Developmental decrease in synaptic facilitation at the mouse hippocampal mossy fibre synapse

Abstract

Transmission at the hippocampal mossy fibre (MF)-CA3 pyramidal cell synapse is characterized by prominent activity-dependent facilitation, which is thought to provide a wide dynamic range in hippocampal informational flow. At this synapse in mice the magnitude of paired-pulse facilitation and frequency-dependent facilitation markedly decreased with postnatal development from 3 weeks (3W) to 9 weeks (9W). Throughout this period the mean amplitude and variance of unitary EPSCs stayed constant. By altering extracellular Ca2+/Mg2+ concentrations the paired-pulse ratio could be changed to a similar extent as observed during development. However, this was accompanied by an over 30-fold change in EPSC amplitude, suggesting that the developmental change in facilitation ratio cannot simply be explained by a change in release probability. With paired-pulse stimulation the Ca2+ transients at MF terminals, monitored using mag-fura-5, showed a small facilitation, but its magnitude remained similar between 3W and 9W mice. Pharmacological tests using CNQX, adenosine, LY341495, H-7 or KN-62 suggested that neither presynaptic receptors (kainate, adenosine and metabotropic glutamate) nor protein kinases are responsible for the developmental change in facilitation. Nevertheless, loading the membrane-permeable form of BAPTA attenuated the paired-pulse facilitation in 3W mice to a much greater extent than in 9W mice, resulting in a marked reduction in age difference. These results suggest that the developmental decrease in the MF synaptic facilitation arises from a change associated with residual Ca2+, a decrease in residual Ca2+ itself or a change in Ca2+-binding sites involved in the facilitation. A developmental decline in facilitation ratio reduces the dynamic range of MF transmission, possibly contributing to the stabilization of hippocampal circuitry.

Figure 3. Unitary EPSCs evoked by minimal stimulation in developing mice

A, unitary EPSCs at 3W and 9W showing stochastic fluctuations in amplitude; 40 records are superimposed in each. B, the mean amplitude of EPSCs (left) and CV⁻² (right) compared between 3W and 9W mice. There was no significant difference between 3W and 9W (P > 0.3).

Figure 1. Developmental change in PPR

Aa, sample traces of fEPSP evoked by paired-pulse stimulations at 50 ms ISI in 3W and 9W mice. Traces are averaged fEPSPs from 40 records. Ab, summarized data indicating PPRs of fEPSPs at different ISIs in 3W and 9W mice. Data points and error bars indicate means and S.E.M. derived from 5 slices. The PPRs in 9W mice were significantly smaller than those in 3W mice (P < 0.01) at ISIs of 50 ms (n = 6-7) and 200 ms (n = 6-7). Ac, developmental decrease in PPR of fEPSPs (ISI, 50 ms). Ba, sample traces of EPSCs evoked at 50 ms ISI at 3W and 9W (averaged from 50 records each). Sample records in 3W and 9W mice are normalized at the amplitude of the first EPSCs and superimposed (bottom). Bb, summarized data for PPR of EPSCs at different ISIs in 3W and 9W mice. The PPR in 9W mice was significantly smaller than that in 3W mice (P < 0.01), at both the ISIs of 50 ms and 200 ms (n = 7-14).

Figure 2. Developmental change in FFR

A, the stimulus frequency to evoke fEPSPs was switched from 0.033 Hz (sample trace i) to 1.0 Hz as indicated by the horizontal bar (sample trace ii superimposed) in 4W (•)and 9W (○) mice. The ordinate indicates the fEPSP amplitude normalized to that evoked at the basal frequency. B, the magnitude of FFR at different stimulus frequencies (0.0083, 0.033, 0.1, 0.33 and 1.0 Hz) in 4W and 9W mice. Each data point was derived from 4-6 slices. The FFR magnitude between 4W and 9W at 1 Hz was significantly different (P < 0.01).

Figure 4. Dependence of the EPSC amplitude and PPR on the ratio of extracellular Ca²⁺ and Mg²⁺ concentrations

A, the relationships between the relative EPSC amplitude and the Ca²⁺/Mg²⁺ ratio (0.65-16) in 3W and 9W mice. The EPSC amplitudes were normalized to that at 2.5 mm [Ca²⁺]_o and 1.25 mm [Mg²]_o (Ca²⁺/Mg²⁺ ratio = 2.0). Ca²⁺/Mg²⁺ concentrations at each data point are 1.5/2.3, 2.5/1.25, 3/0.8, 4/0.5, 6/0.5 and 8/0.5, respectively. Each data point derives from 4-8 cells. The lowest two points (at Ca²⁺/Mg²⁺, 1.5/2.3) are 0.13 (3W) and 0.12 (9W), respectively. B, the relationship between PPR and Ca²⁺/Mg²⁺ ratio in 3W and 9W mice.

Figure 5. Ca²⁺ transients in MF terminals evoked by paired-pulse stimulations (ISI, 50 ms, 0.05 Hz) and monitored using mag-fura-5

A, field potentials (top traces) were simultaneously recorded with Ca²⁺ transients (bottom traces) in 3W (left) and 9W (right) mice. B, field potentials and Ca²⁺ transients after blocking excitatory synaptic transmission with CNQX (10 µM) and APV (25 µM) in 3W and 9W mice; recorded from the same slice as in A. C, summary of PPR of fEPSPs and Ca²⁺ transients. There was no significant difference in the amplitude ratio of Ca²⁺ transients (P2/P1) between 3W and 9W mice in the presence or absence of CNQX and APV, whereas the PPR of fEPSPs in 3W mice (3.8 ± 0.1, n = 10) was significantly larger than that in 9W mice (3.0 ± 0.1, n = 8, P < 0.05) in these experiments. The magnitude of fibre volley did not change in response to paired-pulse stimulation at both ages.

Figure 6. Lack of involvement of presynaptic receptors in the developmental decrease of synaptic facilitation

A, AMPA-EPSCs and NMDA-EPSCs evoked by the paired-pulse protocol in 3W and 9W mice (ISI, 50 ms). NMDA-EPSCs (superimposed with AMPA-EPSCs) were recorded after blocking AMPA/kainate receptors with CNQX at a holding potential of +40 mV. The PPR of NMDA-EPSCs in 3W mice was significantly larger than that in 9W mice. B, adenosine (ade, 100 µm) attenuated fEPSPs (fEPSPs at 3W and 9W before and after adenosine application are superimposed in top column), but did not affect PPR at both 3W and 9W (the two records are normalized to the first amplitude and superimposed). C, LY341495 (LY, 100 nm) had no effect on fEPSPs or PPR at both 3W and 9W. Field EPSPs evoked by the paired-pulse protocol, before and after LY341495 application, are superimposed both in 3W and 9W mice.

Figure 7. Differential effects of BAPTA-AM on PPR in 3W and 9W mice

A and B, time plots of the first fEPSP amplitude (•) and PPR(○) (both normalized to control) after bath application of BAPTA-AM (50 µM) in 3W mice (n = 10, A) and in 9W mice (n = 11, B). The upper panels show fEPSPs before and after BAPTA application (superimposed) at 3W (A) and 9W (B). C, aummary data of the mean PPR before and after BAPTA application in 3W and 9W mice. All values are calculated from the entire data set.