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1.
Gene ; 807: 145960, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-34509581

RESUMO

Opsin is a fellow of the G protein-coupled receptors (GPCRs) superfamily. It can be divided into visual and non-visual opsin according to whether it is directly involved in visual imaging. Opsin plays an important role in visual image formation and the regulation of non-image forming functions such as circadian entrainment in the growth, development and evolution of fish. Crimson snapper belongs to Perciforme mainly found in the Indo-West Pacific and the South China Sea. It is one of the most influential economic fishes in the South China Sea. In order to study the existence and expression of opsin gene in Crimson snapper, we sequenced the genome and tissue sample transcriptome of Crimson snapper. In this study, 32 opsin genes were identified from the genome of Crimson snapper. The length of these genes ranged from 1061 bp to 86203 bp and were distributed on 15 different chromosomes. The analysis of opsin gene family of Crimson snapper showed that the sws2 had two extra copies as compared with that of Zebrafish. Domain and motif analysis revealed that all the 32 opsin genes have seven-(pass)-transmembrane domain receptors (7TM receptors) each, and the opsin family contained 10 common motifs. The expression level of opsin gene, confirmed by RT-qPCR, was analyzed by using nine tissues transcriptome databases of Crimson snapper. The results showed that almost all opsin genes were highly expressed in the retina and brain, except opn7a and opn7b which were expressed in intestine and red skin, and almost no expression in other tissues. Our results provide a comprehensive basic knowledge for the opsin gene family of Crimson snapper, which has significance for the study of the function of opsin in Lutjanidaes.


Assuntos
Opsinas/genética , Perciformes/genética , Animais , Sequência de Bases/genética , China , Clonagem Molecular/métodos , Doenças dos Peixes/genética , Expressão Gênica/genética , Opsinas/metabolismo , Perciformes/metabolismo , Receptores Acoplados a Proteínas G/genética , Transcriptoma/genética
2.
Nat Commun ; 12(1): 5871, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34620851

RESUMO

Optogenetics combined with electrical recording has emerged as a powerful tool for investigating causal relationships between neural circuit activity and function. However, the size of optogenetically manipulated tissue is typically 1-2 orders of magnitude larger than that can be electrically recorded, rendering difficulty for assigning functional roles of recorded neurons. Here we report a viral vector-delivery optrode (VVD-optrode) system for precise integration of optogenetics and electrophysiology in the brain. Our system consists of flexible microelectrode filaments and fiber optics that are simultaneously self-assembled in a nanoliter-scale, viral vector-delivery polymer carrier. The highly localized delivery and neuronal expression of opsin genes at microelectrode-tissue interfaces ensure high spatial congruence between optogenetically manipulated and electrically recorded neuronal populations. We demonstrate that this multifunctional system is capable of optogenetic manipulation and electrical recording of spatially defined neuronal populations for three months, allowing precise and long-term studies of neural circuit functions.


Assuntos
Encéfalo , Fenômenos Eletrofisiológicos , Optogenética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microeletrodos , Neurônios/fisiologia , Opsinas/genética , Polímeros
4.
J Exp Biol ; 224(18)2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34587624

RESUMO

In true color vision, animals discriminate between light wavelengths, regardless of intensity, using at least two photoreceptors with different spectral sensitivity peaks. Heliconius butterflies have duplicate UV opsin genes, which encode ultraviolet and violet photoreceptors, respectively. In Heliconius erato, only females express the ultraviolet photoreceptor, suggesting females (but not males) can discriminate between UV wavelengths. We tested the ability of H. erato, and two species lacking the violet receptor, Heliconius melpomene and Eueides isabella, to discriminate between 380 and 390 nm, and between 400 and 436 nm, after being trained to associate each stimulus with a sugar reward. We found that only H. erato females have color vision in the UV range. Across species, both sexes show color vision in the blue range. Models of H. erato color vision suggest that females have an advantage over males in discriminating the inner UV-yellow corollas of Psiguria flowers from their outer orange petals. Moreover, previous models ( McCulloch et al., 2017) suggested that H. erato males have an advantage over females in discriminating Heliconius 3-hydroxykynurenine (3-OHK) yellow wing coloration from non-3-OHK yellow wing coloration found in other heliconiines. These results provide some of the first behavioral evidence for female H. erato UV color discrimination in the context of foraging, lending support to the hypothesis ( Briscoe et al., 2010) that the duplicated UV opsin genes function together in UV color vision. Taken together, the sexually dimorphic visual system of H. erato appears to have been shaped by both sexual selection and sex-specific natural selection.


