use-pre-assembled-plastic-microfluidic-chips-for-compartmentalizing

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Microfabricated methods to compartmentalize neurons have become essential tools for many neuroscientists. This protocol describes the use of a ...

compartmentalization-human-stem-cell-derived-neurons-within-pre

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

UNC Neuroscience Center

Xona Microfluidics, LLC

UNC/NC State Joint Department of Biomedical Engineering

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The precise spatial and temporal control afforded by microfluidic devices make them uniquely suited as experimental tools for cellular neuroscience. Micro-structures have been developed to direct the placement of cells and small organisms within a device. Microfluidics can precisely define pharmacological microenvironments, mimicking conditions found in vivo with the advantage of defined parameters which are usually difficult to control and manipulate in vivo. These devices are compatible with high-resolution microscopy, are simple to assemble, and are reproducible. In this review we will focus on microfluidic devices that have recently been developed for small, whole organisms such as C. elegans and dissociated cultured neurons. These devices have improved control over the placement of cells or organisms and allowed unprecedented experimental access, enabling novel investigations in neurobiology.

Micro-scale and microfluidic devices for neurobiology.

Le contr&#xF4;le pr&#xE9;cis spatiale et temporelle assur&#xE9;e par des dispositifs microfluidiques rendre particuli&#xE8;rement bien adapt&#xE9; comme des outils exp&#xE9;rimentaux pour cellulaire en neurosciences. Micro-structures ont &#xE9;t&#xE9; d&#xE9;velopp&#xE9;es pour diriger le placement de cellules et de petits organismes au sein d&#x26;#39;un appareil. Microfluidique ne peut d&#xE9;finir pr&#xE9;cis&#xE9;ment microenvironnements pharmacologiques, imitant les conditions trouv&#xE9;es in vivo avec l&#x26;#39;avantage de param&#xE8;tres d&#xE9;finis, qui sont g&#xE9;n&#xE9;ralement difficiles &#xE0; contr&#xF4;ler et &#xE0; manipuler in vivo. Ces appareils sont compatibles avec microscopie &#xE0; haute r&#xE9;solution, sont simples &#xE0; assembler, et sont reproductibles. Dans cette revue, nous nous concentrerons sur des dispositifs microfluidiques qui ont &#xE9;t&#xE9; r&#xE9;cemment d&#xE9;velopp&#xE9;s pour les petites, les organismes entiers, tels que C. elegans et dissoci&#xE9;es des neurones en culture. Ces dispositifs ont am&#xE9;lior&#xE9; le contr&#xF4;le sur le placement de cellules ou d&#x26;#39;organismes autoris&#xE9;s et un acc&#xE8;s sans pr&#xE9;c&#xE9;dent exp&#xE9;rimentale, permettant de nouvelles enqu&#xEA;tes en neurobiologie.

Micro Devices &#xE9;chelle et microfluidique pour la neurobiologie

Die genaue r&auml;umliche und zeitliche Kontrolle durch Microfluidic Vorrichtungen gew&auml;hrte machen sie eindeutig als experimentelle Werkzeuge f&uuml;r zellul&auml;re Neurowissenschaften geeignet. Mikrostrukturen sind entwickelt worden, um die Platzierung von Zellen und kleinen Organismen innerhalb eines Ger&auml;ts zu leiten. Mikrofluidik k&ouml;nnen genau definieren pharmakologische Mikroumgebungen Nachahmung gefunden in-vivo mit dem Vorteil der definierten Parameter, die in der Regel schwer zu kontrollieren und manipulieren in-vivo Bedingungen. Diese Ger&auml;te sind kompatibel mit hochaufl&ouml;senden Mikroskopie, sind einfach zu montieren und sind reproduzierbar. In diesem Bericht konzentrieren wir uns auf Microfluidic Vorrichtungen, die vor kurzem entwickelt wurden f&uuml;r kleine, ganze Organismen wie C. Elegans und getrennten kultivierten Neuronen. Diese Ger&auml;te haben verbesserte Kontrolle &uuml;ber die Platzierung von Zellen oder Organismen und beispiellosen experimentellen Zugang, erm&ouml;glicht neuartige Untersuchungen in Neurobiologie gestattet.

Mikroskala- und Microfluidic Vorrichtungen f&uuml;r Neurobiologie.

Il preciso controllo spaziale e temporale offerto da dispositivi microfluidici li rendono adatti in modo univoco come strumenti sperimentali per neuroscienza cellulare. Micro-strutture sono state sviluppate per dirigere il posizionamento delle cellule e piccoli organismi all&#39;interno di un dispositivo. Microfluidics pu&ograve; definire precisamente microambienti farmacologiche, imitando le condizioni presenti in vivo con il vantaggio di parametri definiti, che sono solitamente difficili da controllare e manipolare in vivo. Questi dispositivi sono compatibili con la microscopia ad alta risoluzione, sono semplici da montare e sono riproducibile. In questa recensione ci concentreremo su dispositivi microfluidici che recentemente sono stati sviluppati per piccoli, interi organismi come c. elegans e dissociati neuroni coltivati. Questi dispositivi hanno migliorato controllo sopra il posizionamento delle cellule o organismi e consentito l&#39;accesso sperimentale senza precedenti, permettendo nuove indagini in neurobiologia.

