Samuel L. Pfaff est un professeur dans les laboratoires de l&#39;expression génique au Salk Institute for Biological Studies et un chercheur à l&#39;Institut médical Howard Hughes. Ce travail a été soutenu par la Christopher Reeve et Dana Foundation. Joe Belcovson, Kent et Mike Sullivan Schnoeker de Ressources Multimédia à l&#39;Institut Salk a fourni une aide à la photographie et l&#39;édition.

Enregistrements électrophysiologiques de la moelle épinière isolée peut révéler des changements génétiques et développementales de neurones de circuits 1. Nous avons précédemment démontré une méthode de disséquer les cordons de souris néonatales vertébrale 2. Nous présentons ici une méthode pour préparer les électrodes d&#39;aspiration utile dans l&#39;enregistrement de locomotion fictive dans les régions isolées de la moelle épinière 3. La pointe d&#39;une électrode de tubes en plastique peuvent être tirées d&#39;une pointe très fine à la main en utilisant une lampe à alcool à basse température 3-5. Le tube de plastique (PE90, Clay Adams Intramedic TM) est détenu sur la flamme pour adoucir le tube dans une forme malléable. Comme le tube commence à fondre et devient plus translucide, il est retiré de la source de chaleur et les extrémités sont doucement tiré à part. Le moment de retirer le tube de la flamme est important pour assurer le plastique ne s&#39;effondre ou divisé en cours d&#39;élaboration. La partie mince du tube est coupé avec une lame de rasoir en fonction du diamètre interne désirée, comme dicté par l&#39;âge de l&#39;échantillon ou le niveau segmentaire de la moelle. Pour plus de facilité de placer un joint torique sur le tuyau, une coupe de 45 ° est faite à l&#39;extrémité la plus épaisse de la tubulure avec une lame de rasoir. Une bague et le joint torique sont installés sur la fin d&#39;épaisseur pour fixer le bout de l&#39;électrode sur le canon de l&#39;électrode. Pour permettre électrodes spécifiquement dimensionnés et éviter les difficultés de la main-conseils en tirant des électrodes, électrodes peuvent être construits en utilisant des tubes insérés dans une petite épais tubes (PE90). Les longueurs de la tubulure sont spécifiques aux exigences de la configuration d&#39;enregistrement et sont variables. Nous présentons les mesures utilisées dans nos expériences 1-3,5. Une longueur de 1 cm de tube PTFE fines (Zeus, petites pièces) et une longueur de 10cm du tube PE90 sont coupés avec une lame de rasoir. Les extrémités du tube petits devraient être examinés pour s&#39;assurer qu&#39;ils ne sont pas écrasés ou fermé. Ils peuvent être ouverts à l&#39;aide d&#39;une broche d&#39;insectes (Outils Fine Science). Une goutte de colle (JB Weld) est placée sur le milieu du tube PTFE. Le tube de PTFE et de l&#39;adhésif sont aspirés dans le tuyau d&#39;aspiration en utilisant PE90 bouche sur le tube PE90. Une coupe de 45 ° est faite à l&#39;issue d&#39;épaisseur du tube et un embout et joint torique sont attachés, comme précédent. Le connecteur BNC virole et sont dévissés partir d&#39;une électrode d&#39;aspiration commerciales. Le connecteur BNC est trempé dans du xylène pendant une heure pour enlever le sceau de cire. Le connecteur est ensuite rincé à l&#39;eau et séché. Un port latérale inférieure inclinée doit être faite sur le côté du canon. Une tige de métal est chauffé à l&#39;aide d&#39;un brûleur Bunsen et pressé dans le trou existant du côté du baril à un angle faible. Une longueur de 15cm de 0,010 pouces de fil d&#39;argent (AM Systems) est coupé et imbibé d&#39;eau de javel pendant quinze minutes. Ceci est fait pour créer un revêtement de chlorure d&#39;argent qui aide à 6 de la conduction du signal. Le fil est rincé à l&#39;eau, séché, puis soudé sur le connecteur BNC. Le fil connecteur BNC et l&#39;argent sont insérés dans le canon d&#39;électrodes d&#39;aspiration avec assez de place pour le connecteur BNC de rester sur le côté du canon. Tuyau d&#39;aspiration est insérée dans le port du côté 2-3cm. Mastic silicone est injecté dans le cylindre d&#39;environ 2-3cm. Le connecteur BNC est vissé dans le canon de l&#39;électrode de pousser le mastic avant. La pointe de l&#39;électrode est désormais vissé dans la partie avant de l&#39;électrode. Gaine thermorétractable peuvent également être ajoutés pour renforcer la ligne d&#39;aspiration du côté bâbord. Les électrodes sont montées sur micromanipulateurs qui sont attachés aux stands magnétique, et placé près de l&#39;antenne d&#39;enregistrement. BNC câbles coaxiaux sont attachés à un headstage qui est relié à un amplificateur. L&#39;amplificateur est connecté à un adaptateur (Polyview) qui est connecté à une carte de l&#39;analogique au numérique (National Instruments) dans un ordinateur PC avec le logiciel d&#39;acquisition de données (Polyview). Lors de l&#39;enregistrement des données électrophysiologiques, il est nécessaire d&#39;éliminer toutes les interférences extérieures. Le plat d&#39;enregistrement, un amplificateur, et le stade de la tête sont placés sur une table de l&#39;air intérieur d&#39;une cage de Faraday pour éviter les interférences électriques et mécaniques, respectivement. Les résultats représentatifs: <img src="/files/ftp_upload/2580/2580fig1.jpg" alt="Figure 1" /> Figure 1. Une électrode d&#39;aspiration disponibles dans le commerce peuvent être modifiés par l&#39;ajout d&#39;un fil de plus gros calibre d&#39;argent et un bout de plastique qui est dessiné à la main sur une lampe à alcool, ou construits avec des tubes en plastique disponibles dans le commerce de formats spécifiques. En outre, l&#39;étanchéité de paraffine est remplacé avec plus de mastic silicone calfeutrage durable. <img src="/files/ftp_upload/2580/2580fig2.jpg" alt="Figure 2" /> Figure 2. Un schéma de l&#39;électrode d&#39;aspiration par rapport à la table de l&#39;air (pour l&#39;isolation mécanique), cage de Faraday (électrique isolatile), un amplificateur et un ordinateur. Il faut noter qu&#39;il ya un terrain référencé à l&#39;antenne d&#39;enregistrement et un deuxième motif qui est référencé à la table de l&#39;air et la cage 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>

