Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Optogenetics is a technology using light-sensitive proteins to control signaling pathways and physiological processes in cells and organs and has been applied in neuroscience, cardiovascular sciences, and many other research fields. Most commonly used optogenetic actuators are sensitive to blue and green light, but red-light activation would allow better tissue penetration and less phototoxicity. Cyp27c1 is a recently deorphanized cytochrome P450 enzyme that converts vitamin A1 to vitamin A2, thereby red-shifting the spectral sensitivity of visual pigments and enabling near-infrared vision in some aquatic species. Here, we investigated the ability of Cyp27c1-generated vitamin A2 to induce a shift in spectral sensitivity of the light-gated ion channel Channelrhodopsin-2 (ChR2) and its red-shifted homolog ReaChR. We used patch clamp to measure photocurrents at specific wavelengths in HEK 293 cells expressing ChR2 or ReaChR. Vitamin A2 incubation red-shifted the wavelength for half-maximal currents (λ50%) by 6.8 nm for ChR2 and 12.4 nm for ReaChR. Overexpression of Cyp27c1 in HEK 293 cells showed mitochondrial localization, and HPLC analysis showed conversion of vitamin A1 to vitamin A2. Notably, the λ50% of ChR2 photocurrents was red-shifted by 10.5 nm, and normalized photocurrents at 550 nm were about twofold larger with Cyp27c1 expression. Similarly, Cyp27c1 shifted the λ50% of ReaChR photocurrents by 14.3 nm and increased normalized photocurrents at 650 nm almost threefold. Since vitamin A2 incubation is not a realistic option for in vivo applications and expression of Cyp27c1 leads to a greater red-shift in spectral sensitivity, we propose co-expression of this enzyme as a novel strategy for red-shifted optogenetics. [ABSTRACT FROM AUTHOR]

Introduction: Early afterdepolarizations (EADs) are secondary voltage depolarizations associated with reduced repolarization reserve (RRR) that can trigger lethal arrhythmias. Relating EADs to triggered activity is difficult to study, so the ability to suppress or provoke EADs would be experimentally useful. Here, we use computational simulations to assess the feasibility of subthreshold optogenetic stimulation modulating the propensity for EADs (cell-scale) and EAD-associated ectopic beats (organ-scale). Methods: We modified a ventricular ionic model by reducing rapid delayed rectifier potassium (0.25–0.1 × baseline) and increasing L-type calcium (1.0–3.5 × baseline) currents to create RRR conditions with varying severity. We ran simulations in models of single cardiomyocytes and left ventricles from post-myocardial infarction patient MRI scans. Optogenetic stimulation was simulated using either ChR2 (depolarizing) or GtACR1 (repolarizing) opsins. Results: In cell-scale simulations without illumination, EADs were seen for 164 of 416 RRR conditions. Subthreshold stimulation of GtACR1 reduced EAD incidence by up to 84.8% (25/416 RRR conditions; 0.1 μW/mm2); in contrast, subthreshold ChR2 excitation increased EAD incidence by up to 136.6% (388/416 RRR conditions; 50 μW/mm2). At the organ scale, we assumed simultaneous, uniform illumination of the epicardial and endocardial surfaces. GtACR1-mediated suppression (10–50 μW/mm2) and ChR2-mediated unmasking (50–100 μW/mm2) of EAD-associated ectopic beats were feasible in three distinct ventricular models. Conclusions: Our findings suggest that optogenetics could be used to silence or provoke both EADs and EAD-associated ectopic beats. Validation in animal models could lead to exciting new experimental regimes and potentially to novel anti-arrhythmia treatments. [ABSTRACT FROM AUTHOR]

The dentate gyrus of the hippocampus is targeted by axons from serotonin raphe neurons, where the neurotransmitter modulates adult neurogenesis and antidepressant action, and mediates the neurogenic effect of running. Whether running-induced cell proliferation is directly mediated by serotonin remains unknown. Here, we took advantage of Tph2-ChR2-YFP transgenic mice in which the light-sensitive protein channelrhodopsin-2 (ChR2) is specifically expressed in tryptophan hydroxylase 2 (TPH2)-expressing neurons. We selectively activated serotonin neurons via optogenetics and determined the effect on cell proliferation in the dentate gyrus. Our data reveal a significant reduction in the number of newly generated cells upon overnight raphe stimulation. The decrease in cell proliferation was absent when serotonin neurons were light-activated for six consecutive nights. However, we observed an interhemispheric difference in BrdU-positive cell numbers. We conclude that acute network dynamics occur between serotonin raphe neurons and the hippocampus, directly affecting precursor cell proliferation.

Originating from the brainstem raphe nuclei, serotonin is an important neuromodulator involved in a variety of physiological and pathological functions. Specific optogenetic stimulation of serotonergic neurons results in the divisive suppression of spontaneous, but not sensory evoked activity in the majority of neurons in the primary olfactory cortex and an increase in firing in a minority of neurons. To reveal the mechanisms involved in this dual serotonergic control of cortical activity we used a combination of in vitro electrophysiological recordings from identified neurons in the primary olfactory cortex, optogenetics and pharmacology and found that serotonin suppressed the activity of principal neurons, but excited local interneurons. The results have important implications in sensory information processing and other functions of the olfactory cortex and related brain areas. [ABSTRACT FROM AUTHOR]

Subcellular targeting of opsins in optogenetics provides new possibilities for studying the function of nerve cells. One of the widely used motifs for central targeting of opsins is the motif of the potential-dependent potassium channel Kv2.1. In our study, ChR2-Venus-Kv2.1 construct was expressed in the layer 2/3 pyramidal neurons of murine cerebral cortex by means of in utero electroporation. We found that, although the majority of neurons expressing ChR2-Venus-Kv2.1 demonstrated mainly central localization of fluorescence, limited to the soma, proximal dendrites, and axon, a considerable quantity of neurons remarkably exhibited disrupted targeting, i.e., fluorescent protein distributed far beyond the soma and proximal parts of all processes. We hypothesized that the observed mislocalization might have been caused by overexpression of the construct. It was found that a decrease in the plasmid concentration during the in utero electroporation from 1 to 0.35 μg/μl resulted in almost complete disappearance of the neurons that had altered targeting. Moreover, we found a pronounced correlation between the absolute brightness of the somatic fluorescence and the visible length of the apical dendrite, which also provides evidence that indeed overexpression might be the reason for the mislocalization of ChR2-Venus-Kv2.1 construct. Thus, the possibility of disruption of central targeting of ChR2 by the potassium channel Kv2.1 motif should be taken into account when using this construct in optogenetic experiments when somatic localization of the opsin is necessary. Lowering plasmid concentration during transfection procedure could be a solution for the above problem. [ABSTRACT FROM AUTHOR]

