Fluidic Isolation Research Articles

Screening Alzheimer’s genetic risk factors against amyloid‐βeta synaptotoxicity

AbstractBackgroundSynapse degeneration is one of the earliest events in Alzheimer’s disease (AD) and toxic oligomers of amyloid‐beta (Aβ) peptide, cleavage product of APP, plays a major role in this process. Genome‐wide association studies recently reported 75 genomic risk loci for developing AD, but how the potential risk genes contribute to AD remains unknown. This project aims to identify AD genetic risk factors that mediate Aβ‐induced synaptotoxicity.MethodWe conducted a high‐content shRNA screening to assess the impact of ca. 200 AD risk genes on synaptic density and connectivity. Twenty‐four genes expressed in neurons with highest impact on synapses when silenced were shortlisted for a medium‐throughput screening to assess their capacity to block Aβ‐induced synaptotoxicity. Synaptotoxicity was modelled in microfluidic devices that allow primary postnatal rat neurons to be co‐cultured with Chinese hamster ovary cells, that overexpress wild‐type APP or APP with V717I (or London) mutation with increased secretion of Aβ1‐42 oligomers. Microfluidic devices allow the fluidic isolation of neurites from neuronal cell bodies and therefore provide exclusive access to pre‐ and post‐synaptic neurons. Target genes expression will be modulated either in pre‐ or post‐synaptic neurons and its impact on synaptic connectivity will be assessed.ResultBased on the shRNA screening, we shortlisted 17 detrimental genes, for which we obtained overexpression vectors packed in lentiviruses, and 6 protective genes. We have developed a medium‐throughput co‐culture device that permits screening twelve genes per imaging session using the INCell 6000 automated microscope, as well as an automated image analysis workflow for detecting and assigning presynaptic to postsynaptic puncta based on their relative proximity. Gene expression modulation is validated and the screen is on‐going.ConclusionWe have developed a microfluidic co‐culture model to rapidly and systematically interrogate genetic risk factors for their impact on synaptic connectivity. This study will help us identify AD genetic risk factors with a potential to block Aβ‐induced synaptotoxicity in vitro. Hit genes will be functionally validated via microelectrode array recordings.

Microfluidic Platforms Promote Polarization of Human-Derived Retinal Ganglion Cells That Model Axonopathy.

Axons depend on long-range transport of proteins and organelles which increases susceptibility to metabolic stress in disease. The axon initial segment (AIS) is particularly vulnerable due to the high bioenergetic demand of action potential generation. Here, we prepared retinal ganglion cells derived from human embryonic stem cells (hRGCs) to probe how axonal stress alters AIS morphology. hRGCs were cultured on coverslips or microfluidic platforms. We assayed AIS specification and morphology by immunolabeling against ankyrin G (ankG), an axon-specific protein, and postsynaptic density 95 (PSD-95), a dendrite-specific protein. Using microfluidic platforms that enable fluidic isolation, we added colchicine to the axon compartment to lesion axons. We verified axonopathy by measuring the anterograde axon transport of cholera toxin subunit B and immunolabeling against cleaved caspase 3 (CC3) and phosphorylated neurofilament H (SMI-34). We determined the influence of axon injury on AIS morphology by immunolabeling samples against ankG and measuring AIS distance from soma and length. Based on measurements of ankG and PSD-95 immunolabeling, microfluidic platforms promote the formation and separation of distinct somatic-dendritic versus axonal compartments in hRGCs compared to coverslip cultures. Chemical lesioning of axons by colchicine reduced hRGC anterograde axon transport, increased varicosity density, and enhanced expression of CC3 and SMI-34. Interestingly, we found that colchicine selectively affected hRGCs with axon-carrying dendrites by reducing AIS distance from somas and increasing length, thus suggesting reduced capacity to maintain excitability. Thus, microfluidic platforms promote polarized hRGCs that enable modeling of axonopathy. Microfluidic platforms may be used to assay compartmentalized degeneration that occurs during glaucoma.

