Samuel L. Pfaff es profesor en el Laboratorio de Expresión Génica en el Instituto Salk de Estudios Biológicos y un investigador en el Instituto Médico Howard Hughes. Este trabajo fue apoyado por el Christopher y Dana Reeve Foundation. Joe Belcovson, Schnoeker Kent y Mike Sullivan en Recursos Multimedia en el Instituto Salk proporcionó asistencia con la fotografía y la edición.

Registros electrofisiológicos de la médula espinal aislada puede revelar cambios genéticos y del desarrollo de circuitos neuronales 1. Hemos demostrado previamente un método para analizar los cables neonatal espinal de ratón 2. Aquí presentamos un método para preparar electrodos de succión útil en la grabación de la locomoción ficticia en la médula espinal aislada 3. La punta de un electrodo de un tubo de plástico se pueden extraer de una punta muy fina a mano usando una lámpara de alcohol de baja temperatura 3.5. El tubo de plástico (PE90, Clay Adams Intramedic TM) se lleva a cabo sobre la llama para ablandar la tubería en una forma maleable. Como el tubo comienza a derretirse y se vuelve más transparente que se extrae de la fuente de calor y los extremos se tira suavemente de distancia. El momento de retirar el tubo de la llama es importante para asegurar que el plástico no se derrumbe o se separan, mientras está dibujando. La parte fina de la tubería se corta con una cuchilla de afeitar de acuerdo con el diámetro deseado interna, según lo dictado por la edad de la muestra o el nivel segmentario de la médula. Para facilitar la colocación de una junta tórica en el tubo, un corte de 45 ° se realiza en el extremo grueso de la tubería con una cuchilla de afeitar. Un casquillo y la junta tórica se instalan en el extremo más grueso para conectar el extremo del electrodo en el cañón del electrodo. Para permitir que los electrodos de un tamaño específico y evitar las dificultades de la mano tirando puntas de los electrodos, los electrodos pueden ser construidos usando tubos de pequeño se inserta en un tubo grueso (PE90). Las longitudes de las tuberías son específicas para los requisitos de la configuración de la grabación y son variables. Se presentan las medidas utilizadas en nuestros experimentos 1-3,5. Una longitud de 1 cm de tubo de PTFE fina (Zeus, piezas pequeñas) y una longitud de 10 cm de tubería PE90 se cortan con una cuchilla de afeitar. Los extremos de los tubos pequeños deben ser examinados para asegurarse de que no se trituran o se cierra. Que se pueden abrir con un pin de insectos (Herramientas Artes Ciencia). Una gota de adhesivo (JB Weld) se coloca en el punto medio del tubo de PTFE. Los tubos de PTFE y adhesivo se dibujan en el tubo de succión con la boca PE90 en la tubería PE90. Un corte de 45 ° se realiza en el extremo grueso de la tubería y un casquillo y la junta tórica se adjuntan, que el anterior. El conector BNC férula y se desenrosca de un electrodo de succión comercial. El conector BNC se sumerge en xileno durante una hora para quitar el sello de cera. El conector se enjuaga en agua y se seca. Un puerto del lado inferior en ángulo deben hacerse en el lado del cañón. Una barra de metal se calienta con un mechero de Bunsen y se introduce en el agujero existente en el lado del cañón en un ángulo bajo. Una longitud de 15 cm de alambre de plata .010 pulgadas (AM Systems) se corta y empapado en lejía durante quince minutos. Esto se hace para crear una capa de cloruro de plata que ayuda a la conducción de la señal 6. El cable se enjuaga con agua, se seca, y luego soldar en el conector BNC. El cable conector BNC y la plata se introduce en el cañón de electrodos de aspiración con suficiente espacio para el conector BNC para permanecer al lado del cañón. Tubo de succión se inserta en el orificio lateral de 2-3cm. Sellador de silicona que se inyecta en el cilindro de aproximadamente 2 a 3 cm. El conector BNC se enrosca en el barril del electrodo empujando el sellador hacia adelante. La punta del electrodo está enroscado en la parte delantera del electrodo. Tubo de contracción también se pueden añadir para reforzar la línea de aspiración desde el puerto de lado. Los electrodos se montan en micromanipuladores que sujetarse a la base magnética, y se coloca cerca de la fuente de grabación. BNC cables coaxiales están conectados a un cabezal que está conectado a un amplificador. El amplificador está conectado a un adaptador (PolyView) que está conectado a una tarjeta de analógico a digital (National Instruments) en un ordenador PC con el software de adquisición de datos (PolyView). Al grabar datos electrofisiológicos, es necesario eliminar todas las interferencias externas. El plato de grabación, un amplificador y etapa de la cabeza se colocan en una mesa de aire dentro de una jaula de Faraday para evitar las interferencias eléctricas y mecánicas, respectivamente. Los resultados representativos: <img src="/files/ftp_upload/2580/2580fig1.jpg" alt="Figura 1" /> Figura 1. Un electrodo de succión disponible en el mercado pueden ser modificados mediante la adición de un alambre de calibre pesado plata y una punta de plástico que es dibujado a mano en una lámpara de alcohol, o construido con tubos de plástico disponibles en el mercado de tamaños específicos. Además, el sellador de parafina se sustituye con más sellador de masilla de silicona durable. <img src="/files/ftp_upload/2580/2580fig2.jpg" alt="Figura 2" /> Figura 2. Un esquema de los electrodos de aspiración en relación con la mesa de aire (para aislamiento mecánico), jaula de Faraday (eléctrica isolatide), un amplificador y una computadora. Cabe señalar que hay una referencia a tierra al plato de grabación y un segundo motivo que se hace referencia a la mesa de aire y la jaula de Faraday.

