Fig. 2.

YFP-CaRhGC produces cGMP and not cAMP in hippocampal neurons. a, b Confocal images of neurons 8 days after electroporation with DNA encoding YFP-CaRhGC and mtSapphire; a maximum intensity projection, excitation 405 nm (mtSapphire); b top: maximum projection, excitation 515 nm (YFP); bottom: single plane. scale bars 10 µm. c Top: Whole-cell response to current injections from −400 pA to 400 pA in 100 pA steps. Bottom: The first, second and fifth currents evoked by repeated green light flashes (530 nm, 0.3 mW mm⁻², 100 ms, inter-stimulus interval 40 s). d Sample currents evoked by a 2 s green light pulse (530 nm, 27.3 mW mm⁻²) in neurons expressing YFP-CaRhGC, CaRhGC or the guanylyl cyclase from Blastocladiella emersonii (BeRhGC) together with CNG(cGMP) or the cAMP-sensitive CNGA2 (CNG(cAMP)) channels. Green bar: light application, 2 s. e Peak photocurrents (p) and the sustained response (s) recorded from neurons expressing YFP-CaRhGC, CaRhGC, or BeRhGC and one of the CNG channels. Shown are individual data points, median and 25–75% interquartile range, n’s = 12, 12, 4, 17, 17, 6, 13, 13, 7 left to right; ***p = 0.0001, p = 0.0006, **p = 0.009; Mann–Whitney test, peak vs sustained response of YFP-Ca/Ca/BeRhGC + CNG(cGMP). Median peak current YFP-CaRhGC −0.79 nA, CaRhGC −1.3 nA, BeRhGC −0.6 nA; median sustained current YFP-CaRhGC −0.25 nA, CaRhGC −0.24 nA, BeRhGC −0.11 nA. f Detail of photocurrent onset from neurons expressing YFP-CaRhGC, CaRhGC or BeRhGC, and CNG(cGMP). Graph shows individual data points, median and interquartile range, n’s = 12, 17, 12. median slope YFP-CaRhGC = −8.7 pA ms⁻¹, CaRhGC = −7.2 pA ms⁻¹, BeRhGC = −2.2 pA ms⁻¹. g Sample currents recorded from a neuron expressing YFP-CaRhGC + CNG(cGMP) when stimulated with green light. h Light intensity-response relationship for YFP-CaRhGC + CNG(cGMP) fitted with a quadratic equation. Photocurrents were normalized to the maximum current recorded for each neuron. n = 17. RhGC DNA was electroporated at 10 ng µl⁻¹, CNG channel DNA at 25 ng µl⁻¹ and mtSapphire DNA at 5 ng µl⁻¹