Assuntos
Borboletas , Visão de Cores , Animais , Borboletas/genética , Cor , Feminino , Masculino , Opsinas/genética , Opsinas de Bastonetes , Asas de Animais
5.
Int J Mol Sci ; 22(17)2021 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-34502519

RESUMO

Optogenetics approach is used widely in neurobiology as it allows control of cellular activity with high spatial and temporal resolution. In most studies, optogenetics is used to control neuronal activity. In the present study optogenetics was used to stimulate astrocytes with the aim to modulate neuronal activity. To achieve this goal, light stimulation was applied to astrocytes expressing a version of ChR2 (ionotropic opsin) or Opto-α1AR (metabotropic opsin). Optimal optogenetic stimulation parameters were determined using patch-clamp recordings of hippocampal pyramidal neurons' spontaneous activity in brain slices as a readout. It was determined that the greatest increase in the number of spontaneous synaptic currents was observed when astrocytes expressing ChR2(H134R) were activated by 5 s of continuous light. For the astrocytes expressing Opto-α1AR, the greatest response was observed in the pulse stimulation mode (T = 1 s, t = 100 ms). It was also observed that activation of the astrocytic Opto-a1AR but not ChR2 results in an increase of the fEPSP slope in hippocampal neurons. Based on these results, we concluded that Opto-a1AR expressed in hippocampal astrocytes provides an opportunity to modulate the long-term synaptic plasticity optogenetically, and may potentially be used to normalize the synaptic transmission and plasticity defects in a variety of neuropathological conditions, including models of Alzheimer's disease and other neurodegenerative disorders.


Assuntos
Astrócitos/metabolismo , Rede Nervosa/fisiologia , Optogenética/métodos , Animais , Astrócitos/fisiologia , Encéfalo/metabolismo , Região CA1 Hipocampal/metabolismo , Channelrhodopsins/metabolismo , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Rede Nervosa/metabolismo , Plasticidade Neuronal , Neurônios/metabolismo , Opsinas/genética , Opsinas/metabolismo , Técnicas de Patch-Clamp , Células Piramidais/metabolismo , Transmissão Sináptica
6.
eNeuro ; 8(5)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34417283

RESUMO

Opsin 3 (Opn3) is highly expressed in the adult brain, however, information for spatial and temporal expression patterns during embryogenesis is significantly lacking. Here, an Opn3-eGFP reporter mouse line was used to monitor cell body expression and axonal projections during embryonic and early postnatal to adult stages. By applying 2D and 3D fluorescence imaging techniques, we have identified the onset of Opn3 expression, which predominantly occurred during embryonic stages, in various structures during brain/head development. In addition, this study defines over twenty Opn3-eGFP-positive neural structures never reported before. Opn3-eGFP was first observed at E9.5 in neural regions, including the ganglia that will ultimately form the trigeminal, facial and vestibulocochlear cranial nerves (CNs). As development proceeds, expanded Opn3-eGFP expression coincided with the formation and maturation of critical components of the central and peripheral nervous systems (CNS, PNS), including various motor-sensory tracts, such as the dorsal column-medial lemniscus (DCML) sensory tract, and olfactory, acoustic, and optic tracts. The widespread, yet distinct, detection of Opn3-eGFP already at early embryonic stages suggests that Opn3 might play important functional roles in the developing brain and spinal cord to regulate multiple motor and sensory circuitry systems, including proprioception, nociception, ocular movement, and olfaction, as well as memory, mood, and emotion. This study presents a crucial blueprint from which to investigate autonomic and cognitive opsin-dependent neural development and resultant behaviors under physiological and pathophysiological conditions.


