TK에 G2205 (과학 진흥을 위해 국립 과학 재단 (BAC에 IOS-1050701), 건강의 국립 연구소 (SM에 NS54174과 AML-W에 F30DC0111907.), 우에하라 기념 재단과 일본 사회에 의해 제공하는 자금 조달 ). 우리는 세포 축삭 녹화의 주제에 대한 자신의 지원과지도를 위해 율리우스 믹스 감사합니다. 우리는 프로토 기록 챔버를 제공하기 위해 칼 홉킨스 감사합니다.

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관심 없음 충돌 선언하지 않습니다.

일단 마스터,이 기술 한 여러 모델 시스템에서 생체 기록에 대한 개별 축삭 등의 확인 뉴런, 대상 수있게됩니다. 또한,이 기술은 하나의 안정적 도전 생체 녹화 방법에서 전통적인 만드는 독특한 해부학 적 특성을 가진 뉴런의 스파이크 출력을 기록 할 수 있습니다. 우리는 mormyrid 약하게 전기 물고기 ELA &quot;작은 세포&quot;에서 기록하려면이 기술을 활용했다. &quot;작은 세포&quot;의 튜닝 특성을 연구하는 이전 시도는 어려운 촬영 조건 11, 14로 인해 실패했다. 비슷한 해부학 적 특징은 여러 가지 척추 동물의 청각 electrosensory 신경 8-10에서 단일 단위 기록을 얻기 장벽을 만들 수 있습니다. 우리의 시스템에서 이러한 문제를 극복하기 위해, 우리는 &quot;작은 세포가&quot;그 ELA에서 유일한 세포가 있다는 사실을 이용했다 ELP하는 프로젝트입니다. 따라서, ELP에 ​​배치 염료의 역행 전송 &quot;작은에 ELA의 표시를 제한셀 &quot;somas와 축삭. 축삭의 형광 라벨에 액세스 할 somas에도 불구하고 가능한 확인 된 세포에서 단일 단위 녹음을하고, 레이블 축삭 옆에 정확한 전극의 배치를 허용했다. 우리는 주변 침몰 시냅스 11에 의해 아마, 체세포 녹음을 시도했지만 실패했다 17. 그러나 체세포 라벨은 분명히이 기술은 다른 세포 유형과 다른 회로의 체세포 녹음을 대상으로 사용될 수 있다고 제안 볼 수 있었다. 생체 내에서 역행 전송을 통해 신경 세포의 형광 라벨이 생체 19-21 표적으로 녹음을 안내하는 데 사용되었습니다 . 유사한 기술은 제브라 피쉬의 척수 22 모터 뉴런의 생체 녹음에서 대상을 위해 사용되었다. 우리의 작업 표시와 기록이 모두 뇌에서 생체 내에서 수행되는이 방법의 새로운 확장을 나타냅니다. 우리의 방법을 보여주는 생체 내 역행 T로 CNS 영역의 레이블레이서는 유사 선택적 투사 뉴런과 다른 그대로 회로의 연구에 확장 할 수 있습니다. 예를 들어, 포유류의 청각 처리, 하부 둔덕 (IC)는 여러 rhombencephalic 구조 23 입력의 중요한 중계 센터의 역할을합니다. IC에 염료를 주입 선택적으로 이러한 핵에서 각각 투사 세포를 레이블합니다. 우수한 둔덕 (SC)는 비전 24 유사한 기능을 제공합니다. 척수 준비는 특히 척수 쉽게 액세스로, 염료 주입까지 녹화 현장에서 발생할 수 있습니다,이 기술에 적합, 그것은 더 자세한 해부학 적 정보에게 25를 획득하도록 선택 뉴런의 세포 내 기록 및 작성과 결합 할 수 있습니다. 마지막으로, 요로-추적 복잡한 회로에게 26 매핑하는 중추 신경계 전반에 걸쳐 사용 잘 설립 된 기술입니다. 우리의 방법은 w를 수행 한 이러한 연구에 대한 기능 정보를 추가하기 위하여 이용 될 수있다고양이 시각 피질 27 i 번째 칼슘에 민감한 지표. 잘 설립 된 수술은, 안정적이고 정기적으로 생체 녹음 16 장님에 사용되는 최소한의 출혈과 동물과 생존을 위해 조직을 허용하는 두뇌의 표면에 손상없이 완료해야합니다. 연습으로, 수술 및 염료 응용 프로그램 30~45분에 완료 할 수 있습니다. 우리는 성공적으로 준비 67 %의 &quot;작은 셀&quot;축삭 표시. 대부분의 준비는 1 개 또는 2 개 표시 레이블 축삭을 가지고 있지만, 일부는 많은 8로되어 있습니다. 시도 119 표시 단위, 우리는 12 준비 (표 2)에 분산 된 26 대에서 단일 단위 기록을 얻었다. 따라서, 데이터는 28 %의 성공률에 대한 레이블 축삭과 준비의 41 %에서 수집되었다. 염료 응용 프로그램의 중요한 측면은 깊이 라벨입니다. 텅스텐 와이어의 얕은 삽입 세척 아우되는 염료가 발생합니다AY. 