Short-term plasticity at recurrent PN synapses is unaltered in CB−/− and PV−/−
The axonal arborizations of individual PNs were visualized by two-photon microscopy after single-cell electroporation with red Atto-594 dye (Fig. 1A; see Methods). The morphology of axon collaterals yielded data for the WT that were consistent with previous descriptions (Orduz & Llano, 2007; Watt et al. 2009) and were not altered in CB−/− and PV−/− mice (Table 1). Dual whole-cell recordings were established on the dye-filled presynaptic cell and the putative postsynaptic PN identified with brightfield optics. In total we recorded from 128 pairs of connected PNs (WT and mutants). In the WT, presynaptic APs elicited PSCs strongly scattering in amplitude (Fig. 1B and C) with a median amplitude of 90 pA (IQR 52–299 pA, n= 37 pairs) and a median fraction of synaptic failures in response to the first AP (F1) of 0.12 (0.02–0.36; Fig. 1D). The amplitude histograms were skewed towards larger values with no indication of discrete groups of amplitudes (Fig. 1E). Quantal peaks were not clearly evident, which precluded determination of miniature PSC amplitudes from the histograms. Frequency-dependent STP was evident at a 5 ms ISI with PPRs covering a range from moderate paired pulse depression (PPD) to PPF (n= 8; Fig. 1F). On average (n= 37), moderate PPF prevailed at the 5 ms ISI (PPR = 1.21, 1.00–1.49). PPF was of presynaptic nature, since it was associated with fewer failures in the second response (F2, 0.07, 0–0.23), indicating increased release probability for the second release process (cf. Orduz & Llano, 2007). PPR showed no dependency on recording time; rather, it was essentially stable over 100 min whole-cell recording time (Fig. 1G; r=−0.076 ± 0.089, n= 5; P > 0.05).
To test the idea that saturation of CB underlies PPF (Blatow et al. 2003; Orduz & Llano, 2007) we focused on the 5 ms ISI and performed paired-pulse experiments in pairs of connected PNs from CB−/− mice. The amplitude distributions of PSCs were similar to the WT with a median value of 114 pA (61–447 pA, n= 34) that was statistically not different from the WT (Fig. 1C; P= 0.26). Unexpectedly, also the PPR in CB−/− was not significantly different from the WT, i.e. moderate PPF was present at the 5 ms ISI (1.32, 1.03–1.51; n= 34, P= 0.2; Fig. 1H). In addition, neither F1 (0.16, 0.01–0.38; P= 0.66; Fig. 1D) nor F2 (0.08, 0–0.27; P= 0.94) were altered compared to the WT, which argues against the idea of CB saturation as causing PPF.
Buffering of [Ca2+]res by PV (Caillard et al. 2000) or its interference with saturation of unidentified fixed buffers (Eggermann & Jonas, 2012) could have been another factor in PPF. This was tested by paired-pulse experiments in PV mutants at the 5 ms ISI. However, again neither PSC amplitudes (208 pA, 72–712 pA; P= 0.26, n= 23, Fig. 1C), PPR (1.05, 0.89–1.37; P= 0.2; Fig. 1H) nor failure rates (F1: 0.10, 0–0.38, P= 0.66; F2: 0.07, 0–0.37, P= 0.94; Fig. 1D) were significantly affected by the absence of PV. At the Calyx of Held it has been observed that PV does not affect the amount of PPF at 4 ms ISI but does at longer ISI, resulting in a prolonged time-course of facilitation (Müller et al. 2007). Therefore, we also compared PPF between PV−/− and WT at ISIs of 10 and 20 ms but again found no significant difference between the two groups (ISI 10 ms: WT, PPR 1.0, 0.46–1.18, PV−/− 1.07, 0.9–1.3; P= 0.4; ISI 20 ms: WT, PPR 1.1, 0.9–1.2, PV−/− 0.98, 0.86–1.2; P= 0.7). These findings provide evidence against an involvement of PV in PPF and also argue against a significant involvement of [Ca2+]res (Caillard et al. 2000; Müller et al. 2007) or saturation of a fixed high-affinity Ca2+ buffer (Eggermann & Jonas, 2012) in PPF.
