Figure 1.

Cis-Retinal supplement is required for reliable Optoα1AR activation by brief illumination in vivo. (A) Optoα1AR, optically-activatable Gq-GPCR, is a chimeric molecule of mammalian rhodopsin and Gq-coupled α1 adrenergic receptor (Airan et al., 2009), which induces intracellular Ca²⁺ elevation upon activation. For astrocyte-selective expression, a TG vector was constructed with the BAC GLT1 DNA (Regan et al., 2007). (B) Line #941 (“Strong”) shows intense EYFP fluorescence (green) throughout the brain. High magnification view in white rectangle shows that EYFP is expressed in S100β-positive astrocytes (red). Relatively high EYFP signals are visible in astrocytic somata and endfeet. By contrast, hardly any NeuN signals (red) from neurons overlap with EYFP signals. Scale bar: 1 mm (left), 50 μm (middle and right). (C) Line #877 (“Patchy”) shows visible EYFP fluorescence (green) in a patchy pattern throughout the cortex, hippocampus and striatum. High magnification view in white rectangle shows that EYFP signals colocalize with S100β signals (red) in roughly half of the astrocytes. There are EYFP-negative but S100β-positive domains. Relatively high EYFP signals are visible in astrocytic somata and endfeet. NeuN signals (red) do not overlap with EYFP signals. Scale bar: 1 mm (left), 50 μm (middle and right). (D) Sketch of in vivo astrocytic Ca²⁺ imaging with LED illumination in cortical superficial layers of urethane-anesthetized patchy-TG mice. Astrocytes are loaded with the red Ca²⁺ indicator Rhod-2. (E–G) Example plots of in vivo astrocytic Ca²⁺ imaging with optical stimulation. Optoα1AR-positive and negative astrocytes (white circles and arrowheads, respectively) are analyzed based on the EYFP expression. Green and black traces correspond to EYFP-positive and -negative astrocytes, respectively. Insets: images of cells analyzed in the respective plots. Scale bars: 50 μm. (E) Astrocytic Ca²⁺ imaging without retinal addition. Strong blue LED illumination (1 mW, 5 s) did not induce Ca²⁺ elevations in all the encircled six astrocytes. (F) Astrocytic Ca² imaging with retinal. Weak LED illumination (0.1 mW, 1 s) induced a transient Ca²⁺ increase in EYFP-positive astrocytes, but not in EYFP-negative astrocytes. (G) Astrocytic Ca² imaging with retinal. Strong LED illumination (1 mW, 1 s) induced a rapid Ca²⁺ increase in EYFP-positive astrocytes. Delayed Ca²⁺ elevation was observed in EYFP-negative astrocytes. (H–L) Analysis of Ca²⁺ response in EYFP-positive and -negative astrocytes upon weak or strong LED illumination with retinal addition. Each symbol represents an individual imaging session (Weak LED: 35 sessions, nine patchy TG mice; Strong LED: 17 sessions, nine patchy TG mice). (H) Proportion of responsive cells. The weak-negative group was the least responsive (***p < 0.001, Tukey's test after one-way ANOVA). (I) Peak amplitude was similar for all groups (p > 0.11, one-way ANOVA). (J,K) Onset time and onset-to-peak time were shorter in the positive group for both stimulation strengths (***p < 0.001, **p < 0.01, Dunn's test after Kruskal-Wallis one-way ANOVA). (L) Peak-to-offset time was shorter in the strong-negative group (*p < 0.05, Dunn's test after Kruskal-Wallis one-way ANOVA). (M–P) Comparison of spontaneous, tail-pinch-induced, and optogenetically induced (strong illumination) Ca²⁺ response. (M) Mean and SEM trace of optogenetically induced (green) and tail-pinch-induced (gray) Ca²⁺ increase. Time 0 corresponds to onset time, when F/F₀ reaches 120%. (N) Peak amplitude was similar among the 3 groups (p > 0.32, one-way ANOVA). (O,P) Onset-to-peak and peak-to-offset times of Ca²⁺ events were similar between the tail pinch and optogenetically induced groups (p > 0.05, Tukey's test after one-way ANOVA) and distinct from spontaneously observed Ca²⁺ events (***p < 0.001, **p < 0.01, *p < 0.05, Tukey's test after one-way ANOVA). Each symbol represents an individual imaging session (Spontaneous: 14 sessions, 10 TG mice; Tail-pinch: six sessions, four TG mice).