Doc2-mediated superpriming supports synaptic augmentation

Abstract

Various forms of synaptic plasticity underlie aspects of learning and memory. Synaptic augmentation is a form of short-term plasticity characterized by synaptic enhancement that persists for seconds following specific patterns of stimulation. The mechanisms underlying this form of plasticity are unclear but are thought to involve residual presynaptic Ca²⁺ Here, we report that augmentation was reduced in cultured mouse hippocampal neurons lacking the Ca²⁺ sensor, Doc2; other forms of short-term enhancement were unaffected. Doc2 binds Ca²⁺ and munc13 and translocates to the plasma membrane to drive augmentation. The underlying mechanism was not associated with changes in readily releasable pool size or Ca²⁺ dynamics, but rather resulted from superpriming a subset of synaptic vesicles. Hence, Doc2 forms part of the Ca²⁺-sensing apparatus for synaptic augmentation via a mechanism that is molecularly distinct from other forms of short-term plasticity.

Keywords: Doc2; munc13; short-term plasticity; superpriming; synaptic augmentation.

Fig. 1.

Doc2 selectively regulates synaptic augmentation. (A) Test pulses (0.2 Hz) were used to monitor synaptic transmission for 80 s; augmentation was induced via a stimulus train (10 Hz, 5 s) at t = 30–35 s. Shown are representative evoked EPSC traces recorded from WT (black), Doc2α/β DKO (red), and DKO neurons expressing WT-Doc2β (light blue), at 25, 40 and 55 s. (B) The peak amplitudes of evoked EPSCs were normalized to the baseline and plotted as mean ± SEM; the stimulus train was omitted for clarity (gray bar; detailed in SI Appendix, Fig. S1). Augmentation was impaired in Doc2α/β DKO neurons (P = 0.012, WT versus DKO, Kruskal–Wallis test followed by Dunn’s post hoc test); expression of WT-Doc2β completely rescued augmentation. (C) Representative EPSCs showing responses to a paired pulse stimulus (25-ms interval). (D) PPF (amplitude of second EPSC over that of the first EPSC) was quantified and graphed using box plots. No difference was found using Mann–Whitney U test (P = 0.0719). (E) Representative EPSC traces recorded before, and 20 s after, PTP was induced via a stimulus train (100 Hz, 1 s). (F) The normalized peak amplitudes of evoked EPSCs were quantified and plotted as mean ± SEM. The arrow indicates the stimulus train. WT and Doc2α/β DKO neurons exhibited no differences in PTP. (G) WT, Doc2α/β DKO, and DKO neurons expressing WT-Doc2β were pretreated with EGTA-AM (100 μM, 20 min) and subjected to the augmentation protocol as shown in A. Shown are representative evoked EPSC traces recorded at 25 and 40 s with (Lower) or without (control; Upper) EGTA-AM pretreatment. (H) Extent of augmentation was calculated as the normalized EPSC amplitude 5 s after the induction train (10 Hz, 5 s). EGTA-AM pretreatment significantly decreased the extent for augmentation in WT neurons and DKO neurons expressing WT-Doc2β, but failed to further reduce augmentation in Doc2α/β DKO neurons. *P < 0.05, unpaired t test.

Fig. 2.

Ca²⁺•Doc2β mediates munc13-1 translocation to the plasma membrane to drive augmentation. (A) WT-Doc2β, Doc2β_clm in which two acidic Ca²⁺ ligands were neutralized to disrupt Ca²⁺ binding to the C2B domain (clm; Ca²⁺ ligand mutant), and Doc2β_MID-scrm in which the MID domain was scrambled, were expressed in neurons. (B) Upon depolarization with 60 mM KCl, both munc13-1–mCherry (magenta) and WT Doc2β-GFP (green) translocated to the plasma membrane. (Scale bar: 10 μm.) (C) Magnified images are shown. (D) The ratio of fluorescence intensity (plasma membrane/cytosol) was quantified and normalized to baseline, as detailed in SI Appendix, Fig. S3, and plotted versus time. (E) Upon depolarization, Doc2β_clm-GFP neither translocates to the plasma membrane nor recruits munc13-1–mCherry (Upper); Doc2β_MID-scrm-GFP translocates but was also unable to recruit munc13-1–mCherry (Lower). (F) Translocation data from E were quantified and plotted. (G) Normalized peak amplitudes of EPSCs before and after the augmentation protocol, as described in Fig. 1, recorded from Doc2α/β DKO neurons expressing Doc2β_clm (Upper) or Doc2β_MID-scrm (Lower) are plotted as mean ± SEM versus time. Data from WT and Doc2α/β DKO neurons (Fig. 1) are shown again as controls. Both Doc2β mutants failed to rescue synaptic augmentation.

Fig. 3.

