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Throughout the animal kingdom, several members of the basic helix-loop-helix (bHLH) family act as proneural genes during early steps of nervous system development. Roles of bHLH genes in specifying terminal differentiation of postmitotic neurons have been less extensively studied. We analyze here the function of five C. elegans bHLH genes, falling into three phylogenetically conserved subfamilies, which are continuously expressed in a very small number of postmitotic neurons in the central nervous system. We show (a) that two orthologs of the vertebrate bHLHb4/b5 genes, called hlh-17 and hlh-32, function redundantly to specify the identity of a single head interneuron (AUA), as well as an individual motor neuron (VB2), (b) that the PTF1a ortholog hlh-13 acts as a terminal selector to control terminal differentiation and function of the sole octopaminergic neuron class in C. elegans, RIC, and (c) that the NHLH1/2 ortholog hlh-15 controls terminal differentiation and function of the peptidergic AVK head interneuron class, a known neuropeptidergic signaling hub in the animal. Strikingly, through null mutant analysis and cell-specific rescue experiments, we find that loss of hlh-15/NHLH in the peptidergic AVK neurons and the resulting abrogation of neuropeptide secretion causes a substantially expanded lifespan of the animal, revealing an unanticipated impact of a central, peptidergic hub neuron in regulating lifespan, which we propose to be akin to hypothalamic control of lifespan in vertebrates. Taken together, our functional analysis reveals themes of bHLH gene function during terminal differentiation that are complementary to the earlier lineage specification roles of other bHLH family members. However, such late functions are much more sparsely employed by members of the bHLH transcription factor family, compared to the function of the much more broadly employed homeodomain transcription factor family.
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An animal's locomotor rate is an important indicator of its motility. In studies of the nematode C. elegans, assays of the frequency of body bending waves have often been used to discern the effects of mutations, drugs, or aging. Traditional manual methods for measuring locomotor frequency are low in throughput and subject to human error. Most current automated methods depend on image segmentation, which requires high image quality and is prone to errors. Here, we describe an algorithm for automated estimation of C. elegans locomotor frequency using image invariants, i.e., shape-based parameters that are independent of object translation, rotation, and scaling. For each video frame, the method calculates a combination of 8 Hu's moment invariants and a set of Maximally Stable Extremal Regions (MSER) invariants. The algorithm then calculates the locomotor frequency by computing the autocorrelation of the time sequence of the invariant ensemble. Results of our method show excellent agreement with manual or segmentation-based results over a wide range of frequencies. We show that compared to a segmentation-based method that analyzes a worm's shape and a method based on video covariance, our technique is more robust to low image quality and background noise. We demonstrate the system's capabilities by testing the effects of serotonin and serotonin pathway mutations on C. elegans locomotor frequency.
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Large-scale assays of behavior in model organisms play an important role in genetic screens, drug testing, and the elucidation of gene-behavior relationships. We have developed an automated, high-throughput imaging and analysis method for assaying behaviors of the nematode C. elegans . We use high-resolution optical imaging to longitudinally record the behaviors of 96 animals at a time in multi-well plates, and computer vision software to quantify the animals' locomotor activity, behavioral states, and egg laying events. To demonstrate the capabilities of our system we used it to examine the role of serotonin in C. elegans behavior. We found that egg-laying events are preceded by a period of reduced locomotion, and that this decline in movement requires serotonin signaling. In addition, we identified novel roles of serotonin receptors SER-1 and SER-7 in regulating the effects of serotonin on egg laying across roaming, dwelling, and quiescent locomotor states. Our system will be useful for performing genetic or chemical screens for modulators of behavior.
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An animal's locomotor rate is an important indicator of its motility. In studies of the nematode C. elegans, assays of the frequency of body bending waves have often been used to discern the effects of mutations, drugs, or aging. Traditional manual methods for measuring locomotor frequency are low in throughput and subject to human error. Most current automated methods depend on image segmentation, which requires high image quality and is prone to errors. Here, we describe an algorithm for automated estimation of C. elegans locomotor frequency using image invariants, i.e., shape-based parameters that are independent of object translation, rotation, and scaling. For each video frame, the method calculates a combination of 8 Hu's moment invariants and a set of Maximally Stable Extremal Regions (MSER) invariants. The algorithm then calculates the locomotor frequency by computing the autocorrelation of the time sequence of the invariant ensemble. Results of our method show excellent agreement with manual or segmentation-based results over a wide range of frequencies. We show that compared to a segmentation-based method that analyzes a worm's shape and a method based on video covariance, our technique is more robust to low image quality and background noise. We demonstrate the system's capabilities by testing the effects of serotonin and serotonin pathway mutations on C. elegans locomotor frequency.
