사무엘 L. 파프는 생물 학적 연구 솔크 연구소에서 유전자 발현 연구소에서 교수와 하워드 휴즈 의학 연구소의 탐정이다. 이 작품은 크리스토퍼와 다나 리브 재단​​에 의해 지원되었다. 조 Belcovson, 켄트 Schnoeker와 솔크 연구소에서 멀티미디어 자원에 마이크 설리반은 사진 촬영 및 편집 지원을 제공했습니다.

절연 척추 코드의 Electrophysiological 녹음 신경 회로 1 유전자 및 개발 변경 사항을 확인할 수 있습니다. 우리는 이전에 신생아 마우스 척추 코드 2을 해부하다하는 방법을 보여주었다. 여기 우리는 고립 척추 코드 3 상상의 운동을 기록에 유용 흡입 전극을 준비하는 방법을 제시한다. 플라스틱 튜브 전극의 끝은 저온 알코올 램프 3-5 사용 손으로 아주 좋은 팁을 그립 수 있습니다. 플라스틱 관 (PE90, 클레이 애덤스 Intramedic TM)는 가단 형태로 튜브를 부드럽게하기 위해 화염을 통해 실시하고 있습니다. 튜브가 녹기 시작하고 더 투명 해짐에 따라 그것은 열원과 종료가 부드럽게 떨어져 행진에서 제거됩니다. 불꽃에서 튜브를 제거하는시기는 플라스틱이 붕괴하거나 그려하면서 분할하지 않습니다 확보하는 것이 중요합니다. 튜빙의 얇은 부분은 원하는 내경에 따라 면도날로 절단 등 표본이나 코드의 segmental 수준의 나이에 의해 결정. 튜브에 O - 링을 배치의 용이성을 위해, 45 ° 삭감은 면도날로 튜브의 두께 끝에 이루어집니다. 물미 및 O - 링은 전극의 배럴당 전극 팁을 첨부하기 위해 두꺼운 끝에 설치되어 있습니다. 특히 중소 전극에 대한 허용의 어려움 피하려면 손을 당기는 전극 팁, 전극은 두꺼운 튜브 (PE90)에 삽입 작은 튜브를 사용하여 건설 수 있습니다. 튜브의 길이는 녹화 설정의 요구 사항을 특정하고 변수입니다. 우리는 실험 1-3,5에 사용되는 측정을 제시한다. PTFE 미세 관 (제우스, 소형 부품) 및 PE90 튜브의 10cm 길이 1cm 길이는 면도날로 절단됩니다. 작은 튜브의 끝을들은이 조각 또는 폐쇄되지 않도록하는 검사해야합니다. 그들은 곤충 핀 (파인 과학 도구)를 사용하여 열 수 있습니다. 접착제 (JB 용접)의 드롭은 PTFE 튜빙의 미드에 배치됩니다. PTFE 튜빙과 접착제는 PE90 튜브에 입 흡입을 사용하여 PE90 튜브로 그려져 있습니다. 45 ° 컷은 튜브의 두께 끝에 만들어진이며 물미 및 O - 링은 같은 이전, 첨부합니다. 물미 및 BNC 커넥터는 상업 흡입 전극에서 unscrewed 있습니다. BNC 커넥터는 왁스 봉인을 제거 한 시간 크실렌에 배어있다. 커넥터는 다음 물로 씻어서 건조합니다. 낮은 직각 사이드 포트 통의 측면에하셔야합니다. 금속 막대는 알콜 램프를 사용하여 가열하고 낮은 각도로 통의 측면에있는 기존 구멍에 누르면됩니다. 0.010 인치 실버 와이어 (AM 시스템)의 15cm 길이를 15 분 동안 잘라 표백제에 배어있다. 이것은 신호 전도 6 에이즈되는 은색 염화물 코팅을 만들 수 이루어집니다. 와이어는 물로 씻어서 건조하고, BNC 커넥터에 다음 납땜합니다. BNC 커넥터와 실버 와이어는 통의 측면을 유지하기 위해 BNC 커넥터에 대한 충분한 공간과 흡입 전극 배럴에 삽입됩니다. 흡입 튜브는 사이드 포트 2-3cm에 삽입됩니다. 실리콘 밀봉제는 배럴당 약 2-3cm로 주입됩니다. BNC 커넥터는 앞으로 밀봉 재를 밀어 전극의 총구에 스레드입니다. 전극의 팁 이제 전극의 프런트 엔드로 스레드입니다. 축소 관은 또한 측면 포트의 흡입 라인을 강화하기 위해 추가할 수 있습니다. 전극 자기 서에 첨부하고, 녹음 요리 근처에 배치 micromanipulators에 탑재하고 있습니다. BNC 동축 케이블은 앰프에 설치된 headstage에 첨부됩니다. 증폭기는 데이터 수집 소프트웨어 (Polyview)와 PC의 컴퓨터에 디지털 카드에 아날로그 (국가 악기)에 연결된 어댑터 (Polyview)에 연결되어 있습니다. electrophysiological 데이터를 기록하면, 모든 외부 간섭을 제거하기 위해 필요합니다. 각각, 전기 및 기계적 간섭을 방지하기 위해 녹음 요리, 증폭기, 그리고 머리 단계는 패러데이 케이지 내에서 공기 테이블에 배치됩니다. 대표 결과 : <img src="/files/ftp_upload/2580/2580fig1.jpg" alt="그림 1" /> 그림은 1. 상용 흡입 전극은 알콜 램프에 그려져, 또는 특정 크기의 상용 플라스틱 튜브와 건설 무거운 게이지 실버 와이어 손으로하는 플라스틱 팁을 추가하여 수정할 수 있습니다. 또한, 파라핀의 밀봉제 더 내구성이 실리콘 편자에 박는 뾰족한 징을 박다 밀봉제로 대체됩니다. <img src="/files/ftp_upload/2580/2580fig2.jpg" alt="그림 2" /> 그림 2. 공기 테이블과 관련하여 흡입 전극의 구조 (기계 격리를 위해), 패러데이 케이지 (전기 isolati)에 앰프 및 컴퓨터. 이것은 공기 테이블과 패러데이 케이지에 참조된 기록 요리와 두 번째 접지 참조 지상이있다는 것을 주목하여야한다.

