1-Undecene from Pseudomonas aeruginosa is an olfactory signal for flight-or-fight response in Caenorhabditis elegans.

Animals possess conserved mechanisms to detect pathogens and to improve survival in their presence by altering their own behavior and physiology. Here, we utilize Caenorhabditis elegans as a model host to ask whether bacterial volatiles constitute microbe-associated molecular patterns. Using gas chromatography-mass spectrometry, we identify six prominent volatiles released by the bacterium Pseudomonas aeruginosa. We show that a specific volatile, 1-undecene, activates nematode odor sensory neurons inducing both flight and fight responses in worms. Using behavioral assays, we show that worms are repelled by 1-undecene and that this aversion response is driven by the detection of this volatile through AWB odor sensory neurons. Furthermore, we find that 1-undecene odor can induce immune effectors specific to P. aeruginosa via AWB neurons and that brief pre-exposure of worms to the odor enhances their survival upon subsequent bacterial infection. These results show that 1-undecene derived from P. aeruginosa serves as a pathogen-associated molecular pattern for the induction of protective responses in C. elegans.

C. elegans foraging as a model for understanding the neuronal basis of decision-making.

Animals have evolved to seek, select, and exploit food sources in their environment. Collectively termed foraging, these ubiquitous behaviors are necessary for animal survival. As a foundation for understanding foraging, behavioral ecologists established early theoretical and mathematical frameworks which have been subsequently refined and supported by field and laboratory studies of foraging animals. These simple models sought to explain how animals decide which strategies to employ when locating food, what food items to consume, and when to explore the environment for new food sources. These foraging decisions involve integration of prior experience with multimodal sensory information about the animal's current environment and internal state. We suggest that the nematode Caenorhabditis elegans is well-suited for a high-resolution analysis of complex goal-oriented behaviors such as foraging. We focus our discussion on behavioral studies highlighting C. elegans foraging on bacteria and summarize what is known about the underlying neuronal and molecular pathways. Broadly, we suggest that this simple model system can provide a mechanistic understanding of decision-making and present additional avenues for advancing our understanding of complex behavioral processes.

Intestine-to-neuronal signaling alters risk-taking behaviors in food-deprived Caenorhabditis elegans.

Animals integrate changes in external and internal environments to generate behavior. While neural circuits detecting external cues have been mapped, less is known about how internal states like hunger are integrated into behavioral outputs. Here, we use the nematode C. elegans to examine how changes in internal nutritional status affect chemosensory behaviors. We show that acute food deprivation leads to a reversible decline in repellent, but not attractant, sensitivity. This behavioral change requires two conserved transcription factors MML-1 (MondoA) and HLH-30 (TFEB), both of which translocate from the intestinal nuclei to the cytoplasm during food deprivation. Next, we identify the insulin-like peptide INS-31 as a candidate ligand relaying food-status signals from the intestine to other tissues. Further, we show that neurons likely use the DAF-2 insulin receptor and AGE-1/PI-3 Kinase, but not DAF-16/FOXO to integrate these intestine-released peptides. Altogether, our study shows how internal food status signals are integrated by transcription factors and intestine-neuron signaling to generate flexible behaviors via the gut-brain axis.

Well-free agglomeration and on-demand three-dimensional cell cluster formation using guided surface acoustic waves through a couplant layer.

Three-dimensional cell agglomerates are broadly useful in tissue engineering and drug testing. We report a well-free method to form large (1.4-mm) multicellular clusters using 100-MHz surface acoustic waves (SAW) without direct contact with the media or cells. A fluid couplant is used to transform the SAW into acoustic streaming in the cell-laden media held in a petri dish. The couplant transmits longitudinal sound waves, forming a Lamb wave in the petri dish that, in turn, produces longitudinal sound in the media. Due to recirculation, human embryonic kidney (HEK293) cells in the dish are carried to the center of the coupling location, forming a cluster in less than 10 min. A few minutes later, these clusters may then be translated and merged to form large agglomerations, and even repeatedly folded to produce a roughly spherical shape of over 1.4 mm in diameter for incubation-without damaging the existing intercellular bonds. Calcium ion signaling through these clusters and confocal images of multiprotein junctional complexes suggest a continuous tissue construct: intercellular communication. They may be formed at will, and the method is feasibly useful for formation of numerous agglomerates in a single petri dish.