T3 Enters Axon Terminals of Mouse Cortical Neurons, Is Retrogradely Transported to the Cell Nucleus and Activates Gene Expression

Thyroid hormone (TH) is critical for brain development and function. T3 enters neurons through membrane transporters and reaches the cell nucleus where it binds to receptors (TR) to regulate gene transcription. However, neurons also express the type 3 deiodinase (D3), which is located in the cellular and nuclear membranes and inactivates T3. Here, we investigated the fate and biological impact of T3 that enters neurons through axons. Primary cortical neurons were isolated from E16.5 embryos of the TH action indicator (THAI) mice, which were engineered with a TH-responsive transgene where three copies of a T3-responsive element drive a luciferase (Luc) reporter. Neurons were seeded on a microfluidic device consisting of two independent compartments: (i) cellular, where about 70-90,000 cell bodies were located, and (ii) axonal, where a few hundred distal axons were located. Fluidic isolation of the compartments was monitored with Alexa Fluor 594 hydrazide. In the first set of experiments (repeated 3 times), 8-10-day old cultures were incubated for 48h with medium containing 1% charcoal-stripped serum (Tx-medium). Subsequently, 10nM T3 was added to the axonal compartment, and 24h later cell bodies were harvested and Luc mRNA measured by RT-qPCR. There was a 2.4 ± 0.7-fold increase in Luc mRNA levels, but the addition of 2uM Silychristin (MCT8 inhibitor) to the axonal compartment reduced T3 induction of Luc mRNA by 32 ± 4.2%. In the second set of experiments (repeated 3 times), 4.9 ± 2.2pM 125I-T3 (final concentration) was added to the cellular or axonal compartments. Medium was sampled and 125I-T3 and its metabolites were separated/quantified via UPLC linked to a flow scintillation detector. After 72h of adding 125I-T3 to the axonal compartment, about 0.73 % 125I-T3 (0.052 ± 0.025pM) was found in the cellular compartment. In addition, 3,3’-125I-T2 and 125I (0.011 ± 0.003 and 0.052 ± 0.023pM, respectively) were also detected. When 125I-T3 was added to the cellular compartment, about 1.6% 125I-T3 (0.048 ± 0.027pM), no metabolites, was detected in the axonal compartment. Only background radioactivity was detected in the opposing compartment when 125I-T3 was added in the absence of cells. We conclude that T3 can be taken up by neuronal axons, partly via MCT8, and transported retrogradely to the cell nucleus to initiate TH signaling. D3-generated T3 metabolites exit the cell body alongside with small amounts of intact T3. This pathway could explain how D2-generated T3 in tanycytes is taken up by TRH-secreting neurons to mediate negative T3 feedback. Anterograde T3 transport was also detected, the significance of which remains unknown.

A Fluidic Isolation-Assisted Homogeneous-Flow-Pressure Chip-Solid Phase Extraction-Mass Spectrometry System for Online Dynamic Monitoring of 25-Hydroxyvitamin D3 Biotransformation in Cells.

It is well known that cell can response to various chemical and mechanical stimuli. Therefore, flow pressure variation induced by sample loading and elution should be small enough to ignore the physical impact on cells when we use a Chip-SPE-MS system for cells. However, most existent Chip-SPE-MS systems ignored the pressure alternation because it is extremely difficult to develop a homogeneous-flow-pressure hyphenated module. Herein, we developed an interesting fluidic isolation-assisted homogeneous-flow-pressure Chip-SPE-MS system and demonstrated that it is adequate for online high-throughput determination and quantification of the 25-hydroxyvitamin D3 (25(OH)D3) biotransformation in different cells. Briefly, the homogeneous ambient flow pressure is achieved by fluidic isolation between the cell culture channel and the SPE column, and an automatic sampling probe could accomplish the sample loading and dispensing to fulfill online pretreatment of the sample. Through this new system, the expression levels of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) can be determined in real time with a detection limit of 2.54 nM. In addition, the results revealed that 25(OH)D3 metabolic activity differed significantly between normal L-02 cells and cancerous HepG2 cells. Treatment of L-02 cells with a high dose of 25(OH)D3 was found to increase significant formation of 24,25(OH)2D3, but this change was not apparent in HepG2 cells. The presented system promises to be a versatile tool for online accurate molecule biotransformation investigation and drug screening processes.

