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

Los autores agradecen a Ulrich Marggraf (ISAS) para SU-8 de fabricación y Maria Becker (ISAS) para obtener imágenes de SEM. La investigación fue apoyada financieramente por la Deutsche Forschungsgemeinschaft (DFG WE3737/3-1), una Bundesministerium für Bildung und Forschung subvención (BMBF 0101-31P6541) y por el Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein-Westfalen. Heike Hardelauf agradece al Leibniz Escuela Internacional &quot;Systems Biology Lab-on-a-Chip&quot; para el apoyo financiero.

Los protocolos están destinados a los neurocientíficos, y se resumen en la Figura 2. Como tal, es recomendar que la microfabricación de los SU-8 maestros utilizados para la replicación de PDMS se externaliza a las instalaciones comerciales o institucionales. Los dos diseños de máscara de capa están disponibles gratuitamente como información complementaria (CP) en la Real Sociedad de Química página web 12. Es importante destacar que la primera capa de SU-8 debe ser fabricado a una pr...

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La técnica de agrupación de microfluidos es el primero de su tipo que permite precisión simple manejo de las neuronas para establecer culturas minimalistas. Junto con el método in situ patrón biomaterial, un enfoque poderoso para alinear patrones celulares con estructuras de microfluidos, estas culturas minimalistas tienen niveles de conectividad de alta intercompartimiento con reducida entrelazamiento local. Estas características se pueden utilizar para el estudio eficiente de transmisión inter-comparti...

<html> Tejido neuronal es muy complejo; una mezcla celular heterogénea que se ordena espacialmente dentro de las capas y los compartimentos definidos y con conectividad de plástico a través de contactos celulares y en especial a través de los axones y dendritas excrecencias. Se requieren nuevas técnicas para conferir una mayor libertad experimental para obtener una visión más profunda y los mecanismos centrales para desentrañar la enfermedad, el desarrollo y la función saludable. La cámara de Campenot 1,2 y, más recientemente microfabricado realizaciones 3,4 se pueden utilizar para la preparación ex vivo de co-cultivos neuronales conectados en red con la capacidad para perturbar de forma selectiva las diferentes poblaciones somáticas y también sus excrecencias de neuritas. Estos dispositivos de microfluidos han, por ejemplo, han utilizado para estudiar la degeneración axonal y la regeneración siguiente químico 5,6 o láser axotomía 6-8, tauopatía 9, difusión viral 10,11, y la localización del mRNA en los axones 4. ent &quot;&gt; Para ampliar el alcance de los neurobiólogos, se requieren desarrollos tecnológicos para preparar co-cultivos neuronales minimalistas. Esto permite que el desenmarañado de la red neuronal para la investigación del sistema con una sola célula y la precisión de sub-celular. El requisito para el número de células mínimas abre la posibilidad de analizar los tipos de células raras, incluyendo células dopaminérgicas de la sustancia negra pertinentes a la enfermedad de Parkinson, los ganglios de espiral desde el oído, las neuronas periféricas, y las células madre. Más allá de esto, la economía celular es relevante a la iniciativa de las 3R. El uso de estas plataformas de microfluidos, pantallas de toxicidad a gran escala u otras de alto rendimiento, rica serie experimental de datos que requieren las neuronas de los animales puede considerarse ahora. En este trabajo se describen los protocolos para la fabricación y el uso de un dispositivo de microfluidos. De agrupación de microfluidos en combinación con un en biomateri situmétodo de modelado al se puede utilizar para el registro de los co-cultivos neuronales altamente interconectadas que utilizan el número de células mínimas. De agrupación de microfluidos se basa en un enfoque de flujo diferencial 12-15, en el que las trampas microestructurados se colocan dentro de un circuito de fluido (ilustrada junto con imágenes de SEM en la Figura 1). La ruta de 0 → 1 tiene la resistencia fluídica inferior (R2&gt; R1) para el transporte de las neuronas a una serie lineal de aberturas microestructurados - las entradas a los canales de desarrollo de neuritas. Ocupación de la trampa por una sola célula impide localmente el flujo de desviar las líneas de corriente para atrapar las células posteriores en trampas vecinos. Ocupación completa de las trampas de la matriz cambia la relación de fluidos (R1&gt; R2) para desviar las líneas de corriente en la trayectoria de serpentina (0 → 2) para producir un modo de derivación de operación para la eliminación del exceso de neuronas. Figura 1. Microfluidic Circuito. A) El circuito de resistencia diferencial de fluido para el solo arraying neurona, con flanqueando cámaras de cultivo interconectados por canales de desarrollo de neuritas. B, las imágenes C) de SEM de la bicapa neurona compartimentada gama co-cultivo con menisco fijando micropilares. Con este diseño, se utilizaron estructuras de captura de neuronas en forma de tridente-para promover fasciculación de las excrecencias de neuritas. La figura y la leyenda reproducido con permiso de la Sociedad Real de Química (RSC) 12. <a href="https://www.jove.com/files/ftp_upload/51389/51389fig1highres.jpg" target="_blank">Haga clic aquí para ver la imagen más grande.</a> La preparación de las redes neuronales micropatterned en sustratos planos se puede conseguir fácilmente (por EJEMPLOS<html> es de nuestro grupo, ver Frimat et al. 16 y Heike et al. 17). Sin embargo, la encapsulación de patrones materiales bioactivos dentro de los dispositivos de PDMS y con el requisito de alineación micrómetro escala de estos a los canales de microfluidos plantea un gran desafío técnico. En la sección 3.1 de un protocolo para la en-chip, o in situ, se presenta la preparación de los patrones de biomateriales. Estos patrones permiten la inscripción de las neuronas durante las largas escalas de tiempo de cultivo y promover crecimientos entre los compartimentos. Microestructuras de menisco-colocación de clavos se utilizan para alinear una llamada máscara de agua con la neurona sitios y canales de desarrollo de neuritas arraying. El agua máscara protege los recubrimientos de moléculas de adhesión durante el tratamiento de plasma, mientras que las superficies expuestas se desintegran para definir el patrón de biomaterial. Además, se proporcionan protocolos para el cultivo celular y para el aislamiento de fluido necesario para el tratamiento selectivo de los diferentes compartimentos de co-cultivo. ve_content &quot;&gt; Los protocolos están diseñados para aprovechar los principios de uso fácil de la litografía suave para la replicación de poli (dimetilsiloxano) (PDMS) dispositivos de microfluidos 18. Del mismo modo, en el patrón de biomaterial in situ es sencillo, la explotación de la superficie de evaporación y fenómenos de tensión, y sólo que requiere una fuente de plasma de mano barata. El circuito de microfluidos con eficacia los programas de carga celular y compartimiento tratamientos específicos que hacen estas operaciones De esta manera, se pretende dar a los neurobiólogos simplemente una cuestión de distribución de materiales en el puerto inferior correcto y aspirando desde arriba. la libertad de preparar y usar los dispositivos de microfluidos en sus propios laboratorios.

<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">
			<td height="21" style="height:21px;width:235px;">Femto Plasma Oven</td>
			<td style="width:163px;">Diener Electronic</td>
			<td style="width:143px;"></td>
			<td></td>
		</tr>
		<tr height="21">
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preparation of neuronal co-cultures with single cell precision

Realizaciones de microfluidos de la cámara Campenot han despertado gran interés por parte de la comunidad de las neurociencias. Estas plataformas de co-cultivo interconectadas se pueden utilizar para investigar una variedad de preguntas, que abarca la neurobiología del desarrollo y funcional a la infección y la propagación de la enfermedad. Sin embargo, los sistemas convencionales requieren insumos significativos celulares (muchos miles por compartimiento), inadecuados para el estudio de las células de baja abundancia, como el nigra del substantia primaria dopaminérgico, ganglios espiral, y las neuronas melanogaster Drosophila y poco práctico para la experimentación de alto rendimiento. Los cultivos densos también se enredan muy localmente, con unos crecimientos (&lt;10%) que interconectan las dos culturas. En este trabajo se describen los protocolos de microfluidos y de modelado sencillas que frente a estos desafíos: (i) una neurona única de microfluidos método arraying, y (ii) un método de enmascaramiento de agua para recubrimientos de biomateriales de modelado de plasma para Register neuronas y promover la excrecencia de entre los compartimentos. Co-cultivos neuronales Minimalistic se prepararon con alto nivel (&gt; 85%) la conectividad intercompartimiento y se pueden utilizar para los de alto rendimiento experimentos neurobiología con precisión simple célula.

Hazañas de enmascaramiento Agua tensión superficial. La tolerancia a la presión en la interfase aire-líquido escalas inversamente con el radio de curvatura [ <img fo:content-width="1in" src="files/ftp_upload/51389/51389eq1.jpg" width="113" /> , La producción de las interfaces altamente estables en las dimensiones de microfluidos. Los micropilares anclar eficazmente el agua máscara en su lugar. Formación de máscara de agua es impulsado por la evaporación de los puertos microfluídicos, con la tasa de aumento de ...

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

Se describen protocolos para sola neurona arraying microfluidos y el enmascaramiento de agua para el patrón de plasma en el chip de recubrimientos de biomateriales. Co-cultivos altamente interconectadas se pueden preparar usando entradas de celda mínimos.

Preparación de Neuronales Co-culturas con precisión Single Cell

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.

Medición única célula de la liberación de dopamina simultánea con fijación de voltaje y Amperometría

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

Investigación

Neurociencias

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.

Grabación óptica de la actividad neural suprathreshold con la resolución de una sola célula y único punta-

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.

Simultánea pre-y post-sináptica de registro electrofisiológico<em&gt; Xenopus</em&gt; Músculo Nervio-Co-culturas

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.

Un procedimiento para la implantación de matrices Organizados de Microhilos para grabaciones de una sola unidad en Awake, Comportarse Animales

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.

La inducción de la plasticidad de astrocítico Receptores por manipulación de Neuronales cocción Precios

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.

La investigación de la regeneración funcional en organotípicos médula espinal Co-culturas cultivadas en Arrays Multi-electrodos

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.

Extracelular de grabación de la actividad neuronal junto con la aplicación de sustancias Microiontophoretic neuroactivos en ratones Awake

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.

La electroporación en el útero métodos para estudiar la excitabilidad de las subpoblaciones neuronales y Conectividad Una sola célula

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.

Co-cultivos de neurona y macrófagos para activar macrófagos secretando factores moleculares con actividad Neurite consecuencia

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.

Generación Libre De Gm De Células Neuronales Derivadas De La Sangre

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