Fusion Competent Synaptic Vesicles Persist upon Active Zone Disruption and Loss of Vesicle Docking

Abstract

In a nerve terminal, synaptic vesicle docking and release are restricted to an active zone. The active zone is a protein scaffold that is attached to the presynaptic plasma membrane and opposed to postsynaptic receptors. Here, we generated conditional knockout mice removing the active zone proteins RIM and ELKS, which additionally led to loss of Munc13, Bassoon, Piccolo, and RIM-BP, indicating disassembly of the active zone. We observed a near-complete lack of synaptic vesicle docking and a strong reduction in vesicular release probability and the speed of exocytosis, but total vesicle numbers, SNARE protein levels, and postsynaptic densities remained unaffected. Despite loss of the priming proteins Munc13 and RIM and of docked vesicles, a pool of releasable vesicles remained. Thus, the active zone is necessary for synaptic vesicle docking and to enhance release probability, but releasable vesicles can be localized distant from the presynaptic plasma membrane.

Figure 1. Genetic removal of RIM and ELKS leads to disruption of the active zone

A. Schematic of the protein complex at the active zone and its connections to other important presynaptic protein families (marked in red). SAM: sterile alpha motif, MUN: Munc13 homology domain, PDZ: PSD-95/Dlg1/ZO-1 homology domain, SH3: Src homology 3 domain, PxxP: proline rich motif, FN3: fibronectin 3 repeat. B, C. Sample images (B) and quantitation (C) of protein levels at RIM and ELKS knockout (cKO^R+E) and control (control^R+E) synapses using confocal microscopy. The synaptic vesicle marker Synaptophysin-1 (Syp-1) was used to define the region of interest (ROI). The black dotted line indicates control levels and the grey dotted line non- specific staining as assessed for RIM. Example images for RIM-BP2, Piccolo, Liprin-α2, Liprin-α3, SNAP-25, and quantitation of puncta number and size are in Fig. S1 (control^R+E n = 3 independent cultures, cKO^R+E n = 3, 10 images per culture). D, E. Quantitative Western blotting for presynaptic proteins using fluorescent secondary antibodies. Some cultures were fractionated into pellet and supernatant (sup.) using Triton X-100 solubilization and ultracentrifugation. Quantitation (E) of total protein levels in cKO^R+E neurons normalized to protein levels in control^R+E neurons are shown. Black and grey dotted as in C. For detailed analysis of protein solubility and protein levels in each fraction see Table S1B (control^R+E n = 6 independent cultures, cKO^R+E n = 6, except for Bassoon where n = 3 for both conditions, Syb-2: synaptobrevin/VAMP-2). F, G. Sample images (F) and quantitation (G) of the fraction of Bassoon puncta containing Munc13-1 or RIM-BP2. The fraction of Bassoon pixels and the fraction of Syp-1 puncta containing Munc13-1 or RIM-BP2 are in Fig. S1 (control^R+E n = 6 independent cultures, cKO^R+E n = 6, 10 images per culture). All data are means ± SEM; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 as determined by Student's t test. All numerical data are in Table S1.

Figure 2. Disruption of the active zone leads to loss of synaptic vesicle docking but PSDs appear normal

A, B. Sample images (A) and quantification (B) of synaptic morphology of high pressure frozen neurons analyzed by electron microscopy of cKO^R+E and control^R+E synapses. For an identical analysis using glutaraldehyde fixed tissue, see Fig. S2 (control^R+E n = 50 synapses, cKO^R+E n = 50). C. Distribution of synaptic vesicles relative to the presynaptic plasma membrane area opposed to the PSD. Vesicle distribution is shown in 100 nm bins (left) in cKO^R+E and control^R+E synapses. Gaussian fits were used to model the vesicle distribution. The two genotypes were significantly different (* p < 0.05) and could not be fit with a single distribution, requiring individual fits. Distribution of synaptic vesicles within the first 100 nm in 10 nm bins and the number of tethered vesicles (defined as vesicles within 100 nm of the presynaptic plasma membrane) are shown in the middle and on the right, respectively (control^R+E n = 50 synapses, cKO^R+E n = 50). D, E. Sample images (D) and quantification (E) of PSD protein synaptic fluorescence levels at cKO^R+E and control^R+E synapses using confocal microscopy as described in Figs. 1B, C. The black dotted line indicates control levels (control^R+E n = 3 independent cultures, cKO^R+E n = 3, 10 images per culture). F, G. Quantitative Western blotting for PSD proteins using fluorescent secondary antibodies. Sample images (F) and quantification (G) of total postsynaptic protein levels in cKO^R+E neurons normalized to protein levels in control^R+E neurons (black dotted line) are shown (control^R+E n = 3 independent cultures, cKO^R+E n = 3). All data are means ± SEM; ***p ≤ 0.001 as determined by Student's t test (B) or *p < 0.05 by extra sum of squares F test (C). All numerical data are in Table S2.

