STUDYING AXON PATHFINDING IN CONTROLLED MICROFLUIDIC ENVIRONMENTS S. Moorjani, * N. Bhattacharjee and A. Folch University of Washington, Seattle, U.S.A. ABSTRACT Developing axons are directed along specific pathways by gradients of a relatively small number of chemical cues to create precise neuronal-wiring patterns. The increasing appreciation of the complexity of signaling interactions between these small numbers of cues emphasizes the need for tools that would allow gradient delivery to growing axons in a controlled combinatorial manner for deciphering their guidance code. We report the development of an open-chamber- microfluidic platform that permits mammalian-neural-tissue-explant culture and provides an isolated environment for exposing axons, emerging from explants, to stable gradients. Our device substantially extends capabilities for chemical interactions with cultured axons enabling quantitative-neurobiological investigations. KEYWORDS: Axon guidance, Axon pathfinding, Neural development, Visual system, Retinal ganglion cells, Axio- somatic separation, Gradient generator, Open-chamber microfluidics INTRODUCTION Visual information is relayed from the eye to the brain by the axons of the retinal ganglion cells (RGCs). During development, these axons navigate unerringly to reach and form connections with targets in the brain. Due to its relatively simple anatomy and topographic mapping pattern (between axonal projections from the retina and visual targets in the brain), the developing visual system has been extensively studied to identify the players involved in axon- pathfinding decisions. Several families of ligand-receptor signaling systems, highly conserved between species, have emerged [1]. Despite identification of many ligand-receptor families and the associated signaling cascades, unraveling the mechanisms guiding developing RGC axons remains challenging. An emerging theme is that a small number of extracellular cues are used reiteratively along intermediate points of the optic pathway to help axons navigate their complex milieu. This is achieved by multiple identities espoused by an individual cue to modulate the axonal response. Chemical cues can be either attractive or repulsive, or both [2], and can act at variable distances, such that the same cue serves as both a short-range and a long-range signal [3]. Guidance cues, more often than not, work in a combinatorial fashion, rather than in isolation, to precisely direct the trajectory of a growing axon. Redundancies are also put in place to help correct any errors that might occur. In summary, a key challenge to understanding RGC axon-guidance mechanisms lies in the unraveling of this complex multi-identity, combinatorial, co-operative and redundant relationship that exists between extracellular cues, their receptors, and triggered signaling pathways. Further refinement of the guidance code used by RGC axons will advance our understanding of the developing visual system and offer insights into the general wiring mechanisms used in the developing nervous system. This paper focuses on the development of microfluidic technologies for exposing developing RGC axons to controlled gradients of multiple guidance factors in an attempt to provide quantitative descriptions of RGC axon- guidance mechanisms. THEORY The discovery of guidance cues followed by the increasing appreciation of the complexity of signaling interactions between these relatively small number of cues emphasizes the need for tools that would allow delivery of gradients of guidance factors to growing RGC axons in a controlled, combinatorial, and quantitative fashion for further deciphering their guidance code. In recent years, a number of researchers have exploited fluid-control capabilities of microfabricated systems to create stable concentration gradients within cell cultures on the micrometer scale [4-7]. In these approaches, laminar-flow streams, with sharp interfacial boundaries, can be targeted to specific subcellular regions [6, 7] or multiple streams can be brought into confluence to create varying concentration profiles [4, 5]. These approaches have been widely used for studying polarized cellular events, such as cell migration [6, 8] and differentiation [9, 10]. Despite these advantages, the microfluidic gradient-generator approach suffers from important limitations. Gradients can only be generated under constant fluid flow conditions, which induces shear and drag forces on cultures under study. These forces can lead to cell detachment, changes in intracellular signaling and migrational biases. They can be particularly detrimental when studying neurons, which are more sensitive to such forces compared to many other cell types (such as neutrophils and endothelial cells). Secondly, downstream cells are exposed to higher concentrations of cell-secreted molecules compared to cells present upstream in the channel, creating differential contributions of secreted and gradient factors on cell responses depending on the placement of cells inside channels. Lastly, the closed microfluidic channels used in these approaches limits gas and pH equilibration, which are critical for cell viability, differentiation and activity. Here, we report the development of an open-chamber microfluidic platform that allows for neural-tissue explant 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 1194 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany