우리는 생활 자금, 오클라호마 대학 학부 연구 기회 프로그램, 그리고이 작품을 자금에 대한 동물학의 부 감사합니다. 또한 편집에 도움 박사 마리엘의 Hoefnagels 감사합니다.

1. 사전 녹음, 라이브 동물 준비 <ol><li> 자사 홈 병의 전갈을 제거하고 미리 차게 유리 컨테이너에 배치, 다음, 두 분 최소 냉동 환경 (-5 ° C)에 전갈이 들어있는 항아리를 놓습니다. 이 시간은 연령에 따라 다를 수 있습니다, 크기, 연구에서 구체적인 전갈의 종류 등. 동물이 움직이지되면 그러나 일반적으로 냉동 환경 단계가 완료되었습니다. </li><li> 이분 후, 차가운 유리 용기에서 고정 전갈을 제거하고, 현미경 슬라이드에 복부 쪽을 놓으십시오. 스팅거, 꼬리, 다리, 그리고 moldable 점토 (석유 기반의 반 아켄 Plastalina)와 pedipalps를 확보. </li><li> 전갈의 pectines 넣어하는 플랫폼을합니다. 펙튼의 크기에 따라 플랫폼의 길이와 너비를 조정합니다. 일반적으로, 우리의 플랫폼은 현미경 커버 유리 밖으로 건설하고 약 10 (L) X 18 (W) mm입니다. </li><li> 에 맞게 (필요한 경우) 테이프를 트리밍, 펙튼 플랫폼에 양면 접착 테이프를 준수합니다. </li><li> pectinal 챔버하려면, 3~4mm의 깊이에 미리 녹인 왁스 (우리는 100 % 밀랍 칩을 사용)으로 플랫폼의 사면 세를 넣어. 우리는 뜨거운 접시에있는 유리 페트리 접시 안에 칩을 배치하여 왁스를 녹여. pectinal 챔버의 가장자리에 왁스가 상온으로 냉각되면, 이것은 높이 1mm에 대해 왁스의 벽을 만들어야합니다. </li><li> 동물, pectinal 삽입 지점을 찾아, 그것을 단지 사후 플랫폼의 취소 왁스칠 가장자리 (테이프 측면까지)를 놓으십시오. 그 후 부드럽게 동물의 다리를 제지하는 데 사용되는 점토 상대로 좌우 주변 가장자리를 눌러 플랫폼을 (이것은 나중에 이동하지 않도록) 안정화. </li><li> 챔버 내부의 펙튼 또는 pectines을 신중하게 배치하기 위해 미세하게 밀고 집게를 사용하십시오. 특히 집게를 사용하여 pectinal 척추 함께 펙튼을 보유하고 부드럽게 커버 유리 아래에서 펙튼을 지참 접착제 플랫폼 바닥에 그것을 낮추십시오. 조심스럽게 pectinal 척추에 압력을 적용하여 펙튼 - 투 - 테이프 본드를 설정합니다. </li><li> 이제 플랫폼의 취소 왁스칠 가장자리에 녹인 왁스를 (우리가 사전에 가열 금속 주걱을 사용하여)이 적용됩니다. 목적은 펙튼 확보하고 완벽하게 미래의 광유 소개 챔버 묶으을 두 배입니다. 너무는 뜨겁다 왁스와 펙튼 손상을 수 있습니다 것처럼, 동물의 펙튼 위에 왁스를 적용할 때주의를 사용합니다. </li><li> 펙튼 챔버 공사가 완료되었습니다. 다음 두 꼬리 세그먼트 사이에 은색 철사를 삽입하여 동물의 hemolymph와 무관심 전극 연결을 설정합니다. </li></ol> 2. 동시 세포외 녹화 및 화학적 자극 <ol><li> 우리는 다른 이유 중 (고정 위치에 즉, 동물성 조정), 수, 세포, 팁 - 녹음을 수행하기 전에 동물 24 H를 준비하는 시간을 지정하게되면 특정 (sensillum -에 따라 다름) 팁 - 직경의 자극제의 pipettes합니다. 우리는 이러한 사양을 달성 micropipette의 풀러를 사용합니다. 이상적으로, pipettes의 직경은 sensillar의 기공 직경보다 ≈ 2 μm의 큰 것입니다. 우리는 각 동물에 대해 적절하게 우리 micropipette 사양을 조정합니다. </li><li> 녹음하기 전에 1 H, 우리는 석유에서 펙튼을 잠수함이 잠수, pectinal 실로 광유 5 μl를 추가합니다. 그렇다면, 우리는 장거리 목표와 에피 조명 일을 가진 고성능 현미경, 아래 동물을 위치. </li><li> 화학적 페그 sensilla을 촉진하기 위해, 우리는 석유 매체를 통해 수성 tastants (예 : 구연산과 소금) 소개합니다. 첫째, 우리는 자극의 피펫에 자극제를 주입하는 micropipette 필러를 사용합니다. 참고 : 모든 자극제 솔루션은 전기 전도성에 대한 전해질을 포함해야합니다. </li><li> 다음 micromanipulator (녹화 장비)에 흥분제의 피펫을 위치 후, 흥분제의 피펫의 무딘 오픈 엔드에 기록 전극을 삽입합니다. 금속 전극과 전해 흥분제 솔루션 사이의 접촉을 보장하는 것이 중요합니다. </li><li> 일단 준비는 전기 오일 매체 통해 sensillar의 구멍에 피펫 단순히 낮은 (무관심 전극을 통해) 접지입니다. pectinal sensillum 당 화학 자극의 기간은 많은 서른로 분 거리로 작은로 두 번째 다를 수 있습니다. 일반적으로, 우리는 피펫마다 여러 펙스 (&gt; 30)을 샘플 수 있습니다. 흥분제의 피펫는 분 파편들로 가득 찼어되고있는 경우, 단순히 새로운 않는 피펫에 대한 막힌 피펫을 교환. </li></ol> 3. 실험의 여러 일 동안 동물 준비 <ol><li> 동물이 확장 기간 동안 사용할 수있다면, 우리는 pectinal 챔버를 재사용하고 각 레코딩 세션에 대해 미네랄 오일 드롭을 대체하는 것이 좋습니다. 특정 일의 레코딩 세션이 완료되면 설정 녹음에서 동물을 제거하고 미네랄 오일 세척을 수행합니다. </li><li> Specifically, 실로 미네랄 오일의 다른 드롭 (5 μl)을 추가하고 모든 기름을 제거합니다. 이것은 레코딩 세션 동안 피펫 밖으로 유출 있습니다 잔여 자극의 존재를 최소화하는 데 도움이됩니다. </li><li> 다음 세션 전에, 앞에서 설명한 것처럼 기름 한 방울을 추가합니다. </li></ol> 4. 대표 결과 : 적어도 3시 1분의 N 비율 : (특정 앰프 예를 들어, 하드웨어 등 디지타이징) 사용 녹음 장비에 따라 대표적인 결과는 S와 세포외 기록이다. 그것은 극지 (물 기준) 화학 tastants 8를 전달하기위한 매체를 제공하는 등 미네랄 오일 챔버의 성공적인 건설은 개인 sensilla에 화학 자극을 분리 필요합니다. 전혀 너무 작은 기름이나 없음이있다면, 흥분제 솔루션은 화학 자극 8 장기간, 통제 한판 승부를 일으킬 가능성이 그 이웃 sensilla에 기록된 sensillum에서 전염됩니다.