Assuntos
Opsinas , Opsinas de Bastonetes , Animais , Embrião de Mamíferos , Desenvolvimento Embrionário , Camundongos , Medula Espinal
7.
Nat Neurosci ; 24(10): 1356-1366, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34400843

RESUMO

Optogenetics ushered in a revolution in how neuroscientists interrogate brain function. Because of technical limitations, the majority of optogenetic studies have used low spatial resolution activation schemes that limit the types of perturbations that can be made. However, neural activity manipulations at finer spatial scales are likely to be important to more fully understand neural computation. Spatially precise multiphoton holographic optogenetics promises to address this challenge and opens up many new classes of experiments that were not previously possible. More specifically, by offering the ability to recreate extremely specific neural activity patterns in both space and time in functionally defined ensembles of neurons, multiphoton holographic optogenetics could allow neuroscientists to reveal fundamental aspects of the neural codes for sensation, cognition and behavior that have been beyond reach. This Review summarizes recent advances in multiphoton holographic optogenetics that substantially expand its capabilities, highlights outstanding technical challenges and provides an overview of the classes of experiments it can execute to test and validate key theoretical models of brain function. Multiphoton holographic optogenetics could substantially accelerate the pace of neuroscience discovery by helping to close the loop between experimental and theoretical neuroscience, leading to fundamental new insights into nervous system function and disorder.


Assuntos
Holografia/instrumentação , Holografia/métodos , Neurociências/métodos , Optogenética/instrumentação , Optogenética/métodos , Animais , Encéfalo/fisiologia , Humanos , Rede Nervosa/fisiologia , Opsinas , Estimulação Luminosa , Fótons
8.
Nat Commun ; 12(1): 4478, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294698

RESUMO

Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd3(Al,Ga)5O12 (Ce:GAGG), can effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles are non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level is sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables minimally invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.


Assuntos
Encéfalo/fisiologia , Animais , Comportamento Animal/fisiologia , Comportamento Animal/efeitos da radiação , Encéfalo/efeitos da radiação , Cério , Feminino , Células HEK293 , Humanos , Luminescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Opsinas/metabolismo , Opsinas/efeitos da radiação , Optogenética/instrumentação , Contagem de Cintilação , Tecnologia sem Fio/instrumentação , Raios X
9.
Nat Commun ; 12(1): 4488, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301944

RESUMO

Opn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the Gi/o pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light. Consistent with this prediction, illumination of recombinant expressed Opn7b in cortical pyramidal cells results in increased neuronal activity. In awake mice, light stimulation of Opn7b expressed in pyramidal cells of somatosensory cortex reliably induces generalized epileptiform activity within a short (<10 s) delay after onset of stimulation. Our study demonstrates a reversed mechanism for G protein-coupled receptor control and Opn7b as a tool for controlling neural circuit properties.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Opsinas/metabolismo , Optogenética/métodos , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Opsinas/genética , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Sinapses/genética , Sinapses/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
10.
Nat Commun ; 12(1): 4527, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312384

RESUMO

Optogenetic manipulation of neuronal activity through excitatory and inhibitory opsins has become an indispensable experimental strategy in neuroscience research. For many applications bidirectional control of neuronal activity allowing both excitation and inhibition of the same neurons in a single experiment is desired. This requires low spectral overlap between the excitatory and inhibitory opsin, matched photocurrent amplitudes and a fixed expression ratio. Moreover, independent activation of two distinct neuronal populations with different optogenetic actuators is still challenging due to blue-light sensitivity of all opsins. Here we report BiPOLES, an optogenetic tool for potent neuronal excitation and inhibition with light of two different wavelengths. BiPOLES enables sensitive, reliable dual-color neuronal spiking and silencing with single- or two-photon excitation, optical tuning of the membrane voltage, and independent optogenetic control of two neuronal populations using a second, blue-light sensitive opsin. The utility of BiPOLES is demonstrated in worms, flies, mice and ferrets.