Développement du système nerveux peut être étudiée en utilisant des cordons rongeurs isolés épinière. En présence de neurotransmetteurs, la locomotion fictive peut être généré à partir de la moelle épinière dans la forme de motifs activité électrique 1,3. Ces bouffées rythmiques sont produites à 0,2 à 0,5 Hz et sont modelés en alternances gauche-droite et fléchisseurs-extenseurs. A différents stades de développement, la robustesse et les modèles de cette activité varie 1....

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<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>
</tr>
<tr>
<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>
</tr>
<tr>
<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>
<tr>
<td>Adhesive: Silicone caulk</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Solder and soldering iron</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Bleach</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Xylene</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Silver wire: 0.010&#x201D;</td>
<td>&nbsp;</td>
<td>A-M Systems</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Insect pins: Austerlitz 0.1mm</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Magnetic Stand</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>GJ-8</td>
<td>&nbsp;</td>
<tr>
<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>Narishige</td>
<td>MN 151</td>
<td>&nbsp;</td>
<tr>
<td>Miniboard (Headstage)</td>
<td>&nbsp;</td>
<td>Grass Industries</td>
<td>F-15EB/B1</td>
<td>&nbsp;</td>
<tr>
<td>Polyview Adaptor Unit</td>
<td>&nbsp;</td>
<td>Grass Industries</td>
<td>PVA 8</td>
<td>&nbsp;</td>
<tr>
<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>
		</table>
		</div>

spinal cord electrophysiology ii: extracellular suction electrode fabrication

Développement des circuits de neurones et la locomotion peut être étudiée à l&#39;aide de la moelle épinière des rongeurs néonatale centrale générateur de pattern (CPG) de comportement. Nous démontrons une méthode pour fabriquer des électrodes d&#39;aspiration qui sont utilisés pour examiner l&#39;activité CPG, ou de la locomotion fictive, dans les cordons de rongeurs disséqué épinière. Les cordons de rongeurs épinière sont placés dans le liquide céphalorachidien artificiel et les racines ventrales sont entraînés dans l&#39;électrode d&#39;aspiration. L&#39;électrode est construit en modifiant une électrode d&#39;aspiration disponible commercialement. Un fil plus lourd argent est utilisé au lieu du fil standard donné par l&#39;électrode disponibles commercialement. La pointe de verre sur l&#39;électrode commercial est remplacé par un embout en plastique pour une durabilité accrue. Nous préparons la main électrodes dessinés et fabriqués à partir d&#39;électrodes spécifiques tailles de tubes, ce qui permet la cohérence et la reproductibilité. Les données sont recueillies au moyen d&#39;un amplificateur et un logiciel d&#39;acquisition neurogram. Les enregistrements sont effectués sur une table de l&#39;air intérieur d&#39;une cage de Faraday pour éviter les interférences électriques et mécaniques, respectivement.

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

Une démonstration de la fabrication et l&#39;utilisation d&#39;une électrode extracellulaire aspiration utilisés pour mesurer des enregistrements électrophysiologiques des cordons de rongeur néonatal épinière<em&gt; In vitro</em

La moelle épinière électrophysiologie II: fabrication des électrodes extracellulaires d&#39;aspiration

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

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15.0K vues.  The Salk Institute for Biological Studies. Une démonstration de la fabrication et l'utilisation d'une électrode extracellulaire aspiration utilisés pour mesurer des enregistrements électrophysiologiques des cordons de rongeur néonatal épinière In vitro

Vidéo: 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.

Embryon de poulet moelle épinière culture Tranche Protocole

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

Recherche

Neurosciences

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.

Spinal Cord Transection dans le poisson zèbre larvaire

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.

Une<em&gt; Ex Vivo</em&gt; Induite par laser Spinal Cord Injury modèle pour évaluer les mécanismes de la dégénérescence axonale dans en temps réel

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.

Électrophysiologie sur isolés Cord Préparatifs du tronc cérébral-spinal de la naissance Rongeurs Permet Neural respiratoire Réseau d&#39;enregistrement de sortie

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.

Enquêter Régénération fonctionnelle en organotypiques médullaires Co-cultures Cultivé sur des tableaux multi-électrodes

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.

Spiral Ganglion Neuron Explantation Culture et électrophysiologie sur multi-électrodes Arrays

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.

Imagerie de l’activité neuronale dans le Cortex somatosensoriel primaire utilisant Thy1-GCaMP6s des souris transgéniques

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.

La formation axée sur l’activité sur un tapis roulant avec la moelle épinière blessée des Rats 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.

Livraison intra-artérielle de cellules souches neuronales au cerveau du rat et de la souris : application à l’ischémie cérébrale

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.

Absorption de petites vésicules extracellulaires étiquetées par fluorescence in vitro et dans la moelle épinière

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