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. 2010 Apr 26;588(Pt 12):2133–2145. doi: 10.1113/jphysiol.2009.184929

Impaired development of hippocampal mossy fibre synapses in mouse mutants for the presynaptic scaffold protein Bassoon

Frederic Lanore 1, Christophe Blanchet 1, Anna Fejtova 2, Paulo Pinheiro 1, Karin Richter 3, Detlef Balschun 2, Eckart Gundelfinger 2, Christophe Mulle 1
PMCID: PMC2911216  PMID: 20421286

Abstract

Bassoon, a protein highly concentrated at the synaptic active zone, is thought to participate in the organization of the cytomatrix at the site of neurotransmitter release. Bassoon is amongst the first proteins to accumulate at newly formed synaptic junctions, raising the question of the functional role of this protein in the early stages of synaptic development. Here we show that the course of synaptic maturation of hippocampal mossy fibre (MF) synapses (glutamatergic synapses with multiple release sites) is markedly altered during the first 2 weeks of postnatal development in mutant mice lacking the central region of Bassoon (Bsn−/− mice). At postnatal day 7 (P7), Bsn−/− mice display large amplitude MF-EPSCs with decreased paired pulse ratios, an abnormality which may be linked to deficits in the organization of the presynaptic active zone. Surprisingly, 1 week later, decreased MF-EPSCs amplitude is observed in Bsn−/− mice, consistent with the inactivation of a subset of synaptic release sites. Finally, at more mature states a decreased posttetanic potentiation is observed at MF-synapses. These results support the notion that Bassoon is important for organizing the presynaptic active zone during the postnatal maturation of glutamatergic synapses.

Introduction

Synapse formation within the central nervous system is initiated by the formation of axodendritic contacts followed by the differentiation and maturation of pre- and postsynaptic functional domains at the contact site. The differentiation of the presynaptic bouton includes the formation of active zones, i.e. regions of the synaptic plasma membrane specialized for neurotransmitter release. The electron-dense cytoskeletal matrix associated with the active zone (CAZ) is thought to organize the synaptic vesicles that are lined up in close vicinity to the cytoplasmic face of the terminal. The CAZ contains several multimeric protein complexes, including the major scaffolding proteins Bassoon and Piccolo (tom Dieck et al. 1998; Fenster et al. 2000; Schoch & Gundelfinger, 2006). The role of Bassoon in the formation and function of active zones has been addressed by analyzing a mouse mutant lacking the central exons of the Bassoon (Bsn) gene. Although this central region is critical for anchoring Bassoon to the CAZ, mutant hippocampal synapses look structurally normal (Altrock et al. 2003). In the adult hippocampus, neither synapse density nor morphological parameters defining presynaptic nerve terminals are significantly altered in Bsn−/− mice as compared to Bsn+/+ mice. However, a fraction of glutamatergic synapses in CA1 pyramidal cells appears inactivated in Bsn−/− mice (Altrock et al. 2003).

Bassoon is expressed at early stages of neuronal differentiation and is one of the first proteins to be found at nascent synapses, at least in cultured neurons, suggesting a role in the assembly of the active zone (Zhai, 2000, 2001; Shapira et al. 2003). To evaluate whether Bassoon may perform a key role in the postnatal development of synaptic properties, we investigated the functional maturation of a model glutamatergic synapse during the first postnatal weeks in Bsn−/− mice. Guided by the need to understand how presynaptic parameters might be impaired in the absence of functional Bassoon, we have studied hippocampal mossy fibre synapses, which display unique presynaptic properties (Henze et al. 2000). In the mature rodent brain, mossy fibre synapses onto CA3 pyramidal cells (MF–CA3 synapses) display robust forms of presynaptic short-term plasticity (Nicoll & Schmitz, 2005) and express high levels of Bassoon close to release sites (Richter et al. 1999; Zhang et al. 2000). MF–CA3 synapses constitute an attractive system to investigate patterns of synaptic maturation. In rodents, MF synaptic contacts are established following concurrent pre- and postsynaptic morphological changes that take place during the first weeks of development (Amaral & Dent, 1981). During a critical period of postnatal development, between postnatal days 6 and 9, rapid changes in presynaptic and postsynaptic electrophysiological properties of MF–CA3 synapses occur in parallel (Marchal & Mulle, 2004). Here we show that pre- and postsynaptic parameters of synaptic transmission at MF–CA3 pyramidal cell synapses are clearly and biphasically impaired at earlier stages of maturation indicating that Bassoon might be important for organizing the presynaptic active zone during synaptic development.

Methods

Experimental procedures followed the recommendations of the CNRS ethics committee and the French Ministry of Agriculture and Forestry concerning animal care (authorization number, A33093). Bsn−/− mice were generated as described earlier (Altrock et al. 2003), backcrossed into the C57/Bl6 mouse strain, and then crossed with the SV129 strain yielding mice with a mixed C57/bl6/SV129 genetic background. Mutant and wild-type mice used were littermates of heterozygote breeding pairs.

