RESUMO
BACKGROUND: Identification of biallelic CNPY3 mutations in patients with epileptic encephalopathy and abnormal electroencephalography findings of Cnpy3 knock-out mice have indicated that the loss of CNPY3 function causes neurological disorders such as epilepsy. However, the basic property of CNPY3 in the brain remains unclear. NEW METHOD: We generated C-terminal 2xHA-tag knock-in Cnpy3 mice by i-GONAD in vivo genome editing system to investigate the expression and function of Cnpy3 in the mouse brain. RESULTS: 2xHA-tagged Cnpy3 was confirmed by immunoblot analysis using anti-HA and CNPY3 antibodies, although HA tagging caused the decreased Cnpy3 protein level. Immunohistochemical analysis of Cnpy32xHA knock-in mice showed that Cnpy3-2xHA was predominantly expressed in the neuron. In addition, Cnpy3 and Cnpy3-2xHA were both localized in the endoplasmic reticulum and synaptosome and showed age-dependent expression changes in the brain. COMPARISON WITH EXISTING METHODS: Conventional Cnpy3 antibodies could not allow us to investigate the distribution of Cnpy3 expression in the brain, while HA-tagging revealed the expression of CNPY3 in neuronal cells. CONCLUSIONS: Taken together, we demonstrated that Cnpy32xHA knock-in mice would be useful to further elucidate the property of Cnpy3 in brain function and neurological disorders.
Assuntos
Epilepsia , Neurônios , Animais , Camundongos , Neurônios/fisiologia , Encéfalo/metabolismo , Eletroencefalografia , Camundongos Knockout , AnticorposRESUMO
Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIß mice by combining CRISPR-Cas9 genome editing with electroporation (i-GONAD). With i-GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and ß proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i-GONAD is a useful and convenient choice, especially if specific antibodies are unavailable.
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Eletroporação , Edição de Genes , Animais , Anticorpos/metabolismo , Western Blotting , Sistemas CRISPR-Cas/genética , Epitopos/genética , Epitopos/metabolismo , Gônadas/metabolismo , CamundongosRESUMO
Recently, we have identified CaMKIIα and CaMKIIß mutations in patients with neurodevelopmental disorders by whole exome sequencing study. Most CaMKII mutants have increased phosphorylation of Thr286/287, which induces autonomous activity of CaMKII, using cell culture experiments. In this study, we explored the pathological mechanism of motor dysfunction observed exclusively in a patient with Pro213Leu mutation in CaMKIIß using a mouse model of the human disease. The homozygous CaMKIIß Pro213Leu knockin mice showed age-dependent motor dysfunction and growth failure from 2 weeks after birth. In the cerebellum, the mutation did not alter the mRNA transcript level, but the CaMKIIß protein level was dramatically decreased. Furthermore, in contrast to previous result from cell culture, Thr287 phosphorylation of CaMKIIß was also reduced. CaMKIIß Pro213Leu knockin mice showed similar motor dysfunction as CaMKIIß knockout mice, newly providing evidence for a loss of function rather than a gain of function. Our disease model mouse showed similar phenotypes of the patient, except for epileptic seizures. We clearly demonstrated that the pathological mechanism is a reduction of mutant CaMKIIß in the brain, and the physiological aspects of mutation were greatly different between in vivo and cell culture.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Cerebelo , Animais , Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cerebelo/metabolismo , Humanos , Camundongos , Mutação/genética , FosforilaçãoRESUMO
Brain malformations have heterogeneous genetic backgrounds. Tubulinopathies are a wide range of brain malformations caused by variants in tubulin and microtubules-associated genes. Recently biallelic variants in TTC5, also known as stress responsive activator of p300, have been reported in 11 patients from seven families with developmental delay, intellectual disability, and brain malformations. Here, we report compound heterozygous frameshift variants in TTC5 in a Japanese boy who showed severe psychomotor developmental delay and pseudobulbar palsy with growth failure. Brain magnetic resonance imaging showed a simplified gyral pattern and undetectable anterior limb of the internal capsule, suggesting tubulinopathies. Immunoblotting using lymphoblastoid cells derived from the patient showed undetectable TTC5 protein. Ttc5 silencing by RNA interference in Neuro2a cells reduced Tubulin ß3 protein level and caused abnormal cell cycle. Our report suggests a possible link between TTC5-related brain malformation and tubulinopathies.