The functional state of denervated muscle is a critical factor in the ability to restore movement after injury- or disease-related paralysis. Here we used peripheral optogenetic stimulation and transcriptome profiling in the mouse whisker system to investigate the time course of changes in neuromuscular function following complete unilateral facial nerve transection. While most skeletal muscles rapidly lose functionality after lower motor neuron denervation, optogenetic muscle stimulation of the paralyzed whisker pad revealed sustained increases in the sensitivity, velocity, and amplitude of whisker movements, and reduced fatigability, starting 48 h after denervation. RNA-seq analysis showed distinct regulation of multiple gene families in denervated whisker pad muscles compared with the atrophy-prone soleus, including prominent changes in ion channels and contractile fibers. Together, our results define the unique functional and transcriptomic landscape of denervated facial muscles and have general implications for restoring movement after neuromuscular injury or disease. NEW & NOTEWORTHY Optogenetic activation of muscle can be used to noninvasively induce movements and probe muscle function. We used this technique in mice to investigate changes in whisker movements following facial nerve transection. We found unexpectedly enhanced functional properties of whisker pad muscle following denervation, accompanied by unique transcriptomic changes. Our findings highlight the utility of the mouse whisker pad for investigating the restoration of movement after paralysis. [ABSTRACT FROM AUTHOR]

L-dopa-induced dyskinesias (LIDs) are a serious complication of L-dopa therapy for Parkinson's disease. Emerging evidence indicates that the nicotinic cholinergic system plays a role in LIDs, although the pathways and mechanisms are poorly understood. Here we used optogenetics to investigate the role of striatal cholinergic interneurons in LIDs. Mice expressing cre-recombinase under the control of the choline acetyltransferase promoter (ChAT-Cre) were lesioned by unilateral injection of 6-hydroxydopamine. AAV5-ChR2-eYFP or AAV5-control-eYFP was injected into the dorsolateral striatum, and optical fibers implanted. After stable virus expression, mice were treated with L-dopa. They were then subjected to various stimulation protocols for 2 h and LIDs rated. Continuous stimulation with a short duration optical pulse (1–5 ms) enhanced LIDs. This effect was blocked by the general muscarinic acetylcholine receptor (mAChR) antagonist atropine indicating it was mAChR-mediated. By contrast, continuous stimulation with a longer duration optical pulse (20 ms to 1 s) reduced LIDs to a similar extent as nicotine treatment (~50%). The general nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine blocked the decline in LIDs with longer optical pulses showing it was nAChR-mediated. None of the stimulation regimens altered LIDs in control-eYFP mice. Lesion-induced motor impairment was not affected by optical stimulation indicating that cholinergic transmission selectively regulates LIDs. Longer pulse stimulation increased the number of c-Fos expressing ChAT neurons, suggesting that changes in this immediate early gene may be involved. These results demonstrate that striatal cholinergic interneurons play a critical role in LIDs and support the idea that nicotine treatment reduces LIDs via nAChR desensitization.

Channelrhodopsin-2 (ChR2) is a light-sensitive ion channel widely used in optogenetics. Photoactivation triggers a trans-to-cis isomerization of a covalently bound retinal. Ensuing conformational changes open a cation-selective channel. We explore the structural dynamics in the early photocycle leading to channel opening by classical (MM) and quantum mechanical (QM) molecular simulations. With QM/MM simulations, we generated a protein-adapted force field for the retinal chromophore, which we validated against absorption spectra. In a 4-µs MM simulation of a dark-adapted ChR2 dimer, water entered the vestibules of the closed channel. Retinal all-trans to 13-cis isomerization, simulated with metadynamics, triggered a major restructuring of the charge cluster forming the channel gate. On a microsecond time scale, water penetrated the gate to form a membrane-spanning preopen pore between helices H1, H2, H3, and H7. This influx of water into an ion-impermeable preopen pore is consistent with time-resolved infrared spectroscopy and electrophysiology experiments. In the retinal 13-cis state, D253 emerged as the proton acceptor of the Schiff base. Upon proton transfer from the Schiff base to D253, modeled by QM/MM simulations, we obtained an early-M/P2 390-like intermediate. Rapid rotation of the unprotonated Schiff base toward the cytosolic side effectively prevents its reprotonation from the extracellular side. From MM and QM simulations, we gained detailed insight into the mechanism of ChR2 photoactivation and early events in pore formation. By rearranging the network of charges and hydrogen bonds forming the gate, water emerges as a key player in light-driven ChR2 channel opening. [ABSTRACT FROM AUTHOR]

Optogenetics has emerged as an important tool in neuroscience, especially in central nervous system research. It allows for the study of the brain's highly complex network with high temporal and spatial resolution. The enteric nervous system (ENS), the brain in the gut, plays critical roles for life. Although advanced progress has been made, the neural circuits of the ENS remain only partly understood because the appropriate research tools are lacking. In this review, I highlight the potential application of optogenetics in ENS research. Firstly, I describe the development of optogenetics with focusing on its three main components. I discuss the applications in vitro and in vivo , and summarize current findings in the ENS research field obtained by optogenetics. Finally, the challenges for the application of optogenetics to the ENS research will be discussed. [ABSTRACT FROM AUTHOR]

The advent of optogenetic tools has allowed unprecedented insights into the organization of neuronal networks. Although recently developed technologies have enabled implementation of optogenetics for studies of brain function in freely moving, untethered animals, wireless powering and device durability pose challenges in studies of spinal cord circuits where dynamic, multidimensional motions against hard and soft surrounding tissues can lead to device degradation. We demonstrate here a fully implantable optoelectronic device powered by near-field wireless communication technology, with a thin and flexible open architecture that provides excellent mechanical durability, robust sealing against biofluid penetration and fidelity in wireless activation, thereby allowing for long-term optical stimulation of the spinal cord without constraint on the natural behaviors of the animals. The system consists of a double-layer, rectangular-shaped magnetic coil antenna connected to a microscale inorganic light-emitting diode (μ-ILED) on a thin, flexible probe that can be implanted just above the dura of the mouse spinal cord for effective stimulation of light-sensitive proteins expressed in neurons in the dorsal horn. Wireless optogenetic activation of TRPV1-ChR2 afferents with spinal μ-ILEDs causes nocifensive behaviors and robust real-time place aversion with sustained operation in animals over periods of several weeks to months. The relatively low-cost electronics required for control of the systems, together with the biocompatibility and robust operation of these devices will allow broad application of optogenetics in future studies of spinal circuits, as well as various peripheral targets, in awake, freely moving and untethered animals, where existing approaches have limited utility. [ABSTRACT FROM AUTHOR]

To maintain energy homeostasis, orexigenic (appetite-inducing) and anorexigenic (appetite suppressing) brain systems functionally interact to regulate food intake. Within the hypothalamus, neurons that express agouti-related protein (AgRP) sense orexigenic factors and orchestrate an increase in food-seeking behavior. In contrast, calcitonin gene-related peptide (CGRP)-expressing neurons in the parabrachial nucleus (PBN) suppress feeding. PBN CGRP neurons become active in response to anorexigenic hormones released following a meal, including amylin, secreted by the pancreas, and cholecystokinin (CCK), secreted by the small intestine. Additionally, exogenous compounds, such as lithium chloride (LiCl), a salt that creates gastric discomfort, and lipopolysaccharide (LPS), a bacterial cell wall component that induces inflammation, exert appetite-suppressing effects and activate PBN CGRP neurons. The effects of increasing the homeostatic drive to eat on feeding behavior during appetite suppressing conditions are unknown. Here, we show in mice that food deprivation or optogenetic activation of AgRP neurons induces feeding to overcome the appetite suppressing effects of amylin, CCK, and LiCl, but not LPS. AgRP neuron photostimulation can also increase feeding during chemogenetic-mediated stimulation of PBN CGRP neurons. AgRP neuron stimulation reduces Fos expression in PBN CGRP neurons across all conditions. Finally, stimulation of projections from AgRP neurons to the PBN increases feeding following administration of amylin, CCK, and LiCl, but not LPS. These results demonstrate that AgRP neurons are sufficient to increase feeding during noninflammatory-based appetite suppression and to decrease activity in anorexigenic PBN CGRP neurons, thereby increasing food intake during homeostatic need. [ABSTRACT FROM AUTHOR]

Background The algal protein Channelrhodopsin-2 (ChR2) has been widely used in recent years in optogenetic technique to investigate the functions of complex neuronal networks through minimally invasive and temporally precise photostimulation of genetically defined neurons. However, as with any other new technique, current optogentic approaches have various limitations. In addition, how ChR2 may behave in response to complex biochemical changes associated with various physiological/pathological conditions is largely unknown. Aim In this study, we investigated whether a change in redox state of the cell affects the activity of ChR2 channels. Methods Whole-cell patch-clamp recordings were used to examine the effect of reducing and oxidizing agents on ChR2 currents activated by blue light. Results We show that the reducing agent dithiothreitol ( DTT) dramatically potentiates the ChR2 currents in a reversible and concentration-dependent manner. Glutathione, an endogenous reducing agent, shows a similar effect on ChR2 currents. The oxidizing agent 5,5′-dithio-bis-(2-nitrobenzoic acid) ( DTNB) has no effect on ChR2 currents by itself; however, it completely reverses the potentiating effect of DTT. DTT also causes a shift in the current-voltage relationship by 23 ± 4.31 mV, suggesting a change in ion selectivity. Conclusion Taken together, these data suggest that redox modification of ChR2 plays an important role in its sensitivity to the light stimulation. Our findings not only help for a better understanding of how ChR2 may behave in physiological/pathological conditions where changes in redox state are common, but also provide a new direction for further optimization of this important opsin. [ABSTRACT FROM AUTHOR]

Summary Cyclic AMP (cAMP) signaling augments synaptic transmission, but because many targets of cAMP and protein kinase A (PKA) may be involved, mechanisms underlying this pathway remain unclear. To probe this mechanism, we used optogenetic stimulation of cAMP signaling by Beggiatoa -photoactivated adenylyl cyclase (bPAC) in Caenorhabditis elegans motor neurons. Behavioral, electron microscopy (EM), and electrophysiology analyses revealed cAMP effects on both the rate and on quantal size of transmitter release and led to the identification of a neuropeptidergic pathway affecting quantal size. cAMP enhanced synaptic vesicle (SV) fusion by increasing mobilization and docking/priming. cAMP further evoked dense core vesicle (DCV) release of neuropeptides, in contrast to channelrhodopsin (ChR2) stimulation. cAMP-evoked DCV release required UNC-31/Ca 2+ -dependent activator protein for secretion (CAPS). Thus, DCVs accumulated in unc-31 mutant synapses. bPAC-induced neuropeptide signaling acts presynaptically to enhance vAChT-dependent SV loading with acetylcholine, thus causing increased miniature postsynaptic current amplitudes (mPSCs) and significantly enlarged SVs. [ABSTRACT FROM AUTHOR]

We present a reconfigurable neural processor for real-time simulation and prediction of opto-neural behaviour. We combined a detailed Hodgkin–Huxley CA3 neuron integrated with a four-state Channelrhodopsin-2 (ChR2) model into reconfigurable silicon hardware. Our architecture consists of a Field Programmable Gated Array (FPGA) with a custom-built computing data-path, a separate data management system and a memory approach based router. Advancements over previous work include the incorporation of short and long-term calcium and light-dependent ion channels in reconfigurable hardware. Also, the developed processor is computationally efficient, requiring only 0.03 ms processing time per sub-frame for a single neuron and 9.7 ms for a fully connected network of 500 neurons with a given FPGA frequency of 56.7 MHz. It can therefore be utilized for exploration of closed loop processing and tuning of biologically realistic optogenetic circuitry. [ABSTRACT FROM PUBLISHER]

