Preparazione E18 neuroni corticali di ratto fetale per compartimentazione Prima di partire, è importante per scaldare tutti i supporti necessari e reagenti a 37 ° C. E &#39;anche importante per sterilizzare tutto ciò che viene utilizzato per la preparazione delle cellule (ad esempio, le lampadine gomma, portaprovette, bottiglie media, ecc), e ciò che è posto nel cofano, con l&#39;annientamento verso il basso con il 70% di etanolo. <ol><li> Mettere due pezzi di corteccia di ratto E18 fetale (un cervello, precedentemente sezionato) in una provetta da 15 ml contenente 1 ml ghiacciata calcio-magnesio libero senza buffer di dissezione. </li><li> Aggiungere 1 ml di 0,25% tripsina-EDTA alla corteccia dissezione nel buffer, portando il volume finale a 2 ml e la concentrazione di tripsina finale a 0,125%. </li><li> Posizionare il tubo di 15 ml in un bagno d&#39;acqua a 37 ° C per 8 minuti. </li><li> Durante questo periodo, il fuoco lucidare 3 pipette Pasteur di vetro, formando aperture sempre più piccoli, in un armadio di biosicurezza per aiutare a mantenere la sterilità. </li><li> Dopo l&#39;incubazione 8 minuti della corteccia, aggiungere 10 ml di DMEM contenente 10% FBS alla corteccia per aiutare a fermare la reazione tripsina. </li><li> Centrifugare i 15 tubo ml contenente la corteccia e DMEM/10% FBS a 2500 rpm per 2 minuti. </li><li> Nell&#39;armadio biosicurezza, rimuovere il sopranatante della corteccia con una pipetta Pasteur in vetro sottovuoto con aspirazione allegato. Fare attenzione a non disturbare o rimuovere il pellet. </li><li> Aggiungere 1 ml di NMB al pellet di corteccia e pipettare delicatamente su e giù. E &#39;molto importante evitare di creare bolle d&#39;aria, mentre pipeting su e giù, come le bolle d&#39;aria può danneggiare le cellule dall&#39;ossidazione. </li><li> Usare il fuoco lucido pipetta Pasteur con la più ampia apertura verso triturare la corteccia ed accompagnato da una lampadina sterile gomma fino alla fine e pipeting su e giù per 5 volte, sempre facendo attenzione a non introdurre bolle d&#39;aria. Continuare questo processo con gli altri 2 pipette di vetro, ciascuna con una dimensione di apertura ridotto. </li><li> Dopo la triturazione, centrifuga giù le cellule di nuovo a 2500 rpm per 2 minuti. </li><li> Dopo la centrifugazione, ancora una volta, rimuovere il supernatante e risospendere il pellet di cellule in 2 ml di NBM. </li><li> Filtrare la soluzione risospeso cellula attraverso un colino 45 celle um. </li><li> Macchia le cellule con Blu tripan, contati, e caricati nel dispositivo. Noi di solito carico 20 ul di cellule per dispositivo. </li></ol> Nota: la concentrazione finale di cellule è in genere tra 2,5 milioni di cellule / ml e 8 milioni di cellule / ml Caricamento in corso le cellule Dopo che le cellule sono state contate, è tempo di caricare le cellule del dispositivo: <ol><li> Portare i dispositivi precedentemente preparato contenente NBM nel mobile biosicurezza per mantenere la sterilità. </li><li> Rimuovere i supporti in eccesso dai pozzi di aspirazione. Tuttavia, fare attenzione a evitare di rimuovere tutti i mezzi di comunicazione - ci deve essere qualche supporto nel canale principale. </li><li> Applicare 20 ul di cellule al serbatoio in alto a sinistra del dispositivo (vedi schema se necessario). Il flusso di cellule nel dispositivo e attaccarsi alla superficie trattata PLL. </li><li> Dopo il caricamento, posizionare i dispositivi contenenti cellule indietro in un incubatore per 10 minuti per consentire alle cellule di allegare. </li><li> Dopo 10 minuti, riempire i serbatoi con i media e dispositivi di luogo indietro nell&#39;incubatrice. </li></ol>

<ol>
	<li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 1, 2128-2136 (2006).</li><li>Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2, 599-605 (2005).</li></ol>

Densità delle cellule può essere variata a seconda dell&#39;applicazione. Tuttavia, semina neuroni primari troppo basso di una densità di porta normalmente alla morte cellulare. A seconda della incubatrice e il livello di umidità, i media può avere bisogno di essere cambiato nei dispositivi ogni 2 o 3 giorni. Quando si cambiano i media, è importante dopo aver rimosso il supporto dal serbatoio, per rimuovere mai i media dai canali principali. Inoltre, è buona abitudine mettere mezzi freschi nei pozzetti superiore e consentire ad alcuni mezzi freschi a fluire attraverso il dispositivo e nei principali canali prima di riempire tutti i serbatoi.

