RESUMEN
Fluorescent calcium sensors are widely used to image neural activity. Using structure-based mutagenesis and neuron-based screening, we developed a family of ultrasensitive protein calcium sensors (GCaMP6) that outperformed other sensors in cultured neurons and in zebrafish, flies and mice in vivo. In layer 2/3 pyramidal neurons of the mouse visual cortex, GCaMP6 reliably detected single action potentials in neuronal somata and orientation-tuned synaptic calcium transients in individual dendritic spines. The orientation tuning of structurally persistent spines was largely stable over timescales of weeks. Orientation tuning averaged across spine populations predicted the tuning of their parent cell. Although the somata of GABAergic neurons showed little orientation tuning, their dendrites included highly tuned dendritic segments (5-40-µm long). GCaMP6 sensors thus provide new windows into the organization and dynamics of neural circuits over multiple spatial and temporal scales.
Asunto(s)
Potenciales de Acción , Proteínas de Unión al Calcio/química , Colorantes Fluorescentes/química , Proteínas Luminiscentes/química , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Células Cultivadas , Espinas Dendríticas/metabolismo , Neuronas GABAérgicas/metabolismo , Proteínas Luminiscentes/genética , Ratones , Imagen Molecular , Mutagénesis , Ingeniería de Proteínas , Células Piramidales/metabolismo , Células Piramidales/fisiología , Corteza Visual/citología , Corteza Visual/fisiologíaRESUMEN
In Caenorhabditis elegans, satiety quiescence mimics behavioral aspects of satiety and postprandial sleep in mammals. On the basis of calcium-imaging, genetics, and behavioral studies, here we report that a pair of amphid neurons, ASI, is activated by nutrition and regulates worms' behavioral states specifically promoting satiety quiescence; ASI inhibits the switch from quiescence to dwelling (a browsing state) and accelerates the switch from dwelling to quiescence. The canonical TGFß pathway, whose ligand is released from ASI, regulates satiety quiescence. The mutants of a ligand, a receptor and SMADs in the TGFß pathway all eat more and show less quiescence than wild-type. The TGFß receptor in downstream neurons RIM and RIC is sufficient for worms to exhibit satiety quiescence, suggesting neuronal connection from ASI to RIM and RIC is essential for feeding regulation through the TGFß pathway. ASI also regulates satiety quiescence partly through cGMP signaling; restoring cGMP signaling in ASI rescues the satiety quiescence defect of cGMP signaling mutants. From these results, we propose that TGFß and cGMP pathways in ASI connect nutritional status to promotion of satiety quiescence, a sleep-like behavioral state.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Ingestión de Alimentos/fisiología , Proteínas Quinasas/fisiología , Saciedad/fisiología , Células Receptoras Sensoriales/fisiología , Animales , Animales Modificados Genéticamente , GMP Cíclico/fisiología , Actividad Motora/fisiología , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
We designed a real-time computer vision system, the Multi-Worm Tracker (MWT), which can simultaneously quantify the behavior of dozens of Caenorhabditis elegans on a Petri plate at video rates. We examined three traditional behavioral paradigms using this system: spontaneous movement on food, where the behavior changes over tens of minutes; chemotaxis, where turning events must be detected accurately to determine strategy; and habituation of response to tap, where the response is stochastic and changes over time. In each case, manual analysis or automated single-worm tracking would be tedious and time-consuming, but the MWT system allowed rapid quantification of behavior with minimal human effort. Thus, this system will enable large-scale forward and reverse genetic screens for complex behaviors.