Optogenetics has revolutionized the study of functional neuronal circuitry (Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K. Nat Neurosci 8: 1263-1268, 2005; Deisseroth K. Nat Methods 8: 26-29, 2011). Although these techniques have been most successfully implemented in rodent models, they have the potential to be similarly impactful in studies of nonhuman primate brains. Common marmosets (Callithrix jacchus) have recently emerged as a candidate primate model for gene editing, providing a potentially powerful model for studies of neural circuitry and disease in primates. The application of viral transduction methods in marmosets for identifying and manipulating neuronal circuitry is a crucial step in developing this species for neuroscience research. In the present study we developed a novel, chronic method to successfully induce rapid photostimulation in individual cortical neurons transduced by adeno-associated virus to express channelrhodopsin (ChR2) in awake marmosets. We found that large proportions of neurons could be effectively photoactivated following viral transduction and that this procedure could be repeated for several months. These data suggest that techniques for viral transduction and optical manipulation of neuronal populations are suitable for marmosets and can be combined with existing behavioral preparations in the species to elucidate the functional neural circuitry underlying perceptual and cognitive processes. [ABSTRACT FROM AUTHOR]

Corticotrophin Releasing Factor (CRF) is a critical stress-related neuropeptide in major output pathways of the amygdala, including the central nucleus (CeA), and in a key projection target of the CeA, the bed nucleus of the stria terminalis (BnST). While progress has been made in understanding the contributions and characteristics of CRF as a neuropeptide in rodent behavior, little attention has been committed to determine the properties and synaptic physiology of specific populations of CRF-expressing (CRF + ) and non-expressing (CRF − ) neurons in the CeA and BnST. Here, we fill this gap by electrophysiologically characterizing distinct neuronal subtypes in CeA and BnST. Crossing tdTomato or channelrhodopsin-2 (ChR2-YFP) reporter mice to those expressing Cre-recombinase under the CRF promoter allowed us to identify and manipulate CRF + and CRF − neurons in CeA and BnST, the two largest areas with fluorescently labeled neurons in these mice. We optogenetically activated CRF + neurons to elicit action potentials or synaptic responses in CRF + and CRF − neurons. We found that GABA is the predominant co-transmitter in CRF + neurons within the CeA and BnST. CRF + neurons are highly interconnected with CRF − neurons and to a lesser extent with CRF + neurons. CRF + and CRF − neurons differentially express tonic GABA currents. Chronic, unpredictable stress increase the amplitude of evoked IPSCs and connectivity between CRF + neurons, but not between CRF + and CRF − neurons in both regions. We propose that reciprocal inhibition of interconnected neurons controls CRF + output in these nuclei. [ABSTRACT FROM AUTHOR]

To investigate excitatory and inhibitory GABA actions in cortical neuronal networks, we present a novel optogenetic approach using a mouse knock-in line with conditional expression of channelrhodopsin-2 (ChR2) in GABAergic interneurons. During whole-cell recordings from hippocampal and neocortical slices from postnatal day (P) 2-P15 mice, photostimulation caused depolarization and excitation of interneurons and evoked barrages of postsynaptic GABAergic currents. Excitatory/inhibitory GABA actions on pyramidal cells were assessed by monitoring the alteration in the frequency of EPSCs during photostimulation of interneurons. We found that in slices from P2-P8 mice, photostimulation evoked an increase in EPSC frequency, whereas in P9-P15 mice the response switched to a reduction in EPSC frequency, indicating a developmental excitatory-to-inhibitory switch in GABA actions on glutamatergic neurons. Using a similar approach in urethane-anesthetized animals in vivo, we found that photostimulation of interneurons reduces EPSC frequency at ages P3-P9. Thus, expression of ChR2 in GABAergic interneurons of mice enables selective photostimulation of interneurons during the early postnatal period, and these mice display a developmental excitatory-to-inhibitory switch in GABA action in cortical slices in vitro, but so far show mainly inhibitory GABA actions on spontaneous EPSCs in the immature hippocampus and neocortex in vivo. [ABSTRACT FROM AUTHOR]

Since the introduction of Channelrhodopsin-2 (ChR2) to neuroscience, optogenetics technology was developed, making it possible to activate specific neurons or circuits with spatial and temporal precision. Various ChR2 transgenic animal models have been generated and are playing important roles in revealing the mechanisms of neural activities, mapping neural circuits, controlling the behaviors of animals as well as exploring new strategy for treating the neurological diseases in both central and peripheral nervous system. An animal including humans senses environments through Aristotle's five senses (sight, hearing, smell, taste and touch). Usually, each sense is associated with a kind of sensory organ (eyes, ears, nose, tongue and skin). Is it possible that one could hear light, smell light, taste light and touch light? When ChR2 is targeted to different peripheral sensory neurons by viral vectors or generating ChR2 transgenic animals, the animals can sense the light as various sensations such as hearing, touch, pain, smell and taste. In this review, we focus on ChR2 transgenic animals in the peripheral nervous system. Firstly the working principle of ChR2 as an optogenetic actuator is simply described. Then the current transgenic animal lines where ChR2 was expressed in peripheral sensory neurons are presented and the findings obtained by these animal models are reviewed. [ABSTRACT FROM AUTHOR]