<ol>
	<li>Hodgson, E. S., Lettvin, J. Y., Roeder, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiology+of+a+Primary+Chemoreceptor+unit.">Physiology of a Primary Chemoreceptor unit.</a> Science. 12, 417-418 (1955).</li><li>Newland, P. L., Christensen, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste+processing+in+the+insect+nervous+system.">Taste processing in the insect nervous system.</a> Methods in Insect Sensory Neuroscience. , 289-318 (2005).</li><li>Foelix, R. F., M&#252;ller-Vorholt, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fine+structure+of+scorpion+sensory+organs+II.+Pecten+sensilla.">The fine structure of scorpion sensory organs II. Pecten sensilla.</a> Bull. Br. Arachnol. Soc. 6, 68-74 (1983).</li><li>Carthy, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+structure+and+function+of+the+sensory+pegs+on+the+scorpion+pecten.">Fine structure and function of the sensory pegs on the scorpion pecten.</a> Experientia. 22, 89-91 (1966).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+properties+of+chemosensory+peg+sensilla+on+the+pectines+of+scorpions.">Response properties of chemosensory peg sensilla on the pectines of scorpions.</a> J. Comp. Physiol. A. 181, 291-300 (1997).</li><li>Wolf, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pectine+organs+of+the+scorpion,+Vaejovis+spinigerus:+Structure+and+(glomerular)+central+projections.">The pectine organs of the scorpion, Vaejovis spinigerus: Structure and (glomerular) central projections.</a> Arthropod Struct. Dev. 37, 67-80 (2008).</li><li>Gaffin, D. D., Brownell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+evidence+of+synaptic+interactions+within+chemosensory+sensilla+of+scorpion+pectines.">Electrophysiological evidence of synaptic interactions within chemosensory sensilla of scorpion pectines.</a> J. Comp. Physiol. A. 181, 301-307 (1997).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+examining+scorpion+peg+sensilla:+the+mineral+oil+flood+technique.">A new approach to examining scorpion peg sensilla: the mineral oil flood technique.</a> J. Arachnol. 37, 379-382 (2009).</li><li>Knowlton, E. D., Gaffin, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+tip-recording+method+to+test+scorpion+pecten+chemoresponses+to+water-soluble+stimulants.">A new tip-recording method to test scorpion pecten chemoresponses to water-soluble stimulants.</a> J. Neurosci Methods. , .</li><li>Haupt, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antennal+sucrose+perception+in+the+honey+bee+(Apis+mellifera+L.):+behaviour+and+electrophysiology.">Antennal sucrose perception in the honey bee (Apis mellifera L.): behaviour and electrophysiology.</a> J. Comp. Physiol. A. 190, 735-745 (2004).</li><li>Bland, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensilla+on+the+antennae,+mouthparts,+and+body+of+the+larva+of+the+alfalfa+weevil,+hypera+postica+(gyllenhal)+(Coleoptera+:+Curculionidae).">Sensilla on the antennae, mouthparts, and body of the larva of the alfalfa weevil, hypera postica (gyllenhal) (Coleoptera : Curculionidae).</a> Int. J. Insect Morphol. Embryol. 12, 261-272 (1983).</li><li>Cate, H. S., Derby, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooded+Sensilla+Homologues:+Structural+Variations+of+a+Widely+Distributed+Bimodal+Chemomechanosensillum.">Hooded Sensilla Homologues: Structural Variations of a Widely Distributed Bimodal Chemomechanosensillum.</a> J. Comp. Neurol. 444, 345-357 (2002).</li></ol>