Figure 3

Single action potential evoked synaptic transmission and release probability are strongly decreased upon disruption of the active zone A, B. NMDAR-EPSCs were evoked by a focal stimulation electrode. Example traces (A) and quantitation of EPSC amplitudes (B) and their coefficient of variation (C.V.) in cKO^R+E and control^R+E neurons are shown (control^R+E n = 24 cells/4 independent cultures, cKO^R+E n = 26/4). C, D. Sample traces (C) and quantitation (D) of EPSC rise times and their C.V. (n as in B). Individual sweeps are shown in grey and the average of all sweeps is shown in black. Traces are normalized to the average response. E-H. Same analysis as in A-D for IPSCs (control^R+E n = 19/3, cKO^R+E n = 19/3). I,J. Analysis of NMDAR-EPSC paired pulse ratios (PPRs) in cKO^R+E and control^R+E neurons. Sample traces (I, traces normalized to the first response) and quantitation (J) of the PPR at 100 ms interstimulus interval (control^R+E n = 23/4 independent cultures, cKO^R+E n= 26/4). K, L. Scaled sample traces (K, traces normalized to the first response) and summary data (L) of IPSC PPRs at variable interstimulus intervals (control^R+E n = 19/3, cKO^R+E n = 19/3). All data are means ± SEM; **p ≤ 0.01, ***p ≤ 0.001 as determined by Student's t test in A-H, or by two-way ANOVA in L (genotype, interstimulus interval, and interaction p ≤ 0.001, p values of post-hoc Holm-Sidak tests are shown). All numerical data are in Table S3.

Figure 4. Impaired Ca²⁺ influx upon active zone disruption

A. Sample images of cKO^R+E and control^R+E neurons filled via patch pipette with Fluo-5F and Alexa 594 (red, top) and enlarged view of boutons (bottom) analyzed in B. B. Somatic action potentials (top) and presynaptic Ca²⁺ transients imaged via Fluo-5F fluorescence (bottom) of the color coded boutons shown in A. C. Summary plots of single action potential-induced Ca²⁺ transients in boutons, inset: same plot for dendrites. Data are shown as mean (line) ± SEM (shaded area). ***p < 0.001 for Ca²⁺ transients during the first 60 ms after the action potential as assessed by two-way ANOVA for genotype and time; interaction n.s. (boutons: control^R+E n = 202 boutons/16 cells/3 independent cultures, cKO^R+E n = 157/13/3; dendrites: control^R+E n = 148 dendrites/16 cells/3 independent cultures, cKO^R+E n = 100/13/3). All numerical data are in Table S4.

Figure 5. Release during action potential trains and mini release are sustained upon disruption of the active zone

