Red Light Optogenetics in Neuroscience

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Red Light Optogenetics in Neuroscience © 2022 Lehtinen, Nokia and Takala. Published version Lehtinen, Kimmo; Nokia, Miriam S.; Takala, Heikki Lehtinen, K., Nokia, M. S., & Takala, H. (2022). Red Light Optogenetics in Neuroscience. Frontiers in Cellular Neuroscience, 15, Article 778900. https://doi.org/10.3389/fncel.2021.778900 2022 fncel-15-778900 December 21, 2021 Time: 15:2 # 1 REVIEW published: 03 January 2022 doi: 10.3389/fncel.2021.778900 Edited by: Andreas Möglich, University of Bayreuth, Germany Reviewed by: Tommaso Patriarchi, University of Zurich, Switzerland Bernd Kuhn, Okinawa Institute of Science and Technology Graduate University, Japan *Correspondence: Heikki Takala [email protected] †These authors have contributed equally to this work Specialty section: This article was submitted to Cellular Neurophysiology, a section of the journal Frontiers in Cellular Neuroscience Received: 17 September 2021 Accepted: 02 December 2021 Published: 03 January 2022 Citation: Lehtinen K, Nokia MS and Takala H (2022) Red Light Optogenetics in Neuroscience. Front. Cell. Neurosci. 15:778900. doi: 10.3389/fncel.2021.778900 Red Light Optogenetics in Neuroscience Kimmo Lehtinen1, Miriam S. Nokia2,3†and Heikki Takala1*† 1Department of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland, 2Department of Psychology, University of Jyväskylä, Jyväskylä, Finland, 3Centre for Interdisciplinary Brain Research, University of Jyväskylä, Jyväskylä, Finland Optogenetics, a field concentrating on controlling cellular functions by means of light-activated proteins, has shown tremendous potential in neuroscience. It possesses superior spatiotemporal resolution compared to the surgical, electrical, and pharmacological methods traditionally used in studying brain function. A multitude of optogenetic tools for neuroscience have been created that, for example, enable the control of action potential generation via light-activated ion channels. Other optogenetic proteins have been used in the brain, for example, to control long-term potentiation or to ablate specific subtypes of neurons. In in vivo applications, however, the majority of optogenetic tools are operated with blue, green, or yellow light, which all have limited penetration in biological tissues compared to red light and especially infrared light. This difference is significant, especially considering the size of the rodent brain, a major research model in neuroscience. Our review will focus on the utilization of red lightoperated optogenetic tools in neuroscience. We first outline the advantages of red light for in vivo studies. Then we provide a brief overview of the red light-activated optogenetic proteins and systems with a focus on new developments in the field. Finally, we will highlight different tools and applications, which further facilitate the use of red light optogenetics in neuroscience. Keywords: optogenetics, neuroscience, brain, neuron, near-infrared, opsin, phytochrome INTRODUCTION Optogenetics is a field of science focusing on controlling cellular functions with light-sensing proteins (Deisseroth et al., 2006). Neuroscience has always been the major target for optogenetic systems, and they are well established as neuroscientific research methods (Mahmoudi et al., 2017). The goal in most neuroscientific studies conducted in living animals in vivo is to find out the role of a certain structure or cell type in cognition and behavior. To get to this goal, researchers try to manipulate the function of a specific cell type within a specific brain structure during a specific time, while maintaining the rest of the brain as intact as possible. For this purpose, the spatiotemporal precision of optogenetics is superior compared to traditional research methods in neuroscience. The temporal resolution of optogenetics is dependent on light exposure (i.e., turning the light on or off). The spatial resolution is a combination of gene technology and the accurate focusing of the Abbreviations: AAV, adeno-associated virus; CBCRs, cyanobacteriochromes; GRIN, gradient index; EEG, electroencephalography; PBM, photobiomodulation; PDB, Protein Data Bank; PIF, phytochrome-interacting factor; NIR, near-infrared; MIR, magnetic resonance imaging; UPNC, upconversion nanoparticles. Frontiers in Cellular Neuroscience | www.frontiersin.org 1January 2022 | Volume 15 | Article 778900 fncel-15-778900 December 21, 2021 Time: 15:2 # 2 Lehtinen et al. Red Light Optogenetics light on the target. Gene technology ensures the expression of the optogenetic system in the specific cells and the localization in targeted cellular compartments (Ziegler and Möglich, 2015). Optogenetic systems are based on light-sensitive proteins which can be divided into indicators and actuators (Rost et al., 2017). Indicators are utilized to observe different cellular processes, as their optical properties are sensitive to changing cellular parameters, like pH and Ca2+(Germond et al., 2016). Our review focuses on actuators. Actuators are mainly natural sensory photoreceptors