침투가 너무 깊은 경우, 레이블 축삭 타겟팅 표시되지 않습니다. 또한, 세포에 약간의 기계적 손상 적절하게 25, 28까지 수행 할 염료 발생해야합니다. 그러나 너무 많은 손상은 세포를 죽일 것입니다. 우리는 ELP에 표시된 축삭 (표 3) 기록과 짝 ELA에 염료를 주입을 통해 선행 성 라벨 등 다른 염료 및 다른 방법 (표 2)로 라벨을 시도했다. 우리는 라벨이 그들에게 응답하고, 신체적 손상과 세포에 국한 되었기 때문에 선행 성 라벨이 성공적으로 완료되지 않은 가설. 또한, 사전 시냅스 터미널이 방해되었을 수 있습니다. 대조적으로, 역행 표지는 이러한 문제를 모두 최소화합니다. 금액과 염료 응용 프로그램의 위치를​​ 연구하는 특정 회로에 따라 변경 될 수 있습니다. 최대 라벨은 우리가 코팅 된 텅스텐 와이어를 사용하여 달성 큰 염료 농도가 발생합니다. 그러나 드와 축삭 레이블을그것은 진보로 경과 예측, 염료 텅스텐 바늘을 올 수 있습니다. 이러한 경우, 압력 주입 더 적합 할 것이다. 염료 흡수 및 수송 2 시간 주입 후 6 시간 후 주사로 늦게 나타나는 레이블이있는 추가 축삭 빠르면으로 표시되는 우리의 준비에 표시된 축삭과 급속한 있습니다. 따라서, 표시 및 기록은 생존 수술과 관련된 기술적 인 문제를 제거, 하루에 수행 할 수 있습니다. 타이밍 염료 전송에 필요한 거리에 따라 각 응용 프로그램에 따라 다를 것입니다. 또 다른 중요한 측면은 전극 배치됩니다. 이 팁의 막힘 방지하기 위해 레이블 축삭의 사이트에 조직 가까이를 입력하는 것이 중요합니다. 같은 ELA에서와 같이 밀도가 조직의 경우, 기록 전극에 긴 얇은 정강이 주변 조직의 과도한 움직임을 최소화합니다. 성공적으로 녹음 기록을 얻을 때까지 신선한 전극 첫 번째 시도, 반복에 달성하거나, 담배 마는되지 않는 경우UE는 축삭이 더 이상 표시되는 시점에 중단됩니다. 그러나 신속하게 표백 광독성을 일으킬 수 형광 노출의 양을 최소화하고 세포 29-31의 생리적 특성에 영향을 미칠 수있는 축삭 옆에 전극을 배치하는 것이 중요하다. 축삭의 세그먼트가 성공적으로 기록 전극에 흡입되면 녹음은 몇 시간 동안 얻을 수 있습니다. 단위는 일관되게 1 시간 미만에 분실되는 경우에, 밖으로 미끄러지는 축삭을 방지하기 위해 작은 전극의 끝 부분을 만드는 것이 좋습니다. 반면에, 팁이 너무 작은 축삭에 막힘 낮은 신호 대 잡음이나 손상을 초래할 수 있습니다. 스파이크 진폭과 추가 흡인 장치의 &#39;리턴&#39;의 꾸준한 감소는 끝이 너무 크다는 표시입니다. 너무 많은 흡입 축삭에 치명적인 손상을 줄 수 있습니다. 하나의 솔루션은 압력이 천천히 제로 반환되도록 공기 라인의 작은 누출을 허용하는 것입니다. 빠르게 일 동점 골전자 압력은 축삭을 추방 될 수있는 일시적인 상대적으로 안쪽에서 바깥쪽으로 &#39;푸시&#39;가 발생합니다. 이 기술은 확인 투사 뉴런에서 대상 레코딩을 얻기위한 중요한 이점을 나타냅니다 있지만 염료를 주입 사이트에있는 모든 세포 유형에 의해 촬영 될 것이라고, 그것은 지역의 interneurons을 구별하는 데 유용하지 않습니다. 이론적으로, 별도의 주사 부위 여러 ​​형광 물질의 사용이 메서드가 확장 할 수 있습니다. 예를 들어, 단일 대 이중 라벨의 비교 회로에 32 개의 지점에서 이중 주사를 따라 투영 뉴런에서의 interneurons을 구별하는 데 사용할 수 있습니다. 마찬가지로, 역행 추적자는 얼룩말 핀치 높은 보컬 센터 (HVC) 32 최근에 수행 된 같은 두 광자 이미징과 같은 다른 고급 이미징 기법과 결합 할 수 있습니다. epifluorescence에 현미경을 사용하는 경우 또한, 녹화 영역은 우리가 단지 &amp; 1를 해결 할 수 있었다로서, 표면 근처로 제한됩니다무, 조직의 처음 30 μm의에서 M 구조. 그러나이 깊이는 이러한 두 광자 현미경 33 또는 목적 결합 평면 조명 현미경 34와 같은 다른 현미경 기술의 사용을 통해 확장 할 수 있습니다. 상대적으로 접근 할 수없는 것을 포함하여 - 그것은 모델 시스템의 다양한 여러 회로에서 하나의 뉴런에서 기록하는 데 사용할 수 있기 때문에 전반적으로,이 기술은 생체 내에서 신경 회로의 연구에 중요한 진보를 나타냅니다.

