Vorremmo ringraziare il Dott. Jeffrey Rothstein per la fornitura di BAC GLT1 topi eGFP e GLT1 anticorpi; Tufts Center for Neuroscience Research (NIH P30 NS047243, PI, Rob Jackson) per la fornitura di servizi di base preziosi, New facoltà di assunzione di assegnazione (NIH 5P30NS069254 P30-02 , PI, Phil Haydon) in Tufts Neuroscienze Dipartimento.

1. Assemblea della Camera Cultura Microfluidic (MCP) <ol><li> MCP (Figura 1) sono progettati per cavità si aprono culture compartimenti di diversi tipi di cellule 4. Ha tipicamente due compartimenti che sono collegati attraverso i canali centrale (3 mm di diametro). Montaggio di MCP con fondo di vetro piatti è necessario per la preparazione di culture e l&#39;analisi di immagini successive. </li><li> Primo, cappotto sterili fondo di vetro piatti con Polyornithine (Sigma-Aldrich, 1 mg / ml) sciolto in tetraborato di sodio (Sigma-Aldrich, 10 mM pH 8,5) e sono state incubate per una notte a 37 ° C. </li><li> Il giorno seguente, il fondo di vetro rivestite piastre sono state lavate tre volte con DDH 2 O ed essiccato sotto cappa sterile fumi. I fondo di vetro piatti erano poi ulteriormente rivestito con laminina (Sigma-Aldrich, 1 mg / ml) ed essiccati sotto luce UV per 1 ora. Piatti rivestiti sono pronti all&#39;uso o può essere conservato a -20 ° C fino all&#39;uso. Questi un rivestimentori necessari per neuroni e astrociti placcatura in co-coltura. </li><li> Per assemblare la piattaforma cultura, piattaforme cultura microfluidici sono stati collocati in cima alle fondo di vetro rivestite con piatti centrali canali di connessione sul suo lato inferiore per formare una tenuta ermetica. Neurone regolare o terreni di coltura sono stati aggiunti astrociti (dalle zone di placcatura di cella) su entrambi i lati (figura 1) nella assemblato MCP e incubate per 2-4 ore a 37 ° C, per garantire l&#39;assenza di fughe tra MCP e il vetro- piatto fondo. Abbiamo testato con il mezzo prima che le cellule placcatura per garantire non ci sono perdite. L&#39;assemblea di MCP sul vetro a fondo piatto deve essere preparata estemporaneamente prima dell&#39;uso. </li></ol> 2. Preparazione della Cultura neuronale e induzione degli assoni neuronali in assemblato MCP <ol><li> Corticali colture di cellule neuronali sono stati preparati da embrionali 14-16 giorni di età cervello dei topi. Il mezzo di coltura neurone è composto medie Neurobasal, 2% B27 neurobasaL integratore, 2 mM glutammina aggiungendo 1% di 100x glutamax, e 1% di penicillina-streptomysin. Seguendo dissezione del cervello di topo, le meningi stato rimosso dalla corteccia sotto il microscopio da dissezione. Il tessuto è stato quindi macinata con una lama di rasoio per fare piccoli blocchi di tessuto al fine di aumentare la superficie esposta alla tripsina. Blocchi di tessuto sono state tripsinizzate (Sigma-Aldrich, 0,05% di tripsina) per 10 min in un bagno di acqua a 37 ° C, e quindi dissociato delicatamente per triturazione con un incendio lucidato pipetta Pasteur. Cellule dissociate sono state filtrate attraverso un filtro 70 micron a raccogliere chiaro sospensione cellulare neuronale. </li><li> Neuroni (2-3 x 10 6 / ml, 150 ml) sono state piastrate su aree placcatura cella sulla destra (Figura 1) del MCP assemblato in modo che i neuroni possono attaccare nella zona di ritenzione cellulare (Figura 1). Neuroni preparate sono state piastrate in mezzo di placcatura neurone che viene integrata con il 5% di siero bovino fetale (Hyclone) nella regular neurone terreno di coltura. Perché solo i neuroni che fissano nella zona ritenzione delle cellule sono in grado di crescere assoni nell&#39;altro lato della assemblato MCP attraverso canali centrali, è importante densità piastra elevato di neuroni nell&#39;area placcatura cella per garantire pochi neuroni sono collegati al cella area di ritenzione. Una immagine rappresentativa di alta densità di assoni neuronali è mostrato nella figura 2A. </li><li> Il giorno seguente, il mezzo placcatura