Optogenetic Tools Research Articles

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

Illuminating the inner workings of a natural protein switch: Blue-light sensing in LOV-activated diguanylate cyclases.

Regulatory proteins play a crucial role in adaptation to environmental cues. Especially for lifestyle transitions, such as cell proliferation or apoptosis, switch-like characteristics are desirable. While nature frequently uses regulatory circuits to amplify or dampen signals, stand-alone protein switches are interesting for applications like biosensors, diagnostic tools, or optogenetics. However, such stand-alone systems frequently feature limited dynamic and operational ranges and suffer from slow response times. Here, we characterize a LOV-activated diguanylate cyclase (LadC) that offers precise temporal and spatial control of enzymatic activity with an exceptionally high dynamic range over four orders of magnitude. To establish this pronounced activation, the enzyme exhibits a two-stage activation process in which its activity is inhibited in the dark by caging its effector domains and stimulated upon illumination by the formation of an extended coiled-coil. These switch-like characteristics of the LadC system can be used to develop new optogenetic tools with tight regulation.

Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor

Optogenetic tools respond to light through one of a small number of behaviors including allosteric changes, dimerization, clustering, or membrane translocation. Here, we describe a new class of optogenetic actuator that simultaneously clusters and translocates to the plasma membrane in response to blue light. We demonstrate that dual translocation and clustering of the BcLOV4 photoreceptor can be harnessed for novel single-component optogenetic tools, including for control of the entire family of epidermal growth factor receptor (ErbB1-4) tyrosine kinases. We further find that clustering and membrane translocation are mechanistically linked. Stronger clustering increased the magnitude of translocation and downstream signaling, increased sensitivity to light by ~threefold-to-fourfold, and decreased the expression levels needed for strong signal activation. Thus light-induced clustering of BcLOV4 provides a strategy to generate a new class of optogenetic tools and to enhance existing ones.

Introduction of reversible cysteine ligation ability to the biliverdin-binding cyanobacteriochrome photoreceptor.

Cyanobacteriochrome (CBCR) photoreceptors are distantly related to the canonical red/far-red reversible phytochrome photoreceptors. In the case of the CBCRs, only the GAF domain is required for chromophore incorporation and photoconversion. The GAF domains of CBCR are highly diversified into many lineages to sense various colors of light. These CBCR GAF domains are divided into two types: those possessing only the canonical Cys residue and those with both canonical and second Cys residues. The canonical Cys residue stably ligates to the chromophore in both cases. The second Cys residue mostly shows reversible adduct formation with the chromophore during photoconversion for spectral tuning. In this study, we focused on the CBCR GAF domain AnPixJg2_BV4, which possesses only the canonical Cys residue. AnPixJg2_BV4 covalently ligates to the biliverdin (BV) chromophore and shows far-red/orange reversible photoconversion. Because BV is a mammalian intrinsic chromophore, BV-binding molecules are advantageous for in vivo optogenetic and bioimaging tool development. To obtain a better developmental platform molecule, we performed site-saturation random mutagenesis and serendipitously obtained a unique variant molecule that showed far-red/blue reversible photoconversion, in which the Cys residue was introduced near the chromophore. This introduced Cys residue functioned as the second Cys residue that reversibly ligated with the chromophore. Because the position of the introduced Cys residue is distinct from the known second Cys residues, the variant molecule obtained in this study would expand our knowledge about the spectral tuning mechanism of CBCRs and contribute to tool development.

Tissue flows are tuned by actomyosin-dependent mechanics in developing embryos.

