Effects of Calcium-Containing Fixation Solutions on Cholinergic Synaptic Vesicles

Abstract

Calcium (Ca)-containing fixation solutions applied to slices of electric organ of the electric ray, Narcine brasiliensis, have been shown to have three distinct ultrastructural effects on cholinergic synaptic vesicles of the nerve terminals.(a) An electron-dense particle (EDS) is observed within the vesicle; the particle is seen in unosmicated, unstained tissues and can be removed from thin sections by Ca-chelating agents. It is concluded that the EDS represents Ca bound by the vesicle. It is suggested that the bound ATP of the vesicle provides anionic Ca binding sites.(b) The vesicle membrane tends to 'crinkle' or collapse depending on the concentration of the other components of the fixative solution. The 'crinkling' or collapse are largely reversed by a wash step in the absence of Ca.(c) The presence of Ca results in the appearance of a population of vesicles which form characteristic fusions or 'tight' junctions with the terminal membrane. This appears to be morphological evidence for the proposal, which has been frequently put forward, that Ca facilitates such a fusion before discharge of vesicle-bound transmitter.With the discovery that the use of Ca-containing fixatives leads to the demonstration of a subpopulation of synaptic vesicles fused to the terminal membrane, we are led to propose that this is the ultrastructural location of the newly synthesized acetylcholine which has been shown by others to be preferentially released by stimulation.Recently, we (4) described fixation conditions which result in the appearance of a single, dense particle in many of the cholinergic vesicles of the neuroplaque junction of the electric ray, Narcine brasiliensis. Now we are presenting additional findings including the demonstration that calcium is necessary in the fixative solutions, if the particle is to be visualized.Oschman and Wall (29) have suggested that calcium-containing fixatives may facilitate the ultrastructural identification of calcium binding sites, which then appear as spots or plaques of high electron density. These electron-dense spots (EDS) were regarded as Ca binding sites in insect intestinal membranes because (a) they occurred only when Ca was included in the fixation solutions, and (b) they were visible in sections from aldehyde fixed, unosmicated tissues, indicating that the EDS have an intrinsic electron density. Hillman and Llinfis (15) and Oschman et al. (28) have recently analyzed Ca-dependent EDS in the membranes of the squid giant axon by energy dispersive X-ray "780

ATP-Dependent calcium uptake by cholinergic synaptic vesicles isolated fromtorpedo electric organ

“…A dense spot can be visualized within the isolated synaptic vesicles. This is similar to what happens with synaptic vesicles in the nerve terminals in situ [6,14,33]. The calcium nature of these spots were strongly suggested by their disappearance after EGTA treatment [33].…”

“…When calcium (100 ~tM) is present in the glutaraldehyde-osmium fixative (Fig. 2a), a dense spot appears within the isolated synaptic vesicles as it has been reported previously on the fraction or in situ [6,14,33]. No evident increase was noticed in the presence of ATP, oxalate, or both.…”

“…The structural localization of the nonmitochondrial Ca § sequestration mechanism is still a matter of discussion. -Synaptic vesicles may be formed from the intraterminal smooth reticulum [12,15], which is known to accumulate calcium [17].When fixed in the presence of calcium, synaptic vesicles exhibited a dense granule, generally considered to be a calcium precipitate [6,14,33,42]. Finally, a Ca ++ dependent ATPase is present in a purified fraction of synaptic vesicles [7].…”

<b>SPECIFICITY OF IONIC FIXATION WITH SILICOTUNGSTIC ACID FOR CYTOCHEMICAL LOCALIZATION AND IDENTIFICATION OF ACETYLCHOLINE IN SYNAPTIC </b><b>VESICLES </b>

“…In addition, we obtained evidence to support the specificity of ionic fixation for ACh localization in synaptic vesicles. Further experiments are needed to obtain a more comprehensive idea on the heteropolyanion reaction product in synaptic vesicles in relation to vesicular biochemistry (4,6,19) and cytochemistry (1,3,7).…”

Liquid nitrogen‐based quick freezing: Experiences with bounce‐free delivery of cholinergic nerve terminals to a metal surface

“…Such accounting is difficult, and one's enthusiasm for the attempt is weakened when the variables to be measured are known to vary with the chemical composition of the fixative (e.g., Rosenbluth, 1963; Heuseir et al, 1974; Boyne et al, 1974).…”