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The nematode Caenorhabditis elegans is one of the most widely studied organisms in biology due to its small size, rapid life cycle, and manipulable genetics. Research with C. elegans depends on labor-intensive and time-consuming manual procedures, imposing a major bottleneck for many studies, especially for those involving large numbers of animals. Here, we describe a general-purpose tool, WormPicker, a robotic system capable of performing complex genetic manipulations and other tasks by imaging, phenotyping, and transferring C. elegans on standard agar media. Our system uses a motorized stage to move an imaging system and a robotic arm over an array of agar plates. Machine vision tools identify animals and assay developmental stage, morphology, sex, expression of fluorescent reporters, and other phenotypes. Based on the results of these assays, the robotic arm selectively transfers individual animals using an electrically self-sterilized wire loop, with the aid of machine vision and electrical capacitance sensing. Automated C. elegans manipulation shows reliability and throughput comparable with standard manual methods. We developed software to enable the system to autonomously carry out complex protocols. To validate the effectiveness and versatility of our methods, we used the system to perform a collection of common C. elegans procedures, including genetic crossing, genetic mapping, and genomic integration of a transgene. Our robotic system will accelerate C. elegans research and open possibilities for performing genetic and pharmacological screens that would be impractical using manual methods.
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Animals with complex nervous systems demand sleep for memory consolidation and synaptic remodeling. Here, we show that, although the Caenorhabditis elegans nervous system has a limited number of neurons, sleep is necessary for both processes. In addition, it is unclear if, in any system, sleep collaborates with experience to alter synapses between specific neurons and whether this ultimately affects behavior. C. elegans neurons have defined connections and well-described contributions to behavior. We show that spaced odor-training and post-training sleep induce long-term memory. Memory consolidation, but not acquisition, requires a pair of interneurons, the AIYs, which play a role in odor-seeking behavior. In worms that consolidate memory, both sleep and odor conditioning are required to diminish inhibitory synaptic connections between the AWC chemosensory neurons and the AIYs. Thus, we demonstrate in a living organism that sleep is required for events immediately after training that drive memory consolidation and alter synaptic structures.
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Caenorhabditis elegans , Odorantes , Animales , Caenorhabditis elegans/fisiología , Olfato , Sueño/fisiología , Sinapsis/fisiologíaRESUMEN
An animal adapts its motor behavior to navigate the external environment. This adaptation depends on proprioception, which provides feedback on an animal's body postures. How proprioception mechanisms interact with motor circuits and contribute to locomotor adaptation remains unclear. Here, we describe and characterize proprioception-mediated homeostatic control of undulatory movement in the roundworm Caenorhabditis elegans. We found that the worm responds to optogenetically or mechanically induced decreases in midbody bending amplitude by increasing its anterior amplitude. Conversely, it responds to increased midbody amplitude by decreasing the anterior amplitude. Using genetics, microfluidic and optogenetic perturbation response analyses, and optical neurophysiology, we elucidated the neural circuit underlying this compensatory postural response. The dopaminergic PDE neurons proprioceptively sense midbody bending and signal to AVK interneurons via the D2-like dopamine receptor DOP-3. The FMRFamide-like neuropeptide FLP-1, released by AVK, regulates SMB head motor neurons to modulate anterior bending. We propose that this homeostatic behavioral control optimizes locomotor efficiency. Our findings demonstrate a mechanism in which proprioception works with dopamine and neuropeptide signaling to mediate motor control, a motif that may be conserved in other animals.