Rapid epithelial tissue flows are essential to building and shaping developing embryos. However, the mechanical properties of embryonic epithelial tissues and the factors that control these properties are not well understood. Actomyosin generates contractile tensions and contributes to the mechanical properties of cells and cytoskeletal networks in vitro, but it remains unclear how the levels and patterns of actomyosin activity contribute to embryonic epithelial tissue mechanics in vivo. To dissect the roles of cell-generated tensions in the mechanics of flowing epithelial tissues, we use optogenetic tools to manipulate actomyosin contractility with spatiotemporal precision in the Drosophila germband epithelium, which rapidly flows during body axis elongation. We find that manipulating actomyosin-dependent tensions by either optogenetic activation or deactivation of actomyosin alters the solid-fluid mechanical properties of the germband epithelium, leading to changes in cell rearrangements and tissue-level flows. Optogenetically activating actomyosin leads to increases in the overall level but decreases in the anisotropy of tension in the tissue, whereas optogenetically deactivating actomyosin leads to decreases in both the level and anisotropy of tension compared to in wild-type embryos. We find that optogenetically activating actomyosin results in more solid-like (less fluid-like) tissue properties, which is associated with reduced cell rearrangements and tissue flow compared to in wild-type embryos. Optogenetically deactivating actomyosin also results in more solid-like properties than in wild-type embryos but less solid-like properties compared to optogenetically activating actomyosin. Together, these findings indicate that increasing the overall tension level is associated with more solid-like properties in tissues that are relatively isotropic, whereas high tension anisotropy fluidizes the tissue. Our results reveal that epithelial tissue flows in developing embryos involve the coordinated actomyosin-dependent regulation of the mechanical properties of tissues and the tensions driving them to flow in order to achieve rapid tissue remodeling.

Optogenetics for controlling seizure circuits for translational approaches

The advent of optogenetic tools has had a profound impact on modern neuroscience research, revolutionizing our understanding of the brain. These tools offer a remarkable ability to precisely manipulate specific groups of neurons with an unprecedented level of temporal precision, on the order of milliseconds. This breakthrough has significantly advanced our knowledge of various physiological and pathophysiological processes in the brain.Within the realm of epilepsy research, optogenetic tools have played a crucial role in investigating the contributions of different neuronal populations to the generation of seizures and hyperexcitability. By selectively activating or inhibiting specific neurons using optogenetics, researchers have been able to elucidate the underlying mechanisms and identify key players involved in epileptic activity. Moreover, optogenetic techniques have also been explored as innovative therapeutic strategies for treating epilepsy. These strategies aim to halt seizure progression and alleviate symptoms by utilizing the precise control offered by optogenetics.The application of optogenetic tools has provided valuable insights into the intricate workings of the brain during epileptic episodes. For instance, researchers have discovered how distinct interneuron populations contribute to the initiation of seizures (ictogenesis). They have also revealed how remote circuits in regions such as the cerebellum, septum, or raphe nuclei can interact with hyperexcitable networks in the hippocampus. Additionally, studies have demonstrated the potential of closed-loop systems, where optogenetics is combined with real-time monitoring, to enable precise, on-demand control of seizure activity.Despite the immense promise demonstrated by optogenetic approaches, it is important to acknowledge that many of these techniques are still in the early stages of development and have yet to reach potential clinical applications. The transition from experimental research to practical clinical use poses numerous challenges. In this review, we aim to introduce optogenetic tools, provide a comprehensive survey of their application in epilepsy research, and critically discuss their current potential and limitations in achieving successful clinical implementation for the treatment of human epilepsy. By addressing these crucial aspects, we hope to foster a deeper understanding of the current state and future prospects of optogenetics in epilepsy treatment.

Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength.

The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.

Opto-RhoGEFs, an optimized optogenetic toolbox to reversibly control Rho GTPase activity on a global to subcellular scale, enabling precise control over vascular endothelial barrier strength

The inner layer of blood vessels consists of endothelial cells, which form the physical barrier between blood and tissue. This vascular barrier is tightly regulated and is defined by cell-cell contacts through adherens and tight junctions. To investigate the signaling that regulates vascular barrier strength, we focused on Rho GTPases, regulators of the actin cytoskeleton and known to control junction integrity. To manipulate Rho GTPase signaling in a temporal and spatial manner we applied optogenetics. Guanine-nucleotide exchange factor (GEF) domains from ITSN1, TIAM1, and p63RhoGEF, activating Cdc42, Rac, and Rho, respectively, were integrated into the optogenetic recruitment tool improved light-induced dimer (iLID). This tool allows for Rho GTPase activation at the subcellular level in a reversible and non-invasive manner by recruiting a GEF to a specific area at the plasma membrane, The membrane tag of iLID was optimized and a HaloTag was applied to gain more flexibility for multiplex imaging. The resulting optogenetically recruitable RhoGEFs (Opto-RhoGEFs) were tested in an endothelial cell monolayer and demonstrated precise temporal control of vascular barrier strength by a cell-cell overlap-dependent, VE-cadherin-independent, mechanism. Furthermore, Opto-RhoGEFs enabled precise optogenetic control in endothelial cells over morphological features such as cell size, cell roundness, local extension, and cell contraction. In conclusion, we have optimized and applied the optogenetic iLID GEF recruitment tool, that is Opto-RhoGEFs, to study the role of Rho GTPases in the vascular barrier of the endothelium and found that membrane protrusions at the junction region can rapidly increase barrier integrity independent of VE-cadherin.