Asunto(s)
Conducta Animal/fisiología , Caenorhabditis elegans/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Animales , Caenorhabditis elegans/genética , Quimiotaxis , Movimiento , Procesos Estocásticos , Factores de TiempoRESUMEN
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by severalfold, creating a family of "GCaMP5" sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2- to 3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
Asunto(s)
Señalización del Calcio , Colorantes Fluorescentes/química , Fluorometría/métodos , Proteínas Fluorescentes Verdes/química , Neuroimagen/métodos , Neuronas/química , Péptidos/química , Transmisión Sináptica , Animales , Astrocitos/química , Astrocitos/ultraestructura , Caenorhabditis elegans , Cristalografía por Rayos X , Drosophila melanogaster/crecimiento & desarrollo , Femenino , Colorantes Fluorescentes/análisis , Genes Sintéticos , Vectores Genéticos , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/aislamiento & purificación , Células HEK293/química , Células HEK293/ultraestructura , Hipocampo/química , Hipocampo/citología , Humanos , Larva , Rayos Láser , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Neuromuscular/química , Unión Neuromuscular/ultraestructura , Neuronas/fisiología , Neuronas/ultraestructura , Neurópilo/química , Neurópilo/fisiología , Neurópilo/ultraestructura , Neuronas Receptoras Olfatorias/química , Neuronas Receptoras Olfatorias/fisiología , Neuronas Receptoras Olfatorias/ultraestructura , Péptidos/análisis , Péptidos/genética , Estimulación Luminosa , Conformación Proteica , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Células Bipolares de la Retina/química , Células Bipolares de la Retina/fisiología , Células Bipolares de la Retina/ultraestructura , Pez Cebra/crecimiento & desarrolloRESUMEN
Aging is characterized by a global decline in physiological function. However, by constructing a complete single-cell gene expression atlas, we find that Caenorhabditis elegans aging is not random in nature but instead is characterized by coordinated changes in functionally related metabolic, proteostasis, and stress-response genes in a cell-type-specific fashion, with downregulation of energy metabolism being the only nearly universal change. Similarly, the rates at which cells age differ significantly between cell types. In some cell types, aging is characterized by an increase in cell-to-cell variance, whereas in others, variance actually decreases. Remarkably, multiple resilience-enhancing transcription factors known to extend lifespan are activated across many cell types with age; we discovered new longevity candidates, such as GEI-3, among these. Together, our findings suggest that cells do not age passively but instead react strongly, and individualistically, to events that occur during aging. This atlas can be queried through a public interface.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Regulación del Desarrollo de la Expresión Génica , Factores de Transcripción , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Envejecimiento , Senescencia Celular , Metabolismo Energético , Longevidad , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Homeostasis , Análisis de Expresión Génica de una Sola Célula , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fenómenos Fisiológicos CelularesRESUMEN
Organisms undergo a variety of characteristic changes as they age, suggesting a substantial commonality in the mechanistic basis of aging. Experiments in model organisms have revealed a variety of cellular systems that impact lifespan, but technical challenges have prevented a comprehensive evaluation of how these components impact the trajectory of aging, and many components likely remain undiscovered. To facilitate the deeper exploration of aging trajectories at a sufficient scale to enable primary screening, we have created the Caenorhabditis elegans Observatory, an automated system for monitoring the behavior of group-housed C. elegans throughout their lifespans. One Observatory consists of a set of computers running custom software to control an incubator containing custom imaging and motion-control hardware. In its standard configuration, the Observatory cycles through trays of standard 6 cm plates, running four assays per day on up to 576 plates per incubator. High-speed image processing captures a range of behavioral metrics, including movement speed and stimulus-induced turning, and a data processing pipeline continuously computes summary statistics. The Observatory software includes a web interface that allows the user to input metadata and view graphs of the trajectory of behavioral aging as the experiment unfolds. Compared to the manual use of a plate-based C. elegans tracker, the Observatory reduces the effort required by close to two orders of magnitude. Within the Observatory, reducing the function of known lifespan genes with RNA interference (RNAi) gives the expected phenotypic changes, including extended motility in daf-2(RNAi) and progeria in hsf-1(RNAi). Lifespans scored manually from worms raised in conventional conditions match those scored from images captured by the Observatory. We have used the Observatory for a small candidate-gene screen and identified an extended youthful vigor phenotype for tank-1(RNAi) and a progeric phenotype for cdc-42(RNAi). By utilizing the Observatory, it is now feasible to conduct whole-genome screens for an aging-trajectory phenotype, thus greatly increasing our ability to discover and analyze new components of the aging program.
