Visualizing recycling synaptic vesicles in hippocampal neurons by FM 1-43 photoconversion

Abstract

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...

The same synaptic vesicles drive active and spontaneous release

“…It has been assumed for almost six decades that identical vesicles exocytose in both cases, an assumption which has been used to generate the quantal (vesicular) release theory 1 . This assumption is in agreement with the fact that prolonged active 2 or spontaneous release 3 can both release essentially all vesicles (see also 4,5 ). The issue of whether the same vesicles can be released both spontaneously and actively has been tested recently by investigating the loading and unloading of styryl (FM) dyes during vesicle recycling 6 .…”

Ultrastructural readout of functional synaptic vesicle pools in hippocampal slices based on FM dye labeling and photoconversion

“…In this way, functional vesicle pools that were previously labelled with FM-dye can be directly identified in electron micrographs. This approach has been used extensively and highly successfully in cultured neurons 7,10,24,28,30,[32][33][34] , a number of large, peripheral terminals [26][27][28][29]31,[35][36][37] , and large central release sites such as calyx of Held 25 revealing important information about organizational principles of vesicle pools. However, only recently 38 has this method been successfully applied to small central synapses in native brain tissue, where neurons are retained in relevant circuits with defined cytoarchitecture.…”

“…Most vesicles can be readily categorized by visual assessment. However, there are also a number of established quantitative approaches to aid in vesicle classification based on comparisons of membrane and lumenal optical densities 24,29,30,32 .…”

Counting the number of releasable synaptic vesicles in a presynaptic terminal

“…The reserve pool, containing the bulk of SVs, seems to be released only in response to intense stimulation. Thus, reserve vesicles may be seldom or never recruited during physiological activity (1,6,7), raising questions about their functional importance.…”

“…Electron microscopy, often used with endocytic markers to label recycling vesicles, offers high spatial resolution but can only provide a static snapshot (2,6,8,9). Fluorescent labeling of SVs (e.g., with FM dyes or synaptopHluorin) can provide excellent spatial and temporal information, but the assay is somewhat indirect, fueling controversy (4,(10)(11)(12).…”