Article Figures & Data
Figures
- Figure 1.
Intracellular Alexa 594 altered the rectification index of spontaneous EPSCs mediated by AMPARs in cerebellar stellate cells. A, Left, Example traces of spontaneous EPSCs recorded at +40 mV (top) and −60 mV (bottom) when 100 µm Alexa 594 was included in the recording electrode. EPSC traces recorded immediately after obtaining the whole configuration (0–5 min) are shown in black, and those recorded at 15–20 min are shown in red. Right, Averaged spontaneous EPSCs. B, RI of sEPSCs recorded with Alexa Fluor 594 in the patch pipette at 0–5 and 15–20 min. Average, bar graphs; individual cells, open circles. Paired t test, p < 0.05 (n = 9 cells, 7 animals). C, Scatter plot of the percentage change in RI at 15–20 min (100 × (RI15–20 min − RI0–5 min)/RI0–5 min) vs the initial RI at 0–5 min of cells filled with Alexa Fluor 594. Stellate cells with a lower initial rectification index have a greater increase in the magnitude of RI. D–G, RI of sEPSC amplitudes recorded at −60 and +40 mV from cells with an initial RI < 0.5 (n = 7). D, RI of EPSCs increased at 15–20 min of recording. Paired t test, p < 0.004. E, The amplitude of sEPSCs at −60 mV did not change. F, sEPSC amplitude at +40 mV increased at 15–20 min. Paired t test: n = 7, p < 0.03. G, The decay time constant of sEPSCs at −60 mV (p = 0.171). H, Time course of average RI (n = 7). One-way repeated-measures ANOVA, p = 0.004; post hoc: Bonferroni t test: 0–5 vs 10–15 min, p < 0.05; 0–5 vs 15–20 min, p < 0.01; n = 7). I, J, Alexa Fluor 594 (100 µm) fluorescence intensity along stellate cell dendrites (5 dendrites, 4 cells, 4 animals) at each time point. I, Plot of the average fluorescence intensity along dendrites from the soma at 0–5 min (open circles) and 15–20 min (red circles). Arrows show the locations where Alexa Fluor 594 fluorescence intensity decreased to the basal level. J, Time course of the diffusion distance of Alexa Fluor 594 along dendrites. One-way repeated-measures ANOVA, p = 0.014; post hoc: Bonferroni t test: 0–5 vs 10–15 min, p < 0.05; 0–5 vs 15–20 min, p < 0.05; n = 5.
- Figure 2.
Alexa 488 increased the rectification index of sEPSCs in stellate cells. A–D, Spontaneous EPSCs were recorded using a pipette solution that did not contain any Alexa Fluor dye (control). A, Left, Example traces of sEPSCs recorded at +40 mV (top) and −60 mV (bottom) at 0–5 min (black trace) and after 15–20 min of recording (blue trace). Right, Average synaptic currents. B–D, Average and individual RIs of sEPSCs (B) and amplitude at −60 mV (C), and +40 mV (D) in control cells with an initial RI < 0.5 (n = 7 cells, 6 animals). Neither the RI nor the amplitude of EPSCs changed within 20 min of recording. E–H, Spontaneous EPSCs were recorded using a pipette solution that included 100 µm Alexa Fluor 488. E, Left, Example traces of sEPSCs recorded with Alexa 488 in the recording electrode (black traces, 0–5 min; green traces, 15–20 min). Right, Average EPSCs. F, RI increased after 15–20 min of recording (paired t test, n = 7 cells, 6 animals, p < 0.004). G, EPSC amplitude at −60 mV did not change with time. H, The amplitude of synaptic currents at +40 mV (paired t test, p = 0.025). Bar graphs represent the average, and open circles represent individual values.
- Figure 3.
Intracellular Alexa Fluor 594 did not alter the rectification index of EPSCs mediated by GluR2-containing AMPA receptors. Mice were exposed to fox urine for 5 min, a treatment that increased GluA2-containing AMPARs and therefore the initial rectification index of EPSCs. Cerebellar slices were prepared 3 h later, and sEPSCs were recorded with a pipette solution that included Alexa 594. A, Example trace of average synaptic currents (top, +40 mV; bottom, −60 mV; lines: black, 0–5 min; orange, 15–20 min). B–D, Average and individual sEPSC rectification index (B) and amplitude (C) recorded at −60 mV and +40 mV (D). Neither the RI nor the amplitude of EPSCs changed after 15–20 min of recording (5 cells, 5 animals). Bar graphs represent the average, and open circles represent individual values.
