RESUMO
BACKGROUND: Suppression of behavioral and physical responses defines the anesthetized state. This is accompanied, in humans, by characteristic changes in electroencephalogram patterns. However, these measures reveal little about the neuron or circuit-level physiologic action of anesthetics nor how information is trafficked between neurons. This study assessed whether entropy-based metrics can differentiate between the awake and anesthetized state in Caenorhabditis elegans and characterize emergence from anesthesia at the level of interneuronal communication. METHODS: Volumetric fluorescence imaging measured neuronal activity across a large portion of the C. elegans nervous system at cellular resolution during distinct states of isoflurane anesthesia, as well as during emergence from the anesthetized state. Using a generalized model of interneuronal communication, new entropy metrics were empirically derived that can distinguish the awake and anesthetized states. RESULTS: This study derived three new entropy-based metrics that distinguish between stable awake and anesthetized states (isoflurane, n = 10) while possessing plausible physiologic interpretations. State decoupling is elevated in the anesthetized state (0%: 48.8 ± 3.50%; 4%: 66.9 ± 6.08%; 8%: 65.1 ± 5.16%; 0% vs. 4%, P < 0.001; 0% vs. 8%, P < 0.001), while internal predictability (0%: 46.0 ± 2.94%; 4%: 27.7 ± 5.13%; 8%: 30.5 ± 4.56%; 0% vs. 4%, P < 0.001; 0% vs. 8%, P < 0.001), and system consistency (0%: 2.64 ± 1.27%; 4%: 0.97 ± 1.38%; 8%: 1.14 ± 0.47%; 0% vs. 4%, P = 0.006; 0% vs. 8%, P = 0.015) are suppressed. These new metrics also resolve to baseline during gradual emergence of C. elegans from moderate levels of anesthesia to the awake state (n = 8). The results of this study show that early emergence from isoflurane anesthesia in C. elegans is characterized by the rapid resolution of an elevation in high frequency activity (n = 8, P = 0.032). The entropy-based metrics mutual information and transfer entropy, however, did not differentiate well between the awake and anesthetized states. CONCLUSIONS: Novel empirically derived entropy metrics better distinguish the awake and anesthetized states compared to extant metrics and reveal meaningful differences in information transfer characteristics between states.
Assuntos
Anestesia , Anestésicos Inalatórios , Isoflurano , Animais , Humanos , Isoflurano/farmacologia , Caenorhabditis elegans , Anestésicos Inalatórios/farmacologia , NeurôniosRESUMO
The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4 µm) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neurons that did not produce exophers. The extruded exopher transits through surrounding tissue in which some contents appear degraded, but some non-degradable materials can subsequently be found in more remote cells, suggesting secondary release. Our observations suggest that exopher-genesis is a potential response to rid cells of neurotoxic components when proteostasis and organelle function are challenged. We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain ageing.
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Caenorhabditis elegans/metabolismo , Micropartículas Derivadas de Células/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Neuroproteção/fisiologia , Agregados Proteicos , Envelhecimento/metabolismo , Envelhecimento/patologia , Animais , Autofagia , Caenorhabditis elegans/citologia , Citoplasma/metabolismo , Chaperonas Moleculares/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Oxirredução , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
LED array microscopy is an emerging platform for computational imaging with significant utility for biological imaging. Existing LED array systems often exploit transmission imaging geometries of standard brightfield microscopes that leave the rich backscattered field undetected. This backscattered signal contains high-resolution sample information with superb sensitivity to subtle structural features that make it ideal for biological sensing and detection. Here, we develop an LED array reflectance microscope capturing the sample's backscattered signal. In particular, we demonstrate multimodal brightfield, darkfield, and differential phase contrast imaging on fixed and living biological specimens including Caenorhabditis elegans (C. elegans), zebrafish embryos, and live cell cultures. Video-rate multimodal imaging at 20 Hz records real time features of freely moving C. elegans and the fast beating heart of zebrafish embryos. Our new reflectance mode is a valuable addition to the LED array microscopic toolbox.