Pups and young mice (<P21) were killed by cervical dislocation. Parasagittal brain slices (350 μm thick) were prepared from P5 to P21 mice (P0 being the day of birth). For the dissection and the storage of the slices, a solution containing: 80 mm NaCl, 2.5 mm KCl, 25 mm NaHCO3, 1.5 mm NaH2PO4, 7 mm MgCl2, 0.5 mm CaCl2, 10 mm glucose and 75 mm sucrose (equilibrated with 95% O2–5% CO2) was used. Slices were incubated at 33°C for 30 min and subsequently stored at room temperature. Whole-cell voltage-clamp recordings (2.6–3.4 MΩ electrodes, −70 mV holding potential) were made from hippocampal CA3 pyramidal cells visualized by infrared video-microscopy. Experiments were performed at room temperature (22–25°C). Slices were superfused with extracellular solution composed of: 125 mm NaCl, 2.5 mm KCl, 1.25 mm NaH2PO4, 25 mm NaHCO3, 4 mm CaCl2, 4 mm MgCl2, 11 mm glucose and equilibrated with 95% O2–5% CO2. Bicuculline (10 μm) was added to the bath to inhibit GABAA receptors. Intracellular solution was composed of: 122 mm cesium methanesulfonate, 2 mm NaCl, 10 mm Hepes, 10 mm EGTA, 2 mm MgCl2, 4 mm ATP-Na (pH 7.3).

A glass microelectrode was placed in the hilus of the dentate gyrus to stimulate MFs. While recording from a pyramidal cell, the stimulating electrode was moved to a position where a sharp EPSC with fixed latency was evoked. Stimulation intensity (200 μs pulse; <20 μA) was adjusted just above the sharp threshold for activation of a synaptic response. Using such low minimal stimulations, no prominent polysynaptic activation was observed. MF stimulation was assessed by its short term facilitation properties (40 ms paired pulse ratio or 1 Hz/0.1 Hz ratio above 3), except for some P5 or P7 recordings where facilitation ratio could be less because of their immature state. In that case, the group II mGluR agonist LCCG-1 (10 μm) was applied to confirm that MFs were stimulated. LCCG-1 was also occasionally applied even when the facilitation ratio was above 3, and, in such cases, it always markedly inhibited EPSCs (Supplemental Fig. 1). Recordings were analysed using IGOR pro (WaveMetrics, Inc., Lake Oswego, OR, USA). Low-frequency facilitation was evoked by stimulating MFs at frequencies of 0.1 and 1 Hz. For Sr2+ experiments, slices were bathed in an extracellular solution containing SrCl2 (2 mm) and a low concentration of CaCl2 (0.03 mm). Asynchronous EPSCs (aEPSCs) were evoked by trains of five stimuli at 50 Hz repeated 100 times at 30 s intervals. Only events with amplitudes larger than 7.5 pA and occurring within a 400 ms time window beginning 50 ms after each train were considered for statistical analysis.

Unless otherwise mentioned, values are expressed as means ±s.e.m. ANOVA followed by Bonferroni's post hoc test, the Mann–Whitney U test or the Kolmogorov–Smirnov (KS) test were used where appropriate.

All drugs were obtained from Tocris Bioscience (Bristol, UK) or Ascent Scientific (Avonmouth, UK).

Immunostaining of brain sections

Mice at P7, P14 and P21 (5 animals of each genotype and age) were deeply anaesthetized using a mixture of Ketavet (Parke-Davis) and Domitor (Pfizer) and perfused transcardially with 0.9% NaCl for 1 min followed by 4% formaldehyde in 0.1 m phosphate buffer (PB) for 10 min. Brains were removed, post-fixed in the same fixative for 20 h at 6°C, freeze-protected with 1 m sucrose in 0.1 m PB and frozen at −40°C in isopentane. Free-floating 25 μm thick coronal sections of dorsal hippocampus were stained 48 h with mouse anti-synaptophysin (1:2000), rabbit anti-Munc13-1 (1:1000, both Synaptic systems, Germany), and guinea pig anti-ProSAP2/Shank3 (1:2000, Bockmann et al. 2002) followed by overnight incubation with Alexa488-labelled anti-rabbit (1:2000, Invitrogen), Cy3-labelled anti-guinea pig (1:1000); Cy5-labelled anti-mouse (1:1000, both Jackson lab) antibodies. The mossy fibre termination zone (stratum lucidum) in the CA3 region of hippocampus was analysed. The images with resolution of 80 × 80 × 300 nm per voxel were taken on CLSM SP2 or SP5 (Leica Microsystems, Wetzlar, Germany) using 63× objective and 6-fold zoom of scanner head. Munc-13 positive puncta were counted manually on around 60 mossy terminals on 5–9 μm thick stacks (minimum 10 stacks per genotype and age) using Image5D plug-in of ImageJ freeware (NIH, USA). In preparing the figures, stacks were deconvolved using Imaris 5.0 software (Bitplane, Zurich, Switzerland), maximal fluorescence Z-projection was created using ImageJ and assembled using Adobe CS2 (Adobe Systems, San Jose, CA, USA).

