Photochemical gating of heterologous ion channels: remote control over genetically designated populations of neurons.

Heterologous proteins capable of transducing physical or chemical stimuli into electrical signals can be used to control the function of excitable cells in intact tissues or organisms. Restricted genetically to circumscribed populations of cellular targets, these selectively addressable sources of depolarizing current can supply distributed inputs to neural circuits, stimulate secretion, or regulate force and motility. In an initial demonstration of this principle, we have used elements of a G protein coupled signaling system, the phototransduction cascade of the fruit fly, to sensitize generalist vertebrate neurons to light [Zemelman, B. V., Lee, G. A., Ng, M. & Miesenböck, G. (2002) Neuron 33, 15-22]. We now describe the use of ectopically expressed ligand-gated ion channels as transducers of optical or pharmacological stimuli. When either the capsaicin receptor, TRPV1, the menthol receptor, TRPM8, or the ionotropic purinergic receptor P2X(2) was introduced into hippocampal neurons, the cells responded to pulsed applications of agonist with characteristic sequences of depolarization, spiking, and repolarization. Responses required cognate matches between receptor and agonist, peaked at firing frequencies of approximately 40 Hz, initiated and terminated rapidly, and did not attenuate. Precise dose-response relationships allowed current amplitudes and firing frequencies to be tuned by varying the concentration of ligand. Agonist could be administered either pharmacologically or, in the cases of TRPV1 and P2X(2), optically, through photorelease of the active compounds from the respective "caged" precursors, 4,5-dimethoxy-2-nitrobenzyl-capsaicin and P(3)-[1-(4,5-dimethoxy-2-nitrophenyl)ethyl]-ATP.

Selective photostimulation of genetically chARGed neurons.

To permit direct functional analyses of neural circuits, we have developed a method for stimulating groups of genetically designated neurons optically. Coexpression of the Drosophila photoreceptor genes encoding arrestin-2, rhodopsin (formed by liganding opsin with retinal), and the alpha subunit of the cognate heterotrimeric G protein--an explosive combination we term "chARGe"--sensitizes generalist vertebrate neurons to light. Illumination of a mixed population of neurons elicits action potentials selectively and cell-autonomously in its genetically chARGed members. In contrast to bath-applied photostimulants or caged neurotransmitters, which act indiscriminately throughout the illuminated volume, chARGe localizes the responsiveness to light. Distributed activity may thus be fed directly into a circumscribed population of neurons in intact tissue, irrespective of the spatial arrangement of its elements.