Visualization of synaptic activity in hippocampal slices with FM1-43 enabled by fluorescence quenching

Fluorescence imaging of presynaptic uptake and release of styryl dyes such as FM1-43 has provided valuable insights into synaptic function. However, in studies of CNS neurons, the utility of these dyes has been severely limited by nonsynaptic background fluorescence. This has thwarted the use of FM dyes in systems more intact than dissociated neuronal cultures. Here, we describe an approach to selectively reduce undesired fluorescence through quenching of the surface-bound FM1-43 signal. The introduction of sulforhodamine, a fluorophore that is not taken up by synaptic vesicles, selectively reduced the nonsynaptic fluorescence in FM1-43-labeled hippocampal cultures. When applied to rat hippocampal slices, this procedure allowed us to observe activity-dependent staining and destaining of functional synapses. Extending the usefulness of styryl dyes to slice preparations may help make functional synaptic networks amenable to optical measurements.     

Properties of fast endocytosis at hippocampal synapses

Regulation of synaptic transmission is a widespread means for dynamic alterations in nervous system function. In several cases, this regulation targets vesicular recycling in presynaptic terminals and may result in substantial changes in efficiency of synaptic transmission. Traditionally, experimental accessibility of the synaptic vesicle cycle in central neuronal synapses has been largely limited to the exocytotic side, which can be monitored with electrophysiological responses to neurotransmitter release. Recently, physiological measurements on the endocytotic portion of the cycle have been made possible by the introduction of styryl dyes such as FM1-43 as fluorescent markers for recycling synaptic vesicles. Here we demonstrate the existence of fast endocytosis in hippocampal nerve terminals and derive its kinetics from fluorescence measurements using dyes with varying rates of membrane departitioning. The rapid mode of vesicular retrieval was greatly speeded by exposure to staurosporine or elevated extracellular calcium. The effective time-constant for retrieval can be < 2 seconds under appropriate conditions. Thus, hippocampal synapses capitalize on efficient mechanisms for endocytosis and their vesicular retrieval is subject to modulatory control.     

Frequency-dependent kinetics and prevalence of kiss-and-run and reuse at hippocampal synapses studied with novel quenching methods

The kinetics of exo-endocytotic recycling could restrict information transfer at central synapses if neurotransmission were entirely reliant on classical full-collapse fusion. Nonclassical fusion retrieval by kiss-and-run would be kinetically advantageous but remains controversial. We used a hydrophilic quencher, bromophenol blue (BPB), to help detect nonclassical events. Upon stimulation, extracellular BPB entered synaptic vesicles and quenched FM1-43 fluorescence, indicating retention of FM dye beyond first fusion. BPB also quenched fluorescence of VAMP (synaptobrevin-2)-EGFP, thus indicating the timing of first fusion of vesicles in the total recycling pool. Comparison with FM dye destaining revealed that kiss-and-run strongly prevailed over full-collapse fusion at low frequency, giving way to a near-even balance at high frequency. Quickening of kiss-and-run vesicle reuse was also observed at higher frequency in the average single vesicle fluorescence response. Kiss-and-run and reuse could enable hippocampal nerve terminals to conserve scarce vesicular resources when responding to widely varying input patterns.     

Synapse clusters are preferentially formed by synapses with large recycling pool sizes

Synapses are distributed heterogeneously in neural networks. The relationship between the spatial arrangement of synapses and an individual synapse's structural and functional features remains to be elucidated. Here, we examined the influence of the number of adjacent synapses on individual synaptic recycling pool sizes. When measuring the discharge of the styryl dye FM1-43 from electrically stimulated synapses in rat hippocampal tissue cultures, a strong positive correlation between the number of neighbouring synapses and recycling vesicle pool sizes was observed. Accordingly, vesicle-rich synapses were found to preferentially reside next to neighbours with large recycling pool sizes. Although these synapses with large recycling pool sizes were rare, they were densely arranged and thus exhibited a high amount of release per volume. To consolidate these findings, functional terminals were marked by live-cell antibody staining with anti-synaptotagmin-1-cypHer or overexpression of synaptopHluorin. Analysis of synapse distributions in these systems confirmed the results obtained with FM 1-43. Our findings support the idea that clustering of synapses with large recycling pool sizes is a distinct developmental feature of newly formed neural networks and may contribute to functional plasticity.     

