An efficient behavioral screening platform classifies natural products and other chemical cues according to their chemosensory valence in C. elegans.

Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction, and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multi-well plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals, but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.

Mechanosensitive membrane proteins: Usual and unusual suspects in mediating mechanotransduction.

This Viewpoint, which accompanies a Special Issue focusing on membrane mechanosensors, discusses unifying and unique features of both established and emerging mechanosensitive (MS) membrane proteins, their distribution across protein families and phyla, and current and future challenges in the study of these important proteins and their partners. MS membrane proteins are essential for tissue development, cellular motion, osmotic homeostasis, and sensing external and self-generated mechanical cues like those responsible for touch and proprioception. Though researchers' attention and this Viewpoint focus on a few famous ion channels that are considered the usual suspects as MS mechanosensors, we also discuss some of the more unusual suspects, such as G-protein coupled receptors. As the field continues to grow, so too will the list of proteins suspected to function as mechanosensors and the diversity of known MS membrane proteins.

Visualizing Neurons Under Tension In Vivo with Optogenetic Molecular Force Sensors.

The visualization of mechanical stress distribution in specific molecular networks within a living and physiologically active cell or animal remains a formidable challenge in mechanobiology. The advent of fluorescence-resonance energy transfer (FRET)-based molecular tension sensors overcame a significant hurdle that now enables us to address previously technically limited questions. Here, we describe a method that uses genetically encoded FRET tension sensors to visualize the mechanics of cytoskeletal networks in neurons of living animals with sensitized emission FRET and confocal scanning light microscopy. This method uses noninvasive immobilization of living animals to image neuronal ß-spectrin cytoskeleton at the diffraction limit, and leverages multiple imaging controls to verify and underline the quality of the measurements. In combination with a semiautomated machine-vision algorithm to identify and trace individual neurites, our analysis performs simultaneous calculation of FRET efficiencies and visualizes statistical uncertainty on a pixel by pixel basis. Our approach is not limited to genetically encoded spectrin tension sensors, but can also be used for any kind of ratiometric imaging in neuronal cells both in vivo and in vitro.

Image-based axon model highlights heterogeneity in initiation of damage.

Head injury simulations predict the occurrence of traumatic brain injury by placing a threshold on the calculated strains for axon tracts within the brain. However, a current roadblock to accurate injury prediction is the selection of an appropriate axon damage threshold. While several computational studies have used models of the axon cytoskeleton to investigate damage initiation, these models all employ an idealized, homogeneous axonal geometry. This homogeneous geometry with regularly spaced microtubules, evenly distributed throughout the model, overestimates axon strength because, in reality, the axon cytoskeleton is heterogeneous. In the heterogeneous cytoskeleton, the weakest cross section determines the initiation of failure, but these weak spots are not present in a homogeneous model. Addressing one source of heterogeneity in the axon cytoskeleton, we present a new semiautomated image analysis pipeline for using serial-section transmission electron micrographs to reconstruct the microtubule geometry of an axon. The image analysis procedure locates microtubules within the images, traces them throughout the image stack, and reconstructs the microtubule structure as a finite element mesh. We demonstrate the image analysis approach using a C. elegans touch receptor neuron due to the availability of high-quality serial-section transmission electron micrograph data sets. The results of the analysis highlight the heterogeneity of the microtubule structure in the spatial variation of both microtubule number and length. Simulations comparing this image-based geometry with homogeneous geometries show that structural heterogeneity in the image-based model creates significant spatial variation in deformation. The homogeneous geometries, on the other hand, deform more uniformly. Since no single homogeneous model can replicate the mechanical behavior of the image-based model, our results argue that heterogeneity in axon microtubule geometry should be considered in determining accurate axon failure thresholds.

Engineering Bright and Mechanosensitive Alkaline-Earth Rare-Earth Upconverting Nanoparticles.

Upconverting nanoparticles (UCNPs) are an emerging platform for mechanical force sensing at the nanometer scale. An outstanding challenge in realizing nanometer-scale mechano-sensitive UCNPs is maintaining a high mechanical force responsivity in conjunction with bright optical emission. This Letter reports mechano-sensing UCNPs based on the lanthanide dopants Yb3+ and Er3+, which exhibit a strong ratiometric change in emission spectra and bright emission under applied pressure. We synthesize and analyze the pressure response of five different types of nanoparticles, including cubic NaYF4 host nanoparticles and alkaline-earth host materials CaLuF, SrLuF, SrYbF, and BaLuF, all with lengths of 15 nm or less. By combining optical spectroscopy in a diamond anvil cell with single-particle brightness, we determine the noise equivalent sensitivity (GPa/√Hz) of these particles. The SrYb0.72Er0.28F@SrLuF particles exhibit an optimum noise equivalent sensitivity of 0.26 ± 0.04 GPa/√Hz. These particles present the possibility of robust nanometer-scale mechano-sensing.

