Automated Multimodal Stimulation and Simultaneous Neuronal Recording from Multiple Small Organisms.

Fluorescent genetically encoded calcium indicators have contributed greatly to our understanding of neural dynamics from the level of individual neurons to entire brain circuits. However, neural responses may vary due to prior experience, internal states, or stochastic factors, thus generating the need for methods that can assess neural function across many individuals at once. Whereas most recording techniques examine a single animal at a time, we describe the use of wide-field microscopy to scale up neuronal recordings to dozens of Caenorhabditis elegans or other sub-millimeter-scale organisms at once. Open-source hardware and software allow great flexibility in programming fully automated experiments that control the intensity and timing of various stimulus types, including chemical, optical, mechanical, thermal, and electromagnetic stimuli. In particular, microfluidic flow devices provide precise, repeatable, and quantitative control of chemosensory stimuli with sub-second time resolution. The NeuroTracker semi-automated data analysis pipeline then extracts individual and population-wide neural responses to uncover functional changes in neural excitability and dynamics. This paper presents examples of measuring neuronal adaptation, temporal inhibition, and stimulus crosstalk. These techniques increase the precision and repeatability of stimulation, allow the exploration of population variability, and are generalizable to other dynamic fluorescent signals in small biosystems from cells and organoids to whole organisms and plants.

Monitoring neural activity during sleep/wake events in adult C. elegans by automated sleep detection and stimulation.

Sleep in adult C. elegans occurs spontaneously, making timing of individual sleep/wake state transitions unpredictable. This protocol presents a closed-loop system to automatically detect sleep state transitions, trigger stimulation, and record evoked neural responses. This allows users to assess functional consequences of behavioral states in an unbiased manner and identify state-dependent neuromodulation. This closed-loop system is flexible and can be configured to detect any visible events, such as behavior patterns or optical reporters, and measure corresponding evoked neural responses. For complete details on the use and execution of this protocol, please refer to Lawler et al. (2021).

Microfluidic Devices for Behavioral Analysis, Microscopy, and Neuronal Imaging in Caenorhabditis elegans.

Microfluidic devices offer several advantages for C. elegans research, particularly for presenting precise physical and chemical environments, immobilizing animals during imaging, quantifying behavior, and automating screens. However, challenges to their widespread adoption in the field include increased complexity over conventional methods, operational problems (such as clogging, leaks, and bubbles), difficulty in obtaining or fabricating devices, and the need to characterize biological results obtained from new assay formats. Here we describe the preparation and operation of simple, reusable microfluidic devices for quantifying behavioral responses to chemical patterns, and single-use devices to arrange animals for time-lapse microscopy and to measure neuronal activity. We focus on details that eliminate or reduce the frustrations commonly experienced by new users of microfluidic devices.

A polymer index-matched to water enables diverse applications in fluorescence microscopy.

We demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

Sleep Analysis in Adult C. elegans Reveals State-Dependent Alteration of Neural and Behavioral Responses.

Sleep, a state of quiescence associated with growth and restorative processes, is conserved across species. Invertebrates including the nematode Caenorhabditis elegans exhibit sleep-like states during development, satiety, and stress. Here, we describe behavior and neural activity during sleep and awake states in adult C. elegans hermaphrodites using new microfluidic methods. We observed effects of fluid flow, oxygen, feeding, odors, and genetic perturbations on long-term sleep behavior over 12 h. We developed a closed-loop sleep detection system to automatically deliver chemical stimuli to assess sleep-dependent changes to evoked neural responses in individual animals. Sleep increased the arousal threshold to aversive stimulation, yet the associated sensory neuron and first-layer interneuron responses were unchanged. This localizes adult sleep-dependent neuromodulation within interneurons presynaptic to the premotor interneurons, rather than afferent sensory circuits. However, sleep prolonged responses in appetitive chemosensory neurons, suggesting that sleep modulates responsiveness specifically across sensory systems rather than broadly damping global circuit activity.SIGNIFICANCE STATEMENT Much is known about molecular mechanisms that facilitate sleep control. However, it is unclear how these pathways modulate neural circuit-level sensory processing or how misregulation of neural activity contributes to sleep disorders. The nematode Caenorhabditis elegans provides the ability to study neural circuitry with single-neuron resolution, and recent studies examined sleep states between developmental stages and when stressed. Here, we examine an additional form of spontaneous sleep in adult C. elegans at the behavioral and neural activity levels. Using a closed-loop system, we show that delayed behavioral responses to aversive chemical stimulation during sleep arise from sleep-dependent sensorimotor modulation localized presynaptic to the premotor circuit, rather than early sensory circuits.