Differential labelling of bulk endocytosis in nerve terminals by FM dyes

Bulk endocytosis is triggered in central nerve terminals during intense physiological stimulation. This endocytosis pathway can be labelled by the dye FM1-43 but not its more hydrophilic counterpart FM2-10. This selective labelling was proposed to be due to the retention of FM1-43, but not FM2-10, in slowly retrieving structures after washout of the dye. However, this explanation assumed that bulk endocytosis was a slow process that persisted after stimulation. We have recently shown that the great majority of bulk endocytosis occurs during stimulation, therefore another explanation for the specific labelling of this pathway by FM1-43 must be found. In this paper we show that the ability of FM dyes to label bulk endocytosis is dependent on the concentration of dye used and not their washout properties. When the loading concentration of FM1-43 was reduced 10-fold, its ability to label bulk endocytosis was lost. Conversely when the loading concentration of FM2-10 was increased 10-fold, it now labelled the pathway. This suggests that a difference in affinity of bulk endosome membranes for FM1-43 and FM2-10 underlies the disparity in labelling.     

Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals

We have identified and characterized two vesicle recycling pathways in frog motor nerve terminals. We exploited the differential staining properties of FM dyes of varying hydrophobicity to label selectively two different vesicle pools, using optical imaging and electron microscopy of photoconverted dyes. During a 1 min tetanus, a rapidly recycling route places vesicles selectively into a small readily releasable pool comprising about 20% of vesicles. After the tetanus, a much slower pathway (from which FM2-10 but not FM1-43 can be rinsed) delivers vesicles via infoldings and cisternae selectively to a reserve pool with a halftime of about 8 min. Mixing between the two pools is slow. During stimulation at 30 Hz, 10-15 s is required to mobilize and release dye from the reserve pool.     

FM1-43 measurements of local exocytotic events in rat melanotrophs

We have explored the existence of fusion- and secretion-competent sites on the plasma membrane of peptide secreting rat pituitary melanotrophs at rest, and following stimulation with glutamate. We monitored changes in fluorescence of FM1-43, a styryl dye which labels plasma membrane. The results show spontaneous local increases in FM1-43 reporting changes in membrane surface area due to cumulative exocytosis. Addition of glutamate, further increased the occurrence of these events. Statistical analysis of local FM1-43 fluorescence changes suggests that this is due to the recruitment of inactive exocytotic domains and due to the stimulation of already active exocytotic domains.     

Imaging synaptic activity in intact brain and slices with FM1-43 in C. elegans, lamprey, and rat

The fluorescent probe FM1-43 has been used extensively for imaging vesicle recycling; however, high nonspecific adsorption resulting in elevated background levels has precluded its use in certain tissues, notably brain slices. We have found that a sulfobutylated derivative of beta-cyclodextrin (ADVASEP-7) has a higher affinity for FM1-43 than the plasma membrane. ADVASEP-7 was used as a carrier to remove FM1-43 nonspecifically bound to the outer leaflet of the plasma membrane or extracellular molecules, significantly reducing background staining. This has enabled us to visualize synaptic vesicle recycling in the nematode C. elegans, intact lamprey spinal cord, and rat brain slices.     

Uptake of a fluorescent marker in plant cells is sensitive to brefeldin A and wortmannin

We assessed FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide] as a fluorescent endocytosis marker in intact, walled plant cells. At 4 degrees C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 degrees C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (approximately 65% reduction at 16 degrees C, >90% at 4 degrees C). FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited approximately 0.4-fold by wortmannin. FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was stimulated, and brefeldin A treatment caused the accumulation of large prevacuolar endosomal vesicles heavily labeled with FM1-43. Three-dimensional time lapse imaging revealed that FM1-43-labeled vacuoles and vesicles are highly dynamic. Thus, FM1-43 serves as a fluorescent marker for imaging and quantifying membrane endocytosis in intact plant cells.     

Spatial variability in release at the frog neuromuscular junction measured with FM1-43

We quantified the spatial variability in release properties at different synaptic vesicle clusters in frog motor nerve terminals, using a combination of fluorescence and electron microscopy. Individual synaptic vesicle clusters labeled with FM1-43 varied more than 10-fold in initial intensity (integrated FM1-43 fluorescence) and in absolute rate of dye loss during tetanic electrical nerve stimulation. Most of this variability arose because large vesicle clusters spanned more than one presynaptic active zone (inferred from postsynaptic acetylcholine receptor stripes labeled with rhodamine-conjugated alpha-bungarotoxin); when the rate of dye loss was normalized to the length of receptor stripe covered, variability from spot to spot was greatly reduced. In addition, electron microscopic measurements showed that large vesicle clusters (i.e., those spanning multiple active zones) were also thicker, and the increased depth of vesicles led to increased total spot fluorescence without a corresponding increase in the rate of dye loss during stimulation. These results did not reveal the presence of "hot zones" of secretory activity.     