<table border="1" cellspacing="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Tricaine Methanesulfonate</td>
 <td>Sigma-Aldrich</td>
 <td>E10521</td>
 <td>MS-222</td>
 </tr>
 <tr>
 <td>Gallamine Triethiodide </td>
 <td>Sigma-Aldrich</td>
 <td>G8134</td>
 <td>Flaxedil</td>
 </tr>
 <tr>
 <td>Lidocaine Hydrochloride</td>
 <td>Sigma-Aldrich</td>
 <td>L5647</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hickman's Ringer Solution </td>
 <td>NA</td>
 <td>NA</td>
 <td>NaCl (6.48 g/l), KCl (0.15 g/l), CaCl2&bull;2H20 (0.29 g/l), MgSO4 (0.12 g/l), NaHCO3 (0.084 g/l), NaH2PO4 (0.06 g/l)</td>
 </tr>
 <tr>
 <td>Peristaltic Pump</td>
 <td>Gilson or Rainin</td>
 <td>Minipulse 3 Model 312; RP-1</td>
 <td>10.0 &plusmn; 1.0 rpm, Alternatively, a gravity-fed line with a flow-meter</td>
 </tr>
 <tr>
 <td>Dissection Microscope </td>
 <td>Nikon</td>
 <td>SM2645</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Super Glue</td>
 <td>Super Glue Corporation</td>
 <td>SGH2</td>
 <td>2g tube</td>
 </tr>
 <tr>
 <td>Omni Drill 35</td>
 <td>World Precision Instruments</td>
 <td>503-598</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ball Mill Carbide Drill Bit #1/4</td>
 <td>World Precision Instruments</td>
 <td>501860</td>
 <td>0.19 DIA (bit diameter = 0.48 mm) </td>
 </tr>
 <tr>
 <td>Low Temp Cautery Kit</td>
 <td>World Precision Instruments</td>
 <td>500391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Manual Micromanipulator</td>
 <td>World Precision Instruments</td>
 <td>M3301R</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dextran Conjugated Alexa Fluor 568</td>
 <td>Invitrogen</td>
 <td>D-22912</td>
 <td>2mM concentration; 10,000MW The choice of dye wavelength should be selected to match the microscope filter settings</td>
 </tr>
 <tr>
 <td>Epifluorescent Microscope</td>
 <td>Nikon </td>
 <td>E600 FN</td>
 <td>A remote focus accessory is helpful for fine focus TRITC filter - or appropriate filter to match focal planes for different dye wavelengths</td>
 </tr>
 <tr>
 <td>Low Power Objective</td>
 <td>Nikon</td>
 <td>93182</td>
 <td>CFI Plan Achromat Series, 4X N.A. 0.1, W.D. 30mm</td>
 </tr>
 <tr>
 <td>High Power Objective</td>
 <td>Nikon</td>
 <td>93148</td>
 <td>CFI Fluor Series, 40X WI N.A. 0.8, W.D. 2.0 mm</td>
 </tr>
 <tr>
 <td>White Light Source</td>
 <td>Dolan-Jenner</td>
 <td>Model 190</td>
 <td>Fiber Optic Illuminator</td>
 </tr>
 <tr>
 <td>Fluorescent Light Source </td>
 <td>Lumen Dynamics</td>
 <td>X-Cite 120Q</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Low-light Level Camera</td>
 <td>Photometrics</td>
 <td>CoolSnap ES</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Digital Manometer</td>
 <td>Omega Engineering</td>
 <td>HHP-201</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Motorized Micromanipulator</td>
 <td>Sutter Instruments</td>
 <td>MP-285</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Headstage</td>
 <td>Molecular Devices</td>
 <td>CV-7B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Amplifier</td>
 <td>Molecular Devices</td>
 <td>MultiClamp 700B</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Data Acquisition System</td>
 <td>Molecular Devices</td>
 <td>Digidata 1322A</td>
 <td>Axon Instruments</td>
 </tr>
 <tr>
 <td>Isolated Pulse Stimulator</td>
 <td>A-M Systems</td>
 <td>2100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Flaming/Brown Micropipette Puller</td>
 <td>Sutter Instruments</td>
 <td>P-97</td>
 <td>Program settings for our application: Heat: ramp + 1, Pull: '0', Velocity: 60, Time: 90 Box filament</td>
 </tr>
 <tr>
 <td>Pipette Glass</td>
 <td>World Precision Instruments</td>
 <td>1B100F-4</td>
 <td>Borosilicate capillary glass with filament (1 mm OD, 0.58 mm ID) </td>
 </tr>
 </tbody>
</table>