Results

Electrophysiological properties of MF–CA3 synapses in P21 Bsn−/− mice

Single MF boutons contain multiple active zones (25 on average) and their associated post-synaptic densities (Henze et al. 2000; Rollenhagen et al. 2007). Bsn mutation leads to the inactivation of a fraction of conventional excitatory synapses in CA1 pyramidal cells (Altrock et al. 2003). We sought to explore whether a single MF bouton would show a decreased number of release sites in Bsn mutant (Bsn−/−) mice, i.e. whether all or only some of the active zones would be functionally inactive. For this purpose we have recorded MF excitatory postsynaptic currents (MF-EPSCs) from CA3 pyramidal cells in hippocampal slices from wild-type (Bsn+/+) and Bsn−/− mice at P21. Throughout this study, a minimal stimulation protocol was used to evoke MF-EPSCs. At low frequency (0.1 Hz), we did not observe any significant difference in the average amplitude of MF-EPSCs between Bsn−/− mice and Bsn+/+ littermates (Bsn+/+: 51.1 ± 7.8 pA, n= 11 and Bsn−/−: 43.1 ± 10.0 pA, n= 9, P= 0.32) (KS test for distribution: P= 0.33) (Fig. 1A) as well as in the rate of failures (Bsn+/+: 25.8 ± 6.1%, n= 11 and Bsn−/−: 36.6 ± 6.1%, n= 9, P= 0.23) (Fig. 1B). Potency (average amplitude excluding failures) was not significantly different (Bsn+/+: 80.2 ± 16.1 pA, n= 11 and Bsn−/−: 62.6 ± 9.0 pA, n= 9, P= 0.36).

Figure 1. Normal synaptic transmission and presynaptic short-term plasticity in Bsn−/− mice at P21.

Figure 1

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A, average of 50 traces (left) illustrating LFF of MF-EPSCs when shifting stimulation from 0.1 to 1 Hz for Bsn+/+ and Bsn−/− mice. Cumulative distribution (right) of average MF-EPSC amplitudes at 0.1 Hz (Bsn+/+: n= 11 and Bsn−/−: n= 9) (KS test for distribution, P= 0.33). B, bar graph of failure rate at 0.1 Hz (Bsn+/+: n= 11 and Bsn−/−: n= 9). C, average of 20 traces (left) illustrating PPF at 40 and 400 ms for Bsn+/+ and Bsn−/− mice. Summary graph (right) of PPF ratio at 10, 40, 100 and 400 ms (Bsn+/+: n= 10 and Bsn−/−: n= 6). Isi: interstimulus interval. D, bar graph of mean LFF ratio from 0.1 to 1 Hz (Bsn+/+: n= 9 and Bsn−/−: n= 9).

MF synaptic transmission displays prominent forms of presynaptic short-term plasticity, among which are paired pulse facilitation (PPF) and low frequency facilitation (LFF), which are likely to depend on different presynaptic mechanisms: residual Ca2+ for PPF and activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for LFF (Salin et al. 1996). We did not find any significant difference in PPF ratio between the two genotypes with intervals ranging from 10 to 400 ms (Bsn+/+, n= 10 and Bsn−/−, n= 6; from 10 ms to 400 ms, P > 0.05) (Fig. 1C). LFF develops over a slow time scale with repetitive stimulation in the low frequency range (0.05 to 5 Hz). We tested LFF of MF-EPSCs by increasing the stimulation frequency from 0.1 Hz to 1 Hz for 60 s. LFF developed in a few seconds to the same extent in Bsn+/+ and Bsn−/− mice (ratio 1 Hz/0.1 Hz: Bsn+/+: 9.1 ± 1.4, n= 9 and Bsn−/−: 7.1 ± 0.9, n= 9, P= 0.22) (Fig. 1D). MF–CA3 synapses display a well known PKA-dependent presynaptic form of LTP (MF-LTP) (Nicoll & Schmitz, 2005) that can be induced either chemically with forskolin (10 μm, 15 min) or by a high frequency stimulations (HFS) protocol (100 stimuli at 100 Hz repeated 3 times at 10 s intervals). MF-EPSCs were potentiated to a similar extent in Bsn+/+ and Bsn−/− mice whatever the induction protocol (average LTP between 30 and 35 min; HFS: Bsn+/+: 181 ± 31%, n= 9 and Bsn−/−: 163 ± 23%, n= 12, P= 0.69; and forskolin: Bsn+/+: 205.7 ± 49.1%, n= 6 and Bsn−/−: 190.5 ± 18.8%, n= 6, P= 0.79) (Fig. 2A, C and D). However, we observed that posttetanic potentiation (PTP) was significantly smaller in Bsn−/− mice as compared with Bsn+/+ mice (Bsn+/+: 847 ± 85%, n= 9 and Bsn−/−: 553 ± 71%, n= 12, P= 0.01) (Fig. 2B).

Figure 2. PTP is decreased in Bsn−/− mice.

Figure 2

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A, traces (top) represent MF-EPSCs during baseline (1), PTP (2), LTP (3) and LCCG-1 (10 μm) application (4) in Bsn+/+ and Bsn−/− mice. The LTP protocol was applied at the time indicated by the arrow. Time course (bottom) of MF LTP for Bsn+/+ (n= 9) and Bsn−/− (n= 12) mice. MF-EPSCs were averaged every minute and normalized to the mean amplitude of the 10 min period preceding the LTP induction protocol. B, bar graph of PTP (average first 5 min after the LTP protocol) (Bsn+/+: n= 9 and Bsn−/−: n= 12). C, bar graph of LTP 30–35 min after LTP induction (Bsn+/+: n= 9 and Bsn−/−: n= 12). D, time course of MF-EPSCs potentiation induced by bath application of forskolin (10 μm, 15 min) for Bsn+/+ (n= 6) and Bsn−/− (n= 6). MF-EPSCs were averaged every minute and normalized to the mean amplitude of the 10 min period preceding bath application of forskolin. *P < 0.05.

