Preparative resolution and identification of synaptic components of rat neocortex

Abstract

SUMMARYA varity of density gradient centrifugation techniques have been used with varying degrees of success, for the preparative resolution of pinched off nerve endings, i.e. "synaptosomes," and synaptic components from brain homogenates. The physics of such gradients was critically considered from the standpoint of improving the degree of resolution of particulate classes and preserving morphological integrity. A procedure, using continuous linear Ficoll gradients, is presented which yields improved particulate resolution with good preservation of ultrastructural morphology. Further purification of the membraneous fraction yielded relatively high purity preparations of PSM particulates. The PSM particulates were identified as dissociated postsynaptic membranes by three kinds of electron microscope studies. Broad field electron micrographs, which permit assessment of fraction homogeneity, are presented for all fractions.

Identity of “dumbbell” profiles in synaptosomal fractions from rat brain

“…Structures morphologically similar to the curved, dark-staining profiles seen in the preparation of Garey et al (1972) have also been found in our synaptosome fractions. Elias (1971) has shown that dumbbell profiles in thin sections are most consistent with a cup shape in three dimensions, that is, "a torus with a membrane closing the hole."…”

“…Finally D shows a profile of a free structure similar to that labeled "free postsynaptic membrane" by Garey et al (1972). Figure 2 shows a structure which has a dumbbell-shaped profile in the thin section, also ex- Figure 1 shows a field from the P,B, pellet.…”

“…A recent report (Garey et al, 1972) has identified "dumbbell" shaped profiles in synaptosomal fractions as postsynaptic membranes. I n view of the importance of this finding it is significant that in a different preparation (McGovern, Maguire, Mahler, Moore, and Gurd, in preparation) free mitochrondria may appear deceptively similar.…”

“…Thus while postsynaptic membranes may have dumbbell profiles, dumbbell profiles need not be due to postsynaptic membranes. Garey et al (1972) have found "small electron dense particulates'' designated as postsynaptic membranes (PSM) in their fractions Fl-F4 from continuous Ficoll gradients and in a PSM fraction prepared by purifying fraction F2 on a continuous sucrose gradient. Electron microscopic evidence supporting this interpretation included histochemical staining employing phosphotungstic acid or indium.…”

Effects of cannabis sativa on ultrastructure of the synapse in monkey brain

“…Aggregation of vesicles is also produced by ischemia (Williams and Grossman, 1970) and by experimental allergic encephalomyelitis (Ross and Bornstein, 1969), as well as by toxins (triorthocresylphosphate and methionine sulfoximine -De Robertis et al, 1967b) and certain neuropathies (Alzheimer's disease - Gonatas et al, 1967).…”