Assuntos
Membrana Celular/fisiologia , Opsinas/metabolismo , Optogenética/métodos , Células Piramidais/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Células Cultivadas , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Furões/genética , Furões/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Masculino , Potenciais da Membrana/fisiologia , Camundongos Transgênicos , Opsinas/genética , Técnicas de Patch-Clamp/métodos , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos Wistar , Reprodutibilidade dos Testes
11.
J Neural Eng ; 18(4)2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34229315

RESUMO

Objective.Optogenetics has emerged as a promising technique for neural prosthetics, especially retinal prostheses, with unprecedented spatiotemporal resolution. Newly discovered opsins with high light sensitivity and fast temporal kinetics can provide sufficient temporal resolution at safe light powers and overcome the limitations of presently used opsins. It is also important to formulate accurate mathematical models for optogenetic retinal prostheses, which can facilitate optimization of photostimulation factors to improve the performance.Approach.A detailed theoretical analysis of optogenetic excitation of model retinal ganglion neurons (RGNs) and hippocampal neurons expressed with already tested opsins for retinal prostheses, namely, ChR2, ReaChR and ChrimsonR, and also with recently discovered potent opsins CsChrimson, bReaChES and ChRmine, was carried out.Main results.Under continuous illumination, ChRmine-expressing RGNs begin to respond at very low irradiances ∼10-4mW mm-2, and evoke firing upto ∼280 Hz, highest among other opsin-expressing RGNs, at 10-2mW mm-2. Under pulsed illumination at randomized photon fluxes, ChRmine-expressing RGNs respond to changes in pulse to pulse irradiances upto four logs, although very bright pulses >1014photons mm-2s-1block firing in these neurons. The minimum irradiance threshold for ChRmine-expressing RGNs is lower by two orders of magnitude, whereas, the first spike latency in ChRmine-expressing RGNs is shorter by an order of magnitude, alongwith stable latency of subsequest spikes compared to others. Further, a good set of photostimulation parameters were determined to achieve high-frequency control with single spike resolution at minimal power. Although ChrimsonR enables spiking upto 100 Hz in RGNs, it requires very high irradiances. ChRmine provides control at light powers that are two orders of magnitude smaller than that required with experimentally studied opsins, while maintaining single spike temporal resolution upto 40 Hz.Significance.The present study highlights the importance of ChRmine as a potential opsin for optogenetic retinal prostheses.


Assuntos
Optogenética , Próteses Visuais , Opsinas/genética , Estimulação Luminosa , Células Ganglionares da Retina
12.
BMC Ecol Evol ; 21(1): 114, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34098879

RESUMO

BACKGROUND: As flatfish, turbot undergo metamorphosis as part of their life cycle. In the larval stage, turbot live at the ocean surface, but after metamorphosis they move to deeper water and turn to benthic life. Thus, the light environment differs greatly between life stages. The visual system plays a great role in organic evolution, but reports of the relationship between the visual system and benthic life are rare. In this study, we reported the molecular and evolutionary analysis of opsin genes in turbot, and the heterochronic shifts in opsin expression during development. RESULTS: Our gene synteny analysis showed that subtype RH2C was not on the same gene cluster as the other four green-sensitive opsin genes (RH2) in turbot. It was translocated to chromosome 8 from chromosome 6. Based on branch-site test and spectral tuning sites analyses, E122Q and M207L substitutions in RH2C, which were found to be under positive selection, are closely related to the blue shift of optimum light sensitivities. And real-time PCR results indicated the dominant opsin gene shifted from red-sensitive (LWS) to RH2B1 during turbot development, which may lead to spectral sensitivity shifts to shorter wavelengths. CONCLUSIONS: This is the first report that RH2C may be an important subtype of green opsin gene that was retained by turbot and possibly other flatfish species during evolution. Moreover, E122Q and M207L substitutions in RH2C may contribute to the survival of turbot in the bluish colored ocean. And heterochronic shifts in opsin expression may be an important strategy for turbot to adapt to benthic life.