MF–CA3 synapses show impaired synaptic transmission in Bsn−/− mice at a critical period of postnatal development

Bassoon has been proposed to participate in the initial stages of synapse formation and maturation (Zhai, 2000), and we cannot exclude that compensatory mechanisms occurring progressively during the course of postsynaptic maturation (including possible replacement of Bassoon by Piccolo) could explain the lack of significant functional changes in Bsn−/− mice at P21. We thus evaluated the impact of the Bsn mutation on MF synaptic transmission at earlier stages of postnatal development. We first focused on a critical period of synaptic maturation for MF–CA3 synapses (between P6 and P9 in mice) (Amaral & Dent, 1981; Marchal & Mulle, 2004), during which rapid morphological and functional changes occur at both pre- and postsynaptic levels. MF-EPSCs evoked by minimal stimulation at a rate of 0.1 Hz were recorded at P5, P7 and P9 (Fig. 3A). The mean MF-EPSC amplitude markedly increased between P5 and P9 in both Bsn+/+ and Bsn−/− mice but this increase appears to occur earlier in the absence of Bassoon (Fig. 3B). The average MF-EPSC amplitude was notably larger in Bsn−/− mice as compared to Bsn+/+ mice at P7 (Fig. 3B) although this difference did not reach statistical significance due to high variability of the individual average values (Fig. 3D). Further examination of the distribution of average MF-EPSC amplitudes revealed striking differences between Bsn+/+ and Bsn−/− mice at P5 and at P7 (KS test, comparison of distributions: P5, P= 0.01; P7, P= 0.0001; P9, P= 0.13) (Fig. 3D). Because we average together failures and successful synaptic events, the increase in mean MF-EPSC amplitude at P7 could be due to a decrease in failure rate between Bsn+/+ and Bsn−/− mice. We thus measured the failure rate at 0.1 Hz and found that, at P7 but not at P5 and P9, it was significantly lower in Bsn−/− mice (P5: Bsn+/+: 63.2 ± 9.4% and Bsn−/−: 62.2 ± 7.2%, P= 0.93; P7: Bsn+/+: 68.5 ± 5.8% and Bsn−/−: 53.6 ± 4.4%, P= 0.04; P9: Bsn+/+: 48.8 ± 10.1% and Bsn−/−: 52.4 ± 5.2%, P= 0.73) (Fig. 3C). As for the average amplitude, the mean potency of MF-EPSCs was not significantly different between both genotypes at P5, P7 and P9 (P5: Bsn+/+: 22.9 ± 3.0 pA and Bsn−/−: 41.8 ± 10.7 pA, P= 0.50; P7: Bsn+/+: 48.8 ± 9.7 pA and Bsn−/−: 83.6 ± 14.0 pA, P= 0.13; P9: Bsn+/+: 64.5 ± 13.7 pA and Bsn−/−: 98.7 ± 14.6 pA, P= 0.09). Interestingly, however, we recorded individual MF-EPSCs of large amplitude at 0.1 Hz (>200 pA; ‘maxi’ MF-EPSCs) in a larger proportion of cells from Bsn−/− than from Bsn+/+ mice (12 out of 29 cells, in 8 out of 10 Bsn−/− mice; vs. 2 out 15 cells, in 2 out of 7 Bsn+/+ mice) (Fig. 3A). To ensure that the unusual amplitude of these ‘maxi’ MF-EPSCs did not result from polysynaptic activity in Bsn−/− mice, we analysed the rise time (20–80%) for each individual MF-EPSC recorded at 0.1 and 1 Hz in Bsn+/+ and Bsn−/− mice at P7 and did not find any significant differences between the two genotypes (0.1 Hz: Bsn+/+: 0.61 ± 0.04 ms, n= 10 and Bsn−/−: 0.74 ± 0.07 ms, n= 11, P= 0.16; 1 Hz: Bsn+/+: 0.73 ± 0.04 ms, n= 10 and Bsn−/−: 0.83 ± 0.05 ms, n= 11, P= 0.09) (Supplemental Fig. 2).

Figure 3. Impaired synaptic transmission at immature hippocampal MF–CA3 synapses.

Figure 3

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A, superimposed recordings showing ‘all or none’ unitary MF-EPSCs recorded at P5, P7 and P9 in Bsn+/+ and Bsn−/− mice at 0.1 Hz. B, bar graph of average MF-EPSC amplitudes at 0.1 Hz at P5, P7 and P9 for Bsn+/+ and Bsn−/− mice (P5: Bsn+/+: n= 10 and Bsn−/−: n= 11; P7: Bsn+/+: n= 10 and Bsn−/−: n= 11; P9: Bsn+/+: n= 8 and Bsn−/−: n= 11). C, bar graph of failures at 0.1 Hz at P5, P7 and P9 for Bsn+/+ and Bsn−/− mice (P5: Bsn+/+: n= 10 and Bsn−/−: n= 11; P7: Bsn+/+: n= 10 and Bsn−/−: n= 11; P9: Bsn+/+: n= 8 and Bsn−/−: n= 11). D, cumulative distribution of average MF-EPSC amplitudes at 0.1 Hz (KS test for distribution: P5: P= 0.01; P7: P= 0.0001; P9: P= 0.13). *P < 0.05.

