We have demonstrated that PPRs of both fEPSPs and EPSCs decrease with postnatal development from 3W to 9W at the MF-CA3 synapse in mice. This contrasts with the developmental increase in PPR of EPSCs during earlier postnatal periods at other CNS synapses (Bolshakov & Siegelbaum, 1995; Choi & Lovinger, 1997; Pouzat & Hestrin, 1997; Taschenberger & von Gersdorff, 2000; Iwasaki & Takahashi, 2001). Whereas the developmental changes in PPR have been commonly ascribed to the change in P, our results at the MF synapse cannot be explained by such a mechanism.
Mechanism underlying developmental changes in PPR
The PPR is widely used as an index for P (Manabe et al. 1993), because PPR is generally higher when P is lower and vice versa (Otsuka et al. 1962; Katz & Miledi, 1968). This can be explained by the greater depletion of releasable synaptic vesicles at higher P (Betz, 1970; Weis et al. 1999). The marked facilitation at the MF-CA3 synapse suggests a very low P at this synapse. At the MF-CA3 synapse, stimulation at 100 Hz causes a 20- to 40-fold facilitation of EPSCs (Langdon et al. 1995). Similarly, application of caffeine (5 mm) in combination with a 2.5-fold increase in the Ca2+/Mg2+ ratio causes a 30-fold increase in fEPSP amplitude (K. Kobayashi, unpublished observation). These data suggest that P is less than 0.03 at this synapse. Consistently, unitary EPSCs in the present study showed relatively large fluctuations in amplitude including occasional failures, despite the fact that a single MF bouton makes up to 37 synaptic contacts with a single CA3 pyramidal cell (Chicurel & Harris, 1992). This characteristic behaviour clearly distinguishes the MF-CA3 synapse from other CNS synapses. For example, P estimated for the calyx of Held synapse is 0.25-0.4 (in a 8- to 10-day-old rat; Meyer et al. 2001) and that for the CA3-CA1 synapse is 0.5 (in 2- to 3-week-old rats; Bolshakov & Siegelbaum, 1995). At the MF-CA3 synapse, because of the low P and a large pool size of releasable vesicles (Hallermann et al. 2003), the releasable vesicles cannot be easily depleted by repetitive stimulation. This explains the relatively weak dependence of PPR on P at this synapse upon changes in the Ca2+/Mg2+ ratio or after adenosine application. A large reduction in PPR, despite the unchanged unitary EPSC amplitude during development, suggests that a change in P cannot be the primary mechanism underlying the developmental decrease in MF synaptic facilitation.
Residual Ca2+ is thought to underlie synaptic facilitation (Katz & Miledi, 1968; Zucker & Regehr, 2002). The residual free Ca2+ summates with Ca2+ that enters at the second impulse and facilitates the second synaptic response (Katz & Miledi, 1968), being amplified by the non-linear relationship between Ca2+ and transmitter release (Dodge & Rahamimoff, 1967). In the present study, the Ca2+ chelator BAPTA-AM reduced the PPR more markedly in 3W than in 9W mice, for the entire data set as well as for the selected data after matching the effect of BAPTA-AM on the first EPSP. Assuming no age difference in the sensitivity of the first EPSP to BAPTA, the simplest explanation for these results would be that the residual Ca2+ concentration decreases with development because of the strengthened effect of endogenous Ca2+ buffers. If the occupancy of endogenous Ca2+ buffer by the first Ca2+ entry is greater at 3W than 9W, PPR can be larger at 3W because of Ca2+ buffer saturation (Blatow et al. 2003; Felmy et al. 2003), and intraterminal loading of exogenous Ca2+ buffer will reduce PPR at 3W more efficiently than at 9W. The MF terminal highly expresses the fast Ca2+ binding protein calbindin-D28K (Celio, 1990). A developmental increase in the expression of calbindin-D28K in MF might underlie the developmental decrease in synaptic facilitation.
Whereas a change in the strength of Ca2+ buffer would be expected to alter the time course of Ca2+ transients, our results showed no such difference between 3W and 9W mice. Even if the overall strength of Ca2+ buffer does not change much with development, an increase in the proportion of the fast buffer to slow buffers will affect the Ca2+ dynamics at the Ca2+ channel domain near the release site, thereby possibly affecting the PPR. The strengthening of fast Ca2+ buffers can reduce P as well as PPF. We did not see an age difference in the quantal content. However, this does not necessarily indicate that P was constant because developmental reduction in P can be compensated for by a concomitant increase in the number of releasable quanta or the number of release sites as at the calyx of Held (Iwasaki & Takahashi, 2001). If P decreases with development, however, it would contribute to an increase in the magnitude of synaptic facilitation, in an opposite direction to that observed in the present study.
Recently Blatow et al. (2003) have reported, in contrast to our results, that the magnitude of PPF is positively correlated with the extracellular Ca2+/Mg2+ concentration ratio at the MF synapse. Whereas the reason for this discrepancy is unclear, our experimental conditions are different from theirs in the following respects: (i) in our study mice were 3-9 weeks old, whereas mice in their study were 2 weeks old. The PPF caused by Ca2+ buffer saturation requires optimal Ca2+ buffer strength (Blatow et al. 2003), which might be attained only at a restricted developmental period. (ii) Their data are based entirely upon whole-cell recordings, whereas ours are based upon both whole-cell and field recordings. Changing Ca2+/Mg2+ concentration ratio can alter the excitability and the number of input fibres, and thereby potentially affect the EPSC amplitude. This effect will be greater on EPSCs than fEPSPs because of the fewer number of input fibres involved. (iii) In our study, synaptic responses were evoked by stimulation of the dentate gyrus, whereas in theirs the stratum lucidum was stimulated. Because of the higher number of synapses in the stratum lucidum, stimulation of this region may induce release of transmitters around the stimulation site, which can affect the excitability of MFs via presynaptic receptors. (iv) Inhibitory synaptic transmission was intact in our study, whereas it was blocked in theirs. This makes a difference in the excitability of the cells in the slice, thereby possibly leading to different results.
