UBCs introduce a tunable delay in vestibolo-cerebellar circuits which depends upon Ih

The vestibulo-cerebellum regulates ocular movements and controls head and trunk posture, thus contributing to the correct positioning of the body in space. The cellular network involved is analogous to that of other cerebellar regions, except for the dense presence in the granular layer of unipolar brush cells (UBCs). UBCs are excitatory glutamatergic interneurons endowed with AMPA and NMDA glutamate receptors. It has been shown that each individual UBC receives a single input from one mossy fiber (MF), which forms a giant synapse with the UBC brush-like structure composed of several dendrioles. By using the patch-clamp technique in combination with the rat vestibulo-cerebellum slice preparation, we have investigated the voltage responses of UBCs to MFs excitation. In most UBCs, MFs stimulation evoked an all-or-none response consisting of a burst of action potentials elicited within a short delay (few ms) from the stimulus. Consistent with the literature, this response was blocked by AMPA/NMDA glutamate receptor antagonists APV and NBQX. However, in a significant number of UBCs a different type of postsynaptic response was found, characterized by a very slow membrane depolarization. As a consequence, the time required to reach the voltage threshold for the action potential discharge was considerably long (tens to hundreds of ms). By increasing stimulus intensity to the MFs, the delay of the postsynaptic response decreased, while its duration increased. The delayed response therefore is not an all-or-none phenomenon. Moreover, it was not blocked by APV and NBQX. A basic pharmacological screening revealed that neither metabotropic glutamate, nor GABA or acetylcholine receptors are involved. Additional experiments with ZD 7288 revealed that the slow depolarization depends on Ih, whose level of activation is increased by MFs stimulation. Thus, by setting the slope of the slow depolarization, Ih regulates the time required to fire. The present findings reveal a new modality of synaptic transmission in the vestibulo-cerebellum, characterized by a tunable delay and duration, which may play an important role for vestibular reflexes

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 reduction by 18.2 ± 6.5 % (p<0.05, n=5), while during st-LTD PPR showed a significantly increase by 26.3 ± 9.4% (p<0.01, n=4). During st-LTP, CV showed a significantly reduction by –27.9 ± 5.4% (p<0.005, n=9), while during st-LTD CV showed a significant increase by +37.1 ± 11.9% (p<0.05, n=9). The PPR and CV changes followed the time course of EPSC amplitude (see Fig. 1), suggesting that the a bidirectional modification in quantum content took part to mossy fiber-granule cell STDP. Additional evidence for a pre- or postsynaptic mechanism of expression for STDP can be obtained by analyzing miniature EPSCs (mEPSCs) before and after induction of STDP [14-16]. After EPSP/AP pairing with t=+25 ms, the EPSCs showed +22.0 ± 6.3% increase (p<0.05, n=5) attesting effective st-LTP induction. Interestingly, in the same recordings the mEPSCs showed significant increase in both amplitude (+16.9 ± 6.3 %, n=5; p<0.05; Fig.) and frequency (+18.1 ± 8.7%, n=5; p<0.05). After AP-EPSP pairing with t=-25 ms, the EPSCs showed -43.8 ± 4.0 % change (p<0.05, n=4) attesting effective st-LTD induction. In this case, the mEPSCs showed a decrease in both amplitude (-20.1 ± 8.8%, n=4; p<0.05) and frequency (-29.8 ± 11.1%, n=4; p<0.05; Fig. 7). Altogether, these results suggested that a modification in quantum size took part to mossy fiber - granule cell STDP. This work reveals the existence of STDP at the rat cerebellar mossy fiber – granule cell synapse. While st-LTP was induced when EPSP led AP, st-LTD was induced when AP led EPSP according to the Hebbian principle of coincidence detection between pre- and postsynaptic activity. To our knowledge, this is the first evidence that Hebbian STDP occurs in the cerebellum, since previous observations only reported either non-Hebbian STDP at the parallel fiber-Purkinje-like cell synapse [5] or anti-Hebbian STDP at the corresponding synapses of cerebellum-like structures [6-8]. Mossy fiber - granule cell STDP was detected using repeated cycles on the theta-frequency band and depended on precise positive or negative phase locking, such that it was abolished by random pairing. STDP was optimal in the theta-frequency band (6-10 Hz). This is actually a range of frequencies at which coherent oscillations have been detected in the granular layer [17, 18]. Thus, STDP at mossy fiber – granule cell synapse could provide a mean to coordinate learning at the cerebellar input stage with activity generated in extracerebellar structures like the neocortex and striatum, that show coherent oscillations with the cerebellum [19-21]