<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>

관심 없음 충돌 선언하지 않습니다.

신경 시스템 개발은 격리 쥐 척수 코드를 사용하여 공부하실 수 있습니다. 신경 전달 물질의 존재에 상상의 운동은 패턴 전기 활동을 1,3의 형태로 척수에서 생성할 수 있습니다. 이러한 리듬 파열은 0.2-0.5 Hz에서에서 생산하고 왼쪽, 오른쪽과 flexor - 신근 alternations에서 패턴입니다. 다른 발달 단계에,이 활동의 견고하고 패턴 1 다릅니다. 유전자 변이는 또한이 활동을 3,5,7의</sup...

<!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

신경 circuitries와 운동의 발전은 신생아 쥐 척수 중앙 패턴 발생기 (CPG) 동작을 이용하여 공부하실 수 있습니다. 우리는 해부 쥐 척수 코드에서 흡입 CPG 활동을 검사하는 데 사용되는 전극, 또는 상상의 운동을 조작하는 방법을 보여줍니다. 설치류 척추 코드는 인공 뇌척수에 배치되고 복부 뿌리는 흡입 전극으로 그려져있다. 전극은 상용 흡입 전극을 수정하여 구성되어 있습니다. 무거운 은색 와이어는 상용 전극에 의해 주어진 표준 와이어 대신 사용됩니다. 상업 전극에 유리 팁은 증가 내구성 플라스틱 팁로 대체됩니다. 우리는 일관성과 재현성있게, 튜빙의 특정 크기로 만들어진 손으로 그린​​ 전극과 전극을 준비합니다. 데이터는 증폭기 및 neurogram 수집 소프트웨어를 사용하여 수집됩니다. 각각, 기계 및 전기 간섭을 방지하기 위해 녹음은 패러데이 케이지 내에서 공기 테이블에 수행됩니다.

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

신생아 설치류 척추 코드의 el​​ectrophysiological 녹음을 측정하는 데 사용되는 세포 흡입 전극의 제조 및 사용의 데모<em&gt; 체외에서</em

척수 전기 생리학 II : 세포 흡입 전극 제작

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 조회수.  The Salk Institute for Biological Studies. 신생아 설치류 척추 코드의 el​​ectrophysiological 녹음을 측정하는 데 사용되는 세포 흡입 전극의 제조 및 사용의 데모 체외에서

비디오: 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.

닭의 배아 척수 코드 슬라이스 문화 프로토콜

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

연구

신경과학

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.

애벌레 Zebrafish의 척수 절개

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.

<em&gt; 전의 VIVO</em&gt; 레이저에 의한 척수 손상 모델은 실시간으로 축삭 변성의 메커니즘을 평가하기 위해

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.

신생아 설치류에서 분리 뇌간 - 척수 준비에 전기 생리학은 신경 호흡 네트워크 출력 녹화를 할 수 있습니다

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.

다중 전극 배열에 성장의 Organotypic 척수 공동 문화 기능 재생 조사

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.

다중 전극 배열에 나선 신경절 신경 이식편 문화 및 전기 생리학

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.

Thy1를 사용 하 여 기본 Somatosensory 피 질에서 신경 활동을 이미징-GCaMP6s 유전자 변형 쥐

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.

척수와 디딜 방 아에 활동 기반 교육 부상 Wistar 쥐

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.

쥐와 마우스 두뇌에 신경 줄기 세포의 동맥 내 납품: 대뇌 허혈에 응용 프로그램

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.

체외 및 척수에서 형광 라벨 작은 세포 외 소포의 섭취

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