retrograde fluorescent labeling allows for targeted extracellular single-unit recording from identified neurons in vivo

이 방법의 전반적인 목표는 뉴런의 확인 인구에서 하나의 단위 반응을 기록하는 것입니다. 개별 뉴런의 생체 전기 생리학 녹음에서 신경 회로는 자연 조건 하에서 작동 방법을 이해하기위한 중요합니다. 전통적으로,이 기록은 기록 된 셀의 ID가 녹음 시작시 알 수없는 의미 &#39;블라인드&#39;수행되었다. 세포 신원은 이후 세포 juxtacellular 1, 2, 느슨한 패치 염료의 3 이온 도입을 통해 확인할 수 있지만,이 기록은 기능적으로 서로 다른 세포 유형과 지역의 특정 신경 세포에 사전에 대상이 될 수 없습니다. 형광 단백질은 시각적으로 유도 한 세포 전기 생리학 4-6을 허용 셀 타입 특정 방식으로 표현 될 수 있습니다. 그러나 이러한 유전 적 도구를 사용할 수 없습니다하는 여러 모델 시스템이 있습니다. 심지어 유전자 접근 모델 시스템에서 원하는 프로moter를 알 수있을 수 있습니다 또는 유 전적으로 균일 한 신경 돌기 패턴을 다양한있을 수 있습니다. 마찬가지로, 바이러스 성 벡터를 투사 뉴런 7의 특정 하위 레이블로 사용되었지만,이 방법의 사용은 횡단 시냅스 특이 독성과 부족에 의해 제한됩니다. 따라서, 생체 내에서 확인 된 하나의 뉴런에서 기록하는 특정 사전 시각화를 제공하는 추가적인 기술이 필요합니다. 대상 신경 세포의 사전 시각화 고전 단일 셀 녹음은 종종 8-11 엄청나게 어려운하는 어려운 촬영 조건에 특히 유용합니다. 이 문서에 설명 된 새로운 기술은이를 대상으로 전기 생리학 녹음을 얻기 위해 시각적으로 제공하는 신속하고 선택적으로 그들의 독특한 축삭 돌기를 기반으로 특정 뇌 영역 내에서 세포의 특정 부분 집합에 레이블을 텅스텐 바늘을 사용하여 형광 염료의 역행 전송을 적용된 사용 그대로 회로 withi에서 확인 뉴런NA는 CNS 척추. 우리의 방법의 가장 중요한 새로운 발전이 아닌 유전자 접근 모델 시스템의 특정 세포 유형을 대상으로 형광 라벨의 사용이다. 약한 전기 물고기 동물 12 행동, 깨어있는 신경 회로를 공부를위한 훌륭한 모델 시스템입니다. 우리는 약한 전기 mormyrid 물고기의 전방 exterolateral 핵에서 &quot;작은 세포&quot;(ELA)의 감각 처리를 연구하는이 기술을 활용. &quot;작은 세포는&quot;연접 스파이크 (13)의 도착 시간의 밀리 초 이하의 차이를 검출하기위한 중요한 시간을 비교 뉴런으로 가설되어 있습니다. 그러나, 밀도 수초, 침몰 시냅스 작은 세포 기관으로 해부학 적 기능은 매우 어려운 전통적인 방법 11, 14를 사용하여 이러한 세포에서 기록을 만들었습니다. 여기에 우리가 신뢰할 수있는 강력한 레코딩 및 characte을 허용, 우리의 새로운 방법은 선택적 준비의 28 %에서 이러한 세포를 레이블 입증electrosensory 자극에 대한 응답 rization.

Watch this Scientific Journal Video about Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo at JoVE.com

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

형광 염료의 역행 전송 해부학 적 투사에 따라 신경 세포의 하위 인구 레이블. 레이블 축삭은 시각적 대상이 될 수 있습니다<em&gt; 생체 내</em&gt; 확인, 축색 돌기에서 세포 녹음을 허용. 이 기술은 신경 세포가 유전자 조작을 통해 표시하거나 분리하기 어려운 할 수없는 경우 &#39;블라인드&#39;를 사용하여 녹화를 촉진<em&gt; 생체 내</em&gt; 방법.

역행 형광 라벨이 확인 된 신경 세포의 표적 세포 외 단일 단위 기록을 허용<em&gt; 생체 내</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

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JoVE Journal

Neuroscience

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