As ‘maxi’ MF-EPSCs and the increased mean MF-EPSC amplitude could result, at least partially, from an increased quantal size, we analysed the amplitude of asynchronous EPSCs (aEPSCs) recorded in the presence of Sr2+ in the external solution as previously described (Marchal & Mulle, 2004). At other synapses, it was demonstrated that aEPSCs arise from the same synapses that generated the evoked EPSC and correspond to the quantal release of neurotransmitter (Bekkers & Clements, 1999). We found a significant difference between mean aEPSC amplitude recorded in cells from Bsn+/+ and Bsn−/− mice (Bsn+/+: 31.9 ± 3.8 pA, n= 14 and Bsn−/−: 43.2 ± 3.6 pA, n= 17, P= 0.03) (Fig. 4B), consistent with changes occurring at a postsynaptic level (e.g. changes in the number of activated AMPA receptors). The large amplitude of MF-EPSCs in Bsn−/− mice at P7 may also depend on either the number of functional release sites or the release probability. We thus compared PPF in the two genotypes. With paired MF-EPSCs evoked at intervals ranging from 10 to 400 ms, the paired pulse ratio increased from P5 to P9 for short interstimulus intervals (ISIs) (Fig. 5A and B) in Bsn+/+ mice. This developmental increase appeared to be delayed in Bsn−/− mice at P7 with values of PPF significantly smaller at 10 and 40 ms ISIs (Bsn+/+, n= 10 and Bsn−/−, n= 10, 10 ms P= 0.01; 40 ms P= 0.005), while no significant differences were observed at P5 and P9 at any ISI. We also compared LFF in both genotypes, but we found no significant differences between mean values during this developmental time period (ratio 1 Hz/0.1 Hz: P5: Bsn+/+: 2.9 ± 0.8, n= 10 and Bsn−/−: 2.7 ± 0.3, n= 11, P= 0.40; P7: Bsn+/+: 5.5 ± 0.8, n= 10 and Bsn−/−: 4.4 ± 0.8, n= 11, P= 0.37; P9: Bsn+/+: 4.3 ± 0.6, n= 8 and Bsn−/−: 5.1 ± 1.1, n= 11, P= 0.96). However, plotting mean LFF or PPF at 40 ms as a function of the corresponding mean MF-EPSC amplitudes at 0.1 Hz at P7 (Fig. 5C and D) revealed that cells displaying large mean MF-EPSC amplitudes had also low facilitation ratios. This suggests that the increased MF-EPSC amplitudes in Bsn−/− mice are probably, at least partially, due to higher release probability. Overall, the changes in aEPSCs and facilitation ratios suggest that the lack of a functional Bassoon protein at a critical period of postnatal development impairs normal functional MF synapse maturation both at the pre- and postsynaptic levels, at least in a subpopulation of these synapses.

Figure 4. Increase in average MF-EPSC amplitudes at P7 cannot be attributed to a change in quantal size.

Figure 4

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A, representative recordings of MF-EPSCs in response to a train of 5 MF stimuli (50 Hz, every 10 s) in control conditions (top) or in an extracellular medium where Ca2+ is replaced by Sr2+ (2 mm) in Bsn+/+ (middle) and Bsn−/− mice (bottom). B, bar graph of average aEPSC amplitudes (Bsn+/+: n= 14 and Bsn−/−: n= 17). *P < 0.05.

Figure 5. Developmental increase in PPF is delayed in Bsn−/− mice.

Figure 5

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A, average of 20 traces illustrating PPF at 40 and 400 ms for Bsn+/+ and Bsn−/− mice at P7. B, summary graph of PPF ratio at 10, 40, 100 and 400 ms at P5, P7 and P9 (P5: Bsn+/+: n= 8 and Bsn−/−: n= 8; P7: Bsn+/+: n= 10 and Bsn−/−: n= 10; P9: Bsn+/+: n= 8 and Bsn−/−: n= 7). Isi: interstimulus interval. C, plots of PPF ratio as a function of average MF-EPSCs amplitude for Bsn+/+ (black circles) and Bsn−/− (grey circles) mice. D, plots of LFF ratio from 0.1 to 1 Hz as a function of average MF-EPSC amplitude for Bsn+/+ (black circles) and Bsn−/− (grey circles) mice. *P < 0.05, **P < 0.01.