<table><tbody><tr><td>NBM</td><td>medium</td><td>Invitrogen</td><td>21103-049</td><td>Neural Basal Media is obtained by Invitrogen under their Gibco line of cell culture reagents.</td></tr><tr><td>E18 rat embryos</td><td>Animal</td><td></td><td></td><td>Obtained from pregnant Sprague Dawley Rats. The pregnant rat is sacrificed and the E18 fetal rat pups dissected to obtain the E18 fetal rat cortex.</td></tr><tr><td>CMFM dissection buffer HBSS</td><td>buffer</td><td></td><td></td><td>ice-cold Calcium-free Magnesium-free dissection buffer HBSS based.</td></tr><tr><td>15 ml Tube</td><td>Tool</td><td>BD Biosciences</td><td>Falcon No. 352097</td><td>Available through Fisher Scientific catalog number 14-959-70C</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Reagent</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>37&amp;#730;C water bath</td><td></td><td></td><td></td><td></td></tr><tr><td>Pasteur pipets</td><td></td><td>Fisher Scientific</td><td></td><td>glass</td></tr><tr><td>DMEM</td><td>medium</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>FBS</td><td>Reagent</td><td>Invitrogen</td><td></td><td>serum, used at 10% in DMEM</td></tr><tr><td>centrifuge</td><td></td><td></td><td></td><td>set at 2500 rpm </td></tr><tr><td>Trypan Blue</td><td></td><td>Invitrogen</td><td></td><td>for counting live/dead cells</td></tr><tr><td>BD Falcon Cell Strainer 40 um</td><td>Tool</td><td>BD Biosciences</td><td>Falcon cat# 352340</td><td>Available through Fisher Scientific. Fisher catalog# 08-771-1</td></tr><tr><td>B27</td><td>Reagent</td><td>Invitrogen</td><td>17504-044</td><td>B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents.</td></tr><tr><td>Glutamax</td><td>Reagent</td><td>Invitrogen</td><td>35050-061</td><td>Glutamax is available through Invitrogen under their Gibco line of celll culture reagents.</td></tr></tbody></table>

preparing e18 cortical rat neurons for compartmentalization in a microfluidic device

In questo video, dimostriamo la preparazione della E18 neuroni corticali di ratto. E18 neuroni corticali di ratto sono ottenuti da E18 corteccia fetale di ratto precedentemente sezionati e preparati. La corteccia è E18, dopo la dissezione, immediatamente dissociato in singoli neuroni. E &#39;possibile memorizzare E18 corteccia in un tampone contenente Hibernate E B27 a 4 ° C per un massimo di una settimana prima della dissociazione viene eseguita. Tuttavia, ci sarà un calo di vitalità cellulare. Tipicamente si ottiene la nostra E18 Cortex fresca. E &#39;trasportato al laboratorio a freddo di calcio privo di ghiaccio tampone dissezione di magnesio libero (CMFM). All&#39;arrivo, tripsina viene aggiunto alla corteccia a una concentrazione finale di 0,125%. La corteccia viene quindi incubato a 37 ° C per 8 minuti. DMEM contenente 10% FBS viene aggiunto alla corteccia per arrestare la reazione. La corteccia viene poi centrifugato a 2500 rpm per 2 minuti. Il surnatante viene rimosso e 2 ml di Neural media basale (NBM) contenente 2% B27 (vol / vol) e lo 0,25% si aggiunge Glutamax (vol / vol) verso la corteccia che viene poi nuovamente sospeso pipettando su e giù. Successivamente, la corteccia è triturato con pipette di vetro precedentemente lucidate a fuoco, ciascuno con una successiva apertura più piccola. Dopo la triturazione, la corteccia è ancora una volta centrifugato a 2500 rpm per 2 minuti. Il surnatante viene quindi rimosso e il pellet di corteccia risospese con 2 ml di NBM contenenti B27 e Glutamax. La sospensione cellulare viene fatto passare attraverso un colino 40 um cellule nylon. Avanti le cellule sono contati. I neuroni sono ora pronti per il caricamento nel dispositivo neurone microfluidica.

Watch this Scientific Journal Video about Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device at JoVE.com

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In questo video ci mostrano la preparazione della E18 neuroni corticali di ratto.

Preparazione E18 neuroni corticali di ratto per compartimentazione in un dispositivo a microfluidi

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

preparing-e18-cortical-rat-neurons-for-compartmentalization

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

19.7K Views.  University of California, Irvine (UCI). In questo video ci mostrano la preparazione della E18 neuroni corticali di ratto.

Video: Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Fabbricazione di un dispositivo a microfluidi per la compartimentazione dei Neuron Soma e assoni

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Ricerca

Biologia

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 &mu;m) 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.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un multi-vano SNC Neuron-glia Co-cultura Microfluidic piattaforma

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used to investigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia, and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (&#60;10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (&#62;85%) intercompartment connectivity and can be used for high throughput neurobiology experiments with single cell precision.

Protocols for single neuron microfluidic arraying and water masking for the in-chip plasma patterning of biomaterial coatings are described. Highly interconnected co-cultures can be prepared using minimal cell inputs.

Preparazione della neuronali Co-culture con precisione singola cella

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

Neuroscienze

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Tessutale tridimensionale allineato Astrocyte reti per ricapitolare i meccanismi dello sviluppo e facilitare la rigenerazione del sistema nervoso

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Bioingegneria

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microfluidica di interfacciamento con microelettrodi per studiare comunicazione neuronale e propagazione del segnale assonale

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

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 commercially available pre-assembled plastic chip for compartmentalizing cultured primary rat hippocampal neurons. These plastic chips, contained within the footprint of a standard microscope slide, are compatible with high-resolution, live, and fluorescence imaging. This protocol demonstrates how to retrograde label neurons via isolated axons using a modified rabies virus encoding a fluorescent protein, create isolated microenvironments within one compartment, and perform axotomy and immunocytochemistry on-chip. Neurons are cultured for &gt;3 weeks within the plastic chips, illustrating the compatibility of these chips for long-term neuronal cultures.

This protocol describes the use of plastic chips to culture and compartmentalize primary murine neurons. These chips are preassembled, user-friendly, and compatible with high-resolution, live, and fluorescence imaging. This protocol describes how to plate rat hippocampal neurons within these chips and perform fluidic isolation, axotomy and immunostaining.

Uso di chip microfluidici plastica pre-assemblato per divisione in compartimenti neuroni primari murini

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

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 pre-assembled multi-compartment chip made in a cyclic olefin copolymer (COC) to compartmentalize neurons differentiated from human stem cells. The footprint of these COC chips are the same as a standard microscope slide and are equally compatible with high resolution microscopy. Neurons are differentiated from human neural stem cells (NSCs) into glutamatergic neurons within the chip and maintained for 5 weeks, allowing sufficient time for these neurons to develop synapses and dendritic spines. Further, we demonstrate multiple common experimental procedures using these multi-compartment chips, including viral labeling, establishing microenvironments, axotomy, and immunocytochemistry.

This protocol demonstrates the use of compartmentalized microfluidic chips, injection molded in a cyclic olefin copolymer to cultured neurons differentiated from human stem cells. These chips are preassembled and easier-to-use than traditional compartmentalized poly(dimethylsiloxane) devices. Multiple common experimental paradigms are described here, including viral labeling, fluidic isolation, axotomy, and immunostaining.

Compartimentazione dei neuroni derivati da cellule staminali umane all'interno di chip microfluidici in plastica pre-assemblati

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

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many neurodegenerative diseases. Given some axons' elaborate architecture and extreme length, it is not surprising that mitochondria in axons can experience different environments compared to their cell body counterparts. Interestingly, dysfunction of axonal mitochondria often precedes effects on the cell body. To model axonal mitochondrial dysfunction in vitro, microfluidic devices allow treatment of axonal mitochondria without affecting the somal mitochondria. The fluidic pressure gradient in these chambers prevents diffusion of molecules against the gradient, thus allowing for analysis of mitochondrial properties in response to local pharmacological challenges within axons. The current protocol describes the seeding of dissociated hippocampal neurons in microfluidic devices, staining with a membrane-potential sensitive dye, treatment with a mitochondrial toxin, and the subsequent microscopic analysis. This versatile method to study axonal biology can be applied to many pharmacological perturbations and imaging readouts, and is suitable for several neuronal subtypes.

The present protocol describes the seeding and staining of neuronal mitochondria in microfluidic chambers. The fluidic pressure gradient in these chambers allows for the selective treatment of mitochondria in axons to analyze their properties in response to pharmacological challenges without affecting the cell body compartment.

Depolarizzazione selettiva assistita da microfluidica dei mitocondri assonali

Mitochondria are the primary suppliers of ATP (adenosine triphosphate) in neurons. Mitochondrial dysfunction is a common phenotype in many ...

Compartmentalized Primary Murine Neuron Culture Using Plastic Microfluidic Chips

Source: Nagendran, T., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/58421">Use of Pre-Assembled Plastic Microfluidic Chips for Compartmentalizing Primary Murine Neurons.</a> J. Vis. Exp. (2018).
This video demonstrates the utilization of a pre-assembled plastic microfluidic chip for the compartmentalized culture of primary rat hippocampal neurons. The neurons are plated into one of the microfluidic compartments, where they attach and grow, extending their axons through the chip&#39;s microgrooves into the adjacent compartment. The narrow microgrooves prevent neuronal cell bodies from entering, thereby enabling physical separation between the somatic and axonal compartments.

Coltura di neuroni murini primari compartimentati utilizzando chip microfluidici in plastica

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

Initiating Rapid Neurite Extension and Formation Functional Neuronal Connections

Source: M. H. Magdesian et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55697">Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection,</a> J. Vis. Exp. (2017)
This video demonstrates a technique for the rapid initiation and extension of a neuron and creation of new connections with a target neuron using a microfluidic device and micromanipulation. This could be used for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.