Doc2 dwell time at the plasma membrane coincides with the duration of augmentation. (A and B) Sample images from neurons stimulated using augmentation (10 Hz, 5 s; A) or PTP (100 Hz, 1 s; B) protocols. (Scale bar: 10 μm.) Doc2β-GFP translocated to the plasma membrane upon stimulation; after the stimulus train, it retreated back to the cytosol in a time-dependent manner. (C and D) Under these conditions, low levels of translocation were observed, so representative line scans of Doc2β-GFP fluorescence (yellow line segments in A and B) are shown; the position of the PM in the line-scan data is indicated. (E and F) Translocation of Doc2-GFP was quantified using normalized fluorescence intensity ratios (plasma membrane/cytosol), as detailed in SI Appendix, Fig. S3. To ensure successful activation of neurons, [Ca²⁺]_i was monitored during the experiment using X-Rhod-1 AM (averaged trace of normalized fluorescence intensity shown in Insets). Single exponential fitting of the averaged traces revealed comparable time constants (τ) for the release of Doc2-GFP from plasma membrane, back to cytosol, following augmentation (τ = 8.65 ± 0.22 s; E) and PTP (τ = 7.45 ± 0.33 s, F).

Fig. 4.

The function of Doc2 in augmentation is independent of its role in asynchronous release. (A) Averaged traces of evoked EPSCs; black arrows indicate stimulation. (B and C) The EPSC amplitude (amp; B) and decay time constants (τ; C), calculated by fitting the data with single exponential functions, are represented as mean ± SEM. No difference in peak amplitude was found among any group. The decay time constant was fully rescued by expression of Doc2β_MID-scrm and Doc2β_MID-del, but not Doc2β_clm. *P < 0.05, **P < 0.01, ***P < 0.001 versus Doc2α/β DKO, Kruskal –Wallis test followed by Dunn’s post hoc test.

Fig. 5.

The function of Doc2 in augmentation does not involve changes in the size of the RRP. (A) Representative EPSCs, in response to local perfusion with 500 mM sucrose (black bars). (B) RRP size was evaluated by integrating the EPSC charge transfer in response to hypertonic sucrose. The RRP ratio was calculated by dividing RRP values obtained with and without augmentation. No significant differences were detected among each group (Kruskal–Wallis test). (C) P_vr was calculated by normalizing the total charge of evoked EPSCs (Fig. 1) to the RRP. The P_vr ratio (P_vr after augmentation/P_vr before augmentation) was plotted as mean ± SEM, *P < 0.05 versus Doc2α/β DKO, Kruskal–Wallis test followed by Dunn’s post hoc test. The original data are provided in SI Appendix, Fig. S9.

Fig. 6.

Doc2-promoted augmentation is not mediated by changes in presynaptic Ca²⁺ dynamics. (A) Image of a representative neuron loaded with FM4-64 (magenta), to identify synaptic boutons, and Fluo-5F (heat map), to measure changes in [Ca²⁺]_i. (B) Averaged Fluo-5F ∆F/F₀ versus time traces in response to a single stimulation 5 s before (Upper) and 5 s after (Lower) the augmentation protocol were imaged using WT (340 boutons, four independent litters of mice) or Doc2α/β DKO (214 boutons, four independent litters of mice) neurons. (C) Scatterplots of F_peak/F₀ quantified from individual boutons. **P < 0.01, unpaired t test.

Fig. 7.

Doc2 mediates synaptic augmentation by enhancing SV superpriming. (A) A 40-Hz stimulus train (0.5 s) was delivered 20 s before (Left) and 5 s after (Right) the augmentation protocol. (B–E) The peak amplitude of each EPSC during the two 40-Hz trains before (B) and after (C–E) applying the augmentation protocol was normalized to the first EPSC before augmentation. Data are plotted as mean ± SEM versus the stimulus number. In the first three EPSCs after the augmentation protocol (Inset), depression was steeper in WT versus Doc2α/β DKO neurons while steady-state amplitudes remained the same. These results indicate fewer superprimed SVs in the DKO. Expression of WT-Doc2β (C), but not Doc2β_clm (D) or Doc2β_MID-scrm (E), rescued the superpriming phenotype. (F and G) Normalized peak amplitude of each first response (F) and steady-state responses (G) before and after the augmentation protocol are presented as mean ± SEM. (H) After the augmentation protocol, the extent of SV superpriming was estimated by dividing the amplitude of the third peak by the first peak. Data are shown as mean ± SEM. *P < 0.05 versus Doc2α/β DKO, Kruskal–Wallis test followed by Dunn’s post hoc test.

Fig. 8.

Model of Doc2-dependent synaptic augmentation. In basal conditions, only a small portion of SVs in the RRP are superprimed. Augmentation is induced when synapses are stimulated by a series of action potentials (APs), resulting in the accumulation of residual Ca²⁺. This Ca²⁺ binds to the C2B domain of Doc2, triggering the translocation of the Doc2–munc13 complex to the plasma membrane. At the plasma membrane, the complex drives superpriming of a subset of SVs.