The role of the corticothalamic projection in the ventral motor thalamus remains poorly understood. Therefore, we studied the electrophysiological responses of neurons in the basal ganglia and cerebellar receiving-territories of the motor thalamus (BGMT and CbMT, respectively) using optogenetic activation of corticothalamic projections in awake rhesus macaques. After injections of viral vectors carrying the excitatory opsins ChR2 or C1V1 into the primary motor and premotor cortices of two monkeys, we used optrodes to light activate opsin-expressing neurons in cortex or their terminals in the thalamus while simultaneously recording the extracellular activity of neurons in the vicinity of the stimulation sites. As expected, light activation of opsins in the cerebral cortex evoked robust, short-latency increases in firing of cortical neurons. In contrast, light stimulation of corticothalamic terminals induced small-amplitude, long-latency increases and/or decreases of activity in thalamic neurons. In postmortem material, opsins were found to be expressed in cell bodies and dendrites of cortical neurons and along their corticothalamic projections. At the electron microscopic level, opsin labeling was confined to unmyelinated preterminal axons and small terminals that formed asymmetric synapses with dendrites of projection neurons or GABAergic interneurons in BGMT and CbMT and with neurons in the reticular thalamic nucleus. The morphological features of the transfected terminals, along with the long latency and complex physiological responses of thalamic neurons to their activation, suggest a modulatory role of corticothalamic afferents upon the primate ventral motor thalamus. [ABSTRACT FROM AUTHOR]

This review, one of a series of articles, tries to make sense of optogenetics, a recently developed technology that can be used to control the activity of genetically-defined neurons with light. Cells are first genetically engineered to express a lightsensitive opsin, which is typically an ion channel, pump, or G protein--coupled receptor. When engineered cells are then illuminated with light of the correct frequency, opsin-bound retinal undergoes a conformational change that leads to channel opening or pump activation, cell depolarization or hyperpolarization, and neural activation or silencing. Since the advent of optogenetics, many different opsin variants have been discovered or engineered, and it is now possible to stimulate or inhibit neuronal activity or intracellular signaling pathways on fast or slow timescales with a variety of different wavelengths of light. Optogenetics has been successfully employed to enhance our understanding of the neural circuit dysfunction underlying mood disorders, addiction, and Parkinson's disease, and has enabled us to achieve a better understanding of the neural circuits mediating normal behavior. It has revolutionized the field of neuroscience, and has enabled a new generation of experiments that probe the causal roles of specific neural circuit components. [ABSTRACT FROM AUTHOR]

Conditioned taste aversion (CTA) is a phenomenon in which an individual forms an association between a novel tastant and toxin-induced gastrointestinal malaise. Previous studies showed that the parabrachial nucleus (PBN) contains neurons that are necessary for the acquisition of CTA, but the specific neuronal populations involved are unknown. Previously, we identified calcitonin gene-related peptide (CGRP)-expressing neurons in the external lateral subdivision of the PBN (PBel) as being sufficient to suppress appetite and necessary for the anorexigenic effects of appetite-suppressing substances including lithium chloride (LiCl), a compound often used to induce CTA. Here, we test the hypothesis that PBel CGRP neurons are sufficient and necessary for CTA acquisition in mice. We show that optogenetic activation of these neurons is sufficient to induce CTA in the absence of anorexigenic substances, whereas genetically induced silencing of these neurons attenuates acquisition of CTA upon exposure to LiCl. Together, these results demonstrate that PBel CGRP neurons mediate a gastrointestinal distress signal required to establish CTA. [ABSTRACT FROM AUTHOR]

Optogenetics has become an important research tool and is being considered as the basis for several neural prostheses. However, few studies have applied optogenetics to the auditory brainstem. This study explored whether optical activation of the cochlear nucleus (CN) elicited responses in neurons in higher centers of the auditory pathway and whether it elicited an evoked response. Viral-mediated gene transfer was used to express channelrhodopsin-2 (ChR2) in the mouse CN. Blue light was delivered via an optical fiber placed near the surface of the infected CN and recordings were made in higher-level centers. Optical stimulation evoked excitatory multiunit spiking activity throughout the tonotopic axis of the central nucleus of the inferior colliculus (IC) and the auditory cortex (Actx). The pattern and magnitude of IC activity elicited by optical stimulation was comparable to that obtained with a 50 dB SPL acoustic click. This broad pattern of activity was consistent with histological confirmation of green fluorescent protein (GFP) label of cell bodies and axons throughout the CN. Increasing pulse rates up to 320 Hz did not significantly affect threshold or bandwidth of the IC responses, but rates higher than 50 Hz resulted in desynchronized activity. Optical stimulation also evoked an auditory brainstem response, which had a simpler waveform than the response to acoustic stimulation. Control cases showed no responses to optical stimulation. These data suggest that optogenetic control of central auditory neurons is feasible, but opsins with faster channel kinetics may be necessary to convey information at rates typical of many auditory signals. [ABSTRACT FROM AUTHOR]

With the accumulation of our knowledge about how memories are formed, consolidated, retrieved, and updated, neuroscience is now reaching a point where discrete memories can be identified and manipulated at rapid timescales. Here, we start with historical studies that lead to the modern memory engram theory. Then, we will review recent advances in memory engram research that combine transgenic and optogenetic approaches to reveal the underlying neuronal substrates sufficient for activating mnemonic processes. We will focus on three concepts: isolating memory engrams at the level of single cells to tag them for subsequent manipulation; testing the sufficiency of these engrams for memory recall by artificially activating them; and finally, presenting new stimuli during the artificial activation of these engrams to induce an association between the two to form a false memory. We propose that hippocampal cells that show activity-dependent changes during learning construct a cellular basis for contextual memory engrams.

In rodent motor cortex, the rostral forelimb area (RFA) and the caudal forelimb area (CFA) are major actors in orchestrating the control of complex forelimb movements. However, their intrinsic connectivity and reciprocal functional organization are still unclear, limiting our understanding of how the brain coordinates and executes voluntary movements. Here, we causally probe cortical connectivity and activation patterns triggered by transcranial optogenetic stimulation of ethologically relevant complex movements exploiting a large-scale all-optical method in awake mice. Results show specific activation features for each movement class, providing evidence for a segregated functional organization of CFA and RFA. Importantly, we identify a second discrete lateral grasping representation area, namely the lateral forelimb area (LFA), with unique connectivity and activation patterns. Therefore, we propose the LFA as a distinct forelimb representation in the mouse somatotopic motor map. [Display omitted] • A large-scale single-photon all-optical system is developed • Movement-specific activation maps show a modular organization of the motor cortex • Activity propagation analysis reveals movement-specific spatiotemporal patterns • An independent lateral grasping representation area is identified The intrinsic connections and functional structure of areas in the forelimb motor cortex are unclear. Here, Resta et al. develop an all-optical system to causally dissect cortical activation during movement execution, providing evidence for a segregated organization of RFA and CFA networks and identifying a second grasping representation area. [ABSTRACT FROM AUTHOR]

