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.

A Biochip with a 3D microfluidic architecture for trapping white blood cells.

We present a microfluidic biochip for trapping single white blood cells (WBCs). The novel biochip, microfabricated using standard surface micromachining processes, consists of an array of precisely engineered microholes that confine single cells in a tight, three dimensional space and mechanically immobilize them. A high (> 87%) trapping efficiency was achieved when WBC-containing samples were delivered to the biochip at the optimal pressure of 3 psi. The biochip can efficiently trap up to 7,500 cells, maintaining a high trapping efficiency even when the number of cells is extremely low (~200 cells). We believe that the developed biochip can be used as a standalone unit in a biology/clinical lab for trapping WBCs as well as other cell types and imaging them using a standard fluorescent microscope at the single cell level. Furthermore, it can be integrated with other miniaturized optical modules to construct a portable platform for counting a wide variety of cells and therefore it can be an excellent tool for monitoring human diseases at the point-of-care.

Emerging biotechnologies for evaluating disruption of stress, sleep, and circadian rhythm mechanism using aptamer-based detection of salivary biomarkers.

The internally driven 24-h cycle in humans, called circadian rhythm, controls physiological, metabolic, and hormonal processes, and is tied to the circadian clocks ticking in most of the cells and tissues. The central clock, located in suprachiasmatic nuclei of the hypothalamus, is directly influenced by external cues, particularly light, and entrains the peripheral clocks through neural and hormonal pathways to the external light-dark cycle. However, peripheral clocks also have self-sustained circadian rhythmicity and feeding is the potent synchronizer. The internal clock system regulates the sleep-wake cycle and maintains stress responses through the hypothalamus-pituitary-adrenal axis and autonomic pathways. Any misalignment in this complex network could lead to circadian clock disruption and endocrine and metabolic dysfunction that may induce inflammatory responses. The detrimental consequences of such dysfunction are broad and can lead to serious health problems; however, the extent of the circadian disruption is difficult to assess. New promising techniques based on biosensors and point-of-care devices using aptamers - single-stranded DNA or RNA biorecognition molecules that can measure biomarkers of stress, sleep, and circadian rhythms in bodily fluids such as saliva with high sensitivity and specificity - can provide timely and accurate diagnosis and allow for effective implementation of behavioral and therapeutic interventions. This review provides detailed insight into the complex crosstalk between stress, sleep, and circadian rhythm, their relationship with the body's homeostasis, and the consequences of circadian dysregulation. The review also summarizes the mechanisms of aptamer-based biosensors and/or point-of-care devices developed to date for the detection of salivary biomarkers linked to stress, sleep, and circadian rhythm. Lastly, the review outlines the knowledge gaps in the literature related to the detection of lower concentrations of biomarkers in saliva and discusses the prospects of aptamer-based detection of salivary biomarkers from a high-precision perspective that is crucial for clinical diagnosis, at a time when circadian disruption is evident in unprecedented proportions across the globe.

Femtosecond laser nanoaxotomy lab-on-a-chip for in vivo nerve regeneration studies.

A thorough understanding of nerve regeneration in Caenorhabditis elegans requires performing femtosecond laser nanoaxotomy while minimally affecting the worm. We present a microfluidic device that fulfills such criteria and can easily be automated to enable high-throughput genetic and pharmacological screenings. Using the 'nanoaxotomy' chip, we discovered that axonal regeneration occurs much faster than previously described, and notably, the distal fragment of the severed axon regrows in the absence of anesthetics.

Worm chips: microtools for C. elegans biology.

The study of small-size animal models, such as the roundworm C. elegans, has provided great insight into several in vivo biological processes, extending from cell apoptosis to neural network computing. The physical manipulation of this micron-sized worm has always been a challenging task. Here, we discuss the applications, capabilities and future directions of a new family of worm manipulation tools, the 'worm chips'. Worm chips are microfabricated devices capable of precisely manipulating single worms or a population of worms and their environment. Worm chips pose a paradigm shift in current methodologies as they are capable of handling live worms in an automated fashion, opening up a new direction in in vivo small-size organism studies.

An automated microfluidic platform for calcium imaging of chemosensory neurons in Caenorhabditis elegans.

Functional fluorescence imaging methods are widely used to study cellular physiology. When applied to small organisms, these methods suffer from low-throughput due to the laborious immobilization/stimulus delivery procedure that is typically involved during imaging. Here, we describe the development of an automated microfluidic-based platform for performing automated neuronal functional (calcium) imaging in the roundworm Caenorhabditis elegans. The platform, capable of processing tens to hundreds of worms per hour, immobilizes individual worms, delivers a chemical odor to their nose and collects calcium imaging data from single neurons without any manual intervention. We used the developed platform to obtain a large number of calcium responses from worms of different ages (212 worms were imaged in total). The calcium imaging data revealed significant difference in the responses from young and old worms, indicating that neural functionality is age-dependent. We believe that such a technology will be an essential tool for obtaining repeatable and accurate functional imaging data from a large population of worms, in order to minimize stochastic biological noise and identify statistically significant trends.

CO2 and compressive immobilization of C. elegans on-chip.

We present two microfluidic approaches for immobilizing the roundworm C. elegans on-chip. The first approach creates a CO(2) micro-environment while the second one utilizes a deformable PDMS membrane to mechanically restrict the worm's movement. An on-chip 'behavior' module was used to characterize the effect of these methods on the worm's locomotion pattern. Our results indicate that both methods are appropriate for the short-term (minutes) worm immobilization. The CO(2) method offers the additional advantages of long-term immobilization (1-2 hours) and reduced photobleaching, if fluorescent imaging during immobilization is required. We envision the use of these methods in a wide variety of biological studies in C. elegans, including cell developmental and neuronal regeneration studies.

A high numerical aperture, polymer-based, planar microlens array.

We present a novel microfabrication approach for obtaining arrays of planar, polymer-based microlenses of high numerical aperture. The proposed microlenses arrays consist of deformable, elastomeric membranes that are supported by polymer-filled microchambers. Each membrane/microchamber assembly is converted into a solid microlens when the supporting UV-curable polymer is pressurized and cured. By modifying the microlens diameter (40-60 microm) and curing pressure (7.5-30 psi), we demonstrated that it is possible to fabricate microlenses with a wide range of effective focal lengths (100-400 microm) and numerical apertures (0.05-0.3). We obtained a maximum numerical aperture of 0.3 and transverse resolution of 2.8 microm for 60 microm diameter microlenses cured at 30 psi. These values were found to be in agreement with values obtained from opto-mechanical simulations. We envision the use of these high numerical microlenses arrays in optical applications where light collection efficiency is important.

An automated compound screening for anti-aging effects on the function of C. elegans sensory neurons.

Discovery of molecular targets or compounds that alter neuronal function can lead to therapeutic advances that ameliorate age-related neurodegenerative pathologies. Currently, there is a lack of in vivo screening technologies for the discovery of compounds that affect the age-dependent neuronal physiology. Here, we present a high-throughput, microfluidic-based assay for automated manipulation and on-chip monitoring and analysis of stimulus-evoked calcium responses of intact C. elegans at various life stages. First, we successfully applied our technology to quantify the effects of aging and age-related genetic and chemical factors in the calcium transients of the ASH sensory neuron. We then performed a large-scale screen of a library of 107 FDA-approved compounds to identify hits that prevented the age-dependent functional deterioration of ASH. The robust performance of our assay makes it a valuable tool for future high-throughput applications based on in vivo functional imaging.

On chip cryo-anesthesia of Drosophila larvae for high resolution in vivo imaging applications.

We present a microfluidic chip for immobilizing Drosophila melanogaster larvae for high resolution in vivo imaging. The chip creates a low-temperature micro-environment that anaesthetizes and immobilizes the larva in under 3 minutes. We characterized the temperature distribution within the chip and analyzed the resulting larval body movement using high resolution fluorescence imaging. Our results indicate that the proposed method minimizes submicron movements of internal organs and tissue without affecting the larva physiology. It can be used to continuously immobilize larvae for short periods of time (minutes) or for longer periods (several hours) if used intermittently. The same chip can be used to accommodate and immobilize arvae across all developmental stages (1st instar to late 3rd instar), and loading larvae onto the chip does not require any specialized skills. To demonstrate the usability of the chip, we observed mitochondrial trafficking in neurons from the cell bodies to the axon terminals along with mitochondrial fusion and neuro-synaptic growth through time in intact larvae. Besides studying sub-cellular processes and cellular development, we envision the use of on chip cryo-anesthesia in a wide variety of biological in vivo imaging applications, including observing organ development of the salivary glands, fat bodies and body-wall muscles.

