Post-tetanic potentiation is caused by two signalling mechanisms affecting quantal size and quantal content

Authors


Corresponding author L.-G. Wu: National Institute of Neurological Disorders and Stroke, 35 Convent Drive, Bldg 35, Bethesda, MD 20892, USA. Email: wul@ninds.nih.gov

Abstract

A high-frequency action potential train induces post-tetanic potentiation (PTP) of transmission at many synapses by increasing the intra-terminal calcium concentration, which may increase the quantal content by activation of protein kinase C (PKC). A recent study found that an increase of the mEPSC size, caused by compound vesicle fusion, parallels PTP, suggesting that the quantal size increase also contributes to the PTP generation. However, the strength of this suggestion is somewhat undermined by recent studies suggesting that vesicles responsible for spontaneous and evoked EPSCs may originate from different pools. Furthermore, it is unclear whether the quantal size increase is also mediated by PKC. The present work addressed these issues at a large calyx of Held synapse. We found that PTP was caused by both a PKC-dependent increase of the quantal content and a PKC-independent increase of the quantal size. In addition, we found that mEPSCs and EPSCs were subjected to similar up- and down-regulation, which verifies the basic assumption of quantal analysis – the same mechanism controls the quantal size of spontaneous and evoked release. This verification supports the use of quantal analysis at central synapses. However, unlike the traditional quantal analysis that attributes the quantal size change to a postsynaptic mechanism, the present work, together with one of our previous studies, suggests that the quantal size increase is caused by a presynaptic mechanism, the compound fusion among vesicles that forms large compound vesicles.

Abbreviations 
PTP

post-tetanic potentiation

PTPEPSC and PTPmEPSC

EPSC and mEPSC amplitude potentiation

PTPEPSC_fast and slow

fast and slow component of the PTPEPSC

Train10s

train of stimuli at 100 Hz for 10 s

Introduction

Synaptic plasticity is critical in the neuronal circuit function, neuronal development, and certain forms of learning and memory (Zucker & Regehr, 2002; Abbott & Regehr, 2004; Malenka & Bear, 2004). A high-frequency train of action potential stimulation may induce post-tetanic potentiation (PTP) of synaptic transmission for a few minutes at many synapses (Zucker & Regehr, 2002). Accumulated evidence suggests that this form of synaptic plasticity is mainly caused by a calcium-dependent activation of protein kinase C (PKC), which increases the number of released vesicles (quantal content) by increasing the release probability and/or the readily releasable pool size (Alle et al. 2001; Brager et al. 2003; Habets & Borst, 2005, 2006; Korogod et al. 2005, 2007; Xu et al. 2007).

A recent study showed that PTP of the evoked, AMPA receptor-mediated EPSC was accompanied by calcium-dependent potentiation of the mEPSC amplitude at the calyx of Held synapse (He et al. 2009). The mEPSC amplitude potentiation was smaller than the PTP in the first minute after tetanic stimulation, but became similar to the PTP ∼1 min later, suggesting that in addition to the increased vesicle number, an increase in the quantal size contributes to the generation of PTP (He et al. 2009). However, two issues remained unresolved. First, it is unclear whether the calcium-dependent quantal size increase is also caused by activation of PKC in a similar way as the calcium-dependent increase of the quantal content. Second, recent studies suggest that spontaneous and evoked release originate from different vesicle pools at hippocampal synapses (Sara et al. 2005; Atasoy et al. 2008; Mathew et al. 2008; Fredj & Burrone, 2009; Chung et al. 2010; but see Groemer & Klingauf, 2007). These studies suggested caution in using quantal analysis, a traditional analysis of evoked release based on the assumption that the spontaneous quantal event is the same as the quantal event during evoked release (Zucker, 2005; Rothwell, 2010). Consequently, additional evidence may be needed to strengthen the suggestion that quantal size increase, detected as an increase of the mEPSC amplitude, contributes to the generation of PTP.

