Optogenetics is an optical technique that exploits visible light for selective neuromodulation with spatio‐temporal precision. Despite enormous effort, the effective stimulation of targeted neurons, ...which are located in deeper structures of the nervous system, by visible light, remains a technical challenge. Compared to visible light, near‐infrared illumination offers a higher depth of tissue penetration owing to a lower degree of light attenuation. Herein, an overview of advances in developing new modalities for neural circuitry modulation utilizing upconversion‐nanoparticle‐mediated optogenetics is presented. These developments have led to minimally invasive optical stimulation and inhibition of neurons with substantially improved selectivity, sensitivity, and spatial resolution. The focus is to provide a comprehensive review of the mechanistic basis for evaluating upconversion parameters, which will be useful in designing, executing, and reporting optogenetic experiments.
Optogenetics generally utilizes visible light to modulate opsins in targeted neurons but with poor penetration to deep tissues. Upconversion nanoparticle (UCNP)‐mediated NIR optogenetics offers an attractive alternative due to the improved depth of tissue penetration. The basic principles and feasibility of UCNPs for NIR optogenetics are discussed together with a critical survey of the latest developments in the field.
Neurons become photosensitive by genetically introducing one of green algae-derived protein, channelrhodopsin-2 (ChR2). Here, we quantitatively investigated the rapidness of the light-gated current ...of ChR2 expressed in PC12 cells using blue light-emitting diode (LED) light. The light-gated current consists of two components, inactivating and non-inactivating. The magnitude of inactivating component was almost linearly related to the light intensity. The non-inactivating component showed a tendency to saturate at high illumination. Both the activation and inactivation rates of the light-gated current were linearly dependent on the light intensity. However, the activation rate (turning-on rate) is about 10-fold faster than the inactivation rate. Although the turning-off time constant was little dependent on the light intensity, that at the end of 1
s light pulse was about two-fold larger than that at 20
ms. Neurons are also made photosensitive by the expression of ChR2 in the living animal. Since both the turning-on and turning-off time constants of light-gated current was smaller than the membrane time constant of neurons, the LED light illumination of the photosensitive neurons was enough to evoke action potentials in a pulse-to-pulse manner in an acute slice of hippocampus.
Loss of one sensory modality can cause other types to become more perceptive (cross-modal plasticity). To test the hypothesis that the loss of vision changes the perceptual threshold in the ...somatosensory system, we applied optogenetics to directly manipulate the afferent inputs involved in the whisker-barrel system using a transgenic rat (W-TChR2V4) that expresses channelrhodopsin-2 (ChR2) selectively in the large mechanoreceptive neurons in the trigeminal ganglion (TG) and their peripheral nerve terminals. The licking behavior of W-TChR2V4 rat was conditioned to a blue LED light cue on the whisker area while the magnitude and duration of light pulses were varied. The perceptual threshold was thus quantitatively determined for each rat according to the relationship between the magnitude/duration of light and the reaction time between the LED light cue and the first licking event after it. We found that the perceptual threshold was more significantly reduced than the control non-deprived rats when the rats were visually deprived at postnatal 26-30 days (P26-30, early VD group), but not at P58-66 (late VD group). However, the sensory threshold of a late VD animal was similar to that of a control. Our results suggest the presence of cross-modal plasticity by which the loss of vision at the juvenile period increased the sensitivity of the somatosensory system involved in the touch of whiskers.
The rodent whisker-barrel cortical system is a model for studying somatosensory discrimination at high spatiotemporal precision. Here, we applied optogenetics to produce somatosensory inputs in the ...whisker area using one of transgenic rat lines, W-TChR2V4, which expresses channelrhodopsin-2 (ChR2) in the mechanoreceptive nerve endings around whisker follicles. An awake W-TChR2V4 rat was head-fixed and irradiated by blue LED light on the whisker area with a paradigm conditioned with a reward. The Go task was designed so the rat is allowed to receive a reward, when it licked the nozzle within 5 s after photostimulation. The No-go task was designed so as the rat has to withhold licking for at least 5 s to obtain a reward after photostimulation. The Go-task conditioning was established within 1 hr of training with a reduction in the reaction time and increase of the success rate. To investigate the relationship between the spatiotemporal pattern of sensory inputs and the behavioral output, we designed a multi-optical fiber system that irradiates the whisker area at 9 spots in a 3×3 matrix. Although the Go-task conditioning was established using synchronous irradiation of 9 spots, the success rate was decreased with an increase of the reaction time for the asynchronous irradiation. After conditioning to the Go task, the rat responded to the blue LED flash irradiated on the barrel cortex, where many neurons also express ChR2, or photostimulation of the contralateral whisker area with a similar reaction time and success rate. Synchronous activation of the peripheral mechanoreceptive nerves is suggested to drive a neural circuit in the somatosensory cortex that efficiently couples with the decision. Our optogenetic system would enable the precise evaluation of the psychophysical values, such as the reaction time and success rate, to gain some insight into the brain mechanisms underlying conditioned behaviors.
Integration of organic electrochemical transistors and organic field‐effect transistors is successfully realized on a 600 nm thick parylene film toward an electrophysiology array. A single cell of an ...integrated device and a 2 × 2 electrophysiology array succeed in detecting electromyogram with local stimulation of the motor nerve bundle of a transgenic rat by a laser pulse.
During locomotion in vertebrates, reticulospinal neurons in the hindbrain play critical roles in providing descending excitation to the spinal cord locomotor systems. However, despite the fact that ...many genes that are used to classify the neuronal identities of neurons in the hindbrain have been identified, the molecular identity of the reticulospinal neurons that are critically involved in locomotor drive is not well understood. Chx10-expressing neurons (V2a neurons) are ipsilaterally projecting glutamatergic neurons in the spinal cord and the hindbrain. Many of the V2a neurons in the hindbrain are known to project to the spinal cord in zebrafish, making hindbrain V2a neurons a prime candidate in descending locomotor drive.
