Name: preparation of neuronal co-cultures with single cell precision
Uploaded: 2014-05-20T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Neuronal Co-cultures with Single Cell Precision at JoVE.com

Die Autoren sind dankbar, dass Ulrich Marggraf (ISAS) für SU-8-Herstellung und Maria Becker (ISAS) für SEM-Bildgebung. Die Forschung wurde von der Deutschen Forschungsgemeinschaft (DFG WE3737/3-1), ein Bundesministerium für Bildung und Forschung Zuschuss (BMBF 0101-31P6541) und vom Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen unterstützt. Heike Hardelauf dank der Internationale Leibniz Graduate School &quot;Systems Biology Lab-on-a-Chip&quot; für finanzielle Unterstützung.

Die Protokolle werden für Neurowissenschaftler bestimmt, und sind in Abbildung 2 zusammengefasst. Als solche ist sie empfehlen, die Mikro der SU-8-Master für PDMS-Replikation verwendet wird, um gewerbliche oder institutionelle Einrichtungen ausgelagert. Die beiden Ebenenmaske Entwürfe sind als ergänzende Information (ZIP) auf der Royal Society of Chemistry 12 Website frei verfügbar. Wichtig ist, dass die erste SU-8-Schicht bis zu einer Tiefe von 2,5-3,0 um hergestellt, und die zweite zu ...

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Die mikrofluidische Anordnungs Technik ist die erste ihrer Art, die Präzision einzelnen Neurons Handhabung für die Einrichtung minimalistisch Kulturen ermöglicht. Mit der in-situ-Biomaterial-Strukturierungsverfahren, einem leistungsfähigen Ansatz zur Zellmuster mit mikrofluidischen Strukturen auszurichten Gekoppelt sind diese minimalistischen Kulturen Hoch intercompartment Connectivity-Level mit eingeschränkter lokale Verschränkung. Diese Merkmale können für die effiziente Untersuchung von interFach Üb...

<html> Nervengewebe ist sehr komplex; eine heterogene Zellmischung, die räumlich in definierten Schichten und Fächer und mit Kunststoff-Konnektivität über Zell-Kontakte und vor allem über Axon und Dendriten Auswüchse bestellt wird. Neue Techniken sind erforderlich, um mehr experimentelle Freiheit, tiefere Einblicke und entwirren zentralen Mechanismen der Krankheit, Entwicklung und gesunde Funktion gewinnen verleihen. Die Campenot Kammer 1,2 und kürzlich mikroAusführungs 3,4 kann für die ex vivo Herstellung von vernetzten neuronalen Co-Kulturen mit der Fähigkeit, ihre Neuriten Auswüchse selektiv stören die verschiedenen somatischen Populationen und auch verwendet werden. Diese mikrofluidischen Bauteilen haben beispielsweise verwendet, um Axon-Degeneration und Regeneration zu studieren folgenden chemischen 5,6 oder Laser-Axotomie 6-8, Tauopathie 9, virale Verbreitung 10,11, und mRNA-Lokalisierung in Axone 4. ent &quot;&gt; Um die Reichweite der Neurobiologen erstrecken, sind technologische Entwicklungen erforderlich, um minimalistische neuronalen Co-Kulturen vorzubereiten. Dies ermöglicht die Herauslösung des neuronalen Netzes für die Ermittlung des Systems mit einzelnen Zelle und subzellulären Präzision. Die Anforderung für eine minimale Zellzahlen eröffnet die Möglichkeit, seltenen Zelltypen, einschließlich dopaminergen Substantia nigra-Zellen relevant Parkinson, Spiralganglien aus dem Ohr, peripheren Neuronen zu analysieren, und Stammzellen. Darüber hinaus ist Mobilwirtschaft, die für die 3R-Initiative. Mit Hilfe dieser Mikrofluidik-Plattformen, große Bildschirme Toxizität oder andere Hochdurchsatz, können die Daten reichen Versuchsreihen, die Tier Neuronen betrachtet. In diesem Papier Protokolle für die Herstellung und Verwendung einer Mikrofluid-Vorrichtung beschrieben. Mikrofluidik-Anordnungs in Kombination mit einem in situ Biomaterialienal Musterungsverfahren können für die Registrierung von hochvernetzten neuronalen Co-Kulturen mit minimalem Zellzahlen verwendet werden. Mikrofluidik-Anordnungs auf einem Differenzstrom-Ansatz 12-15, wobei mikro Traps in einem Fluidkreis (mit REM-Bildern in Fig. 1 dargestellt) auf der Basis positioniert. Der Pfad 0 → 1 die untere fluidische Widerstand (R 2&gt; R 1) zum Transportieren Neuronen zu einer linearen Anordnung von mikrostrukturierten Öffnungen - die Einlässe zu den Neuriten-Auswuchs-Kanälen. Belegung der Falle durch eine einzelne Zelle lokal behindert den Durchfluss auf die Stromlinien zum Einfangen nachfolgenden Zellen in benachbarten Fallen abzulenken. Vollständige Belegung der Fallen in der Anordnung schaltet das fluidische Verhältnis (R 1&gt; R 2), um die Stromlinien in den Serpentinenpfad (0 → 2) umzuleiten, um eine Bypass-Betriebsmodus für die Entfernung von überschüssigem Neuronen zu erzeugen. Abbildung 1. Mikrofluidik-Circuit. A) Die differentielle Widerstand Fluidkreis für einzelnes Neuron Anordnungs, mit flankierenden Kulturkammern von Neuritenwachstum Kanäle miteinander verbunden. B, C) ​​SEM-Bilder der Doppelschicht compartmentalized Neuron Co-Kultur-Array mit Meniskus Pinning Mikrosäulen. Mit diesem Entwurf wurden Dreizack-förmigen Neuron Fangstrukturen verwendet, um Faszikulation der Neuriten Auswüchse zu fördern. Abbildung und Legende mit Genehmigung der Royal Society of Chemistry (RSC) wiedergegeben 12. <a href="https://www.jove.com/files/ftp_upload/51389/51389fig1highres.jpg" target="_blank">Klicken Sie hier für eine größere Ansicht.</a> Die Herstellung von mikro neuronaler Netzwerke auf ebenen Substraten kann leicht erreicht werden (exampl<html> es aus unserer Gruppe finden Frimat et al. 16 und Heike et al. 17). Allerdings Verkapselung bioaktiven Material Muster in PDMS-Geräten und mit der Anforderung des Mikrometer-Skala Ausrichtung der diese den mikrofluidischen Kanälen stellt eine große technische Herausforderung. In Abschnitt 3.1 wird ein Protokoll für die in dem Chip oder in situ Herstellung von Biomaterial-Muster dargestellt. Diese Muster ermöglichen Neuron Anmeldung bei längeren Zeitskalen und Kultur zu fördern Auswüchse zwischen Fächern. Meniskus-Pinning Mikrostrukturen verwendet werden, um eine so genannte Wasser-Maske mit dem Neuron Anordnen Sites und Neuritenwachstum Kanälen auszurichten. Das Wasser Maske schützt Adhäsionsmolekül Beschichtungen während der Plasmabehandlung, während exponierten Flächen werden aufgelöst, das Biomaterial Muster definieren. Zusätzlich sind Protokolle für die Zellkultur und für die fluidische Trennung für die selektive Behandlung der verschiedenen Co-Kultur Kammern notwendig ist. ve_content &quot;&gt; Die Protokolle sind auf die benutzerfreundliche Prinzipien der Softlithographie für die Replikation von Poly (dimethylsiloxan) (PDMS) mikrofluidischen Vorrichtungen 18 zu nutzen. Auch in situ Biomaterial Musterung ist einfach, die Nutzung Verdunstung und Oberflächenspannung Phänomene und nur erfordern eine preiswerte Hand-Plasmaquelle. Mikrofluidik-Schaltung effektiv Programme Zelle Laderaum und spezielle Behandlungen machen diese Operationen nur eine Frage der Abgabe von Materialien in die richtige unteren Anschluss und Absaugen von oben. Auf diese Weise beabsichtigt ist, die Neurobiologen geben die Freiheit, in den eigenen Labors herzustellen und zu verwenden mikrofluidischen Bauteilen.

<table border="0" cellpadding="0" cellspacing="0" width="857">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PDMS Sylgard 184</td>
			<td style="width:163px;">Dow Corning</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PDMS Elastosil RT 601</td>
			<td style="width:163px;">Wacker</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Coverslips</td>
			<td style="width:163px;">VWR</td>
			<td>630-1590</td>
			<td>130-160 mm thick</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">3 mm Biopsy Punches</td>
			<td style="width:163px;">Kai Medical</td>
			<td style="width:143px;"></td>
			<td>Handle with care - extremely sharp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon Tubing</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:143px;">S-50-HL</td>
			<td>1.65 mm ID; 3.35 mm OD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL Syringe (Inkjekt and Omnifix)</td>
			<td style="width:163px;">Braun</td>
			<td style="width:143px;">6064204</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">4-Way Tubing Connector</td>
			<td style="width:163px;">VWR or Fisher Scientific</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Flow Regulator</td>
			<td style="width:163px;">Harvard Apparatus</td>
			<td style="width:143px;">722645</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">0.5 mm Pins</td>
			<td style="width:163px;">Dressmaking Departments</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">PLL-g-PEG</td>
			<td style="width:163px;">SuSoS</td>
			<td style="width:143px;">PLL(20)-g[3.5]-PEG(5)</td>
			<td>Stability in storage can be an issue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-D-lysine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P6407</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-lysine-FITC</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P3069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-ornithine</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4957</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">fibronectin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">F2006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">laminin</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">L2020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PBS</td>
			<td style="width:163px;">Sigma-Aldrich</td>
			<td style="width:143px;">P4417</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Inverted Fluorescent Microscope</td>
			<td style="width:163px;"></td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Example Aspiration Pump</td>
			<td style="width:163px;">KNF Neuberger, Laboport</td>
			<td style="width:143px;">N811KVP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Hand Held Corona Discharge Device</td>
			<td style="width:163px;">Leybold-Heraeus, USA</td>
			<td style="width:143px;">VP23</td>
			<td>May not comply with your country&#39;s safety standards</td>
		</tr>
		<tr height="21">
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preparation of neuronal co-cultures with single cell precision

Mikrofluidik-Ausführungsformen der Campenot Kammer haben großes Interesse aus der Neurowissenschaften angezogen. Diese miteinander verbundenen Co-Kultur-Plattformen verwendet werden, um eine Vielzahl von Fragen zu untersuchen, Spanning Entwicklungs-und Funktions Neurobiologie, um Infektionen und Krankheiten Ausbreitungs werden. Herkömmliche Systeme benötigen jedoch erheblichen Zell Eingänge (viele Tausende pro Fach), für das Studium geringer Menge Zellen unzureichend, wie primäre dopaminergen Substantia nigra, Spiralganglien und Drosophila melanogaster Neuronen und unpraktisch für die Hochdurchsatz-Experimente. Die dichten Kulturen sind auch sehr lokal verstrickt, mit wenigen Auswüchse (&lt;10%), die die beiden Kulturen. In diesem Papier einfach mikro-und Musterprotokolle beschrieben, die diese Herausforderungen anzugehen: (i) eine mikrofluidische einzelnen Neurons Anordnungsverfahren, und (ii) eine Wassermaskierungsverfahren für Plasma-Strukturierung Biomaterial Beschichtungen auf register Neuronen und fördern Auswuchs zwischen den Abteilen. Minimalistische neuronalen Co-Kulturen wurden mit High-Level (&gt; 85%) intercompartment Konnektivität bereit und kann für hohe Durchsatz Neurobiologie Experimente mit einzelnen Zelle Präzision verwendet werden.

Wasser Maskierung Exploits Oberflächenspannung. Die Drucktoleranz an der Luft-Flüssigkeit-Grenzfläche skaliert invers mit dem Krümmungsradius [ <img fo:content-width="1in" src="files/ftp_upload/51389/51389eq1.jpg" width="113" /> , Herstellung hochstabiler Schnittstellen an mikrofluidischen Dimensionen. Die Mikrosäulen das Wasser effektiv zu verankern Maske an Ort und Stelle. Wassermaskenbildung wird durch Verdunstung aus den mikrofluidischen Häfen getrieben, mit der Rate erhöht durch Erhitzen. Mit der Wärme aus ...

Watch this Scientific Journal Video about Preparation of Neuronal Co-cultures with Single Cell Precision at JoVE.com

Preparation of Neuronal Co-cultures with Single Cell Precision

Protokolle für die einzelnen Neurons mikrofluidischen Anordnungs und Wasser für die Maskierung in dem Chip Plasma Strukturierung der Biomaterial-Beschichtungen beschrieben. Sehr verbunden Co-Kulturen können mit minimalem Ein-Zelle hergestellt werden.

Vorbereitung der neuronalen Co-Kulturen mit Single Cell Precision

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

preparation-of-neuronal-co-cultures-with-single-cell-precision

Research

JoVE Journal

Neuroscience

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

Single Cell Messung der Freisetzung von Dopamin mit gleichzeitiger Voltage-Clamp-und Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Forschung

Neurowissenschaften

Optical Recording of Suprathreshold Neural Activity with Single-cell and Single-spike Resolution

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also propagation of suprathreshold spiking activity involves populations of neurons. Empirical studies addressing cortical function directly thus require recordings from populations of neurons with high resolution. Here we describe an optical method and a deconvolution algorithm to record neural activity from up to 100 neurons with single-cell and single-spike resolution. This method relies on detection of the transient increases in intracellular somatic calcium concentration associated with suprathreshold electrical spikes (action potentials) in cortical neurons. High temporal resolution of the optical recordings is achieved by a fast random-access scanning technique using acousto-optical deflectors (AODs)1. Two-photon excitation of the calcium-sensitive dye results in high spatial resolution in opaque brain tissue2. Reconstruction of spikes from the fluorescence calcium recordings is achieved by a maximum-likelihood method. Simultaneous electrophysiological and optical recordings indicate that our method reliably detects spikes (&gt;97% spike detection efficiency), has a low rate of false positive spike detection (&lt; 0.003 spikes/sec), and a high temporal precision (about 3 msec) 3. This optical method of spike detection can be used to record neural activity in vitro and in anesthetized animals in vivo3,4.

Understanding the function of the vertebrate central nervous system requires recordings from many neurons because cortical function arises on the level of populations of neurons. Here we describe an optical method to record suprathreshold neural activity with single-cell and single-spike resolution, dithered random-access scanning. This method records somatic fluorescence calcium signals from up to 100 neurons with high temporal resolution. A maximum-likelihood algorithm deconvolves the underlying suprathreshold neural activity from the somatic fluorescence calcium signals. This method reliably detects spikes with high detection efficiency and a low rate of false positives and can be used to study neural populations in vitro and in vivo.

Optische Aufnahme überschwellige neuronale Aktivität mit Single-Cell-und Single-Spike Auflösung

Signaling of information in the vertebrate central nervous system is often carried by populations of neurons rather than individual neurons. Also ...

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse1. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed2-4. The preparation has been used widely to study the biophysical properties of presynaptic ion channels and the regulation of transmitter release5-8. In addition, the preparation has lent itself to other uses including the study of neurite outgrowth and synaptogenesis9-12, molecular mechanisms of neurotransmitter release13-15, the role of diffusible messengers in neuromodulation16,17, and in vitro synaptic plasticity18-19.

Simultaneous Pre- and Post-synaptic Electrophysiological Recording from Xenopus Nerve-muscle Co-cultures

This video demonstrates the procedures used to grow primary cultures of embryonic Xenopus nerve and muscle cells and the usefulness of this preparation for making simultaneous pre- and post-synaptic patch clamp recordings.

Gleichzeitige Pre-und Post-synaptischen Elektrophysiologische Aufnahme von<em&gt; Xenopus</em&gt; Nerv-Muskel-Co-Kulturen

Much information about the coupling of  presynaptic ionic currents with the release of neurotransmitter has been obtained  from invertebrate preparations, ...

