Blue Light-Induced Gene Expression Alterations in Cultured Neurons Are the Result of Phototoxic Interactions with Neuronal Culture Media

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 (470 nm) 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 h 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 postmitotic 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.

An Improved CRISPR/dCas9 Interference Tool for Neuronal Gene Suppression

“…Total RNA was extracted (RNAeasy kit, Qiagen) and reversetranscribed (iScript cDNA Synthesis Kit, Bio-Rad) following the manufacturers' instructions. cDNA was subject to RT-qPCR for genes of interest in duplicate using a CFX96 real-time PCR system (Bio-Rad) at 95 • C for 3 min, followed by 40 cycles of 95 • C for 10 s and 58 • C for 30 s, followed by realtime melt analysis to verify product specificity, as described previously (Savell et al, 2016(Savell et al, , 2019aDuke et al, 2020). Gapdh was used for normalization via the Ct method (Livak and Schmittgen, 2001).…”

An improved CRISPR/dCas9 interference tool for neuronal gene suppression

“…Primary Rat Brain Cultures. Primary rat cell cultures were generated from embryonic day 18 (E18) rat striatal and hippocampal tissue, as described previously 44,[48][49][50] . Briefly, cell culture plates (Denville Scientific Inc.) were coated overnight with poly-L-lysine (Sigma-Aldrich; 50 µg/ml) supplemented with laminin (Sigma-Aldrich; 7.5µg/mL) and rinsed with diH 2 O. Dissected cortical or hippocampal tissue was incubated with papain (Worthington LK003178) for 25 min at 37°C.…”

“…Total RNA was extracted (RNAeasy kit, Qiagen) and reverse-transcribed (iScript cDNA Synthesis Kit, Bio-Rad) following the manufacturers' instructions. cDNA was subject to RT-qPCR for genes of interest in duplicate using a CFX96 real-time PCR system (Bio-Rad) at 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 s and 58 °C for 30 s, followed by real-time melt analysis to verify product specificity, as described previously 44,49,50 . Gapdh was used for normalization via the ΔΔCt method 53 .…”

Optogenetics: Keep Interpretations Light

“…are very sensitive to changes in temperature, the mere action of shining light may alter cell biology/function. Among other things, photons change the biophysical properties of NMDA receptors (Leszkiewicz et al, 2000) and alter gene expression (Cheng et al, 2016;Volkov et al, 2017;Tyssowski and Gray, 2019;Duke et al, 2020). In these conditions, even if activation of cell X leads to observation Y, while light only (X does not express opsins) does not induce Y, it does not rule out the possibility that Y is the result of the activation of X plus the photon effect (i.e., to obtain Y, you need both X activation and the photon effect).…”