<ol>
	<li>Gallarda, B. W., Sharpee, T. O., Pfaff, S. L., Alaynick, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+rhythmic+locomotor+burst+patterns+using+a+continuous+wavelet+transform.">Defining rhythmic locomotor burst patterns using a continuous wavelet transform.</a> Ann N Y Acad Sci. 1198, 133-139 (2010).</li><li>Meyer, A., Gallarda, B. W., Pfaff, S., Alaynick, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+cord+electrophysiology.">Spinal cord electrophysiology.</a> J Vis Exp. , (2010).</li><li>Gallarda, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+axial+motor+and+sensory+pathways+via+heterotypic+trans-axonal+signaling.">Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling.</a> Science. 320, 233-236 (2008).</li><li>Landmesser, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+motor+projection+patterns+in+the+chick+hind+limb.">The development of motor projection patterns in the chick hind limb.</a> J Physiol. 284, 391-414 (1978).</li><li>Myers, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+input+is+required+during+embryonic+development+to+mediate+proper+assembly+of+spinal+locomotor+circuits.">Cholinergic input is required during embryonic development to mediate proper assembly of spinal locomotor circuits.</a> Neuron. 46, 37-49 (2005).</li><li>Chanin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+determination+of+chloride+by+use+of+the+silver-silver+chloride+electrode.">The determination of chloride by use of the silver-silver chloride electrode.</a> Science. 119, 323-324 (1954).</li><li>Goulding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circuits+controlling+vertebrate+locomotion:+moving+in+a+new+direction.">Circuits controlling vertebrate locomotion: moving in a new direction.</a> Nat Rev Neurosci. 10, 507-518 (2009).</li></ol>

Desarrollo del sistema nervioso pueden ser estudiados usando los cables aislados de roedores espinal. En presencia de los neurotransmisores, la locomoción ficticia puede ser generada a partir de la médula espinal en forma de patrones de actividad eléctrica 1,3. Estas explosiones rítmicas se producen en el 0,2 a 0,5 Hz y siguen el modelo de alternancia izquierda-derecha y flexor extensor. En diferentes etapas de desarrollo, la solidez y los patrones de esta actividad varía de 1. Las mutaciones ...

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<td>PTFE Sub Lite Wall Tubing (Small tubing)</td>
<td>&nbsp;</td>
<td>Zeus</td>
<td>36AWG</td>
<td>0.005&#x201D;ID x 0.003&#x201D; Wall (Small Parts) Also available in 0.003&#x201D; to 0.006&#x201D;</td>
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<td>Large tubing (0.86mm (0.34&#x201D;))</td>
<td>&nbsp;</td>
<td>Clay Adams Brand Intramedic Becton Dickinson and Company</td>
<td>427420</td>
<td>0.86mm (0.34&#x201D;) O.D. 1.27mm (.050&#x201D;)</td>
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<td>Electrode Barrel</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>573000</td>
<td>&nbsp;</td>
<tr>
<td>Adhesive</td>
<td>&nbsp;</td>
<td>JB Weld</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Adhesive: Silicone caulk</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Solder and soldering iron</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Xylene</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Silver wire: 0.010&#x201D;</td>
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<td>A-M Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Insect pins: Austerlitz 0.1mm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
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<td>Magnetic Stand</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>GJ-8</td>
<td>&nbsp;</td>
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<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige</td>
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<td>Miniboard (Headstage)</td>
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<td>F-15EB/B1</td>
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<td>Polyview Adaptor Unit</td>
<td>&nbsp;</td>
<td>Grass Industries</td>
<td>PVA 8</td>
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<td>Bipolar Portable Physiodata Amplifier System</td>
<td>&nbsp;</td>
<td>Grass Industries</td>
<td>15LT</td>
<td>&nbsp;</td>
<tr>
<td>ANALOG TO DIGITAL CARD</td>
<td>&nbsp;</td>
<td>National Instruments</td>
<td>6035E</td>
<td>&nbsp;</td>
<tr>
<td>Air Table; Vibraplane</td>
<td>&nbsp;</td>
<td>Kinetic Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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spinal cord electrophysiology ii: extracellular suction electrode fabrication