Chemically induced oxidative stress affects ASH neuronal function and behavior in C. elegans.

Oxidative stress (OS) impact on a single neuron's function in vivo remains obscure. Using C. elegans as a model organism, we report the effect of paraquat (PQ)-induced OS on wild type worms on the function of the ASH polymodal neuron. By calcium (Ca2+) imaging, we quantified ASH activation upon stimulus delivery. PQ-treated worms displayed higher maximum depolarization (peak of the Ca2+ transients) compared to untreated animals. PQ had a similar effect on the ASH neuron response time (rising slope of the Ca2+ transients), except in very young worms. OS effect on ASH was partially abolished in vitamin C-treated worms. We performed octanol and osmotic avoidance tests, to investigate the OS effect on ASH-dependent behaviors. PQ-treated worms have enhanced avoidance behavior compared to untreated ones, suggesting that elevated ASH Ca2+ transients result in enhanced ASH-mediated behavior. The above findings suggest a possible hormetic effect of PQ, as a factor inducing mild oxidative stress. We also quantified locomotion parameters (velocity, bending amplitude), which are not mediated by ASH activation. Bending amplitude did not differ significantly between treated and untreated worms; velocity in older adults decreased. The differential effect of OS on behavioral patterns may mirror a selective impact on the organism's neurons.

On-Demand Isolation and Manipulation of C. elegans by In Vitro Maskless Photopatterning.

Caenorhabditis elegans (C. elegans) is a model organism for understanding aging and studying animal behavior. Microfluidic assay techniques have brought widespread advances in C. elegans research; however, traditional microfluidic assays such as those based on soft lithography require time-consuming design and fabrication cycles and offer limited flexibility in changing the geometric environment during experimentation. We present a technique for maskless photopatterning of a biocompatible hydrogel on an NGM (Agar) substrate, enabling dynamic manipulation of the C. elegans culture environment in vitro. Maskless photopatterning is performed using a projector-based microscope system largely built from off-the-shelf components. We demonstrate the capabilities of this technique by building micropillar arrays during C. elegans observation, by fabricating free-floating mechanisms that can be actuated by C. elegans motion, by using freehand drawing to isolate individual C. elegans in real time, and by patterning arrays of mazes for isolation and fitness testing of C. elegans populations. In vitro photopatterning enables rapid and flexible design of experiment geometry as well as real-time interaction between the researcher and the assay such as by sequential isolation of individual organisms. Future adoption of image analysis and machine learning techniques could be used to acquire large datasets and automatically adapt the assay geometry.

Altered Sensory Code Drives Juvenile-to-Adult Behavioral Maturation in Caenorhabditis elegans.

Adults perform better than juveniles in food-seeking tasks. Using the nematode Caenorhabditis elegans to probe the neural mechanisms underlying behavioral maturation, we found that adults and juveniles require different combinations of sensory neurons to generate age-specific food-seeking behavior. We first show that adults and juveniles differ in their response to and preference for food-associated odors, and we analyze genetic mutants to map the neuronal circuits required for those behavioral responses. We developed a novel device to trap juveniles and record their neuronal activity. Activity measurements revealed that adult and juvenile AWA sensory neurons respond to the addition of diacetyl stimulus, whereas AWB, ASK, and AWC sensory neurons encode its removal specifically in adults. Further, we show that reducing neurotransmission from the additional AWB, ASK, and AWC sensory neurons transforms odor preferences from an adult to a juvenile-like state. We also show that AWB and ASK neurons drive behavioral changes exclusively in adults, providing more evidence that age-specific circuits drive age-specific behavior. Collectively, our results show that an odor-evoked sensory code is modified during the juvenile-to-adult transition in animal development to drive age-appropriate behavior. We suggest that this altered sensory code specifically enables adults to extract additional stimulus features and generate robust behavior.

Circuit mechanisms encoding odors and driving aging-associated behavioral declines in Caenorhabditis elegans.

