RESUMO
Natural goal-directed behaviors often involve complex sequences of many stimulus-triggered components. Understanding how brain circuits organize such behaviors requires mapping the interactions between an animal, its environment, and its nervous system. Here, we use brain-wide neuronal imaging to study the full performance of mating by the C. elegans male. We show that as mating unfolds in a sequence of component behaviors, the brain operates similarly between instances of each component but distinctly between different components. When the full sensory and behavioral context is taken into account, unique roles emerge for each neuron. Functional correlations between neurons are not fixed but change with behavioral dynamics. From individual neurons to circuits, our study shows how diverse brain-wide dynamics emerge from the integration of sensory perception and motor actions in their natural context.
Assuntos
Encéfalo/fisiologia , Caenorhabditis elegans/fisiologia , Sensação/fisiologia , Comportamento Sexual Animal/fisiologia , Animais , Mapeamento Encefálico , Copulação/fisiologia , Corte , Bases de Dados como Assunto , Retroalimentação , Feminino , Masculino , Modelos Biológicos , Movimento , Neurônios/fisiologia , Descanso , Processamento de Sinais Assistido por Computador , Sinapses/fisiologia , Vulva/fisiologiaRESUMO
Comprehensively resolving neuronal identities in whole-brain images is a major challenge. We achieve this in C. elegans by engineering a multicolor transgene called NeuroPAL (a neuronal polychromatic atlas of landmarks). NeuroPAL worms share a stereotypical multicolor fluorescence map for the entire hermaphrodite nervous system that resolves all neuronal identities. Neurons labeled with NeuroPAL do not exhibit fluorescence in the green, cyan, or yellow emission channels, allowing the transgene to be used with numerous reporters of gene expression or neuronal dynamics. We showcase three applications that leverage NeuroPAL for nervous-system-wide neuronal identification. First, we determine the brainwide expression patterns of all metabotropic receptors for acetylcholine, GABA, and glutamate, completing a map of this communication network. Second, we uncover changes in cell fate caused by transcription factor mutations. Third, we record brainwide activity in response to attractive and repulsive chemosensory cues, characterizing multimodal coding for these stimuli.
Assuntos
Atlas como Assunto , Mapeamento Encefálico , Encéfalo/fisiologia , Caenorhabditis elegans/fisiologia , Neurônios/fisiologia , Software , Algoritmos , Pontos de Referência Anatômicos , Animais , Corpo Celular/fisiologia , Linhagem da Célula , Drosophila/fisiologia , Mutação/genética , Rede Nervosa/fisiologia , Fenótipo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Neurotransmissores/metabolismo , Olfato/fisiologia , Paladar/fisiologia , Fatores de Transcrição/metabolismo , TransgenesRESUMO
Reading out neuronal activity from three-dimensional (3D) functional imaging requires segmenting and tracking individual neurons. This is challenging in behaving animals if the brain moves and deforms. The traditional approach is to train a convolutional neural network with ground-truth (GT) annotations of images representing different brain postures. For 3D images, this is very labor intensive. We introduce 'targeted augmentation', a method to automatically synthesize artificial annotations from a few manual annotations. Our method ('Targettrack') learns the internal deformations of the brain to synthesize annotations for new postures by deforming GT annotations. This reduces the need for manual annotation and proofreading. A graphical user interface allows the application of the method end-to-end. We demonstrate Targettrack on recordings where neurons are labeled as key points or 3D volumes. Analyzing freely moving animals exposed to odor pulses, we uncover rich patterns in interneuron dynamics, including switching neuronal entrainment on and off.
Assuntos
Aprendizado Profundo , Animais , Caenorhabditis elegans/fisiologia , Imageamento Tridimensional/métodos , Redes Neurais de Computação , Neurônios/fisiologia , Processamento de Imagem Assistida por Computador/métodosRESUMO
An animal's nervous system changes as its body grows from birth to adulthood and its behaviours mature1-8. The form and extent of circuit remodelling across the connectome is unknown3,9-15. Here we used serial-section electron microscopy to reconstruct the full brain of eight isogenic Caenorhabditis elegans individuals across postnatal stages to investigate how it changes with age. The overall geometry of the brain is preserved from birth to adulthood, but substantial changes in chemical synaptic connectivity emerge on this consistent scaffold. Comparing connectomes between individuals reveals substantial differences in connectivity that make each brain partly unique. Comparing connectomes across maturation reveals consistent wiring changes between different neurons. These changes alter the strength of existing connections and create new connections. Collective changes in the network alter information processing. During development, the central decision-making circuitry is maintained, whereas sensory and motor pathways substantially remodel. With age, the brain becomes progressively more feedforward and discernibly modular. Thus developmental connectomics reveals principles that underlie brain maturation.
Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Caenorhabditis elegans/citologia , Conectoma , Modelos Neurológicos , Vias Neurais , Sinapses/fisiologia , Envelhecimento/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/ultraestrutura , Caenorhabditis elegans/anatomia & histologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/ultraestrutura , Individualidade , Interneurônios/citologia , Microscopia Eletrônica , Neurônios/citologia , Comportamento EstereotipadoRESUMO
Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Ca2+ levels, and increases in cytosolic Ca2+ resemble the Piezo overexpression phenotype, suggesting that Piezo functions through Ca2+ signalling. Further studies suggest that Ca2+ signalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Ca2+ in response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo.
Assuntos
Diferenciação Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Canais Iônicos/metabolismo , Células-Tronco/citologia , Estresse Mecânico , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Linhagem da Célula , Proliferação de Células , Citosol/metabolismo , Sistema Digestório/citologia , Sistema Digestório/metabolismo , Proteínas de Drosophila/genética , Células Enteroendócrinas/citologia , Células Enteroendócrinas/metabolismo , Feminino , Canais Iônicos/genética , MutaçãoRESUMO
We propose a simple, cost-effective method for synchronized phase contrast and fluorescence video acquisition in live samples. Counter-phased pulses of phase contrast illumination and fluorescence excitation light are synchronized with the exposure of the two fields of an interlaced camera sensor. This results in a video sequence in which each frame contains both exposure modes, each in half of its pixels. The method allows real-time acquisition and display of synchronized and spatially aligned phase contrast and fluorescence image sequences that can be separated by de-interlacing in two independent videos. The method can be implemented on any fluorescence microscope with a camera port without needing to modify the optical path.
RESUMO
Understanding the coordination of cell-division timing is one of the outstanding questions in the field of developmental biology. One active control parameter of the cell-cycle duration is temperature, as it can accelerate or decelerate the rate of biochemical reactions. However, controlled experiments at the cellular scale are challenging, due to the limited availability of biocompatible temperature sensors, as well as the lack of practical methods to systematically control local temperatures and cellular dynamics. Here, we demonstrate a method to probe and control the cell-division timing in Caenorhabditis elegans embryos using a combination of local laser heating and nanoscale thermometry. Local infrared laser illumination produces a temperature gradient across the embryo, which is precisely measured by in vivo nanoscale thermometry using quantum defects in nanodiamonds. These techniques enable selective, controlled acceleration of the cell divisions, even enabling an inversion of division order at the two-cell stage. Our data suggest that the cell-cycle timing asynchrony of the early embryonic development in C. elegans is determined independently by individual cells rather than via cell-to-cell communication. Our method can be used to control the development of multicellular organisms and to provide insights into the regulation of cell-division timings as a consequence of local perturbations.
Assuntos
Temperatura Corporal/fisiologia , Divisão Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Pontos Quânticos/química , Termometria , Animais , Caenorhabditis elegans/embriologia , Nanodiamantes/química , Termometria/instrumentação , Termometria/métodosRESUMO
The chemotrophic factor Netrin can simultaneously instruct different neurodevelopmental programs in individual neurons in vivo. How neurons correctly interpret the Netrin signal and undergo the appropriate neurodevelopmental response is not understood. Here we identify MIG-10 isoforms as critical determinants of individual cellular responses to Netrin. We determined that distinct MIG-10 isoforms, varying only in their N-terminal motifs, can localize to specific subcellular domains and are differentially required for discrete neurodevelopmental processes in vivo. We identified MIG-10B as an isoform uniquely capable of localizing to presynaptic regions and instructing synaptic vesicle clustering in response to Netrin. MIG-10B interacts with Abl-interacting protein-1 (ABI-1)/Abi1, a component of the WAVE complex, to organize the actin cytoskeleton at presynaptic sites and instruct vesicle clustering through SNN-1/Synapsin. We identified a motif in the MIG-10B N-terminal domain that is required for its function and localization to presynaptic sites. With this motif, we engineered a dominant-negative MIG-10B construct that disrupts vesicle clustering and animal thermotaxis behavior when expressed in a single neuron in vivo. Our findings indicate that the unique N-terminal domains confer distinct MIG-10 isoforms with unique capabilities to localize to distinct subcellular compartments, organize the actin cytoskeleton at these sites, and instruct distinct Netrin-dependent neurodevelopmental programs.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Tecido Nervoso/genética , Vesículas Sinápticas/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Comportamento Animal/fisiologia , Caenorhabditis elegans/metabolismo , Movimento Celular , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica , Interneurônios/citologia , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/metabolismo , Netrinas , Isoformas de Proteínas , Transporte Proteico/genética , Vesículas Sinápticas/genéticaRESUMO
Animals find mates and food, and avoid predators, by navigating to regions within a favorable range of available sensory cues. How are these ranges set and recognized? Here we show that male Caenorhabditis elegans exhibit strong concentration preferences for sex-specific small molecule cues secreted by hermaphrodites, and that these preferences emerge from the collective dynamics of a single male-specific class of neurons, the cephalic sensory neurons (CEMs). Within a single worm, CEM responses are dissimilar, not determined by anatomical classification and can be excitatory or inhibitory. Response kinetics vary by concentration, suggesting a mechanism for establishing preferences. CEM responses are enhanced in the absence of synaptic transmission, and worms with only one intact CEM show nonpreferential attraction to all concentrations of ascaroside for which CEM is the primary sensor, suggesting that synaptic modulation of CEM responses is necessary for establishing preferences. A heterogeneous concentration-dependent sensory representation thus appears to allow a single neural class to set behavioral preferences and recognize ranges of sensory cues.
Assuntos
Caenorhabditis elegans/fisiologia , Organismos Hermafroditas/fisiologia , Células Receptoras Sensoriais/fisiologia , Atrativos Sexuais/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Sinais (Psicologia) , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Fenômenos Eletrofisiológicos/fisiologia , Feminino , Glicolipídeos/química , Glicolipídeos/farmacologia , Organismos Hermafroditas/citologia , Organismos Hermafroditas/metabolismo , Masculino , Preferência de Acasalamento Animal/fisiologia , Estrutura Molecular , Transmissão Sináptica/fisiologiaRESUMO
We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of â¼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.
Assuntos
Caenorhabditis elegans , Núcleo Celular/metabolismo , Gânglios dos Invertebrados , Locomoção , Neurônios , Imagem Óptica/métodos , Animais , Comportamento Animal , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteína Vermelha FluorescenteRESUMO
Complex animal behaviors are built from dynamical relationships between sensory inputs, neuronal activity, and motor outputs in patterns with strategic value. Connecting these patterns illuminates how nervous systems compute behavior. Here, we study Drosophila larva navigation up temperature gradients toward preferred temperatures (positive thermotaxis). By tracking the movements of animals responding to fixed spatial temperature gradients or random temperature fluctuations, we calculate the sensitivity and dynamics of the conversion of thermosensory inputs into motor responses. We discover three thermosensory neurons in each dorsal organ ganglion (DOG) that are required for positive thermotaxis. Random optogenetic stimulation of the DOG thermosensory neurons evokes behavioral patterns that mimic the response to temperature variations. In vivo calcium and voltage imaging reveals that the DOG thermosensory neurons exhibit activity patterns with sensitivity and dynamics matched to the behavioral response. Temporal processing of temperature variations carried out by the DOG thermosensory neurons emerges in distinct motor responses during thermotaxis.
