Anne Fischer, firstname.lastname@example.org
CAMBRIDGE, Mass. – In the past, scientists could study only one neuron at a time in the nematode Caenorhabditis elegans. Now they can look at how tens of neurons in the roundworm interact with one another in vivo. This nematode, with a total of just 302 neurons, is a good model for studying how activity in the nervous system leads to specific behaviors.
In a Harvard University study reported in the December 2009 issue of Nature Methods, a team led by Sharad Ramanathan, an assistant professor of molecular and cellular biology and applied physics, used genetically encoded light-based electrophysiology to perform circuit-level analysis of neural activity.
Previously, through electrophysiology, cell ablation, in vivo calcium imaging and genetic analysis, the connections between only a few neurons had been inferred. Now the researchers are monitoring calcium activity in another group of neurons, to discover whether priming one neuron with a burst of light activates or inhibits the other neurons. For the first time, they are able to excite both a sensory neuron and an interneuron and measure how activity is propagated.
One problem was the overlap of the excitation spectra of both channelrhodopsins – channelrhodopsin-2 (ChR2) and Volvox carteri channelrhodopsin (VChR1) – and of the genetically encoded calcium sensors G-CaMP and Cameleon. When the fluorescent signal from the calcium sensors is measured, channelrhodopsins are activated. Another difficulty was activating only one of the several neurons expressing channelrhodopsins. Most neuronal promoters drive expression in multiple neurons, so exposure of the worm to the excitation light activates all neurons expressing ChR2 or VChR1. The challenge was to excite just a subset of those neurons expressing ChR2 or VChR1.
The researchers found that using ChR2 and G-CaMP simultaneously allowed them to synchronously activate and record multiple neurons in intact worms; the combination enabled them to determine whether one neuron was exciting another. They used a low-power 488-nm laser and a spinning disc to measure G-CaMP activity and a higher-power epifluorescence source to excite ChR2. A digital light processing mirror array allowed for activation of specific neurons in a field of ChR2-expressing neurons. They combined in vivo optical stimulation with simultaneous calcium imaging. As a result, they found that they could excite specific neurons expressing ChR2 while at the same time monitoring G-CaMP fluorescence in several other neurons.
The team used a piezo Z-drive coordinated with an optical setup to stimulate and monitor neurons in different focal planes. A USB power meter from Newport was used to measure the blue light reaching the nematode growth medium cultivation plate in each assay. The intensity of light reaching the plate was controlled with the click stop iris on the X-Cite Illuminator and the iris on the Zeiss Discovery dissecting microscope.
A next step in this research could be to change the wavelength filter in the excitation light path to incorporate and simultaneously excite halorhodopsin in other neurons. Using the same strategy of low-light intensity for G-CaMP imaging, the researchers could avoid ChR2 activation for other neuronal circuits in C. elegans or in other organisms.