cryo-preservation methods for EM in any experimental setting.
1.3.4. Synaptotagmin-1
The fusion of docked and primed vesicles in response to action potential-evoked elevations in presynaptic Ca2+ concentrations occurs within milliseconds. The neuronal vesicular protein Synaptotagmin-1 has been proposed to be the Ca2+-sensor to trigger evoked release in neurons (Brose et al., 1992; Fernández-Chacón et al., 2001; Geppert et al., 1994).
Synaptotagmin-1 is a 65 kDa vesicular protein comprising one N-terminal TMR, which serves as a vesicular anchor, and two C2 domains, C2A and C2B, which are connected by a flexible linker and able to bind three and two Ca2+ ions, respectively (Fernandez et al., 2001; Fernández-Chacón et al., 2001; Shao et al., 1998; Ubach et al., 1998). The C2
domains can bind anionic phospholipids (e.g. PIP2) in both a Ca2+-independent manner, through a stretch of polybasic amino acids, and in a Ca2+-dependent manner at the Ca2+ -binding pocket (Araç et al., 2006; van den Bogaart et al., 2011; Li et al., 2006;
Radhakrishnan et al., 2009). Moreover, Synaptotagmin-1 has been shown to interact with t-SNAREs and the SNARE complex (Choi et al., 2010; Kim et al., 2012; Lai et al., 2011;
Rickman et al., 2004; Zhou et al., 2013). However, whether this interaction has physiological relevance for the Ca2+-triggering step in neurons in vivo remains unclear since Synaptotagmin-1 binding appeared to be weak and transient through electrostatic interactions, and may actually be indirect through Syntaxin-1-bound PIP2 (Choi et al., 2010; Honigmann et al., 2013).
Early studies showed that Synaptotagmin-1 KO mice die perinatally and that neurons lacking Synaptotagmin-1 exhibit a severe reduction in the fast component of the Ca2+ -evoked EPSC. No changes were observed in the mEPSC frequency or in the size of the RRP after hypertonic sucrose application, a means of triggering release of primed vesicles in a Ca2+-independent manner (Geppert et al., 1994). Since then, many physiological functions have been proposed for Synaptotagmin-1 in addition to its ability to trigger Ca2+ -dependent synchronous neurotransmitter release. However, different organisms or culture systems often revealed conflicting results. Synaptotagmin-1 was proposed to have a role in inhibiting release by acting as a fusion clamp that can be relieved in a Ca2+-dependent manner during SNARE-mediated fusion, reflected by an increase in the mini-frequency of inhibitory and excitatory PSCs in dissociated neuron cultures and hippocampal slice
cultures from Synaptotagmin-1 KO mice (Kerr et al., 2008; Liu et al., 2009; Xu et al., 2009). Moreover, in neurons of C.elegans, Drosophila and mice, as well as in chromaffin cells, Synaptotagmin-1 deletion results in a reduction in the number of membrane-attached synaptic vesicles and LDCVs, respectively, indicating a role of Synaptotagmins in a docking process prior to Ca2+-triggering (Jorgensen et al., 1995; Liu et al., 2009; Reist et al., 1998; de Wit et al., 2009; Yu et al., 2013). In addition, a reduction in total synaptic vesicle numbers was observed in Synaptotagmin-1 KO synapses, possibly related to a role of Synaptotagmin-1 in vesicle recycling (Jorgensen et al., 1995; Kononenko et al., 2013; Maritzen et al., 2010; Reist et al., 1998). In contrast to these morphological findings, cultured mammalian hippocampal neurons lacking Synaptotagmin-1 exhibit no, or only very little changes in the size of the RRP measured after application of hypertonic sucrose solution or during Ca2+-uncaging experiments (Burgalossi et al., 2012; Geppert et al., 1994; Liu et al., 2009; Xu et al., 2009). These results indicate that Synaptotagmin-1 most likely does not play an essential role in the physiological synaptic vesicle priming step, but rather support a model according to which Synaptotagmin-1 has, if any, a regulatory function in synaptic vesicle priming, in addition to its function as a Ca2+-sensor.
In summary, the mechanism by which Synaptotagmin-1 triggers Ca2+-evoked neurotransmitter release and the processes in which it participates prior to Ca2+-triggering (e.g. inhibitory fusion clamp for spontaneous release or membrane-attachment) remains unclear and a point of controversy (reviewed in Chapman, 2008; Jahn and Fasshauer, 2012; Rizo et al., 2006). The observed morphological docking phenotypes in Synaptotagmin-1 null mutant cells and results from recent in vitro studies indicate that the interaction of Synaptotagmin-1 with the plasma membrane and/or t-SNARE acceptor complexes might indeed be the molecular correlate of synaptic vesicle priming. This model challenges the idea of a (partially) assembled SNARE complex prior to fusion and would rather place SNARE complex zippering downstream of priming to result in immediate membrane fusion.
1.3.5. Complexins
Another protein family called Complexins has been shown to function in the Ca2+ -triggering step of evoked neurotransmitter release in neurons (reviewed in Brose, 2008).