P53 is required for nuclear but not mitochondrial DNA damage-induced degeneration

While the consequences of nuclear DNA damage have been well studied, the exact consequences of acute and selective mitochondrial DNA (mtDNA) damage are less understood. DNA damaging chemotherapeutic drugs are known to activate p53-dependent apoptosis in response to sustained nuclear DNA damage. While it is recognized that whole-cell exposure to these drugs also damages mtDNA, the specific contribution of mtDNA damage to cellular degeneration is less clear. To examine this, we induced selective mtDNA damage in neuronal axons using microfluidic chambers that allow for the spatial and fluidic isolation of neuronal cell bodies (containing nucleus and mitochondria) from the axons (containing mitochondria). Exposure of the DNA damaging drug cisplatin selectively to only the axons induced mtDNA damage in axonal mitochondria, without nuclear damage. We found that this resulted in the selective degeneration of only the targeted axons that were exposed to DNA damage, where ROS was induced but mitochondria were not permeabilized. mtDNA damage-induced axon degeneration was not mediated by any of the three known axon degeneration pathways: apoptosis, axon pruning, and Wallerian degeneration, as Bax-deficiency, or Casp3-deficiency, or Sarm1-deficiency failed to protect the degenerating axons. Strikingly, p53, which is essential for degeneration after nuclear DNA damage, was also not required for degeneration induced with mtDNA damage. This was most evident when the p53-deficient neurons were globally exposed to cisplatin. While the cell bodies of p53-deficient neurons were protected from degeneration in this context, the axons farthest from the cell bodies still underwent degeneration. These results highlight how whole cell exposure to DNA damage activates two pathways of degeneration; a faster, p53-dependent apoptotic degeneration that is triggered in the cell bodies with nuclear DNA damage, and a slower, p53-independent degeneration that is induced with mtDNA damage.

Integration of marker-free selection of single cells at a wireless electrode array with parallel fluidic isolation and electrical lysis.

We present integration of selective single-cell capture at an array of wireless electrodes (bipolar electrodes, BPEs) with transfer into chambers, reagent exchange, fluidic isolation and rapid electrical lysis in a single platform, thus minimizing sample loss and manual intervention steps. The whole process is achieved simply by exchanging the solution in a single inlet reservoir and by adjusting the applied voltage at a pair of driving electrodes, thus making this approach particularly well-suited for a broad range of research and clinical applications. Further, the use of BPEs allows the array to be scalable to increase throughput. Specific innovations reported here include the incorporation of a leak channel to balance competing flow paths, the use of 'split BPEs' to create a distinct recapture and electrical lysis point within the reaction chamber, and the dual purposing of an ionic liquid as an immiscible phase to seal the chambers and as a conductive medium to permit electrical lysis at the split BPEs.

Live Imaging of ESCRT Proteins in Microfluidically Isolated Hippocampal Axons.

Live imaging of microfluidically isolated axons permits study of the dynamic behavior of fluorescently tagged proteins and vesicles in these neuronal processes. We use this technique to study the motility and transport of ESCRT proteins in axons of primary hippocampal neurons. This chapter details the preparation of microfluidic chambers, as well as the seeding, fluidic isolation, and lentiviral transduction of hippocampal neurons in these chambers, optimized for the study of ESCRT protein dynamics.

Neurturin is a PGC-1α1-controlled myokine that promotes motor neuron recruitment and neuromuscular junction formation

ObjectiveWe examined whether skeletal muscle overexpression of PGC-1α1 or PGC-1α4 affected myokine secretion and neuromuscular junction (NMJ) formation. MethodsA microfluidic device was used to model endocrine signaling and NMJ formation between primary mouse myoblast-derived myotubes and embryonic stem cell-derived motor neurons. Differences in hydrostatic pressure allowed for fluidic isolation of either cell type or unidirectional signaling in the fluid phase. Myotubes were transduced to overexpress PGC-1α1 or PGC-1α4, and myokine secretion was quantified using a proximity extension assay. Morphological and functional changes in NMJs were measured by fluorescent microscopy and by monitoring muscle contraction upon motor neuron stimulation. ResultsSkeletal muscle transduction with PGC-1α1, but not PGC-1α4, increased NMJ formation and size. PGC-1α1 increased muscle secretion of neurturin, which was sufficient and necessary for the effects of muscle PGC-1α1 on NMJ formation. ConclusionsOur findings indicate that neurturin is a mediator of PGC-1α1-dependent retrograde signaling from muscle to motor neurons.

