Blue light-induced gene expression alterations in cultured neurons are the result of phototoxic interactions with neuronal culture media

Abstract

Blue waveform light is used as an optical actuator in numerous optogenetic technologies employed in neuronal systems. However, the potential side effects of blue waveform 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 waveform light in cultured primary rat cortical neurons. 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. Changes in gene expression induced by blue waveform light were prevented when neurons were cultured 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.

Resonance energy transfer sensitises and monitors in situ switching of LOV2-based optogenetic actuators

“…Moreover, biological end-points of interest may require maintenance of actuation for prolonged periods of time, unlike the milliseconds of activation in channelrhodopsin applications. For popular blue-light photosensors, using endogenous flavins rather than exogenous chromophores like phytochromobilin, this is an additional challenge because even low doses of blue light can perturb biological systems in the long term 1,2 .…”

“…Blue-light photoreceptors such as LOV2 are among the most popular cellular optogenetics photosensors 1 . However, blue light can perturb cells, potentially complicating long-term (hoursdays) optogenetic modulation 1,2 . LOV2 can be indirectly sensitised by tuning relaxation 3 , but this compromises time-resolution.…”

Inducible CRISPR epigenome systems mimic cocaine induced bidirectional regulation of Nab2 and Egr3

“…Between these two conditions, we did not observe significant changes in Nab2 or Egr3 mRNA levels (Student’s t test; mRNA sample n= 3 per group, t (4) = 1.183, p=0.3023; mRNA sample n= 3 per group, t (4) = 2.126, p=0.1006; Fig5 D). To avoid potential blue light-induced gene expression alterations from phototoxicity, we changed the culture media to Photostable NEUMO media at least 3 hours prior to the start of light stimulations in all of the experiments performed in this study (Duke et al, 2019).…”

Repetitive short‐pulsed illumination efficiently activates photoactivatable‐Cre as continuous illumination in embryonic stem cells and pre‐implantation embryos of transgenic mouse

“…In general, strong and long‐period blue light illumination causes phototoxicity in living cells (Allen, Singer, & Boyden, 2015; Duke, Savell, Tuscher, Phillips, & Day, 2020) (Figure S2). To reduce illumination energy while yielding high recombination rates, we tested repetitive pulsed illuminations.…”