Impairment of MF–CA3 synaptic transmission at P14 in Bsn−/− mice

We explored the possibility that the Bsn mutation could alter MF synaptic transmission at a later stage of maturation, P14. Surprisingly, we observed a marked decrease in the mean amplitude of MF-EPSCs recorded at 0.1 Hz in Bsn−/− mice (Bsn+/+: 70.8 ± 11.1 pA, n= 9 and Bsn−/−: 44.6 ± 6.6 pA, n= 15, P= 0.04) (KS test for distribution, P= 0.01) (Fig. 6A). This decrease was accompanied by an increase in the proportion of failures, which could by itself explain the change in mean MF-EPSC amplitude (Bsn+/+: 20.8 ± 4.7%, n= 9 and Bsn−/−: 42.8 ± 5.0%, n= 15, P= 0.01) (Fig. 6B). Indeed, potencies of MF-EPSCs were not significantly different in both genotypes (Bsn+/+: 88.0 ± 11.2 pA, n= 9 and Bsn−/−: 76.4 ± 8.3 pA, n= 15, P= 0.44). However, the Bsn mutation did not affect PPF at any ISI ranging from 10 to 400 ms (Bsn+/+: n= 8 and Bsn−/−: n= 9, P > 0.05) (Fig. 6C), nor LFF (ratio 1 Hz/0.1 Hz: Bsn+/+: 5.3 ± 0.3, n= 8 and Bsn−/−: 5.6 ± 0.7, n= 14, P= 0.58) (Fig. 6D). Hence the decrease of MF-EPSCs and the increase in failure rate in P14 Bsn−/− cannot be easily explained by a change in the probability of release of MF synaptic sites. A change in failure rates may alternatively rely on a change in the number of release sites. The simplest explanation might therefore be that in individual MF boutons, a proportion of synaptic active zones in P14 mutant mice are functionally inactive, similarly to what has been observed in autapses of hippocampal micro-island cultures (Altrock et al. 2003). Finally, to further explore mechanisms underlying changes in the mean amplitude of MF-EPSCs, we evaluated the quantal size of MF-EPSCs by aEPSCs in the presence of Sr2+ in P14 mice, as described above. We did not find any significant difference in the mean amplitude of aEPSCs (Bsn+/+: 40.2 ± 3.3 pA, n= 10 and Bsn−/−: 41.6 ± 3.0 pA, n= 9, P= 0.71) (Supplemental Fig. 3) suggesting that, at this maturation stage, the quantal sizes of MF-EPSCs in Bsn−/− mice were normal. Altogether our electrophysiological data suggest that the Bsn mutation affects MF synaptic maturation in CA3 pyramidal cells (before P14) but reaches apparently almost normal function before adulthood.

Figure 6. Synaptic transmission is decreased in Bsn−/− mice at P14.

Figure 6

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A, average of 50 traces (left) illustrating LFF of MF-EPSCs when shifting stimulation from 0.1 to 1 Hz for Bsn+/+ and Bsn−/− mice. Cumulative distribution (right) of average MF-EPSCs at 0.1 Hz (Bsn+/+: n= 9 and Bsn−/−: n= 15). B, bar graph of failure rate at 0.1 Hz (Bsn+/+: n= 9 and Bsn−/−: n= 15). C, average of 20 traces (left) illustrating PPF at 40 and 400 ms for Bsn+/+ and Bsn−/− mice. Summary graph (right) of PPF ratio at 10, 40, 100 and 400 ms (Bsn+/+: n= 8 and Bsn−/−: n= 9). Isi: interstimulus interval. *P < 0.05. D, bar graph of mean LFF ratio from 0.1 to 1 Hz (Bsn+/+: n= 8 and Bsn−/−: n= 13).

The time course of maturation of MF-EPSCs was characterized in conditions of 4 mm Ca2+/4 mm Mg2+ at room temperature in order to minimize overall neuronal excitability. However, these conditions are known to reduce presynaptic function. We have reexamined the properties of MF synaptic transmission at the main time points of postnatal development, at near physiological temperature (33°C) and in conditions of 2 mm Ca2+/1 mm Mg2+. For these experiments we have monitored NMDAR-dependent MF-EPSCs at +30 mV in the presence of 10 μm NBQX (Supplemental Fig. 4). Essentially, we repeated our observations in conditions of 4 mm Ca2+/4 mm Mg2+ at room temperature. In Bsn−/− mice we observed: at P7, an increase of the mean amplitude of NMDAR-dependent MF-EPSCs, a significant decrease of failure rate and of PPF ratio; at P14, a significant decrease of the mean amplitude of NMDAR-dependent MF-EPSCs, a significant increase of failure rate and no change of PPF ratio; at P21, no significant changes in these parameters (Supplemental Fig. 4).

Morphological correlates of MF synaptic transmission impairment in Bsn−/− mice

To compare the developmental course of assembly of presynaptic cytomatrix in MF synapses we performed immunostaining of brain sections from Bsn−/− and their wild-type littermates at P7, 14 or 21 (Fig. 7A). Anti-synaptophysin antibodies were used to identify the presynapses of MF and anti-Munc13-1 to label active zones. The active zones of MF–CA3 synapses were counted as detectable discrete spots of Munc13-1 staining. The quantitative analysis revealed a slightly but significantly increased number of Munc13-1 puncta per MF–CA3 synapse in Bsn−/− animals at P7 compared to Bsn+/+ (Bsn+/+: 4.7 ± 0.1 sites, n= 59 and Bsn−/−: 5.2 ± 0.1 sites, n= 58, P= 0.01) (Fig. 7B). On the contrary, no significant differences could be found in MF–CA3 synapses at later time points (P14: Bsn+/+: 16.0 ± 0.4 sites, n= 60 and Bsn−/−: 16.6 ± 0.4 sites, n= 60, P= 0.35; P21: Bsn+/+: 16.8 ± 0.4 sites, n= 61 and Bsn−/−: 17.5 ± 0.4 sites, n= 74, P= 0.28). This suggests that at P7 but not at P14 or P21, there is more presynaptic active zone containing Munc13-1, at least large enough to be observed optically. The apparent discrepancy between immunofluorescent and electrophysiological data at P14 might simply indicate that the presence of a presynaptic active zone does not necessarily mean functional release site as has been suggested by Altrock et al. (2003).

Figure 7. Increased number of active zones at P7 in Bsn−/− mice.