Reciprocal interactions between transforming growth factor beta signaling and collagens: Insights from Caenorhabditis elegans.

Studies in genetically tractable organisms such as the nematode Caenorhabditis elegans have led to pioneering insights into conserved developmental regulatory mechanisms. For example, Smad signal transducers for the transforming growth factor beta (TGF-ß) superfamily were first identified in C. elegans and in the fruit fly Drosophila. Recent studies of TGF-ß signaling and the extracellular matrix (ECM) in C. elegans have forged unexpected links between signaling and the ECM, yielding novel insights into the reciprocal interactions that occur across tissues and spatial scales, and potentially providing new opportunities for the study of biomechanical regulation of gene expression.

DEG/ENaC/ASIC channels vary in their sensitivity to anti-hypertensive and non-steroidal anti-inflammatory drugs.

The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, and responding to acidification in the nervous system. They have two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers. Based on studies of selected family members, these channels are assumed to form nonvoltage-gated and sodium-selective channels sensitive to the anti-hypertensive drug amiloride. They are also emerging as a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Caenorhabditis elegans has more than two dozen genes encoding DEG/ENaC/ASIC subunits, providing an excellent opportunity to examine variations in drug sensitivity. Here, we analyze a subset of the C. elegans DEG/ENaC/ASIC proteins to test the hypothesis that individual family members vary not only in their ability to form homomeric channels but also in their drug sensitivity. We selected a panel of C. elegans DEG/ENaC/ASICs that are coexpressed in mechanosensory neurons and expressed gain-of-function or d mutants in Xenopus laevis oocytes. We found that only DEGT­1d, UNC­8d, and MEC­4d formed homomeric channels and that, unlike MEC­4d and UNC­8d, DEGT­1d channels were insensitive to amiloride and its analogues. As reported for rat ASIC1a, NSAIDs inhibit DEGT­1d and UNC­8d channels. Unexpectedly, MEC­4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the putative NSAID-binding site in the extracellular domain. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and that NSAIDs can both inhibit and potentiate these channels.

Touch-induced mechanical strain in somatosensory neurons is independent of extracellular matrix mutations in Caenorhabditis elegans.

Cutaneous mechanosensory neurons are activated by mechanical loads applied to the skin, and these stimuli are proposed to generate mechanical strain within sensory neurons. Using a microfluidic device to deliver controlled stimuli to intact animals and large, immobile, and fluorescent protein-tagged mitochondria as fiducial markers in the touch receptor neurons (TRNs), we visualized and measured touch-induced mechanical strain in Caenorhabditis elegans worms. At steady state, touch stimuli sufficient to activate TRNs induce an average strain of 3.1% at the center of the actuator and this strain decays to near zero at the edges of the actuator. We also measured strain in animals carrying mutations affecting links between the extracellular matrix (ECM) and the TRNs but could not detect any differences in touch-induced mechanical strain between wild-type and mutant animals. Collectively, these results demonstrate that touching the skin induces local mechanical strain in intact animals and suggest that a fully intact ECM is not essential for transmitting mechanical strain from the skin to cutaneous mechanosensory neurons.

Expansion microscopy of C. elegans.

We recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at ~70 nm resolution (with ~4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal.

Parallel Processing of Two Mechanosensory Modalities by a Single Neuron in C. elegans.

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.

Progressive recruitment of distal MEC-4 channels determines touch response strength in C. elegans.

Touch deforms, or strains, the skin beyond the immediate point of contact. The spatiotemporal nature of the touch-induced strain fields depend on the mechanical properties of the skin and the tissues below. Somatosensory neurons that sense touch branch out within the skin and rely on a set of mechano-electrical transduction channels distributed within their dendrites to detect mechanical stimuli. Here, we sought to understand how tissue mechanics shape touch-induced mechanical strain across the skin over time and how individual channels located in different regions of the strain field contribute to the overall touch response. We leveraged Caenorhabditis elegans' touch receptor neurons as a simple model amenable to in vivo whole-cell patch-clamp recording and an integrated experimental-computational approach to dissect the mechanisms underlying the spatial and temporal dynamics we observed. Consistent with the idea that strain is produced at a distance, we show that delivering strong stimuli outside the anatomical extent of the neuron is sufficient to evoke MRCs. The amplitude and kinetics of the MRCs depended on both stimulus displacement and speed. Finally, we found that the main factor responsible for touch sensitivity is the recruitment of progressively more distant channels by stronger stimuli, rather than modulation of channel open probability. This principle may generalize to somatosensory neurons with more complex morphologies.

Somatosensory neurons integrate the geometry of skin deformation and mechanotransduction channels to shape touch sensing.

