Climbing fiber-triggered metabotropic slow potentials enhance dendritic calcium transients and simple spike firing in cerebellar Purkinje cells

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Abstract

Cerebellar Purkinje cells (PCs) receive synaptic input from numerous parallel fibers (PFs) and from a single climbing fiber (CF). At both types of synapses, fast synaptic transmission is mediated by AMPA receptors, while at PF synapses burst activity can additionally recruit metabotropic glutamate receptors (mGluRs) that mediate a slow depolarizing potential. Here, we show that mGluR-activated slow potentials can be evoked throughout the dendrite by CF-evoked complex spike firing in the presence of an mGluR agonist. The CF-triggered mGluR potential was not only blocked by an mGluR antagonist but also when the CF-induced Ca2+ transient was blocked by an AMPA receptor antagonist, suggesting the possibility that the slow potential can be activated by the simultaneous occurrence of agonist binding at mGluRs and a CF-evoked Ca2+ transient. In turn, these CF-triggered slow mGluR potentials enhance the complex spike-associated calcium signals throughout the dendrite. Moreover, they provide a mechanism by which CFs can modulate the simple spike frequency of PCs.

Introduction

PCs have a high intrinsic Na+ spike firing rate in the range of 30–80 Hz but can be synaptically driven to frequencies exceeding 200 Hz. Activity at PF synapses is classically associated with this so-called ‘simple spike’ firing. In contrast, the hallmark of CF activity is a massive, all-or-none ‘complex spike’ which occurs at about 1 Hz in alert mice (Simpson et al., 1996, Goossens et al., 2001), is followed by a pause (Simpson et al., 1996) and evokes widespread dendritic Ca2+ transients (Ross and Werman, 1987, Knöpfel et al., 1991, Miyakawa et al., 1992) that provide a key signal for synaptic plasticity (Konnerth et al., 1992, Hansel et al., 2001, Coesmans et al., 2004). Bursts of PF activity can induce slow depolarizing potentials or corresponding inward currents that result from the activation of metabotropic glutamate receptors (mGluR1), which can last several seconds and are associated with a Ca2+ transient in the PC dendrite (Batchelor and Garthwaite, 1993, Batchelor et al., 1997, Batchelor and Garthwaite, 1997, Tempia et al., 1998, Tempia et al., 2001). Under comparable stimulation conditions, slow mGluR1 potentials cannot be observed at CF synapses (Tempia et al., 1998, Reichelt and Knöpfel, 2002, Dzubay and Otis, 2002), even though morphological studies indicate the presence of mGluR1s close to CF synapses (Nusser et al., 1994, Petralia et al., 1998, Mateos et al., 2000, Lopez-Bendito et al., 2001). The only condition reported to produce mGluR1 potentials following CF stimulation is when glutamate uptake is blocked (Dzubay and Otis, 2002; but see Reichelt and Knöpfel, 2002). This observation can be explained by spillover of glutamate from the cleft of CF synapses and activation of the peri- and extrasynaptically located mGluRs (Dzubay and Otis, 2002). Alternatively, an increase in ambient glutamate concentration in the extracellular space could shift the mGluRs into an agonist bound state that, along with an intracellular Ca2+ transient, activates the slow mGluR potential. Coincident agonist binding and Ca2+ signaling fit the activation conditions for the mGluR1-mediated inward current in PCs (Staub et al., 1992, Batchelor and Garthwaite, 1997) and the responses of the TRPC family of nonspecific cation channels that have been suggested to underlie mGluR1-activated slow inward currents at PF synapses (Kim et al., 2003). In this scenario, CF activity could trigger a slow mGluR1 potential wherever coincident agonist binding and a Ca2+ transient occur. For CF-evoked slow potentials, the trigger factor could be the large complex spike-associated Ca2+ transient. The present experiments were designed to investigate this possibility.

Section snippets

CF activity triggers a slow mGluR1 potential in the continuous presence of an mGluR1 agonist

In control ACSF, activation of CFs by a single stimulus induced the well-characterized complex spike and a dendritic calcium transient in Purkinje cells patch clamped in current-clamp mode (Ross and Werman, 1987, Knöpfel et al., 1990, Knöpfel et al., 1991, Miyakawa et al., 1992, Simpson et al., 1996). To test the effect of tonic agonist binding at mGluR1 on CF-induced responses, we bath applied the mGluR1 agonist 3,5-dihydroxyphenylglycine (DHPG). DHPG applied at 50 μM slightly increased

Discussion

Our data show that CF stimulation can evoke an mGluR1-activated slow depolarizing potential when the dendritic Ca2+ transient associated with the complex spike coincides with agonist binding at mGluR1's.

In a recent study, Dzubay and Otis (2002) reported that a slow mGluR1 EPSC can be activated in response to CF stimulation when glutamate transporters are blocked. These authors discussed whether the mGluR1 responsible for this current is perisynaptically located at CF synapses (Nusser et al.,

Slice preparation and electrophysiology

Sagittal slices of the cerebellar vermis (200-μm thick) were prepared from P18–23 ICR mice using a Vibratome (Leica) and standard ACSF containing (in mM): 124 NaCl, 5 KCl, 1.25 Na2HPO4, 2 MgSO4, 2 CaCl2, 26 NaHCO3, and 10 d-glucose bubbled with 95% O2 and 5% CO2. Following a recovery period of at least 1 h, the slices were placed in a submerged chamber and were perfused at a flow rate of 2–3 ml/min with ACSF either at 22–24 °C or at 30–32 °C. The signals described in this study were observed at

Acknowledgments

This work was supported by an intramural grant from RIKEN (T.K.), NWO-ALW, NWO-VIDI (C.H.). We thank J. Yokoyama for the excellent editing and H. Mutsukawa for the technical help during an initial phase of this study.

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