A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo.
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.
Affiliation: Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.Show MeSH
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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).
Affiliation: Department of Neurophysiology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.