Touch sensation hinges on force transfer across the skin and activation of mechanosensitive ion channels along the somatosensory neurons that invade the skin. This skin-nerve sensory system demands a quantitative model that spans the application of mechanical loads to channel activation. Unlike prior models of the dynamic responses of touch receptor neurons in Caenorhabditis elegans (Eastwood et al., 2015), which substituted a single effective channel for the ensemble along the TRNs, this study integrates body mechanics and the spatial recruitment of the various channels. We demonstrate that this model captures mechanical properties of the worm's body and accurately reproduces neural responses to simple stimuli. It also captures responses to complex stimuli featuring non-trivial spatial patterns, like extended or multiple contacts that could not be addressed otherwise. We illustrate the importance of these effects with new experiments revealing that skin-neuron composites respond to pre-indentation with increased currents rather than adapting to persistent stimulation.

Optically Robust and Biocompatible Mechanosensitive Upconverting Nanoparticles.

Upconverting nanoparticles (UCNPs) are promising tools for background-free imaging and sensing. However, their usefulness for in vivo applications depends on their biocompatibility, which we define by their optical performance in biological environments and their toxicity in living organisms. For UCNPs with a ratiometric color response to mechanical stress, consistent emission intensity and color are desired for the particles under nonmechanical stimuli. Here, we test the biocompatibility and mechanosensitivity of α-NaYF4:Yb,Er@NaLuF4 nanoparticles. First, we ligand-strip these particles to render them dispersible in aqueous media. Then, we characterize their mechanosensitivity (∼30% in the red-to-green spectral ratio per GPa), which is nearly 3-fold greater than those coated in oleic acid. We next design a suite of ex vivo and in vivo tests to investigate their structural and optical properties under several biorelevant conditions: over time in various buffers types, as a function of pH, and in vivo along the digestive tract of Caenorhabditis elegans worms. Finally, to ensure that the particles do not perturb biological function in C. elegans, we assess the chronic toxicity of nanoparticle ingestion using a reproductive brood assay. In these ways, we determine that mechanosensitive UCNPs are biocompatible, i.e., optically robust and nontoxic, for use as in vivo sensors to study animal digestion.

How Caenorhabditis elegans Senses Mechanical Stress, Temperature, and Other Physical Stimuli.

Caenorhabditis elegans lives in a complex habitat in which they routinely experience large fluctuations in temperature, and encounter physical obstacles that vary in size and composition. Their habitat is shared by other nematodes, by beneficial and harmful bacteria, and nematode-trapping fungi. Not surprisingly, these nematodes can detect and discriminate among diverse environmental cues, and exhibit sensory-evoked behaviors that are readily quantifiable in the laboratory at high resolution. Their ability to perform these behaviors depends on <100 sensory neurons, and this compact sensory nervous system together with powerful molecular genetic tools has allowed individual neuron types to be linked to specific sensory responses. Here, we describe the sensory neurons and molecules that enable C. elegans to sense and respond to physical stimuli. We focus primarily on the pathways that allow sensation of mechanical and thermal stimuli, and briefly consider this animal's ability to sense magnetic and electrical fields, light, and relative humidity. As the study of sensory transduction is critically dependent upon the techniques for stimulus delivery, we also include a section on appropriate laboratory methods for such studies. This chapter summarizes current knowledge about the sensitivity and response dynamics of individual classes of C. elegans mechano- and thermosensory neurons from in vivo calcium imaging and whole-cell patch-clamp electrophysiology studies. We also describe the roles of conserved molecules and signaling pathways in mediating the remarkably sensitive responses of these nematodes to mechanical and thermal cues. These studies have shown that the protein partners that form mechanotransduction channels are drawn from multiple superfamilies of ion channel proteins, and that signal transduction pathways responsible for temperature sensing in C. elegans share many features with those responsible for phototransduction in vertebrates.

Synaptic Communication upon Gentle Touch.

Gentle touch sensation in mammals depends on synaptic transmission from primary sensory cells (Merkel cells) to secondary sensory neurons. Hoffman et al. (2018) identify norepinephrine and ß2-adrendergic receptors as the neurotransmitter-receptor pair responsible for sustained touch responses. The findings may deepen understanding of how drugs affect touch and pain sensation.

Loss of CaMKI Function Disrupts Salt Aversive Learning in C. elegans.

