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

RECEIVED 1 APRIL; ACCEPTED 9 JULY; PUBLISHED ONLINE

The nematode C. elegans is an excellent model organism for studying behavior at the neuronal level. Because of the organism's small size, it is challenging to deliver stimuli to C. elegans and monitor neuronal activity in a controlled environment. To address this problem, we developed two microfluidic chips, the 'behavior' chip and the 'olfactory' chip for imaging of neuronal and behavioral responses in C. elegans. We used the behavior chip to correlate the activity of AVA command interneurons with the worm locomotion pattern. We used the olfactory chip to record responses from ASH sensory neurons exposed to high-osmotic-strength stimulus. Observation of neuronal responses in these devices revealed previously unknown properties of AVA and ASH neurons. The use of these chips can be extended to correlate the activity of sensory neurons, interneurons and motor neurons with the worm's behavior. How neural circuits process information to generate behavior is a fundamental question in neuroscience. To address this question, one should observe an animal in a well-controlled environment, in which a specific behavior can be generated and corresponding neuronal activity monitored. Ideally such an environment should not disturb normal neuronal function and should be able to reveal the specific neuronal circuit under study. C. elegans, with its optically accessible, stereotyped and compact nervous system, has drawn great scientific attention because of its diverse repertoire of behavioral outputs and its genetic conservation with vertebrates. Initial efforts to measure activity in the C. elegans nervous system relied on electrophysiological recordings from single neurons in dissected worms 1 . The recent development of genetically encoded fluorescent calcium indicators 2 has spawned an increasing interest in optical imaging approaches that permit the tracking of calcium transients in individual neurons in vivo in intact worms 3 . Although transgenic worms that express neuron-specific indicators can now routinely be generated, the present methods for confining and stimulating the worm during imaging are not ideal. The typical experimental setup involves application of glue onto specific segments of the worm to achieve permanent immobilization on a hydrated agar pad. Fluid-filled pipettes, temperature-controlled plates and sharp electrodes have been used in the past to deliver chemical, thermal and mechanical stimuli, respectively 4,5 . Whether the organic glue is toxic to the worm and how it influences neuronal activity are difficult to determine. Moreover, the delivery of chemical stimuli to the glued worm cannot be precisely controlled or separated from mechanical stimuli associated with fluid flow. More concerns arise when the circuit controlling locomotion is under study. The glue immobilizes the worm, not allowing muscles and stretch-receptor neurons, if any, to contract and relax normally. This mechanically restricted microenvironment might affect the function of the proprioceptive sensory neurons as well as motor neurons. Most importantly, the glue setup does not permit most behaviors to be generated, visualized, quantified or correlated to neuronal activity in real time. A system with two objectives 6 has been a welcome step toward simultaneous neuronal-behavior analysis, as has been a new system for tracking thermosensory neurons (albeit at low optical resolution) in freely moving worms 7 . Recent advances in microfabrication technology permit the construction of well-controllable microenvironments with applications ranging from cell analysis to tissue engineering RESULTS The behavior chip The first microfluidic device, the behavior chi

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

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98694/1/ApplPhysLett_99_053702.pd

A White Blood Cell Capturing Biochip Using a 3D Trapping Architecture

White blood cells (WBCs) and their subtypes are important constituents of the human immune system as their concentration, quantified by a WBC count test, indicates the state of body’s immune response against infections. These cell count tests are important prognostic and diagnostic indicators for a number of human immunological diseases, most prominent of them being AIDS (1).</jats:p

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

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