Fig. 4

Development of membrane-targeted PACmn and characteristics of the light-evoked responses in expressing hippocampal neurons. A cAMP concentrations of whole oocytes expressing soluble or membrane-targeted (Lyn) bPAC variants in dark and light conditions as in Fig. 1A. Mean ± SEM, n = 3 experiments with 5 oocytes each. ***p < 0.0001, *p < 0.05, Dunnett’s multiple comparisons vs control. B Cytosol and membrane distribution of bPAC variants in oocytes indicated by Venus fluorescence, as in Fig. 2D. C PACmn construct design and hippocampal slice culture electroporation strategy. D Confocal projection images of hippocampal neurons co-expressing PACmn (2xLyn-Venus-bPAC(F198Y)) and mKate2, together with close-up of an apical dendrite. Merged image PACmn (yellow) mKate2 (magenta) colocalization appears white. E Whole-cell responses to somatic current injections from −400 pA to 700 pA in a PACmn expressing neuron in the dark (resting membrane potential −70 mV). F Membrane resistance and G holding current of non-transfected (NT), PACmn + mCNG, or bPAC(wt) + mCNG—expressing neurons when clamping the membrane voltage at −70 mV. H Photocurrents evoked by five consecutive 470 nm light flashes (50 ms, 1 mW mm^-2, interval 100 s) in a PACmn + mCNG expressing neuron. I Representative currents recorded from PACmn and mCNG expressing hippocampal neurons in response to 50 ms 470-nm light pulses of varying intensity and the light intensity-response relationship fitted with a quadratic equation. Currents were normalized to the maximum current recorded for each neuron. J Representative currents recorded from PACmn and mCNG expressing hippocampal neurons in response to 1 mW mm^-2 470 nm light pulses of varying duration (5 ms to 15 s). At right is the light duration-response relationship (total charge). The maximum charge from each neuron was set to 100%