우리는 이전에 두 저널, 놀턴 및 Gaffin (2009)와 놀턴 및 Gaffin (보도)에 이러한 방법을 발표했습니다.

위의 프로토콜 electrophysiological 연구 사막 초원 전갈 (Paruroctonus utahensis)를 준비하는 방법을 설명합니다. 특히, 우리는 석유에 따라 pectinal chemosensory 뉴런의 제어 세포 끝 - 녹음을위한 챔버를 빌드하는 방법을 보여줍니다. 기름과 물을 혼합하지 않기 때문에, 그것은 화학 자극 (물 기반 각성제)를 단일 sensilla로 분리 수 있습니다. 그것은 스트레스를해야한다는 P. utahensis는 비교적 작은 동물 (≈ 2.5-5 ㎝)이며 큰 동물이 프로토콜을 적용하면 여러 크기와 같은 동물의 플랫폼, 챔버의 크기 조정, 그리고 적용 기름 양을 요구할 수 있습니다. 우리는 물을 기반의 화학 자극의 전달 제어에 필요한 석유의 최소 금액을 테스트하기 위해 파일럿 연구를 실시하는 것이 좋습니다. 또한, 우리는 pectinal sensilla 8 시간 기준 신경 활동에 기름의 영향을 찾을 수 없습니다. 이것은 이전 chemoresponsiveness를 평가하기 위해,뿐만 아니라 다른 모델 시스템을위한 확인하여야한다. 이 방법은 감각 분야에서 sensilla 간의 chemoresponse 상호 작용 효과에 대해 테스트하는 다른 방법과 함께 사용할 수 있습니다. 예를 들어, 한 sensillum에서 끝 - 기록 (석유 이하)이 가능합니다 같은 우리 이웃 sensillum 9 시부터베이스 레코드 (텅스텐 전극을 통해). 이러한 녹음 구성은 기본 - 기록 sensillum의 신경 활동에 영향을 미치는 지 여부를 화학적으로 한 sensillum을 자극하지 평가하기 위해 사용할 수 있습니다. 지금까지 아무도 전갈 pectinal sensilla이 테스트를하지 있으며, 그것뿐만 아니라 다른 chemosensory 시스템을위한 열린 질문 유지됩니다. 요약하면, 우리는 석유 진보 arthropod 맛보기의 신경 기초로 electrophysiological 조사의 범위를 아래의 팁 - 녹음 방법을 생각합니다. 이 원고에서는, 우리는이 방법에 대한 동물을 준비 프로토콜을 제공하고, 우리는 주변 감각 신경 시스템의 진학 확고한 기반을 제공합니다 바랍니다.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Lift-N-Press tab</td>
<td>&nbsp;</td>
<td>Ted Pella, Inc.</td>
<td>16082</td>
<td>Doubled-sided adhesive</td>
</tr>
<tr>
<td>MicroFil</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>MF34G</td>
<td>Micropipette filler</td>
</tr>
<tr>
<td>Standard Glass Capillaries</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>1B100F-6</td>
<td>Glass pipettes</td>
</tr>
<tr>
<td>Microelectrode Holder</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc. </td>
<td>MEH3SBW10</td>
<td>&nbsp;</td>		</tbody>
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electrophysiology of scorpion peg sensilla

우리는 electrophysiologically 짧은 감각 sensilla 3,4 PEG - 같은 조사에 대한 기존의 팁 - 녹음 기술의 1,2의 수정을 설명합니다. 모든 전갈의 중간 복부 표면의 밀도 분야가 pectines라는 두 개의 부속, 메카 및 chemosensory 말뚝 sensilla 5,6입니다. 이러한 sensilla의 chemoresponsiveness 평가하기위한 한 가지 방법은 휘발성 odorant이 감각 필드 5,7에 소개로 extracellularly sensillum 내에서 신경 활동을 기록을위한 텅스텐 전극을 사용합니다. 이 방법의 제한 속도가 느린 데이터 수집 및 통제 흥분제 소개 및 페그 분야에서 제거를 포함합니다. 이러한 한계를 극복하기 위해, 우리는 각각의 말뚝 sensilla 8,9 물을 기반 tastants를 제공하는을 통해 매체로 무극 성 미네랄 오일을 사용하는 새로운 팁 - 녹화 기술을 개발했습니다. 우리는 성공적으로 구연산 초산, 에탄올, 그리고 소금에 sensillar의 chemoresponses를 얻는이 방법을 적용했습니다. 여기 우리는 이러한 연구 9 실험 프로토콜을 설명합니다. 우리는이 새로운 방법은 곤충 10, 11, 갑각류 12 포함한 다른 arthropod chemosensory 시스템의 응답 특성을 공부하는 데 유용할 수 있습니다 생각합니다.

Watch this Scientific Journal Video about Electrophysiology of Scorpion Peg Sensilla at JoVE.com

Electrophysiology of Scorpion Peg Sensilla

이 문서는 광유에 따라 세포, 팁 - 녹음을 통해 개별 sensilla에 화학 자극을 분리에 대한 electrophysiological 방법을 설명합니다.

전갈 페그 Sensilla의 전기 생리학

We describe a modification of an existing tip-recording technique1,2 for electrophysiologically investigating short, peg-like sensory sensilla3,4. On the ...

electrophysiology-of-scorpion-peg-sensilla

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.0K 조회수.  University of Oklahoma. 이 문서는 광유에 따라 세포, 팁 - 녹음을 통해 개별 sensilla에 화학 자극을 분리에 대한 electrophysiological 방법을 설명합니다.

비디오: Electrophysiology of Scorpion Peg Sensilla

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

쥐 두뇌에 동시 fMRI와 전기 생리학

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

연구

신경과학

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

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 ...