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Microscopia/instrumentação , Fenômenos Ópticos , Espalhamento de Radiação , Semicondutores , Sobrevivência Celular , Células HT29 , HumanosRESUMO
BACKGROUND: Animal studies demonstrate that anesthetic exposure during neurodevelopment can lead to persistent behavioral impairment. The changes in neuronal function underlying these effects are incompletely understood. Caenorhabditis elegans is well suited for functional imaging of postanesthetic effects on neuronal activity. This study aimed to examine such effects within the neurocircuitry underlying C. elegans locomotion. METHODS: C. elegans were exposed to 8% isoflurane for 3 h during the neurodevelopmentally critical L1 larval stage. Locomotion was assessed during early and late adulthood. Spontaneous activity was measured within the locomotion command interneuron circuitry using confocal and light-sheet microscopy of the calcium-sensitive fluorophore GCaMP6s. RESULTS: C. elegans exposed to isoflurane demonstrated attenuation in spontaneous reversal behavior, persisting throughout the animal's lifespan (reversals/min: untreated early adulthood, 1.14 ± 0.42, vs. isoflurane-exposed early adulthood, 0.83 ± 0.55; untreated late adulthood, 1.75 ± 0.64, vs. isoflurane-exposed late adulthood, 1.14 ± 0.68; P = 0.001 and 0.006, respectively; n > 50 animal tracks/condition). Likewise, isoflurane exposure altered activity dynamics in the command interneuron AVA, which mediates crawling reversals. The rate at which AVA transitions between activity states was found to be increased. These anesthetic-induced effects were more pronounced with age (off-to-on activity state transition time (s): untreated early adulthood, 2.5 ± 1.2, vs. isoflurane-exposed early adulthood, 1.9 ± 1.3; untreated late adulthood, 4.6 ± 3.0, vs. isoflurane-exposed late adulthood, 3.0 ± 2.4; P = 0.028 and 0.008, respectively; n > 35 traces acquired from more than 15 animals/condition). Comparable effects were observed throughout the command interneuron circuitry, indicating that isoflurane exposure alters transition rates between behavioral crawling states of the system overall. These effects were modulated by loss-of-function mutations within the FoxO transcription factor daf-16 and by rapamycin-mediated mechanistic Target of Rapamycin (mTOR) inhibition. CONCLUSIONS: Altered locomotive behavior and activity dynamics indicate a persistent effect on interneuron dynamics and circuit function in C. elegansafter developmental exposure to isoflurane. These effects are modulated by a loss of daf-16 or mTOR activity, consistent with a pathologic activation of stress-response pathways.
Assuntos
Anestésicos Inalatórios/efeitos adversos , Comportamento Animal/efeitos dos fármacos , Isoflurano/efeitos adversos , Neurônios/efeitos dos fármacos , Animais , Caenorhabditis elegans , Modelos Animais de Doenças , Locomoção/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: A comprehensive understanding of how anesthetics facilitate a reversible collapse of system-wide neuronal function requires measurement of neuronal activity with single-cell resolution. Multineuron recording was performed in Caenorhabditis elegans to measure neuronal activity at varying depths of anesthesia. The authors hypothesized that anesthesia is characterized by dyssynchrony between neurons resulting in a collapse of organized system states. METHODS: Using light-sheet microscopy and transgenic expression of the calcium-sensitive fluorophore GCaMP6s, a majority of neurons (n = 120) in the C. elegans head were simultaneously imaged in vivo and neuronal activity was measured. Neural activity and system-wide dynamics were