Assuntos
Comportamento Animal/fisiologia , Drosophila melanogaster/fisiologia , Termorreceptores/fisiologia , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio , Gânglios/fisiologia , Larva/fisiologia , Locomoção/fisiologia , Optogenética , Sensação Térmica/fisiologiaRESUMO
Like other ectotherms, the roundworm Caenorhabditis elegans and the fruit fly Drosophila melanogaster rely on behavioral strategies to stabilize their body temperature. Both animals use specialized sensory neurons to detect small changes in temperature, and the activity of these thermosensors governs the neural circuits that control migration and accumulation at preferred temperatures. Despite these similarities, the underlying molecular, neuronal, and computational mechanisms responsible for thermotaxis are distinct in these organisms. Here, we discuss the role of thermosensation in the development and survival of C. elegans and Drosophila, and review the behavioral strategies, neuronal circuits, and molecular networks responsible for thermotaxis behavior.
Assuntos
Comportamento Animal/fisiologia , Caenorhabditis elegans/fisiologia , Drosophila melanogaster/fisiologia , Células Receptoras Sensoriais/fisiologia , Sensação Térmica/fisiologia , Animais , Caenorhabditis elegans/genética , Temperatura Baixa , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Temperatura Alta , Sensação Térmica/genéticaRESUMO
The nematode Caenorhabditis elegans navigates toward a preferred temperature setpoint (Ts) determined by long-term temperature exposure. During thermotaxis, the worm migrates down temperature gradients at temperatures above Ts (negative thermotaxis) and performs isothermal tracking near Ts. Under some conditions, the worm migrates up temperature gradients below Ts (positive thermotaxis). Here, we analyze positive and negative thermotaxis toward Ts to study the role of specific neurons that have been proposed to be involved in thermotaxis using genetic ablation, behavioral tracking, and calcium imaging. We find differences in the strategies for positive and negative thermotaxis. Negative thermotaxis is achieved through biasing the frequency of reorientation maneuvers (turns and reversal turns) and biasing the direction of reorientation maneuvers toward colder temperatures. Positive thermotaxis, in contrast, biases only the direction of reorientation maneuvers toward warmer temperatures. We find that the AFD thermosensory neuron drives both positive and negative thermotaxis. The AIY interneuron, which is postsynaptic to AFD, may mediate the switch from negative to positive thermotaxis below Ts. We propose that multiple thermotactic behaviors, each defined by a distinct set of sensorimotor transformations, emanate from the AFD thermosensory neurons. AFD learns and stores the memory of preferred temperatures, detects temperature gradients, and drives the appropriate thermotactic behavior in each temperature regime by the flexible use of downstream circuits.
Assuntos
Caenorhabditis elegans/fisiologia , Memória de Longo Prazo/fisiologia , Modelos Neurológicos , Movimento/fisiologia , Neurônios/fisiologia , Sensação Térmica/fisiologia , Animais , TemperaturaRESUMO
Monoamines provide chemical codes of behavioral states. However, the neural mechanisms of monoaminergic orchestration of behavior are poorly understood. Touch elicits an escape response in Caenorhabditis elegans where the animal moves backward and turns to change its direction of locomotion. We show that the tyramine receptor SER-2 acts through a Gαo pathway to inhibit neurotransmitter release from GABAergic motor neurons that synapse onto ventral body wall muscles. Extrasynaptic activation of SER-2 facilitates ventral body wall muscle contraction, contributing to the tight ventral turn that allows the animal to navigate away from a threatening stimulus. Tyramine temporally coordinates the different phases of the escape response through the synaptic activation of the fast-acting ionotropic receptor, LGC-55, and extrasynaptic activation of the slow-acting metabotropic receptor, SER-2. Our studies show, at the level of single cells, how a sensory input recruits the action of a monoamine to change neural circuit properties and orchestrate a compound motor sequence.