Neural network features distinguish chemosensory stimuli in Caenorhabditis elegans.

Nervous systems extract and process information from the environment to alter animal behavior and physiology. Despite progress in understanding how different stimuli are represented by changes in neuronal activity, less is known about how they affect broader neural network properties. We developed a framework for using graph-theoretic features of neural network activity to predict ecologically relevant stimulus properties, in particular stimulus identity. We used the transparent nematode, Caenorhabditis elegans, with its small nervous system to define neural network features associated with various chemosensory stimuli. We first immobilized animals using a microfluidic device and exposed their noses to chemical stimuli while monitoring changes in neural activity of more than 50 neurons in the head region. We found that graph-theoretic features, which capture patterns of interactions between neurons, are modulated by stimulus identity. Further, we show that a simple machine learning classifier trained using graph-theoretic features alone, or in combination with neural activity features, can accurately predict salt stimulus. Moreover, by focusing on putative causal interactions between neurons, the graph-theoretic features were almost twice as predictive as the neural activity features. These results reveal that stimulus identity modulates the broad, network-level organization of the nervous system, and that graph theory can be used to characterize these changes.

Intraguild predation between Pristionchus pacificus and Caenorhabditis elegans: a complex interaction with the potential for aggressive behaviour.

The related nematodes Pristionchus pacificus and Caenorhabditis elegans both eat bacteria for nutrition and are therefore competitors when they exploit the same bacterial resource. In addition to competing with each other, P. pacificus is a predator of C. elegans larval prey. These two relationships together form intraguild predation, which is the killing and sometimes eating of potential competitors. In killing C. elegans, the intraguild predator P. pacificus may achieve dual benefits of immediate nutrition and reduced competition for bacteria. Recent studies of P. pacificus have characterized many aspects of its predatory biting behaviour as well as underlying molecular and genetic mechanisms. However, little has been explored regarding the potentially competitive aspect of P. pacificus biting C. elegans. Moreover, aggression may also be implicated if P. pacificus intentionally bites C. elegans with the goal of reducing competition for bacteria. The aim of this review is to broadly outline how aggression, predation, and intraguild predation relate to each other, as well as how these concepts may be applied to future studies of P. pacificus in its interactions with C. elegans.

A hub-and-spoke circuit drives pheromone attraction and social behaviour in C. elegans.

Innate social behaviours emerge from neuronal circuits that interpret sensory information on the basis of an individual's own genotype, sex and experience. The regulated aggregation behaviour of the nematode Caenorhabditis elegans, a simple animal with only 302 neurons, is an attractive system to analyse these circuits. Wild social strains of C. elegans aggregate in the presence of specific sensory cues, but solitary strains do not. Here we identify the RMG inter/motor neuron as the hub of a regulated circuit that controls aggregation and related behaviours. RMG is the central site of action of the neuropeptide receptor gene npr-1, which distinguishes solitary strains (high npr-1 activity) from wild social strains (low npr-1 activity); high RMG activity is essential for all aspects of social behaviour. Anatomical gap junctions connect RMG to several classes of sensory neurons known to promote aggregation, and to ASK sensory neurons, which are implicated in male attraction to hermaphrodite pheromones. We find that ASK neurons respond directly to pheromones, and that high RMG activity enhances ASK responses in social strains, causing hermaphrodite attraction to pheromones at concentrations that repel solitary hermaphrodites. The coordination of social behaviours by RMG suggests an anatomical hub-and-spoke model for sensory integration in aggregation, and points to functions for related circuit motifs in the C. elegans wiring diagram.

Ocean to Tree: Leveraging Single-Molecule RNA-Seq to Repair Genome Gene Models and Improve Phylogenomic Analysis of Gene and Species Evolution.