A Procedure for Implanting Organized Arrays of Microwires for Single-unit Recordings in Awake, Behaving Animals

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell level. The technique allows experimenters to examine temporally and regionally specific firing patterns in order to correlate recorded action potentials with ongoing behavior. Moreover, single-unit recordings can be combined with a plethora of other techniques in order to produce comprehensive explanations of neural function. In this article, we describe the anesthesia and preparation for microwire implantation. Subsequently, we enumerate the necessary equipment and surgical steps to accurately insert a microwire array into a target structure. Lastly, we briefly describe the equipment used to record from each individual electrode in the array. The fixed microwire arrays described are well-suited for chronic implantation and allow for longitudinal recordings of neural data in almost any behavioral preparation. We discuss tracing electrode tracks to triangulate microwire positions as well as ways to combine microwire implantation with immunohistochemical techniques in order to increase the anatomical specificity of recorded results.

Implanting organized arrays of microwires for use in single-unit electrophysiological recordings presents a number of technical challenges.&#160;Methods for performing this technique and the equipment necessary are described.&#160;Also, the beneficial use of organized microwire arrays to record from distinct neural subregions with high spatial selectivity is discussed.

Ein Verfahren für die Implantation von Mikrodrähte Organisierte Arrays für Einzel-Einheit Recordings in Awake, Behaving Tiere

In vivo electrophysiological recordings in the awake, behaving animal provide a powerful method for understanding neural signaling at the single-cell ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

Induktion von Plastizität Astrozytäre Rezeptoren durch Manipulation der neuronalen Feuerraten

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

Untersuchung funktionelle Regeneration im Organotypische Spinal Cord Co-Kulturen auf Multi-Elektroden-Arrays Grown

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

Die extrazelluläre Aufnahme neuronaler Aktivität in Kombination mit Microiontophoretic Anwendung von neuroaktiven Substanzen in Awake Mäuse

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, their distinct dendritic morphologies, their specific patterns of axonal connectivity, and their selective firing responses. The molecular and cellular mechanisms responsible for these aspects of differentiation during development are still poorly understood.
Here, we describe combined protocols for labeling and characterizing the structural connectivity and excitability of cortical neurons. Modification of the in utero electroporation (IUE) protocol allows the labeling of a sparse population of neurons. This, in turn, enables the identification and tracking of the dendrites and axons of individual neurons, the precise characterization of the laminar location of axonal projections, and morphometric analysis. IUE can also be used to investigate changes in the excitability of wild-type (WT) or genetically modified neurons by combining it with whole-cell recording from acute slices of electroporated brains. These two techniques contribute to a better understanding of the coupling of structural and functional connectivity and of the molecular mechanisms controlling neuronal diversity during development. These developmental processes have important implications on axonal wiring, the functional diversity of neurons, and the biology of cognitive disorders.

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

This manuscript provides protocols that use in utero electroporation (IUE) to describe the structural connectivity of neurons at the single-cell level and the excitability of fluorescently labeled neurons. Histology is used to characterize dendritic and axonal projections. Whole-cell recording in acute slices is used to investigate excitability.

In Utero Ansätze Elektroporation die Erregbarkeit neuronaler Subpopulationen und Einzelzell - Konnektivität zu studieren

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

Neuron-Macrophage Co-cultures to Activate Macrophages Secreting Molecular Factors with Neurite Outgrowth Activity

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.

The current protocol presents experimental procedures to stimulate cultured macrophages to be endowed with capacity to release molecular factors that promote neurite outgrowth. Treatment of cAMP to the neuron-macrophage co-cultures induces the macrophages to produce conditioned medium that possesses strong neurite outgrowth activity.

Neuron-Makrophagen Ko-Kulturen, Makrophagen sezernieren molekulare Faktoren mit Neuriten Auswuchs Aktivität zu aktivieren

There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

GEN-freie Erzeugung von blutabgeleiteten neuronalen Zellen

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...