RESUMEN
BACKGROUND: Egg laying in Caenorhabditis elegans has been well studied at the genetic and behavioral levels. However, the neural basis of egg-laying behavior is still not well understood; in particular, the roles of specific neurons and the functional nature of the synaptic connections in the egg-laying circuit remain uncharacterized. RESULTS: We have used in vivo neuroimaging and laser surgery to address these questions in intact, behaving animals. We have found that the HSN neurons play a central role in driving egg-laying behavior through direct excitation of the vulval muscles and VC motor neurons. The VC neurons play a dual role in the egg-laying circuit, exciting the vulval muscles while feedback-inhibiting the HSNs. Interestingly, the HSNs are active in the absence of synaptic input, suggesting that egg laying may be controlled through modulation of autonomous HSN activity. Indeed, body touch appears to inhibit egg laying, in part by interfering with HSN calcium oscillations. CONCLUSIONS: The egg-laying motor circuit comprises a simple three-component system combining feed-forward excitation and feedback inhibition. This microcircuit motif is common in the C. elegans nervous system, as well as in the mammalian cortex; thus, understanding its functional properties in C. elegans may provide insight into its computational role in more complex brains.
Asunto(s)
Conducta Animal/fisiología , Caenorhabditis elegans/fisiología , Neuronas Motoras/fisiología , Oviparidad/fisiología , Acetilcolina/metabolismo , Animales , Homeostasis , Neuropéptidos/metabolismo , Concentración Osmolar , Serotonina/metabolismo , Sinapsis/metabolismo , Factores de Tiempo , TactoRESUMEN
In the nematode C. elegans, genes encoding components of a putative mechanotransducing channel complex have been identified in screens for light-touch-insensitive mutants. A long-standing question, however, is whether identified MEC proteins act directly in touch transduction or contribute indirectly by maintaining basic mechanoreceptor neuron physiology. In this study, we used the genetically encoded calcium indicator cameleon to record cellular responses of mechanosensory neurons to touch stimuli in intact, behaving nematodes. We defined a gentle touch sensory modality that adapts with a time course of approximately 500 ms and primarily senses motion rather than pressure. The DEG/ENaC channel subunit MEC-4 and channel-associated stomatin MEC-2 are specifically required for neural responses to gentle mechanical stimulation, but do not affect the basic physiology of touch neurons or their in vivo responses to harsh mechanical stimulation. These results distinguish a specific role for the MEC channel proteins in the process of gentle touch mechanosensation.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/fisiología , Tacto/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Canales de Calcio Tipo L/fisiología , Células Cultivadas , Mecanotransducción Celular/genética , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/genética , Neuronas/fisiología , Tacto/genéticaRESUMEN
From nematodes to humans, animals employ neuromodulators like serotonin to regulate behavioral patterns and states. In the nematode C. elegans, serotonin has been shown to act in a modulatory fashion to increase the rate and alter the temporal pattern of egg laying. Though many candidate effectors and regulators of serotonin have been identified in genetic studies, their effects on specific neurons and muscles in the egg-laying circuitry have been difficult to determine. Using the genetically encoded Ca(2+) indicator cameleon, we found that serotonin acts directly on the vulval muscles to increase the frequency of Ca(2+) transients. In contrast, we found that the spontaneous activity of the egg-laying motorneurons was silenced by serotonin. Mutations in G protein alpha subunit genes altered the responses of both vulval muscles and egg-laying neurons to serotonin; specifically, mutations in the G(q)alpha homolog egl-30 blocked serotonin stimulation of vulval muscle Ca(2+) transients, while mutations in the G(o)alpha homolog goa-1 prevented the silencing of motorneuron activity by serotonin. These data indicate that serotonin stimulates egg laying by directly modulating the functional state of the vulval muscles. In addition, serotonin inhibits the activity of the motorneurons that release it, providing a feedback regulatory effect that may contribute to serotonin adaptation.
