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The roundworm C. elegans is a mainstay of aging research due to its short lifespan and easily manipulable genetics. Current, widely used methods for long-term measurement of C. elegans are limited by low throughput and the difficulty of performing longitudinal monitoring of aging phenotypes. Here we describe the WorMotel, a microfabricated device for long-term cultivation and automated longitudinal imaging of large numbers of C. elegans confined to individual wells. Using the WorMotel, we find that short-lived and long-lived strains exhibit patterns of behavioral decline that do not temporally scale between individuals or populations, but rather resemble the shortest and longest lived individuals in a wild type population. We also find that behavioral trajectories of worms subject to oxidative stress resemble trajectories observed during aging. Our method is a powerful and scalable tool for analysis of C. elegans behavior and aging. Aging affects almost all living things, yet little is known about the biological changes that occur as we get older. Scientists often study aging in the microscopic roundworm Caenorhabditis elegans because it reproduces quickly and its lifespan is short (about 2–3 weeks on average). To date, investigations have helped to reveal genes that affect overall lifespan. However, it is not known how much these genes also affect the animal’s healthy lifespan or “healthspan”, that is to say, the length of time before advancing age begins to negatively affect health. Until now, studies with worms have often been limited because measuring health and aging required time-consuming and difficult manual experiments. This also meant that worms were studied together as groups, rather than as individuals, providing a simplified picture of what was going on. An automated system in which many single worms can be analyzed and assessed would provide a much more detailed view of the effects of aging on health. Churgin et al. have now developed a device called the WorMotel to allow simultaneous automated examination of 240 worms throughout their entire adult lifespan. The WorMotel is a rectangular slab of clear silicone rubber with small wells in it. A single worm is confined in each well with a source of bacteria for food, and a camera is used to track and monitor each worm’s behavior over time. This device confirmed that worms move more slowly as they get older, which was taken to be a measurement of the worms’ declining health. Worms that lived the longest declined over the first few days and then had a long plateau of very low activity before eventually dying. Short-lived worms became slower and died fairly promptly. Churgin et al. also showed that the worms with mutations that increase lifespan declined in a similar way to the longest-lived normal worms, and that mutants with shorter lifespans declined like the shortest-lived normal worms. Also, normal worms that had been exposed to a chemical called paraquat – which stresses the worm's cells and shortens the worm’s lifespans to a few days – slowed down in a similar manner as aging worms, suggesting that the stress is similar to the aging process. Tools like the WorMotel can improve our understanding of the links between lifespan and healthspan. The tool is designed to be versatile and can be used with standard imaging systems and automated tools, meaning it can be scaled up to deal with tens of thousands of worms at once. Churgin et al. are now using the WorMotel to find other genes that influence healthspan and understand how they contribute to deteriorating health as animals age. Aging affects us all and learning more about healthspan could lead to drugs or interventions to help more people to live healthily for longer.

Behavior varies even among genetically identical animals raised in the same environment. However, little is known about the circuit or anatomical origins of this individuality. Here, we demonstrate a neural correlate of Drosophila odor preference behavior in the olfactory sensory periphery. Namely, idiosyncratic calcium responses in projection neuron (PN) dendrites and densities of the presynaptic protein Bruchpilot in olfactory receptor neuron (ORN) axon terminals correlate with individual preferences in a choice between two aversive odorants. The ORN-PN synapse appears to be a locus of individuality where microscale variation gives rise to idiosyncratic behavior. Simulating microscale stochasticity in ORN-PN synapses of a 3062 neuron model of the antennal lobe recapitulates patterns of variation in PN calcium responses matching experiments. Conversely, stochasticity in other compartments of this circuit does not recapitulate those patterns. Our results demonstrate how physiological and microscale structural circuit variations can give rise to individual behavior, even when genetics and environment are held constant.

