Search Results (51)

Astrocyte activation and gender differences play critical roles in the prognosis following stroke. Recent studies have shown that optogenetic technology can promote brain repair after stroke by activating astrocytes in male rats. However, it remains unclear whether gender differences influence the efficacy of optogenetic activation of astrocytes in regulating post-stroke brain repair and its underlying mechanisms. In this study, we activated astrocytes in the ipsilateral cortex of adult glial fibrillary acidic protein-channelrhodopsin 2-enhanced yellow fluorescent protein (GFAP-ChR2-EYFP) transgenic Sprague Dawley rats using optogenetic stimulation at 24, 36, 48, and 60 h after inducing photothrombosis stroke. Neurobehavioral tests, cresyl violet staining, RT-qPCR, Western blot, and immunofluorescence analysis were performed on both female and male rats. Our results showed that male rats exhibited significant improvements in behavioral scores and reduction in infarct size after optogenetic activation of astrocytes at three days post-stroke (p < 0.05), whereas no significant changes were observed in female rats. Additionally, in female rats, the expression of basic fibroblast growth factor (bFGF) increased after ischemic stroke and astrocytic optogenetic stimulation (p < 0.05), leading to enhanced endothelial cell proliferation compared to male rats (p < 0.05). In vitro experiments further demonstrated that the astrocyte activation was inhibited in the presence of bFGF (p < 0.05). These findings suggest that the increase in bFGF levels in females following stroke may inhibit the optogenetic activation of astrocytes, thereby attenuating the therapeutic effect of astrocyte activation on post-stroke brain repair. This study provides important insights into the gender-specific roles of astrocytes in the acute phase of ischemic stroke. Full article

Retinal degenerations, such as age-related macular degeneration and retinitis pigmentosa, present significant challenges due to genetic heterogeneity, limited therapeutic options, and the progressive loss of photoreceptors in advanced stages. These challenges are compounded by difficulties in precisely targeting residual retinal neurons and ensuring the sustained efficacy of interventions. Optogenetics offers a novel approach to vision restoration by inducing light sensitivity in residual retinal neurons through gene delivery of light-sensitive opsins. This review traces the evolution of opsins in optogenetic therapies, highlighting advancements from early research on channelrhodopsin-2 (ChR2) to engineered variants addressing key limitations. Red-shifted opsins, including ReaChR and ChrimsonR, reduced phototoxicity by enabling activation under longer wavelengths, while Chronos introduced superior temporal kinetics for dynamic visual tracking. Further innovations, such as Multi-Characteristic Opsin 1 (MCO1), optimized opsin performance under ambient light, bridging the gap to real-world applications. Key milestones include the first partial vision restoration in a human patient using ChrimsonR with light-amplifying goggles and ongoing clinical trials exploring the efficacy of opsin-based therapies for advanced retinal degeneration. While significant progress has been made, challenges remain in achieving sufficient light sensitivity for functional vision under normal ambient lighting conditions in a manner that is both effective and safe, eliminating the need for external light-enhancing devices. As research progresses, optogenetic therapies are positioned to redefine the management of retinal degenerative diseases, offering new hope for millions affected by vision loss. Full article

This paper presents the development of a computer simulation framework, designed as a cost–effective and technically efficient alternative to experimental studies. The framework focuses on the Bio–Neural Dust System proposed in our previous works, which consists of two components: a light–emitting bio–nanosensor and an opsin–expressing genetically modified neuron. This innovative system holds significant potential for applications in neuroscience and biotechnology research. Programmed in Python, the framework provides researchers with a virtual tool to test and evaluate the Bio–Neural Dust System, enabling the prediction of outcomes for future in vivo experiments. This approach not only conserves resources, but also offers scientists a flexible and accessible means to investigate the complex interactions within the system prior to real–world applications. The framework’s adaptability and potential for diverse research applications highlight its importance in advancing the field of bio–nanotechnology. Full article

Optogenetics is a combination of optical and genetic technologies used to activate or, conversely, inhibit specific cells in living tissues. The possibilities of using optogenetics approaches for the treatment of epilepsy, Parkinson’s and Alzheimer’s disease (AD) are being actively researched. In recent years, it has become clear that one of the most important players in the development of AD is astrocytes. Astrocytes affect amyloid clearance, participate in the development of neuroinflammation, and regulate the functioning of neural networks. We used an adeno-associated virus carrying the glial fibrillary acidic protein (GFAP) promoter driving the optogenetic channelrhodopsin-2 (ChR2) gene to transduce astrocytes in primary mouse hippocampal cultures. We recorded the bioelectrical activity of neural networks from day 14 to day 21 of cultivation using multielectrode arrays. A single optogenetic stimulation of astrocytes at 14 day of cultivation (DIV14) did not cause significant changes in neural network bioelectrical activity. Chronic optogenetic stimulation from DIV14 to DIV21 exerts a stimulatory effect on the bioelectrical activity of primary hippocampal cultures (the proportion of spikes included in network bursts significantly increased since DIV19). Moreover, chronic optogenetic stimulation over seven days partially preserved the activity and functional architecture of neuronal network in amyloidosis modeling. These results suggest that the selective optogenetic activation of astrocytes may represent a promising novel therapeutic strategy for combating AD. Full article

