The Journal of Neuroscience, June 14, 2006, ():
Glial Glutamate Transporters Maintain One-to-One Relationship at the Climbing Fiber–Purkinje Cell Synapse by Preventing Glutamate Spillover
J. Neurosci. Takayasu et al.
26: 6563
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure S1. Effects of PMB-TBOA on synaptically activated currents (STCs) in Bergmann glia and PCs.
STCs were recorded at −80 mV in the presence of 20 μM NBQX using K-gluconate-based internal solution in Bergmann glia or PCs in cerebellar slices of young rats (P13−P17). A, Example traces of CF-evoked currents in Bergmann glia composed of AMPAR-mediated current and STC. STCs in Bergmann glia were seen as slow currents resistant to 20 μM NBQX, and completely blocked by 200 μM DL-TBOA. B, Example traces of CF-evoked STCs in Bergmann glia in the absence and the presence of 10, 50, 100, 200 nM PMB-TBOA and 200 μM DL-TBOA. C, Example traces of CF-evoked STCs in PCs in the absence and the presence of 200, 500 nM, 1 μM PMB-TBOA, and 200 μM DL-TBOA. The residual currents in the presence of 200 μM DL-TBOA were probably mediated by activation of kainate receptors (Huang et al. 2004). D, Dose-response relationships of the effect of PMB-TBOA on STCs in Bergmann glia (black circles) and PCs (white circles). The amplitude of the STC was defined as the current sensitive to 200 μM DL-TBOA, which was obtained by subtracting the amplitude of the residual current in the presence of 200 μM DL-TBOA from the peak amplitude of each trace in the presence of various concentrations of PMB-TBOA. Each point represents the mean ± SEM of the value obtained from 3-11 cells. Plots were fitted to the equation: I / Icontrol = 1 / {1 + (PMB-TBOA/IC50)n }, where I is the amplitude of STCs in the presence of various concentrations of PMB-TBOA, Icontrol is that in the absence of PMB-TBOA, IC50 is the concentration that inhibits the peak amplitude of STC by half, and n is the Hill coefficient. The best fit for the effect of PMB-TBOA on the STC in Bergmann glia yields an IC50 of 52.0 nM and a Hill coefficient of 1.32. The corresponding values for the STC in PCs were 651 nM and 1.14. E, Example traces of CF-evoked small currents in the late phase in the presence of 200 nM PMB-TBOA in PCs. Their amplitudes were increased by the additional application of 200 μM DL-TBOA. F, MCPG, an antagonist of group I mGluRs, at 1 mM completely blocked the CF-evoked small current in the presence of 200 nM PMB-TBOA in the late phase, whereas it had no effect on the STC that preceded the small current. All current traces in (B) to (F) were recorded at −80 mV in the presence of 20 μM NBQX.
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Supplementary Figure S2. Effects of CTZ and γ-DGG on STCs in PCs.
The application of 100 μM CTZ (left) and 1 mM γ-DGG (right) caused no change in both the amplitude and the kinetics of STCs in PCs in matured WT mice (P37).
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Supplementary Figure S3. Schematic diagram illustrating glutamate spillover between adjacent PCs.
A, Normal condition, in which the functions of glial glutamate transporters are intact. No response occurs in the PC tested when the CF innervating the adjacent PC is stimulated because the glial glutamate transporter restricts the diffusion of released glutamate to neighboring PCs (left). The CF-EPSC is elicited only when the CF innervating the neuron tested is stimulated (right). B, Conditions in which removal of glutamate by the glial glutamate transporter is inhibited by either the deletion of the GLAST gene or the application of PMB-TBOA. Slow-rising, small EPSCs occur in the PC tested when a CF innervating an adjacent PC is stimulated. Glutamate released from the CF innervating the adjacent PC spills over to the neuron tested because of the impairment of glial transporter functions.
