Alkema Lab Approaches to Neuroscience
The Alkema lab uses a variety of cutting edge techniques in our research. This page highlights a sample of the tools we use in the pursuit of science.
Microfluidics
Microfluidic devices are molded PDMS chambers that have micron scale channels which allow the precise control of liquid flow. C. elegans can be loaded into microfluidic devices for imaging or behavioral assays. Custom microfluidic chip designs have been developed to address a variety of experimental needs. Soluble stimuli can be delivered to animals in gradients, stripes, pulses etc. Large arenas allow animals to move freely and are useful for chemotaxis assays. Devices with tapered channels can be used to immobilize animals without anesthetics for calcium imaging.
In this real time demo, the microfluidic device has been set up to deliver a stimulus to half of the arena at a time. Fluorescent dye is used to visualize buffer flow. Computer controlled valves direct flow from one half of the chamber to another with an exponentially increasing speed.
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C. elegans expressing the genetically encoded calcium indicator GCaMP6 in a subset of sensory neurons are exposed to a stimulus solution in a microfluidic chamber. A computer controlled valve system allowed precise timing of stimulus delivery. The stimulus solution contains a fluorescent dye which lights up the chamber when the stimulus is being delivered.
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The video above is an example of a chemotaxis experiment to examine the mechanisms behind nitric oxide sensing in C. elegans. Wild type (Left) and mutant (Right) animals in a microfluidic arena are presented with two concentrations of dissolved nitric oxide. The nitric oxide solution is mixed with a dye and is visible as a stripe in the center of the arena. Playback is sped up 100x, the actual time is indicated in the top left corner. Nitric oxide is delivered at a lower concentration beginning at 10 min, and at a higher concentration beginning at 40 min. The wild type animals on the left avoid the nitric oxide stripe but mutants on the right are defective in avoidance.
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Optogenetics
Transgenic expression of light gated ion channels in neurons has revolutionized the field of neuroscience, allowing researchers to turn on and off neurons with different wavelengths of light. These optogenetic techniques are powerful tools to dissect the function of neurons and neural circuits in intact behaving animals. C. elegans is particularly well suited to optogenetic manipulation due to its optical transparency and a variety of cell specific promoters.
In the video above, animals express the light gated cation channel ChannelRhodopsin in a pair of motor neurons called the RIV. These neurons innervate ventral neck muscle and are important for the initiation of the omega turn during the C. elegans escape response. Shining blue light on the animal causes the RIV to become active and induces a ventral turn. Techniques like this allow us to determine the function of individual neurons in the coordination of complex behavior.
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Calcium Imaging
Calcium influx is a key mediator of neuronal excitation, particularly in C. elegans which lack voltage gated sodium channels. genetically encoded calcium indicators allow researchers to monitor neuronal activity in intact behaving animals. GCaMP is a modified green fluorescent protein (GFP) which is linked to the calcium binding domain of calmodulin. Calcium influx results in a conformational change in the GCaMP protein increasing the fluorescent output of the GFP allowing real time monitoring of neuronal activity.
The above video is a confocal microscope stack of an animal expressing GCaMP6 under a pan-neuronal promoter as well as a nuclear localized red fluorescent protein (RFP) for cell identification. This allows us to do "whole brain imaging" in behaving animals. See Venkatachalam et al. PNAS 2015