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Channelrhodopsins (ChRs) are light-gated ion channels and central optogenetic tools that can control neuronal activity with high temporal resolution at the single-cell level. Although their application in optogenetics has rapidly progressed, it is unsolved how their channels open and close. ChRs transport ions through a series of interlocking elementary processes that occur over a broad time scale of subpicoseconds to seconds. During these processes, the retinal chromophore functions as a channel regulatory domain and transfers the optical input as local structural changes to the channel operating domain, the helices, leading to channel gating. Thus, the core question on channel gating dynamics is how the retinal chromophore structure changes throughout the photocycle and what rate-limits the kinetics. Here, we investigated the structural changes in the retinal chromophore of canonical ChR, C1C2, in all photointermediates using time-resolved resonance Raman spectroscopy. Moreover, to reveal the rate-limiting factors of the photocycle and channel gating, we measured the kinetic isotope effect of all photoreaction processes using laser flash photolysis and laser patch clamp, respectively. Spectroscopic and electrophysiological results provided the following understanding of the channel gating: the retinal chromophore highly twists upon the retinal Schiff base (RSB) deprotonation, causing the surrounding helices to move and open the channel. The ion-conducting pathway includes the RSB, where inflowing water mediates the proton to the deprotonated RSB. The twisting of the retinal chromophore relaxes upon the RSB reprotonation, which closes the channel. The RSB reprotonation rate-limits the channel closing.
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Fenômenos Eletrofisiológicos , Canais Iônicos , Channelrhodopsins/química , Prótons , LuzRESUMO
Microbial rhodopsins are a large family of photoreceptive membrane proteins with diverse light-regulated functions. While the most ubiquitous microbial rhodopsins are light-driven outward proton (H+) pumps, new subfamilies of microbial rhodopsins transporting H+ inwardly, i.e., light-driven inward H+ pumps, have been discovered recently. Although structural and spectroscopic studies provide insights into their ion transport mechanisms, the minimum key element(s) that determine the direction of H+ transport have not yet been clarified. Here, we conducted the first functional conversion study by substituting key amino acids in a natural outward H+-pumping rhodopsin (PspR) with those in inward H+-pumping rhodopsins. Consequently, an artificial inward H+ pump was constructed by mutating only three residues of PspR. This result indicates that these residues govern the key processes that discriminate between outward and inward H+ pumps. Spectroscopic studies revealed the presence of an inward H+-accepting residue in the H+ transport pathway and direct H+ uptake from the extracellular solvent. This finding of the simple element for determining H+ transport would provide a new basis for understanding the concept of ion transport not only by microbial rhodopsins but also by other ion-pumping proteins.
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Bombas de Próton , Rodopsina , Bombas de Próton/química , Rodopsina/química , Rodopsinas Microbianas/metabolismo , Transporte de Íons , Bombas de Íon/metabolismo , Prótons , LuzRESUMO
Microbial rhodopsins are photoreceptive membrane proteins, which are used as molecular tools in optogenetics. Here, a machine learning (ML)-based experimental design method is introduced for screening rhodopsins that are likely to be red-shifted from representative rhodopsins in the same subfamily. Among 3,022 ion-pumping rhodopsins that were suggested by a protein BLAST search in several protein databases, the ML-based method selected 65 candidate rhodopsins. The wavelengths of 39 of them were able to be experimentally determined by expressing proteins with the Escherichia coli system, and 32 (82%, p = 7.025 × 10-5) actually showed red-shift gains. In addition, four showed red-shift gains >20 nm, and two were found to have desirable ion-transporting properties, indicating that they would be potentially useful in optogenetics. These findings suggest that data-driven ML-based approaches play effective roles in the experimental design of rhodopsin and other photobiological studies. (141/150 words).