Involvement of nucleus accumbens D2-medium spiny neurons projecting to the ventral pallidum in anxiety-like behaviour.

The nucleus accumbens (NAcc) is a crucial brain region for emotionally relevant behaviours. The NAcc is mainly composed of medium spiny neurons (MSNs) expressing either dopamine receptor D1 (D1-MSNs) or D2 (D2-MSNs). The D1-MSNs project to the ventral tegmental area (VTA) and the ventral pallidum (VP), whereas the D2-MSNs project only to the VP. The D1- and D2-MSNs have been associated with depression-like behaviours, but their contribution to anxiety remains to be determined. We used optogenetic tools to selectively manipulate D1-MSN projections from the NAcc core to the VP or VTA and D2-MSN projections to the VP during validated anxiety-producing behavioural procedures in naive mice. In addition, we assessed the effects of optical stimulation on neuronal activity using in vivo electrophysiologic recordings in anesthetized animals. Optogenetic activation of D1-MSN projections to the VTA or VP did not trigger anxiety-like behaviour. However, optical activation of D2-MSN projections to the VP significantly increased anxiety-like behaviour. This phenotype was associated with a decrease in the neuronal activity of putative GABAergic neurons in the VP. Importantly, pretreating D2-MSN-VP animals with the γ-aminobutyric acid modulator diazepam prevented the optically triggered anxiety-like behaviour. The exclusive use of males in the behavioural tests limits broader interpretation of the findings. Although we used optogenetic conditions that trigger quasi-physiologic changes, there are caveats associated with the artificial manipulation of neuronal activity. The D2-MSN-VP projections contributed to the development of anxiety-like behaviour, through modulation of GABAergic activity in the VP.

Empirical mode decomposition of local field potential data from optogenetic experiments.

This study investigated the effects of cocaine administration and parvalbumin-type interneuron stimulation on local field potentials (LFPs) recorded in vivo from the medial prefrontal cortex (mPFC) of six mice using optogenetic tools. The local network was subject to a brief 10 ms laser pulse, and the response was recorded for 2 s over 100 trials for each of the six subjects who showed stable coupling between the mPFC and the optrode. Due to the strong non-stationary and nonlinearity of the LFP, we used the adaptive, data-driven, Empirical Mode Decomposition (EMD) method to decompose the signal into orthogonal Intrinsic Mode Functions (IMFs). Through trial and error, we found that seven is the optimum number of orthogonal IMFs that overlaps with known frequency bands of brain activity. We found that the Index of Orthogonality (IO) of IMF amplitudes was close to zero. The Index of Energy Conservation (IEC) for each decomposition was close to unity, as expected for orthogonal decompositions. We found that the power density distribution vs. frequency follows a power law with an average scaling exponent of ~1.4 over the entire range of IMF frequencies 2-2,000 Hz. The scaling exponent is slightly smaller for cocaine than the control, suggesting that neural activity avalanches under cocaine have longer life spans and sizes.

Optogenetic dissection of RET signaling reveals robust activation of ERK and enhanced filopodia-like protrusions of regenerating axons

RET (REarranged during Transfection) is a receptor tyrosine kinase that transduces various external stimuli into biological functions, such as survival and differentiation, in neurons. In the current study, we developed an optogenetic tool for modulating RET signaling, termed optoRET, combining the cytosolic region of human RET with a blue-light–inducible homo-oligomerizing protein. By varying the duration of photoactivation, we were able to dynamically modulate RET signaling. Activation of optoRET recruited Grb2 (growth factor receptor-bound protein 2) and stimulated AKT and ERK (extracellular signal-regulated kinase) in cultured neurons, evoking robust and efficient ERK activation. By locally activating the distal part of the neuron, we were able to retrogradely transduce the AKT and ERK signal to the soma and trigger formation of filopodia-like F-actin structures at stimulated regions through Cdc42 (cell division control 42) activation. Importantly, we successfully modulated RET signaling in dopaminergic neurons of the substantia nigra in the mouse brain. Collectively, optoRET has the potential to be developed as a future therapeutic intervention, modulating RET downstream signaling with light.

