Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Abstract

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

Simple Microfluidic Devices for <em>in vivo</em> Imaging of <em>C. elegans</em>, <em>Drosophila</em> and Zebrafish

“…The rate of heartbeat was measured to be 136.8 ± 1.6 per min (n=8 movies) and was similar to other published reports 25 . Our simple microfluidic device had already been demonstrated for measuring a variety of sub-cellular and cellular events in wild type C. elegans 4 . In our microfluidic device, wild type C. elegans show a larger number of cargo such as synaptic vesicles that move in neurons compared to animals that are immobilized using anesthetics.…”

“…Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals ( Figure 1J and 3C-F 4 ).…”

“…Simple fabrication processes are available for microfluidic devices using soft lithography techniques [1][2][3] . Microfluidic devices have been used for sub-cellular imaging 4,5 , in vivo laser microsurgery 2,6 and cellular imaging 4,7 . In vivo imaging requires immobilization of organisms.…”

“…This has been achieved using suction 5,8 , tapered channels 6,7,9 , deformable membranes [2][3][4]10 , suction with additional cooling 5 , anesthetic gas 11 , temperature sensitive gels 12 , cyanoacrylate glue 13 and anesthetics such as levamisole 14,15 . Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4 .In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies.…”

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in <em>Drosophila</em> Larvae

“…The anesthetics ether 4 and isofluorane [5][6][7][8] have also been used. While anesthetics provide many advantages, they also inhibit neural activity and important physiology (including the heartbeat) [9][10][11] , hence can affect the process studied and create stress on the animal. There are also human safety concerns for working with ether and isofluorane.…”