The role of Complexins in synaptic vesicle docking

Finally, I included a Complexin-1/2/3 triple KO (TKO) mouse line into my analysis (Xue et al., 2008). Complexins are a family of proteins, which can bind with a central α-helix to the assembled SNARE-complex. By this action, murine Complexin isoforms were previously shown to facilitate fast neurotransmitter release in a post-priming step ('super-priming'), since the EPSC amplitude and the vesicular release probability is drastically reduced in hippocampal neurons from Complexin-1/2/3 triple KO mice with no changes in the RRP size or in the number of docked vesicles (Reim et al., 2001; Xue et al., 2007, 2008).

However, a recent study describes a drastic decrease in the RRP size after lentiviral

knock-down of Complexin, proposing a priming role for Complexin upstream of Synaptotagmin-1 function and an additional role as a fusion clamp for asynchronous and spontaneous release in concert with Synaptotagmin-1 (Yang et al., 2010). Moreover, C.

elegans Complexin mutants show a severe reduction in the number of docked vesicles, most likely due to an increase in spontaneous fusion events caused by the absence of Complexin-mediated inhibitory clamping-functions (Hobson et al., 2011).

3.1.3.7.1. 2D-EM analysis of synaptic morphology in Complexin-1/2/3 TKO neurons

Samples from three Complexin TKO animals (Complexin-1^-/-, Complexin-2^-/-, Complexin-3^-/-) were compared with three control animals with the following genotypes (Complexin-1^+/+, Complexin-2^-/-, Complexin-3^-/-), (Complexin-1^+/-, Complexin-2^-/-, Complexin-3^-/-), (Complexin-1^+/-, Complexin-2^-/-, Complexin-3^+/-). In the two-dimensional analysis of electron micrographs, no statistically significant differences were detected in any of the parameters measured (Fig. 3.16 A, B). The total number of synaptic vesicles per synaptic profile (control: 68.75 ± 2.635, n = 222; Complexin-1/2/3 TKO: 67.66 ± 3.241, n = 155 / P = 0.4543, n.s.; Fig. 3.16 C), the synaptic vesicle terminal density (control:

1.539 ± 0.037, n = 222; Complexin-1/2/3 TKO: 1.581 ± 0.043, n = 155 / P=0.4448, n.s.;

Fig. 3.16 D) and the synaptic vesicle cluster density were unchanged between the two groups (control: 3.883 ± 0.038, n = 222; Complexin-1/2/3 TKO: 3.991 ± 0.050, n = 155 / P

= 0.2399, n.s.; Fig. 3.16 E). Moreover, the PSD length (control: 361.3 ± 8.92, n = 222;

Complexin-1/2/3 TKO: 378.2 ± 12.22, n = 155 / P = 0.3383, n.s.; Fig. 3.16 F), the number of endosomes per synaptic profile (control: 1.063 ± 0.108, n = 222; Complexin-1/2/3 TKO:

1.103 ± 0.121, n = 155 / P = 0.7002, n.s.; Fig. 3.16 G) and the number of LDCVs per presynaptic profile did not differ between Complexin-1/2/3 TKO and control neurons (control: 0.176 ± 0.031, n = 222; Complexin-1/2/3 TKO: 0.219 ± 0.047, n = 155 / P = 0.6946, n.s.; Fig. 3.16 H).

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Figure 3.16. Two-dimensional ultrastructural analysis of synaptic morphology in Complexin-1/2/3 TKO neurons

Electron micrographs of control (A) and Complexin-1/2/3 TKO (B) synaptic profiles acquired from 60 nm-thick ultrathin sections. Mean number of synaptic vesicles (SVs) per synaptic profile (C).

Mean number of SVs normalized to synaptic terminal area (SV terminal density; D). Mean number of SVs normalized to SV cluster area (SV cluster density; E). Mean postsynaptic density (PSD) length (F) Mean number of endosomes per synaptic profile (G). Mean number of large dense-core vesicles (LDCVs) per synaptic profile (H). C-H: Control: N=3, n=222; Complexin-1/2/3 TKO: N=3, n=155 (Mean + SEM), P<0.001: ***; P<0.01: **; P<0.05: *. Scale bar: B, 500 nm. *. Imaging and analysis were performed by S. Krinner.

3.1.3.7.2. 3D-ET analysis of synaptic vesicle docking in Complexin-1/2/3 TKO neurons

Next, I analyzed synaptic vesicle docking in presynaptic terminals from electron tomographic reconstructions of 200 nm semithin sections in glutamatergic, excitatory spine synapses of control (N=3) and Complexin-1/2/3 TKO samples (N=3) (Fig. 3.17 A-F).

