As a neuroscientist, one typically becomes accustomed to thinking outside of the “box.” After all, the brain is incredibly complex and cryptic, and some creative thought is required to develop methods to uncover its secrets.
Francis Crick advised that the greatest hurdle standing in the way of neuroscience is the inability to specifically stimulate a single neuron without altering any of its surrounding cells. This daunting task appears to be a thing of fantasy when considering the innumerable intricate connections the brain is composed of. However, Harvard University’s Samuel Lab has made a reality out of a dream with their groundbreaking research involving transgenic C. elegans. Recently published in Nature, the research of Dr. Andrew M Leifer and his team utilizes a manipulatory optogentics technique called ColBeRT to control the nervous system of a worm with the light from a laser.
Optogenetics is the methodology of employing genetics and visible light to manipulate the activity of living cells. In order for optogenetics to be applicable in the laboratory, the insertion of opsin genes, which encode for light sensitive proteins, into an organism’s genome is necessary. The activity of the resulting opsin-containing cells can be regulated by exposure to visible light. Relative to the work of Leifer et al., optogenetics provides a platform by which the genetically altered motor and sensory neurons of C. elegans can be controlled with the use of a precise laser. Why use C. elegans? According to Leifer et al., “the nematode C. elegans is particularly amenable to optogenetics owing to its optical transparency, compact nervous system and ease of genetic manipulation.”
The ColBeRT (Controlling Locomotion and Behavior in Real-Time) technique, which was designed for the optogenetics research being done at The Samuel Lab, provides a way to specifically track a worm’s movement. A video camera with real-time feedback follows an illuminated and moving C. elegans under a dark field. The worm is placed on a motorized stage, which keeps the image of the organism centered in the camera’s view. Once the worm’s movement is recognized and registered by a specialized graphical user interface (GUI) software called MindControl, the image of the C. elegans is processed and the worm’s image is divided into 100 evenly spaced segments. From these segmented portions, specific target cells can be chosen, the locations of which are transferred to a DMD (digital micromirror device). This pattern is then projected onto the worm by the DMD, allowing for the illumination of the targeted points with a laser. This laser can precisely pinpoint the location of a specific target cell by a simple algorithm specifically designed for the movement analysis of C. elegans.
The impressive spatial and temporal resolution (~50 frames per second) of the ColBeRT technique makes the system scientifically applicable and valid. The ColBeRT’s spatial resolution of ~30ms allowed Leifer et al. to utilize the technique for a number of manipulatory actions on their transgenic nematodes. When cholinergic motor neurons of transgenic C. elegans were exposed to laser light, forward motor movement was suppressed and either paralysis or backward movement of the worm was propagated. Similarily, single touch receptors of the worms were also genetically modified to be sensitive to light. In a normal worm, a gentle touch will stimulate these receptors, causing the worm to repel in the opposite direction of movement. In transgenic C. elegans, illuminating these specific receptors with the light from a laser was able to affect the direction of the worm’s movement, just as a physical stimulus would have. Even more astounding, HSNs (hermaphrodite specific neurons), which innervate the vulval region of C. elegans, were also able to be genetically modified and stimulated with light exposure. When a thin laser strip was shone on the HSN region of the worms, involuntary egg-laying was evoked.
Although still in its beginning stages, the ColBeRT technique seems to be a promising solution to overcoming one of the primary difficulties standing in the way of neuroscience. ColBeRT not only highlights cell-to-cell interactions, but also identifies the precise actions of specific neurons, something that had never been thought possible in the past. As technology develops further, perhaps we will soon be able to manipulate the cells of more complex organisms and eventually, even mammals. Optogenetics and techniques like ColBeRT may be the key to discovering the subtleties of different neurons and could even potentially help map out the human brain.
Single Worm Neurons Remotely Controlled with Lasers – Scientific American
The Samuel Lab videos – Vimeo