Figure 7

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A, brain sections of Bsn+/+ and Bsn−/− animals killed at days of P7, P14 and P21 days were stained with antibody against the active zone protein Munc13-1 (first column), the presynaptic marker protein synaptophysin (third column) and the postsynaptic marker ProSAP2/Shank3 (fifth column). Staining of single MF terminals is shown on maximal projections of confocal stacks. Second and fourth columns show overlay of images on their right and left. Scale bar 1 μm. B, bar graph showing the quantification of the numbers of Munc13-1 puncta in MF terminals. *P < 0.05.

Discussion

In this report we show that the course of functional maturation of hippocampal MF–CA3 synapses during the first 2 weeks of postnatal development is markedly impaired in Bsn−/− mice. Interestingly three stages can be distinguished: a gain of function at the end of the first week, a loss of function at the end of the second week and an apparent normalization with some impairment of PTP, but normal presynaptic LTP at the end of the third week (Fig. 7). Our data thus point to a complex function of Bassoon in synaptic maturation that might be due to defects in the structural organization of the presynaptic active zone during key steps of postnatal development.

The maturation of MF–CA3 synapses is likely to extend from the very first postnatal days (P0–P3), when the first synaptic contacts are made, up to delayed changes in morphological and probably physiological properties well into adulthood (Amaral & Dent, 1981; Marchal & Mulle, 2004; Gogolla et al. 2009). During the first days of postnatal development, MF–CA3 synaptic currents can be recorded, although they display very low probability of release and high fatigue and thus appear as presynaptically ‘silent’ (Gasparini et al. 2000). Before P6, MF-EPSCs are of low amplitude, are devoid of a kainate receptor component, and display a moderate extent of presynaptic plasticity (Marchal & Mulle, 2004). At this stage it has also been reported that GABA acts as a neurotransmitter in addition to glutamatergic synaptic signalling (Safiulina, 2006). The beginning of the second week (P6–P9) has been described as a critical period of synaptic development at MF–CA3 synapses because it is characterized by a steep and robust increase in the average amplitude of MF-EPSCs, the appearance of both postsynaptic KARs and prominent forms of short-term plasticity (Marchal & Mulle, 2004). The data we obtained with Bsn+/+ mice are in quite good agreement with this previous report despite differences in mouse strains and experimental conditions. In addition, we report that during a critical period of maturation, between P5 and P9, the paired-pulse ratio increases earlier than basal MF-EPSC amplitudes and success rate at 0.1 Hz (Fig. 8). This suggests that, while the number of release sites increases (Amaral & Dent, 1981), the probability of release at each site decreases. After P9, MF-EPSC amplitude increases, as a possible consequence of an increase in the number of release sites. Between P14 and P21, the properties of basal synaptic transmission seem to stabilize. However, LFF keeps increasing during this period suggesting a delayed maturation of more complex presynaptic properties (Fig. 8). It should be noted that the presynaptic form of LTP specific to this synapse also emerges at this period (Battistin & Cherubini, 1994).

Figure 8. MF-EPSC amplitudes at 0.1 Hz, PPF ratio at 40 ms, success rate at 0.1 Hz and LFF ratio from 0.1 to 1 Hz.

Figure 8

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Summary graphs of average MF-EPSC amplitudes at 0.1 Hz (A), PPF ratio at 40 ms (B), success rate at 0.1 Hz (C) and LFF ratio from 0.1 to 1 Hz (D) in Bsn+/+ (black circles) and Bsn−/− (grey circles) at P5, P7, P9, P14 and P21.

As shown previously (Altrock et al. 2003), the protein Bassoon is not essential for synapse formation and function. However, the maturation of synaptic properties is clearly impaired in the absence of Bassoon, at least at the hippocampal mossy fibre synapse. In Bsn−/− mice, basal MF-EPSC amplitude and success rate increase before paired pulse ratio during the critical period of maturation (P5–P9). This suggests that the number of release site increases before the release probability decreases contrary to wild-type mice (Fig. 8). At P7, we recorded MF-EPSCs of unusual large amplitude (‘maxi’ MF-EPSCs). The slight increase in quantal content revealed by the mean amplitude of Sr2+-evoked asynchronous MF-EPSCs is unlikely to be sufficient to account for the presence of ‘maxi’ MF-EPSCs. In addition, we cannot exclude that these asynchronous MF-EPSCs arise from a different pool of vesicles than the synchronously evoked MF-EPSCs in control conditions (Neher & Sakaba, 2008). The higher release probability and the changes in quantal content are in favour of a deficit at both pre- and postsynaptic levels. A critical structural determinant for synaptic transmission at a multisite synapse, like the MF–CA3 synapse, is the spacing between individual active zones as well as the organization of the pool of vesicles, which has not yet been precisely examined at early stages of postnatal development. However, this organization was reported to differ in young (P28) and adult rats (Rollenhagen et al. 2007), with synaptic vesicles evenly distributed in the MF terminal at P28, and clustered in the adult stage. It could be speculated that unequal distribution of CAZ components to active zones due to the lack of Bassoon might impair the precise organization of vesicles in the presynaptic terminal. Here we report a slight increase in the number of Munc13-1 puncta at P7. This could reflect an increased number of release sites but also an impaired organization of the CAZ. One possibility might be that Bassoon is important for the fine positioning of release ready vesicles and Ca2+ channels. Accordingly, it has been reported that release probability depends on the distance between fusion competent vesicles and Ca2+ channels (Wadel et al. 2007; Wang et al. 2008). Furthermore, as pre- and postsynaptic compartments mature in parallel, presynaptic changes could be linked to an increase in the size of the PSD and of the number of postsynaptic AMPA receptors, as suggested by the observed increase in quantal size in Bsn−/− mice.