Grey matter protoplasmic astrocytes extend very thin processes and establish close contacts with synapses. It has been suggested that the release of neuroactive gliotransmitters at the tripartite synapse contributes to information processing. However, the concept of calcium (Ca2+)-dependent gliotransmitter release from astrocytes, and the release mechanisms are being debated.Studying astrocytes in their natural environment is challenging because: i) astrocytes are electrically silent; ii) astrocytes and neurons express an overlapping repertoire of transmembrane receptors; iii) astrocyte processes in contact with synapses are below confocal and two-photon microscope resolution; iv) bulk-loading techniques using fluorescent Ca2+ indicators lack cellular specificity.In this review, we will discuss some limitations of conventional methodologies and highlight the interest of novel tools and approaches for studying gliotransmission. Genetically encoded Ca2+ indicators (GECIs), light-gated channels, and exogenous receptors are being developed to selectively read out and stimulate astrocyte activity. Our review discusses emerging perspectives on: i) the complexity of astrocyte Ca2+ signalling revealed by GECIs; ii) new pharmacogenetic and optogenetic approaches to activate specific Ca2+ signalling pathways in astrocytes; iii) classical and new techniques to monitor vesicle fusion in cultured astrocytes; iv) possible strategies to express specifically reporter genes in astrocytes.

The advent of optogenetics and genetically-encoded photosensors has provided neuroscience researchers with a wealth of new tools and methods for examining and manipulating neuronal function in vivo. There exists now a wide range of experimentally validated protein tools capable of modifying cellular function, including light-gated ion channels, recombinant light-gated G-protein coupled receptors, and even neurotransmitter receptors modified with tethered photo-switchable ligands. A large number of genetically-encoded protein sensors have also been developed to optically track cellular activity in real time, including membrane-voltage-sensitive fluorophores and fluorescent calcium and pH indicators. The development of techniques for controlled expression of these proteins has also increased their utility by allowing the study of specific populations of cells. Additionally, recent advances in optics technology have enabled both activation and observation of target proteins with high spatiotemporal fidelity. In combination, these methods have great potential in the study of neural circuits and networks, behavior, animal models of disease, as well as in high-throughput ex vivo studies. This review collects some of these new tools and methods and surveys several current and future applications of the evolving field of optophysiology.

Interactions between distinct motor cortical areas are essential for coordinated motor behaviors. In rodents, the motor cortical forelimb areas are divided into at least two distinct areas: the rostral forelimb area (RFA) and the caudal forelimb area (CFA). The RFA is thought to be an equivalent of the premotor cortex in primates, whereas the CFA is believed to be an equivalent of the primary motor cortex. Although reciprocal connections between the RFA and the CFA have been anatomically identified in rats, it is unknown whether there are functional connections between these areas that can induce postsynaptic spikes. In this study, we used an in vivo Channelrhodopsin-2 photostimulation method to trace the functional connections between the mouse RFA and CFA. Simultaneous electrical recordings were utilized to detect spiking activities induced by synaptic inputs originating from photostimulated areas. This method, in combination with anatomical tracing, demonstrated that the RFA receives strong functional projections from layer 2/3 and/or layer 5a, but not from layer 5b, of the CFA. Further, the CFA receives strong projections from layer 5b neurons of the RFA. The onset latency of electrical responses evoked in remote areas upon photostimulation of the other areas was approximately 10 ms, which is consistent with the synaptic connectivity between these areas. Our results suggest that neuronal activities in the RFA and the CFA during movements are formed through asymmetric reciprocal connections.

Excessive food intake leads to lipid accumulation in white adipose tissue, triggering inflammation, cellular stress, insulin resistance, and metabolic syndrome. In contrast, the dynamic energy expenditure and heat generation of brown and beige adipose tissue, driven by specialized mitochondria, render it an appealing candidate for therapeutic strategies aimed at addressing metabolic disorders. This review examines the therapeutic potential of brown and beige adipocytes for obesity and metabolic disorders, focusing on recent studies that employ optogenetics for thermogenesis control in these cells. The findings delve into the mechanisms underlying UCP1-dependent and UCP1-independent thermogenesis and how optogenetic approaches can be used to precisely modulate energy expenditure and induce thermogenesis. The convergence of adipocyte biology and optogenetics presents an exciting frontier in combating metabolic disorders and advancing our understanding of cellular regulation and energy balance., Competing Interests: Conflicts of Interest There are no conflicts of interest.

Distinct subsets of sensory nerve fibres are involved in mediating mechanical and thermal pain hypersensitivity. They may also differentially respond to analgesics. Heat-sensitive C-fibres, for example, are thought to respond to m-opioid receptor (MOR) activation while mechanoreceptive fibres are supposedly sensitive to d-opioid receptor (DOR) or GABAB receptor (GABABR) activation. The suggested differential distribution of inhibitory neurotransmitter receptors on different subsets of sensory fibres is, however, heavily debated. In this study, we quantitatively compared the degree of presynaptic inhibition exerted by opioids and the GABABR agonist baclofen on (1) vesicular glutamate transporter subtype 3-positive (VGluT31) non-nociceptive primary afferent fibres and (2) putative nociceptive C-fibres. To investigate VGluT31 sensory fibres, we evoked excitatory postsynaptic currents with blue light at the level of the dorsal root ganglion (DRG) in spinal cord slices of mice, expressing channelrhodopsin-2. Putative nociceptive C-fibres were explored in VGluT3-knockout mice through electrical stimulation. The MOR agonist DAMGO strongly inhibited both VGluT31 and VGluT32 C-fibres innervating lamina I neurons but generally had less influence on fibres innervating lamina II neurons. The DOR agonist SNC80 did not have any pronounced effect on synaptic transmission in any fibre type tested. Baclofen, in striking contrast, powerfully inhibited all fibre populations investigated. In summary, we report optogenetic stimulation of DRG neurons in spinal slices as a capable approach for the subtype-selective investigation of primary afferent nerve fibres. Overall, pharmacological accessibility of different subtypes of sensory fibres considerably overlaps, indicating that MOR, DOR, and GABABR expressions are not substantially segregated between heat and mechanosensitive fibres. [ABSTRACT FROM AUTHOR]