In response to environments that cause cellular stress, animals engage in sleep behavior that facilitates recovery from the stress. In Caenorhabditis elegans, stress-induced sleep(SIS) is regulated by cytokine activation of the ALA neuron, which releases FLP-13 neuropeptides characterized by an amidated arginine-phenylalanine (RFamide) C-terminus motif. By performing an unbiased genetic screen for mutants that impair the somnogenic effects of FLP-13 neuropeptides, we identified the gene dmsr-1, which encodes a G-protein coupled receptor similar to an insect RFamide receptor. DMSR-1 is activated by FLP-13 peptides in cell culture, is required for SIS in vivo, is expressed non-synaptically in several wake-promoting neurons, and likely couples to a Gi/o heterotrimeric G-protein. Our data expand our understanding of how a single neuroendocrine cell coordinates an organism-wide behavioral response, and suggest that similar signaling principles may function in other organisms to regulate sleep during sickness. People often feel fatigued and sleepy when they are sick. Other animals also show signs of sleepiness when ill – they stop eating, move less, and are less responsive to changes in their environment. Sickness-induced sleep helps both people and other animals to recover, and many scientists believe that this type of sleep is different than nightly sleep. Studies of sickness-induced sleep have made use of a simple worm with a simple nervous system. In this worm, a single nerve cell releases chemicals that cause the worm to fall asleep in response to illness. Animals exposed to one of these chemicals, called FLP-13, fall asleep even when they are not sick. As such, scientists would like to know which cells in the nervous system FLP-13 interacts with, what receptor the cells use to recognize this chemical, and whether it turns on cells that induce sleep or turns off the cells that cause wakefulness. Now, Iannacone et al. show that FLP-13 likely causes sleep by turning down activity in the cells in the nervous system that promote wakefulness. The experiments sifted through genetic mutations to determine which ones cause the worms not to fall asleep when FLP-13 is released. This revealed that worms with a mutation that causes them to lack a receptor protein called DMSR-1 do not become sleepy in response to FLP-13. This suggests that DMSR-1 must be essential for FLP-13 to trigger sleep. About 10% of cells in the worm’s nervous system have the DMSR-1 receptor. Some of these neurons tell the worm to move forward or to forage around for food. The experiments also showed that FLP-13 is probably not the only chemical that interacts with the DMSR-1 receptor, but the identities of these other chemicals remain unknown. Additional experiments are now needed to determine if sickness-induced sleepiness in humans and other mammals is triggered by a similar mechanism. If it is, then drugs might be developed to treat people experiencing fatigue associated with sickness as well as other unexplained cases of fatigue.

Sleep during development is involved in refining brain circuitry, but a role for sleep in the earliest periods of nervous system elaboration, when neurons are first being born, has not been explored. Here we identify a sleep state in Drosophila larvae that coincides with a major wave of neurogenesis. Mechanisms controlling larval sleep are partially distinct from adult sleep: octopamine, the Drosophila analog of mammalian norepinephrine, is the major arousal neuromodulator in larvae, but dopamine is not required. Using real-time behavioral monitoring in a closed-loop sleep deprivation system, we find that sleep loss in larvae impairs cell division of neural progenitors. This work establishes a system uniquely suited for studying sleep during nascent periods, and demonstrates that sleep in early life regulates neural stem cell proliferation. Nearly all animals sleep more while they are still developing, suggesting that sleep is important in early life. Previous studies have shown that sleep may be required for building connections in the brain. However, it has been difficult to study the effects of sleep in earlier stages of brain development, when stem cells divide to create brain cells in a process known as “neurogenesis”. This is partly because, in mammals, most neurogenesis occurs in the womb. Scientists have successfully studied sleep using the common fruit fly. But these studies have so far focused on adult flies, in which neurogenesis is mostly complete. Fly larvae, on the other hand, are widely used to study brain development and neurogenesis. Compared to mammals in the womb, fruit fly larvae are very easy to access and manipulate. However, unlike adult flies, no one had previously looked to see if larvae even display a behaviour that would fit the definition of sleep. To see if fruit fly larvae do sleep, Szuperak et al. created the “LarvaLodge”, an apparatus in which individual larvae can be housed while having their activity monitored over time. In these lodges, a bright light was used to test how hard it is to arouse inactive fruit fly larvae, and further experiments asked what happens when larvae are prevented from resting. Then, to look at neurogenesis in the larvae, Szuperak et al. used a stain that labels dividing stem cells within the nervous system. Those cells could then be seen and counted when a larva was dissected and examined under a microscope. The results from the LarvaLodge showed that fruit fly larvae do indeed sleep: they have extended periods of rest during which they react less to outside disturbances and adopt a particular posture (they retract their heads towards their bodies). Also when larvae were deprived of sleep, by shining a light or shaking, they compensated by sleeping more afterwards. Importantly, depriving the larvae of sleep also led to lower levels of neurogenesis. These findings establish the fruit fly larva as a new and useful system for studying the role of sleep in early development, and may help shed light on the role sleep plays in disorders affecting brain development.