Taken together, we confirm moderate PPF at PN–PN synapses during high-frequency activation. Surprisingly, however, we found no evidence for a substantial contribution of the endogenous buffers CB and PV to PPF.
Lack of CB but not of PV increases the initial release probability
Quantitative estimates of pr, a major determinant of STP, were obtained by MPFA of peak PSC amplitudes recorded at different extracellular Ca2+ concentrations (Silver, 2003). Amplitudes were displayed in variance–mean plots (V–M plots) and fitted by a parabolic function that inferred quantal parameters under consideration of intra- and intersite variability. Despite high [Ca2+]e we found almost linear V–M relationships in all WT pairs (n= 7), indicating small pr in the WT. Due to the limited solubility of Ca2+, we partially blocked potassium currents by bath-applied TEA to increase pr further, assuming that increasing Ca2+ influx by either raising [Ca2+]e or by broadening actions potentials by TEA application has similar effects on intrinsic release sites (Huang et al. 2010; Schmidt et al. 2013). With TEA, V–M plots yielded parabolic fits in 6 out of 7 WT cells (Fig. 2A). On average, the fluctuation analyses yielded a quantal size (q) of 66 pA (43–125 pA, n= 14; pooled with values from the linear relationships, which were statistically not different, P= 0.5) and a binominal parameter N of 11.9 (8.6–15.8; n= 6). For 2 mm[Ca2+]e in the absence of TEA, pr was 0.16 (0.09–0.27).
Fluctuation analyses from CB−/− pairs yielded parabolic V–M plots in 6 out of 8 pairs (Fig. 2B) without application of TEA, indicating an increased pr in the mutant. Indeed, MPFA revealed a pr of 0.30 (0.24–0.43; n= 6), a value significantly larger than in the WT (P= 0.046). Increased pr was accompanied by a significant increase in q (217 pA, 112—278 pA, n= 8; P= 0.004), while N showed a tendency towards lower values that, however, did not reach the level of statistical significance (2.6, 1.2–5.2, P= 0.1). The significant alteration in q possibly reflects postsynaptic compensation, corresponding to spine enlargement in CB−/− PNs (Vecellio et al. 2000). For MPFA of PV−/− connections, TEA supplement was again required and none of the quantal parameters was statistically different from the WT (pr= 0.19, 0.14–0.30; N= 13.6, 6.0–26.0; n= 4; q= 111 pA, 80–193 pA; n= 7; P= 0.6, 0.1, 0.2; Fig. 2C–F).
The pr values might be considered upper limits because the MPFA may be distorted by postsynaptic saturation or desensitization at higher pr (Meyer et al. 2001). However, a substantial postsynaptic involvement appears unlikely because, even in the presence of TEA or high [Ca2+]e, PSC amplitudes remained strongly scattered with some large amplitudes (Fig. 2A–C). In addition, average amplitudes calculated from the quantal parameters were in good accordance with those recorded during the basal characterization of the connections (cf. Methods). Finally, a comparison of second PSCs following a release failure to those following a success revealed no significant difference, with respect to either amplitudes or decays (Fig. 3; Saviane & Silver, 2006b). Taken together, while PV did not significantly influence pr the MPFA data show an almost 2-fold increased pr in CB−/− compared to WT.
Figure 3. No obvious influence of vesicle depletion or postsynaptic effects on second PSC amplitudes A, ratio between second PSC amplitudes following a release failure (0_1) or a success (1_1) recorded in 2 mm[Ca2+]e. Values were statistically not different from a concordant distribution around 1.0 (P= 0.063). B, averaged PSCs recorded at 2 mm[Ca2+]e from a WT, CB−/− and PV−/− pair of 0_1 (black) and 1_1 events (grey) with amplitudes being drawn to scale. Note the identical decay kinetics. The data suggest that receptor desensitization is negligible at near physiological [Ca2+]e.
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Increased presynaptic Ca2+ in CB−/− terminals and model construction
To obtain a deeper understanding of the Ca2+ transients that drive and facilitate vesicular release, we quantified volume-averaged Ca2+ signals in putative presynaptic boutons (Fig. 4A and B) that would serve as templates for adjusting constrained, spatially resolved, kinetic simulations of presynaptic Ca2+ dynamics and transmitter release (Fig. 4C).