The present work addressed the above two issues by recording both the mEPSCs and EPSCs at a large central synapse, the calyx of Held synapse. We found that PTP of the EPSC is caused by two different cellular mechanisms, a PKC-dependent increase of the quantal content and a PKC-independent increase of the quantal size. In addition, our study further supports the use of quantal analysis at central synapses.

Methods

Wistar rats 7–10 days old were rapidly decapitated (Borst et al. 1995; Sun & Wu, 2001). Horizontal brainstem slices (200 μm thick) containing the medial nucleus of the trapezoid body were prepared using a vibratome (He et al. 2006). All animal protocols followed the guidelines of the National Institutes of Health, USA, and complied with The Journal of Physiology policy and UK regulations on animal experimentation (Drummond, 2009). Slices were incubated for 30 min at 37°C and then held at room temperature (22–24°C) for experiments in a solution containing (in mm): 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 25 dextrose, 1.25 NaH2PO4, 0.4 ascorbic acid, 3 myo-inositol, 2 sodium pyruvate, 25 NaHCO3, pH 7.4 when bubbled with 95% O2–5% CO2. A bipolar electrode was placed at the midline of the trapezoid body. A 0.1 ms, 2–20 V voltage pulse was applied to this electrode to evoke an action potential at the axon and calyx, which induced an AMPA receptor-mediated EPSC at the principal neuron of the medial nucleus of the trapezoid body. The principal neuron was selected for recording when an action potential, as detected with an extracellular electrode, was evoked by the axonal stimulation (Borst et al. 1995). Voltage-clamp recordings of AMPA receptor-mediated EPSCs and mEPSCs were performed with an EPC 10 amplifier (HEKA, Lambrecht, Germany) using pipettes (2–3 MΩ) containing (in mm): 125 potassium gluconate, 20 KCl, 4 MgATP, 10 Na2-phosphocreatine, 0.3 GTP, 10 Hepes and 0.5 EGTA, pH 7.2, adjusted with KOH (Xu & Wu, 2005). The series resistance (<10 MΩ) was compensated by 85–95% throughout the experiment, including during recordings of the EPSC and the mEPSC. The control bath solution contained (in mm): 125 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 25 dextrose, 1.25 NaH2PO4, 0.4 ascorbic acid, 3 myo-inositol, 2 sodium pyruvate, 25 NaHCO3, 0.05 d-APV, 0.01 bicuculline and 0.01 strychnine, pH 7.4 when bubbled with 95% O2–5% CO2. Bisindolylmaleimide I (BIS) and Ro31-8220 were purchased from CalBiochem (San Diego, CA, USA). As in our previous study (He et al. 2009), the mEPSC was analysed with a mini program (Mini Analysis Program, Synaptosoft Inc., NJ, USA). The statistical test used is t test, if not mentioned otherwise. The data were expressed as mean ±s.e.m.

Figures 1–4 show sampled EPSCs and mEPSCs with a half-width differing by 2–3 times. The synapse heterogeneity and the age difference of the synapse may account for this difference. It has been shown that the half-width of the EPSC and mEPSC decreased by nearly 3 times on average as the calyx of Held synapse matures from post-natal day (P) 5–7 to P12–14 (Taschenberger & Von Gersdorff, 2000; Joshi & Wang, 2002), owing to a speeding up of AMPA receptor deactivation and desensitization (Koike-Tani et al. 2005). Our data were taken from 7- to 10-day-old rats, in which EPSCs and mEPSCs from 7-day-old rats should be slower than those from 10-day-old rats, on average.

Figure 1.

BIS blocks the PTPEPSC_fast, but not the PTPEPSC_slow or the PTPmEPSC
A, the amplitude of the EPSC (upper, black) and the mEPSC (upper, red), and the frequency of the mEPSC (middle) are plotted versus the time in the control condition (n= 6 synapses). The EPSC was evoked every 10 s, and the mEPSCs within the 10 s period were collected for averaging. The arrow indicates time the Train10s (100 Hz, 10 s) was applied to induce PTP. The PTPEPSC was fitted bi-exponentially (upper, black curve, τ1= 40 s, 62%; τ2= 203 s, 41%; percentages are relative to the baseline), whereas the PTPmEPSC was fitted mono-exponentially (upper, red curve, τ= 212 s). Data were normalized to the baseline before Train10s and expressed as mean ±s.e.m. Sampled EPSCs (lower left, black) and mEPSCs (lower right, red) taken at times labelled before and after Train10s are shown below. B, sampled mEPSCs within 10 s before (upper) and after (lower) Train10s, and sampled EPSCs (middle) induced by Train10s. C, similar to A, except in the presence of BIS (10 μm) for ∼5 min (n= 6). Both the PTPEPSC (black, τ= 239 s) and the PTPmEPSC (red, τ= 193 s) were fitted mono-exponentially.

Figure 2.

Ro31-8220 blocks the PTPEPSC_fast, but not the PTPEPSC_slow or the PTPmEPSC
A, similar to Fig. 1A, but from different neurons (n= 5), and the mEPSC frequency is not plotted. The PTPEPSC was fitted bi-exponentially (black curve, τ1= 49 s, 64%; τ2= 209 s, 39%), whereas the PTPmEPSC in A was fitted mono-exponentially (red curve, τ= 206 s). B, similar to A except that the PKC inhibitor Ro31-8220 (3 μm) was applied for 30 min (n= 5). Both the PTPEPSC (black, τ= 195 s) and the PTPmEPSC (red, τ= 216 s) were fitted mono-exponentially. Scale bar in upper panel in A applies also to B.

Figure 3.

Smaller PTPmEPSC was accompanied by smaller PTPEPSC
A, similar to Fig. 1A, except that the tetanic stimulus was Train4s (100 Hz, 4 s). The PTPEPSC was fitted bi-exponentially (black curve, τ1= 43 s, 44%; τ2= 183 s, 13%), whereas the PTPmEPSC was fitted mono-exponentially (red curve, τ= 180 s). Data were taken from 5 synapses. B, similar to A, except in the presence of the PKC inhibitor BIS (10 μm) for 5 min. Both the PTPEPSC (black, τ= 201 s) and the PTPmEPSC (red, τ= 182 s) were fitted mono-exponentially. Scale bar in upper panel in A applies also to B.

Figure 4.

γ-DGG increased both the PTPmEPSC and the PTPEPSC to a similar extent
A, similar to Fig. 2A, except in the presence of γ-DGG (300–600 μm) and BIS (10 μm, n= 7). Both the PTPEPSC (black, τ= 260 s) and the PTPmEPSC (red τ= 281 s) were fitted mono-exponentially. Scale bar in upper panel in A applies also to B and C. B, similar to A, except that only BIS (10 μm) was in the bath solution (n= 6). This panel is identical to Fig. 1B. We show it here for better comparison with panels A and C. C, similar to A, except in the presence of NBQX (60–80 nm) and BIS (10 μm, n= 6). Both the PTPEPSC (black, τ= 186 s) and the PTPmEPSC (red, τ= 199 s) were fitted mono-exponentially.

Results

PKC-independent potentiation of the mEPSC size and the EPSC are similar in amplitude and time course

A brief stimulus (0.1 ms, 2–20 V) was applied every 10 s via a bipolar electrode positioned at the midline of the trapezoid body, which evoked an action potential at the calyx and thus an EPSC as recorded from the postsynaptic neuron (Fig. 1A) (Borst et al. 1995). While the evoked EPSC was recorded, the mEPSC was collected continuously. Both the EPSC and the mEPSC originated from the same calyx of Held nerve terminal (Von Gersdorff & Borst, 2002). After establishing a stable baseline, a train of stimuli at 100 Hz for 10 s (Train10s) was applied, which induced a PTP of the EPSC (PTPEPSC) (Habets & Borst, 2005; Korogod et al. 2005) and an increase of the mEPSC amplitude (Fig. 1A, n= 6) (He et al. 2009). During Train10s, the EPSC was depressed (Fig. 1B) mostly due to depletion of the readily releasable pool (Von Gersdorff et al. 1997; Wu & Borst, 1999; Schneggenburger et al. 1999; Xu & Wu, 2005). As measured 10–30 s after Train10s, the EPSC amplitude was above the baseline and reach the peak (Fig. 1A, see also Fig. 2A and Fig. 3A), indicating that short-term depression fully recovered within 30 s. The mEPSC amplitude potentiation was observed immediately after Train10s (Fig. 1A, see Fig. 1B for sample raw data).

Similar to our previous report (He et al. 2009), the decay of the PTP of the EPSC amplitude could be fitted with a bi-exponential function with time constants of 40 s, and 203 s, respectively (Fig. 1A). The amplitude for the fast component of the PTPEPSC (PTPEPSC_fast) was 62% as compared to the baseline EPSC amplitude (note that the data were normalized to the baseline before tetanic stimulus), whereas the amplitude of the slow component (PTPEPSC_slow) was 41%. The mEPSC amplitude potentiation (PTPmEPSC) decayed approximately mono-exponentially with an amplitude (39 ± 4%, n= 6) and a time constant (212 s) similar to those of PTPEPSC_slow (Fig. 1A). These results were similar to our previous report (He et al. 2009). Although these results were obtained in P7–10 rats, similar results were obtained in P13–14 rats (n= 3) (He et al. 2009). We also noticed that Train10s induced a transient increase of the mEPSC frequency (Fig. 1A–B), consistent with the finding that the PTPEPSC_fast results from an increase of the released vesicle number (Korogod et al. 2005; Habets & Borst, 2005).

It has been shown that two PKC inhibitors, bisindolylmaleimide I (BIS) and Ro31-8220, did not inhibit the baseline EPSC, but blocked the generation of the PTPEPSC induced by a 4 s stimulation at 100 Hz (Korogod et al. 2007). To determine whether PKC inhibitors block both the PTPEPSC and the PTPmEPSC induced by Train10s, BIS and Ro31-8220 were separately applied to the bath. The effect of BIS was evident within 5 min of BIS application, which allowed us to compare the PTP results before and after BIS application from the same cell. At ∼5 min after BIS (10 μm) application, Train10s induced a PTPEPSC and a PTPmEPSC, both of which decayed mono-exponentially (Fig. 1C). The amplitude of the PTPEPSC (40 ± 11%, n= 6, Fig. 1C) was much smaller than the control before BIS application (104 ± 7%, n= 6, Fig. 1A, P < 0.01). The PTPEPSC decayed mono-exponentially with a time constant (239 s, Fig. 1C) similar to the PTPEPSC_slow in control (203 s, Fig. 1A). In contrast to the partial block of the PTPEPSC, the PTPmEPSC amplitude (34 ± 5%, n= 6, Fig. 1C) and time constant (193 s) were similar to control (amplitude: 39 ± 4%, n= 6, P > 0.2; τ= 212 s, Fig. 1A). Clearly, in the presence of BIS, the PTPmEPSC amplitude and time constant were similar to the PTPEPSC amplitude (40 ± 11%, n= 6, P > 0.1) and time constant (239 s, Fig. 1C). The mEPSC frequency increase induced by Train10s in the presence of BIS (Fig. 1C) was similar to that of control (Fig. 1A), consistent with the previous report (Korogod et al. 2007).

Similar results were obtained for another PKC inhibitor, Ro31-8220 (Fig. 2A and B, n= 5). Since the effect of Ro31-8220 was evident only after ∼30 min application, we obtained the control data (Fig. 2A) from different cells to those subjected to Ro31-8220 application (Fig. 2B). In summary, the PTPEPSC_fast was sensitive to PKC inhibitors, whereas the PTPEPSC_slow and the PTPmEPSC, both of which had a similar amplitude and time constant, were not (Figs 1 and 2). These results suggest that PTPmEPSC was PKC independent and was responsible for the generation of the PKC-independent PTPEPSC_slow.

A change in PTPmEPSC is accompanied by a similar extent of change in PTPEPSC

The use of quantal analysis to study synaptic plasticity has been challenged by recent studies suggesting that spontaneous and evoked release may originate from different vesicle pools at hippocampal synapses (Sara et al. 2005; Atasoy et al. 2008; Mathew et al. 2008; Fredj & Burrone, 2009; Chung et al. 2010; but see Groemer & Klingauf, 2007). Accordingly, the possibility that the similar amplitude and time course of PTPmEPSC and the PTPEPSC_slow is due to co-incidence rather than a causal relation could not be fully excluded. A causal relation between PTPmEPSC and PTPEPSC_slow would predict that a decrease or an increase in PTPmEPSC should cause a proportional change in PTPEPSC. We tested this prediction by either decreasing or increasing PTPmEPSC in the presence of BIS, which blocked PTPEPSC_fast and thus isolated PTPEPSC_slow (Fig. 1).

Reduction of PTPmEPSC was made by decreasing the intensity of the tetanic stimulus from 10 s to 4 s at 100 Hz (Train4s), which induced a much smaller PTPmEPSC (16 ± 6%, n= 5, Fig. 3A) than Train10s in the control condition (He et al. 2009). Train4s induced a PTPEPSC with an amplitude of 57 ± 21% (n= 5), the decay of which was fitted bi-exponentially with time constants of 43 s (44%) and 183 s (13%), respectively (Fig. 3A). The time constant and amplitude of PTPEPSC_slow were similar to those of PTPmEPSC. In the presence of BIS (10 μm), PTPEPSC_fast was abolished (Fig. 3B). The remaining PTPEPSC, PTPEPSC_slow, had a small amplitude (18 ± 7%, n= 5) and a mono-exponential time constant (201 s) similar to those of PTPmEPSC (12 ± 3%, n= 5, τ= 182 s, Fig. 3B). Evidently, in the presence of BIS, reducing the tetanic stimulus intensity from Train10s to Train4s reduced PTPmEPSC and PTPEPSC to a similar extent (compare Fig. 3B with Fig. 1B). This result strengthens the suggestion that PTPmEPSC is responsible for PTPEPSC_slow.

The increase of PTPmEPSC was made by bath application of a competitive AMPA receptor antagonist, γ-DGG (300–600 μm), which relieves AMPA receptor saturation by blocking AMPA receptors to a lesser degree at higher glutamate concentrations (Liu et al. 1999). If PTPmEPSC was due to an increase of the transmitter amount in a vesicle as suggested recently (He et al. 2009), AMPA receptors would be more saturated during PTPmEPSC. Accordingly, γ-DGG should relieve AMPA receptor saturation and thus increase PTPmEPSC. Indeed, in the presence of BIS (10 μm) and γ-DGG (300–600 μm in the bath), the latter of which decreased the baseline mEPSC and EPSC amplitude by ∼24–36% (n= 7, not shown), Train10s induced a PTPmEPSC with an amplitude of 55 ± 5% (n= 7, Fig. 4A) significantly higher than that in the absence of γ-DGG (but in the presence of BIS, 34 ± 5%, n= 6, Fig. 4B, P < 0.01). The PTPEPSC amplitude was 61 ± 10% (n= 7, Fig. 4A), which was similar to PTPmEPSC (Fig. 4A), but significantly higher than PTPEPSC in the absence of γ-DGG (40 ± 11%, P < 0.01, n= 6, Fig. 4B).

As a control, we also examined the effect of a non-competitive AMPA receptor blocker, NBQX. NBQX at 60–80 nm reduced the baseline mEPSC and EPSC by a similar percentage (∼26–40%, n= 6, not shown) as γ-DGG at 300–600 μm, but did not increase either PTPmEPSC or PTPEPSC. In the presence of NBQX and BIS, Train10s induced a PTPmEPSC (32 ± 3%, n= 6) and a PTPEPSC (36 ± 7%, n= 6, Fig. 4C) similar to those in the presence of only BIS (Fig. 4B, P > 0.3). This result suggests that the increase of PTPmEPSC and PTPEPSC by γ-DGG (Fig. 4A) is not caused by a decrease of the mEPSC amplitude itself, but by an increase of the transmitter amount. The similar increase of PTPmEPSC and PTPEPSC in the presence of γ-DGG (Fig. 4), and the similar decrease of both of them when Train10s was changed to Train4s (Fig. 3), suggested that PTPmEPSC is responsible for the generation of PTPEPSC.

Discussion

A presynaptic quantal size increase contributes to the generation of PTP

PTP can be divided into a fast (PTPEPSC_fast) and a slow (PTPEPSC_slow) component (Fig. 1A) (He et al. 2009). A previous study suggests that compound fusion between vesicles is responsible for PTPmEPSC and thus PTPEPSC_slow (He et al. 2009). However, this suggestion needs to be strengthened because the use of quantal analysis has been challenged owing to the observation that spontaneous and evoked release may originate from different vesicle pools (Zucker, 2005; Rothwell, 2010). The current work has strengthened this suggestion by presenting two additional pieces of evidence at the calyx of Held in 7- to 14-day-old rats. First, both the PTPEPSC_slow and PTPmEPSC were resistant to PKC inhibitors BIS and Ro31-8220, whereas PTPEPSC_fast was not (Figs 1 and 2). When PTPEPSC_fast was abolished by PKC inhibitors, PTPEPSC_slow and PTPmEPSC were similar in both amplitude and time course (Figs 1 and 2). Second, both PTPEPSC_slow and PTPmEPSC were reduced to a similar extent when the intensity of the PTP-inducing stimulus was reduced (Fig. 3), and were increased to a similar level during γ-DGG application (Fig. 4). Since γ-DGG is a competitive AMPA receptor blocker that can relieve AMPA receptor saturation, the increase of PTPmEPSC by γ-DGG (Fig. 4) suggests that PTPmEPSC is due to an increase in the glutamate concentration at the synaptic cleft, consistent with the finding that compound fusion causes an increase in the mEPSC size (He et al. 2009). These results significantly strengthen the suggestion that PTPmEPSC is responsible for the generation of PTPEPSC_slow. Together with the previous observation that calcium/synaptotagmin-mediated compound fusion between vesicles occurs during PTP (He et al. 2009), our results suggest that compound fusion increases the vesicle size and transmitter amount, which mediates PTPmEPSC and thus PTPEPSC_slow.

PTP is caused by PKC inhibitor-sensitive and -insensitive mechanisms

Previous studies suggest that PTP, including that induced by Train4s at the calyx, is mediated by a PKC inhibitor-sensitive increase of the quantal content (Alle et al. 2001; Brager et al. 2003; Korogod et al. 2007). The present work confirms this observation, and revealed an additional mechanism, the PKC inhibitor-resistant increase of the quantal size. This additional mechanism, PTPmESPC, was not reported in previous studies at the calyx (Korogod et al. 2007, 2005; Habets & Borst, 2005, 2006). The reason for this discrepancy is mostly due to the difference in the intensity of tetanic stimulation used to induce PTP. We used mostly 100 Hz stimulation for 10 s, whereas previous studies used 100 Hz stimulation for 4 s or a lower frequency of stimulation. When we reduced the tetanic stimulation from 10 to 4 s at 100 Hz, PTPmEPSC was as low as 12 ± 3% (n= 5, Fig. 3B). This minimal increase of PTPmEPSC was in general consistent with previous studies showing that PTP induced by the 100 Hz stimulation for 4 s or a lower frequency of stimulation is mainly not caused by the quantal size increase at the calyx preparation (Habets & Borst, 2005, 2006; Korogod et al. 2005, 2007). The slight difference (between no PTPmEPSC and ∼12% PTPmEPSC) is understandable, because the focus of the studies is different; and the slice condition, the selection of the synapse for recording and the data analysis performed by different labs cannot be exactly the same.