Assuntos
Evolução Biológica , Linguados , Opsinas , Animais , Proteínas de Peixes/genética , Linguados/genética , Opsinas/genética , Filogenia , Seleção Genética , Sintenia
13.
J Exp Biol ; 224(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34151984

RESUMO

Human opsin-based photopigments have great potential as light-sensitisers, but their requirement for phototransduction cascade-specific second messenger proteins may restrict their functionality in non-native cell types. In this study, eight chimeric human opsins were generated consisting of a backbone of either a rhodopsin (RHO) or long-wavelength-sensitive (LWS) opsin and intracellular domains from Gq/11-coupled human melanopsin. Rhodopsin/melanopsin chimeric opsins coupled to both Gi and Gq/11 pathways. Greater substitution of the intracellular surface with corresponding melanopsin domains generally showed greater Gq/11 activity with a decrease in Gi activation. Unlike melanopsin, rhodopsin and rhodopsin/melanopsin chimeras were dependent upon exogenous chromophore to function. By contrast, wild-type LWS opsin and LWS opsin/melanopsin chimeras showed only weak Gi activation in response to light, whilst Gq/11 pathway activation was not detected. Immunocytochemistry (ICC) demonstrated that chimeric opsins with more intracellular domains of melanopsin were less likely to be trafficked to the plasma membrane. This study demonstrates the importance of Gα coupling efficiency to the speed of cellular responses and created human opsins with a unique combination of properties to expand the range of customised optogenetic biotools for basic research and translational therapies.


Assuntos
Opsinas , Optogenética , Quimera , Humanos , Luz , Transdução de Sinal Luminoso , Opsinas/genética , Rodopsina/genética , Opsinas de Bastonetes/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-34126231

RESUMO

It is not well understood how the spectral composition (wavelength) of daylight that varies considerably during the day and seasons affects photoperiodic responses in a seasonal species. Here, we investigated the molecular underpinnings of wavelength-dependent photoperiodic induction in migratory redheaded buntings transferred to 13 h long days in neutral (white), 460 nm (blue), 500 nm (green) or 620 nm (red) wavelength that were compared with one another, and to short day controls for indices of the migratory (body fattening and weight gain, and Zugunruhe) and reproductive (testicular maturation) responses. Buntings showed wavelength-dependent photoperiodic response, with delayed Zugunruhe and slower testis maturation under 620 nm red light. Post-mortem comparison of gene expressions further revealed wavelength-dependence of the photoperiodic molecular response. Whereas there were higher retinal expressions of opn2 (rhodopsin) and opn5 (neuropsin) genes in red daylight, and of rhodopsin-like opsin (rh2) gene in green daylight, the hypothalamic opn2 mRNA levels were higher in blue daylight. Similarly, we found in birds under blue daylight an increased hypothalamic expression of genes involved in the photoperiodic induction (thyroid stimulating hormone subunit beta, tshb; eye absent 3, eya3; deiodinase type 2, dio2) and associated neural responses such as the calcium signaling (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, atp2a2), dopamine biosynthesis (tyrosine hydroxylase, th) and neurogenesis (brain-derived neurotrophic factor, bdnf). These results demonstrate transcriptional changes in parallel to responses associated with migration and reproduction in buntings, and suggest a role of daylight spectrum in photoperiodic induction of the vernal response in obligate spring avian migrants.


Assuntos
Migração Animal , Luz , Fotoperíodo , Rodopsina/fisiologia , Estações do Ano , Aves Canoras/fisiologia , Animais , Comportamento Animal , Encéfalo/metabolismo , Sinalização do Cálcio , Ritmo Circadiano/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hipotálamo/metabolismo , Índia , Masculino , Opsinas , Fenótipo , Retina/metabolismo , Rodopsina/metabolismo , Testículo/crescimento & desenvolvimento
15.
Methods Mol Biol ; 2268: 207-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085271

RESUMO

GPCRs are responsible for activation of numerous downstream effectors. Live cell imaging of these effectors therefore provides a real-time readout of GPCR activity and allows for better understanding of temporal dynamics of GPCR-mediated signaling. Opsins, or optically activatable GPCRs, allow for these signaling pathways to be activated in a spatiotemporally precise and reversible manner. Here, we describe optogenetic methods for activating Gi, Gq, and Gs signaling pathways. Additionally, we present assays for detecting activation of these pathways in real time through live cell imaging of Gßγ translocation, PIP3 increase, PIP2 hydrolysis, cAMP production, and cell migration. These assays can be utilized for GPCR-targeted drug development, as well as for studies of a wide range of GPCR-mediated physiological processes.


