Samuel L. Pfaff Salk Biyolojik Araştırmalar Enstitüsü Gen İfadesi Laboratuvarlarında Profesör ve Howard Hughes Tıp Enstitüsü bir Araştırmacı. Bu çalışma, Christopher ve Dana Reeve Vakfı tarafından desteklenmiştir. Joe Belcovson, Kent Schnoeker Multimedya Kaynakları ve Salk Enstitüsü&#39;nden Mike Sullivan, fotoğraf ve düzenleme ile ilgili yardım sağladı.

Elektrofizyolojik kayıtları izole omurilik, sinir devresi, 1 genetik ve gelişimsel değişiklikler ortaya çıkarabilir . Daha önce yenidoğan fare omurilik 2 incelemek için bir yöntem olduğunu göstermiştir . Burada izole omurilik 3 kurgusal lokomosyon kayıt yararlı emme elektrotlar hazırlamak için bir yöntem mevcut. Düşük sıcaklık alkol lamba 3-5 kullanarak elle çok ince uçlu bir plastik boru elektrot ucu çizilebilir. Plastik boru (PE90, Clay Adams Intramedic TM), uysal bir forma boru yumuşatmak için ateşte tutulur . Boru erimeye başlar ve daha saydam hale geldikçe, ısı kaynağı ve uçları hafifçe dışında çekti kaldırılır. Tüp alev kaldırma zamanlaması plastik daraltmak veya çizilmiş ise bölünmüş değildir sigortalamak için önemlidir. Boru ince kısmı istenen iç çapına uygun bir tıraş bıçağı ile kesilir, numune veya kablosunun segmental düzeyde yaşına tarafından dikte. Hortumun bir o-ring yerleştirme kolaylığı için, 45 ° kesim, kalın bir jilet ile boru sonunda yapılır. Bir halkalı ve o-ring varil elektrot elektrot ucu takmak için kalın sonuna yüklenir. Özellikle büyüklükteki elektrotlar için izin ve zorlukları önlemek için el-çekme elektrot ipuçları, elektrotlar, kalın bir boru (PE90) küçük bir tüp yerleştirilir kullanılarak inşa edilebilir. Boru uzunlukları kayıt kurulum gereksinimleri için özel ve değişkendir. Biz deneyler 1-3,5 kullanılan ölçümler de sunuyorum. PTFE ince borular (Zeus, Küçük Parça) ve PE90 boru 10cm uzunluğu 1 cm uzunluğunda bir tıraş bıçağı ile kesilir. Küçük tüp biter, onlar ezilmiş veya kapalı değil emin olmak için muayene edilmelidir. Bunlar bir böcek pin (Güzel Bilim Araçları) kullanılarak açılabilir. Bir damla yapıştırıcı (JB Kaynak), PTFE tüp orta yerleştirilir. PTFE tüp ve yapıştırıcı PE90 tüp ağız emme kullanarak PE90 tüp içine çekilir. 45 ° &#39;lik bir kesim kalın boru sonunda yapılır ve bir halkalı ve o-ring önceki bağlıdırlar. Halkalı ve BNC konektör ticari bir emme elektrodu vida. BNC konnektör mum mühür kaldırmak için bir saat ksilen batırılmış. Konektörü, sonra suda yıkanır ve kurutulur. Düşük açılı yan port varil tarafında yapılmalıdır. Bir metal çubuk bir Bunsen beki ile ısıtılır ve düşük bir açıda varil tarafında mevcut deliğe basıldığında. 0.010 inç gümüş tel (PM Sistemleri), 15cm uzunluğunda, on beş dakika için kesilir ve çamaşır ıslatılır. Bu sinyal iletim 6 yardımcı olan bir gümüş klorür kaplama oluşturmak için yapılır. Tel, su ile durulanır, kurutulur, sonra BNC konnektörüne lehimlenmiştir. BNC konnektörü ve gümüş tel BNC konnektör varil tarafında kalması için yeterli oda emme elektrot namlu içine eklenir. Emme boru yan port 2-3cm eklenir. Varil başına yaklaşık 2-3cm silikon enjekte edilir. BNC konektörü elektrot dolgu ileri iterek namlu içine dişli. Elektrot ucu elektrot ön ucunu dişli. Shrink tüp de yan port emiş hattını güçlendirmek için eklenebilir. Elektrotlar manyetik standları bağlı ve kayıt çanak yakınına yerleştirilen mikromanipülatörler üzerine monte edilir. BNC koaksiyel kablolar bir amplifikatöre kablolu bir headstage bağlıdırlar. Amplifikatör, veri toplama yazılımı (PolyView) ile bir PC bilgisayar dijital kart için bir analog (National Instruments) bağlı bir adaptör (PolyView) bağlıdır. Elektrofizyolojik verileri kaydederken, tüm dış parazitleri ortadan kaldırmak için gerekli. Sırasıyla, elektrik ve mekanik bir girişimi önlemek için kayıt çanak, amplifikatör ve baş sahne Faraday kafesi içinde bir hava masaya yerleştirilir. Temsilcisi Sonuçlar: <img src="/files/ftp_upload/2580/2580fig1.jpg" alt="Şekil 1" /> Şekil 1: piyasada bulunan emme elektrot alkol lambası çekilmiş, ya da piyasada bulunan belirli büyüklükteki plastik boru ile inşa ağır bir göstergesi gümüş tel ve plastik bir ucu eli ekleyerek değiştirilebilir . Buna ek olarak, parafin dolgu daha dayanıklı silikon kalafat dolgu ile değiştirilir. <img src="/files/ftp_upload/2580/2580fig2.jpg" alt="Şekil 2" /> Şekil 2 emme elektrot hava tabloya ilişkin bir şema (mekanik izolasyonu için), Faraday kafesi (elektrik isolati), amplifikatör ve bilgisayar. Bu hava masa ve Faraday kafesi başvurulan kayıt çanak ve ikinci bir zemin için başvurulan bir zemin olduğunu dikkate alınmalıdır.