Assuntos
Encéfalo/anormalidades , Mutação da Fase de Leitura , Predisposição Genética para Doença , Malformações do Sistema Nervoso/diagnóstico , Malformações do Sistema Nervoso/genética , Fenótipo , Fatores de Transcrição/genética , Tubulina (Proteína)/genética , Encéfalo/diagnóstico por imagem , Estudos de Associação Genética , Humanos , Japão , Imageamento por Ressonância Magnética , Masculino , Tubulina (Proteína)/metabolismoRESUMO
Vacuolar H+-ATPases (V-ATPases) transport protons across cellular membranes to acidify various organelles. ATP6V0A1 encodes the a1-subunit of the V0 domain of V-ATPases, which is strongly expressed in neurons. However, its role in brain development is unknown. Here we report four individuals with developmental and epileptic encephalopathy with ATP6V0A1 variants: two individuals with a de novo missense variant (R741Q) and the other two individuals with biallelic variants comprising one almost complete loss-of-function variant and one missense variant (A512P and N534D). Lysosomal acidification is significantly impaired in cell lines expressing three missense ATP6V0A1 mutants. Homozygous mutant mice harboring human R741Q (Atp6v0a1R741Q) and A512P (Atp6v0a1A512P) variants show embryonic lethality and early postnatal mortality, respectively, suggesting that R741Q affects V-ATPase function more severely. Lysosomal dysfunction resulting in cell death, accumulated autophagosomes and lysosomes, reduced mTORC1 signaling and synaptic connectivity, and lowered neurotransmitter contents of synaptic vesicles are observed in the brains of Atp6v0a1A512P/A512P mice. These findings demonstrate the essential roles of ATP6V0A1/Atp6v0a1 in neuronal development in terms of integrity and connectivity of neurons in both humans and mice.
Assuntos
Encefalopatias/genética , Encéfalo/crescimento & desenvolvimento , Neurônios/fisiologia , Neurotransmissores/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Autofagossomos/patologia , Mapeamento Encefálico/métodos , Catepsina D/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Mutação com Perda de Função/genética , Lisossomos/patologia , Imageamento por Ressonância Magnética/métodos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mutação de Sentido Incorreto/genética , Neurônios/citologia , Vesículas Sinápticas/patologiaRESUMO
Objective: α (CAMK2A) and ß (CAMK2B) isoforms of Calcium/calmodulin-dependent protein kinase II (CaMKII) play a pivotal role in neuronal plasticity and in learning and memory processes in the brain. Here, we explore the possible involvement of α- and ß-CaMKII variants in neurodevelopmental disorders. Methods: Whole-exome sequencing was performed for 976 individuals with intellectual disability, developmental delay, and epilepsy. The effect of CAMK2A and CAMK2B variants on CaMKII structure and firing of neurons was evaluated by computational structural analysis, immunoblotting, and electrophysiological analysis. Results: We identified a total of five de novo CAMK2A and CAMK2B variants in three and two individuals, respectively. Seizures were common to three individuals with CAMK2A variants. Using a minigene splicing assay, we demonstrated that a splice site variant caused skipping of exon 11 leading to an in-frame deletion of the regulatory segment of CaMKII α. By structural analysis, four missense variants are predicted to impair the interaction between the kinase domain and the regulatory segment responsible for the autoinhibition of its kinase activity. The Thr286/Thr287 phosphorylation as a result of release from autoinhibition was increased in three mutants when the mutants were stably expressed in Neuro-2a neuroblastoma cells. Expression of a CaMKII α mutant in primary hippocampal neurons significantly increased A-type K+ currents, which facilitated spike repolarization of single action potentials. Interpretation: Our data highlight the importance of CaMKII α and CaMKII ß and their autoinhibitory regulation in human brain function, and suggest the enhancement of A-type K+ currents as a possible pathophysiological basis.
RESUMO
Early-onset epileptic encephalopathies, including West syndrome (WS), are a group of neurological disorders characterized by developmental impairments and intractable seizures from early infancy. We have now identified biallelic CNPY3 variants in three individuals with WS; these include compound-heterozygous missense and frameshift variants in a family with two affected siblings (individuals 1 and 2) and a homozygous splicing variant in a consanguineous family (individual 3). All three individuals showed hippocampal malrotation. In individuals 1 and 2, electroencephalography (EEG) revealed characteristic fast waves and diffuse sharp- and slow-wave complexes. The fast waves were clinically associated with seizures. CNPY3 encodes a co-chaperone in the endoplasmic reticulum and regulates the subcellular distribution and responses of multiple Toll-like receptors. The amount of CNPY3 in lymphoblastoid cells derived from individuals 1 and 2 was severely lower than that in control cells. Cnpy3-knockout mice exhibited spastic or dystonic features under resting conditions and hyperactivity and anxiolytic behavior during the open field test. Also, their resting EEG showed enhanced activity in the fast beta frequency band (20-35 Hz), which could mimic the fast waves in individuals 1 and 2. These data suggest that CNPY3 and Cnpy3 perform essential roles in brain function in addition to known Toll-like receptor-dependent immune responses.
Assuntos
Chaperonas Moleculares/genética , Mutação , Convulsões/genética , Espasmos Infantis/genética , Adolescente , Sequência de Aminoácidos , Animais , Criança , Consanguinidade , Eletroencefalografia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Família , Feminino , Expressão Gênica , Heterozigoto , Hipocampo/diagnóstico por imagem , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Knockout , Convulsões/diagnóstico por imagem , Convulsões/fisiopatologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Irmãos , Espasmos Infantis/diagnóstico por imagem , Espasmos Infantis/fisiopatologiaRESUMO
Corticotropin-releasing hormone (CRH), which is synthesized in the paraventricular nucleus (PVN) of the hypothalamus, plays an important role in the endocrine stress response. The excitability of CRH neurons is regulated by γ-aminobutyric acid (GABA)-containing neurons projecting to the PVN. We investigated the role of GABA in the regulation of CRH release. The release of CRH was impaired, accumulating in the cell bodies of CRH neurons in heterozygous GAD67-GFP (green fluorescent protein) knock-in mice (GAD67(+/GFP)), which exhibited decreased GABA content. The GABAA receptor (GABAAR) and the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), but not the K(+)-Cl(-) cotransporter (KCC2), were expressed in the terminals of the CRH neurons at the median eminence (ME). In contrast, CRH neuronal somata were enriched with KCC2 but not with NKCC1. Thus, intracellular Cl(-) concentrations ([Cl(-)]i) may be increased at the terminals of CRH neurons compared with concentrations in the cell body. Moreover, GABAergic terminals projecting from the arcuate nucleus were present in close proximity to CRH-positive nerve terminals. Furthermore, a GABAAR agonist increased the intracellular calcium (Ca(2+)) levels in the CRH neuron terminals but decreased the Ca(2+) levels in their somata. In addition, the increases in Ca(2+) concentrations were prevented by an NKCC1 inhibitor. We propose a novel mechanism by which the excitatory action of GABA maintains a steady-state CRH release from axon terminals in the ME.
Assuntos
Hormônio Liberador da Corticotropina/metabolismo , Receptores de GABA-A/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/genética , Ácido gama-Aminobutírico/metabolismo , Animais , Axônios/metabolismo , Sinalização do Cálcio , Neurônios GABAérgicos/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Glutamato Descarboxilase/genética , Proteínas de Fluorescência Verde/genética , Hipotálamo/metabolismo , Eminência Mediana/metabolismo , Camundongos , Núcleo Hipotalâmico Paraventricular/metabolismo , Receptores de GABA-A/genética , Membro 2 da Família 12 de Carreador de Soluto/genética , Simportadores/metabolismo , Cotransportadores de K e Cl-RESUMO
RATIONALE: Monitoring and controlling cardiac myocyte activity with optogenetic tools offer exciting possibilities for fundamental and translational cardiovascular research. Genetically encoded voltage indicators may be particularly attractive for minimal invasive and repeated assessments of cardiac excitation from the cellular to the whole heart level. OBJECTIVE: To test the hypothesis that cardiac myocyte-targeted voltage-sensitive fluorescence protein 2.3 (VSFP2.3) can be exploited as optogenetic tool for the monitoring of electric activity in isolated cardiac myocytes and the whole heart as well as function and maturity in induced pluripotent stem cell-derived cardiac myocytes. METHODS AND RESULTS: We first generated mice with cardiac myocyte-restricted expression of VSFP2.3 and demonstrated distinct localization of VSFP2.3 at the t-tubulus/junctional sarcoplasmic reticulum microdomain without any signs for associated pathologies (assessed by echocardiography, RNA-sequencing, and patch clamping). Optically recorded VSFP2.3 signals correlated well with membrane voltage measured simultaneously by patch clamping. The use of VSFP2.3 for human action potential recordings was confirmed by simulation of immature and mature action potentials in murine VSFP2.3 cardiac myocytes. Optical cardiograms could be monitored in whole hearts ex vivo and minimally invasively in vivo via fiber optics at physiological heart rate (10 Hz) and under pacing-induced arrhythmia. Finally, we reprogrammed tail-tip fibroblasts from transgenic mice and used the VSFP2.3 sensor for benchmarking functional and structural maturation in induced pluripotent stem cell-derived cardiac myocytes. CONCLUSIONS: We introduce a novel transgenic voltage-sensor model as a new method in cardiovascular research and provide proof of concept for its use in optogenetic sensing of physiological and pathological excitation in mature and immature cardiac myocytes in vitro and in vivo.
Assuntos
Potenciais da Membrana/fisiologia , Miócitos Cardíacos/fisiologia , Optogenética/métodos , Animais , Humanos , Camundongos , Camundongos Transgênicos , Imagens com Corantes Sensíveis à Voltagem/métodosRESUMO
The invention of membrane voltage protein indicators widens the reach of optical voltage imaging in cell physiology, most notably neurophysiology, by enabling membrane voltage recordings from genetically defined cell types in chronic and life-long preparations. While the last years have seen a dramatic improvement in the technical performance of these indicators, concomitant innovations in optogenetics, optical axon tracing, and high-speed digital microscopy are beginning to fulfill the age-old vision of an all-optical analysis of neuronal circuits, reaching beyond the limits of traditional electrode-based recordings. We will present our personal account of the development of protein voltage indicators from the pioneering days to the present state, including their applications in neurophysiology that has inspired our own work for more than a decade.
RESUMO
Traditional small molecule voltage sensitive dye indicators have been a powerful tool for monitoring large scale dynamics of neuronal activities but have several limitations including the lack of cell class specific targeting, invasiveness and difficulties in conducting longitudinal studies. Recent advances in the development of genetically-encoded voltage indicators have successfully overcome these limitations. Genetically-encoded voltage indicators (GEVIs) provide sufficient sensitivity to map cortical representations of sensory information and spontaneous network activities across cortical areas and different brain states. In this study, we directly compared the performance of a prototypic GEVI, VSFP2.3, with that of a widely used small molecule voltage sensitive dye (VSD), RH1691, in terms of their ability to resolve mesoscopic scale cortical population responses. We used three synchronized CCD cameras to simultaneously record the dual emission ratiometric fluorescence signal from VSFP2.3 and RH1691 fluorescence. The results show that VSFP2.3 offers more stable and less invasive recording conditions, while the signal-to-noise level and the response dynamics to sensory inputs are comparable to RH1691 recordings.
RESUMO
Complex cognitive processes require neuronal activity to be coordinated across multiple scales, ranging from local microcircuits to cortex-wide networks. However, multiscale cortical dynamics are not well understood because few experimental approaches have provided sufficient support for hypotheses involving multiscale interactions. To address these limitations, we used, in experiments involving mice, genetically encoded voltage indicator imaging, which measures cortex-wide electrical activity at high spatiotemporal resolution. Here we show that, as mice recovered from anesthesia, scale-invariant spatiotemporal patterns of neuronal activity gradually emerge. We show for the first time that this scale-invariant activity spans four orders of magnitude in awake mice. In contrast, we found that the cortical dynamics of anesthetized mice were not scale invariant. Our results bridge empirical evidence from disparate scales and support theoretical predictions that the awake cortex operates in a dynamical regime known as criticality. The criticality hypothesis predicts that small-scale cortical dynamics are governed by the same principles as those governing larger-scale dynamics. Importantly, these scale-invariant principles also optimize certain aspects of information processing. Our results suggest that during the emergence from anesthesia, criticality arises as information processing demands increase. We expect that, as measurement tools advance toward larger scales and greater resolution, the multiscale framework offered by criticality will continue to provide quantitative predictions and insight on how neurons, microcircuits, and large-scale networks are dynamically coordinated in the brain.
Assuntos
Córtex Cerebral/fisiologia , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Vigília/fisiologia , Animais , Córtex Cerebral/química , Eletroencefalografia/métodos , Feminino , Masculino , CamundongosRESUMO
Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviors. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs) has the potential to meet this challenge. Currently prevalent GEVIs are based on the voltage-sensitive fluorescent protein (VSFP) prototypical design or on the voltage-dependent state transitions of microbial opsins. We recently introduced a new VSFP design in which the voltage-sensing domain (VSD) is sandwiched between a fluorescence resonance energy transfer pair of fluorescent proteins (termed VSFP-Butterflies) and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians.
RESUMO
Voltage-sensitive fluorescent proteins (VSFPs) are a family of genetically-encoded voltage indicators (GEVIs) reporting membrane voltage fluctuation from genetically-targeted cells in cell cultures to whole brains in awake mice as demonstrated earlier using 1-photon (1P) fluorescence excitation imaging. However, in-vivo 1P imaging captures optical signals only from superficial layers and does not optically resolve single neurons. Two-photon excitation (2P) imaging, on the other hand, has not yet been convincingly applied to GEVI experiments. Here we show that 2P imaging of VSFP Butterfly 1.2 expresssing pyramidal neurons in layer 2/3 reports optical membrane voltage in brain slices consistent with 1P imaging but with a 2-3 larger ΔR/R value. 2P imaging of mouse cortex in-vivo achieved cellular resolution throughout layer 2/3. In somatosensory cortex we recorded sensory responses to single whisker deflections in anesthetized mice at full frame video rate. Our results demonstrate the feasibility of GEVI-based functional 2P imaging in mouse cortex.
Assuntos
Córtex Cerebral/fisiologia , Potenciais Evocados/fisiologia , Proteínas Luminescentes , Neuroimagem , Fótons , Animais , Feminino , Camundongos , Neurônios/fisiologia , Gravidez , Córtex Somatossensorial/fisiologiaRESUMO
Optical imaging has a long history in physiology and in neurophysiology in particular. Over the past 15 years or so, new methodologies have emerged that combine genetic engineering with light-based imaging methods. This merger has resulted in a tool box of genetically encoded optical indicators that enable nondestructive and long-lasting monitoring of neuronal activities at the cellular, circuit, and system level. This review describes the historical roots and fundamental concepts underlying these new approaches, evaluates current progress in this field, and concludes with a forward-looking perspective on current work and potential future developments in this field.