A GENERIC PACKAGING TECHNIQUE USING FLUIDIC ISOLATION FOR LOW-DRIFT IMPLANTABLE PRESSURE SENSORS.

This paper reports on a generic packaging method for reducing drift in implantable pressure sensors. The described technique uses fluidic isolation by encasing the pressure sensor in a liquid-filled medical-grade polyurethane balloon; thus, isolating it from surrounding aqueous environment that is the major source of baseline drift. In-vitro tests using commercial micromachined piezoresistive pressure sensors show an average baseline drift of 0.006 cmH2O/day (0.13 mmHg/month) for over 100 days of saline soak test, as compared to 0.101 cmH2O/day (2.23 mmHg/month) for a non-fluidic-isolated one soaked for 18 days. To our knowledge, this is the lowest reported drift for an implantable pressure sensor.

A two-compartment organotypic model of mammalian peripheral nerve repair

Schwann cells in the distal stump of transected nerve upregulate growth factors that support regeneration on a modality-specific basis. It is unclear, however, which of these preferentially support motor axon regeneration. Identification of these factors will require a model that can isolate growth factor effects to growing axons while reproducing the complex three-dimensional structure of peripheral nerve. A two-compartment PDMS base is topped by a collagen-coated membrane that supports a spinal cord cross-section above one compartment. Fluorescent motoneurons in this section reinnervate a segment of peripheral nerve that directs axons through a water-tight barrier to the second compartment, where nerve repair is performed. Motoneurons remain healthy for several weeks. The axons they project through the water-tight barrier survive transection and cross a nerve repair in substantial numbers to reinnervate an additional nerve segment. Fluidic isolation of the two compartments was confirmed with a dye leakage test, and the physiologic integrity of the system was tested by retrograde labeling of only those motor neurons to which tracer was exposed and by limitation of toxin effects to a single compartment. Nerve repair cannot be modeled in monolayer cell culture. Our previous organotypic model accurately modeled nerve repair, but did not allow individual control of motoneuron and growth cone environments. This model isolates treatment effects to growing axons while reproducing the complex three-dimensional structure of peripheral nerve. Additionally, it facilitates surgical manipulation of tissues and high-resolution imaging.

A microchip for quantitative analysis of CNS axon growth under localized biomolecular treatments

Growth capability of neurons is an essential factor in axon regeneration. To better understand how microenvironments influence axon growth, methods that allow spatial control of cellular microenvironments and easy quantification of axon growth are critically needed. Here, we present a microchip capable of physically guiding the growth directions of axons while providing physical and fluidic isolation from neuronal somata/dendrites that enables localized biomolecular treatments and linear axon growth. The microchip allows axons to grow in straight lines inside the axon compartments even after the isolation; therefore, significantly facilitating the axon length quantification process. We further developed an image processing algorithm that automatically quantifies axon growth. The effect of localized extracellular matrix components and brain-derived neurotropic factor treatments on axon growth was investigated. Results show that biomolecules may have substantially different effects on axon growth depending on where they act. For example, while chondroitin sulfate proteoglycan causes axon retraction when added to the axons, it promotes axon growth when applied to the somata. The newly developed microchip overcomes limitations of conventional axon growth research methods that lack localized control of biomolecular environments and are often performed at a significantly lower cell density for only a short period of time due to difficulty in monitoring of axonal growth. This microchip may serve as a powerful tool for investigating factors that promote axon growth and regeneration.

Microfluidic construction of minimalistic neuronal co-cultures

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems). The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency Epatt was >75% during lengthy in chip culture, with ∼85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

A microfluidic chip for axonal isolation and electrophysiological measurements

A microfluidic chip for culturing neurons and spatially isolating axons from somas is presented for use with visually guided whole-cell electrophysiological measurements. A modular design consisting of detachable and re-sealable layers is used to satisfy the requirements of both long-term neuron culturing as well as electrophysiological measurements. Whole cell patch clamp recordings indicate functional viability of neurons with isolated axons. Fluidic isolation was used to achieve asymmetric lentiviral infection of neurons on a single side reservoir. Neurons were asymmetrically infected with lentiviruses expressing the light-activated cationic channel channelrhodopsin-2. Light-evoked excitatory postsynaptic responses were detected by whole cell recordings of neurons on the uninfected side showing functional synaptic connectivity between the two isolated but axonally connected sides of the device.