Channelrhodopsins (ChRs) are light-gated ion channels originally discovered in algae and are commonly used in neuroscience for controlling the electrical activity of neurons with high precision. Initially-discovered ChRs were non-selective cation channels, allowing the flow of multiple ions, such as Na⁺, K⁺, H⁺, and Ca²⁺, leading to membrane depolarization and triggering action potentials in neurons. As the field of optogenetics has evolved, ChRs with more specific ion selectivity were discovered or engineered, offering more precise optogenetic manipulation. This review highlights the natural occurrence and engineered variants of sodium-selective channelrhodopsins (NaChRs), emphasizing their importance in optogenetic applications. These tools offer enhanced specificity in Na⁺ ion conduction, reducing unwanted effects from other ions, and generating strong depolarizing currents. Some of the NaChRs showed nearly no desensitization upon light illumination. These characteristics make them particularly useful for experiments requiring robust depolarization or direct Na⁺ ion manipulation. The review further discusses the molecular structure of these channels, recent advances in their development, and potential applications, including a proposed drug delivery system using NaChR-expressing red blood cells that could be triggered to release therapeutic agents upon light activation. This review concludes with a forward-looking perspective on expanding the use of NaChRs in both basic research and clinical settings. Full article

The preBötzinger complex (preBötC) and the Bötzinger complex (BötC) are interconnected neural circuits that are involved in the regulation of breathing in mammals. Fast inhibitory neurotransmission is known to play an important role in the interaction of these two regions. Moreover, the corelease of glycine and GABA has been described in the respiratory network, but the contribution of the individual neurotransmitter in different pathways remains elusive. In sagittal brainstem slices of neonatal mice, we employed a laser point illumination system to activate glycinergic neurons expressing channelrhodopsin-2 (ChR2). This approach allowed us to discern the contribution of glycine and GABA to postsynaptic currents of individual whole-cell clamped neurons in the preBötC and BötC through the application of glycine and GABA receptor-specific antagonists. In more than 90% of the recordings, both transmitters contributed to the evoked IPSCs, with the glycinergic component being larger than the GABAergic component. The GABAergic component appeared to be most prominent when stimulation and recording were both performed within the preBötC. Taken together, our data suggest that GABA–glycine cotransmission is the default mode in the respiratory network of neonatal mice with regional differences that may be important in tuning the network activity. Full article

Channelrhodopsin-2 (ChR2) is an important tool for optogenetics, and some of its mutants are Ca²⁺-selective channels. However, the mechanism for Ca²⁺-selective permeation is still unclear. In this study, molecular dynamic (MD) simulations for the Ca²⁺ permeation of the CatCh mutant were carried out to investigate the fundamental features of the selectivity of Ca²⁺. Research on the conformational changes in the key residues near the central gate (CG) of the channel suggested that E83, E90, and D253 play an important role in Ca²⁺ conductivity. The clustering analysis indicates that the above “EED triad” acts as a filter, and Ca²⁺ can only pass through if the EED is in a certain conformation. It was also found that hydrated Ca²⁺ can be coordinated with carboxyl groups, resulting in the loss of part of the water molecules in the hydrated shell and a reduction in ionic radius, which helps Ca²⁺ enter the channel. Full article

Alzheimer’s disease (AD) is the most common form of dementia, resulting in disability and mortality. The global incidence of AD is consistently surging. Although numerous therapeutic agents with promising potential have been developed, none have successfully treated AD to date. Consequently, the pursuit of novel methodologies to address neurodegenerative processes in AD remains a paramount endeavor. A particularly promising avenue in this search is optogenetics, enabling the manipulation of neuronal activity. In recent years, research attention has pivoted from neurons to glial cells. This review aims to consider the potential of the optogenetic correction of astrocyte metabolism as a promising strategy for correcting AD-related disorders. The initial segment of the review centers on the role of astrocytes in the genesis of neurodegeneration. Astrocytes have been implicated in several pathological processes associated with AD, encompassing the clearance of β-amyloid, neuroinflammation, excitotoxicity, oxidative stress, and lipid metabolism (along with a critical role in apolipoprotein E function). The effect of astrocyte–neuronal interactions will also be scrutinized. Furthermore, the review delves into a number of studies indicating that changes in cellular calcium (Ca²⁺) signaling are one of the causes of neurodegeneration. The review’s latter section presents insights into the application of various optogenetic tools to manipulate astrocytic function as a means to counteract neurodegenerative changes. Full article

