Damian Sendler: As a way to visually influence molecular and cellular activities in target cells, optogenetic methods have just recently appeared. A successful marriage of optics with genetic engineering yields opgenetics, which combines the strengths of both fields. On the millisecond timeframe, these benefits include optical control via the modulation of wavelength and light intensity and subcellular gene expression and product transport. Traditional approaches can’t provide this level of fine-tuned performance. Because of this, optogenetics has revolutionized neuroscience. Psychiatric applications of optogenetics are the topic of this study, which summarizes the field’s history and most recent developments.
Damian Jacob Sendler: Genetic and optical approaches are used in tandem in optogenetics to regulate biological processes in particular tissues and cell types. These light-sensitive photoreceptor molecules, called opsins in the animal world, are essential to the field of optogenetics. Microbiota-specific opsins (bacteriophores, halorophores and channel opsins) are of special interest because of their advantageous gene structure, which encodes both light-sensing and effector domains in one gene (Fig. 1a). The ability to produce light sensitivities in non-light-sensitive cells may be achieved by using microbial opsins. Cellular events, such as an action potential, are triggered as a consequence of the resultant ion flow. Action potential generation and firing may now be controlled optically, which has piqued neuroscience researchers’ interests. Neuronal function can be modulated in a spatially and temporally precise way by leveraging genetic engineering techniques. Opsins may be expressed in particular neuronal populations or targeted subcellular sites via genetic engineering approaches. As a result, optogenetics may be used to study behavior in vivo because of its capacity to adjust the width, frequency and latency of the light pulse on the millisecond timeframe. Traditional approaches, such as electrode stimulation, cannot provide the same level of control. Electrical stimulation using electrodes stimulates a wide range of neuronal subtypes, resulting in limited spatial resolution (Fig. 1b). As a result, optogenetics allows for more creative experimentation with higher levels of accuracy.
Dr. Sendler: It was over 50 years ago that researchers found that certain bacteria possess proteins that control ion movement across the plasma membrane in response to light. Bacteriorhodopsin was discovered in 1971 as a green-light-activated proton pump. Opsins might have a significant influence on neuroscience if successfully implemented. Neuroscientists can manipulate the membrane potential of specific neurons using light-induced ion flow. Neuronal networks and behavioral reactions may now be studied in a new way thanks to this method. Optogenetic methods may also give valuable information regarding brain circuit coding and processing. It was previously anticipated that opsins would not be effective in this application because the photocurrents they create in mammalian neurons are much too sluggish and feeble to trigger an action potential. Channel conductance of channelrhodopsin-2 (ChR2) was shown in 2005 to be adequate to push mammalian neurons over the threshold for action potentials. With millisecond accuracy, blue light can influence the activity of neurons in the brain (Fig. 1a).
Damian Sendler
Additional ChR2 family variations with quicker kinetics have been developed since this breakthrough. In the case of ChR2/E123T (also known as ChETA), a mutation in the E123 residue speeds up channel dynamics, enabling sustained spike trains of at least 200 Hz. Another ChR variation, ChIEF (chimera EF with I170V mutation), was created by combining site-directed mutagenesis with the creation of chimeric ChR from ChR1 and ChR2. Large photocurrents, decreased inactivation during prolonged light stimulation, and enhanced fidelity at frequencies greater than 25 Hz enable the creation of spike trains that more closely match natural rapid spiking patterns. A C128S mutation and C128S/D156A double mutation of ChR2 created step-function opsins (SFO) and stabilized SFO, respectively, despite the fact that millisecond-scale temporal control is one of the key capabilities of ChR variations. Longer channel open duration, improved kinetic stability and bi-stable currents are all features of these SFO. Using these characteristics, the photocurrent may be maintained for a longer period of time until it is deactivated by light of another color. There are five and six It is possible to control the equilibrium between excitation and inhibition (cellular E/I balance) over 30 minutes using stabilized SFO-induced photocurrent, which may be detected in freely moving animals.