Assuntos
Neuroimagem/métodos , Imagem Óptica/métodos , Optogenética/métodos , Animais , História do Século XX , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Potenciais da Membrana , Neuroimagem/tendências , Neurônios/fisiologia , Imagem Óptica/história , Optogenética/tendências , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Membrane proteins that respond to changes in transmembrane voltage are critical in regulating the function of living cells. The voltage-sensing domains (VSDs) of voltage-gated ion channels are extensively studied to elucidate voltage-sensing mechanisms, and yet many aspects of their structure-function relationship remain elusive. Here, we transplanted homologous amino acid motifs from the tetrameric voltage-activated potassium channel Kv3.1 to the monomeric VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) to explore which portions of Kv3.1 subunits depend on the tetrameric structure of Kv channels and which properties of Kv3.1 can be transferred to the monomeric Ci-VSP scaffold. By attaching fluorescent proteins to these chimeric VSDs, we obtained an optical readout to establish membrane trafficking and kinetics of voltage-dependent structural rearrangements. We found that motifs extending from 10 to roughly 100 amino acids can be readily transplanted from Kv3.1 into Ci-VSP to form engineered VSDs that efficiently incorporate into the plasma membrane and sense voltage. Some of the functional features of these engineered VSDs are reminiscent of Kv3.1 channels, indicating that these properties do not require interactions between Kv subunits or between the voltage sensing and the pore domains of Kv channels.
Assuntos
Ciona intestinalis/enzimologia , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Engenharia de Proteínas/métodos , Canais de Potássio Shaw/genética , Canais de Potássio Shaw/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Células PC12 , Monoéster Fosfórico Hidrolases/química , Porosidade , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Canais de Potássio Shaw/químicaRESUMO
Fluorescent membrane voltage indicators that enable optical imaging of neuronal circuit operations in the living mammalian brain are powerful tools for biology and particularly neuroscience. Classical voltage-sensitive dyes, typically low molecular-weight organic compounds, have been in widespread use for decades but are limited by issues related to optical noise, the lack of generally applicable procedures that enable staining of specific cell populations, and difficulties in performing imaging experiments over days and weeks. Genetically encoded voltage indicators (GEVIs) represent a newer alternative that overcomes several of the limitations inherent to classical voltage-sensitive dyes. We critically review the fundamental concepts of this approach, the variety of available probes and their state of development.
Assuntos
Imunofluorescência/métodos , Corantes Fluorescentes/química , Engenharia Genética/métodos , Imagens com Corantes Sensíveis à Voltagem/métodos , Algoritmos , Animais , Fenômenos Fisiológicos Cardiovasculares , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Proteínas Luminescentes , NeurociênciasRESUMO
Population signals from neuronal ensembles in cortex during behavior are commonly measured with EEG, local field potential (LFP), and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here we describe how this goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single-cell synaptic responses, and a high signal-to-noise ratio. Butterfly reports excitatory postsynaptic potentials (EPSPs) in cortical neurons, whisker-evoked responses in barrel cortex, 25-Hz gamma oscillations in hippocampal slices, and 2- to 12-Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics.
Assuntos
Proteínas Luminescentes/genética , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Ondas Encefálicas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Potenciais Pós-Sinápticos Excitadores , Transferência Ressonante de Energia de Fluorescência , Hipocampo/citologia , Hipocampo/fisiologia , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Neurônios/classificação , Optogenética , Células PC12 , Monoéster Fosfórico Hidrolases/química , Estrutura Terciária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The combination of optical imaging methods with targeted expression of protein-based fluorescent probes constitutes a powerful approach for functional analysis of selected cell populations within intact neuronal circuitries. Herein, we lay out the conceptual motivation for optogenetic recording of brain electrical activity using genetically encoded voltage-sensitive fluorescent proteins (VSFPs), describe how the current generation of VSFPs has evolved, and demonstrate how VSFPs report membrane voltage signals in isolated cells, brain slices, and living animals. We conclude with a critical appraisal of VSFPs for voltage recording and highlight promising applications of this emerging methodology for bridging cellular and intact systems biology.
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Encéfalo/fisiologia , Proteínas Luminescentes/genética , Imagem Molecular/métodos , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Opsinas/genéticaRESUMO
Abstract Optogenetics is the latest new subdiscipline in brain sciences that merges optical imaging, protein engineering and genetic dissection of neuronal circuits to optically monitor and control brain activity with high spatial and temporal precision. In this review, the conceptual framework that fueled the development of this methodology is first introduced and the central tools utilized in monitoring and controlling of neuronal circuit elements using light are described. How this innovative approach fosters our understanding of both physiological and pathophysiological properties of brain networks is then discussed. Finally, the potential clinical application of optogenetic approaches is outlined.