At P9, we observed an apparent normalization of synaptic parameters in Bsn−/− mice, which appeared to be transitory. Indeed, at P14, we observed a decrease of MF-EPSC amplitude that is reminiscent of what was observed in CA1 (Altrock et al. 2003). In this earlier work the reason why Bsn mutation only affected a fraction of synapses impinging onto a given neuron was unclear, given the fact that Bassoon is present in ≥90% of hippocampal synapses together with Piccolo. Here we show that a single MF–CA3 synapse with multiple release sites might contain a fraction of non-functional sites. This is suggested by the fact that the decrease in average MF-EPSC amplitude correlates directly with the decrease in success rate without any change in aEPSCs nor short-term facilitation (Fig. 8). These data seem to confirm the earlier interpretation made for CA1 proposing a microheterogeneity of hippocampal synapses (Rosenmund et al. 2002; Altrock et al. 2003). This notion can now be extended to microheterogeneity even within a single multisite presynaptic terminal. A recent study in the calyx of Held showed that within a single presynaptic terminal, only 60% of Bassoon and Piccolo clusters overlapped, while the remaining clusters contained either Bassoon or Piccolo (Dondzillo et al. 2010). A molecular explanation for this observation might be provided by recent data showing that the disturbance of the interaction between Bassoon and dynein light chain impairs the reliability of Bassoon and Piccolo delivery to synapses, as shown by a much higher variance in the content of these proteins in individual synapses/active zones (Fejtova et al. 2009).

Finally, at more mature synapses (P21), we did not observed any significant differences in basal synaptic transmission in Bsn−/− mice. We cannot exclude that some differences persist but did not reach statistical significance because of the overall variability in between MF-synapses. The significantly decreased PTP we observed in Bsn−/− mice might argue in favour of persistent underlying impairments. PTP is a form of presynaptic short-term potentiation that neurons exhibit after high frequency tetanus. PTP is likely to be due to a tetanus-induced elevation in presynaptic Ca2+ which leads to a short-lived increase in release probability (Griffith, 1990; Regehr et al. 1994). At MF–CA3 synapses, the mechanisms of PTP have not been fully characterized. However, changes in PTP could result from alterations in presynaptic Ca2+ entry or in the size of the readily releasable pool (RRP) of vesicles (Zucker & Regehr, 2002). This last hypothesis is supported by the apparent decrease in the RRP at autaptic synapses of cultured hippocampal neurons (Altrock et al. 2003). At P21, Bsn−/− mice could have less fusion-competent synaptic vesicles in MF synapses without significantly affecting synaptic transmission, release probability and presynaptic short-term plasticity except PTP.

Overall, MF–CA3 synaptic transmission shows opposite abnormalities at two distinct phases of postnatal development in Bsn−/− mice. It cannot be predicted whether these early abnormalities in synaptic transmission bear important functional consequences in terms of higher brain function. Whatever, this study highlights the fact that in some instances, the mutation of a gene might be considered as without any effect on synaptic function when analysed at a single time point, whereas the proper course of development is affected. Compensatory mechanisms appear to alleviate the lack of a functional Bassoon protein at MF–CA3 synapses. However, it should be reminded that Bsn−/− mice display an epileptic phenotype, for which there is no direct synaptic explanation (Altrock et al. 2003; Ghiglieri et al. 2009). One can speculate that this epileptic phenotype might lie in a subtle imbalance in hippocampal network activity, which could find a cause in an abnormal synaptic maturation process. A difficult question that lies beyond the scope of this study relates to the question of the nature of the compensatory mechanisms taking place during development in Bsn−/− mice. Hence the postnatal development of the MF synapse is a complex but tightly controlled process, which depends on the organizing properties of presynaptic scaffold proteins, such as Bassoon.

Acknowledgments

This work was supported by the Centre National de la Recherche Scientifique (CNRS), the Ministere de la Recherche of France, the Conseil Regional of Aquitaine, the European Commission (SYNSCAFF program, contract number LSH-2003-511995), the Fondation pour la Recherche Medicale and the Deutsche Forschungsgemeinschaft (SFB779/B9). We thank Alice Vimeney, Audrey Lacquemant, Noëlle Grosjean and Bettina Kracht for technical assistance and mouse breeding.

Glossary

Abbreviations

CAZ

cytoskeletal matrix associated with the active zone

LFF

low frequency facilitation

MF

mossy fibre

PPF

paired pulse facilitation

Author contributions

F.L., C.B. and C.M. designed the experiments. E.D.G. provided the Bsn mutant mice. F.L. and C.B. performed the experiments and analysed the data. F.L., C.B. and C.M. wrote the paper. A.F., D.B. and E.D.G. critically revised the manuscript. A.F., D.B., K.R. and P.P. provided preliminary experiments on Bsn−/− mice. All authors approved the final version of the paper for publication.

Author's present address

D. Balschun: Laboratory of Biological Psychology, University of Leuven, Tiensestraat 102, 3000 Leuven, Belgium.

Supplemental material

Supplementary figure 1

Supplementary figure 2

Supplementary figure 3

Supplementary figure 4

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