Desarrollo de circuitos neuronales y la locomoción pueden ser estudiados usando roedores recién nacidos la médula espinal centro generador de patrones (CPG) de comportamiento. Demostramos un método para la fabricación de electrodos de succión que se utilizan para examinar la actividad de GPC, o la locomoción ficticia, en los cables de roedores disecados espinal. Los cables de la columna vertebral de roedores se colocan en líquido cefalorraquídeo artificial y las raíces ventrales son atraídos hacia el electrodo de succión. El electrodo se construye mediante la modificación de un electrodo de succión disponible en el mercado. Un hilo de plata pesada se utiliza en lugar del cable estándar proporcionada por el electrodo en el comercio. La punta de vidrio del electrodo comercial se sustituye con una punta de plástico para mayor durabilidad. Preparamos la mano electrodos dibujados y electrodos hechos de tamaños específicos de tubos, lo que permite la consistencia y la reproducibilidad. Los datos se recopilaron mediante un amplificador y un software de adquisición de neurogram. Las grabaciones se realizaron en una mesa de aire dentro de una jaula de Faraday para evitar la interferencia mecánica y eléctrica, respectivamente.

Watch this Scientific Journal Video about Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication at JoVE.com

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Una demostración de la fabricación y el uso de un electrodo de succión extracelular para medir los registros electrofisiológicos de los cables de la columna vertebral de roedores recién nacidos<em&gt; In vitro</em

Médula Espinal Electrofisiología II: Fabricación de electrodos de aspiración extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

spinal-cord-electrophysiology-ii-extracellular-suction-electrode

Research

JoVE Journal

Neuroscience

15.0K vistas.  The Salk Institute for Biological Studies. Una demostración de la fabricación y el uso de un electrodo de succión extracelular para medir los registros electrofisiológicos de los cables de la columna vertebral de roedores recién nacidos In vitro

Video: Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Chicken Embryo Spinal Cord Slice Culture Protocol

Slice cultures can facilitate the manipulation of embryo development both pharmacologically and through gene manipulations. In this reduced system, potential lethal side effects due to systemic drug applications can be overcome. However, culture conditions must ensure that normal development proceeds within the reduced environment of the slice. We have focused on the development of the spinal cord, particularly that of spinal motor neurons. We systematically varied culture conditions of chicken embryo slices from the point at which most spinal motor neurons had been born. We assayed the number and type of motor neurons that survived during the culture period and the position of those motor neurons compared to that in vivo. We found that serum type and neurotrophic factors were required during the culture period and were able to keep motor neurons alive for at least 24 hr and allow those motor neurons to migrate to appropriate positions in the spinal cord. We present these culture conditions and the methodology of preparing the embryo slice cultures using eviscerated chicken embryos embedded in agarose and sliced using a vibratome.

Slice cultures facilitate the manipulation of embryo development by gene and pharmacological perturbations. However, culture conditions must ensure that normal development can proceed within the reduced environment of the slice. We illustrate a protocol that facilitates normal spinal cord development to proceed for at least 24 hr.

Embriones de pollo de la Médula Espinal Slice Cultura Protocolo

Slice cultures can facilitate the manipulation of  embryo development both pharmacologically and through gene manipulations. In  this reduced system, ...

Investigación

Neurociencias

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

La transección de la médula espinal en el pez cebra larval

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Una<em&gt; Ex Vivo</em&gt; Médula espinal inducida por láser Lesiones modelo para evaluar los mecanismos de degeneración axonal en tiempo real

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Electrofisiología en aislados Preparativos Cord-tronco cerebral espinal de roedores recién nacidos permite la grabación de salida de red neuronal Respiratorio

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

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 ...

