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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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Simultaneous recordings of calcium transients and APs in neocortical neurons in vivo. (A) Representative traces of simultaneous recordings of calcium transients (upper) and APs (lower). (B) Peak amplitude of calcium transients increased proportionally with the number of APs. Lines show linear fit to the data (Spearman’s ρ, 0.997 for Cal-520 and 0.939 for OGB-1). Values given in parentheses indicate the number of cells. The data for one and two spikes for OGB-1 were obtained by spike-triggered average of fluorescence traces. **P<0.003, ***P<0.0001. (C) The amplitude and (D) SNR of calcium transients normalised by the number of APs (n = 9 cells for Cal-520, n = 4 cells for OGB-1). **P=0.002, ***P<0.001. (E) Average traces of calcium transients in response to a single AP measured with Cal-520 (red, n = 9 cells) or OGB-1 (blue, n = 4 cells). Note that Cal-520 could clearly detect calcium signals by single APs.
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fig05: Simultaneous recordings of calcium transients and APs in neocortical neurons in vivo. (A) Representative traces of simultaneous recordings of calcium transients (upper) and APs (lower). (B) Peak amplitude of calcium transients increased proportionally with the number of APs. Lines show linear fit to the data (Spearman’s ρ, 0.997 for Cal-520 and 0.939 for OGB-1). Values given in parentheses indicate the number of cells. The data for one and two spikes for OGB-1 were obtained by spike-triggered average of fluorescence traces. **P<0.003, ***P<0.0001. (C) The amplitude and (D) SNR of calcium transients normalised by the number of APs (n = 9 cells for Cal-520, n = 4 cells for OGB-1). **P=0.002, ***P<0.001. (E) Average traces of calcium transients in response to a single AP measured with Cal-520 (red, n = 9 cells) or OGB-1 (blue, n = 4 cells). Note that Cal-520 could clearly detect calcium signals by single APs.

Mentions: As both the mean amplitude and mean SNR for each calcium transient, which we quantified above (Figs2F and G, and 4C and D), included different numbers of APs/transient, further quantification was required to compare the true performance of these indicators. Therefore, we performed simultaneous loose-seal cell-attached recordings and high-speed linescan imaging to clarify the relationship between the calcium transients and APs (Fig.5A). The amplitude of the calcium transients linearly correlated with the number of APs (Fig.5B), and the amplitude of calcium transients per spike, which was calculated as the peak amplitude divided by the number of APs, was significantly larger for Cal-520 than for OGB-1 (0.188 ± 0.008 and 0.052 ± 0.009 ΔF/F, nine and four cells, respectively, P=0.0008) (Fig.5C). The SNR per spike was also superior for Cal-520 (1.69 ± 0.08 and 0.54 ± 0.04, nine and four cells, respectively, P=0.002) (Fig.5D). It is notable that Cal-520 could clearly detect calcium signals evoked by single APs, which were barely detectable using OGB-1 (Fig.5E).


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)

Simultaneous recordings of calcium transients and APs in neocortical neurons in vivo. (A) Representative traces of simultaneous recordings of calcium transients (upper) and APs (lower). (B) Peak amplitude of calcium transients increased proportionally with the number of APs. Lines show linear fit to the data (Spearman’s ρ, 0.997 for Cal-520 and 0.939 for OGB-1). Values given in parentheses indicate the number of cells. The data for one and two spikes for OGB-1 were obtained by spike-triggered average of fluorescence traces. **P<0.003, ***P<0.0001. (C) The amplitude and (D) SNR of calcium transients normalised by the number of APs (n = 9 cells for Cal-520, n = 4 cells for OGB-1). **P=0.002, ***P<0.001. (E) Average traces of calcium transients in response to a single AP measured with Cal-520 (red, n = 9 cells) or OGB-1 (blue, n = 4 cells). Note that Cal-520 could clearly detect calcium signals by single APs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: Simultaneous recordings of calcium transients and APs in neocortical neurons in vivo. (A) Representative traces of simultaneous recordings of calcium transients (upper) and APs (lower). (B) Peak amplitude of calcium transients increased proportionally with the number of APs. Lines show linear fit to the data (Spearman’s ρ, 0.997 for Cal-520 and 0.939 for OGB-1). Values given in parentheses indicate the number of cells. The data for one and two spikes for OGB-1 were obtained by spike-triggered average of fluorescence traces. **P<0.003, ***P<0.0001. (C) The amplitude and (D) SNR of calcium transients normalised by the number of APs (n = 9 cells for Cal-520, n = 4 cells for OGB-1). **P=0.002, ***P<0.001. (E) Average traces of calcium transients in response to a single AP measured with Cal-520 (red, n = 9 cells) or OGB-1 (blue, n = 4 cells). Note that Cal-520 could clearly detect calcium signals by single APs.
Mentions: As both the mean amplitude and mean SNR for each calcium transient, which we quantified above (Figs2F and G, and 4C and D), included different numbers of APs/transient, further quantification was required to compare the true performance of these indicators. Therefore, we performed simultaneous loose-seal cell-attached recordings and high-speed linescan imaging to clarify the relationship between the calcium transients and APs (Fig.5A). The amplitude of the calcium transients linearly correlated with the number of APs (Fig.5B), and the amplitude of calcium transients per spike, which was calculated as the peak amplitude divided by the number of APs, was significantly larger for Cal-520 than for OGB-1 (0.188 ± 0.008 and 0.052 ± 0.009 ΔF/F, nine and four cells, respectively, P=0.0008) (Fig.5C). The SNR per spike was also superior for Cal-520 (1.69 ± 0.08 and 0.54 ± 0.04, nine and four cells, respectively, P=0.002) (Fig.5D). It is notable that Cal-520 could clearly detect calcium signals evoked by single APs, which were barely detectable using OGB-1 (Fig.5E).

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