Assuntos
Caenorhabditis elegans/fisiologia , Reação de Fuga/fisiologia , Neurotransmissores/fisiologia , Tiramina/fisiologia , Aldicarb/farmacologia , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/metabolismo , Inibidores da Colinesterase/farmacologia , Neurônios GABAérgicos/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Neurônios Motores/metabolismo , Contração Muscular , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/fisiologia , Neurotransmissores/farmacologia , Receptores de Amina Biogênica/genética , Receptores de Amina Biogênica/metabolismo , Deleção de Sequência , Transmissão Sináptica , Tiramina/farmacologiaRESUMO
The ability of an animal to detect, discriminate, and respond to odors depends on the function of its olfactory receptor neurons (ORNs), which in turn depends ultimately on odorant receptors. To understand the diverse mechanisms used by an animal in olfactory coding and computation, it is essential to understand the functional diversity of its odor receptors. The larval olfactory system of Drosophila melanogaster contains 21 ORNs and a comparable number of odorant receptors whose properties have been examined in only a limited way. We systematically screened them with a panel of â¼500 odorants, yielding >10,000 receptor-odorant combinations. We identify for each of 19 receptors an odorant that excites it strongly. The responses elicited by each of these odorants are analyzed in detail. The odorants elicited little cross-activation of other receptors at the test concentration; thus, low concentrations of many of these odorants in nature may be signaled by a single ORN. The receptors differed dramatically in sensitivity to their cognate odorants. The responses showed diverse temporal dynamics, with some odorants eliciting supersustained responses. An intriguing question in the field concerns the roles of different ORNs and receptors in driving behavior. We found that the cognate odorants elicited behavioral responses that varied across a broad range. Some odorants elicited strong physiological responses but weak behavioral responses or weak physiological responses but strong behavioral responses.
Assuntos
Drosophila melanogaster/genética , Movimento/fisiologia , Odorantes/análise , Condutos Olfatórios/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Compostos Orgânicos/metabolismo , Receptores Odorantes/metabolismo , Potenciais de Ação/fisiologia , Animais , Drosophila melanogaster/citologia , Cromatografia Gasosa-Espectrometria de Massas , Larva/citologiaRESUMO
The avoidance of light by fly larvae is a classic paradigm for sensorimotor behavior. Here, we use behavioral assays and video microscopy to quantify the sensorimotor structure of phototaxis using the Drosophila larva. Larval locomotion is composed of sequences of runs (periods of forward movement) that are interrupted by abrupt turns, during which the larva pauses and sweeps its head back and forth, probing local light information to determine the direction of the successive run. All phototactic responses are mediated by the same set of sensorimotor transformations that require temporal processing of sensory inputs. Through functional imaging and genetic inactivation of specific neurons downstream of the sensory periphery, we have begun to map these sensorimotor circuits into the larval central brain. We find that specific sensorimotor pathways that govern distinct light-evoked responses begin to segregate at the first relay after the photosensory neurons.
Assuntos
Algoritmos , Drosophila/fisiologia , Luz , Modelos Biológicos , Movimento/fisiologia , Vias Neurais/fisiologia , Animais , Larva/fisiologia , Microscopia Confocal , Microscopia de Fluorescência , Movimento/efeitos da radiaçãoRESUMO
Small animals such as nematodes and insects analyze airborne chemical cues to infer the direction of favorable and noxious locations. In these animals, the study of navigational behavior evoked by airborne cues has been limited by the difficulty of precisely controlling stimuli. We present a system that can be used to deliver gaseous stimuli in defined spatial and temporal patterns to freely moving small animals. We used this apparatus, in combination with machine-vision algorithms, to assess and quantify navigational decision making of Drosophila melanogaster larvae in response to ethyl acetate (a volatile attractant) and carbon dioxide (a gaseous repellant).
Assuntos
Fatores Quimiotáticos/administração & dosagem , Sinais (Psicologia) , Drosophila melanogaster/fisiologia , Nebulizadores e Vaporizadores/veterinária , Comportamento Espacial/fisiologia , Animais , Drosophila melanogaster/efeitos dos fármacos , Desenho de Equipamento , Análise de Falha de Equipamento , Comportamento Espacial/efeitos dos fármacos , Estimulação QuímicaRESUMO
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
We present an optogenetic illumination system capable of real-time light delivery with high spatial resolution to specified targets in freely moving Caenorhabditis elegans. A tracking microscope records the motion of an unrestrained worm expressing channelrhodopsin-2 or halorhodopsin in specific cell types. Image processing software analyzes the worm's position in each video frame, rapidly estimates the locations of targeted cells and instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Because each cell in an unrestrained worm is a rapidly moving target, our system operates at high speed (â¼50 frames per second) to provide high spatial resolution (â¼30 µm). To test the accuracy, flexibility and utility of our system, we performed optogenetic analyses of the worm motor circuit, egg-laying circuit and mechanosensory circuits that have not been possible with previous methods.