FIGURE 3.

PMT in iris sphincter myotubes. (A) Iris sphincter myotubes grown in dissociated cell culture display the elongated columnar appearance (left) and diffuse pattern of nAChR puncta expression revealed by AF488-αBgt labeling (right) expected for developing chick striated muscle fibers (Vogel et al., 1972). Scale bars represent 20 μm (left) and 10 μm (right). (B–D) Consistent with the CRY photoactivation spectrum in whole iris (Tu, 2004) the iris myotube PMTR is induced by 435 nm light. (B) Images 1–4 depict Ca²⁺ fluorescence changes mediated by Rhod-2 in a single myotube exposed to 435 nm and 535 nm (435/535 nm) dual excitation light (at 1.5 and 1.8 × 10¹⁶ photons s^–1 cm^–2, respectively to excite CRY and Rhod-2) before (1) and at start (2), peak (3) and end (4) of the PMTR. (C) Specific iris myotube Ca²⁺ fluorescence changes expressed as ΔF/F_B in response to 80 sec exposure to 435/535 nm dual excitation light (blue bar and circles; Arrows 1-4 correspond to images 1–4 in panel (B) and lack of response after exposure to 545 nm single wavelength light (at 3.0 × 10¹⁶ photons s^–1 cm^–2 green bar and circles). Points reflect % change in ΔF/F_B values relative to the value at light onset. Sixteen of 18 iris myotubes tested (89%) displayed PMTRs in response to 435/535 nm dual excitation light. (D) Cumulative iris myotube PMTR expressed as peak% change in ΔF/F_B (± SEM) in response to dual excitation 435/535 nm light (blue bar; 49 ± 9%, n = 16; N = 6) or single wavelength 545 nm light (green bar; 4 ± 7%, n = 10; N = 4).