Epilepsy is a neurological disorder that affects around 1% of the population worldwide. The two main therapies, pharmacology and the electrical stimulation, both have some shortcomings. For instance, pharmacological therapy is frequently accompanied by side effects, and current anticonvulsive drugs fail to be effective to around a third of patients. These patients could suffer astrocyte-related epilepsy, as increasing evidence indicates that dysfunctions of astrocytes can result in epilepsy. However, epilepsy drugs that affect astrocytes are not available currently. Although electrical stimulation has benefited many patients, the electrode stimulates unselective neurons or circuits. All these need to develop new strategies for improving the life of the patients. As channelrhodopsins (ChRs) were discovered, a novel method referred to as “optogenetics” was developed. It has advantages over electrical stimulation of being less-invasiveness and allowing spatiotemporally stimulation. Recently, a number of experiments have explored the treatments for epilepsy with optogenetic control of neurons. Here, we discuss the possibility that an optogenetic approach could be used to control the release of gliotransmitters and improve astrocyte function such as glutamate and K + uptake, and thereby offer a potential strategy to investigate and treat astrocyte-related epilepsy. [ABSTRACT FROM AUTHOR]

Astrocytes modulate synaptic transmission via release of gliotransmitters such as ATP, glutamate, d -serine and l -lactate. One of the main problems when studying the role of astrocytes in vitro and in vivo is the lack of suitable tools for their selective activation. Optogenetic actuators can be used to manipulate astrocytic activity by expression of variants of channelrhodopsin-2 (ChR2) or other optogenetic actuators with the aim to initiate intracellular events such as intracellular Ca 2+ ([Ca 2+ ] i ) and/or cAMP increases. We have developed an array of adenoviral vectors (AVV) with ChR2-like actuators, including an enhanced ChR2 mutant (H134R), and a mutant with improved Ca 2+ permeability (Ca 2+ translocating channelrhodopsin, CatCh). We show here that [Ca 2+ ] i elevations evoked by ChR2(H134R) and CatCh in astrocytes are largely due to release of Ca 2+ from the intracellular stores. The autocrine action of ATP which is released under these conditions and acts on the P2Y receptors also contributes to the [Ca 2+ ] i elevations. We also studied effects evoked using light-sensitive G-protein coupled receptors (opto-adrenoceptors). Activation of optoα1AR (Gq-coupled) and optoβ2AR (G s -coupled) resulted in astrocytic [Ca 2+ ] i increases which were suppressed by blocking the corresponding intracellular signalling cascade (phospholipase C and adenylate cyclase, respectively). Interestingly, the bulk of [Ca 2+ ] i responses evoked using either optoAR was blocked by an ATP degrading enzyme, apyrase, or a P2Y1 receptor blocker, MRS 2179, indicating that they are to a large extent triggered by the autocrine action of ATP. We conclude that, whilst optimal tools for control of astrocytes are yet to be generated, the currently available optogenetic actuators successfully initiate biologically relevant signalling events in astrocytes. [ABSTRACT FROM AUTHOR]

Impairments in social interaction represent a core symptom of a number of psychiatric disease states, including autism, schizophrenia, depression, and anxiety. Although the amygdala has long been linked to social interaction, little is known about the functional role of connections between the amygdala and downstream regions in noncompetitive social behavior. In the present study, we used optogenetic and pharmacological tools in mice to study the role of projections from the basolateral complex of the amygdala (BLA) to the ventral hippocampus (vHPC) in two social interaction tests: the resident-juvenile-intruder home-cage test and the three chamber sociability test. BLA pyramidal neurons were transduced using adeno-associated viral vectors (AAV5) carrying either channelrhodopsin-2 (ChR2) or halorhodopsin (NpHR), under the control of the CaMKIIα promoter to allow for optical excitation or inhibition of amygdala axon terminals. Optical fibers were chronically implanted to selectively manipulate BLA terminals in the vHPC. NpHR-mediated inhibition of BLA-vHPC projections significantly increased social interaction in the resident-juvenile intruder home-cage test as shown by increased intruder exploration. In contrast, ChR2-mediated activation of BLA-vHPC projections significantly reduced social behaviors as shown in the resident-juvenile intruder procedure as seen by decreased time exploring the intruder and in the three chamber sociability test by decreased time spent in the social zone. These results indicate that BLA inputs to the vHPC are capable of modulating social behaviors in a bidirectional manner. [ABSTRACT FROM AUTHOR]

With the accumulation of our knowledge about how memories are formed, consolidated, retrieved, and updated, neuroscience is now reaching a point where discrete memories can be identified and manipulated at rapid timescales. Here, we start with historical studies that lead to the modern memory engram theory. Then, we will review recent advances in memory engram research that combine transgenic and optogenetic approaches to reveal the underlying neuronal substrates sufficient for activating mnemonic processes. We will focus on three concepts: (1) isolating memory engrams at the level of single cells to tag them for subsequent manipulation; (2) testing the sufficiency of these engrams for memory recall by artificially activating them; and (3) presenting new stimuli during the artificial activation of these engrams to induce an association between the two to form a false memory. We propose that hippocampal cells that show activity-dependent changes during learning construct a cellular basis for contextual memory engrams. [ABSTRACT FROM AUTHOR]

Highlights: [•] Light-axon model for peripheral optogenetic neural stimulation. [•] Reproduced the small-to-large diameter axon recruitment order seen experimentally. [•] Hypothesize that the internodal spacing relationship plays an important role in recruitment. [ABSTRACT FROM AUTHOR]