compared in 10 animals, progressively dosed at 0%, 4%, and 8% isoflurane. System-wide neuronal activity was analyzed using principal component analysis. RESULTS: Unanesthetized animals display distinct global neuronal states that are reflected in a high degree of correlation (R = 0.196 ± 0.070) between neurons and low-frequency, large-amplitude neuronal dynamics. At 4% isoflurane, the average correlation between neurons is significantly diminished (R = 0.026 ± 0.010; P < 0.0001 vs. unanesthetized) and neuron dynamics shift toward higher frequencies but with smaller dynamic range. At 8% isoflurane, interneuronal correlations indicate that neuronal activity remains uncoordinated (R = 0.053 ± 0.029; P < 0.0001 vs. unanesthetized) with high-frequency dynamics that are even further restricted. Principal component analysis of unanesthetized neuronal activity reveals distinct structure corresponding to known behavioral states. At 4% and 8% isoflurane this structure is lost and replaced with randomized dynamics, as quantified by the percentage of total ensemble variance captured by the first three principal components. In unanesthetized worms, this captured variance is high (88.9 ± 5.4%), reflecting a highly organized system, falling significantly at 4% and 8% isoflurane (57.9 ± 11.2%, P < 0.0001 vs. unanesthetized, and 76.0 ± 7.9%, P < 0.001 vs. unanesthetized, respectively) and corresponding to increased randomization and collapse of system-wide organization. CONCLUSIONS: Anesthesia with isoflurane in C. elegans corresponds to high-frequency randomization of individual neuron activity, loss of coordination between neurons, and a collapse of system-wide functional organization.
Assuntos
Anestesia por Inalação , Anestésicos Inalatórios/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Isoflurano/farmacologia , Rede Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Animais Geneticamente Modificados , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Corantes Fluorescentes , Interneurônios/efeitos dos fármacos , Rede Nervosa/diagnóstico por imagem , Análise de Componente Principal , Sevoflurano/farmacologiaRESUMO
During development, a neuron transitions from a state of rapid growth to a stable morphology, and neurons within the adult mammalian CNS lose their ability to effectively regenerate in response to injury. Here, we identify a novel form of neuronal regeneration, which is remarkably independent of DLK-1/DLK, KGB-1/JNK, and other MAPK signaling factors known to mediate regeneration in Caenorhabditis elegans, Drosophila, and mammals. This DLK-independent regeneration in C. elegans has direct genetic and molecular links to a well-studied form of endogenous activity-dependent ectopic axon outgrowth in the same neuron type. Both neuron outgrowth types are triggered by physical lesion of the sensory dendrite or mutations disrupting sensory activity, calcium signaling, or genes that restrict outgrowth during neuronal maturation, such as SAX-1/NDR kinase or UNC-43/CaMKII. These connections suggest that ectopic outgrowth represents a powerful platform for gene discovery in neuronal regeneration. Moreover, we note numerous similarities between C. elegans DLK-independent regeneration and lesion conditioning, a phenomenon producing robust regeneration in the mammalian CNS. Both regeneration types are triggered by lesion of a sensory neurite via reduction of neuronal activity and enhanced by disrupting L-type calcium channels or elevating cAMP. Taken as a whole, our study unites disparate forms of neuronal outgrowth to uncover fresh molecular insights into activity-dependent control of the adult nervous system's intrinsic regenerative capacity.