Chemosensory neurons extract information about chemical cues from the environment. How is the activity in these sensory neurons transformed into behavior? Using Caenorhabditis elegans, we map a novel sensory neuron circuit motif that encodes odor concentration. Primary neurons, AWC(ON) and AWA, directly detect the food odor benzaldehyde (BZ) and release insulin-like peptides and acetylcholine, respectively, which are required for odor-evoked responses in secondary neurons, ASEL and AWB. Consistently, both primary and secondary neurons are required for BZ attraction. Unexpectedly, this combinatorial code is altered in aged animals: odor-evoked activity in secondary, but not primary, olfactory neurons is reduced. Moreover, experimental manipulations increasing neurotransmission from primary neurons rescues aging-associated neuronal deficits. Finally, we correlate the odor responsiveness of aged animals with their lifespan. Together, these results show how odors are encoded by primary and secondary neurons and suggest reduced neurotransmission as a novel mechanism driving aging-associated sensory neural activity and behavioral declines.

Using microfluidics chips for live imaging and study of injury responses in Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3(rd) instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Microfluidic chips for in vivo imaging of cellular responses to neural injury in Drosophila larvae.

With powerful genetics and a translucent cuticle, the Drosophila larva is an ideal model system for live imaging studies of neuronal cell biology and function. Here, we present an easy-to-use approach for high resolution live imaging in Drosophila using microfluidic chips. Two different designs allow for non-invasive and chemical-free immobilization of 3(rd) instar larvae over short (up to 1 hour) and long (up to 10 hours) time periods. We utilized these 'larva chips' to characterize several sub-cellular responses to axotomy which occur over a range of time scales in intact, unanaesthetized animals. These include waves of calcium which are induced within seconds of axotomy, and the intracellular transport of vesicles whose rate and flux within axons changes dramatically within 3 hours of axotomy. Axonal transport halts throughout the entire distal stump, but increases in the proximal stump. These responses precede the degeneration of the distal stump and regenerative sprouting of the proximal stump, which is initiated after a 7 hour period of dormancy and is associated with a dramatic increase in F-actin dynamics. In addition to allowing for the study of axonal regeneration in vivo, the larva chips can be utilized for a wide variety of in vivo imaging applications in Drosophila.

Probing the physiology of ASH neuron in Caenorhabditis elegans using electric current stimulation.

Electrical stimulation has been widely used to modulate and study the in vitro and in vivo functionality of the nervous system. Here, we characterized the effect of electrical stimulation on ASH neuron in Caenorhabditis elegans and employed it to probe the neuron's age dependent properties. We utilized an automated microfluidic-based platform and characterized the ASH neuronal activity in response to an electric current applied to the worm's body. The electrically induced ASH neuronal response was observed to be dependent on the magnitude, polarity, and spatial location of the electrical stimulus as well as on the age of the worm.

Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans.

The nematode Caenorhabditis elegans is a widely adopted model organism for studying various neurobiological processes at the molecular and cellular level in vivo. With a small, flexible, and continuously moving body, the manipulation of C. elegans becomes a challenging task. In this review, we highlight recent advances in microfluidic technologies for the manipulation of C. elegans. These new family of microfluidic chips are capable of handling single or populations of worms in a high-throughput fashion and accurately controlling their microenvironment. So far, they have been successfully used to study neural circuits and behavior, to perform large-scale phetotyping and morphology-based screens as well as to understand axon regeneration after injury. We envision that microfluidic chips can further be used to study different aspects of the C. elegans nervous system, extending from fundamental understanding of behavioral dynamics to more complicated biological processes such as neural aging and learning and memory.

Neurons detect increases and decreases in oxygen levels using distinct guanylate cyclases.

Homeostatic sensory systems detect small deviations in temperature, water balance, pH, and energy needs to regulate adaptive behavior and physiology. In C. elegans, a homeostatic preference for intermediate oxygen (O2) levels requires cGMP signaling through soluble guanylate cyclases (sGCs), proteins that bind gases through an associated heme group. Here we use behavioral analysis, functional imaging, and genetics to show that reciprocal changes in O2 levels are encoded by sensory neurons that express alternative sets of sGCs. URX sensory neurons are activated by increases in O2 levels, and require the sGCs gcy-35 and gcy-36. BAG sensory neurons are activated by decreases in O2 levels, and require the sGCs gcy-31 and gcy-33. The sGCs are instructive O2 sensors, as forced expression of URX sGC genes causes BAG neurons to detect O2 increases. Both sGC expression and cell-intrinsic dynamics contribute to the differential roles of URX and BAG in O2-dependent behaviors.