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Canais Iônicos/metabolismo , Aprendizado de Máquina , Optogenética , Rodopsinas Microbianas/metabolismo , Sequência de Aminoácidos , Teorema de Bayes , Cor , Bases de Dados de Proteínas , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Canais Iônicos/genética , Canais Iônicos/efeitos da radiação , Luz , Estudo de Prova de Conceito , Conformação Proteica em alfa-Hélice , Rodopsinas Microbianas/genética , Rodopsinas Microbianas/efeitos da radiação , Análise de Sequência de ProteínaRESUMO
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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Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Células Musculares , Neurônios , OptogenéticaRESUMO
To elucidate neural mechanisms underlying oscillatory phenomena in brain function, we have developed optogenetic tools and statistical methods. Specifically, opto-current-clamp induced oscillation reveals intrinsic frequency preferences in the neural circuits by oscillatory resonance. Furthermore, resonance or entrainment to intrinsic frequency is state-dependent. When resonance phenomena go beyond a certain range, it could even induce epileptic seizure in highly reproducible manner. We are able to study how seizures start, develop, and stop in neural circuits. Therefore, the optogenetics-induced oscillatory activation is a powerful tool in neuroscience research.
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Epilepsia , Optogenética , Humanos , ConvulsõesRESUMO
Microbial rhodopsin is a large family of membrane proteins having seven transmembrane helices (TM1-7) with an all-trans retinal (ATR) chromophore that is covalently bound to Lys in the TM7. The Trp residue in the middle of TM3, which is homologous to W86 of bacteriorhodopsin (BR), is highly conserved among microbial rhodopsins with various light-driven functions. However, the significance of this Trp for the ion transport function of microbial rhodopsins has long remained unknown. Here, we replaced the W163 (BR W86 counterpart) of a channelrhodopsin (ChR), C1C2/ChRWR, which is a chimera between ChR1 and 2, with a smaller aromatic residue, Phe to verify its role in the ion transport. Under whole-cell patch clamp recordings from the ND7/23 cells that were transfected with the DNA plasmid coding human codon optimized C1C2/ChRWR (hWR) or its W163F mutant (hWR-W163F), the photocurrents were evoked by a pulsatile light at 475 nm. The ion-transporting activity of hWR was strongly altered by the W163F mutation in 3 points: (1) the H+ leak at positive membrane potential (V m) and its light-adaptation, (2) the attenuation of cation channel activity and (3) the manifestation of outward H+ pump activity. All of these results strongly suggest that W163 has a role in stabilizing the structure involved in the gating-on and -off of the cation channel, the role of "gate keeper". We can attribute the attenuation of cation channel activity to the incomplete gating-on and the H+ leak to the incomplete gating-off.
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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.
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Optogenética/métodos , Estimulação Luminosa/métodos , Animais , Eletrônica , Luz , Neurônios , Fototerapia/métodos , Ratos , Ratos Wistar , Nervo Isquiático/fisiologiaRESUMO
Physical disuse could lead to a state of chronic pain typified by complex regional pain syndrome type I due to fear of pain through movement (kinesiophobia) or inappropriate resting procedures. However, the mechanisms by which physical disuse is associated with acute/chronic pain and other pathological signs remain unresolved. We have previously reported that inflammatory signs, contractures, disuse muscle atrophy, spontaneous pain-like behaviors, and chronic widespread mechanical hyperalgesia based on central plasticity occurred after 2 weeks of cast immobilization in chronic post-cast pain (CPCP) rat model. In this study, we also demonstrated dystrophy-like changes, both peripheral nociceptive signals and activation of the central pain pathway in CPCP rats. This was done by the following methods: (1) vascular permeability (Evans blue dye) and inflammatory- and oxidative stress-related messenger RNA changes (real-time quantitative polymerase chain reaction); (2) immunofluorescence of pERK and/or c-Fos expression in the spino-parabrachio-amygdaloid pathway; and (3) blockade of nociceptive-related signals using sciatic nerve block. Furthermore, we demonstrated tactile allodynia using an optogenetic method in a transgenic rat line (W-TChR2V4), cold allodynia using the acetone test, and activation of dorsal horn neurons in the chronic phase associated with chronic mechanical hyperalgesia using c-Fos immunofluorescence. In addition, we showed that nociceptive signals in the acute phase are involved in chronic pathological pain-like behaviors by studying the effects of sciatic nerve block. Thus, we conclude that physical disuse contributes to dystrophy-like changes, spontaneous pain-like behavior, and chronic widespread pathological pain-like behaviors in CPCP rats after 2 weeks of cast immobilization.
