The presynaptic scaffolding protein Piccolo organizes the readily releasable pool at the calyx of Held

Abstract

Key points: Bassoon and Piccolo do not mediate basal synaptic vesicle release at a high-frequency synapse. Knockdown of Bassoon increases short-term depression at the calyx of Held. Both Bassoon and Piccolo have shared functions in synaptic vesicle replenishment during high-frequency synaptic transmission. Piccolo organizes the readily releasable pool of synaptic vesicles. It safeguards a fraction of them to be not immediately available for action potential-induced release. This enables the synapse to sustain high-frequency synaptic transmission over long periods.

Abstract: Synaptic vesicles (SVs) are released at the active zone (AZ), a specialized region of the presynaptic plasma membrane organized by a highly interconnected network of multidomain proteins called the cytomatrix of the active zone (CAZ). Two core components of the CAZ are the large, highly homologous scaffolding proteins Bassoon and Piccolo, whose function is not well understood. To investigate their role in synaptic transmission, we established the small hairpin RNA (shRNA)-mediated in vivo knockdown (KD) of Bassoon and Piccolo at the rat calyx of Held synapse. KD of Bassoon and Piccolo, separately or simultaneously, did not affect basic SV release. However, short-term depression (STD) was prominently increased by the KD of Bassoon, whereas KD of Piccolo only had a minor effect. The observed alterations in STD were readily explained by reduced SV replenishment in synapses deficient in either of the proteins. Thus, the regulation of SV refilling during ongoing synaptic activity is a shared function of Bassoon and Piccolo, although Bassoon appears to be more efficient. Moreover, we observed the recruitment of slowly-releasing SVs of the readily-releasable pool (RRP), which are normally not available for action potential-induced release, during high-frequency stimulation in Piccolo-deficient calyces. Therefore, the results obtained in the present study suggest a novel and specific role for Piccolo in the organization of the subpools of the RRP.

Keywords: active zone; replenishment; synaptic vesicles.

Figure 1. Bassoon and Piccolo are efficiently and specifically knocked down at the calyx of Held in vivo

A, representative confocal sections of individual calyces delineated by mGFP (green outline) and immunostained for Bassoon (top) and Piccolo (bottom). Calyces expressing a shRNA were identified by mOrange2 fluorescence (shown below the immunosignal). Scale bar: 10 μm. [Correction made on 23 January 2018 after first online publication: Scale bar measurement was added.] B and C, quantification of Bassoon and Piccolo immunosignal in identified calyces (n = 7–15 calyces). Error bars represent the SEM.

Figure 2. Bassoon and Piccolo KD do not affect basal SV release

A, representative current traces of spontaneous EPSCs. B, quantification of spontaneous EPSC amplitude, frequency and kinetic properties. C, superimposed representative current traces of evoked EPSCs. D, quantification of evoked EPSC amplitude and kinetic properties. E, representative current trace recorded after a train of 50 APs delivered at 100 Hz to induce asynchronous release, which is determined by the number of miniture EPSCs in the time window indicated by the dashed line (left) and quantification of miniture EPSC counts (right) (n = 9–11). Error bars represent the SEM. [Correction made on 23 January 2018 after online publication: Scale bar in Fig. 2 E was corrected from 200 ms to 20 ms.]

Figure 3. KD of Bassoon and Piccolo affects STD

A, superimposed scaled representative current traces recorded in response to 50 stimuli at 100 Hz. B, averaged normalized EPSC amplitudes during 100 Hz stimulation. C–E, quantification of the time constant of EPSC amplitude decay, extent of depression and PPR, respectively (n = 10–11). Error bars represent the SEM.

Figure 4. KD of Bassoon affects STD independent of P_r

A, superimposed scaled representative current traces recorded in response to 50 stimuli at 100 Hz in aCSF containing 1.5 mm extracellular calcium. B–D, quantification of the PPR, the time constant of EPSC amplitude decay and the extent of depression, respectively (n = 9–6). Error bars represent the SEM.

Figure 5. Bassoon and Piccolo regulate SV replenishment during ongoing stimulation

A, averaged cumulative EPSCs recorded during 100 Hz stimulus trains. B and C, quantification of RRP size and normalized SV replenishment rate (slope of the line fit divided by initial EPSC amplitude) (n = 10–12). D, quantification of RRP size as determined by the method described by Elmqvist and Quastel (n = 10–12). E, representative plots of EPSC amplitudes vs. cumulative EPSC amplitudes obtained from single calyces. Error bars represent the SEM.

Figure 6. Piccolo KD results in a biphasic recovery from STD

A, superimposed scaled representative current traces beginning with a pool depleting train (100 Hz, 20 stimuli) followed by a test stimulus at increasing time intervals (0.01 s, 0.064 s, 0.128 s, 0.256 s, 0.512 s, 1.024 s, 2 s, 4 s, 6 s, 8 s, 10 s and 12 s). B, averaged fraction of recovery. Data is shown with fits to average data using mono‐ or bi‐exponential functions (WT, SC, Bsn‐KD and DKD, Pcl‐KD, respectively). Inset: early time points of recovery omitting the fits for clarity. C–F, quantification of recovery time constants and their amplitude fractions obtained from bi‐exponential fits (n = 6–8). Error bars represent the SEM.