that the synaptic vesicles of some of the retina synapses were reduced in size with disuse (6) and changed with dark adaptation and stimulation with light (7). Finally, striking changes in the number of synaptic vesicles were found within the cholinergic nerve endings of the adrenal medulla after stimulation of the splanchnic nerve with supramaximal pulses of different frequencies (8). These experiments led to the conclusion that within the synaptic ending a balance exists between the formation and release of synaptic vesicles, and that this balance depends on the frequency of stimulation (9). At the same time the concept of "synaptic vesicles" was associated with the "quant ized" release of acetylcholine at the neuromuscular junction (10), and it became widely used in the physiological literature on synaptic transmission (see l 1). From the biochemical viewpoint, Feldberg (12) gave some of the first evidence that acetylcholine is associated with a protein containing a particulate component which was generally identified with mitochondria. Recently, it was found that the largest proportion of acetylcholine is bound to the so called mitochondrial fraction (13, 14). However, upon further sedimentation of this fraction on a gradient, Whittaker (13) could demonstrate that most of it is associated with subcellular structures different from mitochondria. He concluded that most of the particle-bound acetylcholine and 5-hydroxytryptamine is localized in a vesicular layer which was interpreted as representing the synaptic vesicles. The fact that synaptic vesicles have a mean size of about 400 A (8) makes it difficult to conceive that it could sediment as such in a fraction of the size of mitochondria. The published electron micrographs of Whittaker (13) were not clear enough to bring out this point, and the mean particle diameter found was larger (650 A) than that of synaptic vesicles. Our laboratory has been engaged in the last year in a similar effort, trying to use several fractionation methods and a close study of each fraction by electron microscopy. While a detailed account of the techniques used and of the results obtained on the submicroscopic analysis of the fractions is being prepared for publication, we would like to present some observations that bear on the work of Whittaker (13) and that may clarify the apparent contradiction between the size of synapfic vesicles and the fraction in which acetylcholine is generally found. As material we used the most superficial layers of the brain cortex of the rat and the dog in which the synaptic endings are very abundant. Using a modification of the method of Berger (15) for isolation of mitochondria, we found that together with the isolated mitochondria there are numerous intact endings filled with synaptic vesicles (Figs. 1 and 2). Some of these endings show small mitochondria within. The surface membrane of the ending is intact and it may even show its synaptic portion, characterized by higher density and attachment to the postsynaptic component. Essentially similar results were found by repeating the isolation with the method used by Whittaker (13) for the mitochondrial fraction, which also shows numerous intact endings filled with synaptic vesicles. These observations were interpreted as indicating that homogenization was not strong enough to produce the disruption of most of synaptic terminals. Using a tighter teflon homogenizer and longer periods, a mitochondrial fraction showing fewer intact nerve endings was obtained. In this case, after isolation of mitochondria, a microsomal fraction was sedimented which is devoid of intact endings. It consists of a complex