At the cerebellar parallel fibre to Purkinje cell synapse, repetitive stimulation of a beam of parallel fibers evokes a fast AMPA receptor mediated synaptic current (EPSCfast), and a slow synaptic current (EPSCslow) mediated by metabotropic glutamate (mGluR1) receptors (Tempia et al. 1998). Here we examine how the EPSCslow magnitude depends on the number of activated synapses, which was determined from the size of the EPSCfast (in GABAzine, 100 µM) evoked by different stimulus strengths (Marcaggi et al. 2003).
We recorded from Purkinje cells in sagittal cerebellar slices from 2-3 week old mice killed humanely by cervical dislocation. Cells were clamped at -70 mV and the internal medium was Cs-gluconate based with 0.1 mM CaCl2 buffered by 0.5 mM EGTA at pH 7.3. Stimulating in the molecular layer (10 pulses at 200 Hz), we observed a supralinear dependence of the amplitude of EPSCslow on the number of activated synapses, implying an interaction between different synapses. We previously showed that the AMPA receptor mediated EPSC was prolonged when more synapses were activated (again implying interaction between synapses), and that this prolongation was mimicked and amplified by inhibition of glial glutamate transporters, suggesting that it was due to glutamate spillover (Marcaggi et al. 2003). Knock-out of the main glial glutamate transporter GLAST approximately doubled the EPSCslow amplitude, and superimposed inhibition of GLT-1 by dihydrokainate further doubled the amplitude. In wild-type slices, superfused with solution containing the broad spectrum glutamate transporter blocker TBOA, when parallel fibres passing close to, but not contacting, the recorded cell were stimulated an EPSCslow could be detected, showing that spillover of glutamate could, in these conditions, elicit an EPSCslow. When stimulation was applied to the granular layer or white matter, rather than the molecular layer, so that activated synapses were more scattered in the Purkinje cell dendritic tree, the EPSCslow was approximately 1/3 of the size of the EPSCslow induced by molecular layer stimulation of the same number of synapses.
We conclude that the EPSCslow is strongly shaped by glial glutamate uptake and by the spatial arrangement of activated synapses on the Purkinje cell dendritic tree.
This work was supported by the European Union, the Wellcome Trust and a Wolfson-Royal Society award. We thank Prof K. Tanaka for the GLAST KO mice.