Automated dual olfactory device for studying head/tail chemosensation in Caenorhabditis elegans.

The correct interpretation of threat and reward is important for animal survival. Often, the decisions underlying these behavioral programs are mediated by volatile compounds in the animal's environment, which they detect and discriminate with specialized olfactory neurons along their body. Caenorhabditis (C.) elegans senses chemical stimuli with neurons located in the head and the tail of the animal, which mediate either attractive or aversive behaviors. How conflicting stimuli are processed in animals navigating different chemical gradients is poorly understood. Here, we conceived, created, and capitalized on a novel microfluidic device to enable automated and precise stimulation of head and tail neurons, either simultaneously or sequentially, while reading out neuronal activity in sensory and interneurons using genetically encoded calcium indicators. We achieve robust and programmable chemical pulses through the modulation of inlet pressures. To evaluate the device performance, we synchronized the flow control with microscopy data acquisition and characterized the flow properties in the fabricated devices. Together, our design has the potential to provide insight into the neural circuits and behavior of C. elegans simulating the experience of natural environments.

Bioluminescence as a functional tool for visualizing and controlling neuronal activity in vivo.

The use of bioluminescence as a reporter for physiology in neuroscience is as old as the discovery of the calcium-dependent photon emission of aequorin. Over the years, luciferases have been largely replaced by fluorescent reporters, but recently, the field has seen a renaissance of bioluminescent probes, catalyzed by unique developments in imaging technology, bioengineering, and biochemistry to produce luciferases with previously unseen colors and intensity. This is not surprising as the advantages of bioluminescence make luciferases very attractive for noninvasive, longitudinal in vivo observations without the need of an excitation light source. Here, we review how the development of dedicated and specific sensor-luciferases afforded, among others, transcranial imaging of calcium and neurotransmitters, or cellular metabolites and physical quantities such as forces and membrane voltage. Further, the increased versatility and light output of luciferases have paved the way for a new field of functional bioluminescence optogenetics, in which the photon emission of the luciferase is coupled to the gating of a photosensor, e.g., a channelrhodopsin and we review how they have been successfully used to engineer synthetic neuronal connections. Finally, we provide a primer to consider important factors in setting up functional bioluminescence experiments, with a particular focus on the genetic model Caenorhabditis elegans, and discuss the leading challenges that the field needs to overcome to regain a competitive advantage over fluorescence modalities. Together, our paper caters to experienced users of bioluminescence as well as novices who would like to experience the advantages of luciferases in their own hand.

Understanding neural circuit function through synaptic engineering.

Synapses are a key component of neural circuits, facilitating rapid and specific signalling between neurons. Synaptic engineering - the synthetic insertion of new synaptic connections into in vivo neural circuits - is an emerging approach for neural circuit interrogation. This approach is especially powerful for establishing causality in neural circuit structure-function relationships, for emulating synaptic plasticity and for exploring novel patterns of circuit connectivity. Contrary to other approaches for neural circuit manipulation, synaptic engineering targets specific connections between neurons and functions autonomously with no user-controlled external activation. Synaptic engineering has been successfully implemented in several systems and in different forms, including electrical synapses constructed from ectopically expressed connexin gap junction proteins, synthetic optical synapses composed of presynaptic photon-emitting luciferase coupled with postsynaptic light-gated channels, and artificial neuropeptide signalling pathways. This Perspective describes these different methods and how they have been applied, and examines how the field may advance.

A MEC-2/stomatin condensate liquid-to-solid phase transition controls neuronal mechanotransduction during touch sensing.

A growing body of work suggests that the material properties of biomolecular condensates ensuing from liquid-liquid phase separation change with time. How this aging process is controlled and whether the condensates with distinct material properties can have different biological functions is currently unknown. Using Caenorhabditis elegans as a model, we show that MEC-2/stomatin undergoes a rigidity phase transition from fluid-like to solid-like condensates that facilitate transport and mechanotransduction, respectively. This switch is triggered by the interaction between the SH3 domain of UNC-89 (titin/obscurin) and MEC-2. We suggest that this rigidity phase transition has a physiological role in frequency-dependent force transmission in mechanosensitive neurons during body wall touch. Our data demonstrate a function for the liquid and solid phases of MEC-2/stomatin condensates in facilitating transport or mechanotransduction, and a previously unidentified role for titin homologues in neurons.

