Optogenetic Light Alters Gene Expression in Wild-type Microglia
New research indicates that the use of optogenetic light for cell-selective control may have effects on surrounding cells, an important consideration for the use of this recently popularized neuroscience technique. Kevin Cheng, a graduate student in the lab of Dr. Jyoti Watters at the University of Wisconsin-Madison discovered this phenomenon while performing research with microglia.
For those unfamiliar with optogenetics, this technique utilizes the ability of light-responsive proteins to control cells. By targeting these transporters to certain cells, scientists can activate cells of their choosing by exposure to blue and other wavelengths of light. This also allows cells to be turned on and off at the researcher’s choosing. This technique was originally used to excite or inhibit neurons, though it has now expanded to various cell types and can also be used to control other functions of the cell such as GPCR activity.
To learn more about the basics of optogenetics and why it is so cool, check out this clip by Nature Video
Originally, Cheng was trying to transduce microglia cells with the construct for the light-responsive protein. They expected that optogenetic light would alter gene expression in the optogenetic protein-containing cells without effects on the wild-type cells lacking the light-responsive transporter. However, what they found was very different. When exposed to the light, control microglia lacking the light-responsive protein showed changes in gene expression. “The first time that I brought this to our weekly lab meeting, they laughed. Do you think it is possible that this is happening?” asked Cheng. “And they said no, it’s not.” After rigorous experimentation, Cheng was able to prove to his labmates that indeed, there were side effects of optogenetic exposure in normal cells.
To prove his hypothesis, Cheng utilized a black-walled 96-well plate aligned with an LED array. This setup allows for control of light duration and intensity in each well. Microglia were grown on the plate, then exposed to blue light at 450 nm, a common wavelength used for optogenetic experiments. Following exposure, pro-inflammatory gene expression was determined by quantitative RT-PCR.
Since the inflammatory response within the central nervous system is mediated by activated microglia, Cheng compared gene expression in both basal and LPS-activated microglia. He also evaluated the difference between a single bolus of light and a light pattern commonly used in optogenetics.
Of the pro-inflammatory genes measured, COX-2 expression was induced in basal cells exposed to both bolus and optogenetic light patterns. In LPS-activated cells, the genes IL-1b, iNOS, COX-2, IL-10, IL-6, and VEGF were decreased and IGF-1 was increased after bolus light exposure. Further, IL-1b, iNOS, COX-2, and IL-10 also exhibited decreases in gene expression following light with LPS activation. To make sure the energy dose delivered was not harmful to the cells Cheng also measured the potential for blue light to induce cell death and DNA damage in microglia, and found no effect. This suggests that wild-type microglia respond to blue light and that it may exert a surprisingly anti-inflammatory effect.It also demonstrates that optogenetic patterened light exposure can have effects on wild-type cells near the cells of interest.
To date, this is the first report of non-specific effects of optogenetic light. This study has important implications on the field of optogenetics, a technique which has named “Method of the Year” in 2010 by Nature Methods. Cheng plans to expand his experiments to include other cell types and to explore the mechanism behind this unexpected phenomenon.
For the abstract of this work, visit here.
To learn more about research in the Watters Lab, click here.