Schistosomiasis, caused by parasitic flatworms of the genus Schistosoma, is a neglected tropical disease affecting hundreds of millions globally. Praziquantel (PZQ), the only drug currently available for treatment and control, is largely ineffective against juvenile worms, and reports of PZQ resistance lend added urgency to the need for development of new therapeutics. Ion channels, which underlie electrical excitability in cells, are validated targets for many current anthelmintics. Transient receptor potential (TRP) channels are a large family of non-selective cation channels. TRP channels play key roles in sensory transduction and other critical functions, yet the properties of these channels have remained essentially unexplored in parasitic helminths. TRP channels fall into several (7-8) subfamilies, including TRPA and TRPV. Though schistosomes contain genes predicted to encode representatives of most of the TRP channel subfamilies, they do not appear to have genes for any TRPV channels. Nonetheless, we find that the TRPV1-selective activators capsaicin and resiniferatoxin (RTX) induce dramatic hyperactivity in adult worms; capsaicin also increases motility in schistosomula. SB 366719, a highly-selective TRPV1 antagonist, blocks the capsaicin-induced hyperactivity in adults. Mammalian TRPA1 is not activated by capsaicin, yet knockdown of the single predicted TRPA1-like gene (SmTRPA) in S. mansoni effectively abolishes capsaicin-induced responses in adult worms, suggesting that SmTRPA is required for capsaicin sensitivity in these parasites. Based on these results, we hypothesize that some schistosome TRP channels have novel pharmacological sensitivities that can be targeted to disrupt normal parasite neuromuscular function. These results also have implications for understanding the phylogeny of metazoan TRP channels and may help identify novel targets for new or repurposed therapeutics.

Innate behavioral biases and preferences can vary significantly among individuals of the same genotype. Though individuality is a fundamental property of behavior, it is not currently understood how individual differences in brain structure and physiology produce idiosyncratic behaviors. Here we present evidence for idiosyncrasy in olfactory behavior and neural responses in Drosophila. We show that individual female Drosophila from a highly inbred laboratory strain exhibit idiosyncratic odor preferences that persist for days. We used in vivo calcium imaging of neural responses to compare projection neuron (second-order neurons that convey odor information from the sensory periphery to the central brain) responses to the same odors across animals. We found that, while odor responses appear grossly stereotyped, upon closer inspection, many individual differences are apparent across antennal lobe (AL) glomeruli (compact microcircuits corresponding to different odor channels). Moreover, we show that neuromodulation, environmental stress in the form of altered nutrition, and activity of certain AL local interneurons affect the magnitude of interfly behavioral variability. Taken together, this work demonstrates that individual Drosophila exhibit idiosyncratic olfactory preferences and idiosyncratic neural responses to odors, and that behavioral idiosyncrasies are subject to neuromodulation and regulation by neurons in the AL.