neurone è stato sostituito dalla media regolare cultura neurone. Medie cambiando è stata compiuta da aspirando accuratamente il terreno dalla superficie cellulare placcatura della camera, ma non dalla zona di ritenzione della cella assemblata MCP, per evitare bolle d&#39;aria nella zona di ritenzione cellulare ei canali centrali di collegamento. Le bolle d&#39;aria si inibiscono gravemente la conseguenza assone e la successiva entrata in canali centrali del MCP. Cellule gliali fattore neurotrofico derivato (GDNF, 10-20 ng / ml) è stato aggiunto anche dall&#39;altro (sinistro)della camera il giorno stesso per facilitare l&#39;induzione della crescita degli assoni 5 dal lato neuronale e trasversale attraverso canali centrali assemblato MCP (Figura 2A). Discreta quantità di crescita dei neuriti spontanea senza GDNF si osserva anche dal lato neuronale e trasversale attraverso i canali centrali della assemblato MCP. Assoni che entrano nel canale centrale sono spesso osservati 2-3 giorni dopo la placcatura. Una volta che gli assoni entra nel canale centrale, di solito immettere lato entro un giorno. Assoni sono normalmente intatto per almeno una settimana dopo aver inserito l&#39;altro lato. </li></ol> 3. L&#39;aggiunta di astrociti in coltura per MCP per stabilire una co-cultura a compartimenti stagni del sistema Colture primarie di astrociti sono stati preparati da P1-3 cervello di topo cucciolo. La procedura di dissociazione cervello è simile alla procedura di isolamento cellulare neuronale descritto sopra. Astrociti sono stati piastrati in piatto 10 cm che sono stati pre-Rivestito con Polyornithine (Sigma-Aldrich, 1 mg / ml). Il mezzo di coltura astrociti (DMEM, 10% di siero fetale bovino, 1% di penicillina-streptomysin) è stato cambiato ogni giorno per i due giorni successivi per rimuovere i detriti. Dopo di che, il mezzo è stato cambiato ogni tre giorni. <ol><li> Astrociti diventano 90% confluenti e formano un monostrato dopo 7 giorni. Astrociti confluenti sono state tripsinizzate e 150 pl di ri-sospese astrociti (1x10 6 / ml) sono stati ri-piastrate nell&#39;area cella fasciame del lato sinistro della assemblato MCP quando assoni stanno per entrare o appena entrato nel lato sinistro l&#39;assemblato MCP. Gli astrociti sono stati di solito 4-5 giorni dopo piastrate i neuroni erano placcato. GDNF è stato rimosso prima della re-placcatura dei astrociti nel lato sinistro della MCP. Re placcati astrociti sono stati attaccati nella zona di ritenzione cellulare, come mostrato dalla immunostaining GFAP (Figura 1). Solo flusso minimo di mezzo tra entrambi i lati delle camere (determinata dalla fluorescenza dsì) è stato trovato nel MCP, come precedentemente illustrato 4,6. </li><li> Astrociti diventano 90% confluenti e formano un monostrato dopo 7 giorni. Astrociti confluenti sono state tripsinizzate e 150 pl di ri-sospese astrociti (1 x 10 6 / ml) sono stati ri-piastrate nell&#39;area cella fasciame del lato sinistro della assemblato MCP quando assoni stanno per entrare o sono entrate in sinistra lato del MCP assemblato. Gli astrociti sono stati di solito 4-5 giorni dopo piastrate i neuroni erano placcato. GDNF è stato rimosso prima della re-placcatura dei astrociti nel lato sinistro della MCP. Re placcati astrociti sono stati attaccati nella zona di ritenzione cellulare, come mostrato dalla immunostaining GFAP (Figura 1). Solo flusso minimo di mezzo tra entrambi i lati delle camere (determinata da coloranti fluorescenti) è stato trovato nel MCP, come precedentemente illustrato 4,6. </li><li> Dopo la ri-placcatura dei astrociti, assoni che sono entrate nel lato sinistro della assemblato MCP interagisce direttamente con il astrocytes di contatto diretto assonale o il rilascio di fattori solubili. Immunocolorazione astrogliale GLT1 membrana plasmatica trasportatore e glutammato neuronale βIII-tubulina è stata eseguita per visualizzare l&#39;interazione tra gli assoni e astrociti, come mostrato nella Figura 2D-F. </li></ol>

