Since its invention in the 1970s, the patch clamp has been a valuable tool for understanding how neurons work. For stimulation, however, each neuron requires its own patch clamp, and the brains of even the simplest organisms consist of hundreds to millions of nerve cells. That fact has made studying the organized activity of groups of brain cells extremely challenging. Now, researchers at the University of California, Berkeley, have developed an optical method for delivering an electrical stimulus to a neuron that allows researchers to remotely stimulate many neurons at the same time.Researchers have developed an all-optical method for stimulating multiple neurons simultaneously. It could lead to a better understanding of neural networks. The method relies on a glutamate-based chemical photoswitch that can be turned on with 380-nm light and turned off with 505-nm light. DMD =digital micromirror device. Images reprinted with permission of Nano Letters. Led by Xiang Zhang, Ehud Y. Isacoff and Dirk Trauner, the group demonstrated the parallel stimulation method, which uses multiple tiny beams individually controlled by millions of digital micromirrors to stimulate genetically engineered neurons. The neurons contain ion channels modified with a synthetic light switch molecule for activation.The chemical switch is a version of the neurotransmitter glutamate that is attached to a glutamate neurotransmitter receptor. A linker molecule containing azobenzene tethers the glutamate near the glutamate binding site on the receptor. Azobenzene is key to the system because its form can change from bent to straight. When excited with 380-nm light, azobenzene causes the linker molecule to bend, enabling the glutamate to bind to the receptor and open the ion channel, triggering the neuron to fire. When excited with 505-nm light, the linker molecule straightens, pulling the glutamate out of its binding site, closing the ion channel and deactivating the neuron. The group studied the new method first with human embryonic kidney cells and then continued with cultured rat hippocampal neurons. Using Fluo-4 calcium dye, the group imaged neuron activity, which increases calcium entry into the cell and is detected as increased Fluo-4 fluorescence. Researchers tested the new optical method on cultured hippocampal neurons (a). Illuminating the entire field with 380-nm light triggered activity in cells A and B (b). Stimulating only cell A, 380-nm light triggered the fluorescence seen in slide (c), and 505-nm light produced the image in slide (d). Stimulating only cell B with 380-nm light triggered the fluorescence in slide (e), and 505-nm light triggered it in slide (f). Lastly, slides (g) and (h) show the effects of stimulating both cells with 380-nm and 505-nm light, respectively. In research published in the December issue of Nano Letters, the researchers tested the method by using it to open calcium channels in human embryonic kidney cells and then measuring the channels’ opening with fluorescence from Fluo-4. They reported a successful optical stimulation rate of 98.8 percent, with increases in Fluo-4 fluorescence that were precisely correlated to cycles of optical stimulation with 380-nm light. The experiments on the rat hippocampal neurons showed success similar to that seen when stimulation directed at the soma of the cells selectively turned activity on and off in individual neurons.The optical stimulation system consisted of two LEDs. A Nichia LED at 380 nm opened the channel, and a Lumileds LED at 505 nm closed it. Light from the LEDs was coupled into a common beam path and modulated using a Texas Instruments digital micromirror device that projected patterned light through a 20× microscope objective. The system was set up on a Nikon epifluorescence microscope. The researchers collected fluorescence images using the microscope’s 488-nm epifluorescence light source and a Photometrics CCD camera.Controlling and recording a large number of cells in parallel required that the digital micromirror device be synchronized with the LED illumination and the CCD imaging. Such a setup could be useful for longitudinal studies of neural circuits or of network plasticity. Isacoff explained that, although the new method involves genetic engineering, it is not particularly challenging. “It consists of simply transfecting the gene encoding the receptor into the neurons, waiting a couple of days for the protein to get made and delivered to the surface of the cell, and then washing on the chemical photoswitch, letting it attach and washing away the excess,” he said. Once this is done, the cell is ready to fire in response to light.The technique may not replace patch clamps completely, but it has the potential to open up avenues of research for scientists wishing to study cell activation in multiple cells at once. It also will open the door to the study of light-activated proteins that can be expressed and activated at specific locations within a cell, in specific cells or in multiple cells simultaneously. Zhang added that the method could be used to study the neuronal network, which could play a key role in understanding how the brain works.Isacoff said that the method is compatible with designing and studying three-dimensional neural patterns. He added that it also is compatible with living organisms. However, at present, azobenzene is not compatible with two-photon activation, and Isacoff said that confocal activation does not offer any advantages over their present epifluorescence system.The group already has begun working on developing new light-based switches, including two-photon switches and ones for other proteins -- receptors, enzymes and motors.“Three groups are working closely to apply this method to living animals and explore new ways to do 3-D stimulations,” Zhang added. “One of the futuristic possibilities is that this parallel method may enable information flows from a man-made computer to the neuronal network or vice versa.”