used by various life forms to adjust their behavior to the present light conditions. Most actuators require an extrinsic chromophore to function. Chromophores are small molecules capable of absorbing light at a certain wavelength range between UV and infrared light. In optogenetic systems, absorbance by the chromophore triggers conformational changes in the actuator, which in turn usually activates the function. This function can be based on several mechanisms, like light-induced dimerization, ion channeling, or enzymatic activity (Figure 1). Modification of the actuators through protein engineering enables control of a vast variety of different cellular processes (Rost et al., 2017). One of the major obstacles in optogenetics is the fact that light does not easily penetrate biological tissues (Chernov et al., 2017). Biological tissues have several different structures and molecules which readily scatter, absorb, or reflect visible light (Ash et al., 2017). In vivo studies suffer greatly from this light penetration problem. The problem can be circumvented, for example, by intracranial light sources that significantly increase the invasiveness of the procedure, and by using higher light intensities that damage the brain tissue (Hu et al., 2017;Pisanello et al., 2018). Different wavelengths of light have different abilities to penetrate tissues. Generally, the light penetration increases as the energy of the light decreases. For example, in the visible light spectrum, tissue penetration by blue light is lower compared to red light (Ash et al., 2017). Most of the existing optogenetic systems still utilize blue light (Losi et al., 2018). The advances of red light in optogenetics can alleviate the light penetration problem (Chernov et al., 2017). In this review, we first describe the advantages of red light-operated optogenetic actuators compared to blue light-based systems in neuroscience. Then we describe the current state of red light optogenetics in detail and highlight the recent advances in the field. Lastly, we introduce attempts to improve the applicability of optogenetics in neuroscientific research. RED VERSUS BLUE In the first section, we compare red light to blue light in terms of their physical and biological properties for neuroscientific optogenetic in vivo studies. The reason for the comparison is to highlight the beneficial properties of red light. A comparison is made with blue light-operated systems, which comprise the majority of the published optogenetic systems. Three relevant matters to be compared between optogenetics utilizing blue vs. red light are light penetration properties, possible side effects, and chromophore availability. It should also be noted that shorter wavelengths of visible light have lower tissue penetration than red light (Figure 2). Here we define the wavelength ranges for blue (∼430–500 nm), red (∼630–710 nm), and near-infrared (NIR) light (∼710– 1400 nm), as depicted in Figure 2. In discussing specific illumination schemes, we consider red and NIR light separately to inform about the specific illumination ranges. Once red light in optogenetics is discussed in general, red and NIR wavelengths are not specified separately. Light Penetration Properties In in vitro studies, illumination is relatively easy, as the light path is not obstructed. However, attaining sufficient illumination to activate optogenetic systems in vivo can be challenging. Various molecules and structures in tissues scatter, absorb, and reflect light differently, which leads to large variations in light penetration (Jacques, 2013). When targeting the brain for illumination of optogenetic systems, the biological tissues to be penetrated are the skin and bone with connective and neural tissues underneath and blood circulating through them. Scattering is the most significant factor diminishing light penetration in biological tissues. First, scattering of light is wavelength-dependent, with shorter wavelengths scattering more. Therefore, the tissue penetration of blue light is low compared to red light (Al-Juboori et al., 2013;Azimipour et al., 2014;Yona et al., 2016). Second, due to constant variation of the refraction index at liquid-lipid interfaces, the composition of the biological tissue affects scattering (Taddeucci et al., 1996; Xu and Alfano, 2005;Jacques, 2013). For example, bone induces less scattering than skin due to its more homogenous structure (Lister et al., 2012;Soleimanzad et al., 2017). Interestingly, scattering of the skull significantly increases as the animal matures and the skull composition becomes more inorganic (Soleimanzad et al., 2017;Zhao et al., 2017). Furthermore, light scattering varies significantly between different brain structures. The main determining factor is the density of cells in the area, as cell nuclei scatter light much more than axons and dendrites (Al-Juboori et al., 2013;Favre-Bulle et al., 2015). However, also the directional organization and myelination of axons increase scattering, for example, in the spinal cord (De Paoli et al., 2020). Absorption is the other main physical phenomenon decreasing light