Assuntos
Caenorhabditis elegans/genética , Regeneração Nervosa , Animais , Axônios/metabolismo , Proteínas de Caenorhabditis elegans/genética , Canais de Cálcio Tipo LRESUMO
WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Previous work on the action of volatile anesthetics has focused at either the molecular level or bulk neuronal measurement such as electroencephalography or functional magnetic resonance imaging. There is a distinct gulf in resolution at the level of cellular signaling within neuronal systems. The authors hypothesize that anesthesia is caused by induced dyssynchrony in cellular signaling rather than suppression of individual neuron activity. METHODS: Employing confocal microscopy and Caenorhabditis elegans expressing the calcium-sensitive fluorophore GCaMP6s in specific command neurons, the authors measure neuronal activity noninvasively and in parallel within the behavioral circuit controlling forward and reverse crawling. The authors compare neuronal dynamics and coordination in a total of 31 animals under atmospheres of 0, 4, and 8% isoflurane. RESULTS: When not anesthetized, the interneurons controlling forward or reverse crawling occupy two possible states, with the activity of the "reversal" neurons AVA, AVD, AVE, and RIM strongly intercorrelated, and the "forward" neuron AVB anticorrelated. With exposure to 4% isoflurane and onset of physical quiescence, neuron activity wanders rapidly and erratically through indeterminate states. Neuron dynamics shift toward higher frequencies, and neuron pair correlations within the system are reduced. At 8% isoflurane, physical quiescence continues as neuronal signals show diminished amplitude with little correlation between neurons. Neuronal activity was further studied using statistical tools from information theory to quantify the type of disruption caused by isoflurane. Neuronal signals become noisier and more disordered, as measured by an increase in the randomness of their activity (Shannon entropy). The coordination of the system, measured by whether information exhibited in one neuron is also exhibited in other neurons (multiinformation), decreases significantly at 4% isoflurane (P = 0.00015) and 8% isoflurane (P = 0.0028). CONCLUSIONS: The onset of anesthesia corresponds with high-frequency randomization of individual neuron activity coupled with induced dyssynchrony and loss of coordination between neurons that disrupts functional signaling.
Assuntos
Anestésicos Inalatórios/farmacologia , Interneurônios/efeitos dos fármacos , Isoflurano/farmacologia , Rede Nervosa/efeitos dos fármacos , Imagem Óptica/métodos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Feminino , Interneurônios/química , Interneurônios/metabolismo , Masculino , Microscopia Confocal/métodos , Rede Nervosa/química , Rede Nervosa/metabolismo , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
Regulated calcium signals play conserved instructive roles in neuronal repair, but how localized calcium stores are differentially mobilized, or might be directly manipulated, to stimulate regeneration within native contexts is poorly understood. We find here that localized calcium release from the endoplasmic reticulum via ryanodine receptor (RyR) channels is critical in stimulating initial regeneration following traumatic cellular damage in vivo. Using laser axotomy of single neurons in Caenorhabditis elegans, we find that mutation of unc-68/RyR greatly impedes both outgrowth and guidance of the regenerating neuron. Performing extended in vivo calcium imaging, we measure subcellular calcium signals within the immediate vicinity of the regenerating axon end that are sustained for hours following axotomy and completely eliminated within unc-68/RyR mutants. Finally, using a novel optogenetic approach to periodically photo-stimulate the axotomized neuron, we can enhance its regeneration. The enhanced outgrowth depends on both amplitude and temporal pattern of excitation and can be blocked by disruption of UNC-68/RyR. This demonstrates the exciting potential of emerging optogenetic technology to beneficially manipulate cell physiology in the context of neuronal regeneration and indicates a link to the underlying cellular calcium signal. Taken as a whole, our findings define a specific localized calcium signal mediated by RyR channel activity that stimulates regenerative outgrowth, which may be dynamically manipulated for beneficial neurotherapeutic effects.