September 2011: This Month in JoVE

The Journal of Visualized Experiments (JoVE) closes 2011 with a December issue that begins by honoring World's AIDS Day with an article that deals with visualizing the structure of HIV envelope glycoproteins, which are a critical component of the viral infection pathway. One key method for determining virion envelope structure is cryo-electron tomography. Prior to using this technique, soluble virus must be vitrified on a gold grid - a process that involves adding gold to a viral suspension, transferring that suspension to the grid, and plunging the grid into liquid ethane. The virus particles, present in a thin film of liquid atop the grid, are frozen at a rate greater than 100,000 K per second. Following vitrification, collected samples are loaded into the electron microscope and precisely defined regions on the grid are hit with a focused electron beam, while the grid is tilted. The tilt series that results from these imaging sessions is comprised of hundreds of individual images, which must be aligned and averaged to resolve individual envelope proteins. This computationally intense process often requires computer clusters, capable of remote-access parallel processing, like NIH's Biowulf. Once completed, 3D models can not only provide insight into the structure of envelope glycoproteins themselves, but can also reveal the manner in which they interact other proteins, like the neutralizing antibody B12, which is information that can greatly influence vaccine design. This article, from the laboratory of Dr. Sriram Subramaniam involves contributions from twenty authors, including not only graduates students and postdocs, but also middle and highschool students, showing that you are never too young to make an impact in the fight against a disease that has infected over 60 million people, worldwide. In Clinical and Translational Medicine, JoVE presents an article from the University College London and the University of Oxford that investigates the physiological correlates of nociception, or pain sensation, in infants. Typically, infants respond to noxious stimuli by crying and changing their facial expressions - reactions considered to be autonomic and reliant on subcortical regions of the brain. In order to understand the involvement of the cerebral cortex and spinal cord in infant nociception our authors first prepare infants for EEG and EMG recordings. They also set up physiological monitoring and video recording, so that the infant's facial expressions can be linked to physiological data. Following assessment of infant responses to neutral tactile stimuli, the baby is subjected to heel lancing - a clinical procedure used to obtain blood samples from infants. After data processing, electrical potentials can be compared between noxious and non-noxious stimuli in these infants, which will hopefully lead to meaningful conclusions, regarding how nociceptive information is interpreted by newborns. JoVE's Bioengineering section takes a more clinical focus in December with an article from the University of Wisconsin, Madison, which involves the fabrication of a compartmentalized microfluidic device for studying cancer stem cell migration. Following device design, master molds are created by photolithography for the application of poly dimethyl siloxane (PDMS)- an organosilicon compound that is transparent and biologically inert - making it suitable for microfluidics applications with live cells. Specifically, our authors load tumor stem cells derived from multiform glioblastoma, a highly malignant brain cancer, into the compartmentalized devices and then place them in a specialized imaging system that acquires data over a few days. Time lapse video from these authors show that cancer stem cells are able to regulate their morphology in the size-constrained microfluidic channels much like they would in the interstitial space, and fluidic isolation made possible by chambers like these facilitates drug screening for potential therapeutics that can affect cancer stem cell migration. Late on in the month, JoVE travels to France, where investigators from the Institute Pasteur and the Centre National de la Recherche Scientifique, illustrate a method for inducing expression of channel-rhodopsin, a light-activated ion channel, into the olfactory bulb of mice, as well as subsequent neuronal stimulation using a miniature light emitting diode (LED). Our authors give detailed step-by-step instructions for preparing injection needles, and for precise injection of virus into the rostral migratory stream, a site of migration for adult born neuroblasts. Furthermore, they show how miniature LED stimulation devices are fabricated, calibrated, and implanted atop a cranial window prepared over the bulb. In particular, our authors are interested in the way that adult born neurons integrate into existing olfactory neuronal circuits, and they show that LED stimulation in awake behaving animals, can lead to activation of virally-tranduced neuroblasts in the olfactory bulb, which have migrated from the rostral migratory stream. These video-articles comprise JoVE's highlights for the month of December. Other noteworthy upcoming articles illustrate methods for patterning conductive inks into microelectrodes, measuring cytosolic calcium in contractile lymphatics, and imaging neuronal responses to pheromones in the vomeronasal organ.