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	<li>Stevens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+signaling+in+the+development+and+plasticity+of+neural+circuits.">Neuron-astrocyte signaling in the development and plasticity of neural circuits.</a> Neuro-Signals. 16, 278-288 (2008).</li><li>Paixao, S., Klein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-astrocyte+communication+and+synaptic+plasticity.">Neuron-astrocyte communication and synaptic plasticity.</a> Current opinion in neurobiology. 20, 466-473 (2010).</li><li>Fields, R. 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Nessun conflitto di interessi dichiarati.

Il neurone MCP based e astrociti co-coltura sistema consente di dissezione neurone dettagliata astroglia vie di segnalazione permettendo solo gli assoni passare i canali centrali e interagire con le cellule astrogliali. Questo co-coltura sistema può essere comodamente impostato con neurone convenzionale e procedure di coltura astrociti. Abbiamo anche descritto un applicazione pratica di questo sistema di co-coltura con l&#39;impiego di un reporter eGFP based per dimostrare assone-dipendente attivazione promotore GLT1 n...

<table border="1"><tbody><tr><td> Siero fetale bovino </td><td> Hyclone </td><td> SH30070.03 </td><td> per la placcatura neurone per neurone medio cutlure </td></tr><tr><td> Siero fetale bovino </td><td> Sigma-Aldrich </td><td> F4135 </td><td> per il terreno di coltura astrociti </td></tr><tr><td> Gliale fattore nervoso derivato </td><td> R &amp; D Systems </td><td> 212-GD </td><td> Applicare 10-20 ng / ml a lato neurone della camera </td></tr><tr><td> Dulbecco modificato aquila medio alte concentrazioni di glucosio </td><td> Sigma-Aldrich </td><td> 11995 </td><td></td></tr><tr><td> 70 millimetri filtro cella </td><td> BD Falcon </td><td> 352350 </td><td></td></tr><tr><td> Sterile piatto di vetro basso </td><td> MatTek Corporation </td><td></td><td></td></tr><tr><td> Piattaforme cultura microfluidici </td><td> Xona microfluidica LLC </td><td> SND150 </td><td></td></tr><tr><td> 6 pozzetti della piastra di coltura </td&gt; <td> Cellstar </td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> Neuron terreno di coltura <ul><li> Media Neurobasal </li><li> 2% B27 supplemento Neurobasal </li><li> 2 mM glutammato aggiungendo 1% 100x glutamax </li><li> 1% di penicillina-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Neuron terreno di coltura per cellule placcatura <ul><li> Media Neurobasal </li><li> 2% B27 supplemento Neurobasal </li><li> 2 mM glutammato aggiungendo 1% 100x glutamax </li><li> 1% di penicillina-streptomysin </li><li> 5% di siero fetale bovino SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> Terreno di coltura astrociti <ul><li> Dulbecco modificato eagle glucosio medio alto </li><li> 10% di siero bovino fetale F4135 </li><li> 1% di penicillina-streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> Materiali Tabella 1. Utilizzati per la microfluidica cultura piattaforma basata su assone neuronale e glia di co-cultura di sistema. </td></tr></tbody></table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