Integration of spatially opposing cues by a single interneuron guides decision-making in C. elegans.

The capacity of animals to respond to hazardous stimuli in their surroundings is crucial for their survival. In mammals, complex evaluations of the environment require large numbers and different subtypes of neurons. The nematode C. elegans avoids hazardous chemicals they encounter by reversing their direction of movement. How does the worms' compact nervous system process the spatial information and direct motion change? We show here that a single interneuron, AVA, receives glutamatergic excitatory and inhibitory signals from head and tail sensory neurons, respectively. AVA integrates the spatially distinct and opposing cues, whose output instructs the animal's behavioral decision. We further find that the differential activation of AVA stems from distinct localization of inhibitory and excitatory glutamate-gated receptors along AVA's process and from different threshold sensitivities of the sensory neurons. Our results thus uncover a cellular mechanism that mediates spatial computation of nociceptive cues for efficient decision-making in C. elegans.

Neural engineering with photons as synaptic transmitters.

Neuronal computation is achieved through connections of individual neurons into a larger network. To expand the repertoire of endogenous cellular communication, we developed a synthetic, photon-assisted synaptic transmission (PhAST) system. PhAST is based on luciferases and channelrhodopsins that enable the transmission of a neuronal state across space, using photons as neurotransmitters. PhAST overcomes synaptic barriers and rescues the behavioral deficit of a glutamate mutant with conditional, calcium-triggered photon emission between two neurons of the Caenorhabditis elegans nociceptive avoidance circuit. To demonstrate versatility and flexibility, we generated de novo synaptic transmission between two unconnected cells in a sexually dimorphic neuronal circuit, suppressed endogenous nocifensive response through activation of an anion channelrhodopsin and switched attractive to aversive behavior in an olfactory circuit. Finally, we applied PhAST to dissect the calcium dynamics of the temporal pattern generator in a motor circuit for ovipositioning. In summary, we established photon-based synaptic transmission that facilitates the modification of animal behavior.

FLInt: single shot safe harbor transgene integration via Fluorescent Landmark Interference.

The stable incorporation of transgenes and recombinant DNA material into the host genome is a bottleneck in many bioengineering applications. Due to the low efficiency, identifying the transgenic animals is often a needle in the haystack. Thus, optimal conditions require efficient screening procedures, but also known and safe landing sites that do not interfere with host expression, low input material and strong expression from the new locus. Here, we leverage an existing library of ≈300 different loci coding for fluorescent markers that are distributed over all 6 chromosomes in Caenorhabditis elegans as safe harbors for versatile transgene integration sites using CRISPR/Cas9. We demonstrated that a single crRNA was sufficient for cleavage of the target region and integration of the transgene of interest, which can be easily followed by loss of the fluorescent marker. The same loci can also be used for extrachromosomal landing sites and as co-CRISPR markers without affecting body morphology or animal behavior. Thus, our method overcomes the uncertainty of transgene location during random mutagenesis, facilitates easy screening through fluorescence interference and can be used as co-CRISPR markers without further influence in phenotypes.

Axonal plasticity in response to active forces generated through magnetic nano-pulling.

Mechanical force is crucial in guiding axon outgrowth before and after synapse formation. This process is referred to as "stretch growth." However, how neurons transduce mechanical input into signaling pathways remains poorly understood. Another open question is how stretch growth is coupled in time with the intercalated addition of new mass along the entire axon. Here, we demonstrate that active mechanical force generated by magnetic nano-pulling induces remodeling of the axonal cytoskeleton. Specifically, the increase in the axonal density of microtubules induced by nano-pulling leads to an accumulation of organelles and signaling vesicles, which, in turn, promotes local translation by increasing the probability of assembly of the "translation factories." Modulation of axonal transport and local translation sustains enhanced axon outgrowth and synapse maturation.

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.

Volumetric imaging of fast cellular dynamics with deep learning enhanced bioluminescence microscopy.

