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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.

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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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In vivo two-photon calcium imaging of layer 2/3 neurons in the barrel cortex labeled by multi-cell bolus loading of Cal-520 AM or OGB-1 AM. (A) Three-dimensional projection image of Cal-520 AM-loaded barrel cortex. Depth, 80–250 μm from pia. Scale bars, 50 μm. Neurons in layer 2/3 of the left barrel cortex labeled with Cal-520 AM (B) or OGB-1 AM (C). Upper left: representative images of the field of view (FOV) showing the somata of labeled neurons. Upper right: neurons producing calcium transients were automatically detected by the pixel correlation method and are shown in the color map. Lower: representative traces of calcium transients from corresponding regions of interest shown in the upper left panels, which were defined as the area showing a higher correlation coefficient than the predetermined threshold. Gray dots indicate calcium transients detected from the baseline noise. Scale bars, 20 μm. (D–G) Bar graphs showing the frequency of APs (D) and number of active cells per FOV (E), amplitude of calcium transients (F), and SNR (G) by multi-cell bolus loading of Cal-520 (filled columns, n = 171 cells in 12 mice) or OGB-1 (open columns, n = 12 cells in four mice). *P<0.05, ***P<0.001. Error bars indicate SEM.
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fig02: In vivo two-photon calcium imaging of layer 2/3 neurons in the barrel cortex labeled by multi-cell bolus loading of Cal-520 AM or OGB-1 AM. (A) Three-dimensional projection image of Cal-520 AM-loaded barrel cortex. Depth, 80–250 μm from pia. Scale bars, 50 μm. Neurons in layer 2/3 of the left barrel cortex labeled with Cal-520 AM (B) or OGB-1 AM (C). Upper left: representative images of the field of view (FOV) showing the somata of labeled neurons. Upper right: neurons producing calcium transients were automatically detected by the pixel correlation method and are shown in the color map. Lower: representative traces of calcium transients from corresponding regions of interest shown in the upper left panels, which were defined as the area showing a higher correlation coefficient than the predetermined threshold. Gray dots indicate calcium transients detected from the baseline noise. Scale bars, 20 μm. (D–G) Bar graphs showing the frequency of APs (D) and number of active cells per FOV (E), amplitude of calcium transients (F), and SNR (G) by multi-cell bolus loading of Cal-520 (filled columns, n = 171 cells in 12 mice) or OGB-1 (open columns, n = 12 cells in four mice). *P<0.05, ***P<0.001. Error bars indicate SEM.

Mentions: Multi-cell bolus loading of the acetoxymethyl ester derivative of the indicator dye was performed to monitor the population activity of neurons in the barrel cortex (Stosiek et al., 2003; Golshani et al., 2009). Cal-520 AM or OGB-1 AM was injected into layer 2/3 of the mouse barrel cortex (200–300 μm from the surface). At about 30 min after dye loading, Cal-520 or OGB-1 was penetrated into the neurons and glia. Spontaneous calcium transients in multiple neurons in the field of view were reliably detected using Cal-520 (Fig.2A and B) as well as OGB-1 (Fig.2C). The mean frequency of spontaneous calcium transients was indistinguishable between Cal-520 and OGB-1 (0.0458 ± 0.002 and 0.0410 ± 0.0022 Hz; 171 cells in 12 mice and 12 cells in four mice, respectively, P=0.64, Mann–Whitney U-test) (Fig.2D). The average number of active neurons in a field of view of the Cal-520-filled cortex (147 × 147 μm), which showed at least one spontaneous AP in 2 min, was significantly larger than that of the OGB-1-filled cortex (7.77 ± 0.27 and 1.71 ± 0.14 cells/field of view, 12 and four mice, respectively, P=0.0002, Mann–Whitney U-test) (Fig.2E). This result reflects the fact that Cal-520-filled cells showed a larger amplitude of calcium transients than OGB-1-filled cells (0.318 ± 0.001 and 0.218 ± 0.008 ΔF/F, 171 cells in 12 mice and 12 cells in four mice, respectively, P=0.03, Mann–Whitney U-test) (Fig.2F), and thus the mean SNR of individual calcium transients by Cal-520 was significantly higher than that by OGB-1 (14.62 ± 0.04 and 8.65 ± 0.28, P=0.0002, Mann–Whitney U-test) (Fig.2G). Calcium transients evoked by sensory stimulation to contralateral whiskers were also clearly observed in multiple neurons (Fig.3).