The fundamental process of information processing and memory formation in the brain is associated with complex neuron firing patterns, which can occur spontaneously or be triggered by sensory inputs. Optogenetics has revolutionized neuroscience by enabling precise manipulation of neuronal activity patterns in specified neural populations using light. However, the light pulses used in optogenetics have been primarily restricted to square waveforms. Here, we present a detailed theoretical analysis of the temporal shaping of light pulses in optogenetic excitation of hippocampal neurons and neocortical fast-spiking interneurons expressed with ultrafast (Chronos), fast (ChR2), and slow (ChRmine) channelrhodopsins. Optogenetic excitation has been studied with light pulses of different temporal shapes that include square, forward-/backward ramps, triangular, left-/right-triangular, Gaussian, left-/right-Gaussian, positive-sinusoidal, and left-/right-positive sinusoidal. Different light shapes result in significantly different photocurrent amplitudes and kinetics, spike-timing, and spontaneous firing rate. For short duration stimulations, left-Gaussian pulse results in larger photocurrent in ChR2 and Chronos than square pulse of the same energy density. Time to peak photocurrent in each opsin is minimum at right-Gaussian pulse. The optimal pulse width to achieve peak photocurrent for non-square pulses is 10 ms for Chronos, and 50 ms for ChR2 and ChRmine. The pulse energy to evoke spike in hippocampal neurons can be minimized on choosing square pulse with Chronos, Gaussian pulse with ChR2, and positive-sinusoidal pulse with ChRmine. The results demonstrate that non-square waveforms generate more naturalistic spiking patterns compared to traditional square pulses. These findings provide valuable insights for the development of new optogenetic strategies to better simulate and manipulate neural activity patterns in the brain, with the potential to improve our understanding of cognitive processes and the treatment of neurological disorders. Full article

Both hemispheres connect with each other by excitatory callosal projections, and whether inhibitory interneurons, usually believed to have local innervation, engage in transcallosal activity modulation is unknown. Here, we used optogenetics in combination with cell-type-specific channelrhodopsin-2 expression to activate different inhibitory neuron subpopulations in the visual cortex and recorded the response of the entire visual cortex using intrinsic signal optical imaging. We found that optogenetic stimulation of inhibitory neurons reduced spontaneous activity (increase in the reflection of illumination) in the binocular area of the contralateral hemisphere, although these stimulations had different local effects ipsilaterally. The activation of contralateral interneurons differentially affected both eye responses to visual stimuli and, thus, changed ocular dominance. Optogenetic silencing of excitatory neurons affects the ipsilateral eye response and ocular dominance in the contralateral cortex to a lesser extent. Our results revealed a transcallosal effect of interneuron activation in the mouse visual cortex. Full article

Channelrhodopsin-2 (ChR2) has been one of the most important objects in the study of optogenetics. The retinal chromophore molecule absorbs photons and undergoes an isomerization reaction, which triggers the photocycle, resulting in a series of conformational changes. In this study, a series of intermediate structures (including D470, P500, P390-early, P390-late, and P520 states) of ChR2 in the photocycle were modeled, and molecular dynamics (MD) simulations were performed to elucidate the mechanism of ion channel opening of ChR2. The maximum absorption wavelength of these intermediates calculated by time-dependent density function theory (TD-DFT) is in general agreement with the experimental values, the distribution of water density gradually increases in the process of photocycle, and the radius of the ion channel is larger than 6 Å. All these results indicate that our structural models of the intermediates are reasonable. The evolution of protonation state of E90 during the photocycle is explained. E90 will deprotonate when the P390-early transforms into P390-late, in which the two conformations of P390-early and P390-late obtained from the simulations are consistent with the experimental descriptions. To validate the conductive P520 state, the potential mean force (PMF) of Na${}^{+}$ ions passing through the P520 intermediate was calculated by using steered molecular dynamics (SMD) simulation combined with umbrella sampling. The result shows that the Na${}^{+}$ ions passing through the channel with a very low energy barrier, especially in the central gate, is almost barrierless. This indicates that the channel is open in the P520 state. Full article

