During development of the nervous system the response of growing axons to their environment is critical to the formation of the complex wiring pattern between neurons. Growth and guidance factors combined with extracellular matrices influence the speed and direction of axonal growth. Although much progress has been made in identifying the factors that influence axonal growth, as well as how axons respond to these factors individually, much less is known about how axons behave in response to the combined effects of multiple factors. To fully understand how axonal growth is regulated ideally this should be studied in vivo. The ability to express fluorescent protein markers in neurons has made such an approach possible. However, it is still limited by a variety of factors. Imaging individual live axons deep in brain tissue is still restricted to layers less than 600 microns. Phototoxicity limits the time lengths of such observations. More importantly, the environment is difficult to characterize or control.
As a complementary approach, we are developing in vitro environments that potentially mimic some of the complexity found in vivo, in particular the development of the anterior visual pathway. In this system, the axon trajectories are simple, multiple relevant guidance molecules have been identified already (many tested with explants in vitro), and a common cause of blindness (Optic Nerve Hypoplasia) is associated with defects in this process. Additionally, the patterns of guidance molecules found on the flat anatomy of the retina is ideally suited to mimicking by micropatterning and microfluidics techniques. This mimicry is accomplished by combining microfluidics patterning of diffusible gradients and substrate patterning of axon pathfinding cues, including axon guidance factors and extracellular matrix molecules. As a source of highly homogeneous cell populations, we use isolated mouse retinal ganglion cells (RGCs), a cell type that responds to Netrin-1 gradients. For experiments designed to maximize the integrity of the cells (isolation procedures are damaging to cells), we will use retinal explants and we microfluidically isolate the axons from their somas. RGCs (or their axons) are exposed to various soluble factors that have previously been shown to affect their axon growth in vivo. Combinatorial microfluidic mixers allow us to test the effects of multiple factors on the direction and speed of axonal growth of RGCs. These experiments allow us to quantitatively examine the basic principles that govern axon pathfinding in the development of the anterior visual pathway and help to better understand the basis of developmental defects in axon growth that alter the organization and function of the nervous system.
Bhattacharjee, N., Li, N., Keenan, T.M., and Folch, A. “A Neuron-Benign Microfluidic Gradient Generator for Studying the Growth of Mammalian Neurons towards Axon Guidance Factors”, Integrative Biology 2, 669 (2010).
Keenan, T.M. and Folch, A. “Biomolecular gradients in cell culture systems”, Lab Chip 8, 34 (2008).
Keenan, T.M., Hsu, C.-H., and Folch, A. “Microfluidic “Jets” for Generating Steady-State Gradients of Soluble Molecules on Open Surfaces”, Appl. Phys. Lett. 89: 114103 (2006).
Keenan, T.M., Hooker, A., Spilker, M. E., Boggy, G. J., Li, N., Vicini, P., and Folch, A. “Automated Identification of Axonal Growth Cones in Time-Lapse Image Sequences”, J. Neurosci. Methods 151: 232 (2006).
Li, N. and Folch, A. “Integration of topographical and biochemical cues by axons during growth on microfabricated 3-D substrates”, Experimental Cell Research 311: 307 (2005).
A major goal in neuroscience is to understand the formation and development of synapses, the tiny membrane specializations that enable nerve cells to communicate with each other. The sequence of molecular signals leading to synapse formation (“synaptogenesis”) is qualitatively well known for the more accessible neuromuscular junction (NMJ). However, very little is known of the quantities (concentration, duration, onset, etc.) of the various neurochemical signals involved in synaptogenesis. Intriguingly, all but one of the axons innervating a given myotube at birth retract after a period of ~1 week as a result of a synaptic competition process that remains, for lack of quantitative methods, poorly understood. Our overall objective is to uncover some of the rules governing the formation and elimination of synapses at the NMJ using a microfluidic cell culture system. Our approach is based on substituting the presynaptic neuron by an artificial microfluidic device that delivers known doses of various synaptogenic neurochemicals to micrometer-scale areas of the membrane of cultured myotubes. We focus on the three key factors – agrin, neuregulin, and the neurotransmitter acetylcholine (ACh) – secreted by the nerve tip during synaptogenesis. We measure muscle cell responses that are specific to ACh receptors (AChRs), such as AChR aggregation/disaggregation, degradation/synthesis, insertion, co-localization with other receptors and cytoskeletal proteins, intracellular signaling pathways, etc.
We have developed a microfluidic mimic of the innervation process that allows for focally stimulating microarrays of single, isolated (“microengineered”) myotubes using laminar flow streams (orthogonal to the myotubes). We have found that a) focal application of agrin entices myotubes to recruit new AChRs to the stimulated area; b) when the microtracks are formed with Matrigel, a basal lamina extract, the microengineered myotubes display AChR clusters of complex, in-vivo-like morphologies even before agrin is applied, similarly to what happens in vivo; and c) when agrin is focally applied to those agrin-predating clusters, AChRs are degraded at reduced rates, suggesting that a putative role for agrin in vivo is to help stabilize AChRs. We seek to continue these investigations by studying the dynamics and spatial patterns of various AChR-specific responses upon (competitive, synergistic, or combinatorial) stimulation with agrin, neuregulin, and ACh.
Tourovskaia, A., Li, N., and Folch, A., “Localized acetylcholine receptor clustering dynamics in response to microfluidic focal stimulation with agrin”, Biophys. J. 95: 3009 (2008).
Tourovskaia, A., Figueroa-Masot, X., and Folch, A. “Long-term Microfluidic Cultures of Myotube Microarrays for High-Throughput Focal Stimulation”, Nature Protocols 1: 1092 (2006).
Kosar, T.F. Tourovskaia, A., Figueroa-Masot, X., Adams, M. and Folch, A. “A Nanofabricated Planar Aperture as a Mimic of the Nerve-Muscle Contact During Synaptogenesis”, Lab Chip 6: 632-638 (2006).
Tourovskaia, A., T.F. Kosar, and Folch, A. “Local Induction of Acetylcholine Receptor Clustering in Myotube Cultures Using Microfluidic Application of Agrin”, Biophys. J. 90: 2192-2198 (2006).
Tourovskaia, A., Figueroa-Masot, X. and Folch, A., “Differentiation-on-a-chip: A Microfluidic Platform for Long-Term Cell Culture Studies”, Lab on a Chip 5:14 (2005).
We owe our sense of smell to the ability of odorant compounds to activate odorant receptors (ORs) on the membrane of the cilia of olfactory sensory neurons (OSNs) present in the nose’s main olfactory epithelium (MOE). Interestingly, the MOE also responds to pheromones, a role previously thought to be limited to the vomeronasal organ (VNO). Our present knowledge indicates that, in mammals, there are around 1,000 types of ORs and that any given neuron only expresses one type of OR, to which different odorants can bind with different affinities. Thus, any odorant (at a given concentration) can be thought of as being “encoded” in the olfactory system as a set of combinatorial affinities between itself and each of the ORs. To date, extensive searches for matches between ORs and odorants have yielded great insight into the olfactory code, albeit with a limited functional characterization of the ORs. Several questions remain to be addressed in depth with more quantitative, higher-throughput methods. What is the full OR space sampled by each odorant and how does it overlap with other odorants or complex odors? How do the dynamics of adaptation affect the olfactory code? Can we find and characterize a sub-population of “pheromone-specialist” OSNs in the MOE?
We have developed a “smell-on-a-chip” platform that combines large-area calcium imaging and microfluidic perfusion for simultaneously screening thousands of OSNs responding to soluble chemicals. We are extending the capabilities of this platform to measure the dynamic response, specificity, and adaptation of OSNs to a larger spectrum of odorants, complex odors, and pheromones.
Figueroa, X.A., Cooksey, G.A., Votaw, S.V., Horowitz, L.F., and Folch, A. “Large-Scale Investigation of the Olfactory Receptor Space Using a Microfluidic Microwell Array”, Lab Chip 10: 1120 (2010).
Cooksey, G.A., Sip, C.G., and Folch, A., “A Multi-Purpose Microfluidic Perfusion System with Combinatorial Choice of Inputs, Mixtures, Gradient Patterns, and Flow Rates”, Lab Chip 9, 417 (2009).
Rettig, J. R. and Folch, A. “Large-Scale Single-Cell Trapping and Imaging Using Microwell Arrays”, Analytical Chemistry 77: 5628-5634 (2005).
Sidorova, J.M. Li, N., Schwartz, D.C., Folch, A., and Monnat Jr., R.J. “Microfluidic-assisted analysis of replicating DNA molecules”, Nature Protocols 4: 849 (2009).
Sidorova, J.M., Li, N., Folch, A., and Monnat Jr., R.J. “The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest”, Cell Cycle 7, 796 (2008).