Bioluminescence microscopy is an appealing alternative to fluorescence microscopy, because it does not depend on external illumination, and consequently does neither produce spurious background autofluorescence, nor perturb intrinsically photosensitive processes in living cells and animals. The low photon emission of known luciferases, however, demands long exposure times that are prohibitive for imaging fast biological dynamics. To increase the versatility of bioluminescence microscopy, we present an improved low-light microscope in combination with deep learning methods to image extremely photon-starved samples enabling subsecond exposures for timelapse and volumetric imaging. We apply our method to image subcellular dynamics in mouse embryonic stem cells, epithelial morphology during zebrafish development, and DAF-16 FoxO transcription factor shuttling from the cytoplasm to the nucleus under external stress. Finally, we concatenate neural networks for denoising and light-field deconvolution to resolve intracellular calcium dynamics in three dimensions of freely moving Caenorhabditis elegans.

Sexually dimorphic architecture and function of a mechanosensory circuit in C. elegans.

How sensory perception is processed by the two sexes of an organism is still only partially understood. Despite some evidence for sexual dimorphism in auditory and olfactory perception, whether touch is sensed in a dimorphic manner has not been addressed. Here we find that the neuronal circuit for tail mechanosensation in C. elegans is wired differently in the two sexes and employs a different combination of sex-shared sensory neurons and interneurons in each sex. Reverse genetic screens uncovered cell- and sex-specific functions of the alpha-tubulin mec-12 and the sodium channel tmc-1 in sensory neurons, and of the glutamate receptors nmr-1 and glr-1 in interneurons, revealing the underlying molecular mechanisms that mediate tail mechanosensation. Moreover, we show that only in males, the sex-shared interneuron AVG is strongly activated by tail mechanical stimulation, and accordingly is crucial for their behavioral response. Importantly, sex reversal experiments demonstrate that the sexual identity of AVG determines both the behavioral output of the mechanosensory response and the molecular pathways controlling it. Our results present extensive sexual dimorphism in a mechanosensory circuit at both the cellular and molecular levels.

Exploring cell and tissue mechanics with optical tweezers.

Cellular and tissue biosystems emerge from the assembly of their constituent molecules and obtain a set of specific material properties. To measure these properties and understand how they influence cellular function is a central goal of mechanobiology. From a bottoms-up, physics or engineering point-of-view, such systems are a composition of basic mechanical elements. However, the sheer number and dynamic complexity of them, including active molecular machines and their emergent properties, makes it currently intractable to calculate how biosystems respond to forces. Because many diseases result from an aberrant mechanotransduction, it is thus essential to measure this response. Recent advances in the technology of optical tweezers have broadened their scope from single-molecule applications to measurements inside complex cellular environments, even within tissues and animals. Here, we summarize the basic optical trapping principles, implementations and calibration procedures that enable force measurements using optical tweezers directly inside cells of living animals, in combination with complementary techniques. We review their versatility to manipulate subcellular organelles and measure cellular frequency-dependent mechanics in the piconewton force range from microseconds to hours. As an outlook, we address future challenges to fully unlock the potential of optical tweezers for mechanobiology.

Mechanosensitive body-brain interactions in Caenorhabditis elegans.

Proprioception and visceral mechanosensation provide important information about the location and deformation of the body parts in space and time. These deformations arise from muscle contraction during locomotion, but also from volume changes in organs that are subjected to stresses as a part of their physiological function. These internal morphodynamics give rise to periodic contraction-relaxation cycles with surprisingly constant amplitudes and the maintenance of these optimal driving patterns is remarkably robust against external and internal perturbations. One of the underlying reason for this robustness is an internal feedback mechanism in which specialized sensory cells and neurons signal the mechanical deformation of the inner workings of our organs, from the body to the brain, which subsequently adjust the driver to a predetermined physiological setpoint. Here, we review recent progress in the field of visceral mechanosensation and proprioception in Caenorhabditis elegans and discuss how future studies with this model can be used to gain insight into mechanosensory body-brain interactions in mammals.

An asymmetric mechanical code ciphers curvature-dependent proprioceptor activity.

A repetitive gait cycle is an archetypical component within the behavioral repertoire of many animals including humans. It originates from mechanical feedback within proprioceptors to adjust the motor program during locomotion and thus leads to a periodic orbit in a low-dimensional space. Here, we investigate the mechanics, molecules, and neurons responsible for proprioception in Caenorhabditis elegans to gain insight into how mechanosensation shapes the orbital trajectory to a well-defined limit cycle. We used genome editing, force spectroscopy, and multiscale modeling and found that alternating tension and compression with the spectrin network of a single proprioceptor encodes body posture and informs TRP-4/NOMPC and TWK-16/TREK2 homologs of mechanosensitive ion channels during locomotion. In contrast to a widely accepted model of proprioceptive "stretch" reception, we found that proprioceptors activated locally under compressive stresses in-vivo and in-vitro and propose that this property leads to compartmentalized activity within long axons delimited by curvature-dependent mechanical stresses.