Selective replenishment of two vesicle pools depends on the source of Ca2+ at the Drosophila synapse

After synaptic vesicles (SVs) undergo exocytosis, SV pools are replenished by recycling SVs at nerve terminals. At Drosophila neuromuscular synapses, there are two distinct SV pools (i.e., the exo/endo cycling pool (ECP), which primarily maintains synaptic transmission, and the reserve pool (RP), which participates in synaptic transmission only during tetanic stimulation). Labeling endocytosed vesicular structures with a fluorescent styryl dye, FM1-43, and measuring intracellular Ca2+ concentrations with a Ca2+ indicator, rhod-2, we show here that the ECP is replenished by SVs endocytosed during stimulation, and this process depends on external Ca2+. In contrast, the RP is refilled after cessation of tetanus by a process mediated by Ca2+ released from internal stores.     

Endocytosis against high turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labelled plasma membrane and GFP-tagged K-channel KAT1

The relevance of endocytosis in plants against high turgor pressure has frequently been questioned on the basis of energetic considerations. Here, we examine the dynamics of the plasma membrane (PM) in turgid guard cells of Vicia faba by monitoring with confocal microscopy the fate of fluorescent styryl dyes (FM1-43, FM2-10 and FM4-64). As a second marker, we also observe the retrieval of a fluorescent chimaera of the K(+)-inward rectifying channel from Arabidopsis thaliana and the green fluorescent protein (KAT1::GFP). Analysis of cytoplasmic structures, which became labelled by the different styryl dyes, revealed that only FM4-64, the most hydrophobic dye, was a reliable marker of endocytosis, whereas the two other styryl dyes resulted also in an unspecific labelling of different cytoplasmic structures including mitochondria. Over some minutes of incubation in continuous presence of these dyes, endocytic vesicles in the cortical cytoplasm beneath the PM were fluorescently labelled. The identification is based on the observation that the size distribution of these structures is very similar to that of endocytic vesicles obtained from patch-clamp capacitance recordings. Also, these structures are frequently co-labelled with KAT1::GFP. Taken together, the data show that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K(+)-channel KAT1 from the PM via endocytic vesicles.     

Behavioural and morphological evidence for the involvement of glial cell activation in delta opioid receptor function: implications for the development of opioid tolerance

The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated currents. Blockade and dye uptake is voltage dependent and regulated by external Ca²⁺. The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them.     

Long-term staining of live Merkel cells with FM dyes

Live Merkel cells in the skin and hair follicles are known to incorporate a fluorescence dye, quinacrine, which has been utilized to identify and dissect the cells for experiments. Quinacrine fluorescence of the cells is, however, quickly lost and quinacrine-stained Merkel cells soon become difficult to identify in tissue culture. To find dyes that remain in the cells for a long period of time, we tested many fluorescence dyes and found that FM dyes (such as FM1-43) are useful markers for live Merkel cells. In the rat footpad skin, FM1-43 was shown to stain 95% of live Merkel cells that were already stained with quinacrine. FM4-64 stained 98% of quinacrine-stained Merkel cells. Merkel cells in sinus hair follicles were also stained with FM dyes. The fluorescence intensity of FM dyes was stronger than that of quinacrine, and the shape of the cells was more distinct in the FM-dye-stained cells. To test how long FM dyes remain in live cells, FM-dye-stained Merkel cells in hair follicles were embedded in collagen gel and were cultured in a serum-free medium. FM-dye-stained cells were easily identified even after 7 days of culture. During the culture, Merkel cells changed their shape, moved in the preparation and tended to aggregate on the surface. We conclude that FM dyes are powerful tools for tracing live Merkel cells in in vitro experiments. Moreover, the finding that Merkel cells incorporate FM dyes suggests that vesicles in the cells are likely to have mechanisms of recycling in a manner similar to those in neurons and secretory cells.     

Botulinum neurotoxins: from paralysis to recovery of functional neuromuscular transmission.

The neuromuscular junction is one of the most accessible mammalian synapses which offers a useful model to study long-term synaptic modifications occurring throughout life. It is also the natural target of botulinum neurotoxins (BoNTs) causing a selective blockade of the regulated exocytosis of acetylcholine thereby triggering a profound albeit transitory muscular paralysis. The scope of this review is to describe the principal steps implicated in botulinum toxin intoxication from the early events leading to a paralysis to the cellular response implementing an impressive synaptic remodelling culminating in the functional recovery of neuromuscular transmission. BoNT/A treatment promotes extensive sprouting emanating from intoxicated motor nerve terminals and the distal portion of motor axons. The current view is that sprouts have the ability to form functional synapses as they display a number of key proteins required for exocytosis: SNAP-25, VAMP/synaptobrevin, syntaxin-I, synaptotagmin-II, synaptophysin, and voltage-activated Na+, Ca2+ and Ca2+-activated K+ channels. Exo-endocytosis was demonstrated (using the styryl dye FM1-43) to occur only in the sprouts in vivo, at the time of functional recovery emphasising the direct role of nerve terminal outgrowth in implementing the restoration of functional neurotransmitter release (at a time when nerve stimulation again elicited muscle contraction). Interestingly, sprouts are only transitory since a second distinct phase of the rehabilitation process occurs with a return of synaptic activity to the original nerve terminals. This is accompanied by the elimination of the dispensable sprouts. The growth or elimination of these nerve processes appears to be strongly correlated with the level of synaptic activity at the parent terminal. The BoNT/A-induced extension and later removal of "functional" sprouts indicate their fundamental importance in the rehabilitation of paralysed endplates, a finding with ramifications for the vital process of nerve regeneration.     

