Hebbian spike-timing dependent plasticity at the cerebellar input stage

Spike-timing dependent plasticity (STDP) is a form of long-term synaptic plasticity exploiting the time relationship between postsynaptic action potentials (AP) and EPSPs [1-4]. Surprisingly enough, very little was known about STDP in the cerebellum [5-8], although it is thought to play a critical role for learning appropriate timing of actions. We speculated that low-frequency oscillations observed in the granular layer may provide a reference for repetitive EPSP/AP phase coupling. Here we show that EPSP-spike pairing at 6Hz ( 60 times) can optimally induce STDP at the mossy fiber - granule cell synapse (Fig. 1). When the AP followed the EPSP, EPSP/AP pairing with 0<t<25 ms induced long- term potentiation of EPSC amplitude (st-LTP: +47.4 ± 11.7%, n=11, p<0.05). When the AP preceded the EPSP, EPSP/AP pairing with 0<t<-25 ms induced long-term depression of EPSC amplitude (st-LTD: -37.7 ± 8.5%, n=13, p<0.005). In order to verify the STDP requirement for a phased-locked EPSP/AP pairing, in a series of recordings the time between the EPSP onset and the AP peak was varied randomly (Fig. 2). After random EPSP/AP pairing, EPSC amplitudes were not significantly changed (-1.4 ± 1.9%, n=5; p=0.8), showing that STDP induction was critically dependent on the maintenance of a precise EPSP/AP phase relationship. Since EPSPs led APs in st-LTP while APs led EPSPs in st-LTD, STDP was Hebbian in nature. STDP occurred at 10 Hz but vanished below 1 Hz. Figure 3 shows the time course of EPSC changes with 10 Hz and 1 Hz pairing. With 10 Hz pairing, STDP was still present showing st-LTP at positive EPSP/AP pairing (t=+25 ms, 23.9 ± 5.4%, n=4; p<0.05) and st-LTD at negative EPSP/AP pairing (t=-25 ms, -18.0 ± 3.1%, n=5; p<0.05). Conversely, with 1 Hz pairing, STDP disappeared leaving only LTD both at positive EPSP/AP pairing (t=+25 ms, -33.5 ± 12.1%, n=5; p<0.05) and at negative EPSP/AP pairing (t=-25 ms, -30.9 ± 8.0%, n=5; p<0.005). In a different series of recordings, in order to investigate whether STDP depended on postsynaptic Ca2+ concentration ([Ca2+]i) changes, the pipette intracellular solution was supplemented with the calcium buffer, 10 mM BAPTA. Figure 4 shows the time course of EPSC changes. The high concentration of BAPTA prevented both st-LTP (-5.3 ± 5.7%, n=4; p=0.4) and st-LTD (+3.7 ± 10.1%, n=4; p=0.6). In order to examine the induction mechanism underlying STDP (Fig. 5), we evaluated the involvement of NMDARs and MGluRs, which are primarily responsible for the postsynaptic [Ca2+]i changes required for both LTP and LTD at several glutamatergic synapses [9-11]. Both st-LTP and st-LTD required NMDA receptors, but st-LTP also required reinforcing signals mediated by mGluRs. When the NMDAR blockers D-APV (50 μM) and 7-Cl-Kyn acid (50 μM), were added to the extracellular solution, STDP protocols failed to induce either st-LTP (+4.7 ± 2.9%, n=5; p=0.2) or st-LTD (-10.6 ± 5.5%, n=6; p=0.2). During extracellular application of the mGluR antagonist AIDA (15 μM), protocols used for st-LTD induction still caused a significant EPSC reduction (-60.4 ± 11.3%, n=4; p<0.05. However, during a similar AIDA application, protocols used for st-LTP induction proved inefficient and a significant st-LTD was observed in turn (-16.9 ± 5.4%, n=5; p<0.05). Importantly, st-LTP and st-LTD were significantly larger than LTP and LTD obtained by modulating the frequency and duration of mossy fiber bursts [9, 10], probably because STDP expression involved postsynaptic in addition to presynaptic mechanisms. The mechanism of STDP expression was first assessed by analyzing the paired-pulse ratio (PPR, interstimulus interval 20 ms) and the coefficient of variation of EPSCs (CV) [12, 13](Fig. 6). .During st-LTP, PPR showed a significantly r