Dual-compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture

We developed a dual-compartment neurofluidic system with inter-connecting microchannels to connect neurons from their respective compartments, placed on a planar microelectrode arrays. The design and development of the compartmented microfluidic device for neuronal cell culture, protocol for sustaining long-term cultures, and neurite growth through microchannels in such a closed compartment device are presented. Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated. The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining. In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation (CC) based techniques. Bidirectional signal propagation between the compartments is demonstrated using functional connectivity maps. CC analysis and connectivity maps demonstrate that the two neuronal populations are not only functionally connected within each compartment but also with each other and a well connected functional network was formed between the compartments despite the physical barrier introduced by the microchannels.

Development of a novel pseudorabies virus-based method for monosynaptic neuronal network tracing

Event Abstract Back to Event Development of a novel pseudorabies virus-based method for monosynaptic neuronal network tracing Alex S. Ferecskó1*, Zsolt Boldogköi2, Dóra Tombácz2, Balázs Ördög2, Hajime Hirase3, Paul Tiesinga4 and Attila Sík1 1 University of Birmingham, Neuronal Networks Group, School of Clinical and Experimental Medicine, United Kingdom 2 University of Szeged, Department of Medical Biology, Faculty of Medicine, Hungary 3 RIKEN Brain Science Institute, Hirase Research Unit, Japan 4 Radboud University Nijmegen, Donders Centre of Neuroscience, Department of Computational Neuroscience/Neuroinformatics, Netherlands Transsynaptically spreading neurotropic viruses are widely used for analyzing complex neural circuits. The attenuated Bartha (Ba) strain of pseudorabies virus is used as a retrograde transneuronal tracer which is transported to the cell body through axons. We have developed a genetically engineered strain of Ba virus allowing tracing of presynaptic monosynaptic neuronal connections. To restrict labelling to primary connections, the thymidine kinase (TK) gene of the virus has been deleted (TK is a key enzyme for the replication of virus DNA). Cultured cortical neurons were co-transfected with an amplicon-GFP (amp-GFP) construct along with a TK-expression cassette and subsequently infected with the TK-defected Ba (Ba-dTK) virus. The amp-GFP plasmid is packaged into the viral particles as concatamers, and crosses synapses together with the virus labelling presynaptic neurons with GFP. The virus replicates only in the neurons which were transfected with a plasmid containing a TK expressing cassette prior to virus infection. The TK gene containing plasmid does not cross the synapse which results both in the inability of the TK-defected virus to replicate and the inability of the amplicon to be encapsidated in the presynaptic neurons. Amp-GFP was used to visualize presynaptic neurons connected to the targeted neurons. Neurons were cultured into multifluidic cell platforms allowing pre- and postsynaptic neurons to be spatially segregated. Neurons are connected via microgrooves that are only accessible for axons, permitting complete fluidic isolation of neighbouring wells. Fluorescent microscopy revealed amp-GFP expressing transsynaptically labelled neurons contralateral to the infected well. Quantitative assessment of the retrogradely labelled neurons confirmed that the number of labelled neurons were constant and did not show further increases 48-72hours after Ba-dTK infection indicating monosynaptic transsynaptic spreading of the virus. This work was supported by HFSP. Conference: IBRO International Workshop 2010, Pécs, Hungary, 21 Jan - 23 Jan, 2010. Presentation Type: Poster Presentation Topic: Abstracts Citation: Ferecskó AS, Boldogköi Z, Tombácz D, Ördög B, Hirase H, Tiesinga P and Sík A (2010). Development of a novel pseudorabies virus-based method for monosynaptic neuronal network tracing. Front. Neurosci. Conference Abstract: IBRO International Workshop 2010. doi: 10.3389/conf.fnins.2010.10.00203 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 May 2010; Published Online: 03 May 2010. * Correspondence: Alex S Ferecskó, University of Birmingham, Neuronal Networks Group, School of Clinical and Experimental Medicine, Birmingham, United Kingdom, a.ferecsko@bham.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alex S Ferecskó Zsolt Boldogköi Dóra Tombácz Balázs Ördög Hajime Hirase Paul Tiesinga Attila Sík Google Alex S Ferecskó Zsolt Boldogköi Dóra Tombácz Balázs Ördög Hajime Hirase Paul Tiesinga Attila Sík Google Scholar Alex S Ferecskó Zsolt Boldogköi Dóra Tombácz Balázs Ördög Hajime Hirase Paul Tiesinga Attila Sík PubMed Alex S Ferecskó Zsolt Boldogköi Dóra Tombácz Balázs Ördög Hajime Hirase Paul Tiesinga Attila Sík Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability. The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput. Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 microm) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master. Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

