Exo-endocytotic turnover of synaptic vesicles (SVs) at synapses between hippocampal neurons in culture was examined by electron microscopy (EM). We carried out photoconversion (PC) of the fluorescent endocytotic marker FM 1-43 by using 3,3 -diaminobenzidine to convert the dye signal into an electron-dense product. Electron-dense products were located almost exclusively in SVs, whose densities were bimodally distributed in two sharply demarcated populations, PC-positive (PC؉) and PC-negative (PC؊). The median densities of these populations did not vary with the proportion of vesicles stained within a presynaptic terminal (bouton). The proportion of PC؉ SVs remained constant across consecutive thin sections of single boutons, but varied greatly from one bouton to another, indicating marked heterogeneity in exo-endocytotic activity. Our experiments indicated that only a minority of SVs were stained in most boutons after stimuli known to cause complete turnover of the functional vesicular pool. A direct spatial correlation was found between FM 1-43 fluorescent spots seen with light microscopy and PC؉ boutons by EM. The correlation was clearer in isolated boutons than in clusters of boutons. Photoconversion in combination with FM dyes allows clarification of important aspects of vesicular traffic in central nervous system nerve terminals.S ynaptic vesicle (SV) turnover in presynaptic terminals of central nervous system (CNS) neurons is critical for information transfer within the brain. Continued progress in understanding exocytosis and endocytosis at the level of single vesicles depends critically on physiological studies using light microscopy and ultrastructural analysis using electron microscopy (EM). FM 1-43 and structurally related styryl dyes offer a way to tie such approaches together. These molecules undergo a large increase in quantum yield when taken up into membranes. Activitydependent sequestration of the dyes in presynaptic vesicles and release on subsequent exocytosis have been monitored as increases and decreases in nerve terminal fluorescence (1-4). Key properties of FM 1-43 as a fluorescent probe were demonstrated by Betz and colleagues (1, 5), working at the frog neuromuscular junction (NMJ), the same preparation used by Heuser and Reese (6) in their pioneering studies of vesicle turnover. Betz and colleagues went on to perform photoconversion of FM 1-43 and FM 2-10 by using photolysis of the dyes in the presence of 3,3Ј-diaminobenzidine (DAB) to cause an electron-dense reaction product in synaptic vesicles (7,8). At CNS synapses, like the NMJ, FM 1-43 has been widely used as an optical marker for studies of vesicle dynamics by the light microscope (2, 9, 10). Photoconversion of FM 1-43 has also provided valuable information about the number of vesicles in the readily releasable pool (11).This paper describes our efforts to use FM 1-43 photoconversion to strengthen links between light microscopic and EM approaches to vesicular properties. Working with rat hippocampal neurons in culture, we modified...

We have studied the fine structure of nerve-muscle contacts during the first few hours of synaptogenesis in embryonic Xenopus cell cultures. The precise timing of contact was achieved by manipulating isolated spherical myocytes (myoballs) into contact with growth cones or neurites of co-cultured spinal neurons. The contacts were shown to be functional by whole-cell voltage-clamp recording of nerve-evoked synaptic currents in the muscle cell. The ultrastructure of these functional contacts was examined by thin-section electron microscopy. In total, 20 nerve-muscle pairs were studied with contact periods ranging from 20 min to 12 hr, during which time a substantial increase in the amplitude of synaptic currents occurred. The structure of noncontacting cells and of nerve-muscle contacts formed between the cells by natural encounters in 1-d-old cultures were also examined in order to identify the features and the time course of morphological differentiation of early functional contacts. Prominent features of the contact area during the first few hours included: close apposition of the nerve and muscle membranes, greater frequency of coated pits and vesicles, and thickening of postsynaptic muscle membrane. Occasionally, clusters of clear vesicles occurred near presynaptic membrane, but no further sign of active zone differentiation was observed. In comparison, definitive active zone structure, well-formed extracellular basal lamina, and widened cleft were seen in natural contacts less than 24 hr old. This study of the identified functional contacts may help us to understand the structural basis for early nerve-muscle interaction and the functional significance of various synaptic specializations.

No abstract

No abstract