Thomas Schikorski

Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function

Synaptic vesicle (SV) size is one parameter that controls the amount of neurotransmitter released from individual SVs and, therefore, is fundamental to our understanding of synaptic function. The recently discovered variability of mean SV size among excitatory hippocampal synapses—if actively regulated—is a potential mechanism for the regulation of transmitter release. Here, we investigated which parameters influence mean SV size. First, we revealed that synapse‐to‐synapse variability of SV size is a general phenomenon in several species and brain regions. In addition, we determined the relationship between mean SV size and synaptic morphology. In three‐dimensional reconstructions from serial ultrathin sections, we found that SV size did not correlate with the area of the postsynaptic density (a measure for synaptic size and synaptic cleft volume) nor with the total number of SVs within a bouton or bouton volume. We tested the long‐held hypothesis that a change in osmotic pressure (potentially caused by a change in neurotransmitter concentration) affects SV size. When we reduced the osmotic pressure, SVs became significantly smaller; however, an increase in osmotic pressure had no effect on SV size. Furthermore, we found that SV size does not adapt to chronic changes in activity and that the SV cycle is capable of providing constant SV size during long‐lasting, high‐frequency stimulation. J. Comp. Neurol. 514:343–352, 2009.

Readily releasable vesicles recycle at the active zone of hippocampal synapses

Significance Nerve cells communicate via the release of a neurotransmitter that is stored in synaptic vesicles. The availability of synaptic vesicles is thus crucial for synaptic function. Availability of synaptic vesicles is ensured by vesicle retrieval after fusion with the plasma membrane; however, the underlying mechanisms of membrane retrieval at hippocampal synapses are still debated. Here we show that synaptic vesicles are retrieved at the active zone without the assistance of coated vesicles. Such a mechanism is similar to “kiss and run” but contradicts the classical pathway via clathrin-coated vesicles. During the synaptic vesicle cycle, synaptic vesicles fuse with the plasma membrane and recycle for repeated exo/endocytic events. By using activity-dependent N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide dye uptake combined with fast (<1 s) microwave-assisted fixation followed by photoconversion and ultrastructural 3D analysis, we tracked endocytic vesicles over time, “frame by frame.” The first retrieved synaptic vesicles appeared 4 s after stimulation, and these endocytic vesicles were located just above the active zone. Second, the retrieved vesicles did not show any sign of a protein coat, and coated pits were not detected. Between 10 and 30 s, large labeled vesicles appeared that had up to 5 times the size of an individual synaptic vesicle. Starting at around 20 s, these large labeled vesicles decreased in number in favor of labeled synaptic vesicles, and after 30 s, labeled vesicles redocked at the active zone. The data suggest that readily releasable vesicles are retrieved as noncoated vesicles at the active zone.

Pre-embedding Immunogold Localization of Antigens in Mammalian Brain Slices

The detection of proteins with antibodies that are conjugated to gold particles has been a major asset to cell biology and the neurosciences, and knowledge about the subcellular location of antigens has formed the basis for many hypotheses regarding protein function. Many protocols have been developed since the introduction of colloidal gold to immunocytochemistry. The two most widely used techniques, however, are based on transmission electron microscopy and consist of either immunolabeling before the specimens are embedded in resin (pre-embedding immunogold labeling) or immunolabeling after embedding in resin (post-embedding immunogold labeling). The following protocol describes a pre-embedding procedure that gives reliable results with all antibodies that produce adequate staining as observed with a light microscope. This procedure results in almost perfect preservation of the ultrastructure. The procedure employs thick sectioning using a vibratome, permeabilization of membranes with Triton X-100, and immunolabeling with fluorescently conjugated Nanogold antibodies, followed by gold enhancement and embedding for electron microscopy. We also discuss some limitations inherent to pre-embedding immunogold labeling.

Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.

Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane. There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct. In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli. By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa. Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission.

Monitoring Rapid Endocytosis in the Electron Microscope via Photoconversion of Vesicles Fluorescently Labeled with FM1-43

Cells communicate via endo- and exocytosis with their environment and neighboring cells. At synapses of the nervous system, fast exocytosis is coupled to fast endocytosis, which forms the basis for neurotransmitter release. The introduction of the unique fluorescent FM dyes allowed the monitoring of fast synaptic vesicle exo-endocytic cycling during live imaging sessions and after photoconversion of FM dyes into an electron-dense diaminobenzidine polymer synaptic vesicle cycling can be studied in the electron microscope. This protocol describes FM dye labeling of synaptic vesicles of cultured hippocampal neurons and photoconversion of the fluorescent synaptic vesicles for analysis in the electron microscope (EM).

Horseradish Peroxidase as a Reporter Gene and as a Cell-Organelle-Specific Marker in Correlative Light-Electron Microscopy

A modern electron microscopic approach to the investigation of the structural organization of proteins and subcellular structures demands the use of molecular genetic techniques. The successful implementation of genetic techniques is closely tied to a reporter gene such as the green fluorescent protein (GFP). Although GFP has been widely used for light microscopy, it has many limitations for use in electron microscopy. In the search for a reporter gene for electron microscopy, interest in the use of horseradish peroxidase (HRP) DNA has recently increased, and several studies already have proven the feasibility of HRP expression in mammalian cells. Here, we describe a protocol that uses a HRP chimera to label the endoplasmic reticulum of HEK cells.

