Fig. 2.
Functional expression and characterization of vertebrate rhodopsin in cultured hippocampal neurons. (A) Colocalization of RO4 and synaptobrevin in cultured hippocampal neurons. (Left) Fluorescence patterns of neurons from low-density hippocampal cultures transfected with RO4 reveal a punctate staining. RO4 was detected with an anti-RO4 antibody and visualized with an Alexa 488-coupled secondary antibody. (Center) Hippocampal cells were stained with an antisynaptobrevin II antibody and visualized with an Alexa 568-coupled secondary antibody. (Right) Overlay of RO4 and synaptobrevin II staining. Yellow indicates colocalization. (B) RO4 induced voltage change during a long (Upper) and short (Lower) light pulse. (C) Average GPCR (RO4, GABAB)-induced hyperpolarization of cultured hippocampal neurons. Throughout the experiments GABAB receptors were activated by application of 50 μM baclofen (Bacl). (D) Time course of GPCR (RO4, GABAB)-induced hyperpolarization and recovery from hyperpolarization after switching off the light or washing out baclofen. (E) Voltage traces of current-induced (30 pA) neuronal firing of cultured hippocampal neurons before and during light activation of RO4. (F) Comparison of the number of action potentials measured after current injection for a neuron before and during light activation of RO4. (G) Comparison of EPSC amplitude before, during, and after light application for EPSCs measured in autaptic hippocampal cultures expressing RO4. EPSCs in autaptic hippocampal neurons were elicited by 2-ms voltage pulses from -60 to +10 mV. (H) Comparison of GPCR (RO4, GABAB)-induced EPSC inhibition measured in autaptic hippocampal neurons. (I) Time constants of GPCR (RO4, GABAB)-induced EPSC inhibition and release from inhibition. EPSCs were elicited every 5 s as described in G. (J) Autaptic EPSC traces elicited by 2-ms voltage pulses from -60 to +10 mV separated by 50 ms (20-Hz stimulation) before and after light activation of RO4. (K) Comparison of paired-pulse facilitation before and after GPCR (RO4, GABAB) activation for a 20-Hz stimulation protocol. The amplitude of the second EPSC was compared with the first EPSC.