Culturing and Electrophysiology of Cells on NRCC Patch-clamp Chips

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard of electrophysiology applied to disease models and pharmaceutical screens alike 1. The method traditionally involves gently contacting a cell with a glass pipette filled by a physiological solution in order to isolate a patch of the membrane under its apex 2. An electrode inserted in the pipette captures ion-channel activity within the membrane patch or, when ruptured, for the whole cell. In the last decade, patch-clamp chips have been proposed as an alternative 3, 4: a suspended film separates the physiological medium from the culture medium, and an aperture microfabricated in the film replaces the apex of the pipette. Patch-clamp chips have been integrated in automated systems and commercialized for high-throughput screening 5. To increase throughput, they include the fluidic delivery of cells from suspension, their positioning on the aperture by suction, and automated routines to detect cell-to-probe seals and enter into whole cell mode. We have reported on the fabrication of a silicon patch-clamp chip with optimized impedance and orifice shape that permits the high-quality recording of action potentials in cultured snail neurons 6; recently, we have also reported progress towards interrogating mammalian neurons 7. Our patch-clamp chips are fabricated at the Canadian Photonics Fabrication Centre 8, a commercial foundry, and are available in large series. We are eager to engage in collaborations with electrophysiologists to validate the use of the NRCC technology in different models. The chips are used according to the general scheme represented in Figure 1: the silicon chip is at the bottom of a Plexiglas culture vial and the back of the aperture is connected to a subterranean channel fitted with tubes at either end of the package. Cells are cultured in the vial and the cell on top of the probe is monitored by a measuring electrode inserted in the channel.The two outside fluidic ports facilitate solution exchange with minimal disturbance to the cell; this is an advantage compared to glass pipettes for intracellular perfusion.
<img alt="Figure 1" src="/files/ftp_upload/3288/3288fig1.jpg" />
Figure 1. Principle of measurement using the NRCC patch-clamp chip
We detail here the protocols to sterilize and prime the chips, load them with medium, plate them with cells, and finally use them for electrophysiological recordings.

We show how planar patch-clamp chips fabricated at the National Research Council of Canada are sterilized, primed, loaded with medium, plated with cells, and used for electrophysiological recordings.

NRCC 패치 - 클램프 칩에 대한 세포의 Culturing 및 전기 생리학

Due to its exquisite sensitivity and the ability to monitor and control individual cells at the level of ion channels, patch-clamping is the gold standard ...

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

를 사용하여 화합물 Bioactivity위한 이점 Nanoinjection 및 화면에 대한 전기 생리학 분석 Drosophila melanogaster 거대 섬유 시스템

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

An Isolated Semi-intact Preparation of the Mouse Vestibular Sensory Epithelium for Electrophysiology and High-resolution Two-photon Microscopy

Understanding vestibular hair cells function under normal conditions, or how trauma, disease, and aging disrupt this function is a vital step in the development of preventative approaches and/or novel therapeutic strategies. However, the majority of studies looking at abnormal vestibular function have not been at the cellular level but focused primarily on behavioral assays of vestibular dysfunction such as gait analyses and vestibulo-ocular reflex performance. While this work has yielded valuable data about what happens when things go wrong, little information is gleaned regarding the underlying causes of dysfunction. Of the studies that focus on the cellular and subcellular processes that underlie vestibular function, most have relied on acutely isolated hair cells, devoid of their synaptic connections and supporting cell environment. Therefore, a major technical challenge has been access to the exquisitely sensitive vestibular hair cells in a preparation that is least disrupted, physiologically. Here we demonstrate a semi-intact preparation of the mouse vestibular sensory epithelium that retains the local micro-environment including hair cell/primary afferent complexes.

Analysis of vestibular hair cell function is complicated by their location deep within the hardest part of the skull, the petrous temporal bone. Most functional hair cell studies have used acutely isolated hair cells. Here we describe a semi-intact preparation of mouse vestibular epithelium for electrophysiological and two-photon microscopy studies.

전기 생리학 및 고해상도 두 광자 현미경을위한 마우스 전정 감각 상피의 격리 된 세미 그대로 준비

Understanding vestibular hair cells function  under normal conditions, or how trauma, disease, and aging disrupt this  function is a vital step in the ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

에서 전기 생리학 기록<em&gt; 초파리</em&gt; Labellar는 Sensilla 맛

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Real-time Electrophysiology: Using Closed-loop Protocols to Probe Neuronal Dynamics and Beyond

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where the stimulus applied depends in real-time on the response of the system. Recent applications range from the implementation of virtual reality systems for studying motor responses both in mice1 and in zebrafish2, to control of seizures following cortical stroke using optogenetics3. A key advantage of closed-loop techniques resides in the capability of probing higher dimensional properties that are not directly accessible or that depend on multiple variables, such as neuronal excitability4 and reliability, while at the same time maximizing the experimental throughput. In this contribution and in the context of cellular electrophysiology, we describe how to apply a variety of closed-loop protocols to the study of the response properties of pyramidal cortical neurons, recorded intracellularly with the patch clamp technique in acute brain slices from the somatosensory cortex of juvenile rats. As no commercially available or open source software provides all the features required for efficiently performing the experiments described here, a new software toolbox called LCG5 was developed, whose modular structure maximizes reuse of computer code and facilitates the implementation of novel experimental paradigms. Stimulation waveforms are specified using a compact meta-description and full experimental protocols are described in text-based configuration files. Additionally, LCG has a command-line interface that is suited for repetition of trials and automation of experimental protocols.

