RESUMEN
The ability to perceive temperature is crucial for most animals. It enables them to maintain their body temperature and swiftly react to noxiously cold or hot objects. Caenorhabditis elegans is a powerful genetic model for the study of thermosensation as its simple nervous system is well characterized and its transparent body is suited for in vivo functional imaging of neurons. The behavior triggered by experience-dependent thermosensation has been well studied in C. elegans under temperature-gradient environments. However, how C. elegans senses temperature via its nervous system is not well understood due to the limitations of currently available technologies. One major bottleneck is the difficulty in creating fast temperature changes, especially cold stimuli. Here, we developed a microfluidic-based platform that allowed the in vivo functional imaging of C. elegans responding to well-controlled temporally varying temperature stimulation by rapidly switching fluid streams at different temperatures. We used computational models to enable rational design and optimization of experimental conditions. We validated the design and utility of our system with studies of the functional role of thermosensory neurons. We showed that the responses of PVD polymodal nociceptor neurons observed in previous studies can be recapitulated. Further, we highlighted how this platform may be used to dissect neuronal circuits with an example of activity recording in PVC interneurons. Both of these neuron types show sensitization phenotypes. We envision that both the engineered system and the findings in this work will spur further studies of molecular and cellular mechanisms underlying cold-sensing through the nervous system.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Temperatura , Caenorhabditis elegans/genética , Microfluídica , Sensación Térmica/fisiología , Frío , Proteínas de Caenorhabditis elegans/genéticaRESUMEN
Animals' perception and behavior involve integration of multiple sensory modalities. Caenorhabditis elegans is a useful model for studying multimodal sensory integration, as it has well-characterized neuronal circuits in a relatively simple nervous system. However, most studies based on functional imaging have only been conducted on single modal stimuli, because well-controlled multimodal experiments for C. elegans are technically difficult. For instance, no single systems currently deliver precise stimuli with spatial, temporal, and intensity control, despite prior hypotheses that interneurons do integrate these sensory inputs to control behavior. Here, a microfluidic platform that can easily deliver spatially and temporally controlled combination stimuli to C. elegans is presented. With this platform, both sensory and interneuron activity is measured in response to mechanical and chemical stimulations in a quantitative and high-throughput manner. It is found that the activity of command interneuron PVC can be modulated by prior stimulation both within the same and across different modalities. The roles of monoaminergic and peptidergic signaling are further examined on the process of multimodal integration through PVC activity. The approach exemplified here is envisioned to be broadly applicable in different contexts to elucidate underlying mechanisms and identify genes affecting multisensory integration.
Asunto(s)
Caenorhabditis elegans , Interneuronas , Microfluídica , Animales , Caenorhabditis elegans/fisiología , Interneuronas/fisiología , Microfluídica/instrumentación , Percepción/fisiologíaRESUMEN
Although identifying cell names in dense image stacks is critical in analyzing functional whole-brain data enabling comparison across experiments, unbiased identification is very difficult, and relies heavily on researchers' experiences. Here, we present a probabilistic-graphical-model framework, CRF_ID, based on Conditional Random Fields, for unbiased and automated cell identification. CRF_ID focuses on maximizing intrinsic similarity between shapes. Compared to existing methods, CRF_ID achieves higher accuracy on simulated and ground-truth experimental datasets, and better robustness against challenging noise conditions common in experimental data. CRF_ID can further boost accuracy by building atlases from annotated data in highly computationally efficient manner, and by easily adding new features (e.g. from new strains). We demonstrate cell annotation in Caenorhabditis elegans images across strains, animal orientations, and tasks including gene-expression localization, multi-cellular and whole-brain functional imaging experiments. Together, these successes demonstrate that unbiased cell annotation can facilitate biological discovery, and this approach may be valuable to annotation tasks for other systems.
Asunto(s)
Caenorhabditis elegans/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Análisis de la Célula Individual , Animales , Encéfalo/fisiología , Modelos EstadísticosRESUMEN
Mechanotransduction channels have been proposed as force sensors in various physiological processes, such as hearing and touch. In particular, TMC1 has been shown to constitute the pore of hair cell mechanotransduction channels, but little is known about how force is sensed by TMC channels. Here, we identify UNC-44/ankyrin as an essential component of the TMC-1 mechanotransduction channel complex in the sensory cilia of Caenorhabditis elegans mechanoreceptor neurons. Ankyrin binds indirectly to TMC-1 via evolutionarily conserved CIB proteins, which are required for TMC-1-mediated mechanosensation in C. elegans OLQ neurons and body wall muscles. Mechanosensory activity conferred by ectopically expressed TMCs in mechanoinsensitive neurons depends on both ankyrin and CIB proteins, indicating that the ankyrin-CIB subcomplex is required for TMC mechanosensitivity. Our work indicates that ankyrin is a long-sought intracellular tether that transmits force to TMC mechanotransduction channels.