Understanding gene evolution across genomes and organisms, including ctenophores, can provide unexpected biological insights. It enables powerful integrative approaches that leverage sequence diversity to advance biomedicine. Sequencing and bioinformatic tools can be inexpensive and user-friendly, but numerous options and coding can intimidate new users. Distinct challenges exist in working with data from diverse species but may go unrecognized by researchers accustomed to gold-standard genomes. Here, we provide a high-level workflow and detailed pipeline to enable animal collection, single-molecule sequencing, and phylogenomic analysis of gene and species evolution. As a demonstration, we focus on (1) PacBio RNA-seq of the genome-sequenced ctenophore Mnemiopsis leidyi, (2) diversity and evolution of the mechanosensitive ion channel Piezo in genetic models and basal-branching animals, and (3) associated challenges and solutions to working with diverse species and genomes, including gene model updating and repair using single-molecule RNA-seq. We provide a Python Jupyter Notebook version of our pipeline (GitHub Repository: Ctenophore-Ocean-To-Tree-2023 https://github.com/000generic/Ctenophore-Ocean-To-Tree-2023 ) that can be run for free in the Google Colab cloud to replicate our findings or modified for specific or greater use. Our protocol enables users to design new sequencing projects in ctenophores, marine invertebrates, or other novel organisms. It provides a simple, comprehensive platform that can ease new user entry into running their evolutionary sequence analyses.

Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans.

Although many properties of the nervous system are shared among animals and systems, it is not known whether different neuronal circuits use common strategies to guide behaviour. Here we characterize information processing by Caenorhabditis elegans olfactory neurons (AWC) and interneurons (AIB and AIY) that control food- and odour-evoked behaviours. Using calcium imaging and mutations that affect specific neuronal connections, we show that AWC neurons are activated by odour removal and activate the AIB interneurons through AMPA-type glutamate receptors. The level of calcium in AIB interneurons is elevated for several minutes after odour removal, a neuronal correlate to the prolonged behavioural response to odour withdrawal. The AWC neuron inhibits AIY interneurons through glutamate-gated chloride channels; odour presentation relieves this inhibition and results in activation of AIY interneurons. The opposite regulation of AIY and AIB interneurons generates a coordinated behavioural response. Information processing by this circuit resembles information flow from vertebrate photoreceptors to 'OFF' bipolar and 'ON' bipolar neurons, indicating a conserved or convergent strategy for sensory information processing.

Conserved autism-associated genes tune social feeding behavior in C. elegans.

Animal foraging is an essential and evolutionarily conserved behavior that occurs in social and solitary contexts, but the underlying molecular pathways are not well defined. We discover that conserved autism-associated genes (NRXN1(nrx-1), NLGN3(nlg-1), GRIA1,2,3(glr-1), GRIA2(glr-2), and GLRA2,GABRA3(avr-15)) regulate aggregate feeding in C. elegans, a simple social behavior. NRX-1 functions in chemosensory neurons (ADL and ASH) independently of its postsynaptic partner NLG-1 to regulate social feeding. Glutamate from these neurons is also crucial for aggregate feeding, acting independently of NRX-1 and NLG-1. Compared to solitary counterparts, social animals show faster presynaptic release and more presynaptic release sites in ASH neurons, with only the latter requiring nrx-1. Disruption of these distinct signaling components additively converts behavior from social to solitary. Aggregation induced by circuit activation is also dependent on nrx-1. Collectively, we find that aggregate feeding is tuned by conserved autism-associated genes through complementary synaptic mechanisms, revealing molecular principles driving social feeding.

Dopamine signaling regulates predator-driven changes in Caenorhabditis elegans' egg laying behavior.

Prey respond to predators by altering their behavior to optimize their own fitness and survival. Specifically, prey are known to avoid predator-occupied territories to reduce their risk of harm or injury to themselves and their progeny. We probe the interactions between Caenorhabditis elegans and its naturally cohabiting predator Pristionchus uniformis to reveal the pathways driving changes in prey behavior. While C. elegans prefers to lay its eggs on a bacteria food lawn, the presence of a predator inside a lawn induces C. elegans to lay more eggs away from that lawn. We confirm that this change in egg laying is in response to bites from predators, rather than to predatory secretions. Moreover, predator-exposed prey continue to lay their eggs away from the dense lawn even after the predator is removed, indicating a form of learning. Next, we find that mutants in dopamine synthesis significantly reduce egg laying behavior off the lawn in both predator-free and predator-inhabited lawns, which we can rescue by transgenic complementation or supplementation with exogenous dopamine. Moreover, we find that dopamine is likely released from multiple dopaminergic neurons and requires combinations of both D1- (DOP-1) and D2-like (DOP-2 and DOP-3) dopamine receptors to alter predator-induced egg laying behavior, whereas other combinations modify baseline levels of egg laying behavior. Together, we show that dopamine signaling can alter both predator-free and predator-induced foraging strategies, suggesting a role for this pathway in defensive behaviors.