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Cálcio/metabolismo , Regeneração Nervosa/fisiologia , Neurônios/fisiologia , Optogenética/métodos , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Channelrhodopsins , Mecanotransdução Celular/fisiologia , Frações Subcelulares/fisiologiaRESUMO
A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. In C. elegans, individual visualized axons can be laser-cut in vivo and neuronal responses to damage can be monitored to decipher genetic requirements for regeneration. With an initial interest in how local environments manage cellular debris, we performed femtosecond laser axotomies in genetic backgrounds lacking cell death gene activities. Unexpectedly, we found that the CED-3 caspase, well known as the core apoptotic cell death executioner, acts in early responses to neuronal injury to promote rapid regeneration of dissociated axons. In ced-3 mutants, initial regenerative outgrowth dynamics are impaired and axon repair through reconnection of the two dissociated ends is delayed. The CED-3 activator, CED-4/Apaf-1, similarly promotes regeneration, but the upstream regulators of apoptosis CED-9/Bcl2 and BH3-domain proteins EGL-1 and CED-13 are not essential. Thus, a novel regulatory mechanism must be utilized to activate core apoptotic proteins for neuronal repair. Since calcium plays a conserved modulatory role in regeneration, we hypothesized calcium might play a critical regulatory role in the CED-3/CED-4 repair pathway. We used the calcium reporter cameleon to track in vivo calcium fluxes in the axotomized neuron. We show that when the endoplasmic reticulum calcium-storing chaperone calreticulin, CRT-1, is deleted, both calcium dynamics and initial regenerative outgrowth are impaired. Genetic data suggest that CED-3, CED-4, and CRT-1 act in the same pathway to promote early events in regeneration and that CED-3 might act downstream of CRT-1, but upstream of the conserved DLK-1 kinase implicated in regeneration across species. This study documents reconstructive roles for proteins known to orchestrate apoptotic death and links previously unconnected observations in the vertebrate literature to suggest a similar pathway may be conserved in higher organisms.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Caspases/metabolismo , Regeneração Nervosa , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Apoptose , Axônios/metabolismo , Axônios/patologia , Axônios/fisiologia , Axotomia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Calreticulina/metabolismo , Caspases/genética , Ativação Enzimática , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Imagem com Lapso de TempoRESUMO
Cellular metabolism plays an essential role in the regrowth and regeneration of a neuron following physical injury. Yet, our knowledge of the specific metabolic pathways that are beneficial to neuron regeneration remains sparse. Previously, we have shown that modulation of O-linked ß-N-acetylglucosamine (O-GlcNAc) signaling, a ubiquitous post-translational modification that acts as a cellular nutrient sensor, can significantly enhance in vivo neuron regeneration. Here, we define the specific metabolic pathway by which O-GlcNAc transferase (ogt-1) loss of function mediates increased regenerative outgrowth. Performing in vivo laser axotomy and measuring subsequent regeneration of individual neurons in C. elegans, we find that glycolysis, serine synthesis pathway (SSP), one-carbon metabolism (OCM), and the downstream transsulfuration metabolic pathway (TSP) are all essential in this process. The regenerative effects of ogt-1 mutation are abrogated by genetic and/or pharmacological disruption of OCM and the SSP linking OCM to glycolysis. Testing downstream branches of this pathway, we find that enhanced regeneration is dependent only on the vitamin B12 independent shunt pathway. These results are further supported by RNA sequencing that reveals dramatic transcriptional changes by the ogt-1 mutation, in the genes involved in glycolysis, OCM, TSP, and ATP metabolism. Strikingly, the beneficial effects of the ogt-1 mutation can be recapitulated by simple metabolic supplementation of the OCM metabolite methionine in wild-type animals. Taken together, these data unearth the metabolic pathways involved in the increased regenerative capacity of a damaged neuron in ogt-1 animals and highlight the therapeutic possibilities of OCM and its related pathways in the treatment of neuronal injury.
Assuntos
Caenorhabditis elegans , Transdução de Sinais , Animais , Caenorhabditis elegans/fisiologia , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional , Carbono/metabolismo , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Acetilglucosamina/metabolismoRESUMO
Ultrafast 3D imaging is indispensable for visualizing complex and dynamic biological processes. Conventional scanning-based techniques necessitate an inherent trade-off between acquisition speed and space-bandwidth product (SBP). Emerging single-shot 3D wide-field techniques offer a promising alternative but are bottlenecked by the synchronous readout constraints of conventional CMOS systems, thus restricting data throughput to maintain high SBP at limited frame rates. To address this, we introduce EventLFM, a straightforward and cost-effective system that overcomes these challenges by integrating an event camera with Fourier light field microscopy (LFM), a state-of-the-art single-shot 3D wide-field imaging technique. The event camera operates on a novel asynchronous readout architecture, thereby bypassing the frame rate limitations inherent to conventional CMOS systems. We further develop a simple and robust event-driven LFM reconstruction algorithm that can reliably reconstruct 3D dynamics from the unique spatiotemporal measurements captured by EventLFM. Experimental results demonstrate that EventLFM can robustly reconstruct fast-moving and rapidly blinking 3D fluorescent samples at kHz frame rates. Furthermore, we highlight EventLFM's capability for imaging of blinking neuronal signals in scattering mouse brain tissues and 3D tracking of GFP-labeled neurons in freely moving C. elegans. We believe that the combined ultrafast speed and large 3D SBP offered by EventLFM may open up new possibilities across many biomedical applications.