Asunto(s)
Caenorhabditis elegans/fisiología , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Músculo Esquelético/inervación , Oviposición/fisiología , Serotonina/metabolismo , Vulva/inervación , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Unión al Calcio/metabolismo , Electromiografía , Femenino , Músculo Esquelético/fisiología , Vulva/fisiologíaRESUMEN
Cells often measure their local environment via the interaction of diffusible chemical signals with cell surface receptors. At the level of a single receptor, this process is inherently stochastic, but cells can contain many such receptors which can reduce the variability in the detected signal by suitable averaging. Here, we use explicit Monte Carlo simulations and analytical calculations to characterize the noise level as a function of the number of receptors. We show that the residual level approaches zero and that the correlation time, i.e., the waiting time needed to obtain statistically independent data, diverges, both for large receptor numbers. This result has important implications for such processes as eukaryotic chemotaxis.
Asunto(s)
Quimiotaxis , Receptores de Superficie Celular/fisiología , Transducción de Señal , Algoritmos , Método de Montecarlo , Procesos EstocásticosRESUMEN
The goal of aging research is to extend healthy, active life. For decades, C. elegans daf-2 insulin/insulin-like growth factor 1 (IGF-1) receptor mutants have served as a model for extended lifespan and youthfulness. However, a recent report suggested that their longevity is associated with an undesirable phenotype: a disproportionately long period of decrepitude at the end of life. In the human population, such an outcome would be a burden to society, bringing into question the relevance of daf-2 mutants as a model for life extension. However, here we report that, following an extended period of movement, daf-2 mutants survive longer in a decrepit state because of a beneficial trait: they are resistant to colonization of the digestive tract by dietary bacteria, a condition that leads to premature death in the wild-type and prevents their manifestation of decrepitude. If bacterial colonization is prevented, then daf-2 mutants lead both chronologically and proportionately healthier lives relative to the wild-type.
Asunto(s)
Envejecimiento/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Mutación/genética , Animales , Caenorhabditis elegans/microbiología , Proteínas de Caenorhabditis elegans/metabolismo , Recuento de Colonia Microbiana , Escherichia coli/crecimiento & desarrollo , Factores de RiesgoRESUMEN
Optical methods provide a noninvasive way to monitor calcium transients in Caenorhabditis elegans. Imaging techniques are particularly appealing in C. elegans because worms are optically transparent and can be imaged while fully intact. Furthermore, a variety of genetically encoded calcium indicators are available that can be targeted to cells of interest with appropriate tissue-specific promoters. Here, we describe a specific protocol, suitable for monitoring neuronal activity, for rapid calcium imaging in C. elegans using the cameleon indicator. Notes are provided to assist with adapting this protocol for use with other indicators and slower time scales.
Asunto(s)
Caenorhabditis elegans/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Procesamiento de Imagen Asistido por Computador , Neuronas/metabolismo , Programas Informáticos , Animales , Animales Modificados Genéticamente/fisiología , Caenorhabditis elegans/citología , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Procesamiento de Imagen Asistido por Computador/métodos , Indicadores y Reactivos/química , Indicadores y Reactivos/metabolismo , Indicadores y Reactivos/farmacología , Microscopía Fluorescente/métodos , Neuronas/citología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower. Theoretical treatment of observed behaviour has suggested a large change in force-posture relationships at different viscosities, but analysis of bend propagation suggests that short-range proprioceptive feedback is used to control and generate body bends. How muscles could be activated in a way consistent with both these results is unclear. We therefore combined automated worm tracking with calcium imaging to determine muscle activation strategy in a variety of external substrates. Remarkably, we observed that across locomotion patterns spanning a threefold change in wavelength, peak muscle activation occurs approximately 45° (1/8th of a cycle) ahead of peak midline curvature. Although the location of peak force is predicted to vary widely, the activation pattern is consistent with required force in a model incorporating putative length- and velocity-dependence of muscle strength. Furthermore, a linear combination of local curvature and velocity can match the pattern of activation. This suggests that proprioception can enable the worm to swim effectively while working within the limitations of muscle biomechanics and neural control.
Asunto(s)
Caenorhabditis elegans/fisiología , Músculos/fisiología , Natación , Alelos , Animales , Conducta Animal , Fenómenos Biomecánicos , Calcio/metabolismo , Cruzamientos Genéticos , Fenómenos Electrofisiológicos , Proteínas Fluorescentes Verdes/metabolismo , Procesamiento de Imagen Asistido por Computador , Modelos Lineales , Microscopía Fluorescente , Modelos Biológicos , Neuronas Motoras/metabolismo , Movimiento , Neuronas/metabolismo , Plásmidos/metabolismo , PropiocepciónRESUMEN
A single nervous system can generate many distinct motor patterns. Identifying which neurons and circuits control which behaviors has been a laborious piecemeal process, usually for one observer-defined behavior at a time. We present a fundamentally different approach to neuron-behavior mapping. We optogenetically activated 1054 identified neuron lines in Drosophila larvae and tracked the behavioral responses from 37,780 animals. Application of multiscale unsupervised structure learning methods to the behavioral data enabled us to identify 29 discrete, statistically distinguishable, observer-unbiased behavioral phenotypes. Mapping the neural lines to the behavior(s) they evoke provides a behavioral reference atlas for neuron subsets covering a large fraction of larval neurons. This atlas is a starting point for connectivity- and activity-mapping studies to further investigate the mechanisms by which neurons mediate diverse behaviors.
Asunto(s)
Conducta Animal , Drosophila melanogaster/fisiología , Neuronas/fisiología , Animales , Inteligencia Artificial , Encéfalo/fisiología , Mapeo Encefálico , Drosophila melanogaster/citología , Larva/fisiología , Locomoción , Neuronas Motoras/fisiología , Movimiento , OptogenéticaRESUMEN
The ability to learn and remember is critical for all animals to survive in the ever-changing environment. As we age, many of our biological faculties decay and of these, decline in learning and memory can be the most distressing. To carefully define age-dependent changes in learning during reproductive age in the nematode Caenorhabditis elegans, we performed a parametric behavioral study of habituation to nonlocalized mechanical stimuli (petri plate taps) over a range of intensities in middle-aged worms. We found that as worms age (from the onset of reproduction to the end of egg laying), response probability habituation increases (at both 10- and 60-second interstimulus intervals) and that these age-related changes were associated with a decrease in the discrimination between stimuli of different intensities. We also used optogenetics to investigate where these age-dependent changes occur. Our data suggest that the changes occur upstream of mechanosensory neuron depolarization. These data support the idea that declines in stimulus intensity discrimination abilities during aging may be one variable underlying age-related cognitive deficits.
Asunto(s)
Envejecimiento/fisiología , Conducta Animal/fisiología , Discriminación en Psicología/fisiología , Habituación Psicofisiológica/fisiología , Factores de Edad , Animales , Caenorhabditis elegans , Memoria a Corto Plazo/fisiología , Estimulación FísicaRESUMEN
All organisms react to noxious and mechanical stimuli but we still lack a complete understanding of cellular and molecular mechanisms by which somatosensory information is transformed into appropriate motor outputs. The small number of neurons and excellent genetic tools make Drosophila larva an especially tractable model system in which to address this problem. We developed high throughput assays with which we can simultaneously expose more than 1,000 larvae per man-hour to precisely timed noxious heat, vibration, air current, or optogenetic stimuli. Using this hardware in combination with custom software we characterized larval reactions to somatosensory stimuli in far greater detail than possible previously. Each stimulus evoked a distinctive escape strategy that consisted of multiple actions. The escape strategy was context-dependent. Using our system we confirmed that the nociceptive class IV multidendritic neurons were involved in the reactions to noxious heat. Chordotonal (ch) neurons were necessary for normal modulation of head casting, crawling and hunching, in response to mechanical stimuli. Consistent with this we observed increases in calcium transients in response to vibration in ch neurons. Optogenetic activation of ch neurons was sufficient to evoke head casting and crawling. These studies significantly increase our understanding of the functional roles of larval ch neurons. More generally, our system and the detailed description of wild type reactions to somatosensory stimuli provide a basis for systematic identification of neurons and genes underlying these behaviors.