- Figure 4.
A low intracellular concentration of Alexa Fluor 594 did not alter the EPSC rectification index. A, Example trace of average synaptic currents when 10 µm Alexa Fluor 594 was included in the recording electrode (top, +40 mV; bottom, −60 mV; lines: black, 0–5 min; pink, 15–20 min). B–D, sEPSC rectification index (B) and amplitude (C) recorded at −60 mV and +40 mV (D). Neither the RI nor the amplitude of EPSCs changed at 15–20 min compared with initial values (5 cells, 4 animals). Bar graphs represent the average, and open circles represent individual values.
Tables
Figure Distribution Statistical test Statistics p Values N Figure 1 aB Normal Paired Student’s t test t(8) = −2.587 0.032 9 bD Normal Paired t test t(6) = −4.469 0.004 7 cE Normal Paired t test t(6) = −1.889 0.108 7 dF Normal Paired t test t(6) = −2.926 0.026 7 gG Normal Paired t test t(6) = −1.555 0.171 7 eH Normal One-way repeated-measures ANOVABonferroni post hoc test:0-5 vs 5–10 min0-5 vs 10–15 min0-5 vs 15–20 min F(2,23) = 6.920 t(6) = 1.59t(6) = 3.646t(6) = 3.693 0.004 0.8040.0160.008 7 fJ Normal One-way repeated-measures ANOVABonferroni post hoc test:5 vs 10 min5 vs 15 min5 vs 20 min F(3,19) = 5.375 t(4) = 1.413t(4) = 4.115t(4) = 4.453 0.014 0.9590.0430.025 5 Figure 2 hB Normal Paired t test t(6) = −0.181 0.862 7 iC Normal Paired t test t(6) = −0.651 0.539 7 jD Normal Paired t test t(6) = −0.099 0.924 7 kF Normal Paired t test t(6) = −4.545 0.004 7 lG Non-normal Wilcoxon signed rank test z = 1.183 0.297 7 mH Normal Paired t test t(6) = −2.957 0.025 7 Figure 3 nB Normal Paired t test t(4) = 1.551 0.196 5 oC Normal Paired t test t(4) = −0.296 0.782 5 pD Normal Paired t test t(4) = 1.511 0.205 5 Figure 4 qB Normal Paired t test t(4) = −0.051 0.962 5 rC Normal Paired t test t(4) = 0.741 0.500 5 sD Normal Paired t test t(4) = −1.801 0.146 5 Group Distri-
butionAmplitude
at −60 mV (pA)t Test
(vs control)p Value Amplitude
at +40 mV (pA)t Test
(vs control)p Value RI N Control n.a. −50.4 ± 5.1 n.a. n.a. 11.7 ± 0.7 n.a. n.a. 0.37 ± 0.04 7 tA594 (100 µm) Normal −36.8 ± 3.6 t(12) = −2.192 0.049 9.2 ± 1.1 t(12) = 1.928 0.078 0.38 ± 0.03 7 uA488 (100 µm) Normal −39.4 ± 3.1 t(12) = −1.871 0.086 9.8 ± 0.4 t(12) = 2.347 0.037 0.38 ± 0.02 7 vA594 (10 µm) Normal −39.6 ± 3.8 t(10) = −1.586 0.144 8.4 ± 0.7 t(10) = 3.188 0.01 0.33 ± 0.03 5 n.a., Not applicable.
Group Time 0–5 min p Value
(Paired
t test)Time 15–20 min p Value
(Paired
t test)N Frequency at
−60 mV (Hz)Frequency at
+40 mV (Hz)Frequency at
−60 mV (Hz)Frequency at
+40 mV (Hz)Control 0.14 ± 0.02 0.12 ± 0.03 0.36 0.21 ± 0.05 0.22 ± 0.12 0.94 7 A594 (100 µm) 0.12 ± 0.03 0.10 ± 0.02 0.12 0.12 ± 0.02 0.08 ± 0.01 0.10 7 A488 (100 µm) 0.23 ± 0.10 0.27 ± 0.11 0.06 0.25 ± 0.1 0.22 ± 0.1 0.04 7 A594 +FxU 0.15 ± 0.03 0.17 ± 0.06 0.42 0.14 ± 0.02 0.12 ± 0.04 0.73 5 A594 (10 µm) 0.18 ± 0.02 0.15 ± 0.01 0.13 0.14 ± 0.02 0.13 ± 0.02 0.58 5
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