Monitoring Synaptic Vesicle Protein Sorting with Enhanced Horseradish Peroxidase in the Electron Microscope.

Protein sorting is the fundamental cellular process that creates and maintains cell organelles and subcellular structures. The synaptic vesicle (SV) is a unique cell organelle that contains a plethora of specific SV proteins and its protein composition is crucial for its function. Thus understanding the mechanisms that sort proteins to SVs and other cell organelles is central to neuroscience and cell biology.While in the past protein sorting was studied in the fluorescence and confocal microscope, we here present a protocol that reveals SV protein trafficking and sorting in the electron microscope (EM). The protocol exploits tagging SV proteins with a new genetically encoded label for EM: enhanced horseradish peroxidase (eHRP). eHRP gained its high sensitivity through direct evolution of its catalytic activity and is detectable in the EM and LM after expression in neurons and other mammalian cells. The protocol describes the use of eHRP, labeling of SVs in cultured hippocampal neurons, and analysis via serial section reconstruction.

Functional Nanoscale Imaging of Synaptic Vesicle Cycling with Superfast Fixation.

Functional imaging is the measurement of structural changes during an ongoing physiological process over time. In many cases, functional imaging has been implemented by tracking a fluorescent signal in live imaging sessions. Electron microscopy, however, excludes live imaging which has hampered functional imaging approaches on the ultrastructural level. This barrier was broken with the introduction of superfast fixation. Superfast fixation is capable of stopping and fixing membrane traffic at sufficient speed to capture a physiological process at a distinct functional state. Applying superfast fixation at sequential time points allows tracking of membrane traffic in a step-by-step fashion.This technique has been applied to track labeled endocytic vesicles at central synapses as they pass through the synaptic vesicle cycle. At synapses, neurotransmitter is released from synaptic vesicles (SVs) via fast activity-dependent exocytosis. Exocytosis is coupled to fast endocytosis that retrieves SVs components from the plasma membrane shortly after release. Fluorescent FM dyes that bind to the outer leaflet of the plasma membrane enter the endocytic vesicle during membrane retrieval and remain trapped in endocytic vesicles have been widely used to study SV exo-endocytic cycling in live imaging sessions. FM dyes can also be photoconverted into an electron-dense diaminobenzidine polymer which allows the investigation of SV cycling in the electron microscope. The combination of FM labeling with superfast fixation made it possible to track the fine structure of endocytic vesicles at 1 s intervals. Because this combination is not specialized to SV cycling, many other cellular processes can be studied. Furthermore, the technique is easy to set up and cost effective.This chapter describes activity-dependent FM dye labeling of SVs in cultured hippocampal neurons, superfast microwave-assisted fixation, photoconversion of the fluorescent endocytic vesicles, and the analysis of individual synapses after serial section 3D reconstruction of individual synapses from electron micrographs.

Presynaptic size of associational/commissural CA3 synapses is controlled by fibroblast growth factor 22 in adult mice

Associational/commissural CA3–CA3 synapses define the recurrent CA3 network that generates the input to CA1 pyramidal neurons. We quantified the fine structure of excitatory synapses in the stratum radiatum of the CA3d area in adult wild type (WT) and fibroblast growth factor 22 knock‐out (FGF22KO) mice by using serial 3D electron microscopy. WT excitatory CA3 synapses are rather small yet range 10 fold in size. Spine size, however, was small and uniform and did not correlate with the size of the synaptic junction. To reveal mechanisms that regulate presynaptic structure, we investigated the role of FGF22, a target‐derived signal specific for the distal part of area CA3 (CA3d). In adult FGF22KO mice, postsynaptic properties of associational CA3 synapses were unaltered. Presynaptically, the number of synaptic vesicles (SVs), the bouton volume, and the number of vesicles in axonal regions (the super pool) were reduced. This concurrent decrease suggests concerted control by FGF22 of presynaptic size. This hypothesis is supported by the finding that WT presynapses in the proximal part of area CA3 (CA3p) that do not receive FGF22 signaling in WT mice were smaller than presynapses in CA3d in WT but of comparable size in CA3d of FGF22KO mice. Docked SV density was decreased in CA1, CA3d, and CA3p in FGF22KO mice. Because CA1 and CA3p are not directly affected by the loss of FGF22, the smaller docked SV density may be an adaptation to activity changes in the CA3 network. Thus, docked SV density potentially is a long‐term regulator for the synaptic release probability and/or the strength of short‐term depression in vivo.

Catching synaptic vesicles in action

The synaptic vesicle (SV) cycle was initially discovered at the neuromuscular junction using electron microscopy (EM) analysis.1 With the introduction of fluorescent probes that are able to monitor real-time cellular events in live cells, EM analysis was pushed to the side lines because it could not provide meaningful kinetic analyses of the various steps in the synaptic vesicle cycle.