Figure 4. Presynaptic Ca2+ dynamics: experiments and simulation A, two-photon image of two putative presynaptic collateral terminals from a PN loaded with OGB1 (200 μm pipette concentration). B, fluorescence transients (ΔF/F0) elicited by a train of 10 APs at 200 Hz (evoked by somatic depolarizing voltage steps, top trace) in the putative boutons shown in A. Bottom traces, linescan recordings at 500 Hz (five repetitions in grey, averages in black). C, scheme of the spatially resolved, reaction–diffusion model, covering Ca2+ influx, buffers (including CB, PV, CaM and OGB1), Ca2+ extrusion and the release sensor. D, left panels, averages of 3–5 fluorescence transients per individual bouton (grey) from WT (23 boutons from 7 PNs), CB−/− (18/7) and PV−/− (21/10). Grand averages per strain in dark grey, corresponding simulations in black, red, and blue, respectively. Right, summary of measured peak fluorescence changes (ΔF/F0; median and IQR; ***P < 0.001). E, results of numerical simulations in response to two presynaptic APs in the absence of OGB1. Note differences in scaling from Ea to Ef. Ea, temporal profile of volume-averaged free [Ca2+]i. Eb–d, the spatio-temporal profiles of Ca2+-bound fractions of PV (Eb), mobile (0.8) plus immobile (0.2) fractions of CB (Ec), and mobile CaM (Ed) simulated at increasing distances from the site of the Ca2+ influx (10 to 100 nm in 10 nm increments). Ee, Ca2+ occupancy of the fraction (0.2) of CaM assumed to be immobilized in the influx shell. Ef, spatio-temporal profile of free [Ca2+]i (10–100 nm in 10 nm increments).
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PNs were equilibrated for 30 min in the whole-cell configuration with the indicator dye Oregon Green 488 BAPTA-1 (OGB1). Single APs typically did not reliably increase fluorescence above noise level due to their small amplitude of only ∼0.08 ΔF/F0 in WT (cf. Orduz & Llano, 2007) but were previously shown to sum linearly from 1 to 10 APs (Orduz & Llano, 2007). Therefore, to monitor the buffering action of CB and PV, we focused on the robust fluorescence signals that could be elicited by 10 APs at 200 Hz and analysed the transients in boutons of the collaterals. Trains of APs in a given bouton were recorded 3–5 times at different whole-cell times and showed stable signals. The median amplitude in WT was 0.72 ΔF/F0 (0.46–1.32; n= 23 boutons from 7 PNs), corresponding to a volume-averaged [Ca2+]i elevation of only ∼110 nm (Schmidt et al. 2003), which gives an approximate upper limit for [Ca2+]res after 10 APs. Based on linearity (Orduz & Llano, 2007) the upper limit for elevation in [Ca2+]res between the first and the second AP would be 11 nm, an unlikely source for PPF. As expected from our previous work on dendritic Ca2+ signals in the same mutants (Airaksinen et al. 1997; Schmidt et al. 2003), in CB−/− boutons Ca2+ amplitudes were significantly increased (ΔF/F0= 1.58, 1.23–2.20, n= 18/7; P < 0.001), while the absence of PV had no statistical influence on the amplitude compared to the WT (ΔF/F0= 0.51, 0.42–0.71, n= 21/10; Fig. 4D).
The average WT signal was used to adjust numerical simulations that were highly constrained by published parameters (Fig. 4C, D; Supplementary Table S1). The model considered known endogenous buffers (CB, PV, CaM, ATP), the indicator dye or otherwise EGTA, non-linear effects such as buffer competition and saturation, diffusion (including CB, PV, CaM, ATP, dye or EGTA), and a correction for experimental whole-cell wash-out of endogenous proteins. Fixed buffers were represented by the 20% immobilized fractions of CB (Schmidt et al. 2005) and CaM (Lee et al. 1999; Schmidt et al. 2007), with immobile CB being present throughout the terminal, while CaM was immobilized only at the site of the influx, reflecting an upper limit for CaM bound to Ca2+ channels (Lee et al. 1999). Together, the buffer capacity (κ) of the simulated proteins (with the wash-out correction omitted, κnative, Table S1) accounts for κ published for PNs of the age analysed here (Fierro & Llano, 1996) with the uncertainty, however, that Fierro & Llano (1996) did not consider wash-out effects during their experiments.