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Proteínas de Caenorhabditis elegans , Neuropéptidos , Animales , Caenorhabditis elegans/fisiología , Dopamina/farmacología , Retroalimentación Sensorial , Locomoción/fisiología , Proteínas de Caenorhabditis elegans/genética , Neuropéptidos/genéticaRESUMEN
Understanding how we can age healthily is a challenge at the heart of biogerontological interest. Whereas myriad genes are known to affect the lifespan of model organisms, effects of such interventions on healthspan-the period of life where an animal is considered healthy, rather than merely alive-are less clear. To understand relationships between life- and healthspan, in recent years several platforms were developed with the purpose of assessing both readouts simultaneously. We here relied on one such platform, the WorMotel, to study effects of adulthood-restricted knock-down of 130 Caenorhabditis elegans genes on the locomotive health of the animals along their lifespans. We found that knock-down of six genes affected healthspan while lifespan remained unchanged. For two of these, F26A3.4 and chn-1, knock-down resulted in an improvement of healthspan. In follow-up experiments we showed that knockdown of F26A3.4 indeed improves locomotive health and muscle structure at old age.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/fisiología , Técnicas de Silenciamiento del Gen , Longevidad/fisiología , Proteínas de Caenorhabditis elegans/genéticaRESUMEN
The nematode Caenorhabditis elegans is among the most common model systems used in aging research owing to its simple and inexpensive culture techniques, rapid reproduction cycle (~3 days), short lifespan (~3 weeks), and numerous available tools for genetic manipulation and molecular analysis. The most common approach for conducting aging studies in C. elegans, including survival analysis, involves culturing populations of tens to hundreds of animals together on solid nematode growth media (NGM) in Petri plates. While this approach gathers data on a population of animals, most protocols do not track individual animals over time. Presented here is an optimized protocol for the long-term culturing of individual animals on microfabricated polydimethylsiloxane (PDMS) devices called WorMotels. Each device allows up to 240 animals to be cultured in small wells containing NGM, with each well isolated by a copper sulfate-containing moat that prevents the animals from fleeing. Building on the original WorMotel description, this paper provides a detailed protocol for molding, preparing, and populating each device, with descriptions of common technical complications and advice for troubleshooting. Within this protocol are techniques for the consistent loading of small-volume NGM, the consistent drying of both the NGM and bacterial food, options for delivering pharmacological interventions, instructions for and practical limitations to reusing PDMS devices, and tips for minimizing desiccation, even in low-humidity environments. This technique allows the longitudinal monitoring of various physiological parameters, including stimulated activity, unstimulated activity, body size, movement geometry, healthspan, and survival, in an environment similar to the standard technique for group culture on solid media in Petri plates. This method is compatible with high-throughput data collection when used in conjunction with automated microscopy and analysis software. Finally, the limitations of this technique are discussed, as well as a comparison of this approach to a recently developed method that uses microtrays to culture isolated nematodes on solid media.
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Envejecimiento , Caenorhabditis elegans , Animales , Caenorhabditis elegans/genética , Envejecimiento/fisiología , Longevidad , Medios de Cultivo , Programas InformáticosRESUMEN
The nematode Caenorhabditis elegans uses rhythmic muscle contractions (pumps) of the pharynx, a tubular feeding organ, to filter, transport, and crush food particles. A number of feeding mutants have been identified, including those with slow pharyngeal pumping rate, weak muscle contraction, defective muscle relaxation, and defective grinding of bacteria. Many aspects of these pharyngeal behavioral defects and how they affect pharyngeal function are not well understood. For example, the behavioral deficits underlying inefficient particle transport in "slippery" mutants have been unclear. Here we use high-speed video microscopy to describe pharyngeal pumping behaviors and particle transport in wild-type animals and in feeding mutants. Different "slippery" mutants exhibit distinct defects including weak isthmus contraction, failure to trap particles in the anterior isthmus, and abnormal timing of contraction and relaxation in pharyngeal compartments. Our results show that multiple deficits in pharyngeal timing or contraction can cause defects in particle transport. NEW & NOTEWORTHY The nematode C. elegans uses rhythmic contractions of its pharynx (feeding organ) to filter, transport, and crush food bacteria. Genetic analyses have identified mutants with defective pharyngeal motions, but many details of these movements and how they affect feeding are poorly understood. We use high-speed video microscopy to describe pharyngeal pumping behaviors and particle transport in feeding mutants. We find that multiple deficits in pharyngeal timing or contraction can cause defects in particle transport.
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Caenorhabditis elegans , Faringe , Animales , Caenorhabditis elegans/fisiología , Conducta Alimentaria/fisiología , Microscopía por Video , Contracción Muscular/fisiologíaRESUMEN
Optogenetic approaches have proven to be powerful for examining the roles of specific neurons in generating behaviors, especially in systems where electrophysiological manipulation is not possible. Here we describe a method for optogenetically manipulating single pharyngeal neurons in intact C. elegans while monitoring pharyngeal behavior. This approach provides bidirectional and dynamic control of pharyngeal neural activity while quantitatively assessing behavior and has allowed us to test hypotheses about the roles of individual pharyngeal neurons in feeding behavior.
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Caenorhabditis elegans , Conducta Alimentaria , Optogenética , Fisiología , Animales , Caenorhabditis elegans/genética , Neuronas/fisiología , Faringe/fisiología , Fisiología/métodosRESUMEN
Many experiments in C. elegans neurobiology rely on imaging its behavior. Here we describe procedures for building a flexible and inexpensive imaging system using standard optical and mechanical components.