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In the aging brain, many of the alterations underlying cognitive and behavioral decline remain opaque. Caenorhabditis elegans offers a powerful model for aging research, with a simple, well-studied nervous system to further our understanding of the cellular modifications and functional alterations accompanying senescence. We perform multi-neuronal functional imaging across the aged C. elegans nervous system, measuring an age-associated breakdown in system-wide functional organization. At single-cell resolution, we detect shifts in activity dynamics toward higher frequencies. In addition, we measure a specific loss of inhibitory signaling that occurs early in the aging process and alters the systems' critical excitatory/inhibitory balance. These effects are recapitulated with mutation of the calcium channel subunit UNC-2/CaV2α. We find that manipulation of inhibitory GABA signaling can partially ameliorate or accelerate the effects of aging. The effects of aging are also partially mitigated by disruption of the insulin signaling pathway, known to increase longevity, or by a reduction of caspase activation. Data from mammals are consistent with our findings, suggesting a conserved shift in the balance of excitatory/inhibitory signaling with age that leads to breakdown in global neuronal dynamics and functional decline.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Longevidade , Mamíferos/metabolismo , Neurônios/fisiologiaRESUMO
BACKGROUND: Conventional light sheet fluorescence microscopy (LSFM), or selective plane illumination microscopy (SPIM), enables high-resolution 3D imaging over a large volume by using two orthogonally aligned objective lenses to decouple excitation and emission. The recent development of oblique plane microscopy (OPM) simplifies LSFM design with only one single objective lens, by using off-axis excitation and remote focusing. However, most reports on OPM have a limited microscopic field of view (FOV), typically within 1×1 mm2. Our goal is to overcome the limitation with a new variant of OPM to achieve a mesoscopic FOV. METHODS: We implemented an optical design of mesoscopic scanning OPM to allow the use of low numerical aperture (NA) objective lenses. The angle of the intermediate image before the remote focusing system was increased by a demagnification under Scheimpflug condition such that the light collecting efficiency in the remote focusing system was significantly improved. A telescope composed of cylindrical lenses was used to correct the distorted image caused by the demagnification design. We characterized the 3D resolutions and imaging volume by imaging fluorescent microspheres, and demonstrated the volumetric imaging on intact whole zebrafish larvae, mouse cortex, and multiple Caenorhabditis elegans (C. elegans). RESULTS: We demonstrate a mesoscopic FOV up to ~6×5×0.6 mm3 volumetric imaging, the largest reported FOV by OPM so far. The angle of the intermediate image plane is independent of the magnification as long as the size of the pupil aperture of the objectives is the same. As a result, the system is highly versatile, allowing simple switching between different objective lenses with low (10×, NA 0.3) and median NA (20×, NA 0.5). Detailed microvasculature in zebrafish larvae, mouse cortex, and neurons in C. elegans are clearly visualized in 3D. CONCLUSIONS: The proposed mesoscopic scanning OPM allows using low NA objectives such that centimeter-level FOV volumetric imaging can be achieved. With the extended FOV, simple sample mounting protocol, and the versatility of changeable FOVs/resolutions, our system will be ready for the varieties of applications requiring in vivo volumetric imaging over large length scales.