The advent of optogenetics and genetically encoded photosensors has provided neuroscience researchers with a wealth of new tools and methods for examining and manipulating neuronal function in vivo. There exists now a wide range of experimentally validated protein tools capable of modifying cellular function, including light-gated ion channels, recombinant light-gated G protein-coupled receptors, and even neurotransmitter receptors modified with tethered photo-switchable ligands. A large number of genetically encoded protein sensors have also been developed to optically track cellular activity in real time, including membrane-voltage-sensitive fluorophores and fluorescent calcium and pH indicators. The development of techniques for controlled expression of these proteins has also increased their utility by allowing the study of specific populations of cells. Additionally, recent advances in optics technology have enabled both activation and observation of target proteins with high spatiotemporal fidelity. In combination, these methods have great potential in the study of neural circuits and networks, behavior, animal models of disease, as well as in high-throughput ex vivo studies. This review collects some of these new tools and methods and surveys several current and future applications of the evolving field of optophysiology. [ABSTRACT FROM AUTHOR]

Abstract: Optogenetic tools comprise a variety of different light-sensitive proteins from single-cell organisms that can be expressed in mammalian neurons and effectively control their excitability. Two main classes of optogenetic tools allow to either depolarize or hyperpolarize, and respectively generate or inhibit action potentials in selective populations of neurons. This opens unprecedented possibilities for delineating the role of certain neuronal populations in brain processing and diseases. Moreover, optogenetics may be considered for developing potential treatment strategies for brain diseases, particularly for excitability disorders such as epilepsy. Expression of the inhibitory halorhodopsin NpHR in hippocampal principal cells has been recently used as a tool to effectively control chemically and electrically induced epileptiform activity in slice preparations, and to reduce in vivo spiking induced by tetanus toxin injection in the motor cortex. In this review we give a comprehensive summary of what has been achieved so far in the field of epilepsy using optogenetics, and discuss some of the possible strategies that could be envisaged in the future. We also point out some of the challenges and pitfalls in relation to possible outcomes of using optogenetics for controlling network excitability, and associated brain diseases. This article is part of the Special Issue entitled ‘New Targets and Approaches to the Treatment of Epilepsy’. [Copyright &y& Elsevier]

Optogenetics is a rapidly evolving field of technology that allows optical control of genetically targeted biological systems at high temporal and spatial resolution. By heterologous expression of light-sensitive microbial membrane proteins, opsins, cell type-specific depolarization or silencing can be optically induced on a millisecond time scale. What started in a petri dish is applicable today to more complex systems, ranging from the dissection of brain circuitries in vitro to behavioral analyses in freely moving animals. Persistent technical improvement has focused on the identification of new opsins, suitable for optogenetic purposes and genetic engineering of existing ones. Optical stimulation can be combined with various readouts defined by the desired resolution of the experimental setup. Although recent developments in optogenetics have largely focused on neuroscience it has lately been extended to other targets, including stem cell research and regenerative medicine. Further development of optogenetic approaches will not only highly increase our insight into health and disease states but might also pave the way for a future use in therapeutic applications. [ABSTRACT FROM AUTHOR]

The recent development of optogenetic techniques has generated considerable excitement in neuroscience research. Optogenetics uses light to control the activity of neurons which have been modified to express light-sensitive proteins. Some proteins, such as channelrhodopsin, are cation channels that produce depolarization of neurons when illuminated. In other cases, neuronal activity can be inhibited through illumination of proteins, such as the chloride pump halorhodopsin, that hyperpolarize neurons. Because these proteins can be selectively expressed in specific cell types and/or in specific locations, optogenetics avoids several of the non-specific effects of electrical or pharmacological brain stimulation. This short review will explain the physiology of this technique, describe the basic and technical aspects of the method, and highlight some of the research as well as the clinical potential of optogenetics. [ABSTRACT FROM AUTHOR]

Recordings of large neuronal ensembles and neural stimulation of high spatial and temporal precision are important requisites for studying the real-time dynamics of neural networks. Multiple-shank silicon probes enable large-scale monitoring of individual neurons. Optical stimulation of genetically targeted neurons expressing light-sensitive channels or other fast (milliseconds) actuators offers the means for controlled perturbation of local circuits. Here we describe a method to equip the shanks of silicon probes with micron-scale light guides for allowing the simultaneous use of the two approaches. We then show illustrative examples of how these compact hybrid electrodes can be used in probing local circuits in behaving rats and mice. A key advantage of these devices is the enhanced spatial precision of stimulation that is achieved by delivering light close to the recording sites of the probe. When paired with the expression of light-sensitive actuators within genetically specified neuronal populations, these devices allow the relatively straightforward and interpretable manipulation of network activity. [ABSTRACT FROM AUTHOR]

• ChR2 optical stimulation induces a fine-tuned control of calcium activity in murine primary myotubes. • Calcium signature of primary myotubes, followed with Gcamp6, evolves with differentiation. • Mimicking myotube spontaneous calcium oscillations through ChR2 stimulation enhances their myogenic differentiation. Calcium takes part in numerous cellular processes such as proliferation, migration, differentiation, or cell death and plays a particular role in myogenesis of skeletal muscle. Indeed, intracellular calcium signaling participates, in a non-negligeable manner, to the "on" signal of muscle differentiation from undifferentiated cells to differentiated myotubes. Therefore, this differentiation can be modulated by controlling calcium activity with electrical or optogenetic stimulation approaches. In this study, we used the optogenetic tool channelrhodopsin 2 (ChR2) to control calcium activity and to modulate skeletal muscle differentiation. Using primary cultures of mouse myotubes, we showed that ChR2 stimulation was well-adapted to control intracellular calcium activity at the single cell or whole culture scale. To modulate the calcium-dependent myotube differentiation, we used an optical stimulation protocol based on GCAMP6s-decoded spontaneous calcium activity patterns of differentiated myotubes. The optical training of myotubes increased the fusion index and their contractile ability. This study demonstrates that handling a mature calcium signature with such optogenetic tool improves the differentiation of primary murine myotubes. The channelrhodopsin 2 (ChR2) constitutes a well-adapted tool to control calcium activity by activating excitation-contraction coupling of myotubes in differentiation. The application of an optical stimulation protocol developed on spontaneous calcium patterns of differentiated myotubes enhanced their differentiation state by increasing fusion processes and their contractile ability. [Display omitted] [ABSTRACT FROM AUTHOR]