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Dor Crônica , Hiperalgesia , Animais , Hiperalgesia/etiologia , Inflamação Neurogênica , Medição da Dor , Ratos , Ratos Sprague-DawleyRESUMO
Optogenetic stimulation of neural stem cells (NSCs) enables their activity-dependent photo-modulation. This provides a spatio-temporal tool for studying activity-dependent neurogenesis and for regulating the differentiation of the transplanted NSCs. Currently, this is mainly driven by viral transfection of channelrhodopsin-2 (ChR2) gene, which requires high irradiance and complex in vivo/vitro stimulation systems. Additionally, despite the extensive application of optogenetics in neuroscience, the transcriptome-level changes induced by optogenetic stimulation of NSCs have not been elucidated yet. Here, we made transformed NSCs (SFO-NSCs) stably expressing one of the step-function opsin (SFO)-variants of chimeric channelrhodopsins, ChRFR(C167A), which is more sensitive to blue light than native ChR2, via a non-viral transfection system using piggyBac transposon. We set up a simple low-irradiance optical stimulation (OS)-incubation system that induced c-fos mRNA expression, which is activity-dependent, in differentiating SFO-NSCs. More neuron-like SFO-NCSs, which had more elongated axons, were differentiated with daily OS than control cells without OS. This was accompanied by positive/negative changes in the transcriptome involved in axonal remodeling, synaptic plasticity, and microenvironment modulation with the up-regulation of several genes involved in the Ca2+-related functions. Our approach could be applied for stem cell transplantation studies in tissue with two strengths: lower carcinogenicity and less irradiance needed for tissue penetration.
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Células-Tronco Neurais/efeitos da radiação , Neurogênese/efeitos da radiação , Optogenética , Sinalização do Cálcio , Linhagem Celular Transformada , Channelrhodopsins/biossíntese , Channelrhodopsins/genética , Channelrhodopsins/efeitos da radiação , Elementos de DNA Transponíveis , Regulação da Expressão Gênica/efeitos da radiação , Ontologia Genética , Genes Reporter , Genes fos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Plasticidade Neuronal/efeitos da radiação , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcriptoma/efeitos da radiação , Regulação para Cima/efeitos da radiaçãoRESUMO
Paired-like homeobox gene Phox2b is predominantly expressed in pre-inspiratory neurons in the parafacial respiratory group (pFRG) in newborn rat rostral ventrolateral medulla. To analyse detailed local networks of the respiratory centre using optogenetics, the effects of selective activation of Phox2b-positive neurons in the ventral medulla on respiratory rhythm generation were examined in brainstem-spinal cord preparations isolated from transgenic newborn rats with Phox2b-positive cells expressing channelrhodopsin variant ChRFR(C167A). Photostimulation up to 43 s increased the respiratory rate > 200% of control, whereas short photostimulation (1.5 s) of the rostral pFRG reset the respiratory rhythm. At the cellular level, photostimulation depolarised Phox2b-positive pre-inspiratory, inspiratory and respiratory-modulated tonic neurons and Phox2b-negative pre-inspiratory neurons. In contrast, changes in membrane potential of Phox2b-negative inspiratory and expiratory neurons varied depending on characteristics of ongoing synaptic connections in local respiratory networks in the rostral medulla. In the presence of tetrodotoxin, photostimulation depolarised Phox2b-positive cells, but caused no significant changes in membrane potential of Phox2b-negative cells. We concluded that depolarisation of Phox2b-positive neurons was due to cell-autonomous photo-activation and summation of excitatory postsynaptic potentials, whereas membrane potential changes of Phox2b-negative neurons depended on the network configuration. Our findings shed further light on local networks among respiratory-related neurons in the rostral ventrolateral medulla and emphasise the important role of pre-inspiratory neurons in respiratory rhythm generation in the neonatal rat en bloc preparation.