19.8K 조회수.  Washington University in St. Louis. 형광 염료의 역행 전송 해부학 적 투사에 따라 신경 세포의 하위 인구 레이블. 레이블 축삭은 시각적 대상이 될 수 있습니다 생체 내 확인, 축색 돌기에서 세포 녹음을 허용. 이 기술은 신경 세포가 유전자 조작을 통해 표시하거나 분리하기 어려운 할 수없는 경우 '블라인드'를 사용하여 녹화를 촉진 생체 내 방법.

비디오: Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).
Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.
The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.

Dual Somatic Recordings from Gonadotropin-Releasing Hormone GnRH Neurons Identified by Green Fluorescent Protein GFP in Hypothalamic Slices

Activity in neuronal systems often requires synchronous action potential discharges from neurons within a specific population. For example, pulses of gonadotropin-releasing hormone (GnRH) likely require coordinated activity between GnRH neurons. We present our methodological approach for reliably obtaining simultaneous electrophysiological recordings from the diffusely distributed GnRH neurons.

에서 듀얼 레코딩 체세포 생식 샘 자극 호르몬 - 방출 Hypothalamic 조각에 녹색 형광 단백질 (GFP)에 의해 확인된 호르몬 (GnRH) 신경을

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating ...

연구

신경과학

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic nerve. In vivo recordings of ganglion cell spike trains in several animal models have revealed much of what is known about how the early visual system processes and encodes visual information. However, such recordings have been rare in one of the most common animal models, the rat, possibly owing to difficulty in detecting spikes fired by small diameter axons. The many retinal disease models involving rats motivate a need for characterizing the functional properties of ganglion cells without disturbing the eye, as with intraocular or in vitro recordings. Here, we demonstrate a method for recording ganglion cell spike trains from the optic chiasm of the anesthetized rat. We first show how to fabricate tungsten-in-glass electrodes that can pick up electrical activity from single ganglion cell axons in rat. The electrodes outperform all commercial ones that we have tried. We then illustrate our custom-designed stereotaxic system for in vivo visual neurophysiology experiments and our procedures for animal preparation and reliable and stable electrode placement in the optic chiasm.