Neurone propria interazione glia è fondamentale per funzione fisiologica del sistema nervoso centrale (CNS). La comunicazione bidirezionale è sofisticato mediata da specifiche vie di segnalazione tra neuroni e glia 1,2. Identificazione e caratterizzazione di queste vie di segnalazione è essenziale per la comprensione di come l&#39;interazione neurone a glia forme CNS fisiologia. In precedenza, le colture di neuroni e glia misti sono stati ampiamente utilizzati per il test e la caratterizzazione di vie di segnalazione tra neuroni e glia. Quello che abbiamo imparato da questi preparativi e altri strumenti in vivo, tuttavia, ha suggerito che la segnalazione reciproci tra neuroni e glia sono presentati spesso in settori specifici all&#39;interno dei neuroni (cioè, assoni, dendriti, o soma) 3. Per questo è importante per sviluppare un nuovo sistema di cultura che permette la separazione dei compartimenti neuronali ed esamina in particolare l&#39;interazione tra glia e neuronali assoni / dendriti. Inoltre, il sistema convenzionale coltura mista non è in grado di differenziare i fattori solubili e segnali in membrana di contatto tra neuroni e glia. Inoltre, la grande quantità di neuroni e cellule gliali del convenzionale sistema di co-coltura manca la risoluzione necessaria per osservare l&#39;interazione tra un singolo assone e cellule gliali. In questo studio, si descrive un assone romanzo e glia sistema di co-coltura con l&#39;uso di una piattaforma microfluidica cultura (MCP). In questo sistema di co-coltura, neuroni e cellule gliali sono coltivati ​​in due camere separate che sono collegati attraverso molteplici canali centrali. In questa piattaforma microfluidica cultura, solo i processi neuronali (soprattutto assoni) può entrare nel lato gliali attraverso i canali centrali. In combinazione con potente etichettatura proteina fluorescente, questo sistema permette esame diretto di percorsi di segnalazione tra interazioni assonali / dendritiche e gliali, tales assone-mediata regolazione trascrizionale nelle cellule gliali, glia-mediata traffico recettore in terminali neuronali e glia-mediata della crescita degli assoni. Il diametro ridotto della camera vieta anche significativamente il flusso del neurone arricchito medio nella camera gliale, facilitando sondaggio della diretta membrana-proteina tra assoni / dendriti e superfici gliali.

Time-lapse imaging di analisi assone indotta da attivazione del promotore GLT1 negli astrociti Il neurone compartimenti e astrociti co-coltura sistema consente solo i processi neuronali, in particolare gli assoni, di interagire selettivamente con astrociti. Seguendo la costituzione ottimale di assoni e astrociti (o altre cellule gliali) co-coltura in assemblata MCP, diversi tipi di assone-glia interazioni possono essere studiate come; assone indotta attivazione di attivazione astrogliale gene p...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Questo studio descrive le procedure di creazione di un nuovo assone neuronale e (astro) glia co-coltura piattaforma. In questo sistema di co-coltura, la manipolazione di interazione diretta tra un singolo assone (e singole cellule gliali) diventa possibile, consentendo l&#39;analisi meccanicistica del neurone reciproco segnalazione gliali.

Analisi Imaging di Neuron Interaction Glia in Platform Cultura Microfluidic (MCP) a base di Axon neurone e glia co-cultura di sistema

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

imaging-analysis-neuron-to-glia-interaction-microfluidic-culture

Research

JoVE Journal

Neuroscience

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

14.9K Views.  Tufts University. Questo studio descrive le procedure di creazione di un nuovo assone neuronale e (astro) glia co-coltura piattaforma. In questo sistema di co-coltura, la manipolazione di interazione diretta tra un singolo assone (e singole cellule gliali) diventa possibile, consentendo l'analisi meccanicistica del neurone reciproco segnalazione gliali.