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Channelrhodopsins/metabolismo , Proteínas de Homeodomínio/metabolismo , Bulbo/metabolismo , Neurônios/metabolismo , Centro Respiratório/metabolismo , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Tronco Encefálico/metabolismo , Feminino , Masculino , Potenciais da Membrana/fisiologia , Optogenética/métodos , Ratos , Respiração , Tetrodotoxina/metabolismoRESUMO
We investigated the relationship between whisker mechanoreceptive inputs and the neural responses to optical stimulation in layer 2/upper 3 (L2/U3) of the barrel cortex using optogenetics since, ideally, we should investigate interactions among inputs with spatiotemporal acuity. Sixteen whisker points of a transgenic rat (W-TChR2V4), that expresses channelrhodopsin 2 (ChR2)-Venus conjugate (ChR2V) in the peripheral nerve endings surrounding the whisker follicles, were respectively connected one-by-one with 16 LED-coupled optical fibres, which illuminated the targets according to a certain pattern in order to evaluate interactions among the inputs in L2/U3. We found that the individual L2/U3 neurons frequently received excitatory inputs from multiple whiskers that were arrayed in a row. Although the interactions among major afferent inputs (MAIs) were negligible, negative interactions with the surrounding inputs suggest that the afferent inputs were integrated in the cortical networks to enhance the contrast of an array to its surroundings. With its simplicity, reproducibility and spatiotemporal acuity, the optogenetic approach would provide an alternative way to understand the principles of afferent integration in the cortex and should complement knowledge obtained by experiments using more natural stimulations.
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Optogenética/métodos , Córtex Somatossensorial/fisiologia , Animais , Feminino , Luz , Masculino , Mecanorreceptores/citologia , Mecanorreceptores/fisiologia , Neurônios Aferentes/citologia , Neurônios Aferentes/fisiologia , Optogenética/instrumentação , Estimulação Física , Ratos , Ratos Transgênicos , Córtex Somatossensorial/citologia , Vibrissas/inervaçãoRESUMO
The calyx-type synapse is a giant synaptic structure in which a presynaptic terminal wraps around a postsynaptic neuron in a one-to-one manner. It has been used for decades as an experimental model system of the synapse due to its simplicity and high accessibility in physiological recording methods. In particular, the calyx of the embryonic chick ciliary ganglion (CG) has enormous potential for synapse science because more flexible genetic manipulations are available compared with other synapses. Here, we describe methods to study presynaptic morphology, physiology, and development using CGs and cutting-edge molecular tools. We outline step-by-step protocols for presynaptic gene manipulation using in ovo electroporation, preparation of isolated CGs, 3-D imaging for single-axon tracing in transparent CGs, electrophysiology of the presynaptic terminal, and an all-optical approach using optogenetic molecular reagents. These methods will facilitate studies of the synapse and neuronal circuits in the future. © 2019 by John Wiley & Sons, Inc.
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Gânglios Parassimpáticos/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Axônios/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Optogenética/métodos , Terminações Pré-Sinápticas/fisiologiaRESUMO
Optogenetics requires implantation of light-delivering optical fibers, as current light-sensitive opsins are activated by visible light, which cannot effectively penetrate biological tissues. Insertion of optical fibers and subsequent photostimulation inherently damages brain tissue, and fiber tethering can restrict animal behavior. To overcome these technical limitations, we developed minimally invasive "fiberless" optogenetics using lanthanide micro-particles (LMPs), which emit up-conversion luminescence in the visible spectrum in response to irradiation with tissue-penetrating near-infrared light. Depolarizing (C1V1) and hyperpolarizing (ACR1) opsins were strongly activated by up-conversion luminescence from green-emitting LMPs both in vitro and in vivo. Using this technique, we successfully manipulated locomotive behavior of mice by activating and inhibiting neurons in the dorsal striatum, at a depth of 2 mm from the brain surface. LMPs were retained and remained functional for >8 weeks at the injection site. Fiberless optogenetics offers opportunities to control neuronal function over longer time frames using freely behaving animals.
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Comportamento Animal/efeitos dos fármacos , Encéfalo , Elementos da Série dos Lantanídeos/farmacologia , Opsinas/metabolismo , Nervo Óptico , Optogenética , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Camundongos , Camundongos Transgênicos , Nervo Óptico/metabolismo , Nervo Óptico/fisiopatologiaRESUMO
To investigate how the functional architecture is organized in layer 5 (L5) of the somatosensory cortex of a mouse in vivo, the input-output relationship was investigated using an all-optical approach. The neural activity in L5 was optically recorded using a Ca2+ sensor, R-CaMP2, through a microprism inserted in the cortex under two-photon microscopy, while the L5 was regionally excited using optogenetics. The excitability was spread around the blue-light irradiated region, but the horizontal propagation was limited to within a certain distance (λ < 130 µm from the center of the illumination spot). When two regions were photostimulated with a short interval, the excitability of each cluster was reduced. Therefore, a column-like architecture had functionally emerged with reciprocal inhibition through a minimal number of synaptic relays. This could generate a synchronous output from a region of L5 with simultaneous enhancement of the signal-to-noise ratio by silencing of the neighboring regions.