Spiral Ganglion Neuron Explant Culture and Electrophysiology on Multi Electrode Arrays

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in the brain stem. Loss of hair cells is a major cause of sensory hearing loss. Prosthetic devices such as cochlear implants function by bypassing lost hair cells and directly stimulating SGNs electrically, allowing for restoration of hearing in deaf patients. The performance of these devices depends on the functionality of SGNs, the implantation procedure and on the distance between the electrodes and the auditory neurons. 
We hypothesized, that reducing the distance between the SGNs and the electrode array of the implant would allow for improved stimulation and frequency resolution, with the best results in a gapless position. Currently we lack in vitro culture systems to study, modify and optimize the interaction between auditory neurons and electrode arrays and characterize their electrophysiological response. To address these issues, we developed an in vitro bioassay using SGN cultures on a planar multi electrode array (MEA). With this method we were able to perform extracellular recording of the basal and electrically induced activity of a population of spiral ganglion neurons. We were also able to optimize stimulation protocols and analyze the response to electrical stimuli as a function of the electrode distance. This platform could also be used to optimize electrode features such as surface coatings.

We present a protocol to culture primary murine spiral ganglion neuron explants on multi electrode arrays to study neuronal response profiles and optimize stimulation parameters. Such studies aim to improve the neuron-electrode interface of cochlear implants to benefit hearing in patients as well as the energy consumption of the device.

Ganglio espiral neurona explante cultura y Electrofisiología de multi electrodos matrices

Spiral ganglion neurons (SGNs) participate in the physiological process of hearing by relaying signals from sensory hair cells to the cochlear nucleus in ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

Imágenes de la actividad neuronal en la corteza somatosensorial primaria usando Thy1-ratones transgénicos GCaMP6s

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Activity-based Training on a Treadmill with Spinal Cord Injured Wistar Rats

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) on a treadmill is widely used as a rehabilitation tool in the SCI population with many benefits and improvements to daily life. We utilize this method of activity-based task-specific training (ABT) in rodents after SCI to both elucidate the mechanisms behind such improvements and to enhance and improve upon existing clinical rehabilitation protocols. Our current goal is to determine the mechanisms underlying ABT-induced improvements in urinary, bowel, and sexual function in SCI rats after a moderate to severe level of contusion. After securing each individual animal in a custom-made adjustable vest, they are secured to a versatile body weight support mechanism, lowered to a modified three-lane treadmill and assisted in step-training for 58 minutes, once a day for 10 weeks. This setup allows for the training of both quadrupedal and forelimb-only animals, alongside two different non-trained groups. Quadrupedal-trained animals with body weight support are aided by a technician present to assist in stepping with proper hind limb placement as necessary, while forelimb-only trained animals are raised at the caudal end to ensure no hind limb contact with the treadmill and no weight-bearing. One non-trained SCI group of animals is placed in a harness and rests next to the treadmill, while the other control SCI group remains in its home cage in the training room nearby. This paradigm allows for the training of multiple SCI animals at once, thus making it more time-efficient in addition to ensuring that our pre-clinical animal model mimics the clinical representation as close as possible, particularly with respect to the body weight support with manual assistance.

This protocol demonstrates our model of activity-based locomotor treadmill training for rats with spinal cord injury (SCI). Included is both quadrupedal and forelimb-only groups, in addition to two distinct types of non-trained control groups. Investigators are able to assess training effects on SCI rats using this protocol.

Formación basada en una cinta rodante con la médula espinal lesionado ratas Wistar

Spinal cord injury (SCI) results in lasting deficits that include both mobility and a multitude of autonomic-related dysfunctions. Locomotor training (LT) ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Entrega intra-arterial de células madre neuronales a la rata y el cerebro del ratón: aplicación a la isquemia cerebral

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, proteins, and lipids from donor to acceptor cells, making them key signaling mediators between cells. In the central nervous system (CNS), sEVs can mediate intercellular signaling, including neuroimmune interactions. sEV functions can be studied by tracking the uptake of labeled sEVs in recipient cells both in vitro and in vivo. This paper describes the labeling of sEVs from the conditioned media of RAW 264.7 macrophage cells using a PKH membrane dye. It shows the uptake of different concentrations of labeled sEVs at multiple time points by Neuro-2a cells and primary astrocytes in vitro. Also shown is the uptake of sEVs delivered intrathecally in mouse spinal cord neurons, astrocytes, and microglia visualized by confocal microscopy. The representative results demonstrate time-dependent variation in the uptake of sEVs by different cells, which can help confirm successful sEVs delivery into the spinal cord.

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

We describe a protocol to label macrophage-derived small extracellular vesicles with PKH dyes and observe their uptake in vitro and in the spinal cord after intrathecal delivery.

Captación de pequeñas vesículas extracelulares con marca fluorescente in vitro y en la médula espinal

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, ...