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Envejecimiento , Caenorhabditis elegans , Animales , Conducta Animal , Diagnóstico por Imagen , NeurobiologíaRESUMEN
Ultracold storage is widely used to preserve genetic stocks. Standard cryopreservation methods for the nematode C. elegans are vulnerable to refrigeration failures, which can result in the loss of stock viability due to freeze-thaw damage. In previous work our laboratory developed a method for cryopreserving worms in a dehydrated form that remains viable after multiple freeze-thaw cycles. However, strains preserved in this manner can be recovered only once from each cryopreservation tube. Here we describe a cryopreservation method in which C. elegans are dehydrated in a granular medium (cornmeal) prior to freezing. To recover worms, a small fraction (~1%) of the medium may be removed with the remainder returned to cold storage. Our improved cryopreservation method is not only resistant to refrigeration failures but also greatly increases the number of recoveries per tube compared to current methods.
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Neural circuits coordinate with muscles and sensory feedback to generate motor behaviors appropriate to an animal's environment. In C. elegans, the mechanisms by which the motor circuit generates undulations and modulates them based on the environment are largely unclear. We quantitatively analyzed C. elegans locomotion during free movement and during transient optogenetic muscle inhibition. Undulatory movements were highly asymmetrical with respect to the duration of bending and unbending during each cycle. Phase response curves induced by brief optogenetic inhibition of head muscles showed gradual increases and rapid decreases as a function of phase at which the perturbation was applied. A relaxation oscillator model based on proprioceptive thresholds that switch the active muscle moment was developed and is shown to quantitatively agree with data from free movement, phase responses, and previous results for gait adaptation to mechanical loadings. Our results suggest a neuromuscular mechanism underlying C. elegans motor pattern generation within a compact circuit.
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Caenorhabditis elegans/fisiología , Locomoción , Actividad Motora , Animales , Relojes Biológicos , PeriodicidadRESUMEN
Many cell types display the remarkable ability to alter their cellular phenotype in response to specific external or internal signals. Such phenotypic plasticity is apparent in the nematode Caenorhabditis elegans when adverse environmental conditions trigger entry into the dauer diapause stage. This entry is accompanied by structural, molecular, and functional remodeling of a number of distinct tissue types of the animal, including its nervous system. The transcription factor (TF) effectors of 3 different hormonal signaling systems, the insulin-responsive DAF-16/FoxO TF, the TGFß-responsive DAF-3/SMAD TF, and the steroid nuclear hormone receptor, DAF-12/VDR, a homolog of the vitamin D receptor (VDR), were previously shown to be required for entering the dauer arrest stage, but their cellular and temporal focus of action for the underlying cellular remodeling processes remained incompletely understood. Through the generation of conditional alleles that allowed us to spatially and temporally control gene activity, we show here that all 3 TFs are not only required to initiate tissue remodeling upon entry into the dauer stage, as shown before, but are also continuously required to maintain the remodeled state. We show that DAF-3/SMAD is required in sensory neurons to promote and then maintain animal-wide tissue remodeling events. In contrast, DAF-16/FoxO or DAF-12/VDR act cell-autonomously to control anatomical, molecular, and behavioral remodeling events in specific cell types. Intriguingly, we also uncover non-cell autonomous function of DAF-16/FoxO and DAF-12/VDR in nervous system remodeling, indicating the presence of several insulin-dependent interorgan signaling axes. Our findings provide novel perspectives into how hormonal systems control tissue remodeling.
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Proteínas de Caenorhabditis elegans/fisiología , Comunicación Celular/genética , Plasticidad de la Célula/genética , Factores de Transcripción Forkhead/fisiología , Receptores Citoplasmáticos y Nucleares/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Factores de Transcripción Forkhead/genética , Regulación del Desarrollo de la Expresión Génica , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Especificidad de Órganos/genética , Organogénesis/genética , Comunicación Paracrina/genética , Receptores de Calcitriol/genética , Receptores de Calcitriol/fisiología , Receptores Citoplasmáticos y Nucleares/genética , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiología , Transducción de Señal/genéticaRESUMEN
Laser microsurgery has long been an important means of assessing the functions of specific cells and tissues. Most laser ablation systems use short, highly focused laser pulses to create plasma-mediated lesions with dimensions on the order of the wavelength of light. While the small size of the lesion enables ablation with high spatial resolution, it also makes it difficult to ablate larger structures. We developed an infrared laser ablation system capable of thermally lesioning tissues with spot sizes tunable by the duration and amplitude of laser pulses. We used our laser system in the roundworm C. elegans to kill single neurons and to sever the dorsal and ventral nerve cords, structures that are difficult to lesion using a plasma-based ablation system. We used these ablations to investigate the source of convulsions in a gain-of-function mutant for the acetylcholine receptor ACR-2. Severing the ventral nerve cord caused convulsions to occur independently anterior and posterior to the lesion, suggesting that convulsions can arise independently from distinct subsets of the motor circuit.