In mammals, the Complexin family consists of four members (Complexin-1 to -4), each of
16
2008). Complexins are small molecules (~20 kDa) that can bind with a central α-helix in an antiparallel orientation to the at least partially assembled SNARE complex in a groove between the SNARE motifs of Synaptobrevin-2 and Syntaxin-1 (Chen et al., 2002). By this mode of action, murine Complexin isoforms have previously been shown to facilitate fast neurotransmitter release in a post-priming step ('super-priming') since the EPSC amplitude and the vesicular release probability was drastically reduced with no concomitant changes in the RRP size or in the number of docked vesicles in mouse hippocampal neurons (Reim et al., 2001; Xue et al., 2007, 2008). According to this model, Complexins might stabilize partially assembled SNARE complexes in the primed state prior to fusion, thus enabling rapid zippering after Ca2+-triggering. However, structure-function analyses of murine and Drosophila Complexin isoforms revealed that distinct domains might execute different but conserved functions, with the central and an accessory α-helix coordinating inhibitory effects of the fast, synchronous release and an unstructured N-terminal sequence facilitating Ca2+-evoked release (Cho et al., 2010; Xue et al., 2007, 2009, 2010). Recent studies described a drastic decrease in the RRP size and a massive increase in the mEPSC frequency after lentiviral knock-down of Complexins in mammalian neuron mass culture systems, which was not observed in similar knock-down experiments for Synaptotagmin-1, indicating a priming role for Complexins upstream of Synaptotagmin-1 function and an additional role as a fusion clamp for asynchronous and spontaneous release in concert with Synaptotagmin-1 (Cao et al., 2013; Kaeser-Woo et al., 2012; Tang et al., 2006; Yang et al., 2010). According to this model, Complexins would inhibit full SNARE complex zippering and therefore prevent synaptic vesicle fusion prior to Ca2+-triggering, suggesting a dominantly inhibitory role for Complexins. This model is supported by data obtained from C. elegans Complexin null mutants, which revealed a severe reduction in evoked release and in the number of docked vesicles, most likely due to an increase in spontaneous fusion events caused by an absence of inhibitory Complexin-mediated clamping-functions (Hobson et al., 2011). In Drosophila Complexin mutants, Complexin seems to regulate synchronous and asynchronous release with no changes in the number of membrane-attached synaptic vesicles at the neuromuscular junction synapse, thereby supporting the model of Complexins being inhibitors of spontaneous release and facilitators of evoked release, most likely by stabilizing partially assembled SNARE complexes and preventing premature synaptic vesicle fusion by preventing Synaptotagmin-1 / SNARE complex interactions (Jorquera et al., 2012).In summary, the functional role of Complexins in mammalian neurons is still heavily debated. Most of the recent discussion has focused primarily on: (1) understanding the
physiological relevance of a multiple role for Complexins in synaptic vesicle priming as well as in facilitating and inhibiting neurotransmitter release, and (2) resolving the molecular interactions of Complexins with the SNARE complex and with the Ca2+-sensor Synaptotagmin-1 (reviewed in Brose, 2008; Jahn and Fasshauer, 2012; Neher, 2010;
Sørensen, 2009; Südhof and Rothman, 2009).
1.4. Ultrastructural analysis of synaptic vesicle docking
Transmission electron microscopy is the key method in studying synaptic morphology and the only method permitting the distances between synaptic vesicles and the active zone membrane to be measured accurately. EM enables the analysis of synaptic ultrastructure in the nanometer range, and due to the fact that plastic-embedded EM samples can be cut to sections as thin as 20 nm, it has the highest z-resolution of any cellular microscopy techniques to date.
1.4.1. Classical aldehyde-based fixation methods for electron microscopy
Classically, EM studies have employed aldehyde-based (e.g. paraformaldehyde and glutaraldehyde) chemical fixation methods of the tissue followed by heavy metal membrane contrasting, dehydration and embedding in a plastic resin that allows ultrathin (20 – 100 nm) sectioning (Hayat, 200; Sabatini et al., 1963). Aldehydes crosslink networks of proteins, thus preserving much of the ultrastructure of biological samples. In addition, the cell membranes are stained by heavy metals, such as osmium tetroxide (OsO4), uranyl acetate or lead citrate, which readily react with phospholipids in lipid bilayers, resulting in an electron-dense precipitate on cell membranes for excellent contrast for EM (Hayat 2000). However, these traditional methods cause several problems when studying synaptic ultrastructure and in particular synaptic vesicle docking at the active zone. First, fixative diffusion into the tissue is a rather slow process (minutes to hours), which results in gradual rather than rapid fixation of the sample (Smith and Reese, 1980). Moreover, aldehydes have been shown to even trigger synaptic vesicle release during the fixation process, which makes reliable quantification of membrane-attached synaptic vesicles rather difficult (Smith and Reese, 1980). Second, the crosslinking process by aldehydes