The ability to adapt behavior to environmental fluctuations is critical for survival of organisms ranging from invertebrates to mammals. Caenorhabditis elegans can learn to avoid sodium chloride when it is paired with starvation. This behavior may help animals avoid areas without food. Although some genes have been implicated in this salt-aversive learning behavior, critical genetic components, and the neural circuit in which they act, remain elusive. Here, we show that the sole worm ortholog of mammalian CaMKI/IV, CMK-1, is essential for salt-aversive learning behavior in C. elegans hermaphrodites. We find that CMK-1 acts in the primary salt-sensing ASE neurons to regulate this behavior. By characterizing the intracellular calcium dynamics in ASE neurons using microfluidics, we find that loss of cmk-1 has subtle effects on sensory-evoked calcium responses in ASE axons and their modulation by salt conditioning. Our study implicates the expression of the conserved CaMKI/CMK-1 in chemosensory neurons as a regulator of behavioral plasticity to environmental salt in C. elegansSIGNIFICANCE STATEMENT Like other animals, the nematode Caenorhabditis elegans depends on salt for survival and navigates toward high concentrations of this essential mineral. In addition to its role as an essential nutrient, salt also causes osmotic stress at high concentrations. A growing body of evidence indicates that C. elegans balances the requirement for salt with the danger it presents through a process called salt-aversive learning. We show that this behavior depends on expression of a calcium/calmodulin-dependent kinase, CMK-1, in the ASE salt-sensing neurons. Our study identifies CMK-1 and salt-sensitive chemosensory neurons as key factors in this form of behavioral plasticity.

Bright, Mechanosensitive Upconversion with Cubic-Phase Heteroepitaxial Core-Shell Nanoparticles.

Lanthanide-doped nanoparticles are an emerging class of optical sensors, exhibiting sharp emission peaks, high signal-to-noise ratio, photostability, and a ratiometric color response to stress. The same centrosymmetric crystal field environment that allows for high mechanosensitivity in the cubic-phase (α), however, contributes to low upconversion quantum yield (UCQY). In this work, we engineer brighter mechanosensitive upconverters using a core-shell geometry. Sub-25 nm α-NaYF4:Yb,Er cores are shelled with an optically inert surface passivation layer of ∼4.5 nm thickness. Using different shell materials, including NaGdF4, NaYF4, and NaLuF4, we study how compressive to tensile strain influences the nanoparticles' imaging and sensing properties. All core-shell nanoparticles exhibit enhanced UCQY, up to 0.14% at 150 W/cm2, which rivals the efficiency of unshelled hexagonal-phase (ß) nanoparticles. Additionally, strain at the core-shell interface can tune mechanosensitivity. In particular, the compressive Gd shell results in the largest color response from yellow-green to orange or, quantitatively, a change in the red to green ratio of 12.2 ± 1.2% per GPa. For all samples, the ratiometric readouts are consistent over three pressure cycles from ambient to 5 GPa. Therefore, heteroepitaxial shelling significantly improves signal brightness without compromising the core's mechano-sensing capabilities and further, promotes core-shell cubic-phase nanoparticles as upcoming in vivo and in situ optical sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans.

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Ultrasound Elicits Behavioral Responses through Mechanical Effects on Neurons and Ion Channels in a Simple Nervous System.

Focused ultrasound has been shown to stimulate excitable cells, but the biophysical mechanisms behind this phenomenon remain poorly understood. To provide additional insight, we devised a behavioral-genetic assay applied to the well-characterized nervous system of Caenorhabditis elegans nematodes. We found that pulsed ultrasound elicits robust reversal behavior in wild-type animals in a pressure-, duration-, and pulse protocol-dependent manner. Responses were preserved in mutants unable to sense thermal fluctuations and absent in mutants lacking neurons required for mechanosensation. Additionally, we found that the worm's response to ultrasound pulses rests on the expression of MEC-4, a DEG/ENaC/ASIC ion channel required for touch sensation. Consistent with prior studies of MEC-4-dependent currents in vivo, the worm's response was optimal for pulses repeated 300-1000 times per second. Based on these findings, we conclude that mechanical, rather than thermal, stimulation accounts for behavioral responses. Further, we propose that acoustic radiation force governs the response to ultrasound in a manner that depends on the touch receptor neurons and MEC-4-dependent ion channels. Our findings illuminate a complete pathway of ultrasound action, from the forces generated by propagating ultrasound to an activation of a specific ion channel. The findings further highlight the importance of optimizing ultrasound pulsing protocols when stimulating neurons via ion channels with mechanosensitive properties.SIGNIFICANCE STATEMENT How ultrasound influences neurons and other excitable cells has remained a mystery for decades. Although it is widely understood that ultrasound can heat tissues and induce mechanical strain, whether or not neuronal activation depends on heat, mechanical force, or both physical factors is not known. We harnessed Caenorhabditis elegans nematodes and their extraordinary sensitivity to thermal and mechanical stimuli to address this question. Whereas thermosensory mutants respond to ultrasound similar to wild-type animals, mechanosensory mutants were insensitive to ultrasound stimulation. Additionally, stimulus parameters that accentuate mechanical effects were more effective than those producing more heat. These findings highlight a mechanical nature of the effect of ultrasound on neurons and suggest specific ways to optimize stimulation protocols in specific tissues.