Closed-loop protocols are becoming increasingly widespread in modern day electrophysiology. We present a simple, versatile and inexpensive way to perform complex electrophysiological protocols in cortical pyramidal neurons in vitro, using a desktop computer and a digital acquisition board.

실시간 전기 생리학 : 프로브 신경 역학과 저쪽​​에 폐쇄 루프 프로토콜을 사용하여

Experimental neuroscience is witnessing an increased interest in the development and application of novel and often complex, closed-loop protocols, where ...

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 ...

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 ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the experimental time, and increases the number of animals that are utilized per study. This limitation specifically impacts protocols such as calcium imaging that require prolonged pre-incubation with bath-applied dyes. Exponential bacterial growth within 3 - 4 h after slicing is tightly correlated with a decrease in tissue health. This study describes a method for&#160;limiting the proliferation of bacteria in acute preparations to maintain viable neuronal tissue for prolonged periods of time (&#62;24 h) without the need for antibiotics, sterile procedures, or tissue culture media containing growth factors. By cycling the extracellular fluid through UV irradiation and keeping the tissue in a custom holding chamber at 15 - 16 &#176;C, the tissue shows no difference in electrophysiological properties, or calcium signaling through intracellular calcium dyes at &#62;24 h postdissection. These methods will not only extend experimental time for those using acute neuronal tissue, but will reduce the number of animals required to complete experimental goals, and will set a gold standard for acute neuronal tissue incubation.

Once removed from the body, neuronal tissue is greatly affected by environmental conditions, leading to eventual degradation of the tissue after 6 - 8 h. Using a unique incubation method, which closely monitors and regulates the extracellular environment of the tissue, tissue viability can be significantly extended for &#62;24 h.

전기 생리학 및 칼슘 이미징에 대한 급성 신경 조직의 장기간 배양

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

전갈 페그 Sensilla의 전기 생리학

요약

더 많은 비디오 탐색

이 비디오의 챕터

쥐 두뇌에 동시 fMRI와 전기 생리학

척수 전기 생리학 II : 세포 흡입 전극 제작

NRCC 패치 - 클램프 칩에 대한 세포의 Culturing 및 전기 생리학

를 사용하여 화합물 Bioactivity위한 이점 Nanoinjection 및 화면에 대한 전기 생리학 분석 Drosophila melanogaster 거대 섬유 시스템

전기 생리학 및 고해상도 두 광자 현미경을위한 마우스 전정 감각 상피의 격리 된 세미 그대로 준비

에서 전기 생리학 기록

실시간 전기 생리학 : 프로브 신경 역학과 저쪽에 폐쇄 루프 프로토콜을 사용하여

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

다중 전극 배열에 나선 신경절 신경 이식편 문화 및 전기 생리학

전기 생리학 및 칼슘 이미징에 대한 급성 신경 조직의 장기간 배양

전갈 페그 Sensilla의 전기 생리학

요약

더 많은 비디오 탐색

이 비디오의 챕터

쥐 두뇌에 동시 fMRI와 전기 생리학

척수 전기 생리학 II : 세포 흡입 전극 제작

NRCC 패치 - 클램프 칩에 대한 세포의 Culturing 및 전기 생리학

를 사용하여 화합물 Bioactivity위한 이점 Nanoinjection 및 화면에 대한 전기 생리학 분석 Drosophila melanogaster 거대 섬유 시스템

전기 생리학 및 고해상도 두 광자 현미경을위한 마우스 전정 감각 상피의 격리 된 세미 그대로 준비

에서 전기 생리학 기록

실시간 전기 생리학 : 프로브 신경 역학과 저쪽​​에 폐쇄 루프 프로토콜을 사용하여

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

다중 전극 배열에 나선 신경절 신경 이식편 문화 및 전기 생리학

전기 생리학 및 칼슘 이미징에 대한 급성 신경 조직의 장기간 배양

실시간 전기 생리학 : 프로브 신경 역학과 저쪽에 폐쇄 루프 프로토콜을 사용하여