The residual Ca2+ may bind with a putative high-affinity site, thereby increasing the Ca2+ sensitivity of transmitter release (Atluri & Regehr, 1996; Bertram et al. 1996; Tang et al. 2000). If the amount or Ca2+ sensitivity of such a ‘facilitation site’ decreases with development, the PPR and its BAPTA sensitivity would be reduced without changes in Ca2+ buffers or residual Ca2+ concentrations. Although the molecular identity of the ‘facilitation site’ remains open, CaMKII is one of the candidates. In heterozygous mice with their α-CaMKII genetically ablated, PPR at the hippocampal CA1 synapse is reduced with no change in basal synaptic efficacy (Chapman et al. 1995). Also, at the immature MF-CA3 synapse in the guinea-pig hippocampus, the CaMKII inhibitor KN-62 reduces FFR (Salin et al. 1996). However, our present results disagree with this report because KN-62 had no effect on synaptic facilitation in mice.
Our results also indicate that PPF remains after application of BAPTA-AM. Similar BAPTA-resistant PPF reported at the crayfish neuromuscular junction has been attributed to the effect of residual bound Ca2+ (Winslow et al. 1994). Presynaptic Ca2+ transients in the MF terminals, when repeated at a 50 ms interval, are facilitated by 10 % (this study) or 20 % (Kamiya et al. 2002). Assuming a fourth power relationship between Ca2+ concentration and transmitter release (Dodge & Rahamimoff, 1967), this corresponds to a synaptic facilitation of 46-107 %. Kamiya et al. (2002) have also shown that the facilitation of Ca2+ transients at the MF terminal is attenuated by blocking kainate receptors with CNQX, and they concluded that this facilitation was in part mediated by kainate autoreceptors. In our present study, in contrast, CNQX had no effect on the magnitude of facilitation of the Ca2+ transients, suggesting that kainate receptors are not involved in the facilitation of the Ca2+ transient. This discrepancy might arise from the difference in the experimental conditions (e.g. mag-fura-5 vs. rhod-2 for Ca2+ indicators) or the age of mice used (> 21 days old in our study vs. 14-20 days old in their study). The facilitation of the Ca2+ transient observed in the present study may be mediated by the activity-dependent broadening of presynaptic action potentials (Geiger & Jonas, 2000), or by the Ca2+ current facilitation, which may be dependent on neuronal calcium sensor-1 (NCS-1, Tsujimoto et al. 2002) or calmodulin (DeMaria et al. 2001). Whatever the mechanism, given no age difference in the facilitation of Ca2+ transients at the MF terminal, it does not account for the developmental change in synaptic facilitation.
There are other mechanisms that can potentially affect PPR. These include desensitization of postsynaptic receptors (Trussell et al. 1993; Rozov et al. 2001), and feedback modulation via presynaptic autoreceptors (von Gersdorff et al. 1997; Schmitz et al. 2001). At the MF-CA3 synapse, PPR of NMDA-EPSCs underwent a developmental decrease similar to that of AMPA-EPSCs. This parallel change in AMPA- and NMDA-EPSCs suggests that postsynaptic factors such as desensitisation of AMPA receptors may not contribute significantly to the developmental change in PPR. Our results also suggest that presynaptic receptors such as kainate receptors, adenosine receptors or mGluRs do not play a crucial role in the developmental change in PPR.
Physiological role of synaptic facilitation and its developmental change
The MF terminal is one of the largest nerve terminals in the CNS. Despite its large size, unitary EPSPs at a low firing frequency would not exceed the action potential threshold of the postsynaptic CA3 pyramidal cell. However, this synapse possesses a wide dynamic range of synaptic efficacy because of its prominent facilitation. Occasional bursts of action potentials in a single dentate granule cell give rise to suprathreshold EPSPs in CA3 pyramidal cells (Henze et al. 2002), thereby enabling the informational flow from granule cells to CA1 pyramidal cells. Furthermore, burst activation of a single MF input can provide postsynaptic activity sufficient for the induction of long-term potentiation at converging associational/ commissural inputs (Kobayashi & Poo, 2002). Our present results indicate a developmental decrease in this dynamic range of transmission at the MF-CA3 synapse. This indicates a decrease in the ability of MF to affect the CA3 circuitry with development, which may potentially contribute to a decrease in the capacity of memory formation and/or recall. However, because dentate granule cells are continuously generated during postnatal development and their survival is regulated by environment and behaviour (Gould et al. 1999), newly formed synapses may compensate for the developmental decrease in the dynamic range of facilitation of MF synapses.
Epilepsy is more common in infants than adults (Wong & Yamada, 2001) and temporal lobe epilepsy is related to changes in the excitability of CA3 pyramidal cells associated with MF sproutings (Anderson et al. 1999). Experimentally induced MF sproutings lead to epileptic seizures in animals (Anderson et al. 1999). In these respects, a developmental decrease in the gain of facilitation at the MF-CA3 synapse may protect CA3 pyramidal cells from over-excitation, thereby contributing to the stability of the hippocampal neuronal network.