Assuntos
Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Opsinas/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Opsinas/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais
16.
Exp Eye Res ; 209: 108669, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34126082

RESUMO

M-opsin, encoded by opn1mw gene, is involved in green-light perception of mice. The role of M-opsin in emmetropization of mice remains uncertain. To answer the above question, 4-week-old wild-type (WT) mice were exposed to white light or green light (460-600 nm, a peak at 510 nm) for 12 weeks. Refractive development was estimated biweekly. After treatment, retinal function was assessed using electroretinogram (ERG). Dopamine (DA) in the retina was evaluated by high-performance liquid chromatography, M-opsin and S-opsin protein levels by Western blot and ELISA, and mRNA expressions of opn1mw and opn1sw by RT-PCR. Effects of M-opsin were further verified in Opn1mw-/- and WT mice raised in white light for 4 weeks. Refractive development was examined at 4, 6, and 8 weeks after birth. The retinal structure was estimated through hematoxylin and eosin staining (H&E) and transmission electron microscopy (TEM). Retinal wholemounts from WT and Opn1mw-/- mice were co-immunolabeled with M-opsin and S-opsin, their distribution and quantity were then assayed by immunofluorescence staining (IF). Expression of S-opsin protein and opn1sw mRNA were determined by Western blot, ELISA, or RT-PCR. Retinal function and DA content were analyzed by ERG and liquid chromatography tandem-mass spectrometry (LC-MS/MS), respectively. Lastly, visual cliff test was used to evaluate the depth perception of the Opn1mw-/- mice. We found that green light-treated WT mice were more myopic with increased M-opsin expression and decreased DA content than white light-treated WT mice after 12-week illumination. No electrophysiologic abnormalities were recorded in mice exposed to green light compared to those exposed to white light. A more hyperopic shift was further observed in 8-week-old Opn1mw-/- mice in white light with lower DA level and weakened cone function than the WT mice under white light. Neither obvious structural disruption of the retina nor abnormal depth perception was found in Opn1mw-/- mice. Together, these results suggested that the M-opsin-based color vision participated in the refractive development of mice. Overexposure to green light caused myopia, but less perception of the middle-wavelength components in white light promoted hyperopia in mice. Furthermore, possible dopaminergic signaling pathway was suggested in myopia induced by green light.


Assuntos
Visão de Cores/genética , Regulação da Expressão Gênica , Opsinas/genética , Refração Ocular/genética , Erros de Refração/genética , Retina/metabolismo , Animais , Modelos Animais de Doenças , Eletrorretinografia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Opsinas/biossíntese , RNA/genética , Erros de Refração/diagnóstico , Erros de Refração/metabolismo , Retina/ultraestrutura , Tomografia Óptica
17.
Nat Commun ; 12(1): 2730, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980868

RESUMO

Neurostimulant drugs or magnetic/electrical stimulation techniques can overcome attention deficits, but these drugs or techniques are weakly beneficial in boosting the learning capabilities of healthy subjects. Here, we report a stimulation technique, mid-infrared modulation (MIM), that delivers mid-infrared light energy through the opened skull or even non-invasively through a thinned intact skull and can activate brain neurons in vivo without introducing any exogeneous gene. Using c-Fos immunohistochemistry, in vivo single-cell electrophysiology and two-photon Ca2+ imaging in mice, we demonstrate that MIM significantly induces firing activities of neurons in the targeted cortical area. Moreover, mice that receive MIM targeting to the auditory cortex during an auditory associative learning task exhibit a faster learning speed (~50% faster) than control mice. Together, this non-invasive, opsin-free MIM technique is demonstrated with potential for modulating neuronal activity.


