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Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

Kelsey M. Tyssowski, Jesse M. Gray

Preprint posted on March 11, 2019 https://www.biorxiv.org/content/10.1101/572370v2

Why doing the right controls matter: Exposure to blue light triggers changes in neuronal gene expression even in cells without light-sensitive channelrhodopsins

Selected by Zheng-Shan Chong

Background

In 2005, Boyden and colleagues published a paper showing that an algal light-sensing ion channel called Channelrhodopsin-2 could be expressed in neurons and used to control neuronal activity with light [1]. 14 years later, this stimulation paradigm, also known as optogenetics, has established itself as a mainstay of most modern neurobiological studies due to its simplicity and utility in causally dissecting neuronal circuits. Consequently, a large repertoire of tools have been developed around this idea, including new channelrhodopsins sensitive to different wavelengths of light [2, 3] and capable of inhibiting rather than activating neurons [4].

Deciding on a stimulation protocol best suited for an experiment is a multifaceted decision involving choosing a suitable channelrhodopsin, as well as determining the ideal frequency, power and duration of light exposure. As with any other experiment, a key consideration is how the stimulation itself will affect the subject under investigation, independent of the intended channelrhodopsin-mediated response. This preprint describes the effect of high-powered light exposure on Fos, Npas4 and Bdnf transcription in cultured mice cortical neurons which have not been engineered to express channelrhodopsins. Fos is an immediate-early transcription factor in neurons which rapidly increases in expression upon neuronal activity [5], whilst Npas4 and Bdnf are known to regulate synaptic plasticity [6, 7].

Key findings

Using quantitative reverse transcription PCR (RT-qPCR), the authors found that transcript abundance of Fos was increased by approximately 2.5 and 4-fold in cultured mouse cortical neurons after exposure to blue (475 nm) light for 1 or 6 hours, respectively. In contrast, neurons exposed to red (612 nm) or green (530 nm) light did not show a significant change in levels of Fos transcripts. Stimulation at a higher frequency of 100 Hz (up from 10 Hz) resulted in a 12-fold induction of Fos expression with blue light, but not red light. The authors then show that this increase in transcript abundance is not accompanied by increased neuronal activity using multi-electrode array recordings. Finally, they investigate the transcription of more neuron-specific genes Npas4 and Bdnf, which showed a 2 to 2.5-fold increase in transcript abundance after 6 h exposure to blue light, but not red and green light.

Why is it important?

To be clear, this isn’t the first manuscript to show that short wavelength light has an effect on cell physiology. As the authors point out, previous studies have shown that prolonged exposure to high-powered blue light is cytotoxic [8], and can trigger inflammatory factor expression by microglia in the absence of channelrhodopsins [9]. However, this preprint does demonstrate that these changes do not occur in response to longer wavelength light, possibly providing a way to circumvent the unwanted effects of blue light stimulation.

In addition, the engagement of this preprint by the optogenetics and wider neuroscience community on Twitter has illustrated how preprints can greatly facilitate the dissemination of data – especially troubleshooting data which did not make it into the actual paper, in this case.

Questions arising:

One thing to note is that the effect sizes of transcriptional changes shown in this preprint are quite small, as transcript levels typically have to differ by more than 4 times (log2 fold change >= 2) for a gene to be considered differentially expressed. However, as stimulation paradigms can go on for up to 48 hours, it would be interesting to know if transcription linearly correlates with light exposure, or if the induction plateaus off. Conversely, optogenetic experiments that use readouts other than transcription may use much shorter stimulation durations than an hour. Therefore, data on transcript levels after a larger variety of stimulation times could help delineate more clearly the relationship between gene expression and blue light exposure.

Something that isn’t so clear is whether the multi-electrode recordings were made after the same duration of light exposure as the experiments that measured transcript abundance. This is important as recordings showing no difference in neuronal firing taken after 6 hours of stimulation would be much more persuasive than recordings taken at the start of stimulation in showing that the increase in Fos transcription was not accompanied by an increase in neuronal activity.

Lastly, the most interesting question is whether blue light-induced transcriptional changes occur in vivo as well. Since exposure to blue light has been shown to induce macroscopic effects like changes in cerebral blood flow in mice [10], it is not unimaginable that such stimulation could have other effects on cell biology as well.

References

 

[1]        Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Millisecond-timescale,    genetically targeted optical control of neural activity. Nat Neurosci. 2005;8: 1263–        1268.

[2]        Lin JY, Knutsen PM, Muller A, Kleinfeld D, Tsien RY. ReaChR: a red-shifted variant            of             channelrhodopsin enables deep transcranial optogenetic excitation. Nat Neurosci.           2013;16: 1499–1508.

[3]        Berndt A, Schoenenberger P, Mattis J, Tye KM, Deisseroth K, Hegemann P, et al. High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels.            Proc Natl Acad Sci U S A. 2011;108: 7595–7600.

[4]        Wietek J, Wiegert JS, Adeishvili N, Schneider F, Watanabe H, Tsunoda SP, et al.     Conversion of channelrhodopsin into a light-gated chloride channel. Science.       2014;344: 409–412.

[5]        Ghosh A, Ginty DD, Bading H, Greenberg ME. Calcium regulation of gene expression       in neuronal cells. J Neurobiol. 1994;25: 294–303.

[6]        Lu B, Nagappan G, Lu Y. BDNF and synaptic plasticity, cognitive function, and        dysfunction. Handb Exp Pharmacol. 2014;220: 223–250.

[7]        Sun X, Lin Y. Npas4: Linking Neuronal Activity to Memory. Trends Neurosci.                       2016;39: 264–275.

[8]        Stoien JD, Wang RJ. Effect of near-ultraviolet and visible light on mammalian cells in       culture II. Formation of toxic photoproducts in tissue culture medium by blacklight. Proc Natl Acad Sci U S A. 1974;71: 3961–3965.

[9]        Cheng KP, Kiernan EA, Eliceiri KW, Williams JC, Watters JJ. Blue Light Modulates Murine Microglial Gene Expression in the Absence of Optogenetic Protein    Expression. Sci Rep. 2016;6: 21172.

[10]      Rungta RL, Osmanski B-F, Boido D, Tanter M, Charpak S. Light controls cerebral   blood flow in naive animals. Nat Commun. 2017;8: 14191.

Tags: neuroscience, optogenetics, reproducibility

Posted on: 21st March 2019

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