Video: Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

Bilaminar co-coltura di neuroni corticali primari di ratto e Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

Ricerca

Neuroscienze

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Analisi di sviluppo di denti Germ innervazione Uso di dispositivi Microfluidic Co-cultura

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

La indiretto Neuron-astrociti co-coltura saggio: An<em&gt; In Vitro</em&gt; Set-up per l&#39;indagine dettagliata delle Interazioni neuroni-glia

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

Isolamento e cultura dei neuroni primari e degli glia dalla vescica urinaria del ratto adulto

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

Lesioni neuronali mirate per la disconnessione non invasiva dei circuiti cerebrali

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

Dissezione e isolamento della glia murina da più regioni del sistema nervoso centrale

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

Un modello di coltura cellulare per studiare il ruolo delle interazioni Neuron-Glia in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.

In vivo Imaging del calcio nell'oliva inferiore del topo

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the ...

Visualizing Shifts on Neuron-Glia Circuit with the Calcium Imaging Technique

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral (dorsal root ganglia) and central tissues (cortex, subventricular zone, organoid) that are dynamically studied in terms of calcium shifts. The model chosen to illustrate the results is the retina, a simple tissue with complex cellular interactions. Calcium is a universal messenger involved in most of the important cellular roles. We explain in a step-by-step protocol how retinal neuron-glial cells in culture can be prepared and evaluated, envisioning calcium shifts. In this model, we differentiate neurons from glia based on their selective response to KCl and ATP. Calcium permeable receptors and channels are selectively expressed in different compartments. To analyze calcium responses, we use ratiometric fluorescent dies such as Fura-2. This probe quantifies free Ca2+ concentration based on Ca2+-free and Ca2+-bound forms, presenting two different peaks, founded on the fluorescence intensity perceived on two wavelengths.

Cell calcium imaging is a versatile methodology to study dynamic signaling of individual cells, on mixed populations in culture or even on awakened animals, based on the expression of calcium-permeable channels/receptors that gives unique functional signatures.

Visualizzazione dei cambiamenti sul circuito Neuron-Glia con la tecnica di imaging del calcio

Here, we report on selective in vitro models of circuits based on glia (astrocytes, oligodendrocytes, and microglia) and/or neurons from peripheral ...

Transplantation of Human Induced Pluripotent Stem Cell-Derived Microglia in Immunocompetent Mice Brain via Non-Invasive Transnasal Route

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain functions. Most of our current understanding of microglia is based on experiments performed in the mouse. Human microglia differ from mouse microglia, and thus response and characteristics of mouse microglia may not always represent that of human microglia. Further, due to ethical and technical difficulties, research on human microglia is restricted to in vitro culture system, which does not capitulate in vivo characteristics of microglia. To overcome these issues, a simplified method to non-invasively transplant induced pluripotent stem cell-derived human microglia (iPSMG) into the immunocompetent mice brain via a transnasal route in combination with pharmacological depletion of endogenous microglia using a colony-stimulating factor 1 receptor (CSF1R) antagonist is developed. This protocol provides a way to non-invasively transplant cells into the mouse brain and may therefore be valuable for evaluating the in vivo role of human microglia in physiological and pathological brain functions.

The protocol presented here allows the transplantation of induced pluripotent stem cell-derived human microglia (iPSMG) into the brain via a transnasal route in immunocompetent mice. The method for the preparation and transnasal transplantation of cells and the administration of cytokine mixture for the maintenance of iPSMG is shown.

Trapianto di microglia derivata da cellule staminali pluripotenti indotte umane nel cervello di topi immunocompetenti tramite via transnasale non invasiva

Microglia are the specialized population of macrophage-like cells of the brain. They play essential roles in both physiological and pathological brain ...