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.

Biochemistry has led the way in reducing what once were considered irreducible biological phenomena to fundamental physico-chemical principles. Keys to this accomplishment were the ability to trace the transformations of defined chemical substrates in cellular extracts or intact cells and the reconstitution of biochemical processes with pure components. For neuroscience to follow this reductionist lead, analogous experimental strategies need to be developed: an ability to trace the transformations of defined neuronal activity patterns in explanted neural tissues or intact nervous systems and a capacity to reconstitute neurally encoded information and the behaviors it guides with synthetic neural signals.The initial hurdle to both of these strategies lies in the difficulty of feeding artificial neural signals into functionally circumscribed but anatomically dispersed populations of neurons. An organic solution to this problem is to harness proteins mediating neuronal excitation as conduits for artificial stimuli and to restrict the expression of these transducers genetically to a predetermined group of target neurons (1, 2). If, for example, a set of generalist neurons could be programmed to express signal transduction machinery that normally is present only in specialized sensory cells, these neurons also might acquire the capacity to respond selectively to the adequate physical or chemical triggers. Not only could all members of a population of neurons then be addressed simultaneously, susceptibility to stimulation rather than the stimulus itself would be localized, and even diffusely broadcast stimuli could elicit precise, patterned responses (1, 2).Neurons offer two principal routes for the transduction of excitatory signals (3). Metabotropic signaling systems consist of heptahelical receptors that communicate with their effectors through heterotrimeric G proteins. The activation of some of these effectors is coupled to changes in membrane potential (3-7). Ionotropic signaling systems effect changes in membrane potential directly, via chemically or physically gated conductances (3). Previously, we have used components of a metabotropic system, the phototransduction cascade of the fruit fly (7), to sensitize vertebrate neurons to light (2). Expression of what was termed ''chARGe'' to commemorate the essential elements (arrestin-2, rhodopsin, and a G protein ␣-subunit) created a light-controlled source of depolarizing current whose activation sparked action potentials (2). We now introduce the use of ionotropic mechanisms for the selective chemical and optical stimulation of genetically designated populations of neurons. In addition to multiple trigger modes, these ionotropic systems possess numerous practical advantages over chARGe, including simplicity, fast kinetics, and broad tunability. Materials and MethodsHeterologous Expression of Ion Channels. Candidate ion channels were expressed under the control of the cytomegalovirus promoter in pCI-fluor, a derivative of the mammalian expression vector pCI-neo (Pr...