Optogenetics can make effective use of the blue-light-gated cation channel ChR2 and its variations. In spite of this, there has been a tremendous amount of work put into building new tools. Neuronal networks may be seen by constructing another cation channel that has a much redder activation spectrum than ChR2 in order to see how they interact. Volvox carteri has been shown to have a hitherto undiscovered microbial ChR with a peak response wavelength of 530 nm, according to the genome database (VChR1). Newer red-shifted ChR variants need to be improved on a number of fronts, including a sluggish channel kinetics, poor membrane trafficking, and inadequate recovery after desensitization. VChR1 has been successfully engineered using the same gene-engineering techniques that were utilized to create ChIEF, such as sequence substitution and mutagenesis (ReaChR). In order to activate ReaChR, red light (wavelengths 590–630 nm) should be used. Improved membrane trafficking, greater photocurrents, quicker kinetics, and higher photocurrents are all advantages of ReaChR over VChR1. This means that different cell types may be labeled with ReaChR and traditional ChR, allowing for the visual control of the corresponding cell types. In addition, red light is less dispersed by tissue and less absorbed by blood than light of shorter wavelengths used to excite conventional ChR variations, giving ReaChR a significant edge when it comes to stimulating neurons in deeper brain areas. One of the most common causes of optical fiber loss is Rayleigh scattering. Inversely proportionate to light’s fourth power, scattering occurs. Since blue light’s wavelength is 3 times shorter than red light’s, it scatters three times as much (longer wavelength of 617 nm). Transcranial stimulation of ReaChR in the vibrissal motor cortex evoked vibrissae movements in conscious mice, as the first report of ReaChR application neatly demonstrated. Because microglia and astrocytes are activated as part of the inflammatory response after cranial window surgery, this non-invasive transcranial stimulation approach is ideal for long-term investigations. Because of this, optogenetic techniques employing red-shifted ChR might significantly lessen the invasiveness of therapeutic treatments..
Damian Jacob Sendler
It is possible to block the target neuron using optogenetics as well as to excite it. NpHR is a light-driven chloride pump that pumps chloride anions into the cells of Natronomonas pharaonic Light-induced hyperpolarization of NpHR-expressing neurons results in suppression of neuronal activity (Fig. 1a). NpHR is best stimulated at 580 nm, which is red-shifted from ChR2 (maximum at around 460 nm). As a result, ChR2 and NpHR may be engaged individually or in tandem. In fact, Zhang et al. demonstrated that it is feasible to concurrently express NpHR and ChR2 in Caenorhabditis worms cholinergic motor neurons to regulate locomotion bidirectionally. Excitatory and inhibitory neurons may be controlled individually, allowing direct and causal connections between certain neural circuits and their associated behaviors. It has been reported that Halorubrum sodomense (archaerhodopsin-3, or Arch) and Leptosphaeria maculans (known as Mac) both contain new photoinhibitory membrane proteins that have been identified lately. By pumping protons out of the cell and hyperpolarizing it, Arch silences neuronal activity quickly and reversibly when expressed in neurons. Because the Arch photocurrent is stronger than the NpHR photocurrent, it is capable of virtually suppressing brain activity in awake, active mice. The anion ChR (ACR), a class of light-gated anion channels produced in cryptophyte algae, is another sort of optogenetic inhibitory instrument. Optogenetic inhibitors with anion selectivity and fast kinetics, such as ACR, are the most light-sensitive of the presently available optogenetic inhibitors. Inhibitory optoprobes are becoming more diverse in their light absorption spectra, which means we may be able to mute various neuronal populations using different light wavelengths. Optoprobe and variations are being constantly improved, and new variants are also being developed to generate variable pump/channel kinetics, ion conductance, and light absorption features. Neuronal activity synchronization and (de)synchronization using multiple-color optical activation, silencing, and (de)synchronization should enable us to causally examine how certain portions of neurons networks contribute to neuronal coding and associated behaviors.