Single-unit In vivo Recordings from the Optic Chiasm of Rat

Retinal ganglion cells transmit visual information from the eye to the brain with sequences of action potentials. Here, we demonstrate how to record the action potentials of single ganglion cells in vivo from anesthetized rats.

단일 유닛 생체내에서</em암흑의 광학 Chiasm에서> 레코딩

Information about the visual world is transmitted to the brain in sequences of action potentials in retinal ganglion cell axons that make up the optic ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

단일 유닛 활동과 Electrocorticographic 신호를 녹음하기 위해 만성 신경 전극 외과 이식

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

확인된 뉴런과 먹이 행동에서 칼슘 신호의 동시 녹화 Drosophila melanogaster</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

신경, 책자, 그리고 형광 염료와 척수 뿌리 역행로드

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

형질 전환 생선의 본래 뇌에서 발견 뉴런의 전기적 활동을 기록

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of neurophysiology, neuroethology, and developmental biology. The goal of this report is to consolidate experimental techniques from the leech system into a single article that will be of use to physiologists with expertise in other nervous system preparations, or to biology students with little or no electrophysiology experience. We demonstrate how to dissect the leech for recording intracellularly from identified neural circuits in the ganglion. Next we show how individual cells of known function can be removed from the ganglion to be cultured in a Petri dish, and how to record from those neurons in culture. Then we demonstrate how to prepare a patch of innervated skin to be used for mapping sensory or motor fields. These leech preparations are still widely used to address basic electrical properties of neural networks, behavior, synaptogenesis, and development. They are also an appropriate training module for neuroscience or physiology teaching laboratories.

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, Hirudo medicinalis

This article describes three nervous system preparations using leeches: intracellular recording from neurons in ventral ganglia, culturing neurons from ventral ganglia, and recording from a patch of innervated skin to map sensory fields.

세포 내 기록, 감각이 필드를 매핑하고, 거머리의 배양 확인 된 뉴런, Hirudo의 medicinalis</i

The freshwater leech, Hirudo medicinalis, is a versatile model organism that has been used to address scientific questions in the fields of ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

형광 염료와 성인 Zebrafish의 망막 신경절 세포의 퇴행성 라벨링

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

에 가이드<em&gt; 생체</emOptogenetically 확인 된 대뇌 피질의 억제의 interneurons에서&gt; 단일 단위 기록

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of olfactory-driven behaviors and their modulation. These coding properties depend heavily on the initial interaction between odor molecules and the olfactory receptor (OR) expressed in the OSNs. The identity, specificity and ligand spectrum of the expressed OR are critical. The probability to find the ligand of the OR expressed in an OSN chosen randomly within the epithelium is very low. To address this challenge, this protocol uses genetically tagged mice expressing the fluorescent protein GFP under the control of the promoter of defined ORs. OSNs are located in a tight and organized epithelium lining the nasal cavity, with neighboring cells influencing their maturation and function. Here we describe a method to isolate an intact olfactory epithelium and record through patch-clamp recordings the properties of OSNs expressing defined odorant receptors. The protocol allows one to characterize OSN membrane properties while keeping the influence of the neighboring tissue. Analysis of patch-clamp results yields&#160;a precise quantification of ligand/OR interactions, transduction pathways and pharmacology, OSNs&#39; coding properties and their modulation at the membrane level.&#160;

Analyzing the physiological properties of olfactory sensory neurons still faces technical limitations. Here we record them through perforated patch-clamp in an intact preparation of the olfactory epithelium in gene-targeted mice. This technique allows the characterization of membrane properties and responses to specific ligands of neurons expressing defined olfactory receptors.

본래 Neuroepithelium에서 마우스 강한 감각 뉴런의 천공 패치 클램프 녹음 : 확인 된 부 취제 수용체를 발현 뉴런의 기능 분석

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...