Micro-scala e microfluidic dispositivi per la neurobiologia.

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&#xC815;&#xD655; &#xD558; &#xAC8C; &#xACF5;&#xAC04; &#xBC0F; &#xC2DC;&#xAC04; &#xC81C;&#xC5B4; &#xACB0;&#xD569; &#xC7A5;&#xCE58;&#xC5D0; &#xC758;&#xD574; &#xC5EC;&#xC720; &#xC218; &#xC788;&#xB3C4;&#xB85D; &#xACE0;&#xC720; &#xD558; &#xAC8C; &#xC138;&#xD3EC; &#xC2E0;&#xACBD; &#xACFC;&#xD559;&#xC744; &#xC704;&#xD55C; &#xC2E4;&#xD5D8; &#xB3C4;&#xAD6C;&#xB85C; &#xC801;&#xD569;. &#xB9C8;&#xC774;&#xD06C;&#xB85C; &#xAD6C;&#xC870; &#xC140; &#xBC0F; &#xC18C;&#xC790; &#xB0B4;&#xC5D0;&#xC11C; &#xC791;&#xC740; &#xC0DD;&#xBB3C;&#xC758; &#xBC30;&#xCE58;&#xB97C; &#xC9C1;&#xC811; &#xAC1C;&#xBC1C; &#xB418;&#xC5C8;&#xC2B5;&#xB2C8;&#xB2E4;. Microfluidics &#xC815;&#xD655; &#xD558; &#xAC8C; &#xC815;&#xC758;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC57D;&#xB9AC; microenvironments &#xC870;&#xAC74; vivo&#xC5D0;&#xC11C; &#xC815;&#xC758; &#xB41C; &#xB9E4;&#xAC1C; &#xBCC0;&#xC218;&#xAC00; &#xC77C;&#xBC18;&#xC801;&#xC73C;&#xB85C; &#xC81C;&#xC5B4; &#xD558; &#xACE0; vivo&#xC5D0;&#xC11C; &#xC870;&#xC791; &#xD558;&#xAE30; &#xC5B4;&#xB824;&#xC6B4; &#xC7A5;&#xC810;&#xC774; &#xBC1C;&#xACAC;&#xC744; &#xD749;&#xB0B4; &#xB0B8;. &#xC774; &#xC18C;&#xC790; &#xB4E4;&#xC740; &#xACE0;&#xD574;&#xC0C1;&#xB3C4; &#xD604;&#xBBF8;&#xACBD;, &#xC870;&#xB9BD;, &#xAC04;&#xB2E8; &#xD558; &#xACE0; &#xC7AC;&#xD604;&#xD560; &#xC218;&#xC640; &#xD638;&#xD658; &#xB429;&#xB2C8;&#xB2E4;. &#xC774; &#xB9AC;&#xBDF0;&#xC5D0;&#xC11C; &#xC6B0;&#xB9AC; C. elegans &#xB04A;&#xB41C; &#xAD50;&#xC591;&#xB41C; &#xB274;&#xB7F0; &#xB4F1; &#xC791;&#xC740;, &#xC804;&#xCCB4; &#xC720;&#xAE30; &#xCCB4;&#xC5D0; &#xB300; &#xD55C; &#xCD5C;&#xADFC;&#xC5D0; &#xAC1C;&#xBC1C; &#xB41C; &#xBBF8;&#xC138; &#xC7A5;&#xCE58;&#xC5D0; &#xCD08;&#xC810;&#xC744; &#xAC83;&#xC785;&#xB2C8;&#xB2E4;. &#xC774; &#xC18C;&#xC790; &#xB4E4;&#xC740; &#xC140; &#xB610;&#xB294; &#xC0DD;&#xBB3C;&#xC758; &#xBC30;&#xCE58;&#xB97C; &#xD1B5;&#xD574; &#xD5A5;&#xC0C1; &#xB41C; &#xC81C;&#xC5B4; &#xBC0F; &#xC2E0;&#xACBD; &#xC0DD;&#xBB3C;&#xD559;&#xC5D0;&#xC11C; &#xC18C;&#xC124; &#xC870;&#xC0AC; &#xD65C;&#xC131;&#xD654; &#xC804;&#xB840; &#xC5C6;&#xB294; &#xC2E4;&#xD5D8;&#xC801;&#xC778; &#xC811;&#xADFC; &#xD5C8;&#xC6A9;.

&#xC2E0;&#xACBD; &#xC0DD;&#xBB3C;&#xD559;&#xC744; &#xC704;&#xD55C; &#xB9C8;&#xC774;&#xD06C;&#xB85C; &#xC2A4;&#xCF00;&#xC77C; &#xBC0F; &#xACB0;&#xD569; &#xC7A5;&#xCE58;&#xC785;&#xB2C8;&#xB2E4;.