Ca2+ influx was assumed to occur at the central hemi-shell (cf. Bucurenciu et al. 2008; Eggermann & Jonas, 2012; Schmidt et al. 2013). While this may represent an oversimplification, a Ca2+ point source predicts the maximal local Ca2+ increase and buffer saturation possible; that is, if under these conditions buffer saturation does not occur, it cannot occur with more distributed influx since this would be associated with smaller local [Ca2+]i (Schmidt et al. 2013). The amplitude of the Ca2+ influx and the pump velocity were the only free parameters during model adjustment; all other parameters were taken from the literature and kept invariant. A further demand on the model was that it needed to fit data from the buffer mutants with the complete parameter set (including influx and pump) kept invariant compared to WT, except removal of CB or PV to mimic the mutant. Notably, this demand was excellently fulfilled by the simulations (Fig. 4D), indicating that major Ca2+ signalling processes were reproduced well by the model.
To account for recent findings that, in pyramidal neurons, the concentrations of PV (Eggermann & Jonas, 2012) or CaM (Faas et al. 2011) are higher than previously expected, we also tested models with increased PV and CaM concentrations (500 and 100 μm, respectively). However, under our constrained conditions simulations with a higher PV and/or CaM concentration did not reproduce the experimental Ca2+ transients accurately (Fig. S1).
Following model adjustment, paired AP-evoked Ca2+ transients were simulated under conditions reflecting the paired electrophysiological recordings, i.e. in particular the Ca2+ indicator dye was replaced by EGTA which was present in the pipette solution during paired recordings (Fig. 4E). In these simulations spatially resolved changes in free Ca2+ and in the Ca2+ occupancies of all buffers were analysed. The fractional Ca2+ occupancy of PV, mobile CaM, mobile CB and ATP showed no signs of saturation during both APs (defined as ≥50% occupancy; Schmidt, 2012), even close to the site of Ca2+ influx (Fig. 4Eb–d). The immobile fractions of CB and CaM became saturated with Ca2+ already during the first AP (Fig. 4Ec,e). However, this did not result in increased [Ca2+]i during the second AP, either in the volume average or at any distance from the site of influx (Fig. 4Ea,f). The reason for this is mainly that additional free Ca2+ ions were readily buffered by the large amount of mobile binding sites. In consequence, [Ca2+]i had virtually identical amplitudes during both pulses and declined to 50 nm between APs. Thus, [Ca2+]res was elevated by only ∼5 nm above the resting level of 45 nm (Wilms et al. 2006) prior to the second AP. A more substantial [Ca2+]res of ∼ 100 nm will build up only after 10 APs.
The simulations are in line with the experimental findings and support the notion that in particular neither CB saturation nor a substantial [Ca2+]res underlies PPF. If, nevertheless, a more substantial [Ca2+]res from the first AP were responsible for PPF, at least excess amounts of EGTA should interfere with PPF (Caillard et al. 2000; Rozov et al. 2001). We therefore performed paired-pulse experiments on pairs of connected WT PNs that were dialysed for 60–70 min with 10 mm EGTA. We estimate that after this time 70–80% of the pipette concentration was reached in the terminal (Schmidt et al. 2003; Bucurenciu et al. 2010), which corresponds to an added buffer capacity (κB) of ∼40,000. Even under these conditions of strong exogenous buffering, PPF remained unaltered compared to controls (PPREGTA= 1.18, 0.98–1.31, PPRcontrol= 1.23, 0.78–1.40, n= 5, P= 0.5; Fig. 5A), substantiating the notion that [Ca2+]res is not causal for PPF here.