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A major challenge in regenerative medicine is the repair of injured neurons. Regeneration of laser-cut C. elegans neurons requires early action of core apoptosis activator CED-4/Apaf1 and CED-3/caspase. While testing models for CED-4 as a candidate calcium-sensitive activator of repair, we unexpectedly discovered that amino acid substitutions affecting alpha-helix-6 within the CED-4 caspase recruitment domain (CARD) confer a CED-4 gain-of-function (gf) activity that increases axonal regrowth without disrupting CED-4 apoptosis activity. The in vivo caspase reporter CA-GFP reveals a rapid localized increase in caspase activity upon axotomy, which is absent in ced-4 and ced-3 loss-of-function mutants but present in the ced-4(gf) mutant. The ced-3 loss-of-function mutation can significantly suppress the axonal regrowth of the ced-4(gf) mutant, indicating that CED-4(gf) regeneration depends on CED-3 caspase. Thus, we identified a subdomain within the CED-4 CARD that regulates the dynamic and controlled caspase activity required for efficient regeneration.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Regeneração Nervosa/fisiologia , Animais , Apoptose/genética , Axônios/metabolismo , Caenorhabditis elegans , Caspase 3/genética , Caspase 3/metabolismo , Domínio de Ativação e Recrutamento de Caspases , Caspases/metabolismo , Mutação com Ganho de Função , Neurônios/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Multichannel (multicolor) imaging has become a powerful technique in biology research for performing in vivo neuronal calcium imaging, colocalization of fluorescent labels, non-invasive pH measurement, and other procedures. We describe a novel add-on approach for simultaneous multichannel optical microscopy based on simple wedge prisms. Our device requires no alignment and is simple, robust, user-friendly, and less expensive than current commercial instruments based on switchable filters or dual-view strategies. Point spread function measurements and simulations in Zemax indicate a reduction in resolution in the direction orthogonal to the wedge interface and in the axial direction, without introducing aberration. These effects depend on the objective utilized and are most significant near the periphery of the field of view. We tested a two-channel device on C. elegans neurons in vivo and demonstrated comparable signals to a conventional dual-view instrument. We also tested a four-channel device on fixed chick embryo Brainbow samples and identified individual neurons by their spectra without extensive image postprocessing. Therefore, we believe that this technology has the potential for broad use in microscopy.
Assuntos
Caenorhabditis elegans/citologia , Microscopia/métodos , Neurônios/metabolismo , Imagem Óptica/métodos , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Embrião de Galinha , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Imagem Óptica/instrumentação , Proteína Vermelha FluorescenteRESUMO
Fast, volumetric imaging over large scales has been a long-standing challenge in biological microscopy. To address this challenge, we report an augmented variant of confocal microscopy that uses a series of reflecting pinholes axially distributed in the detection space, such that each pinhole probes a different depth within the sample. We thus obtain simultaneous multiplane imaging without the need for axial scanning. Our microscope technique is versatile and configured here to provide two-color fluorescence imaging with a field of view larger than a millimeter at video rate. Its general applicability is demonstrated with neuronal imaging of both Caenorhabditis elegans and mouse brains in vivo.
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Epithelial wound healing requires the coordination of cells to migrate as a unit over the basement membrane after injury. To understand the process of this coordinated movement, it is critical to study the dynamics of cell-cell communication. We developed a method to characterize the injury-induced sustained Ca2+ mobilizations that travel between cells for periods of time up to several hours. These events of communication are concentrated along the wound edge and are reduced in cells further away from the wound. Our goal was to delineate the role and contribution of these sustained mobilizations and using MATLAB analyses, we determined the probability of cell-cell communication events in both in vitro models and ex vivo organ culture models. We demonstrated that the injury response was complex and represented the activation of a number of receptors. In addition, we found that pannexin channels mediated the cell-cell communication and motility. Furthermore, the sustained Ca2+ mobilizations are associated with changes in cell morphology and motility during wound healing. The results demonstrate that both purinoreceptors and pannexins regulate the sustained Ca2+ mobilization necessary for cell-cell communication in wound healing.