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Córtex Somatossensorial/fisiologia , Animais , Cálcio/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Optogenética/métodos , Córtex Somatossensorial/metabolismoRESUMO
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.
Assuntos
Plasticidade Neuronal/fisiologia , Limiar Sensorial/fisiologia , Córtex Somatossensorial/fisiologia , Tato/fisiologia , Vibrissas/fisiologia , Visão Ocular/fisiologia , Animais , Condicionamento Psicológico , Feminino , Asseio Animal/fisiologia , Luz , Masculino , Mecanorreceptores/citologia , Mecanorreceptores/fisiologia , Optogenética/métodos , Nervos Periféricos/citologia , Nervos Periféricos/fisiologia , Ratos , Ratos Transgênicos , Córtex Somatossensorial/citologia , Fatores de Tempo , Gânglio Trigeminal/citologia , Gânglio Trigeminal/fisiologia , Vibrissas/citologiaRESUMO
As one of the ubiquitous second messengers, the intracellular Ca2+, has been revealed to be a pivotal regulator of various cellular functions. Two major sources are involved in the initiation of Ca2+-dependent signals: influx from the extracellular space and release from the intracellular Ca2+ stores such as the endoplasmic/sarcoplasmic reticulum (ER/SR). To manipulate the Ca2+ release from the stores under high spatiotemporal precision, we established a new method termed "organelle optogenetics." That is, one of the light-sensitive cation channels (channelrhodopsin-green receiver, ChRGR), which is Ca2+-permeable, was specifically targeted to the ER/SR. The expression specificity as well as the functional operation of the ER/SR-targeted ChRGR (ChRGRER) was evaluated using mouse skeletal myoblasts (C2C12): (1) the ChRGRER co-localized with the ER-marker KDEL; (2) no membrane current was generated by light under whole-cell clamp of cells expressing ChRGRER; (3) an increase of fluorometric Ca2+ was evoked by the optical stimulation (OS) in the cells expressing ChRGRER in a manner independent on the extracellular Ca2+ concentration ([Ca2+]o); (4) the ΔF/F0 was sensitive to the inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and (5) the store-operated Ca2+ entry (SOCE) was induced by the OS in the ChRGRER-expressing cells. Our organelle optogenetics effectively manipulated the ER/SR to release Ca2+ from intracellular stores. The use of organelle optogenetics would reveal the neuroscientific significance of intracellular Ca2+ dynamics under spatiotemporal precision.
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As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra CâC bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.
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Channelrhodopsins/química , Channelrhodopsins/metabolismo , Chlamydomonas reinhardtii/metabolismo , Retinaldeído/análogos & derivados , Retinaldeído/metabolismo , Animais , Channelrhodopsins/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Conformação ProteicaRESUMO
Rats are excellent animal models for experimental neuroscience. However, the application of optogenetics in rats has been hindered because of the limited number of established transgenic rat strains. To accomplish cell-type specific targeting of an optimized optogenetic molecular tool, we generated ROSA26/CAG-floxed STOP-ChRFR(C167A)-Venus BAC rats that conditionally express the step-function mutant channelrhodopsin ChRFR(C167A) under the control of extrinsic Cre recombinase. In primary cultured cortical neurons derived from this reporter rat, only Cre-positive cells expressing ChRFR(C167A) became bi-stable, that is, their excitability was enhanced by blue light and returned to the baseline by yellow~red light. In bigenic pups carrying the Phox2B-Cre driver, ChRFR(C167A) was specifically expressed in the rostral parafacial respiratory group (pFRG) in the medulla, where endogenous Phox2b immunoreactivity was detected. These neurons were sensitive to blue light with an increase in the firing frequency. Thus, this transgenic rat actuator/reporter system should facilitate optogenetic studies involving the effective in vivo manipulation of the activities of specific cell fractions using light of minimal intensity.