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)

In vivo two-photon calcium imaging of layer 2/3 neurons in the barrel cortex labeled by multi-cell bolus loading of Cal-520 AM or OGB-1 AM. (A) Three-dimensional projection image of Cal-520 AM-loaded barrel cortex. Depth, 80–250 μm from pia. Scale bars, 50 μm. Neurons in layer 2/3 of the left barrel cortex labeled with Cal-520 AM (B) or OGB-1 AM (C). Upper left: representative images of the field of view (FOV) showing the somata of labeled neurons. Upper right: neurons producing calcium transients were automatically detected by the pixel correlation method and are shown in the color map. Lower: representative traces of calcium transients from corresponding regions of interest shown in the upper left panels, which were defined as the area showing a higher correlation coefficient than the predetermined threshold. Gray dots indicate calcium transients detected from the baseline noise. Scale bars, 20 μm. (D–G) Bar graphs showing the frequency of APs (D) and number of active cells per FOV (E), amplitude of calcium transients (F), and SNR (G) by multi-cell bolus loading of Cal-520 (filled columns, n = 171 cells in 12 mice) or OGB-1 (open columns, n = 12 cells in four mice). *P<0.05, ***P<0.001. Error bars indicate SEM.
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fig02: In vivo two-photon calcium imaging of layer 2/3 neurons in the barrel cortex labeled by multi-cell bolus loading of Cal-520 AM or OGB-1 AM. (A) Three-dimensional projection image of Cal-520 AM-loaded barrel cortex. Depth, 80–250 μm from pia. Scale bars, 50 μm. Neurons in layer 2/3 of the left barrel cortex labeled with Cal-520 AM (B) or OGB-1 AM (C). Upper left: representative images of the field of view (FOV) showing the somata of labeled neurons. Upper right: neurons producing calcium transients were automatically detected by the pixel correlation method and are shown in the color map. Lower: representative traces of calcium transients from corresponding regions of interest shown in the upper left panels, which were defined as the area showing a higher correlation coefficient than the predetermined threshold. Gray dots indicate calcium transients detected from the baseline noise. Scale bars, 20 μm. (D–G) Bar graphs showing the frequency of APs (D) and number of active cells per FOV (E), amplitude of calcium transients (F), and SNR (G) by multi-cell bolus loading of Cal-520 (filled columns, n = 171 cells in 12 mice) or OGB-1 (open columns, n = 12 cells in four mice). *P<0.05, ***P<0.001. Error bars indicate SEM.
Mentions: Multi-cell bolus loading of the acetoxymethyl ester derivative of the indicator dye was performed to monitor the population activity of neurons in the barrel cortex (Stosiek et al., 2003; Golshani et al., 2009). Cal-520 AM or OGB-1 AM was injected into layer 2/3 of the mouse barrel cortex (200–300 μm from the surface). At about 30 min after dye loading, Cal-520 or OGB-1 was penetrated into the neurons and glia. Spontaneous calcium transients in multiple neurons in the field of view were reliably detected using Cal-520 (Fig.2A and B) as well as OGB-1 (Fig.2C). The mean frequency of spontaneous calcium transients was indistinguishable between Cal-520 and OGB-1 (0.0458 ± 0.002 and 0.0410 ± 0.0022 Hz; 171 cells in 12 mice and 12 cells in four mice, respectively, P=0.64, Mann–Whitney U-test) (Fig.2D). The average number of active neurons in a field of view of the Cal-520-filled cortex (147 × 147 μm), which showed at least one spontaneous AP in 2 min, was significantly larger than that of the OGB-1-filled cortex (7.77 ± 0.27 and 1.71 ± 0.14 cells/field of view, 12 and four mice, respectively, P=0.0002, Mann–Whitney U-test) (Fig.2E). This result reflects the fact that Cal-520-filled cells showed a larger amplitude of calcium transients than OGB-1-filled cells (0.318 ± 0.001 and 0.218 ± 0.008 ΔF/F, 171 cells in 12 mice and 12 cells in four mice, respectively, P=0.03, Mann–Whitney U-test) (Fig.2F), and thus the mean SNR of individual calcium transients by Cal-520 was significantly higher than that by OGB-1 (14.62 ± 0.04 and 8.65 ± 0.28, P=0.0002, Mann–Whitney U-test) (Fig.2G). Calcium transients evoked by sensory stimulation to contralateral whiskers were also clearly observed in multiple neurons (Fig.3).

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