The coupling of transgenes to heat shock promoters is a widely applied method for regulating gene expression. In C. elegans, gene induction can be controlled temporally through timing of heat shock and spatially via targeted rescue in heat shock mutants. Here, we present a method for evoking gene expression in arbitrary cells, with single-cell resolution. We use a focused pulsed infrared laser to locally induce a heat shock response in specific cells. Our method builds on and extends a previously reported method using a continuous-wave laser. In our technique, the pulsed laser illumination enables a much higher degree of spatial selectivity because of diffusion of heat between pulses. We apply our method to induce transient and long-term transgene expression in embryonic, larval, and adult cells. Our method allows highly selective spatiotemporal control of transgene expression and is a powerful tool for model organism biology.

Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. To address these limitations, we developed methods for quantitative imaging of individual animals cultivated in custom microfabricated multiwell substrates, and used them to elucidate molecular mechanisms underlying sleep. Here, we describe the steps necessary to design, produce, and image these plates, as well as analyze the resulting behavioral data. We also describe approaches for experimentally manipulating sleep. Following these procedures, after ~2 h of experimental preparation, we are able to simultaneously image 24 C. elegans from the second larval stage to adult stages or 20 Drosophila larvae during the second instar life stage at a spatial resolution of 10 or 27 µm, respectively. Although this system has been optimized to measure activity and quiescence in Caenorhabditis larvae and adults and in Drosophila larvae, it can also be used to assess other behaviors over short or long periods. Moreover, with minor modifications, it can be adapted for the behavioral monitoring of a wide range of small animals.Individual animals are cultivated in custom microfabricated multiwell substrates. This protocol describes how to design, produce, and image the plates, as well as analyze the resulting behavioral data.

Behavior varies even among genetically identical animals raised in the same environment. However, little is known about the circuit or anatomical origins of this individuality. Here, we demonstrate a neural correlate of Drosophila odor preference behavior in the olfactory sensory periphery. Namely, idiosyncratic calcium responses in projection neuron (PN) dendrites and densities of the presynaptic protein Bruchpilot in olfactory receptor neuron (ORN) axon terminals correlate with individual preferences in a choice between two aversive odorants. The ORN-PN synapse appears to be a locus of individuality where microscale variation gives rise to idiosyncratic behavior. Simulating microscale stochasticity in ORN-PN synapses of a 3062 neuron model of the antennal lobe recapitulates patterns of variation in PN calcium responses matching experiments. Conversely, stochasticity in other compartments of this circuit does not recapitulate those patterns. Our results demonstrate how physiological and microscale structural circuit variations can give rise to individual behavior, even when genetics and environment are held constant.

Schistosomiasis, caused by parasitic flatworms of the genus Schistosoma, is a neglected tropical disease affecting hundreds of millions globally. Praziquantel (PZQ), the only drug currently available for treatment and control, is largely ineffective against juvenile worms, and reports of PZQ resistance lend added urgency to the need for development of new therapeutics. Ion channels, which underlie electrical excitability in cells, are validated targets for many current anthelmintics. Transient receptor potential (TRP) channels are a large family of non-selective cation channels. TRP channels play key roles in sensory transduction and other critical functions, yet the properties of these channels have remained essentially unexplored in parasitic helminths. TRP channels fall into several (7-8) subfamilies, including TRPA and TRPV. Though schistosomes contain genes predicted to encode representatives of most of the TRP channel subfamilies, they do not appear to have genes for any TRPV channels. Nonetheless, we find that the TRPV1-selective activators capsaicin and resiniferatoxin (RTX) induce dramatic hyperactivity in adult worms; capsaicin also increases motility in schistosomula. SB 366719, a highly-selective TRPV1 antagonist, blocks the capsaicin-induced hyperactivity in adults. Mammalian TRPA1 is not activated by capsaicin, yet knockdown of the single predicted TRPA1-like gene (SmTRPA) in S. mansoni effectively abolishes capsaicin-induced responses in adult worms, suggesting that SmTRPA is required for capsaicin sensitivity in these parasites. Based on these results, we hypothesize that some schistosome TRP channels have novel pharmacological sensitivities that can be targeted to disrupt normal parasite neuromuscular function. These results also have implications for understanding the phylogeny of metazoan TRP channels and may help identify novel targets for new or repurposed therapeutics.