Damian Jacob Markiewicz Sendler: Many technological advancements (particularly in the area of invasiveness) need to be made before optogenetics methods may be used to bigger and more complicated animal models, such as zebrafish. These hydrophobic heterologous proteins may be harmful and affect plasma membrane characteristics when they are overexpressed at high quantities. This calls for much more work on optimizing opsins in order to reduce their toxicity, enhance membrane trafficking, and boost their kinetics and photocurrents even more. Most significantly, it is critical to reduce the level of intrusion. Optical fibers are now used to send light to the brain after surgery. Chronic injections and tissue damage might occur as a result of this procedure. An external laser beam (480 nm) is diluted by 50% at 200 m depth and by 90% at 1-mm depth while passing through the brain because to light scattering and tissue absorption, making it ineffective. Wireless implants with a remote control system are now being developed to get over the difficulties of external light supply and instead use this technology. Up-conversion optogenetics is one example of this kind of study. Low-energy near-infrared photons are absorbed by lanthanide nanoparticles, which then release high-energy visible light with a peak wavelength of 543 nm (up-conversion). Lanthanide nanoparticles may be utilized to generate visible light that activates ChR to elicit photocurrents in the cells since near-infrared photons have been observed to penetrate through skin and bone deep into the brain. In another research example, chemical genetics and opsin have been used together in a study. For the first time, scientists have created a luciferase fusion protein that contains both excitatory ChR and inhibitory NpHR. Luminopsin (LMO) and inhibitory LMO are the names given to these two types of constructs, respectively. In the case of opsins, luciferase serves as a light-generating protein. After activation of luciferase by coelenterazine, ionic currents may be generated. Optogenetic approaches may be easily scaled, less intrusive, and hardware-independent if LMO and inhibitory LMO can activate or suppress neuronal activity in response to coelenterazine. In the same way as traditional chemogenetic probes, such as designer receptors exclusively triggered by designer drugs, employ diffusible exogenous molecules to modulate neuronal activity, LMO do the same (DREADD). To regulate the timing of neuronal activity, it is necessary to know how quickly the chemical reaches the brain and is expelled from the brain.. After a chemical injection, the LMO effect has been observed to begin within one to several minutes, lasting from a few minutes to more than an hour. Because of this, the kinetics of LMO are quicker than those of DREADD (20 min to several hours of action). LMO is also easier to operate since it generates its own electricity. Instead of relying on G protein-coupled receptors, DREADD use intracellular second messenger pathways that may have unforeseen side effects. Optogenetics and DREADD benefits are combined in LMO, which is a novel method to neural activation that does not need invasiveness or wide activation.
Neuronal firing is controlled by the passage of ions across the cell membrane with the help of the optoprobes explained above. In order to affect neuronal activity in various ways, further optogenetic methods have been created. It is a photoreceptor and an effector, accelerating the synthesization of the neurotransmitter cAMP, in the case of the unique protein (Pac) that was isolated from Euglena gracilis and works as both. By altering intracellular signaling, blue light stimulation induces a quick rise in cAMP levels and a change in neurotransmitter release. Phototropin from Avena sativa, for example, has a light-oxygen-voltage (LOV) domain that may be used to build optogenetic instruments. The flavin-binding core domain and the carboxy-terminal J-helical extension make up the LOV2 domain. In the dark, the LOV2 domain binds to the J-helix. The core domain forms a covalent adduct with the activated flavin mononucleotide, resulting in a conformational shift inside the core, followed by dissociation and unwinding of the J helix after blue light exposure. Photoactivatable Rac1 (PaRac1), in which the LOV2 domain is fused with Rac1, is a successful use of the LOV2 domain (Fig. 1c). Rac1 cannot attach to its effector molecules when the LOV domain is closed. Rac1 signaling is triggered by the LOV domain’s J helix unwinding in response to light. Dendritic spines have a high concentration of Rac1. Rac1 modulates dendritic spine size by rearranging the actin cytoskeleton. 20-22 For most excitatory synapse formation, spine structural plasticity is closely associated with synaptic function. To modulate synaptic transmission through structural plasticity, optical modulation of PaRac1 may be necessary. Although the shrinking in the spines associated with learned skills was effectively generated by optical stimulation of potentiated spine-specific PaRac1 (AS-PaRac1) in learning-related spines, this learning was not preserved. Dendritic spines’ structural flexibility is a primary mechanism for learning and memory, according to this synaptic optogenetics. Disrupted synaptic plasticity has been implicated in a number of mental illnesses, including schizophrenia, autism, depression, and post-traumatic stress disorder. 24 to 25 Using AS-PaRac1 in proven illness animal models, it may be feasible to get mechanistic understanding into synaptic dysfunction with synaptic resolution by seeing and manipulating it.
Damien Sendler: Like the LOV domain, protein activity and interaction may be controlled by the LOV domain. Light-switchable transcription factor Gal4-VVD-p65 was engineered using the LOV domain by Wang et al., for example (GAVPO). As soon as GAVPO’s well-known DNA-binding domain dimersizes in the presence of blue light, the LOV domain begins to activate the transcription of particular genes that possess Gal4-binding sites in their promoter regions”” (Fig. 1d). Spatiotemporal control over the gene of interest might be achieved by keeping GAVPO in the active state for two hours. J helix may be joined to any cytosolic protein in this way, in theory. The linker sequence between the J helix and the protein of interest may be optimized to construct a wide variety of photoactivatable enzymes, inhibitory peptides, and DNA-binding proteins. Disease-related gene variants have been revealed to enhance the likelihood of developing mental disorders in the last few years. 24 to 25 However, genetic research alone will not be able to reveal the underlying molecular and cellular pathways that alter brain function due to genetic variants. The use of optogenetic technologies, such as LOV domains, might give a novel approach to study the causative function of each candidate gene and the probable link to psychiatric symptoms in a new and innovative manner.