Assuntos
Cálcio/metabolismo , Comunicação Celular/genética , Córnea/metabolismo , Cicatrização/genética , Membrana Basal/metabolismo , Membrana Basal/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/patologia , Movimento Celular/genética , Córnea/patologia , Córnea/ultraestrutura , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Microscopia Confocal , Técnicas de Cultura de Órgãos , Transdução de Sinais/genéticaRESUMO
Our understanding of the operation of neurons and neuronal circuits has come primarily from probing their activity in dissected, anesthetized, or restrained animals. However, the behaviorally relevant operation of neurons and neuronal circuits occurs within intact animals as they freely perform behavioral tasks. The small size and transparency of the nematode Caenorhabditis elegans make it an ideal system for noninvasive, optical measurements of neuronal activity. Here, we use a high signal-to-noise version of cameleon, a fluorescent calcium-binding protein, to quantify the activity of the AFD thermosensory neuron of individual worms freely navigating spatial thermal gradients. We find that AFD activity is directly coupled to the worm's exploratory movements in spatial thermal gradients. We show that the worm is able, in principle, to evaluate and guide its own thermotactic behaviors with respect to ambient spatial thermal gradients by monitoring the activity of this single thermosensory neuron.
Assuntos
Caenorhabditis elegans/fisiologia , Atividade Motora/fisiologia , Neurônios Aferentes/fisiologia , Sensação Térmica/fisiologia , Animais , Movimento/fisiologia , Rede Nervosa/fisiologia , Temperatura , Fatores de TempoRESUMO
The nematode Caenorhabditis elegans deliberately crawls toward the negative pole in an electric field. By quantifying the movements of individual worms navigating electric fields, we show that C. elegans prefers to crawl at specific angles to the direction of the electric field in persistent periods of forward movement and that the preferred angle is proportional to field strength. C. elegans reorients itself in response to time-varying electric fields by using sudden turns and reversals, standard reorientation maneuvers that C. elegans uses during other modes of motile behavior. Mutation or laser ablation that disrupts the structure and function of amphid sensory neurons also disrupts electrosensory behavior. By imaging intracellular calcium dynamics among the amphid sensory neurons of immobilized worms, we show that specific amphid sensory neurons are sensitive to the direction and strength of electric fields. We extend our analysis to the motor level by showing that specific interneurons affect the utilization of sudden turns and reversals during electrosensory steering. Thus, electrosensory behavior may be used as a model system for understanding how sensory inputs are transformed into motor outputs by the C. elegans nervous system.
Assuntos
Comportamento Animal/fisiologia , Caenorhabditis elegans/fisiologia , Estimulação Elétrica , Atividade Motora/fisiologia , Neurônios Aferentes/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Estimulação Elétrica/métodos , Vias Neurais/fisiologia , Fatores de TempoRESUMO
Regrowth of an axon after injury is an inherently metabolic undertaking. Yet the mechanisms of metabolic regulation that influence repair following injury are not well understood. O-linked ß-N-acetylglucosamine (O-GlcNAc) is a post-translational modification of serines and threonines that functions as a sensor of cellular nutrients. Performing in vivo laser axotomies in Caenorhabditis elegans, we find that neuronal regeneration is substantially increased by disruptions of either the O-GlcNAc transferase or the O-GlcNAcase that decrease and increase O-GlcNAc levels, respectively. A lack of O-GlcNAc induces the AKT-1 branch in the insulin-signaling pathway to use glycolysis. In contrast, increased O-GlcNAc levels activate an opposing branch of the insulin-signaling pathway whereby SGK-1 modulates the FOXO transcription factor DAF-16 to influence mitochondrial function. The existence of this toggle-like mechanism between metabolic pathways suggests that O-GlcNAc signaling conveys cellular nutrient status to orchestrate metabolism in a damaged neuron and maximize the regenerative response.