Nikos Chronis

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.

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.

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.

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 3rd 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.

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.

A doublet microlens array for imaging micron-sized objects

We present a high-numerical aperture, doublet microlens array for imaging micron-sized objects. The proposed doublet architecture consists of glass microspheres trapped on a predefined array of silicon microholes and covered with a thin polymer layer. A standard silicon microfabrication process and a novel fluidic assembly technique were combined to obtain an array of 56 μm diameter microlenses with a numerical aperture of ~0.5. Using such an array, we demonstrated brightfield and fluorescent image formation of objects directly on a CCD sensor without the use of intermediate lenses. The proposed technology is a significant advancement toward the unmet need of inexpensive, miniaturized optical modules which can be further integrated with lab-on-chip microfluidic devices and photonic chips for a variety of high-end imaging/detection applications.

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, AWCON 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. DOI: http://dx.doi.org/10.7554/eLife.10181.001

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 neurons 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 worms 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.

A Near-Infrared Optomechanical Intracranial Pressure Microsensor

We present a wireless and power-free, optomechanical, implantable microsensor that can potentially be used to accurately monitor intracranial pressure (ICP) over long periods of time. The developed microsensor vertically integrates a glass mini-lens with a two-wavelength quantum dot (QD) micropillar that is photolithographically patterned on an ICP-exposed silicon nitride membrane. The operation principle is based on a novel optomechanical transduction scheme that converts ICP changes into changes in the intensity ratio of the two-wavelength, near-infrared fluorescent light emitted from the QDs. The microsensor is microfabricated using silicon bulk micromachining, and it operates at an ICP clinically relevant pressure dynamic range (0-40 mmHg). The microsensor has a maximum error of less than 15% throughout its dynamic range, and it is extremely photostable. We believe that the proposed microsensor will open up a new direction not only in ICP monitoring but in other pressure-related biomedical applications.

An X-ray detectable pressure microsensor for monitoring coronary in-stent restenosis

We present a novel implantable X-ray-addressable MEMS Blood Pressure sensor, the X-BP, for the noninvasive and cost-effective surveillance of coronary in-stent restenosis. We successfully fabricated and tested the X-BP sensor and its pressure response curve. We placed the X-BP sensor in a coronary stent and prove adequate visibility in a clinically realistic scenario.