Circuits underlying social function and dysfunction.

Substantial advances have been made toward understanding the genetic and environmental risk factors for autism, a neurodevelopmental disorder with social impairment as a core feature. In combination with optogenetic and chemogenetic tools to manipulate neural circuits in vivo, it is now possible to use model systems to test how specific neural circuits underlie social function and dysfunction. Here, we review the literature that has identified circuits associated with social interest (sociability), social reward, social memory, dominance, and aggression, and we outline a preliminary roadmap of the neural circuits driving these social behaviors. We highlight the neural circuitry underlying each behavioral domain, as well as develop an interactive map of how these circuits overlap across domains. We find that some of the circuits underlying social behavior are general and are involved in the control of multiple behavioral aspects, whereas other circuits appear to be specialized for specific aspects of social behavior. Our overlapping circuit map therefore helps to delineate the circuits involved in the various domains of social behavior and to identify gaps in knowledge.

GluCl.Cre ON enables selective inhibition of molecularly defined pain circuits.

Insight into nociceptive circuits will ultimately build our understanding of pain processing and aid the development of analgesic strategies. Neural circuit analysis has been advanced greatly by the development of optogenetic and chemogenetic tools, which have allowed function to be ascribed to discrete neuronal populations. Neurons of the dorsal root ganglion, which include nociceptors, have proved challenging targets for chemogenetic manipulation given specific confounds with commonly used DREADD technology. We have developed a cre/lox dependant version of the engineered glutamate-gated chloride channel (GluCl) to restrict and direct its expression to molecularly defined neuronal populations. We have generated GluCl.Cre ON that selectively renders neurons expressing cre-recombinase susceptible to agonist-induced silencing. We have functionally validated our tool in multiple systems in vitro, and subsequently generated viral vectors and tested its applicability in vivo. Using Nav1.8 Cre mice to restrict AAV-GluCl.Cre ON to nociceptors, we demonstrate effective silencing of electrical activity in vivo and concomitant hyposensitivity to noxious thermal and noxious mechanical pain, whereas light touch and motor function remained intact. We also demonstrated that our strategy can effectively silence inflammatory-like pain in a chemical pain model. Collectively, we have generated a novel tool that can be used to selectively silence defined neuronal circuits in vitro and in vivo. We believe that this addition to the chemogenetic tool box will facilitate further understanding of pain circuits and guide future therapeutic development.

Optogenetic urothelial cell stimulation induces bladder contractions and pelvic nerve afferent firing.

Urothelial cells, which play an essential role in barrier function, are also thought to play a sensory role in bladder physiology by releasing signaling molecules in response to sensory stimuli that act upon adjacent sensory neurons. However, it is challenging to study this communication due to the overlap in receptor expression and proximity of urothelial cells to sensory neurons. To overcome this challenge, we developed a mouse model where we can directly stimulate urothelial cells using optogenetics. We crossed a uroplakin II (UPK2) cre mouse with a mouse that expresses the light-activated cation channel channelrhodopsin-2 (ChR2) in the presence of cre expression. Optogenetic stimulation of urothelial cells cultured from UPK2-ChR2 mice initiates cellular depolarization and release of ATP. Cystometry recordings demonstrated that optical stimulation of urothelial cells increases bladder pressure and pelvic nerve activity. Increases in bladder pressure persisted, albeit to a lesser extent, when the bladder was excised in an in vitro preparation. The P2X receptor antagonist PPADS significantly reduced optically evoked bladder contractions in vivo and ex vivo. Furthermore, corresponding nerve activity was also inhibited with PPADS. Our data suggest that urothelial cells can initiate robust bladder contractions via sensory nerve signaling or contractions through local signaling mechanisms. These data support a foundation of literature demonstrating communication between sensory neurons and urothelial cells. Importantly, with further use of these optogenetic tools, we hope to scrutinize this signaling mechanism, its importance for normal micturition and nociception, and how it may be altered in pathophysiological conditions.NEW & NOTEWORTHY Urothelial cells play a sensory role in bladder function. However, it has been particularly challenging to study this communication as both sensory neurons and urothelial cells express similar sensory receptors. Here we demonstrate using an optogenetic technique, that specific urothelial stimulation alone resulted in bladder contractions. This approach will have a long-lasting impact on how we study urothelial-to-sensory neuron communication and the changes that occur under disease conditions.

Lactate as a supplemental fuel for synaptic transmission and neuronal network oscillations: Potentials and limitations.

Lactate shuttled from the blood circulation, astrocytes, oligodendrocytes or even activated microglia (resident macrophages) to neurons has been hypothesized to represent a major source of pyruvate compared to what is normally produced endogenously by neuronal glucose metabolism. However, the role of lactate oxidation in fueling neuronal signaling associated with complex cortex function, such as perception, motor activity, and memory formation, is widely unclear. This issue has been experimentally addressed using electrophysiology in hippocampal slice preparations (ex vivo) that permit the induction of different neural network activation states by electrical stimulation, optogenetic tools or receptor ligand application. Collectively, these studies suggest that lactate in the absence of glucose (lactate only) impairs gamma (30-70 Hz) and theta-gamma oscillations, which feature high energy demand revealed by the cerebral metabolic rate of oxygen (CMRO2, set to 100%). The impairment comprises oscillation attenuation or moderate neural bursts (excitation-inhibition imbalance). The bursting is suppressed by elevating the glucose fraction in energy substrate supply. By contrast, lactate can retain certain electric stimulus-induced neural population responses and intermittent sharp wave-ripple activity that features lower energy expenditure (CMRO2 of about 65%). Lactate utilization increases the oxygen consumption by about 9% during sharp wave-ripples reflecting enhanced adenosine-5'-triphosphate (ATP) synthesis by oxidative phosphorylation in mitochondria. Moreover, lactate attenuates neurotransmission in glutamatergic pyramidal cells and fast-spiking, γ-aminobutyric acid (GABA)ergic interneurons by reducing neurotransmitter release from presynaptic terminals. By contrast, the generation and propagation of action potentials in the axon is regular. In conclusion, lactate is less effective than glucose and potentially detrimental during neural network rhythms featuring high energetic costs, likely through the lack of some obligatory ATP synthesis by aerobic glycolysis at excitatory and inhibitory synapses. High lactate/glucose ratios might contribute to central fatigue, cognitive impairment, and epileptic seizures partially seen, for instance, during exhaustive physical exercise, hypoglycemia and neuroinflammation.

Optogenetic manipulation of inhibitory interneurons can be used to validate a model of spatiotemporal sequence learning.

The brain uses temporal information to link discrete events into memory structures supporting recognition, prediction, and a wide variety of complex behaviors. It is still an open question how experience-dependent synaptic plasticity creates memories including temporal and ordinal information. Various models have been proposed to explain how this could work, but these are often difficult to validate in a living brain. A recent model developed to explain sequence learning in the visual cortex encodes intervals in recurrent excitatory synapses and uses a learned offset between excitation and inhibition to generate precisely timed "messenger" cells that signal the end of an instance of time. This mechanism suggests that the recall of stored temporal intervals should be particularly sensitive to the activity of inhibitory interneurons that can be easily targeted in vivo with standard optogenetic tools. In this work we examined how simulated optogenetic manipulations of inhibitory cells modifies temporal learning and recall based on these mechanisms. We show that disinhibition and excess inhibition during learning or testing cause characteristic errors in recalled timing that could be used to validate the model in vivo using either physiological or behavioral measurements.

Adult-born neurons in critical period maintain hippocampal seizures via local aberrant excitatory circuits

Temporal lobe epilepsy (TLE), one common type of medically refractory epilepsy, is accompanied with altered adult-born dentate granule cells (abDGCs). However, the causal role of abDGCs in recurrent seizures of TLE is not fully understood. Here, taking advantage of optogenetic and chemogenetic tools to selectively manipulate abDGCs in a reversible manner, combined with Ca2+ fiber photometry, trans-synaptic viral tracing, in vivo/vitro electrophysiology approaches, we aimed to test the role of abDGCs born at different period of epileptogenic insult in later recurrent seizures in mouse TLE models. We found that abDGCs were functionally inhibited during recurrent seizures. Optogenetic activation of abDGCs significantly extended, while inhibition curtailed, the seizure duration. This seizure-modulating effect was attributed to specific abDGCs born at a critical early phase after kindled status, which experienced specific type of circuit re-organization. Further, abDGCs extended seizure duration via local excitatory circuit with early-born granule cells (ebDGCs). Repeated modulation of “abDGC-ebDGC” circuit may easily induce a change of synaptic plasticity, and achieve long-term anti-seizure effects in both kindling and kainic acid-induced TLE models. Together, we demonstrate that abDGCs born at a critical period of epileptogenic insult maintain seizure duration via local aberrant excitatory circuits, and inactivation of these aberrant circuits can long-termly alleviate severity of seizures. This provides a deeper and more comprehensive understanding of the potential pathological changes of abDGCs circuit and may be helpful for the precise treatment in TLE.

