塞缪尔百福是在索尔克生物研究所的基因表达实验室教授和霍华德休斯医学研究所研究员。这项工作是由克里斯托弗和Dana里夫基金会的支持。乔Belcovson，肯特Schnoeker和迈克沙利文在索尔克研究所的多媒体资源提供援助，摄影和剪辑。

隔离脊髓电生理记录可以揭示遗传和发育的变化的神经电路1。以前，我们表现出一个方法来解剖新生小鼠脊髓2。在这里，我们现在准备吸电极记录在孤立的 3个脊髓虚构运动有用的方法。 塑料管电极的尖端，可以得出一个手工非常精细的提示使用低温酒精灯3-5。塑料管材（PE90，粘土亚当斯Intramedic TM）举行了火焰软化成可塑性形式的油管。由于油管开始融化，变得更加透亮，它是从热源和两端轻轻拉开。从火焰中取出管的时机很重要，以确保塑料不会崩溃或分裂而正在起草。油管薄的部分是用刀片切到所需的内部直径，作为标本的年龄段线的水平决定的。为了便于放置油管O形圈，45 °切割油管用刀片粗末。一个密封圈和O形圈安装在粗末附加电极尖端电极桶。 为了让特定大小的电极，和避免的困难手，拉电极秘诀，电极可使用小管插入一个较厚的管材（PE90）。管材的长度是具体的录音设置的要求，是可变的。我们目前在我们的实验 1-3,5中使用的测量。一个聚四氟乙烯细管（宙斯，小零件）和一个PE90管材10厘米的长度1厘米的长度是用刀片切割。小管的两端应进行检查，以确保他们不会压碎或关闭。他们用昆虫针（精细科学的工具），可以打开。一滴粘合剂（巴顿焊接）被放在PTFE管的中点。 PTFE管和胶画到PE90 PE90管材管口吸。削减45 °油管粗末和套圈和O型圈连接，正如前面。 套圈和BNC接头拧开商业吸电极。 BNC连接器是在二甲苯中浸泡一小时，以去除蜡封。连接器，然后在水中漂洗，晒干。 一个低角度的侧端口必须在桶边。使用本生灯加热一根金属棒，并压入现有的枪管在低角度的侧孔。一个0.010英寸的银线（AM系统）15厘米长度被切断，在漂白水浸泡十五分钟。这样做是为了建立一个氯化银涂层，在第 6信号传导艾滋病。导线是用清水漂洗，晾干，然后到BNC接头的焊接。 BNC连接器及银线插入吸电极桶桶侧面的BNC连接器，以保持足够的房间。吸油管插入端端口2 - 3cm的。硅酮密封胶注入到每桶约2 - 3cm的。 BNC接头螺纹枪管向前推的密封胶的电极。 现在到前端的电极螺纹电极尖端。热缩管，也可以从侧面端口添加到加强吸线。 电极安装附加磁场的立场，并放在附近的录音碟micromanipulators。 BNC同轴电缆连接到一个有线放大器探头。放大器连接到一个适配器（Polyview）是在数据采集软件（Polyview）的PC电脑连接到模拟到数字存储卡（美国国家仪器）。 录制时的电生理数据，它是必要的，以消除一切外界干扰。录音盘，放大器，和头部阶段都被放置在一个法拉第笼，以防止电气和机械干扰，分别对空气表。 代表性的成果: <img src="/files/ftp_upload/2580/2580fig1.jpg" alt="图1" /> 图1。一个市售的吸电极可以修改加入较重的计银线和一个酒精灯上的一个塑料尖，是手工绘制，或特定大小与市售塑料管材建造。此外，石蜡密封胶是取代硅填缝密封胶更耐用。 <img src="/files/ftp_upload/2580/2580fig2.jpg" alt="图2" /> 图2:一个涉及到空气表吸电极示意图（机械隔离），法拉第笼（电气isolati上），放大器和计算机。应该指出的是，录音盘和第二个理由是引用到空气中的表和法拉第笼参考地面。

<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>

可以研究使用孤立的啮齿类动物脊髓神经系统的发育。在神经递质的存在，虚构的运动可以产生脊髓在图案的电气活动 1,3形式。这些节奏的阵阵生产0.2〜0.5 Hz时，在左，右和屈，伸交替图案。在不同的发展阶段，这项活动的稳定性和模式有所不同1。基因突变，也可以扰乱了这一活动的 3,5,7的图案。这项活动的转基因和发育的研究提供了组织的中枢模式产生电路的见解和?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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

使用新生儿啮齿类动物脊髓中枢模式发生器（CPG）的行为，可以研究神经设计电路和运动的发展。我们证明的方法来制造吸电极，用于检查中央人民政府的活动，或虚构的运动，在解剖啮齿类动物脊髓。被放置在人工脑脊液中的啮齿类动物脊髓和腹侧根绘制成吸电极。通过修改一个市售的吸电极的电极构造。较重的银线，是用来代替市售电极的标准线。为提高耐久性的塑料提示取代商业电极的玻璃尖。我们准备了手工绘制的电极和电极特定大小的管，允许的一致性和可重复性。数据收集，使用的放大器和neurogram数据采集软件。录音内执行一个法拉第笼，以防止机械和电气干扰，分别对空气表。

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

外吸用来衡量新生儿啮齿类动物脊髓的电生理记录电极的制造和使用示范<em&gt;在体外</em

脊髓电二:外吸电极的制作

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 次观看.  The Salk Institute for Biological Studies. 外吸用来衡量新生儿啮齿类动物脊髓的电生理记录电极的制造和使用示范在体外

视频: 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.

鸡胚胎脊髓片培养协议

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

科研

神经科学

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.

在斑马鱼幼虫脊髓横断

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.

一个<em&gt;离体</em&gt;激光诱导脊髓损伤模型，以评估轴索变性的机制在实时

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.

在大鼠颈脊髓弥散成像

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.

电从新生儿鼠害隔离脑干，脊髓准备允许呼吸中枢网络输出记录

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.

调查器官脊髓联合培养物中生长多电极阵列功能再生

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.

螺旋神经节神经元外植体培养及电多电极阵列

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.

脊髓神经元从新生小鼠中分离和培养

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.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

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.

脊髓损伤 wistar 大鼠跑步机的作业训练

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