Microfluidic compartmentalized co-culture platform for CNS axon myelination research

This paper presents a circular microfluidic compartmentalized co-culture platform that can be used for central nervous system (CNS) axon myelination research. The microfluidic platform is composed of a soma compartment and an axon/glia compartment connected through arrays of axon-guiding microchannels. Myelin-producing glia, oligodendrocytes (OLs), placed in the axon/glia compartment, interact with only axons but not with neuronal somata confined to the soma compartment, reminiscent to in vivo situation where many axon fibres are myelinated by OLs at distance away from neuronal cell bodies. Primary forebrain neurons from embryonic day 16-18 rats were cultured inside the soma compartment for two weeks to allow them to mature and form extensive axon networks. OL progenitors, isolated from postnatal day 1-2 rat brains, were then added to the axon/glia compartment and co-cultured with neurons for an additional two weeks. The microdevice showed fluidic isolation between the two compartments and successfully isolated neuronal cell bodies and dendrites from axons growing through the arrays of axon-guiding microchannels into the axon/glia compartment. The circular co-culture device developed here showed excellent cell loading characteristics where significant numbers of cells were positioned near the axon-guiding microchannels. This significantly increased the probability of axons crossing these microchannels as demonstrated by the more than 51 % of the area of the axon/glia compartment covered with axons two weeks after cell seeding. OL progenitors co-cultured with axons inside the axon/glia compartment successfully differentiated into mature OLs. These results indicate that this device can be used as an excellent in vitro co-culture platform for studying localized axon-glia interaction and signalling.

Electrokinetic pumping of liquid propellants for small satellite microthruster applications

For most orbital maneuvers, small satellites weighing less than 10 kg require propulsion systems capable of producing thrust in the micro-Newton to milli-Newton force range. At this scale, electrokinetic (EK) pumping offers a method to precisely meter liquid propellants under purely electrical control at pressures and flow rates well-suited for microthruster applications. After exploring a variety of materials and surface treatments for the electrokinetic pumping media, we have demonstrated EK pumping of anhydrous hydrazine using both packed-capillary and larger sintered-monolith pump designs. Hydrogen peroxide has proven difficult to directly pump electrokinetically, but we have shown the utility of delivering peroxide and other electrokinetically incompatible liquids indirectly using in-line reservoirs with fluidic isolation to separate the pump working fluid from the propellant. Directly- and indirectly-pumped propellants have been delivered to novel capillary microthrusters with integrated catalyst beds and plasma-formed micronozzle structures. Specific impulses up to 190 s have been shown for hydrazine in non-optimized capillary thrusters at a mass flow rate of 1.5 mg/s. Controlled thrust pulses with a maximum continuous thrust of 1.5 mN, minimum impulse bit of 7 μN-s, and average specific impulse of 110 s have been demonstrated with the electrokinetically pumped microthruster assembly.

BioMEMS: Forging New Collaborations Between Biologists and Engineers

This video describes the fabrication and use of a microfluidic device to culture central nervous system (CNS) neurons. This device is compatible with live-cell optical microscopy (DIC and phase contrast), as well as confocal and two photon microscopy approaches. This method uses precision-molded polymer parts to create miniature multi-compartment cell culture with fluidic isolation. The compartments are made of tiny channels with dimensions that are large enough to culture neurons in well-controlled fluidic microenvironments. Neurons can be cultured for 2-3 weeks within the device, after which they can be fixed and stained for immunocytochemistry. Axonal and somal compartments can be maintained fluidically isolated from each other by using a small hydrostatic pressure difference; this feature can be used to localize soluble insults to one compartment for up to 20 h after each medium change. Fluidic isolation enables collection of pure axonal fraction and biochemical analysis by PCR. The microfluidic device provides a highly adaptable platform for neuroscience research and may find applications in modeling CNS injury and neurodegeneration.