New therapeutic avenues are opened up by optogenetics as well. Optical reactivation of reward-associated neurons in the hippocampus led in the recovery of stress-induced depression-like behaviors in a chronic stress model of depression. However, natural exposure to a gratifying event did not alleviate depressive-like behaviors, unlike optical stimulation of reward-related neurocircuits. It reminds me a lot of anhedonia that spontaneous exposure to a pleasurable event is useless in treating depression symptoms (the inability to derive pleasure from activities usually found enjoyable). Summarizing, studies from optogenetics show that manipulating neurocircuits to treat mental problems is successful.
Using optogenetic stimulation in people might open up new therapeutic possibilities. Using a mouse model of refractory epilepsy, researchers have shown the therapeutic potential of optogenetics. NpHR expression and on-demand light stimulation in hippocampus excitatory neurons effectively suppressed spontaneous seizures in this paradigm, according to the study’s authors. Retinitis pigmentosa mice models may be restored to normal light sensitivity by using ChR2 as a replacement for the phototransduction cascade (a group of hereditary diseases that lead to incurable blindness). However, there are significant ethical questions about the advantages and unforeseen effects of brain intervention. Because of this, the transition of optogenetics from fundamental to clinical research is difficult. Despite this, significant progress is being made: Optogenetic retinitis pigmentosa therapy has begun a phase I/II clinical study. ChR2 gene therapy is injected into the eye as part of this treatment. Neurological and ophthalmological problems have established the causal involvement of certain neural circuits, but mental disorders are still shrouded in mystery. Because of this, using optogenetics to treat mental health conditions will be more difficult. Additional fundamental research using optogenetics is required to have a better understanding of these illnesses. Deep brain stimulation (DBS) and transcranial magnetic stimulation (TMS) are examples of causal treatments that have been refined through time (TMS). Many mental illnesses have been successfully treated with DBS and TMS. There are still many questions regarding how DBS really works, but it seems to be effective because it alters neural activity in such a way that problematic firing patterns are replaced with less distressing ones.
Cocaine addiction study is a great illustration of this kind of outcome. The nucleus accumbens contains medium-sized spiny neurons that express the dopamine D1 receptor (D1R) and are implicated in the development of cocaine addiction due to behavioral sensitization mediated by cocaine-evoked potentiation of excitatory afferents. High-frequency DBS procedures (>100 Hz) failed to change cocaine-induced plasticity and had very modest effects on behavioral sensitization, according to a study. A long-term depression (LTD) strategy would have to be used to reverse cocaine-induced synaptic potentiation in order to alleviate behavioral sensitization. However, LTD is unlikely to be induced by high-frequency stimulation procedures (>100 Hz). Authors compared optogenetic and electrical stimulation in a comprehensive study. As compared to an otherwise comparable procedure, ChR2 stimulation at 12 Hz produced strong LTD of excitatory transmission into D1R medium-sized spiny neurons. They investigated whether D1R antagonists and electrical stimulation might induce LTD, following recent findings that found that D1R blockage is required for mGluR-dependent LTD. Both 12-Hz DBS and D1R antagonist treatment alone had no effect on the sensitivity of the patient. However, when combined, sensitization was effectively eliminated. As a result, optogenetics research was able to contribute to the formulation of the optimal stimulation regimen for reducing behavioral sensitization. Following approval by the US Food and Drug Administration for the treatment of mental health diseases such as OCD, DBS is currently being investigated for use in treating PTSD, mood disorders, and schizophrenia. As with any treatment, there are risks associated with deep brain stimulation (DBS). Refinement of DBS techniques to restore normal transmission with enduring benefits may be achieved by an optogenetics-driven circuit-centric approach. As a result, optogenetics might be a big breakthrough in the treatment of mental health issues.
Karl Deisseroth, a pioneer in optogenetics, received his medical training in Germany. In light of these facts, it is only natural that his team has achieved significant progress in the field of optogenetics and has amassed vital data for psychiatric research. The study of disease-induced changes in neural circuit formation and function is one of the primary aims of neuroscience research. It should be difficult to directly apply optogenetic methods to mental treatment. While molecular (and chemistry-based) medication designs have their limits, new breakthroughs in optogenetics will allow circuit-centric treatments to be developed.