Mutational analysis of mechanosensitive ion channels in the carnivorous Venus flytrap plant.

How the Venus flytrap (Dionaea muscipula) evolved the remarkable ability to sense, capture, and digest animal prey for nutrients has long puzzled the scientific community.1 Recent genome and transcriptome sequencing studies have provided clues to the genes thought to play a role in these tasks.2,3,4,5 However, proving a causal link between these and any aspect of the plant's hunting behavior has been challenging due to the genetic intractability of this non-model organism. Here, we use CRISPR-Cas9 methods to generate targeted modifications in the Venus flytrap genome. The plant detects prey using touch-sensitive trigger hairs located on its bilobed leaves.6 Upon bending, these hairs convert mechanical touch signals into changes in the membrane potential of sensory cells, leading to rapid closure of the leaf lobes to ensnare the animal.7 Here, we generate mutations in trigger-hair-expressed MscS-like (MSL)-family mechanosensitive ion channel genes FLYCATCHER1 (FLYC1) and FLYCATCHER2 (FLYC2)5 and find that double-mutant plants have a reduced leaf-closing response to mechanical ultrasound stimulation. While we cannot exclude off-target effects of the CRISPR-Cas9 system, our genetic analysis is consistent with these and other functionally redundant mechanosensitive ion channels acting together to generate the sensory system necessary for prey detection.

Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.

Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.

Flexible reprogramming of Pristionchus pacificus motivation for attacking Caenorhabditis elegans in predator-prey competition.

Animals with diverse diets must adapt their food priorities to a wide variety of environmental conditions. This diet optimization problem is especially complex for predators that compete with prey for food. Although predator-prey competition is widespread and ecologically critical, it remains difficult to disentangle predatory and competitive motivations for attacking competing prey. Here, we dissect the foraging decisions of the omnivorous nematode Pristionchus pacificus to reveal that its seemingly failed predatory attempts against Caenorhabditis elegans are actually motivated acts of efficacious territorial aggression. While P. pacificus easily kills and eats larval C. elegans with a single bite, adult C. elegans typically survives and escapes bites. However, non-fatal biting can provide competitive benefits by reducing access of adult C. elegans and its progeny to bacterial food that P. pacificus also eats. We show that the costs and benefits of both predatory and territorial outcomes influence how P. pacificus decides which food goal, prey or bacteria, should guide its motivation for biting. These predatory and territorial motivations impose different sets of rules for adjusting willingness to bite in response to changes in bacterial abundance. In addition to biting, predatory and territorial motivations also influence which search tactic P. pacificus uses to increase encounters with C. elegans. When treated with an octopamine receptor antagonist, P. pacificus switches from territorial to predatory motivation for both biting and search. Overall, we demonstrate that P. pacificus assesses alternate outcomes of attacking C. elegans and flexibly reprograms its foraging strategy to prioritize either prey or bacterial food.

Fear conditioning in invertebrates.

Learning to identify and predict threats is a basic skill that allows animals to avoid harm. Studies in invertebrates like Aplysia californica, Drosophila melanogaster, and Caenorhabditis elegans have revealed that the basic mechanisms of learning and memory are conserved. We will summarize these studies and highlight the common pathways and mechanisms in invertebrate fear-associated behavioral changes. Fear conditioning studies utilizing electric shock in Aplysia and Drosophila have demonstrated that serotonin or dopamine are typically involved in relaying aversive stimuli, leading to changes in intracellular calcium levels and increased presynaptic neurotransmitter release and short-term changes in behavior. Long-term changes in behavior typically require multiple, spaced trials, and involve changes in gene expression. C. elegans studies have demonstrated these basic aversive learning principles as well; however, fear conditioning has yet to be explicitly demonstrated in this model due to stimulus choice. Because predator-prey relationships can be used to study learned fear in a naturalistic context, this review also summarizes what is known about predator-induced behaviors in these three organisms, and their potential applications for future investigations into fear conditioning.

Two pathways are required for ultrasound-evoked behavioral changes in Caenorhabditis elegans.