A-D. Sample traces (A) and quantitation of amplitudes (B), synchronous charge (C), and steady state EPSC amplitude (D, average of the last ten EPSCs) of NMDAR-EPSCs evoked by stimulation trains (10 Hz, 50 stimuli) in cKO^R+E and control^R+E neurons (control^R+E n = 17/3, cKO^R+E n = 18/3). E-H. Analysis as in A-D but for IPSCs evoked by stimulation trains (10 Hz, 50 stimuli, control^R+E n = 19/3, cKO^R+E n = 19/3). I-K. Example traces (I) of action-potential evoked IPSCs at [Ca²⁺]_ex of 0.5, 1, 2, 5, and 7 mM in cKO^R+E and control^R+E neurons. Absolute IPSC amplitudes (J) and amplitudes normalized to the response at 7 mM [Ca²⁺]_ex (K) are shown (control^R+E n = 8/3, cKO^R+E n = 8/3). L-N. Recordings of mEPSCs in synapses lacking RIM (cKO^R), ELKS (cKO^E), or both (cKO^R+E). In each experiment, control neurons are identical to the respective cKO neurons except that the cre lentivirus is inactive in the control neurons. Sample traces (L) and quantitative analysis of mEPSC frequencies (M) and amplitudes (N) are shown (control^R n = 20/3, cKO^R n = 21/3; control^E n = 25/3, cKO^E n = 24/3; control^R+E n = 32/5, cKO^R+E n = 31/5). For expanded mEPSC traces and mIPSC data, see Fig. S3. All data are means ± SEM; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 as determined by Student's t test (A-H, L-N) or two-way ANOVA in J (genotype: ***p < 0.001; [Ca²⁺]_ex: ***p < 0.001, interaction: *p ≤ 0.05; p values of post-hoc Holm-Sidak tests are shown in J). For analyses of release components during trains, see Figs. S3A-D. All numerical data are in Table S5.

Figure 6. Uniform disruption of active zone composition and function in cKO^R+E neurons

A. Histograms of the distribution of fluorescence intensity levels in cKO^R+E and control^R+E synapses (normalized to the average fluorescence in control). Data are from the experiments shown in Figs. 1B, C. For histograms of RIM, ELKS, Piccolo, RIM-BP2, Liprin-α3, and PSD-95 see Fig. S4C. B. Pseudocolored images of SypHy expressing cultures stimulated with 40 and 200 action potentials (APs), and dequenched with NH₄Cl. Images represent peak fluorescence change. C-E. Quantification of fluorescence changes in cKO^R+E and control^R+E neurons stimulated with 40 APs, including the time course of the mean fluorescence change in active synapses as a percentage of the fluorescence increase upon NH₄Cl application (C), the peak response (D) of active synapses, and frequency distribution of the % response in active synapses at the end of the stimulus train (E; control^R+E n = 3493 NH₄Cl responsive synapses/ 2486 active synapses/ 9 coverslips/3 independent cultures, cKO^R+E n = 2192/1272/9/3, the number of coverslips is used as a basis for statistics). F-H. Quantification as in C-D but for neurons stimulated with 200 APs (control^R+E n = 3493/2640/9/3, cKO^R+E n = 2291/1405/9/3). All data are means ± SEM; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 as determined by Student's t test. All numerical data are in Table S6.

Figure 7. Persistence of a readily releasable pool upon loss of synaptic vesicle tethering and docking

A-D. Sample images (A) and quantitative analyses of synaptic vesicle docking (B) in glutaraldehyde fixed neuronal cultures, and sample traces (C) and quantitation (D) of RRP of RIM deficient cKO^R and corresponding control^R synapses. Focal application of hypertonic sucrose for 10 s was used to deplete the RRP (D). E-H. Analyses as outlined in A-D, but of ELKS deficient cKO^E and control^E synapses. I-L. Analyses as outlined in A-D, but of RIM/ELKS deficient cKO^R+E and control^R+E synapses. For analyses of vesicle numbers, bouton size, PSD length, and vesicle distribution, see Figs. S5A-F. All data are means ± SEM; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 as determined by Student's t test (analysis of vesicle docking and tethering: control^R n = 25 synapses, cKO^R n = 25; control^E n = 25, cKO^E n = 25; control^R+E n = 25, cKO^R+E n = 25; analysis of RRP: control^R n = 20 cells/3 independent cultures, cKO^R n = 20/3; control^E n = 17/3, cKO^E n = 17/3; control^R+E n = 20/3, cKO^R+E n = 20/3). All numerical data are in Table S7. M. Schematic of synaptic architecture and function upon disruption of the active zone. Structures and processes that are strongly disrupted upon RIM and ELKS deletion are labeled in yellow (active zone, docking, single action potential mediated release). Synaptic structures and functions that remain at least partially intact are labeled in green (the synaptic vesicle cluster, the postsynaptic density containing neurotransmitter receptors, mini release, and release in response RRP depleting stimuli such as action potential trains or hypertonic sucrose). Our experiments indicate that at least some vesicles can be recruited from vesicle pools distant from the presynaptic plasma membrane for release, and that these vesicles may be released immediately or undergo a transient docking state (dotted arrow) that is initiated after the onset of stimulation.