Light-switchable transcription factors obtained by direct screening in mammalian cells

Optogenetic tools can provide fine spatial and temporal control over many biological processes. Yet the development of new light-switchable protein variants remains challenging, and the field still lacks general approaches to engineering or discovering protein variants with light-switchable biological functions. Here, we adapt strategies for protein domain insertion and mammalian-cell expression to generate and screen a library of candidate optogenetic tools directly in mammalian cells. The approach is based on insertion of the AsLOV2 photoswitchable domain at all possible positions in a candidate protein of interest, introduction of the library into mammalian cells, and light/dark selection for variants with photoswitchable activity. We demonstrate the approach’s utility using the Gal4-VP64 transcription factor as a model system. Our resulting LightsOut transcription factor exhibits a > 150-fold change in transcriptional activity between dark and blue light conditions. We show that light-switchable function generalizes to analogous insertion sites in two additional Cys6Zn2 and C2H2 zinc finger domains, providing a starting point for optogenetic regulation of a broad class of transcription factors. Our approach can streamline the identification of single-protein optogenetic switches, particularly in cases where structural or biochemical knowledge is limited.

OPTO-BLUE: An Integrated Bidirectional Optogenetic Lentiviral Platform for Controlled Light-Induced Gene Expression.

Regulated systems for transgene expression are useful tools in basic research and a promising platform in biomedicine due to their regulated transgene expression by an inducer. The emergence of optogenetics expression systems enabled the construction of light-switchable systems, enhancing the spatial and temporal resolution of a transgene. The LightOn system is an optogenetic tool that regulates the expression of a gene of interest using blue light as an inducer. This system is based on a photosensitive protein (GAVPO), which dimerizes and binds to the UASG sequence in response to blue light, triggering the expression of a downstream transgene. Previously, we adapted the LightOn system to a dual lentiviral vector system for neurons. Here, we continue the optimization and assemble all components of the LightOn system into a single lentiviral plasmid, the OPTO-BLUE system. For functional validation, we used enhanced green fluorescent protein (EGFP) as an expression reporter (OPTO-BLUE-EGFP) and evaluated the efficiency of EGFP expression by transfection and transduction in HEK293-T cells exposed to continuous blue-light illumination. Altogether, these results prove that the optimized OPTO-BLUE system allows the light-controlled expression of a reporter protein according to a specific time and light intensity. Likewise, this system should provide an important molecular tool to modulate gene expression of any protein by blue light.

Self-assembled ultraflexible probes for long-term neural recordings and neuromodulation.

Ultraflexible microelectrode arrays (MEAs) that can stably record from a large number of neurons after their chronic implantation offer opportunities for understanding neural circuit mechanisms and developing next-generation brain-computer interfaces. The implementation of ultraflexible MEAs requires their reliable implantation into deep brain tissues in a minimally invasive manner, as well as their precise integration with optogenetic tools to enable the simultaneous recording of neural activity and neuromodulation. Here, we describe the process for the preparation of elastocapillary self-assembled ultraflexible MEAs, their use in combination with adeno-associated virus vectors carrying opsin genes and promoters to form an optrode probe and their in vivo experimental use in the brains of rodents, enabling electrophysiological recordings and optical modulation of neuronal activity over long periods of time (on the order of weeks to months). The procedures, including device fabrication, probe assembly and implantation, can be completed within 3 weeks. The protocol is intended to facilitate the applications of ultraflexible MEAs for long-term neuronal activity recording and combined electrophysiology and optogenetics. The protocol requires users with expertise in clean room facilities for the fabrication of ultraflexible MEAs.