The Journal of Neuroscience, November 8, 2006, ():
Myosin Light Chain Kinase Is Not a Regulator of Synaptic Vesicle Trafficking during Repetitive Exocytosis in Cultured Hippocampal Neurons
J. Neurosci. Tokuoka and Goda
26: 11606
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Fig. S1 Perfusion of 0.1% DMSO does not significantly affect short-term depression
A, B, Short-term depression of EPSC amplitude (A) and total charge transfer (B) elicited by 40 pulses at 20 Hz is shown before and after 0.1% DMSO perfusion in same cells (n = 6). Data are normalized to the first response of the train.
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Fig. S2 ML-7 increases mEPSC frequency nonspecifically
A, Representative traces of mEPSC before (top) and 10 min after perfusion of 10 ?M ML-7 (bottom). mEPSC was recorded in bath solution containing 0.5 ?M TTX and without CNQX and APV. The massive increase in mEPSC frequency was similarly reproduced in two other experiments. B, Time course of mEPSC frequency increase by ML-7 in the same cell shown in (A). C, Summary of the mEPSC amplitude distribution before and after ML-7 perfusion from an example neuron. A slight increase in the mEPSC amplitude by ML-7 is likely due to the unavoidable overlap of peaks caused by the increase in frequency. D, Effect of 10 ?M wortmannin on mESPC. Wortmannin had no effect on mEPSC frequency, whereas the same neuron responded to ML-7.
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Fig. S3 Effects of ML-9 and low concentration of wortmannin on AP amplitude
A, Number of APs generated upon current injection in neurons pretreated with 0.1% DMSO (n = 8), 30 ?M ML-9 (n = 4) or 0.1 ?M wortmannin (n = 4) for 15 min (cf. Fig. 3). Neuronal excitability is attenuated by ML-9 but not by 0.1 ?M wortmannin (repeated-measures ANOVA, interaction, **p < 0.01. *p < 0.05; Dunnett’s test, DMSO vs. ML-9). B, Peak AP amplitude during repetitive stimulation in neurons pretreated with DMSO (n = 9), 30 ?M ML-9 (n = 4) or 0.1 ?M wortmannin (n = 4). Data are normalized to the first peak. AP amplitude progressively declines in the presence of ML-9 whereas only a small decline is observed in control DMSO and wortmannin (repeated-measures ANOVA, interaction, p < 0.001. **p < 0.01; Dunnett’s test, DMSO vs. ML-9). C, Summary of the amplitude of first AP shows a significant inhibition by ML-9 compared to DMSO or wortmannin (*p < 0.05; Dunnett’s test, DMSO vs. ML-9). D, Summary of half-maximal width of the first AP. ML-9 broadened the AP wave form similarly to ML-7 (** p < 0.01; Dunnett’s test, DMSO vs. ML-9).
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Fig. S4 Effect of ML-9 on VGCC current
Summary of VGCC current induced by a 30 ms voltage step in neurons pretreated with 0.1% DMSO (n = 9) or 30 ?M ML-9 (n = 3) for 15 min. ML-9 severely inhibited VGCC current (*p < 0.05, **p < 0.01; t test).
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Fig. S5 RRP recovery measured by pairwise application of hypertonic sucrose solution
A, RRP recovery after depletion was estimated by paired puffs of hypertonic solution to local dendritic area. Example traces of EPSC from neurons pretreated with DMSO (control) and ML-7 are shown (cf. Fig. 5D). Each puff to deplete the RRP was for 4 sec, and the inter-pulse interval was 3.6 sec. B, Dose-dependency of RRP recovery on ML-7 and wortmannin. Each data point represents the mean RRP recovery from 3 to 5 recordings. There is no statistically significant difference between control and drug treatment across all concentration ranges tested (p > 0.9; one-way ANOVA).
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Fig. S6 Effect of wortmannin on the morphology of astrocytes
Cultured astrocytes were treated with 0.1% DMSO or 10 ?M wortmannin for indicated times, and fixed. Actin filaments were stained with phalloidin-Alexa568 (bottom panels). Wortmannin disrupted actin filament organization and induced a morphological change in astrocytes that was evident in bright field images (top panels). These effects were not apparent with 0.1 ?M wortmannin (data not shown). Scale bar, 50 ?m.