Channelrhodopsins have been utilized in gene therapy to restore vision in patients with retinitis pigmentosa and their channel kinetics are an important factor to consider in such applications. We investigated the channel kinetics of ComV1 variants with different amino acid residues at the 172nd position. Patch clamp methods were used to record the photocurrents induced by stimuli from diodes in HEK293 cells transfected with plasmid vectors. The channel kinetics (τon and τoff) were considerably altered by the replacement of the 172nd amino acid and was dependent on the amino acid characteristics. The size of amino acids at this position correlated with τon and decay, whereas the solubility correlated with τon and τoff. Molecular dynamic simulation indicated that the ion tunnel constructed by H172, E121, and R306 widened due to H172A variant, whereas the interaction between A172 and the surrounding amino acids weakened compared with H172. The bottleneck radius of the ion gate constructed with the 172nd amino acid affected the photocurrent and channel kinetics. The 172nd amino acid in ComV1 is a key residue for determining channel kinetics as its properties alter the radius of the ion gate. Our findings can be used to improve the channel kinetics of channelrhodopsins. Full article

Guillardia theta anion channelrhodopsin 1 (GtACR1) is a widely used inhibitor of optogenetics with unique conductance mechanisms and photochemistry. However, the molecular mechanism of light-gated anion conduction is poorly understood without a crystal structure for the intermediate state. In this study, we built the dark-state model based on the crystal structure of retinal and isomerized the model by twisting the C12-C13=C14-C15 dihedral step by step using molecular dynamics simulation. The conformational changes revealed the all-trans to 13-cis photoisomerization of the retinal chromophore cannot open the channel. There is no water influx, and a pre-opened K-like intermediate after photoisomerization of retinal is formed. During the opening of the ion channel, proton transfer occurs between E68 and D234. Steered molecular dynamics (SMD) and umbrella sampling indicated that the E68 and D234 were the key residues for chloride-ion conducting. We propose a revised channel opening pathway model of GtACR1 after analyzing (de)protonation of E68 and D234. Reprotonation of D234 will result in two different early L intermediates, named L₁-like and L${}_1$‘-like, which correspond to the L${}_1$ and L${}_1$‘ intermediates reported in a recent study. Simulation results showed that L${}_1$-like may convert by parallel paths into L${}_1$‘-like and L${}_2$-like states. This model provides conformational details for the intermediate as well. Full article

Low-frequency electrical stimulation is used to treat some drug-resistant forms of epilepsy. Despite the effectiveness of the method in suppressing seizures, there is a considerable risk of side effects. An optogenetic approach allows the targeting of specific populations of neurons, which can increase the effectiveness and safety of low-frequency stimulation. In our study, we tested the efficacy of the suppression of ictal activity in entorhinal cortex slices in a 4-aminopyridine model with three variants of low-frequency light stimulation (LFLS): (1) activation of excitatory and inhibitory neurons (on Thy1-ChR2-YFP mice), (2) activation of inhibitory interneurons only (on PV-Cre mice after virus injection with channelrhodopsin2 gene), and (3) hyperpolarization of excitatory neurons (on Wistar rats after virus injection with archaerhodopsin gene). Only in the first variant did simultaneous LFLS of excitatory and inhibitory neurons replace ictal activity with interictal activity. We suggest that LFLS caused changes in the concentration gradients of K⁺ and Na⁺ cations across the neuron membrane, which activated Na-K pumping. According to the mathematical modeling, the increase in Na-K pump activity in neurons induced by LFLS led to an antiepileptic effect. Thus, a less specific and generalized optogenetic effect on entorhinal cortex neurons was more effective in suppressing ictal activity in the 4-aminopyridine model. Full article

Optogenetics is a recent breakthrough in neuroscience, and one of the most promising applications is the treatment of retinal degenerative diseases. Multiple clinical trials are currently ongoing, less than a decade after the first attempt at visual restoration using optogenetics. Optogenetic therapy has great value in providing hope for visual restoration in late-stage retinal degeneration, regardless of the genotype. This alternative gene therapy consists of multiple elements including the choice of target retinal cells, optogenetic tools, and gene delivery systems. Currently, there are various options for each element, all of which have been developed as a product of technological success. In particular, the performance of optogenetic tools in terms of light and wavelength sensitivity have been improved by engineering microbial opsins and applying human opsins. To provide better post-treatment vision, the optimal choice of optogenetic tools and effective gene delivery to retinal cells is necessary. In this review, we provide an overview of the advancements in optogenetic therapy for visual restoration, focusing on available options for optogenetic tools and gene delivery methods. Full article