Assuntos
Córtex Auditivo/metabolismo , Neurônios/metabolismo , Opsinas/metabolismo , Animais , Eletrofisiologia , Imuno-Histoquímica , Masculino , Camundongos , Opsinas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo
18.
Mol Ecol Resour ; 21(7): 2388-2406, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34003602

RESUMO

Wrasses are dominant components of major coral reef systems. Among wrasses, Cheilinus undulatus is an endangered species with high economic and ecological value that exhibits sex reversal of females to males, while sexual selection occurs in breeding aggregations. However, the molecular-associated mechanism underlying this remains unclear. Opsin gene diversification is regarded as a potent force in sexual selection. Here we present a genome assembly of C. undulatus, using Illumina, Nanopore and Hi-C sequencing. The 1.17 Gb genome was generated from 328 contigs with an N50 length of 16.5 Mb and anchored to 24 chromosomes. In total, 22,218 genes were functionally annotated, and 96.36% of BUSCO genes were fully represented. Transcriptomic analyses showed that 96.79% of the predicted genes were expressed. Transposons were most abundant, accounting for 39.88% of the genome, with low divergence, owing to their evolution with close species ~60.53 million years ago. In total, 567/1,826 gene families were expanded and contracted in the reconstructed phylogeny, respectively. Forty-six genes were under positive selection. Comparative genomic analyses with other fish revealed expansion of opsin SWS2B, LWS1 and Rh2. The elevated duplicates of SWS2B were generated by gene conversions via transposition of transposons followed by nonallelic homologous recombination. Amino acid substitutions of opsin paralogues occurred at key tuning sites, causing a spectral shift in maximal absorbance of visual pigment to capture functional changes. Among these opsin genes, SWS2B-3 and 4 and Rh1 are expressed in the retina. The genome sequence of C. undulatus provides valuable resources for future investigation of the conservation, evolution and behaviour of fishes.


Assuntos
Opsinas , Perciformes , Animais , Cromossomos , Feminino , Peixes/genética , Genoma , Humanos , Masculino , Opsinas/genética , Perciformes/genética , Filogenia
19.
BMC Ecol Evol ; 21(1): 81, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33971810

RESUMO

BACKGROUND: Vision is a crucial sense for the evolutionary success of many animal groups. Here we explore the diversity of visual pigments (opsins) in the transcriptomes of amphipods (Crustacea: Amphipoda) and conclude that it is restricted to middle (MWS) and long wavelength-sensitive (LWS) opsins in the overwhelming majority of examined species. RESULTS: We evidenced (i) parallel loss of MWS opsin expression in multiple species (including two independently evolved lineages from the deep and ancient Lake Baikal) and (ii) LWS opsin amplification (up to five transcripts) in both Baikal lineages. The number of LWS opsins negatively correlated with habitat depth in Baikal amphipods. Some LWS opsins in Baikal amphipods contained MWS-like substitutions, suggesting that they might have undergone spectral tuning. CONCLUSIONS: This repeating two-step evolutionary scenario suggests common triggers, possibly the lack of light during the periods when Baikal was permanently covered with thick ice and its subsequent melting. Overall, this observation demonstrates the possibility of revealing climate history by following the evolutionary changes in protein families.


Assuntos
Anfípodes , Opsinas , Anfípodes/genética , Animais , Evolução Biológica , Lagos , Opsinas/genética , Filogenia
20.
Elife ; 102021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-34032211

RESUMO

All-optical methods for imaging and manipulating brain networks with high spatial resolution are fundamental to study how neuronal ensembles drive behavior. Stimulation of neuronal ensembles using two-photon holographic techniques requires high-sensitivity actuators to avoid photodamage and heating. Moreover, two-photon-excitable opsins should be insensitive to light at wavelengths used for imaging. To achieve this goal, we developed a novel soma-targeted variant of the large-conductance blue-light-sensitive opsin CoChR (stCoChR). In the mouse cortex in vivo, we combined holographic two-photon stimulation of stCoChR with an amplified laser tuned at the opsin absorption peak and two-photon imaging of the red-shifted indicator jRCaMP1a. Compared to previously characterized blue-light-sensitive soma-targeted opsins in vivo, stCoChR allowed neuronal stimulation with more than 10-fold lower average power and no spectral crosstalk. The combination of stCoChR, tuned amplified laser stimulation, and red-shifted functional indicators promises to be a powerful tool for large-scale interrogation of neural networks in the intact brain.


Assuntos
Córtex Cerebral/efeitos da radiação , Luz , Opsinas/metabolismo , Optogenética , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Camundongos , Neurônios/efeitos da radiação , Fótons
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