Asunto(s)
Drosophila melanogaster/fisiología , Reacción de Fuga/fisiología , Ensayos Analíticos de Alto Rendimiento/métodos , Aire , Animales , Proteínas de Drosophila/genética , Calor , Canales Iónicos/genética , Larva/fisiología , Mutación/genética , Neuronas/patología , Optogenética , Estimulación Física , Programas Informáticos , VibraciónRESUMEN
Fluorescent protein-based sensors for detecting neuronal activity have been developed largely based on non-neuronal screening systems. However, the dynamics of neuronal state variables (e.g., voltage, calcium, etc.) are typically very rapid compared to those of non-excitable cells. We developed an electrical stimulation and fluorescence imaging platform based on dissociated rat primary neuronal cultures. We describe its use in testing genetically-encoded calcium indicators (GECIs). Efficient neuronal GECI expression was achieved using lentiviruses containing a neuronal-selective gene promoter. Action potentials (APs) and thus neuronal calcium levels were quantitatively controlled by electrical field stimulation, and fluorescence images were recorded. Images were segmented to extract fluorescence signals corresponding to individual GECI-expressing neurons, which improved sensitivity over full-field measurements. We demonstrate the superiority of screening GECIs in neurons compared with solution measurements. Neuronal screening was useful for efficient identification of variants with both improved response kinetics and high signal amplitudes. This platform can be used to screen many types of sensors with cellular resolution under realistic conditions where neuronal state variables are in relevant ranges with respect to timing and amplitude.
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Señalización del Calcio , Calcio/metabolismo , Genes Reporteros , Neuronas/metabolismo , Potenciales de Acción/fisiología , Animales , Células Cultivadas , Estimulación Eléctrica , Fluorescencia , Ácido Glutámico/metabolismo , Humanos , Indicadores y Reactivos , Ratas , Receptores de GABA/metabolismo , SolucionesRESUMEN
Neurobiological research in genetically tractable organisms relies heavily on robust assays for behavioral phenotypes. The simple body plan of the nematode Caenorhabditis elegans makes it particularly amenable to the use of automated microscopy and image analysis to describe behavioral patterns quantitatively. This protocol first describes the preparation and use of media for growing and maintaining worms for tracking. The second part of the protocol describes how to prepare a single young adult worm for recording during video analysis. Although the protocol was developed for use in a single-worm tracker, it addresses factors important for the generation of reproducible, standardized images in all systems.
Asunto(s)
Conducta Animal , Caenorhabditis elegans/fisiología , Entomología/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía por Video/métodos , Coloración y Etiquetado/métodos , Animales , Automatización/métodos , LocomociónRESUMEN
Neurobiological research in genetically tractable organisms relies heavily on robust assays for behavioral phenotypes. The simple body plan of the nematode Caenorhabditis elegans makes it particularly amenable to the use of automated microscopy and image analysis to describe behavioral patterns quantitatively. This protocol provides an approach for obtaining uniform illumination during worm tracking. Good lighting can be more of an art than a science. Once the system is set up, it will be necessary to play with it, testing the results after each adjustment to ensure that the analysis software is able to clearly identify the worm and its boundaries. Although the protocol was developed for use in a single-worm tracker, it addresses factors important for the generation of reproducible, standardized images in all systems.