Vesicle docking criteria

A potential source of discrepancy between studies examining the functional relevance of synaptic vesicle organization is the use of different criteria to classify vesicle docking in an electron micrograph. Relevant questions in such analyses become: (i) How accurately can the

‘true’ position of a synaptic vesicle be resolved? (ii) How close to the membrane does a vesicle need to be in order to be considered docked? (iii) How do the applied sample preparation techniques (i.e. fixation, dehydration) influence vesicle organization? Indeed, the definition of a morphologically docked synaptic vesicle is often adapted to take the limitations of the applied methodology into account.

For example, in a 3D serial section EM analysis of vesicle docking in perfusion fixed mossy fiber synapses, in order to compensate for the associated limitations in z-resolution in 50-60 nm-thick plastic sections, Rollenhagen and colleagues measured distances between the center of vesicles to the presynaptic membrane and the mean synaptic vesicle radius measured was subtracted to calculate the closest approach (Rollenhagen et al., 2007). Since the total number of synaptic vesicles within 60 nm of the active zone membrane (~1200 vesicles) were found to correlate to the number of vesicles predicted to fuse upon depletion of the RRP as assessed by presynaptic capacitance recordings (~1400 vesicles; Hallermann et al., 2003), these vesicles were classified as belonging to the putative RRP (Rollenhagen et al., 2007). However, this putative RRP would include all vesicles within 40 nm of the active zone membrane resulting in an overestimation of docked synaptic vesicles.

More stringent docking criteria have been used in other 3D serial section EM studies. In an analysis of synaptic vesicle docking in the stratum oriens of the CA3 from juvenile rat hippocampi, Holderith and colleagues reconstructed entire active zones from serial 20 nm-thick sections (Holderith et al., 2012). For a synaptic vesicle to be considered docked in this study, the distance between the middle of the lipid bilayers on the presynaptic membrane and synaptic vesicle was required to be less than 5 nm (Holderith et al., 2012). Since my electron tomographic analyses reveal lipid bilayers to be approximately 4 nm thick from center-to-center of inner and outer leaflets, the criteria used by Holderith and colleagues,

105 coupled with the very thin sections imaged, provides a stringent analysis of the number of docked synaptic vesicles (Holderith et al., 2012).

In the present study and in previous work from our group using HPF, AFS, and electron tomography, docked vesicles were defined as those with no measurable distance between the synaptic vesicle membrane and the active zone membrane (Imig et al., 2014). In both studies, tomograms were acquired at 30,000x magnification and a 3x binning factor was used when reconstructing synaptic subvolumes, generating tomographic slices with isotropic voxel dimensions of x, y, and z = ~1.6 nm (Imig et al., 2014). Morphologically docked vesicles were reported as vesicles within 0-2 nm of the active zone membrane. This study of Schaffer collateral synapses indicated that docked vesicles classified according to this criterion represent morphological correlates of functionally primed and fusion-competent vesicles (Imig et al., 2014). Docked vesicle pools within 0-2 nm of the membrane are massively reduced in priming and SNARE protein deficient genetic mutants (Imig et al., 2014) and the extent of reduction is highly comparable to published reductions in sucrose-evoked measurements of RRP size in respective mutants (Arancillo et al., 2013; Augustin et al., 1999;

Bronk et al., 2007; Schoch et al., 2001; Varoqueaux et al., 2002; Washbourne et al., 2002).

Although our workflow combining HPF and AFS circumvents many potential limitations of aldehyde fixation and room-temperature dehydration, the tissue is nevertheless dehydrated, albeit at sub-zero temperatures, and heavy metals are deposited on the lipid bilayers to enhance membrane contrast. Consequently, the possibility that these procedures could induce subtle changes in the distribution of vesicles at active zones, or even occlude the detection of very small gaps between vesicles and the presynaptic membrane, should be taken into consideration.

An alternative perspective of how synaptic vesicles interact with the presynaptic membrane has evolved with the technological development of cryo-EM, which allows the ultrastructure of synapses to be viewed in a frozen-hydrated state (Fernández-Busnadiego et al., 2010, 2013;

Lučić et al., 2005; Schrod et al., 2018; Tao et al., 2018b; Zuber and Lučić, 2019). Studies employing cryo-electron tomography to resolve the active zone organization of plunge-frozen synaptosome preparations demonstrated that direct synaptic vesicle contact with the plasma membrane was only rarely observed (Fernández-Busnadiego et al., 2010). Rather, active zone proximal vesicles were connected to the membrane by multiple short filaments (<5 nm)

106 (Fernández-Busnadiego et al., 2010). The authors interpreted vesicles in this state as potentially primed and analogous to the docked vesicles in direct contact observed in heavy metal contrasted and dehydrated preparations (Fernández-Busnadiego et al., 2010).

Although aspects of the synaptosome preparation protocol likely induce some degree of structural reorganization in presynaptic vesicle pools, more recent studies corroborate several observations made in synaptosomes using cultured neurons (Schrod et al., 2018; Tao et al., 2018a, 2018b). In these experiments, dissociated neuron cultures were maintained on EM grids and vitrified by plunge freezing prior to cryo-electron tomographic analysis (Schrod et al., 2018; Tao et al., 2018b). These studies of intact synaptic boutons confirmed that direct vesicle-membrane contact was rare, and that vesicles closest to the active zone membrane were situated at a small distance spanned by multiple, short filamentous tethers (Schrod et al., 2018; Tao et al., 2018b). The molecular identity of the short filaments remains unknown and future experiments quantifying their abundance in appropriate genetic null mutants (i.e.

synapses lacking SNARE components or priming proteins), will be informative.

In summary, it is important to understand the advantages and limitations of the employed methodologies when assessing synaptic vesicle docking. In my study, I used 3D electron tomography which resulted in isotropic voxel dimensions compatible to the accurate assessment of synaptic vesicle docking. I set a stringent docking criterion ̶ no measurable distance between the membranes of synaptic vesicles and the presynaptic active zone. While electron tomography of plastic-embedded samples provides better 3D resolution than 2D transmission EM, cryo-electron tomography images biological samples with no additives such as those used for plastic embedding. An additional comparison between the spatial organization of synaptic vesicles in synapses imaged with cryo-electron tomography and electron tomography of plastic-embedded synapses would be highly informative.

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