The present work showed that PTP is mediated by two mechanisms, a PKC inhibitor-sensitive fast component owing to an increase of the quantal content, and a PKC inhibitor-insensitive slow component owing to an increase of the quantal size. Both mechanisms are initiated by calcium influx, because the calcium buffer EGTA abolished both PTPESPC (Habets & Borst, 2005; Korogod et al. 2005) and PTPmESPC (He et al. 2009). The increase of the released vesicle number for the PKC inhibitor-sensitive fast component is due to an increased sensitivity of the release machinery to calcium, an increased calcium influx during an action potential and/or an increased readily releasable pool size (Korogod et al. 2005; Habets & Borst, 2006, 2007).

At the calyx of Held in 7- to 10-day-old rats, a high-frequency train of action potential stimulation causes depletion of the readily releasable pool and inactivation of calcium currents after the stimulation, which causes subsequent short-term depression of release for about 10–20 s (Von Gersdorff et al. 1997; Wu & Borst, 1999; Schneggenburger et al. 1999; Xu & Wu, 2005). Replenishment of the readily releasable pool with vesicles having a lower release probability (reluctant vesicles), conversion of reluctant vesicles to regular vesicles, and recovery of the calcium current inactivation contribute to the recovery of short-term depression (Wu & Borst, 1999; Wadel et al. 2007). The present work did not focus on the first 10–20 s after the PTP-inducing stimulus, the Train10s or Train4s. However, we noticed that PTPEPSC reached a peak within 20–30 s after Train10s or Train4s (Figs 1A, 2A and 3A). Thus, within the first 20–30 s after the PTP-inducing stimulus, the mechanisms that cause PTP and short-term depression must take place simultaneously. The EPSC within this time period may thus reflect the net change between PTP and short-term depression of the EPSC.

A recent study at the calyx showed that MLCK inhibitors largely blocked PTP induced by Train4s, suggesting the involvement of MLCK (Lee et al. 2008). This study also showed that a BIS analogue that could not inhibit PKC partly mimics BIS in blocking PTP, suggesting that at least part of the PTP fast component is not mediated by the PKC signalling pathway. Distinguishing whether MLCK or PKC is involved in the PTP fast component is not the focus of the present work. Rather, the present work aimed at determining whether the PTP slow component is mediated by a mechanism different from the PTP fast component. BIS and Ro31-8220 served well for this purpose by blocking only the fast, but not the slow, component of the PTP. We therefore cautiously concluded that the PTP fast component is PKC inhibitor sensitive, which may involve PKC and/or MLCK, whereas the PTP slow component is not.

Quantal analysis at central synapses

The traditional quantal analysis often interprets the quantal size change as the postsynaptic change. The present work, together with our previous work (He et al. 2009), suggests that the quantal size change during synaptic plasticity may also reflect a presynaptic change.

Recent studies at cultured hippocampal synapses suggest that spontaneous and evoked release originate from different vesicle pools (Sara et al. 2005; Atasoy et al. 2008; Mathew et al. 2008; Fredj & Burrone, 2009; Chung et al. 2010; but see Groemer & Klingauf, 2007). These studies suggest caution in the use of quantal analysis, a tool that has been used to analyse the evoked release for many decades, based on the assumption that the quantal size of the spontaneous and evoked release is the same (Zucker, 2005; Rothwell, 2010). The present work showed that the mEPSC size potentiation paralleled the PTP slow component in amplitude and time course in various conditions that decreased or increased the PTP amplitude. The parallel changes of the mEPSC and the EPSC size do not necessarily exclude the possibility that spontaneous and evoked release come from different vesicle pools, but do suggest that the same mechanism controls the quantal size of spontaneous and evoked release. This suggestion supports the assumption of quantal analysis that the mEPSC size reflects the quantal size during evoked release. Thus, regardless of the origin of spontaneous and evoked release, quantal analysis can still be used to study changes in synaptic strength at central synapses, at least at the calyx of Held synapse.

Appendix

Author contributions

L.X. designed and performed experiments and analysed data. L.-G.W. designed experiments and wrote the paper. Both authors approved the final version.

Acknowledgements

We thank Drs Liming He and Fujun Luo for comments on the manuscript. This work was supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program.

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