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A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo.

Tada M, Takeuchi A, Hashizume M, Kitamura K, Kano M - Eur. J. Neurosci. (2014)

Bottom Line: Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo.Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo.These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

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Properties of calcium transients in neocortical neurons in vivo measured with Cal-520 and OGB-1. Representative linescan imaging from somata of layer 2/3 neurons labeled with Cal-520 (A) or OGB-1 (B). Scale bars, 20 μm. Upper right: linescan images obtained at the position indicated by the orange lines in the upper left panels. Lower: spontaneous calcium transients recorded at the regions of interest shown as the rectangles in the upper right panels. Gray dots indicate calcium transients detected from the baseline noise. (C–F) Bar graphs showing the properties of calcium transients measured with Cal-520 (filled columns) or OGB-1 (open columns). Cal-520 showed higher mean amplitude of calcium transients than OGB-1 (C), resulting in higher SNR (D). Rise times (E) and decay time constants (F) of calcium transients were indistinguishable between Cal-520 and OGB-1. *P<0.05, ***P<0.001.
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fig04: Properties of calcium transients in neocortical neurons in vivo measured with Cal-520 and OGB-1. Representative linescan imaging from somata of layer 2/3 neurons labeled with Cal-520 (A) or OGB-1 (B). Scale bars, 20 μm. Upper right: linescan images obtained at the position indicated by the orange lines in the upper left panels. Lower: spontaneous calcium transients recorded at the regions of interest shown as the rectangles in the upper right panels. Gray dots indicate calcium transients detected from the baseline noise. (C–F) Bar graphs showing the properties of calcium transients measured with Cal-520 (filled columns) or OGB-1 (open columns). Cal-520 showed higher mean amplitude of calcium transients than OGB-1 (C), resulting in higher SNR (D). Rise times (E) and decay time constants (F) of calcium transients were indistinguishable between Cal-520 and OGB-1. *P<0.05, ***P<0.001.

Mentions: To quantitatively compare the signal amplitude and SNR, we next performed high-speed linescan imaging (sampling rate, 500 Hz) (Fig.4A and B). The mean amplitude of individual calcium transients using Cal-520 was significantly larger than that using OGB-1 (0.696 ± 0.010 and 0.434 ± 0.011 ΔF/F, 49 cells in nine mice and 14 cells in four mice, respectively, P=0.02, Mann–Whitney U-test) (Fig.4C), which resulted in higher SNRs (5.390 ± 0.052 for Cal-520 and 3.465 ± 0.077 for OGB-1, P=0.0004) (Fig.4D). The kinetic properties of calcium transients were not different between Cal-520 and OGB-1 [10–90 rise time (Fig.4E): 0.053 ± 0.001 and 0.099 ± 0.006 s, 49 cells in nine mice and 14 cells in four mice, respectively, P=0.09; decay time constants by double-exponential fitting (Fig.4F): 0.755 ± 0.009 and 1.055 ± 0.010 s for Cal-520 (46 cells in nine mice), 0.675 ± 0.037 and 1.196 ± 0.043 s for OGB-1 (12 cells in four mice), P=0.60 and 0.28, respectively].


A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo.

Tada M, Takeuchi A, Hashizume M, Kitamura K, Kano M - Eur. J. Neurosci. (2014)

Properties of calcium transients in neocortical neurons in vivo measured with Cal-520 and OGB-1. Representative linescan imaging from somata of layer 2/3 neurons labeled with Cal-520 (A) or OGB-1 (B). Scale bars, 20 μm. Upper right: linescan images obtained at the position indicated by the orange lines in the upper left panels. Lower: spontaneous calcium transients recorded at the regions of interest shown as the rectangles in the upper right panels. Gray dots indicate calcium transients detected from the baseline noise. (C–F) Bar graphs showing the properties of calcium transients measured with Cal-520 (filled columns) or OGB-1 (open columns). Cal-520 showed higher mean amplitude of calcium transients than OGB-1 (C), resulting in higher SNR (D). Rise times (E) and decay time constants (F) of calcium transients were indistinguishable between Cal-520 and OGB-1. *P<0.05, ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232931&req=5

fig04: Properties of calcium transients in neocortical neurons in vivo measured with Cal-520 and OGB-1. Representative linescan imaging from somata of layer 2/3 neurons labeled with Cal-520 (A) or OGB-1 (B). Scale bars, 20 μm. Upper right: linescan images obtained at the position indicated by the orange lines in the upper left panels. Lower: spontaneous calcium transients recorded at the regions of interest shown as the rectangles in the upper right panels. Gray dots indicate calcium transients detected from the baseline noise. (C–F) Bar graphs showing the properties of calcium transients measured with Cal-520 (filled columns) or OGB-1 (open columns). Cal-520 showed higher mean amplitude of calcium transients than OGB-1 (C), resulting in higher SNR (D). Rise times (E) and decay time constants (F) of calcium transients were indistinguishable between Cal-520 and OGB-1. *P<0.05, ***P<0.001.
Mentions: To quantitatively compare the signal amplitude and SNR, we next performed high-speed linescan imaging (sampling rate, 500 Hz) (Fig.4A and B). The mean amplitude of individual calcium transients using Cal-520 was significantly larger than that using OGB-1 (0.696 ± 0.010 and 0.434 ± 0.011 ΔF/F, 49 cells in nine mice and 14 cells in four mice, respectively, P=0.02, Mann–Whitney U-test) (Fig.4C), which resulted in higher SNRs (5.390 ± 0.052 for Cal-520 and 3.465 ± 0.077 for OGB-1, P=0.0004) (Fig.4D). The kinetic properties of calcium transients were not different between Cal-520 and OGB-1 [10–90 rise time (Fig.4E): 0.053 ± 0.001 and 0.099 ± 0.006 s, 49 cells in nine mice and 14 cells in four mice, respectively, P=0.09; decay time constants by double-exponential fitting (Fig.4F): 0.755 ± 0.009 and 1.055 ± 0.010 s for Cal-520 (46 cells in nine mice), 0.675 ± 0.037 and 1.196 ± 0.043 s for OGB-1 (12 cells in four mice), P=0.60 and 0.28, respectively].

Bottom Line: Therefore, it is difficult to detect signals caused by single action potentials (APs) particularly from neurons in vivo.Here we showed that a recently developed calcium indicator dye, Cal-520, is sufficiently sensitive to reliably detect single APs both in vitro and in vivo.These characteristics of Cal-520 are a great advantage over those of Oregon Green BAPTA-1, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.

Show MeSH
Related in: MedlinePlus