penetration in biological tissues. Small tissue-specific molecules play an important role in that: skin contains natural photoprotective pigment, melanin, which significantly decreases the penetration of blue light (Jacques and McAuliffe, 1991). Another specific lightabsorbing molecule to be considered is blood hemoglobin, which has higher blue absorption than red (Zijlstra et al., 1991). Besides the tissue-specific molecules, fat and water also affect light absorption (Jacques, 2013). Absorption by fat is relatively similar under both red and blue light. Still, absorption characteristics can vary significantly within these wavelength ranges (Hale and Querry, 1973;Anderson et al., 2006). Water mainly absorbs light at longer NIR wavelengths, but the wavelengths applied in optogenetics Frontiers in Cellular Neuroscience | www.frontiersin.org 2January 2022 | Volume 15 | Article 778900 fncel-15-778900 December 21, 2021 Time: 15:2 # 3 Lehtinen et al. Red Light Optogenetics FIGURE 1 | Types of optogenetic actuator systems. The six major actuator systems and the example studies related to them pertain to ion channels (Klapoetke et al., 2014), homodimerization (Schmidl et al., 2019), heterodimerization (Kaberniuk et al., 2016), enzyme activity (Gasser et al., 2014), allosteric control (Strickland et al., 2008), and signaling proteins (Kim et al., 2005). The components of the systems with similar function are depicted with a similar shape. The shapes are not accurate depictions of the actual proteins and are not to scale. The illumination of each actuator is depicted as lightning and colored according to each system. The figure was created with BioRender.com. are in the low absorption ranges (Hale and Querry, 1973; Sordillo et al., 2017). The third physical factor, reflection, occurs mainly in the skin. However, its total effect on light penetration is minuscule (Lister et al., 2012;Favre-Bulle et al., 2015) and is not therefore considered further here. Taken together, red and NIR light penetrate both skin and bone better than blue light. For example, red light penetration in skin is 4–5 mm (Ash et al., 2017), whereas for blue light this is only ∼1 mm (Jacques, 2013). Of course, the optical properties of the scalp and the skull can be altered by thinning, or the tissues can be removed altogether and replaced with transparent material to create a window into the brain (Cramer et al., 2021). However, the use of red or NIR light could enable in vivo manipulation of the brain in animal models without the need for this, as the mouse scalp is 0.3–0.6 mm (Azzi et al., 2005) and the skull is 0.3– 0.4 mm thick (Jacques, 2013;Park et al., 2015). Light penetration into brain tissue itself is dependent on the targeted structure but is generally more effective than in skin (Jacques, 2013). As the maximal sagittal length of the mouse brain is approximately 5–6 mm (Diem et al., 2016), red light can enable non-invasive optogenetic manipulation of even the deepest brain structures in mice (Figure 2). This puts the advantages of red light penetration into perspective, if at first glance they seemed minuscule. Possible Side-Effects The energy transfer from light can cause side effects in living tissues. UV light has long been known to cause phototoxic damage when absorbed, due to the high energy transfer (Hu et al., 2017). This suggests a higher probability of phototoxic effects with blue light in optogenetic experiments, compared to the lower energy of red light. The actual phototoxic effects can be well mitigated by phototoxicity controls and careful illumination protocols, which address the total power applied to the target (Ziegler and Möglich, 2015;Smart et al., 2017). Similarly, absorbance by water in the red and infrared spectral range can heat up the tissue and cause thermal damage (Ash et al., 2017). Again, a well-planned illumination scheme and pulsed light can significantly mitigate the risk of overheating (Ziegler and Möglich, 2015;Zheng et al., 2017). Natural light reception has emerged as a potential cause of significant disturbance in optogenetic studies. The brain and other non-retinal tissues contain UV and blue light photoreceptors, which apparently can even be activated Frontiers in Cellular Neuroscience | www.frontiersin.org 3January 2022 | Volume 15 | Article 778900 fncel-15-778900 December 21, 2021 Time: 15:2 # 4 Lehtinen et al. Red Light Optogenetics FIGURE 2 | Tissue penetration of different wavelengths of light. Tissue penetration is illustrated from the coronal view of the rodent brain. The light penetration depths, scales, and shown wavelengths are indicative (Jacques, 2013;Diem et al., 2016;Ash et al., 2017). The figure was created with BioRender.com. transcranially (Flyktman et al., 2015;Nissilä et al., 2016;Zhang K. et al., 2020). This could become a problem when interpreting the outcome of experimental manipulation. For example, in optogenetic apoptosis studies (Nihongaki et al., 2014;Hughes et al., 2016;Ji et al., 2019), the activation of cell survival or cell death pathways through natural photoreceptors could protect the target cells from experimental manipulation or cause unwanted damage, respectively (Yoshimoto et al., 2018;de Assis et al., 2020). Blue light can also activate several natural pathways in neural cells even without applied optogenetic actuators (Tyssowski and Gray, 2019). When it comes to red light, the mechanisms behind photobiomodulation (PBM) can cause similar problems. PBM is a field that applies low-power red light illumination for therapeutic purposes. The mechanisms of PBM are still under debate, but are possibly related to mitochondrial enzymes, the production of reactive oxygen species, and endogenous red light-sensing proteins (Hamblin, 2016). Outside of different survival pathways, transcranial PBM has been shown to modulate neural activity, which was apparent through changes in electroencephalography (EEG) even in humans (Zomorrodi et al., 2019;Wang et al., 2021). To summarize, the effects of both endogenous opsin activity (induced by blue light) and PBM (induced by red light) need to be considered in each study. Chromophore Availability The utility of an optogenetic actuator is highly dependent on the availability of a suitable chromophore (i.e., light-absorbing molecule) in the target. In principle, actuators without a need for an extrinsic chromophore would be ideal, but the existing actuators with intrinsic chromophores utilize only strongly scattering and phototoxic UV light (Wu et al., 2012;Zhou et al., 2012). Most of the blue light-sensing actuators utilize different flavin molecules (Losi et al., 2018). Flavins are essential to major redox reactions, like NADH/NAD metabolism, and are readily available in all cell types (Tomiki and Saitou, 2004). Different widely used opsin proteins are activated by either blue or red light (Nagel et al., 2003;Chuong et al., 2014). Their chromophore, a derivate of vitamin A called retinal, seems to be available in most in vivo models in broad utilization in neuroscience (Deisseroth, 2015). Most red light-driven optogenetic actuators utilize different bilin molecules as their chromophores, the availability of which varies between the target cells (Chernov et al., 2017). Bacterial phytochromes utilize biliverdin, a breakdown product of heme, which should be available in sufficient concentrations for in vivo experiments in most animal models (Tenhunen et al., 1969;Bhoo et al., 2001). Plant phytochromes, cyanobacterial phytochromes, and cyanobacteriochromes (CBCRs) depend on bilins, phytochromobilin, or phycocyanobilin, which are absent from animal cells. Therefore, their utilization in animal studies is limited. The utilization of these phytochromes in animal cells therefore requires the external addition of chromophores or the implementation of a genetically engineered chromophoreproducing machinery (Buckley et al., 2016;Uda et al., 2017). For example, phycocyanobilin can be made available in Frontiers in Cellular Neuroscience | www.frontiersin.org 4January 2022 | Volume 15 | Article 778900 fncel-15-778900 December 21, 2021 Time: 15:2 # 5 Lehtinen et al. Red Light Optogenetics mammalian cells by co-expressing the enzymes heme oxygenase 1 (HO1) and phycocyanobilin:ferredoxin oxidoreductase (PcyA) (Uda et al., 2017). Blue light-driven optogenetic actuators seem to have a slight edge in chromophore availability. Still, several advances have been made to ease the bilin availability for red light-sensing optogenetic actuators. For example, the genetically engineered production of bilins has been further improved (Kyriakakis et al., 2018;Uda et al., 2020), and biliverdin-utilizing CBCRs have been either discovered or created by mutations (Fushimi et al., 2019; Moreno et al., 2020;Tachibana et al., 2021). RED LIGHT-SENSING OPTOGENETIC ACTUATORS As stated in the previous chapters, red light has its advantages in optogenetic research in neuroscience. In this chapter, we introduce three classes of red light-sensing optogenetic actuators: rhodopsins, phytochromes, and cyanobacteriochromes (Figure 3). We first give a short introduction of the structure and function of these proteins and then present examples of how to create improved optogenetic actuators. We also highlight the development of specific binding partners of these actuators, which constitute an important part of the red light optogenetic toolbox. Rhodopsins The field of optogenetics has been built on neuroscientific research with opsins (Deisseroth, 2015). The first optogenetic systems were based on opsins and new applications are still routinely published (Zemelman et al., 2002;Essig et al., 2021; Zhang et al., 2021). Opsins are heptahelical transmembrane proteins that are called rhodopsins once covalently bound to their chromophore, retinal (Palczewski et al., 2000;Figure 3). Optogenetics has mainly utilized opsin types that act as lightactivated ion channels and ion pumps (Deisseroth, 2015). These opsins show specificity to certain ions, based on the size and the charge of the channel (Luecke et al., 1999;Wietek et al., 2014). Different opsin channels match perfectly with the electrical signal transmission of the neurons in the central nervous system. A positive or negative ion flow into a neuron changes the membrane potential and can either facilitate or inhibit the firing of an action potential (Vierock et al., 2021). Besides ion channels, some rhodopsins can act as light-activated G-protein coupled receptors or enzymes, like phosphodiesterases (Dixon et al., 1986;Yoshida et al., 2017). These rhodopsins could be utilized in the manipulation of signaling pathways or cellular concentrations of second messengers with light (Jastrzebska et al., 2016;Tian Y. et al., 2021). Phytochromes and Cyanobacteriochromes Phytochromes are natural dimers of two photosensory modules (Figure 3), which are accompanied by enzymatic effector domains (Karniol et al., 2005). The photosensory module is composed of a PAS (Period/ARNT/Single-minded), a GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA), and a PHY (Phytochrome-specific) domain, which are linked together by an α-helical backbone (Burgie et al., 2014). The light-activated conformational changes of phytochromes are particularly interesting among optogenetic actuators (Takala et al., 2020). During the conformational changes, PHY domains are moved in relation to each other, accompanied by changes in their secondary structure (Takala et al., 2014, 2018;Björling et al., 2016). The activity of phytochromes, which can be the activity of an attached effector domain or changes in protein interactions, is controlled by these changes in the photosensory module. In optogenetics, these changes are harnessed by different effector domains or binding partners (Dubreuil et al., 2017;Multamäki et al., 2021). The binding partners allow the creation of different localization systems, which, for example, can modulate the local concentrations of the attached signaling proteins (Chernov et al., 2017). Non-photochromic actuators are proteins that can only be activated with light. Therefore, the duration of their active period is not dependent only on the duration of the light exposure. After the light has been switched off, the non-photochromic actuator will return to the most thermodynamically stable conformation, called a dark state, through dark reversion (i.e., thermal motion) (Ziegler and Möglich, 2015). Different actuators have been designed with desired dark reversion times in relation to biological functions, like submillisecond reversion for neural firing (Klapoetke et al., 2014). Phytochromes, however, are photochromic proteins, that is, they can be shuttled between active and inactive conformations by different wavelengths of red light (Rockwell et al., 2006). The photochromic actuators therefore allow increased control of the signal length. Most phytochromes adopt a red light-absorbing (Pr) state as their dark state, whereas red light causes them to convert to a far-red light-absorbing (Pfr) state. The Pfr state can then absorb NIR light and return the phytochrome back to the Pr state (Takala et al., 2014). Some phytochromes have adopted the Pfr state as their dark-adapted state, leading to a reverted photocycle in these bathy phytochromes (Rottwinkel et al., 2010). The photochromic capacity of the phytochromes has been only marginally utilized in optogenetics, although it has obvious potential to study different reversible biological functions. For example, photochromic optogenetic apoptosis systems could be used to study anastasis, the natural reversion of the cell death (Tang and Tang, 2018). Cyanobacteriochromes have been utilized relatively little as optogenetic actuators compared to the other two red light-sensing actuator classes described above. CBCRs are photochromic relatives of the phytochromes, as they are composed of a singular GAF domain and bind a bilin chromophore (Figure 3;Xu et al., 2020). The smaller size and the increased photochromic capabilities can drive their utilization in future research. Development of the Proteins One important way to improve the utilization of red light in optogenetics is to gain more knowledge about the actuator proteins. New knowledge can then be utilized by genetically Frontiers in Cellular Neuroscience | www.frontiersin.org 5January 2022 | Volume 15 | Article 778900 fncel-15-778900 December 21, 2021 Time: 15:2 # 6 Lehtinen et al. Red Light Optogenetics FIGURE 3 | Red light optogenetic actuator classes. Example structures of the three classes of the red light optogenetic actuators. Note that only the photosensory fragment of each protein is shown. The structures are to scale relative to each other. Each structure was derived from the following Protein Data Bank (Berman et al., 2000) coordinates: 3AYN (rhodopsin) (Murakami and Kouyama, 2011), 4O0P (phytochrome) (Takala et al., 2014), and 6UV8 (cyanobacteriochrome) (Bandara et al., 2021). The figure was created with the PyMOL Molecular Graphics System version 2.3.3 (Schrödinger, LLC). modifying the protein for more desirable attributes, like lower dark activity. Novel actuators and binding partners can also be found from nature, which can offer unprecedented options for optogenetic control. New knowledge has been gained on the structure of red light-sensing opsins (Oda et al., 2018) and the activation dynamics of CBCRs (Wang D. et al., 2020;Ruf and Buhrke, 2021). Novel advances with phytochromes include the structural characterization of the plant phytochrome (Tahir ul Qamar et al., 2021) and increased knowledge about signal transduction in the bacterial phytochromes (Böhm et al., 2021). One intriguing discovery has revealed that a bacterial phytochrome is capable of two-photon absorption. Therefore, instead of a single higherenergy photon, two lower-energy photons can be used to drive the conformational changes in bacterial phytochromes (Sokolovski et al., 2021). This means that even NIR light with a long wavelength (∼1200–1300 nm) could be used in phytochromebased studies. Different dimerization-based localization systems (Figure 1) are an important subset of the optogenetic toolbox for neuroscience. They can be based on homodimerization (i.e., actuator-actuator interaction) or heterodimerization (i.e., actuator-binding partner interaction). Red light-controlled homodimerization has been mainly based on a cyanobacterial phytochrome Cph1 (Schmidl et al., 2019;Sentürk et al., 2019; Rasoulinejad et al., 2020). Homodimerization systems only need one vector to be transported to the target, which decreases the complexity of the study. Heterodimerization systems usually require two vectors, but the small size of certain binding partners may enable the use of a single vector for both components (Kaberniuk et al., 2021;Kuwasaki et al., 2021). For red light-controlled heterodimerization systems, the development of binding partners for phytochromes and CBCRs is crucial for attaining improved optogenetic systems. The first systems were based on the plant phytochrome B and its binding partner Phytochrome-Interacting Factor (PIF) (Shimizu-Sato et al., 2002). These systems are still routinely utilized despite the hindrance of their larger size and complicated application due to the non-mammalian chromophore (Raghavan et al., 2020;Fonin et al., 2021). However, successful efforts have been made to decrease the size of PIF and increase its binding affinity to phytochrome (Golonka et al., 2019;Yousefi et al., 2019). The first bacterial heterodimerization system, which is based on the bacterial phytochrome form Rhodopseudomonas palustris (RpBphP1) and its binding to PpsR2, suffers from a large size and self-oligomerization (Kaberniuk et al., 2016). PpsR2 has been improved by determining the crucial structures required for the actuator binding. The result, QPAS1, is three times smaller and does not self-oligomerize (Redchuk et al., 2017). However, the QPAS1 system has been shown to have relatively high dark activity, which significantly hinders its future utilization (Kuwasaki et al., 2021). Fortunately, two very promising, smaller partners that bind to the photosensory module of a bacterial phytochrome from Deinococcus radiodurans have been published: nanoREDs are optimized nanobodies, that is, engineered singledomain antibodies from camelids (Huang et al., 2020); MagRED is an optimized Affibody, that is, a domain of an immunoglobulin-binding bacterial surface protein (Kuwasaki et al., 2021). Direct comparison of nanoREDs and MagRED has demonstrated their superiority to older QPAS1-based system. Both MagRED and nanoREDs are shown to function in vivo, although the MagRED-based applications were more complex. The D. radiodurans phytochrome has slow dark reversion time, which enables long sustained activation of the optogenetic system (minutes vs. hours). Therefore, their activity is also sustained longer, compared to the QPAS1 system (Huang et al., 2020; Kuwasaki et al., 2021). Frontiers in Cellular Neuroscience | www.frontiersin.org 6January 2022 | Volume 15 | Article 778900 fncel-15-778900 December 21, 2021 Time: 15:2 # 7 Lehtinen et al. Red Light Optogenetics The optogenetic toolbox based on CBCRs has remained very light compared to the one based on phytochromes. Jang et al. (2019) have published in bioRxiv the first CBCR-based binding systems, collectively termed as BAm. BAm has been optimized from a domain of an albumin-binding bacterial surface protein. Different variants of BAm selectively bind different states of the CBCRs but require green light for the photochromic function. In addition, they have only been shown to function in yeasts (Jang et al., 2019). Lastly, it is always wonderful to find something completely new: new opsin chloride pumps, RubyACR, have been discovered from aquatic protists. They are now one of the most red-shifted anion-passing opsins, which makes their future application in optogenetics relatively likely (Govorunova et al., 2020). An even more curious finding is the new cationic fungal rhodopsinguanylyl cyclase, NeoR. It is the only known opsin to be active solely as a hetero-oligomer with two other cyclases of the same species. NeoR is also photochromic, as it is activated by UV light but deactivated by NIR. Interestingly, its NIR-absorbing state is highly fluorescent at NIR wavelengths. The cyclase activity can also be induced by blue light through the oligomerizing opsin (Broser et al., 2020). The application of NeoR for optogenetics can have its problems, but new applications are expected due to its variant qualities. RED LIGHT-SENSING SYSTEMS AND THEIR APPLICATIONS In this section, we focus on red light-sensing actuator systems, which have been published during the last few years. The reason for the selected time period is the excellent review of the older systems by Chernov et al. (2017) and the fact that during this time the utilization of red light in optogenetics has increased rapidly. We have a special focus on the actuator systems already applied in neuroscience, but we also speculate on the potential neuroscientific uses for other systems. Still, it is worth mentioning that new red optogenetic indicators are also constantly developed. For example, a new calcium indicator and a new voltage indicator have been published in the last 2 years (Beck and Yiyang, 2019; Shemetov et al., 2020). Opsin-Based Applications Opsins are the most utilized and developed systems in optogenetics. This also reflects on the volume of new publications with red light-activated opsins (Table 1). Sensory restoration with Chrimson opsins has been one of the most published areas (Bali et al., 2021;Huet et al., 2021). The cation channel Chrimson has variants with faster activation/inactivation kinetics than the original. They are therefore more suited for the high temporal resolution required to coincide with human senses (Klapoetke et al., 2014;Mager et al., 2018). Restoration of hearing and sight can be achieved by light activation of ganglion cells, which normally relay input signal from intact sensory receptors to the brain (Bali et al., 2021;Gauvain et al., 2021). Restoration of sight has also been studied with other intermediate cells and opsins: for example, with the inhibition of certain inhibitory neurons by the red light-sensing chloride pump opsin eNpHR 3.0 (Masuda and Takahashi, 2021). Restoration of hearing is still in its preliminary stages, while restoration of sight has shown utility in non-human primates and even proceeded to clinical trials (Bali et al., 2021;Gauvain et al., 2021;Sahel et al., 2021). The most remarkable advance is the partial restoration of sight in a blind patient with retinitis pigmentosa. This was achieved by ChrimsonR activation of the retinal ganglion cells with light-stimulating goggles. The goggles created a visual stimulus by means of a neuromorphic camera. It was also the first reported case of partial functional recovery in a neurodegenerative disease after optogenetic therapy (Sahel et al., 2021). Other red light opsins have still been actively present in neuroscientific research. ReaChR and MCO have been utilized in the restoration of sight in mice (Batabyal et al., 2020;Lindner et al., 2020). They have also had interesting utilities in pain research (Narcisse et al., 2021;Yi et al., 2021). Microglia, which are neural macrophages, function partly by ionotropic signaling. Prolonged signaling can induce a further inflammatory response, which has been linked to chronic pain. ReaChR stimulation in the spinal cords of mice has also induced an inflammatory response with chronic neural pain (Narcisse et al., 2021). MCO II has been used for pain modulation based on interfering with pain signaling at the limbic cortex. Activation of the inhibitory neurons in the anterior cingulate cortex has induced reduction in the pain response in mice (Yi et al., 2021). At the same time, the anion opsin Jaws and cationic ChRmine have been used to study epilepsy (Chen R. et al., 2020;Farzaneh et al., 2020). In a study featuring Jaws, mesial temporal lobe epilepsy -mimicking seizures has been induced in rats. Jaws activation in the neurons of the hippocampal CA3 region has nullified the seizures (Farzaneh et al., 2020). ChRmine has been used to activate hippocampal inhibitory GABAergic interneurons in mice, which has significantly shortened the seizures. The most impressive part of the study is that light has been administrated non-invasively with transcranial illumination. In the same study, dopamine neurons of the limbic ventral tegmental area have been activated by ChRmine. This has induced feeding-related behavioral changes in the mice. Here, the transcranial activation of ChRmine has been successfully achieved even at the depth of 7 mm (Chen R. et al., 2020). Phytochrome-Based Applications The majority of the new red light-sensing actuator systems outside of opsins are based on the plant phytochrome B and its binding partner PIF. For example, the T cell function has been studied on the basis of this system (Yousefi et al., 2019, 2021). Recent plant phytochrome-based systems have not been utilized in a neuroscientific context, although some of them still have interesting properties for neuroscience research. Integrins have an important role in regulating neuronal connectivity, like neurite growth (Lilja and Ivaska, 2018). The OptoMatrix– OptoIntegrin system can offer a reversible cell-matrix interaction. It utilizes a small PIF variant, PIFs, attached to αVβ3-integrin. After red light illumination, cells expressing the fusion protein on their surface are bound to the phytochrome-coated glass Frontiers