We investigated the roles of hindbrain V2a neurons using optogenetic and electrophysiological approaches. The forced activation of hindbrain V2a neurons using channelrhodopsin efficiently evoked swimming, whereas the forced inactivation of them using Archearhodopsin3 or Halorhodpsin reliably stopped ongoing swimming. Electrophysiological recordings of two populations of hindbrain reticulospinal V2a neurons showed that they were active during swimming. One population of neurons, small V2a neurons in the caudal hindbrain, fired with low rhythmicity, whereas the other population of neurons, large reticulospinal V2a neurons, called MiV1 neurons, fired more rhythmically.
These results indicated that hindbrain reticulospinal V2a neurons play critical roles in providing excitation to the spinal locomotor circuits during swimming by providing both tonic and phasic inputs to the circuits.
► Optogenetical activation of hindbrain V2a neurons elicits swimming in zebrafish ► Optogenetical silencing of hindbrain V2a neurons stops swimming ► Hindbrain V2a neurons provide both tonic and phasic excitation to locomotor circuits
Myoblasts can be differentiated into multinucleated myotubes, which provide a well-established and reproducible muscle cell model for skeletal myogenesis in vitro. However, under conventional ...differentiation conditions, each myotube rarely exhibits robust contraction as well as sarcomere arrangement. Here, we applied trains of optical stimulation (OS) to C2C12 myotubes, which were genetically engineered to express a channelrhodopsin variant, channelrhodopsin-green receiver (ChRGR), to investigate whether membrane depolarization facilitates the maturation of myotubes. We found that light pulses induced membrane depolarization and evoked action potentials in ChRGR-expressing myotubes. Regular alignments of sarcomeric proteins were patterned periodically after OS training. In contrast, untrained control myotubes rarely exhibited the striated patterns. OS-trained and untrained myotubes also differed in terms of their resting potential. OS training significantly increased the number of contractile myotubes. Treatment with nifedipine during OS training significantly decreased the fraction of contractile myotubes, whereas tetrodotoxin was less effective. These results suggest that oscillations of membrane potential and intracellular Ca(2+) accompanied by OS promoted sarcomere assembly and the development of contractility during the myogenic process. These results also suggest that optogenetic techniques could be used to manipulate the activity-dependent process during myogenic development.
Organic electronic devices implemented on flexible thin films are attracting increased attention for biomedical applications because they possess extraordinary conformity to curved surfaces. A ...neuronal device equipped with an organic light-emitting diode (OLED), used in combination with animals that are genetically engineered to include a light-gated ion channel, would enable cell type-specific stimulation to neurons as well as conformal contact to brain tissue and peripheral soft tissue. This potential application of the OLEDs requires strong luminescence, well over the neuronal excitation threshold in addition to flexibility. Compatibility with neuroimaging techniques such as MRI provides a method to investigate the evoked activities in the whole brain. Here, we developed an ultrathin, flexible, MRI-compatible OLED device and demonstrated the activation of channelrhodopsin-2–expressing neurons in animals. Optical stimulation from the OLED attached to nerve fibers induced contractions in the innervated muscles. Mechanical damage to the tissues was significantly reduced because of the flexibility. Owing to the MRI compatibility, neuronal activities induced by direct optical stimulation of the brain were visualized using MRI. The OLED provides an optical interface for modulating the activity of soft neuronal tissues.
Krokinobacter eikastus rhodopsin 2 (KR2) is the first light-driven Na(+) pump discovered, and is viewed as a potential next-generation optogenetics tool. Since the positively charged Schiff base ...proton, located within the ion-conducting pathway of all light-driven ion pumps, was thought to prohibit the transport of a non-proton cation, the discovery of KR2 raised the question of how it achieves Na(+) transport. Here we present crystal structures of KR2 under neutral and acidic conditions, which represent the resting and M-like intermediate states, respectively. Structural and spectroscopic analyses revealed the gating mechanism, whereby the flipping of Asp116 sequesters the Schiff base proton from the conducting pathway to facilitate Na(+) transport. Together with the structure-based engineering of the first light-driven K(+) pumps, electrophysiological assays in mammalian neurons and behavioural assays in a nematode, our studies reveal the molecular basis for light-driven non-proton cation pumps and thus provide a framework that may advance the development of next-generation optogenetics.
This chapter describes the current progress of basic research, and potential therapeutic applications primarily focused on the optical manipulation of muscle cells and neural stem cells using ...microbial rhodopsin as a light-sensitive molecule. Since the contractions of skeletal, cardiac, and smooth muscle cells are mainly regulated through their membrane potential, several studies have been demonstrated to up- or downregulate the muscle contraction directly or indirectly using optogenetic actuators or silencers with defined stimulation patterns and intensities. Light-dependent oscillation of membrane potential also facilitates the maturation of myocytes with the development of T tubules and sarcomere structures, tandem arrays of minimum contractile units consists of contractile proteins and cytoskeletal proteins. Optogenetics has been applied to various stem cells and multipotent/pluripotent cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate light-sensitive neurons and to facilitate neuroscience. The chronic optical stimulation of the channelrhodopsin-expressing neural stem cells facilitates their neural differentiation. There are potential therapeutic applications of optogenetics in cardiac pacemaking, muscle regeneration/maintenance, locomotion recovery for the treatment of muscle paralysis due to motor neuron diseases such as amyotrophic lateral sclerosis (ALS). Optogenetics would also facilitate maturation, network integration of grafted neurons, and improve the microenvironment around them when applied to stem cells.
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