Phosphofructokinase relocalizes into subcellular compartments with liquid-like properties in vivo.

Although much is known about the biochemical regulation of glycolytic enzymes, less is understood about how they are organized inside cells. We systematically examine the dynamic subcellular localization of glycolytic protein phosphofructokinase-1/PFK-1.1 in Caenorhabditis elegans. We determine that endogenous PFK-1.1 localizes to subcellular compartments in vivo. In neurons, PFK-1.1 forms phase-separated condensates near synapses in response to energy stress from transient hypoxia. Restoring animals to normoxic conditions results in cytosolic dispersion of PFK-1.1. PFK-1.1 condensates exhibit liquid-like properties, including spheroid shapes due to surface tension, fluidity due to deformations, and fast internal molecular rearrangements. Heterologous self-association domain cryptochrome 2 promotes formation of PFK-1.1 condensates and recruitment of aldolase/ALDO-1. PFK-1.1 condensates do not correspond to stress granules and might represent novel metabolic subcompartments. Our studies indicate that glycolytic protein PFK-1.1 can dynamically form condensates in vivo.

Automated Functional Screening for Modulators of Optogenetically Activated Neural Responses in Living Organisms.

All-optical methods of probing in vivo brain function are advantageous for their compatibility with automated microscopy and fast spatial targeting of neural circuit excitation and response. Recent advances in optogenetic technologies allow simultaneous light activation of specific neurons and optical readout of neural activity via fluorescent calcium reporters, providing an attractive opportunity for high-throughput screening assays that directly assess dynamic neural function in vivo. Here we describe a method to automatically record optogenetically activated neural responses in living, hydrogel-embedded organisms over many hours in a multiwell plate format. This method is suitable for screening the neural effects of hundreds of chemical compounds and assessing the time course of bioactivity over 12 h or more. As examples, we show the suppression of neural responses over time with various concentrations of two voltage-gated calcium channel blockers and a full-plate screen of 320 chemicals with positive and negative controls in a single experiment.

Silk Particle Production Based on silk/PVA Phase Separation Using a Microfabricated Co-flow Device.

Polymeric particles are ideal drug delivery systems due to their cellular uptake-relevant size. Microparticles could be developed for direct injection of drug formulations into a diseased site, such as a tumor, allowing for drug retention and slow drug exposure over time through sustained release mechanisms. Bombyx mori silk fibroin has shown promise as a biocompatible biomaterial both in research and the clinic. Silk has been previously used to make particles using an emulsion-based method with poly(vinyl alcohol) (PVA). In this study, polydimethylsiloxane-based microfluidic devices were designed, fabricated, and characterized to produce silk particles through self-association of silk when exposed to PVA. Three main variables resulted in differences in particle size and size distribution, or polydispersity index (PDI). Utilizing a co-flow microfluidic device decreased the PDI of the silk particles as compared to an emulsion-based method (0.13 versus 0.65, respectively). With a flow-focusing microfluidics device, lowering the silk flow rate from 0.80 to 0.06 mL/h resulted in a decrease in the median particle size from 6.8 to 3.0 µm and the PDI from 0.12 to 0.05, respectively. Lastly, decreasing the silk concentration from 12% to 2% resulted in a decrease in the median particle size from 5.6 to 2.8 µm and the PDI from 0.81 to 0.25, respectively. Binding and release of doxorubicin, a cytotoxic drug commonly used for cancer treatment, with the fabricated silk particles was evaluated. Doxorubicin loading in the silk particles was approximately 41 µg/mg; sustained doxorubicin release occurred over 23 days. When the cytotoxicity of the released doxorubicin was tested on KELLY neuroblastoma cells, significant cell death was observed. To demonstrate the potential for internalization of the silk particles, both KELLY and THP-1-derived macrophages were exposed to fluorescently labelled silk particles for up to 24 h. With the macrophages, internalization of the silk particles was observed. Additionally, THP-1 derived macrophages exposure to silk particles increased TNF-α secretion. Overall, this microfluidics-based approach for fabricating silk particles utilizing PVA as a means to induce phase separation and silk self-assembly is a promising approach to control particle size and size distribution. These silk particles may be utilized for a variety of biomedical applications including drug delivery to multiple cell types within a tumor microenvironment.

Rapid and gentle hydrogel encapsulation of living organisms enables long-term microscopy over multiple hours.

Imaging living organisms at high spatial resolution requires effective and innocuous immobilization. Long-term imaging places further demands on sample mounting with minimal perturbation of the organism. Here we present a simple, inexpensive method for rapid encapsulation of small animals of any developmental stage within a photo-crosslinked polyethylene glycol (PEG) hydrogel, gently restricting movement within their confined spaces. Immobilized animals maintain their original morphology in a hydrated environment compatible with chemical treatment, optical stimulation, and light-sheet microscopy. We demonstrate prolonged three-dimensional imaging of neural responses in the nematode Caenorhabditis elegans, recovery of viable organisms after 24 h, and imaging of larger squid hatchlings. We characterize a range of hydrogel and illumination conditions for immobilization quality, and identify paralytic-free conditions suitable for high-resolution single-cell imaging. Overall, PEG hydrogel encapsulation provides fast, versatile, and gentle mounting of small living organisms, from yeast to zebrafish, for continuous observation over hours.

Automated fluid delivery from multiwell plates to microfluidic devices for high-throughput experiments and microscopy.

High-throughput biological and chemical experiments typically use either multiwell plates or microfluidic devices to analyze numerous independent samples in a compact format. Multiwell plates are convenient for screening chemical libraries in static fluid environments, whereas microfluidic devices offer immense flexibility in flow control and dynamics. Interfacing these platforms in a simple and automated way would introduce new high-throughput experimental capabilities, such as compound screens with precise exposure timing. Whereas current approaches to integrate microfluidic devices with multiwell plates remain expensive or technically complicated, we present here a simple open-source robotic system that delivers liquids sequentially through a single connected inlet. We first characterized reliability and performance by automatically delivering 96 dye solutions to a microfluidic device. Next, we measured odor dose-response curves of in vivo neural activity from two sensory neuron types in dozens of living C. elegans in a single experiment. We then identified chemicals that suppressed optogenetically-evoked neural activity, demonstrating a functional screening platform for neural modulation in whole organisms. Lastly, we automated an 85-minute, ten-step cell staining protocol. Together, these examples show that our system can automate various protocols and accelerate experiments by economically bridging two common elements of high-throughput systems: multiwell plates and microfluidics.

A Single-Neuron Chemosensory Switch Determines the Valence of a Sexually Dimorphic Sensory Behavior.

Biological sex, a fundamental dimension of internal state, can modulate neural circuits to generate behavioral variation. Understanding how and why circuits are tuned by sex can provide important insights into neural and behavioral plasticity. Here we find that sexually dimorphic behavioral responses to C. elegans ascaroside sex pheromones are implemented by the functional modulation of shared chemosensory circuitry. In particular, the sexual state of a single sensory neuron pair, ADF, determines the nature of an animal's behavioral response regardless of the sex of the rest of the body. Genetic feminization of ADF causes males to be repelled by, rather than attracted to, ascarosides, whereas masculinization of ADF has the opposite effect in hermaphrodites. When ADF is ablated, both sexes are weakly repelled by ascarosides. Genetic sex modulates ADF function by tuning chemosensation: although ADF is functional in both sexes, it detects the ascaroside ascr#3 only in males, a consequence of cell-autonomous action of the master sexual regulator tra-1. This occurs in part through the conserved DM-domain gene mab-3, which promotes the male state of ADF. The sexual modulation of ADF has a key role in reproductive fitness, as feminization or ablation of ADF renders males unable to use ascarosides to locate mates. Our results reveal an economical mechanism in which sex-specific behavioral valence arises through the cell-autonomous regulation of a chemosensory switch by genetic sex, allowing a social cue with salience for both sexes to elicit navigational responses commensurate with the differing needs of each.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons.

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Optical Method to Quantify Mechanical Contraction and Calcium Transients of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

Differentiation of human pluripotent stem cells into cardiomyocytes (hPS-CMs) holds promise for myocardial regeneration therapies, drug discovery, and models of cardiac disease. Potential cardiotoxicities may affect hPS-CM mechanical contraction independent of calcium signaling. Herein, a method using an image capture system is described to measure hPS-CM contractility and intracellular calcium concurrently, with high spatial and temporal resolution. The image capture system rapidly alternates between brightfield and epifluorescent illumination of contracting cells. Mechanical contraction is quantified by a speckle tracking algorithm applied to brightfield image pairs, whereas calcium transients are measured by a fluorescent calcium reporter. This technique captured changes in contractile strain, calcium transients, and beat frequency of hPS-CMs over 21 days in culture, as well as acute responses to isoproterenol and Cytochalasin D. The technique described above can be applied without the need to alter the culture platform, allowing for determination of hPS-CM behavior over weeks in culture for drug discovery and myocardial regeneration applications.

Anti-Fas conjugated hyaluronic acid microsphere gels for neural stem cell delivery.

Central nervous system (CNS) injuries and diseases result in neuronal damage and loss of function. Transplantation of neural stem cells (NSCs) has been shown to improve locomotor function after transplantation. However, due to the immune and inflammatory response at the injury site, the survival rate of the engrafted cells is low. Engrafted cell viability has been shown to increase when transplanted within a hydrogel. Hyaluronic acid (HA) hydrogels have natural anti-inflammatory properties and the backbone can be modified to introduce bioactive agents, such as anti-Fas, which we have previously shown to promote NSC survival while suppressing immune cell activity in bulk hydrogels in vitro. Although bulk HA hydrogels have shown to promote stem cell survival, microsphere gels for NSC encapsulation and delivery may have additional advantages. In this study, a flow-focusing microfluidic device was used to fabricate either vinyl sulfone-modified HA (VS-HA) or anti-Fas-conjugated HA (anti-Fas HA) microsphere gels encapsulated with NSCs. The majority of encapsulated NSCs remained viable for at least 24 h in the VS-HA and anti-Fas HA microsphere gels. Moreover, T-cells cultured in suspension with the anti-Fas HA microsphere gels had reduced viability after contact with the microsphere gels compared to the media control and soluble anti-Fas conditions. This approach can be adapted to encapsulate various cell types for therapeutic strategies in other physiological systems in order to increase survival by reducing the immune response. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 608-618, 2017.

Microfluidic Devices for Behavioral Analysis, Microscopy, and Neuronal Imaging in Caenorhabditis elegans.

Microfluidic devices offer several advantages for C. elegans research, particularly for presenting precise physical and chemical environments, immobilizing animals during imaging, quantifying behavior, and automating screens. However, challenges to their widespread adoption in the field include increased complexity over conventional methods, operational problems (such as clogging, leaks, and bubbles), difficulty in obtaining or fabricating devices, and the need to characterize biological results obtained from new assay formats. Here we describe the preparation and operation of simple, reusable microfluidic devices for quantifying behavioral responses to chemical patterns, and single-use devices to arrange animals for time-lapse microscopy and to measure neuronal activity. We focus on details that eliminate or reduce the frustrations commonly experienced by new users of microfluidic devices.

A Circuit for Gradient Climbing in C. elegans Chemotaxis.