To obtain further insight into the interplay between facilitation and depletion, we plotted PPR versus pr values for different [Ca2+]e (Fig. 5B; cf. Valera et al. 2012). The plot shows a decline in PPR with increasing pr, which is in accordance with reports from other synapses (e.g. Murthy et al. 1997; Valera et al. 2012). The relationship between pr and PPR was similar in WT and PV−/− and shifted towards higher PPR in CB−/−. As deduced from a simple calculus for maximum PPR (PPRmax= (1 –pr)pr,2/pr; with a max. pr,2= 1) and consistent with the above similarity of second PSC amplitudes following a release failure to those following a success (Fig. 3A), no signs of compensation for vesicle depletion (e.g. by replenishment or extra sites, Valera et al. 2012) were evident at near physiological [Ca2+]e. To address this point further, we analysed the variance of second PSC amplitudes recorded at 2 mm[Ca2+]e for all genotypes and included their V–M relationship into the V–M plots for the first amplitudes (Fig. 5C). If recruitment of extra release sites were involved in PPF, the corresponding V–M relationship of the second amplitudes could deviate from the initial parabola towards larger variances (Valera et al. 2012), whereas an increased second pr in the absence of significant depletion compensation results in data points falling close to the initial parabola (Clements & Silver, 2000). We found that for all three genotypes and all pairs recorded the V–M relationships of the second PSCs lay close to the parabolic fits to the V–M relationships of the first amplitudes. This substantiates the above notion that depletion compensation or extra release sites are unlikely to make a strong contribution to PPF at 2 mm[Ca2+]e. At high pr settings, by contrast, some compensation for the loss of vesicles during the first AP probably occurred, as experimental PPR slightly exceeded the theoretical PPRmax for a given pr (Fig. 5B). Taken together, the data presented so far do not suggest substantial contributions of depletion or its compensation or extra release sites (at [Ca2+]e of 2 mm) of CB, PV and [Ca2+]res to PPF.
Facilitated release sensor as a source for PPF
A remaining possibility for explaining PPF is a slow relaxation of the vesicular release sensor from its Ca2+-occupied states. We will refer to a partially occupied sensor as facilitated release sensor, which is reminiscent of the ‘active Ca2+’ hypothesis by Katz & Miledi (1968). To test the hypothesis that PPF could result from a facilitated release sensor we used the estimated Ca2+ transients to drive four different five-site release sensor models (Bollmann et al. 2000; Schneggenburger & Neher, 2000; Lou et al. 2005a; Sakaba, 2008). In all models (Table S2), release rate and pr dropped rapidly with increasing influx–release coupling distance, with best matches between experimental and theoretical values being obtained at coupling distances between 20 and 35 nm (Fig. S2), indicating that PN–PN synapses operate at nanodomain coupling similar to other inhibitory synapses (Bucurenciu et al. 2008; Eggermann & Jonas, 2012) or the excitatory cerebellar parallel fibre synapse (Schmidt et al. 2013).
In all models we found varying degrees of STP due to differences in the durability of their Ca2+ bound states (Fig. S2). The experimental data were best matched by the models of Schneggenburger & Neher (2000) and Sakaba (2008) (models 2 and 3 in Table S3, respectively). These models generated PPF due to long-lived Ca2+ bound states, thereby facilitating the sensor for the second release process during the 5 ms ISI. Model 2 gave a good description of the data at 2 mm[Ca2+]e (Fig. S2B), but underestimated PPR at 10 mm[Ca2+]e since it did not include a vesicle replenishment mechanism. Model 3 was developed to describe release at cerebellar basket cell terminals and included Ca2+-dependent vesicle replenishment (Sakaba, 2008). While such a replenishment step was not required to describe the data at 2 mm[Ca2+]e, we found that it was necessary to account for PPR recorded at the higher pr settings in 10 mm[Ca2+]e (Fig. 5B). With a slightly increased sensor off-rate (koff) and for CB−/− also an increased refilling rate (kfill) the model accounted well for PPR from high to low [Ca2+]e for WT and mutant PNs (Fig. 5B, D–F; model 5 in Table S2). The adjusted simulations reproduced the experimental pr and PPR values at a coupling distance of 24–29 nm under consideration of the data scatter by bootstrap analysis (Fig. 5E, Fig. S2). They show PPF in the absence of substantially elevated [Ca2+]i in the second pulse and indicate that PPF can indeed result from long-lived Ca2+-bound states of the release sensor.
In summary, experiments in mutant mice and whole-cell dialysis of terminals with or without EGTA show that high-frequency PPF is largely independent of the major endogenous Ca2+ buffers CB and PV and of [Ca2+]res at PN–PN synapses. Numerical simulations indicate that a release sensor facilitated by a residue of the active Ca2+ could provide an explanation for PPF here.