Methods of inhibiting and activating neurons are essential to understand how neurons and neuronal circuits produce behavior. Optogenetics is a recently developed technology, which uses light to manipulate the time accurate neuronal activities of behavioral animals, and is widely used by neuroscience researchers. In short, optogenetics uses transgene method to express specific photoactivated ion channels that naturally exist in the cells required by model organisms in algae and archaea. Depending on the type of channel expressed, cells can be depolarized or hyperpolarized by light of a specific wavelength. Optogenetics allows manipulation of the functions of specific neurons or groups of neurons and real-time measurement of behavioral effects.
Caenorhabditis elegans is a powerful undergraduate teaching organism because they are cheap and easy to maintain, and there are many ready-made strains, reagents and information resources. Since the frozen inventory of nematode strains can be stored in a refrigerator at - 80°C or liquid nitrogen, the strains only need to be actively maintained during use. Creative BioMart developed a construction protocol for C.elegans model. The model can produce serotonin under the induction of blue light, which can be used to study the role of serotonin in the neural regulation of behavioral state.
In the strain, we used channelrhodopsin, a cation channel directly gated by blue light. When blue light activates the channelrhodopsin, the transgenic expression channel opens and neurons depolarize. The genetically modified worm expressed channelrhodopsin and GFP label in all serotonin neurons. The serotonergic neurons were activated by blue light, so that the channelrhodopsin with GFP label was expressed in all serotonergic neurons, and the behavioral effects of worms were measured.
| Step | Description |
| C. elegans strains and maintenance | C. elegans are maintained on Nematode Growth Media (NGM) plates with E. coli HB101 or E. coli OP50.Transgenic nematodes express channelrhodopsin with GFP label in all serotonin producing neurons. |
| Exploration assay | Excess worm paths to measure exploration. Place a grid with a square on the transparency under the agar plate. Examine the plate under a microscope and calculate the number of squares the worm tracks into. |
| Blue light stimulation | It is preferable to use computer-controlled LED lights to emit blue light (wavelength about 455-470). Although the time and intensity of stimulation are not as accurate as when using LED, strains expressing rhodopsin should still respond. |
| Scoring behavior | The response speed of animals to blue channel rhodopsin stimulation was measured by calculating the number of body bending every 30 seconds before, during and after stimulation. Body bending describes the sinusoidal motion of the worm. A body bend is defined as "each time the worm part behind the pharynx reaches the maximum bend in the direction opposite to the bend counted last time". This behavior can be rated in real time, or you can record a video and then rate a movie. |
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