Asunto(s)
Ancirinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Canales Iónicos/metabolismo , Mecanorreceptores/metabolismo , Mecanotransducción Celular/fisiología , Animales , Caenorhabditis elegansRESUMEN
Neurons convert synaptic or sensory inputs into cellular outputs. It is not well understood how a single neuron senses, processes multiple stimuli, and generates distinct neuronal outcomes. Here, we describe the mechanism by which the C. elegans PVD neurons sense two mechanical stimuli: external touch and proprioceptive body movement. These two stimuli are detected by distinct mechanosensitive DEG/ENaC/ASIC channels, which trigger distinct cellular outputs linked to mechanonociception and proprioception. Mechanonociception depends on DEGT-1 and activates PVD's downstream command interneurons through its axon, while proprioception depends on DEL-1, UNC-8, and MEC-10 to induce local dendritic Ca2+ increase and dendritic release of a neuropeptide NLP-12. NLP-12 directly modulates neuromuscular junction activity through the cholecystokinin receptor homolog on motor axons, setting muscle tone and movement vigor. Thus, the same neuron simultaneously uses both its axon and dendrites as output apparatus to drive distinct sensorimotor outcomes.
Asunto(s)
Mecanotransducción Celular , Neuropéptidos/metabolismo , Propiocepción , Células Receptoras Sensoriales/metabolismo , Animales , Axones/metabolismo , Axones/fisiología , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Calcio/metabolismo , Dendritas/metabolismo , Dendritas/fisiología , Canales Epiteliales de Sodio/metabolismo , Retroalimentación Fisiológica , Canales Iónicos/metabolismo , Proteínas de la Membrana/metabolismo , Unión Neuromuscular/metabolismo , Unión Neuromuscular/fisiología , Células Receptoras Sensoriales/fisiologíaRESUMEN
Mechanosensation is fundamentally important for the abilities of an organism to experience touch, hear sounds, and maintain balance. Caenorhabditis elegans is a powerful system for studying mechanosensation as this worm is well suited for in vivo functional imaging of neurons. Many years of research using labor-intensive methods have generated a wealth of knowledge about mechanosensation in C. elegans, and the recent microfluidic-based platforms continue to push the boundary for this field. However, developmental aspects of sensory biology, including mechanosensation, are still not fully understood. One current bottleneck is the difficulty in assaying larvae because they are much smaller than adult worms. Microfluidic devices with features small enough for larvae, especially actuators for the delivery of mechanical stimulation, are difficult to design and fabricate. Here, we present a series of automatic microfluidic platforms that allow for in vivo functional imaging of C. elegans responding to controlled mechanical stimulation at different developmental stages. Using a novel fabrication method, we designed highly deformable pneumatically actuated on-chip structures that can deliver mechanical stimulation to larval worms. The PDMS actuator allows for quantitatively controlled mechanical stimulation of both gentle and harsh touch neurons, by simply changing the actuation pressure, which makes this device easily translatable to other labs. We validated the design and utility of our systems with studies of the functional role of mechanosensory neurons in developing worms; we showed that gentle and harsh touch neurons function similarly in early larvae as they do in the adult stage, which would not have been possible previously. Finally, we investigated the effect of a sleep-like state on neuronal responses by imaging C. elegans in the lethargus state.
Asunto(s)
Caenorhabditis elegans , Neuroimagen Funcional , Dispositivos Laboratorio en un Chip , Larva/citología , Larva/metabolismo , Técnicas Analíticas Microfluídicas , Estrés Mecánico , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismoRESUMEN
The field-gradient, superficial photo fluidization of azomaterials allows a specific 3D nano-silhouette to be shaped over a large area, so as to get easy access to a 3D-tapered, deep sub-wavelength Au nanohole (20 nm spatial size) array. The squeezing of visible light into the deep sub-wavelength point and the relevant extraordinary optical transmission (EOT) are achieved using this 3D-tapered, 20 nm Au nanohole.
RESUMEN
The Field-Gradient Effect extends the photofluidization of azobenzene materials to 3D, multi-level micro/nanotexturing with a newly conceptualized design strategy based on "field-gradient photofluidization". In particular, we successfully characterized the vertical gradient optical absorption within the azobenzene material and the resulting field-gradient photofluidization both theoretically and experimentally. Furthermore, we could create the heterogeneously integrated micro/nanotextures at any desired surface heights, capability that is potentially beneficial for plasmonic applications.