in Cellular Neuroscience | www.frontiersin.org 7January 2022 | Volume 15 | Article 778900 fncel-15-778900 December 21, 2021 Time: 15:2 # 8 Lehtinen et al. Red Light Optogenetics slices (Baaske et al., 2019). In the future, the system could be utilized, for example, to research the plasticity of the brain through controlling axon growth. Bacteriobots can be utilized to release cargo, like nanoparticles or drugs, in precise locations. In a study by Sentürk et al. (2020),Escherichia coli was coated with phytochrome and polystyrene particles with PIF6. Red and NIR light illumination was successfully used to attach and release the cargo as desired in vitro (Sentürk et al., 2020). In neuroscientific research, this new method could help achieve more spatiotemporally precise drug administration. Instead of plant phytochromes, recent bacterial phytochromebased systems have been utilized in neuroscience. In the most relevant study, new phytochrome-activated adenylate cyclases were designed (Figure 4C and Table 1;Fomicheva et al., 2019). Adenylate cyclases produce the second messenger cAMP, which is an important signaling molecule for a variety of biological functions (Khannpnavar et al., 2020). In the study by Fomicheva et al. (2019), the best performing cyclase, ilaM5, was transfected into the thalamic ventral posteromedial nucleus and the nucleus reticularis in mice. The cyclase was then activated using transcranial NIR light illumination. The activation of the system reversibly suppresses the formation of cAMP-associated electrophysiological events in the brain called sleep spindles. These oscillations have been associated with memory formation. Unfortunately, this application has had problems with dark activity and the suppression of the other signals. Still, the system has potential to be utilized in memory research after optimization (Fomicheva et al., 2019). The photosensory module from the Deinococcus radiodurans phytochrome has also been utilized by other groups to create different cyclases (Etzl et al., 2018;Fomicheva et al., 2019;Kaberniuk et al., 2021). Etzl et al. (2018) created novel optogenetic guanylate and adenylate cyclases from which the latter were utilized to alter the locomotion of Caenorhabditis elegans through their cholinergic motor neurons (Etzl et al., 2018). In the iLIGHT system by Kaberniuk et al. (2021), the photosensory module was optimized and linked to diguanylyl cyclase. It was utilized in primary neurons and in vivo for transcription modulation in mice (Kaberniuk et al., 2021). Phosphodiesterases are the natural counterparts of the adenylate cyclases in controlling the second messenger concentrations (Figure 4D;Francis et al., 2011). Therefore, red light-controlled cAMP production and destruction has an intriguing potential for neuroscience. By coincidence, new and TABLE 1 | Red light optogenetics in neuroscientific animal studies. Application Target Subject System References Opsin Sight restoration Retinal ganglion Human ChrimsonR Sahel et al., 2021 Sight restoration Retinal ganglion Non-human primate ChrimsonR Gauvain et al., 2021 Sight restoration Retinal ganglion Mongolian gerbil ChrimsonR Huet et al., 2021 Sight restoration Retinal ganglion Mouse ReaChR Lindner et al., 2020 Sight restoration Retinal ganglion Mouse MCO Batabyal et al., 2020 Sight restoration Excitatory neurons in primary visual cortex Rat ChrimsonR Masuda and Takahashi, 2021 Sight restoration Inhibitory neurons in primary visual cortex Rat eNpHR 3.0 Masuda and Takahashi, 2021 Hearing restoration Spiral ganglion Mouse f-Chrimson and vf-Chrimson Bali et al., 2021 Pain research Microglia in spinal cord Mouse ReaChR Narcisse et al., 2021 Pain research Inhibitory neurons in the anterior cingulate cortex Mouse MCO II Yi et al., 2021 Epilepsy research Hippocampal CA3 region Mouse Jaws Farzaneh et al., 2020 Epilepsy research Hippocampal inhibitory GABAergic interneurons Mouse ChRmine Chen R. et al., 2020 Feeding -behavior alteration Dopamine neurons of the ventral tegmental area Mouse ChRmine Chen R. et al., 2020 Bidirectional neural activity control Dopaminergic neurons of the ventral tegmental area Mouse ChrimsonR and stGtACR2 (blue) Li et al., 2021 Bidirectional neural activity control GABAergic neurons of the secondary visual cortex Ferret BiPOLES Vierock et al., 2021 Pupil size control Locus coeruleus Mouse BiPOLES Vierock et al., 2021 Phytochrome Sleep spindle modulation Ventral posteromedial nucleus and nucleus reticularis Mouse Adenylate cyclase ilaM5 Fomicheva et al., 2019 The table contains in vivo applications for red light optogenetic systems in neuroscience, which are studied in primates and rodents. 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Pulsed near infrared transcranial and intranasal photobiomodulation significantly modulates neural oscillations: a pilot exploratory study. Sci. Rep. 9, 1–11. doi: 10.1038/s41598-019-42693-x Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Lehtinen, Nokia and Takala. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Cellular Neuroscience | www.frontiersin.org 20 January 2022 | Volume 15 | Article 778900