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior.

The physical microenvironment regulates cell behavior during tissue development and homeostasis. How single cells decode information about their geometrical shape under mechanical stress and physical space constraints within tissues remains largely unknown. Here, using a zebrafish model, we show that the nucleus, the biggest cellular organelle, functions as an elastic deformation gauge that enables cells to measure cell shape deformations. Inner nuclear membrane unfolding upon nucleus stretching provides physical information on cellular shape changes and adaptively activates a calcium-dependent mechanotransduction pathway, controlling actomyosin contractility and migration plasticity. Our data support that the nucleus establishes a functional module for cellular proprioception that enables cells to sense shape variations for adapting cellular behavior to their microenvironment.

Cortical anchoring of the microtubule cytoskeleton is essential for neuron polarity.

The development of a polarized neuron relies on the selective transport of proteins to axons and dendrites. Although it is well known that the microtubule cytoskeleton has a central role in establishing neuronal polarity, how its specific organization is established and maintained is poorly understood. Using the in vivo model system Caenorhabditis elegans, we found that the highly conserved UNC-119 protein provides a link between the membrane-associated Ankyrin (UNC-44) and the microtubule-associated CRMP (UNC-33). Together they form a periodic membrane-associated complex that anchors axonal and dendritic microtubule bundles to the cortex. This anchoring is critical to maintain microtubule organization by opposing kinesin-1 powered microtubule sliding. Disturbing this molecular complex alters neuronal polarity and causes strong developmental defects of the nervous system leading to severely paralyzed animals.

Transient pressure modeling in jetting animals.

There are many marine animals that employ a form of jet propulsion to move through the water, often creating the jets by expanding and collapsing internal fluid cavities. Due to the unsteady nature of this form of locomotion and complex body/nozzle geometries, standard modeling techniques prove insufficient at capturing internal pressure dynamics, and hence swimming forces. This issue has been resolved with a novel technique for predicting the pressure inside deformable jet producing cavities (M. Krieg and K. Mohseni, J. Fluid Mech., 769, 2015), which is derived from evolution of the surrounding fluid circulation. However, this model was only validated for an engineered jet thruster with simple geometry and relatively high Reynolds number (Re) jets. The purpose of this manuscript is twofold: (i) to demonstrate how the circulation based pressure model can be used to analyze different animal body motions as they relate to propulsive output, for multiple species of jetting animals, (ii) and to quantitatively validate the pressure modeling for biological jetting organisms (typically characterized by complicated cavity geometry and low/intermediate Re flows). Using jellyfish (Sarsia tubulosa) as an example, we show that the pressure model is insensitive to complex cavity geometry, and can be applied to lower Re swimming. By breaking down the swimming behavior of the jellyfish, as well as that of squid and dragonfly larvae, according to circulation generating mechanisms, we demonstrate that the body motions of Sarsia tubulosa are optimized for acceleration at the beginning of pulsation as a survival response. Whereas towards the end of jetting, the velar morphology is adjusted to decrease the energetic cost. Similarly, we show that mantle collapse rates in squid maximize propulsive efficiency. Finally, we observe that the hindgut geometry of dragonfly larvae minimizes the work required to refill the cavity. Date Received: 10-18-2019, Date Accepted: 99-99-9999 *kriegmw@hawaii.edu, UHM Ocean and Res Eng, 2540 Dole St, Honolulu, HI 96822.

Neuronal stretch reception - Making sense of the mechanosense.

The sensation of mechanical force underlies many of our daily activities. As the sense of touch determines the quality of life, the subconscious sense of proprioception and visceral mechanosensation is indispensible for survival. Many internal organs change shape, either as an active part of their physiology or passively due to body movements. Importantly, these shape changes need to be sensed and balanced properly to prevent organ failure and dysfunction. Consequently, a failure to properly sense volume changes of internal organs has a huge clinical relevance, manifested by a plethora of congenital and age-related diseases. Here we review novel data on mammalian stretch reception as well as classical studies from insect and nematode proprioceptors with the aim to highlight the missing link between organ-level deformation and mechanosensing on the molecular level.