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Caenorhabditis elegans/efectos de la radiación , Rayos Infrarrojos/uso terapéutico , Terapia por Láser/métodos , Microcirugia/métodos , Convulsiones/terapia , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Neuronas Motoras/efectos de la radiación , Mutación , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Convulsiones/genéticaRESUMEN
For most metazoans, oxygen deprivation leads to cell dysfunction and if severe, death. Sublethal stress prior to a hypoxic or anoxic insult ("preconditioning") can protect cells from subsequent oxygen deprivation. The molecular mechanisms by which sublethal stress can buffer against a subsequent toxic insult and the role of the nervous system in the response are not well understood. We studied the role of neuronal activity preconditioning to oxygen deprivation in Caenorhabditis elegans. Animals expressing the histamine gated chloride channels (HisCl1) in select cell populations were used to temporally and spatially inactivate the nervous system or tissue prior to an anoxic insult. We find that inactivation of the nervous system for 3 h prior to the insult confers resistance to a 48-h anoxic insult in 4th-stage larval animals. Experiments show that this resistance can be attributed to loss of activity in cholinergic and GABAergic neurons as well as in body wall muscles. These observations indicate that the nervous system activity can mediate the organism's response to anoxia.
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Condicionamiento Psicológico/fisiología , Neuronas GABAérgicas/metabolismo , Hipoxia/fisiopatología , Músculos/fisiopatología , Animales , Caenorhabditis elegans/metabolismo , Colinérgicos/metabolismo , Músculos/metabolismoRESUMEN
People tend to fall asleep when gently rocked or vibrated. Experimental studies have shown that rocking promotes sleep in humans and mice. However, the mechanisms underlying the phenomenon are not well understood. A habituation model proposes that habituation, a form of non-associative learning, mediates sleep induction by monotonous stimulation. Here, we show that gentle vibration promotes sleep in Drosophila in part through habituation. Vibration-induced sleep (VIS) leads to increased homeostatic sleep credit and reduced arousability, and can be suppressed by heightened arousal or reduced GABA signaling. Multiple mechanosensory organs mediate VIS, and the magnitude of VIS depends on vibration frequency and genetic background. Sleep induction improves over successive blocks of vibration. Furthermore, training with continuous vibration does not generalize to intermittent vibration, demonstrating stimulus specificity, a characteristic of habituation. Our findings suggest that habituation plays a significant role in sleep induction by vibration.
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Habituación Psicofisiológica/fisiología , Fármacos Inductores del Sueño/uso terapéutico , Sueño/fisiología , Animales , Drosophila , Fármacos Inductores del Sueño/farmacologíaRESUMEN
Ultracold preservation is widely used for storage of genetic stocks of Caenorhabditis elegans Current cryopreservation protocols are vulnerable to refrigeration failures, which can result in the loss of stock viability due to damage during re-freezing. Here we present a method for preserving worms in a dehydrated and frozen form that retains viability after multiple freeze-thaw cycles. After dehydration in the presence of trehalose or glycerol, C. elegans stocks can be frozen and thawed multiple times while maintaining viability. While both dauer and non-dauer larvae survive desiccation and freezing, the dauer defective mutant daf-16 does not survive desiccation. Our technique is useful for storing stocks in a manner robust to freezer failures, and potentially for shipping strains between laboratories.
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Caenorhabditis elegans , Trehalosa , Animales , Caenorhabditis elegans/genética , Congelación , LarvaRESUMEN
Getting a grip on how we may age healthily is a central interest of biogerontological research. To this end, a number of academic teams developed platforms for life- and healthspan assessment in Caenorhabditis elegans. These are very appealing for medium- to high throughput screens, but a broader implementation is lacking due to many systems relying on custom scripts for data analysis that others struggle to adopt. Hence, user-friendly recommendations would help to translate raw data into interpretable results. The aim of this communication is to streamline the analysis of data obtained by the WorMotel, an economically and practically appealing screening platform, in order to facilitate the use of this system by interested researchers. We here detail recommendations for the stepwise conversion of raw image data into activity values and explain criteria for assessment of health in C. elegans based on locomotion. Our analysis protocol can easily be adopted by researchers, and all needed scripts and a tutorial are available in S1 and S2 Files.