A membrane marker leaves synaptic vesicles in milliseconds after exocytosis in retinal bipolar cells

Perhaps synaptic vesicles can recycle so rapidly because they avoid complete exocytosis, and release transmitter through a fusion pore that opens transiently. This view emerges from imaging whole terminals where the fluorescent lipid FM1-43 seems unable to leave vesicles during transmitter release. Here we imaged single, FM1-43-stained synaptic vesicles by evanescent field fluorescence microscopy, and tracked the escape of dye from single vesicles by watching the increase in fluorescence after exocytosis. Dye left rapidly and completely during most or all exocytic events. We conclude that vesicles at this terminal allow lipid exchange soon after exocytosis, and lose their dye even if they connected with the plasma membrane only briefly. At the level of single vesicles, therefore, observations with FM1-43 provide no evidence that exocytosis of synaptic vesicles is incomplete.     

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye ¹; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K⁺). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1A_i-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1A_iv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining). Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy ^2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.Open in a separate windowClick here to view.^{(49M, flv)}     

Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals

The fluorophore FM1-43 appears to stain membranes of recycled synaptic vesicles. We used FM1-43 to study mechanisms of synaptic vesicle clustering and mobilization in living frog motor nerve terminals. FM1- 43 staining of these terminals produces a linear series of fluorescent spots, each spot marking the cluster of several hundred synaptic vesicles at an active zone. Most agents we tested did not affect staining, but the phosphatase inhibitor okadaic acid (OA) disrupted the fluorescent spots, causing dye to spread throughout the terminal. Consistent with this, electron microscopy showed that vesicle clusters were disrupted by OA treatment. However, dye did not spread passively to a uniform spatial distribution. Instead, time lapse movies showed clear evidence of active dye movements, as if synaptic vesicles were being swept along by an active translocation mechanism. Large dye accumulations sometimes occurred at sites of Schwann cell nuclei. These effects of OA were not significantly affected by pretreatment with colchicine or cytochalasin D. Electrophysiological recordings showed that OA treatment reduced the amount of acetylcholine released in response to nerve stimulation. The results suggest that an increased level of protein phosphorylation induced by OA treatment mobilizes synaptic vesicles and unmasks a powerful vesicle translocation mechanism, which may function normally to distribute synaptic vesicles between active zones.     

Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 — a comparison with annexin V-FITC

The styryl dye FM1-43 becomes highly fluorescent upon binding to cell membranes. The breakdown of membrane phospholipid asymmetry in ionophore-stimulated T-lymphocytes further increases this fluorescence [Zweifach, 2000]. In this study, the capacity of FM1-43 to monitor membrane phospholipid scrambling was explored using flow cytometry in human erythrocytes and human erythrocyte progenitor K562 cells. The Ca²⁺-dependent phosphatidylserine-specific probe annexin V-FITC was used for comparison. The presented data show that the loss of phospholipid asymmetry that could be induced in human erythrocytes by elevated intracellular Ca²⁺ or by structurally different membrane intercalated amphiphilic compounds increases the FM1-43 fluorescence two- to fivefold. The profile of FM1-43 fluorescence for various treatments resembles that of phosphatidylserine exposure reported by annexin V-FITC. FM1-43 detected the onset of scrambling more efficiently than annexin V-FITC. The amphiphile-induced scrambling was shown to be a Ca²⁺-independent process. Monitoring of scrambling in K562 cells caused by NEM-induced Ca²⁺-release from intracellular stores and by Ca²⁺ and ionophore A23187 treatment showed that the increase in FM1-43 fluorescence correlated well with the number of annexin V-FITC-detected phosphatidylserine-positive cells. The results presented here show the usefulness of FM1-43 as a Ca²⁺-independent marker of dissipation in asymmetric membrane phospholipid distribution induced by various stimuli in both nucleated and non-nucleated cells.