Ultrasound has been shown to affect the function of both neurons and non-neuronal cells, but, the underlying molecular machinery has been poorly understood. Here, we show that at least two mechanosensitive proteins act together to generate C. elegans behavioral responses to ultrasound stimuli. We first show that these animals generate reversals in response to a single 10 msec pulse from a 2.25 MHz ultrasound transducer. Next, we show that the pore-forming subunit of the mechanosensitive channel TRP-4, and a DEG/ENaC/ASIC ion channel MEC-4, are both required for this ultrasound-evoked reversal response. Further, the trp-4;mec-4 double mutant shows a stronger behavioral deficit compared to either single mutant. Finally, overexpressing TRP-4 in specific chemosensory neurons can rescue the ultrasound-triggered behavioral deficit in the mec-4 null mutant, suggesting that both TRP-4 and MEC-4 act together in affecting behavior. Together, we demonstrate that multiple mechanosensitive proteins likely cooperate to transform ultrasound stimuli into behavioral changes.

Microscale concert hall acoustics to produce uniform ultrasound stimulation for targeted sonogenetics in hsTRPA1-transfected cells.

The field of ultrasound neuromodulation has rapidly developed over the past decade, a consequence of the discovery of strain-sensitive structures in the membrane and organelles of cells extending into the brain, heart, and other organs. Notably, clinical trials are underway for treating epilepsy using focused ultrasound to elicit an organized local electrical response. A key limitation to this approach is the formation of standing waves within the skull. In standing acoustic waves, the maximum ultrasound intensity spatially varies from near zero to double the mean in one half a wavelength, and has lead to localized tissue damage and disruption of normal brain function while attempting to evoke a broader response. This phenomenon also produces a large spatial variation in the actual ultrasound exposure in tissue, leading to heterogeneous results and challenges with interpreting these effects. One approach to overcome this limitation is presented herein: transducer-mounted diffusers that result in spatiotemporally incoherent ultrasound. Herein, we numerically and experimentally quantified the effect of a diffuser in an enclosed domain, and show that adding the diffuser leads to a two-fold increase in ultrasound responsiveness of hsTRPA1 transfected HEK cells. Furthermore, we demonstrate the diffuser allow us to produce an uniform spatial distribution of pressure in the rodent skull. Collectively, we propose that our approach leads to a means to deliver uniform ultrasound into irregular cavities for sonogenetics.

Ultrasound Mediated Cellular Deflection Results in Cellular Depolarization.

Ultrasound has been used to manipulate cells in both humans and animal models. While intramembrane cavitation and lipid clustering have been suggested as likely mechanisms, they lack experimental evidence. Here, high-speed digital holographic microscopy (kiloHertz order) is used to visualize the cellular membrane dynamics. It is shown that neuronal and fibroblast membranes deflect about 150 nm upon ultrasound stimulation. Next, a biomechanical model that predicts changes in membrane voltage after ultrasound exposure is developed. Finally, the model predictions are validated using whole-cell patch clamp electrophysiology on primary neurons. Collectively, it is shown that ultrasound stimulation directly defects the neuronal membrane leading to a change in membrane voltage and subsequent depolarization. The model is consistent with existing data and provides a mechanism for both ultrasound-evoked neurostimulation and sonogenetic control.

Sonogenetic control of mammalian cells using exogenous Transient Receptor Potential A1 channels.

Ultrasound has been used to non-invasively manipulate neuronal functions in humans and other animals. However, this approach is limited as it has been challenging to target specific cells within the brain or body. Here, we identify human Transient Receptor Potential A1 (hsTRPA1) as a candidate that confers ultrasound sensitivity to mammalian cells. Ultrasound-evoked gating of hsTRPA1 specifically requires its N-terminal tip region and cholesterol interactions; and target cells with an intact actin cytoskeleton, revealing elements of the sonogenetic mechanism. Next, we use calcium imaging and electrophysiology to show that hsTRPA1 potentiates ultrasound-evoked responses in primary neurons. Furthermore, unilateral expression of hsTRPA1 in mouse layer V motor cortical neurons leads to c-fos expression and contralateral limb responses in response to ultrasound delivered through an intact skull. Collectively, we demonstrate that hsTRPA1-based sonogenetics can effectively manipulate neurons within the intact mammalian brain, a method that could be used across species.