El control preciso espacial y temporal que ofrece dispositivos de microfluidos hacen especialmente adecuada como herramientas experimentales para celular, la neurociencia. Las micro-estructuras han sido desarrollados para dirigir la colocaci&#xF3;n de las c&#xE9;lulas y organismos peque&#xF1;os dentro de un dispositivo. Microflu&#xED;dica puede definir con precisi&#xF3;n microambientes farmacol&#xF3;gicos, imitando las condiciones encontradas en vivo con la ventaja de los par&#xE1;metros definidos, que normalmente son dif&#xED;ciles de controlar y manipular in vivo. Estos dispositivos son compatibles con microscop&#xED;a de alta resoluci&#xF3;n, son f&#xE1;ciles de montar, y son reproducibles. En esta revisi&#xF3;n se centrar&#xE1; en los dispositivos de microfluidos que recientemente han sido desarrollados para organismos peque&#xF1;os, enteros, tales como C. elegans y cultivos de neuronas disociadas. Estos dispositivos han mejorado el control sobre la colocaci&#xF3;n de las c&#xE9;lulas u organismos y permite el acceso experimental sin precedentes, permitiendo a las investigaciones novedosas en la neurobiolog&#xED;a.

Micro Devices escala y microflu&#xED;dica para Neurobiolog&#xED;a

The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles, we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform, mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred, thus increasing screening throughput by >30-fold. Compared to conventional methods, micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience. 

Transferable neuronal mini-cultures to accelerate screening in primary and induced pluripotent stem cell-derived neurons.

Neuronal growth cone filopodia contain guidance receptors and contribute to axon guidance; however, the mechanism by which the guidance cue netrin increases filopodia density is unknown. Here, we demonstrate that TRIM9, an E3 ubiquitin ligase that localizes to filopodia tips and binds the netrin receptor DCC, interacts with and ubiquitinates the barbed-end polymerase VASP to modulate filopodial stability during netrin-dependent axon guidance. Studies with murine Trim9(+/+) and Trim9(-/-) cortical neurons, along with a non-ubiquitinatable VASP mutant, demonstrate that TRIM9-mediated ubiquitination of VASP reduces VASP filopodial tip localization, VASP dynamics at tips, and filopodial stability. Upon netrin treatment, VASP is deubiquitinated, which promotes VASP tip localization and filopodial stability. Trim9 deletion induces axon guidance defects in vitro and in vivo, whereas a gradient of deubiquitinase inhibition promotes axon turning in vitro. We conclude that a gradient of TRIM9-mediated ubiquitination of VASP creates a filopodial stability gradient during axon turning.

The E3 Ubiquitin Ligase TRIM9 Is a Filopodia Off Switch Required for Netrin-Dependent Axon Guidance.

The identification of mRNAs in distal projections of model organisms has led to the discovery of multiple proteins that are locally synthesized for functional roles such as axon guidance, injury signaling and regeneration. The extent to which local protein synthesis is conserved in human neurons is unknown. Here we used compartmentalized microfluidic chambers to characterize the transcriptome of distal projections of human embryonic stem cells differentiated using a protocol which enriched for glutamatergic neurons (hESC-neurons). Using gene expression analysis, we identified mRNAs proportionally enriched in these projections, representing a functionally unique local transcriptome as compared to the human neuronal transcriptome inclusive of somata. Further, we found that the most abundant mRNAs within these hESC-neuron projections were functionally similar to the axonal transcriptome of rat cortical neurons. We confirmed the presence of two well characterized axonal mRNAs in model organisms, β-actin and GAP43, within hESC-neuron projections using multiplexed single molecule RNA-FISH. Additionally, we report the novel finding that oxytocin mRNA localized to these human projections and confirmed its localization using RNA-FISH. This new evaluation of mRNA within human projections provides an important resource for studying local mRNA translation and has the potential to reveal both conserved and unique translation dependent mechanisms.

Messenger RNAs localized to distal projections of human stem cell derived neurons.

Injury of CNS nerve tracts remodels circuitry through dendritic spine loss and hyper-excitability, thus influencing recovery. Due to the complexity of the CNS, a mechanistic understanding of injury-induced synaptic remodeling remains unclear. Using microfluidic chambers to separate and injure distal axons, we show that axotomy causes retrograde dendritic spine loss at directly injured pyramidal neurons followed by retrograde presynaptic hyper-excitability. These remodeling events require activity at the site of injury, axon-to-soma signaling, and transcription. Similarly, directly injured corticospinal neurons in vivo also exhibit a specific increase in spiking following axon injury. Axotomy-induced hyper-excitability of cultured neurons coincides with elimination of inhibitory inputs onto injured neurons, including those formed onto dendritic spines. Netrin-1 downregulation occurs following axon injury and exogenous netrin-1 applied after injury normalizes spine density, presynaptic excitability, and inhibitory inputs at injured neurons. Our findings show that intrinsic signaling within damaged neurons regulates synaptic remodeling and involves netrin-1 signaling.Spinal cord injury can induce synaptic reorganization and remodeling in the brain. Here the authors study how severed distal axons signal back to the cell body to induce hyperexcitability, loss of inhibition and enhanced presynaptic release through netrin-1.

Anne Marion Taylor

UNC Neuroscience Center

Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips