Article Figures & Data
Figures
- Figure 1.
CF-EPSCs and PF-EPSCs in wild-type, GLAST-deficient, and EAAT4-deficient mice. A, B, Representative traces of CF-EPSCs (A) and PF-EPSCs (B). The insets show EPSCs in EAAT4-deficient mice on a log scale. C, CF-EPSCs (top) and PF-EPSCs (bottom) in EAAT4-deficient mice evoked by a paired pulse. D, Degrees of paired-pulse depression of CF-EPSCs (filled circles) and facilitation of PF-EPSCs (open circles). The values for CF-EPSCs and PF-EPSCs were not significantly different among 11 and 10 cells in wild-type mice, 9 and 12 cells in GLAST-deficient mice, and 12 and 12 cells in EAAT4-deficient mice, respectively. Error bars represent SEM. E, CF-EPSCs in EAAT4-deficient mice in the absence and presence of 100 μm SYM 2206 or 20 μm NBQX (top) and PF-EPSCs in EAAT4-deficient mice in the absence and presence of 1 mm MCPG or 20 μm NBQX (bottom). Note that a small component mediated by kainate receptor remained in the CF-EPSC trace in the presence of SYM 2206 (Huang et al., 2004).
- Figure 2.
Dependence of the amplitude of tail current of the PF-EPSC on stimulus intensity in EAAT4-deficient mice and effects of antagonists of glutamate receptors. A, PF-EPSCs evoked by paired-pulse stimulations of various intensities in EAAT4-deficient (top and middle) and wild-type (bottom) mice. The middle traces show the initial part of the top traces on a fast time scale. B, Plots of tail current ratios of the EPSCs shown in A. The tail current ratios (b1/a1 and b2/a2) for the first and the second stimuli with a 50 ms interval were defined as shown in the inset. Stim., Stimulus. C, D, Effects of 1 mm γ-DGG (C) and 250 nm NBQX (D) on PF-EPSCs. The bottom traces show EPSCs scaled to their peaks to the second stimuli.
- Figure 3.
Age-dependent appearance of the tail current at CF synapses in EAAT4-deficient mice. A, Comparison of the incidence of the tail current in the three age groups and the mean of the tail current ratio of CF-EPSCs. The incidence represents the number of Purkinje cells with tail currents as percentage of the total cells tested. Filled circles, 0 of 24 at P12–P14, 7 of 22 at P18–P21, and 17 of 26 at P30–P37. The mean of the tail current ratio was obtained from cells with tail currents for the two age groups. Open circles, n = 7 at P18–P21 and n = 17 at P30–P37. Error bars represent SEM. B, Representative traces of CF-EPSCs in EAAT4-deficient mice in the three age groups.
- Figure 4.
Comparison of EPSCs among wild-type, GLAST-deficient, and EAAT4-deficient mice in the presence of 100 μm CTZ. A–C, EPSCs in the absence (black) and presence (red) of 100 μm CTZ in wild-type (A), GLAST-deficient (B), and EAAT4-deficient (C) mice. D, Superimposition of the scaled EPSCs in control solution in wild-type, GLAST-deficient, and EAAT4-deficient mice (gray) and the corresponding EPSC traces in the presence of 100 μm CTZ (black, red, and blue lines, respectively). The scaled traces in control solution are the averages of 13, 19, and 13 cells for CF-EPSCs and those of 11, 19, and 7 cells for PF-EPSCs, and the averages of the corresponding EPSC traces during the application of 100 μm CTZ are further superimposed on them. In the presence of CTZ, the traces of CF-EPSC and PF-EPSC in GLAST-deficient mice crossed those in EAAT4-deficient mice at 76 and 75 ms after the onset of the EPSCs, respectively. E, F, Schematic diagrams illustrating differential roles of glial and neuronal glutamate transporters in Purkinje cell synapses. Synaptically released glutamate taken up by GLAST and EAAT4 in early (E) and late (F) phases of the synaptic event. Under physiological conditions, glutamate that escapes from the uptake by GLAST in the active synapse (left) is almost completely removed by EAAT4 and does not spill over to the nonactive synapse (right).
Additional Files
Supplemental data
Files in this Data Supplement:
- supplemental material - Supplemental table.
- supplemental material - Supplementary Figure S1. Western blot of cerebellar tissues from wild-type, heterozygous and homozygous EAAT4-deficient mice using antibodies to EAAT4, GLAST, EAAC1 and GLT1. EAAT4 protein was not detected in homozygous EAAT4 mutant mice. The apparent equal intensity of the GLAST, EAAT4 and EAAC1 bands indicates that no overexpression of the other glutamate transporter subtypes has occurred in EAAT4-deficient mice. +/+ wild-type; +/� heterozygote EAAT4 mutant mouse; �/� homozygote EAAT4 mutant mouse.
- supplemental material - Supplementary Figure S2. Cerebellar anatomy in wild-type and EAAT4-deficient mice at P35. A, No differences in histology by hematoxylin staining as well as cytology of PCs and Bergmann glia by immunofluorescence for calbindin and GFAP between wild-type and EAAT4-deficient mice. Cerebellar lobules are indicated as 1-10. Dotted line indicates the pial surface. ML,molecular layer; PL, Purkinje cell layer; GL, granule cell layer. B, No significant difference in PF-PC synapse density per 100 ?m2 of the neuropil area (18.9 � 0.7, n = 3 for wild-type and 17.1 � 0.5, n = 3 for EAAT4-deficient mice). The number of synaptic profiles was counted on electron micrographs printed at the final magnification of �16,000.
- supplemental material - Supplementary Figure S3. Effects of pharmacological blockade of transporters on EPSCs. A, B, C, EPSCs in wild-type (A), GLAST-deficient (B) and EAAT4-deficient mice (C) in the absence (black) and presence (gray) of 200 ?M TBOA. D, E, Comparison of tail current ratios of CF-EPSCs (D) and PF-EPSCs (E) in the absence and presence of TBOA. Plots in the graph of CF-EPSCs and PF-EPSCs show the averages of 4 and 3 cells in wild-type mice, 5 and 4 cells in GLAST-deficient mice, and 3 and 3 cells in EAAT4-deficient mice.
- supplemental material - Supplementary Figure S4. Age-dependent changes in the amplitude of STCs. A, Comparison of the mean amplitude of the STC elicited by CF stimulus in wild-type mice in the three age groups. In this experiment, the amplitude of the STC was defined as that of the component of synaptic currents inhibited by 200 ?M TBOA in the presence of 100 ?M NBQX. Filled circles: 9.5 � 1.3 pA at P12?14 (n = 5); 11.0 � 1.7 pA at P18?21 (n = 5); 17.3 � 1.5 pA at P30?37 (n = 7). B, Representative traces of CF-evoked STCs in the absence and the presence of 200 ?M TBOA in wild-type mice in the three age groups.
- supplemental material - Supplementary Figure S5. Comparison of effects of ?-DGG on CF-EPSCs among wild-type, GLAST-deficient and EAAT4-deficient mice. A, Comparison of % inhibition of the peak amplitude of CF-EPSCs induced by 2 mM ?-DGG. B, Comparison of the weighted decay of CF-EPSCs in the presence of 2 mM ?-DGG in wild-type, GLAST- and EAAT4-deficient mice. C, Representative traces of CF-EPSCs in the absence and the presence of 2 mM ?-DGG.