Animals have a remarkable ability to track dynamic sensory information. For example, the nematode Caenorhabditis elegans can locate a diacetyl odor source across a 100,000-fold concentration range. Here, we relate neuronal properties, circuit implementation, and behavioral strategies underlying this robust navigation. Diacetyl responses in AWA olfactory neurons are concentration and history dependent; AWA integrates over time at low odor concentrations, but as concentrations rise, it desensitizes rapidly through a process requiring cilia transport. After desensitization, AWA retains sensitivity to small odor increases. The downstream AIA interneuron amplifies weak odor inputs and desensitizes further, resulting in a stereotyped response to odor increases over three orders of magnitude. The AWA-AIA circuit drives asymmetric behavioral responses to odor increases that facilitate gradient climbing. The adaptation-based circuit motif embodied by AWA and AIA shares computational properties with bacterial chemotaxis and the vertebrate retina, each providing a solution for maintaining sensitivity across a dynamic range.

High-throughput imaging of neuronal activity in Caenorhabditis elegans.

Neuronal responses to sensory inputs can vary based on genotype, development, experience, or stochastic factors. Existing neuronal recording techniques examine a single animal at a time, limiting understanding of the variability and range of potential responses. To scale up neuronal recordings, we here describe a system for simultaneous wide-field imaging of neuronal calcium activity from at least 20 Caenorhabditis elegans animals under precise microfluidic chemical stimulation. This increased experimental throughput was used to perform a systematic characterization of chemosensory neuron responses to multiple odors, odor concentrations, and temporal patterns, as well as responses to pharmacological manipulation. The system allowed recordings from sensory neurons and interneurons in freely moving animals, whose neuronal responses could be correlated with behavior. Wide-field imaging provides a tool for comprehensive circuit analysis with elevated throughput in C. elegans.

Serotonin and the neuropeptide PDF initiate and extend opposing behavioral states in C. elegans.

Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete behavioral states. Here, we characterize a neuromodulatory circuit that generates long-lasting roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces prolonged dwelling states. PDF promotes roaming states through a Gαs-coupled PDF receptor; optogenetic activation of cAMP production in PDF receptor-expressing cells induces prolonged roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of this neuromodulatory circuit supplement fast motor circuits to organize long-lasting behavioral states.

Oxytocin/vasopressin-related peptides have an ancient role in reproductive behavior.

Many biological functions are conserved, but the extent to which conservation applies to integrative behaviors is unknown. Vasopressin and oxytocin neuropeptides are strongly implicated in mammalian reproductive and social behaviors, yet rodent loss-of-function mutants have relatively subtle behavioral defects. Here we identify an oxytocin/vasopressin-like signaling system in Caenorhabditis elegans, consisting of a peptide and two receptors that are expressed in sexually dimorphic patterns. Males lacking the peptide or its receptors perform poorly in reproductive behaviors, including mate search, mate recognition, and mating, but other sensorimotor behaviors are intact. Quantitative analysis indicates that mating motor patterns are fragmented and inefficient in mutants, suggesting that oxytocin/vasopressin peptides increase the coherence of mating behaviors. These results indicate that conserved molecules coordinate diverse behavioral motifs in reproductive behavior.

High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments.

To quantitatively understand chemosensory behaviors, it is desirable to present many animals with repeatable, well-defined chemical stimuli. To that end, we describe a microfluidic system to analyze Caenorhabditis elegans behavior in defined temporal and spatial stimulus patterns. A 2 cm × 2 cm structured arena allowed C. elegans to perform crawling locomotion in a controlled liquid environment. We characterized behavioral responses to attractive odors with three stimulus patterns: temporal pulses, spatial stripes and a linear concentration gradient, all delivered in the fluid phase to eliminate variability associated with air-fluid transitions. Different stimulus configurations preferentially revealed turning dynamics in a biased random walk, directed orientation into an odor stripe and speed regulation by odor. We identified both expected and unexpected responses in wild-type worms and sensory mutants by quantifying dozens of behavioral parameters. The devices are inexpensive, easy to fabricate, reusable and suitable for delivering any liquid-borne stimulus.