Abstract
Blue wavelength light is used as an optical actuator in numerous optogenetic technologies employed in neuronal systems. However, the potential side effects of blue light in neurons has not been thoroughly explored, and recent reports suggest that neuronal exposure to blue light can induce transcriptional alterations in vitro and in vivo. Here, we examined the effects of blue wavelength light in cultured primary rat cortical cells. Exposure to blue light (470nm) resulted in upregulation of several immediate early genes (IEGs) traditionally used as markers of neuronal activity, including Fos and Fosb, but did not alter the expression of circadian clock genes Bmal1, Cry1, Cry2, Clock, or Per2. IEG expression was increased following 4 hours of 5% duty cycle light exposure, and IEG induction was not dependent on light pulse width. Elevated levels of blue light exposure induced a loss of cell viability in vitro, suggestive of overt phototoxicity. Induction of IEGs by blue light was maintained in cortical cultures treated with AraC to block glial proliferation, indicating that induction occurred selectively in post-mitotic neurons. Importantly, changes in gene expression induced by blue wavelength light were prevented when cultures were maintained in a photoinert media supplemented with a photostable neuronal supplement instead of commonly utilized neuronal culture media and supplements. Together, these findings suggest that light-induced gene expression alterations observed in vitro stem from a phototoxic interaction between commonly used media and neurons, and offer a solution to prevent this toxicity when using photoactivatable technology in vitro.
Significance Statement Technology utilizing blue wavelength light is increasingly utilized in neuroscience, and recent reports have noted unintended gene expression alterations during light exposure in vitro. Here, we identify light-induced gene expression alterations in rat cortical cultures, illustrate that this induction coincides with a loss of cell viability, and show that light induced gene induction is dependent on the culture media utilized in these experiments. We demonstrate that these unintended effects can be prevented by using phototinert media during to light exposure in vitro, opening the door for extended light exposure experiments when utilizing powerful optical techniques in neuronal cultures.
Footnotes
The authors declare no competing financial interests.
This work was supported by NIH grants DA039650, DA034681, and MH114990 (J.J.D.), NS061788 (C.G.D.), DA042514 (K.E.S.). Additional assistance to J.J.D. was provided by the UAB Pittman Scholars Program.
NIH grants DA039650, DA034681, and MH114990 (J.J.D.), NS061788 (C.G.D.), DA042514 (K.E.S.). Additional assistance to J.J.D. was provided by the UAB Pittman Scholars Program.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.
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