In three-dimensional electron tomograms, no significant changes were detected in the number of active zone membrane-attached synaptic vesicles (0-2 nm; control: 0.871 ± 0.147, n = 19; Complexin-1/2/3 TKO: 1.116 ± 0.145, n = 25 / P = 0.248, n.s.; Fig. 3.17 I) or in the number of closely-tethered synaptic vesicles (0-4 nm; control: 1.219 ± 0.168, n = 19;

Complexin-1/2/3 TKO: 1.602 ± 0.159, n = 25 / P = 0.1586, n.s.; Fig. 3.17 J). However, there was a tendency towards a slightly higher number of membrane-attached vesicles in Complexin-1/2/3 TKO synapses. The number of membrane proximal vesicles within 40

nm distance of the active zone did not differ between the two groups (control: 2.386 ± 0.180, n = 19; Complexin-1/2/3 TKO: 2.728 ± 0.140, n = 25 / P = 0.135, n.s.; Fig. 3.17 H), although a slight increase in the number of vesicles within 100 nm of the active zone in Complexin-1/2/3 synaptic profiles was observed (control: 6.005 ± 0.319, n = 19;

Complexin-1/2/3 TKO: 6.861 ± 0.247, n = 25 / P = 0.0371, *; Fig. 3.17 G). The synaptic vesicle distribution within 100 nm distance from the presynaptic active zone membrane revealed no major differences between control and Complexin-1/2/3 KO synaptic profiles, with the largest percentage of vesicles being membrane-attached in both groups (Fig.

3.17 M). Neither the mean outer synaptic vesicle diameter (control: 44.71 ± 0.621, n = 19;

Complexin-1/2/3 TKO: 44.11 ± 0.407, n = 25 / P = 0.409, n.s.; Fig. 3.17 K, N) nor the mean synaptic vesicle volume were altered in synapses lacking all Complexin isoforms (control: 47600 ± 2017, n = 19; Complexin-1/2/3 TKO: 45536 ± 1198, n = 25 / P = 0.3596, n.s.; Fig. 3.17 L).

In summary, I could not detect any statistically significant differences in either the number of synaptic vesicles in presynaptic terminals or in the number of membrane-attached synaptic vesicles in Complexin-1/2/3 TKO synapses. These findings correlate well with electrophysiological data for neurons lacking all three Complexin isoforms, which showed no changes in the size of RRP measured after the application of hyperosmolaric sucrose solution (Reim et al., 2001; Xue et al., 2007, 2008). It was possible to reveal a slight increase in the membrane-proximal vesicles in Complexin-1/2/3 TKO neurons. One possible explanation could be that neurons devoid of all Complexin isoforms have an impaired ability to release synaptic vesicles in response to action potential stimuli, leading to an accumulation of synaptic vesicles close to the active zone membrane. However, my data does not support the hypothesis of a clamping function for Complexins in mouse neurons, since in such a scenario I would have expected to see rather a slight decrease in the number of docked vesicles at the membrane, as had been shown in C. elegans Complexin mutants (Hobson et al., 2011). Taken together, the results of the present study support the hypothesis that Complexins are facilitators of synaptic vesicle release, which act on the assembled SNARE-complex after synaptic vesicle docking/priming.

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Figure 3.17. Three-dimensional electron tomographic analysis of synaptic vesicle docking in Complexin-1/2/3 TKO neurons

Tomographically reconstructed subvolumes of control (A) and Complexin-1/2/3 TKO (B) synapses from 200 nm-thick sections.Three-dimensional models of tomographically reconstructed control (C) and Complexin-1/2/3 TKO (D) synaptic profiles. Ultrastructural features reconstructed in the models include the active zone plasma membrane (white), synaptic vesicles (membrane-attached, green;

non-attached, grey) and endosomes (light blue). E, F Orthogonal views of control (E) and Complexin-1/2/3 TKO (F) tomographic models displaying the spatial arrangement of membrane-attached synaptic vesicles within the reconstructed active zone area. Mean number of SVs within 100 nm of the AZ normalized to AZ area (G). Mean number of SVs within 40 nm of the AZ normalized to AZ area (H). Mean number of membrane-attached SVs (within 0-2 nm of the AZ) normalized to AZ area (I). Mean number of membrane-attached and closely-tethered SVs (within 0-4 nm of the AZ) normalized to AZ area (J). Mean outer SV diameter (K). Mean SV volume including the membrane bilayer (L). Spatial distribution of SVs within a 100 nm distance of the AZ membrane (M). Distribution of synaptic vesicle diameters of vesicles within 100 nm of the AZ (N).

G-L: Control: N=3, n=19; Complexin-1/2/3 TKO: N=3, n=25 (Mean + SEM), P<0.001: ***; P<0.01:

**; P<0.05: *. M, N: (617 SVs in control and 953 SVs in Complexin-1/2/3 TKO synaptic profiles).

Scale bar: B, 100 nm.