<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>

Sinir sistemi geliştirme izole kemirgen omurilik kullanarak incelenebilir. Nörotransmitter varlığı, kurgusal lokomosyon desenli elektriksel aktivite 1,3 şeklinde spinal kord oluşturulabilir. Bu ritmik patlamaları 0,2 - 0,5 Hz üretilir ve sol-sağ ve fleksör ekstansör ardalanmasından desenli. Farklı gelişim aşamalarında, bu faaliyetin sağlamlık ve desenler 1 değişir. Genetik mutasyonlar da bu etkinliği 3,5,7 desenlendirme rahatsız. Bu faaliyetin Transgenik ve gelişi...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<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

Nöral devrelerin ve hareket gelişimi, yenidoğan kemirgen omurilik merkezi desen üreteci (CPG) davranışını kullanarak incelenebilir. Biz disseke kemirgen omurilik, emme, CPG aktivitesinin incelemek için kullanılan elektrotlar, ya da kurgusal lokomosyon imal etmek için bir yöntem ortaya koymaktadır. Kemirgen omurilik yapay beyin omurilik sıvısı konur ve ventral kökleri emme elektrot içine çekilir. Elektrot, piyasada yaygın olarak bulunan emme elektrot değiştirerek inşa edilmiştir. Ağır bir gümüş tel, piyasada bulunan elektrot tarafından verilen standart tel yerine kullanılır. Ticari elektrot cam ucu artan dayanıklılık için plastik bir ucu ile değiştirilir. Biz tutarlılık ve tekrarlanabilirlik sağlayan boru belirli boyutlarda yapılan elle çizilmiş elektrotlar ve elektrot hazırlar. Veri, bir amplifikatör ve neurogram kazanım yazılım kullanarak toplanır. Sırasıyla, mekanik ve elektrik girişimi önlemek için Kayıtlar Faraday kafesi içinde bir hava tablo yapılır.

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

Yenidoğan kemirgen omurilik elektrofizyolojik kayıtlar ölçmek için kullanılan ekstraselüler emme elektrot üretimi ve kullanımı bir gösteri<em&gt; In vitro</em

Omurilik Elektrofizyoloji II: Ekstrasellüler Emiş Elektrot İmalatı

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 Görüntüleme.  The Salk Institute for Biological Studies. Yenidoğan kemirgen omurilik elektrofizyolojik kayıtlar ölçmek için kullanılan ekstraselüler emme elektrot üretimi ve kullanımı bir gösteri 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.

Tavuk embriyo Omurilik Dilim Kültür Protokolü

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

Nörobilim

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.

Larva Zebra balığı Omurilik Kesisi

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.

Bir<em&gt; Ex vivo</em&gt; Lazer kaynaklı Omurilik Felçlileri Modeli Gerçek zamanlı aksonal Dejenerasyon mekanizmaları değerlendirmek için

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 ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

Sıçan Servikal Spinal Kord difüzyon Görüntüleme

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

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.

Yenidoğan Kemirgenler İzole Beyin Sapı-spinal Kordon Müstahzarlar Elektrofizyoloji Sinir Solunum Ağı Çıktı Kayıt verir

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.

Çok elektrot Dizileri Yetiştirilen Organotipik Omurilik Co-kültürler Fonksiyonel Rejenerasyon incelenmesi

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.

Çok elektrot dizilerde Spiral Ganglion Nöron Eksplantasyon Kültür ve Elektrofizyoloji

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

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Yenidoğan Faresinden İzole Edilen Spinal Kord Nöronları İzolasyonu ve Kültürü

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

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.

Sinirsel aktivite Thy1kullanarak birincil somatosensor kortekste Imaging-GCaMP6s transgenik fareler

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.

Etkinlik tabanlı eğitim omurilik ile bir koşu bandı üzerinde Wistar fareler yaralı

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