Name: electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording
Uploaded: 2015-11-19T13:00:00.000+00:00
Duration: 5 min 28 s
Description: Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children&#8217;s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.

이 방법은 라발 대학 동물 윤리위원회 (프로토콜 # 2,012에서 170 사이)에 의해 허용되는 동물 환자의 사용을 요구했다. 1. 설치 및 준비 <ol><li> 솔루션 <ol><li> 다음과 같은 조리법 7,17에 따라 ACSF 재고 솔루션을 준비합니다. 농도 변화와 다른 조리법은 문학에서 사용할 수 있습니다. 최대 1 개월 4 ℃에서 보관 재고 솔루션을 제공합니다. <ol><li> 염 용액 : 염화나트륨 75.39 g의 (129 mM의 최종)를 추가; 의 KCl 2.5 g (3.35 mM의 최종); NaH2PO4의 0.81 G (0.58 mM의 최종) 의 MgCl2의 2.33 G (1.15 mM의 최종) CaCl2를 1.85 g (1.26 mm)이다. 다음 증류수 1 L에 기입 증류수 800 ㎖에 용해. </li><li> 중탄산염 용액 :의 NaHCO3 (21 mM의 최종)의 17.65 g을 추가합니다. 증류수 1 L에 기입 증류수 900 mL에 녹인다. 중탄산염 농도의 변화는 산도의 변화가 발생합니다. </li><li> 포도당 용액 : 54.06 추가포도당의 G (30 mM의 최종). 다음 증류수 500㎖의 증류수로 채워 400 ml의 용해. </li></ol></li><li> 염 용액 100 ㎖, 중탄산 용액 100 ㎖, 증류수 1 L의 글루코스 용액 50 mL로 희석하여 준비 ACSF. 사용까지 4 ℃에서 보관. </li><li> 가온하고 휴식까지 유리관을 연신하여 유리 전극을 준비한다. 사용하기 전에 팁 아래로 모래. 전극은 오랫동안 청결 유지로 많은 시간을 재사용 할 수 있습니다. </li></ol></li><li> 실험 설정 참고 : 설정 세부 사항은 그림 2에 제시되어있다. <ol><li> 평균 기, 데이터 수집 시스템, 온도 컨트롤러와 펌프를 이동, 앰프의 전원을 켭니다. 및 원시 신호 (2.5 kHz로)의 아날로그 디지털 변환의 샘플링 속도는 신호 증폭 (이득 = 10,000), 여과 (높은 문턱 값, 5 kHz의 낮은 임계 값, 10 Hz로)를 확인한다. </li><li> ACSF과 carbogen (95 % O 2와 거품을 가진 병 채우기 </suB&gt;, 5 % CO 2). / 분에서 4 ml의 기록 챔버 (부피 5 ㎖) 및 가온 bubbled--ACSF 흐름을 유도한다. 26 ° C에서 기록 챔버 내 온도 (± 1)을 잡아. pH는이 표준 조건에서 7.4이어야한다. </li><li> 해부 실 근처 50 ML의 주사기에 RT와 carbogen - 버블 ACSF 50 ㎖를 유지합니다. </li><li> 컴퓨터를 켜고 레코딩 소프트웨어를 시작합니다. </li></ol></li></ol> 2. 해부 <ol><li> 무게를 시각적으로 동물의 성별을 결정 (여성 짧은 항문 생식기의 거리를 반면 남성, 긴 항문 생식기의 거리를 가지고, 여성 성기 핑크 동안 성기 종종 어두운 수컷). </li><li> 다음 옵션 중 하나를 사용하여 신생아 쥐를 마취 : cryoanesthesia을 (완전히 4 얼음 동물을 담그지 - 5 분 18) (4 ㎖ / ㎏에서 equitensine), 주입 휘발성 마취제 (이소 플루 란 (20) 또는 에테르 21)의 19 또는 흡입을. 눌러 확인RM 발 철수 반사의 부재에 의한 마취의 적절한 비행기. </li><li> 아래 벤치에 동물, 복부 얼굴을 놓습니다. 제 (피부와 두개골을 통해 볼 수) 브레 그마 수준에서 메스와 coronally 머리의 주동이의 부분. 동물이 마취 후 즉시이 단계를 수행합니다. </li><li> 제 몸 coronally 전방 회원에서 메스와. </li><li> 수술 얇은 가위와 집게와 피부, 근육, 지방 조직 및 viscus를 제거합니다. 뼈가이 나이에 부드러운 때문에, 신경 시스템에 아무런 영향을 미치지에주의. 벤치에서이 단계를 수행합니다. </li><li> 해부 실의 준비를 놓습니다. 준비 산소를 위해 주사기에 저장된 ACSF를 사용합니다. 기록의 끝이 시점에서 현미경을 사용합니다. </li><li> 준비 등의면에서 수술 얇은 가위와 집게와 매체 축을 따라 꼬리 부분에 주동이의 두개골과는 척추를 잘라. 절단 된 두개골을 열고그리고 순서는 척추가 신경 조직을 노출. 준비 산소를 위해 주사기에 strored ACSF를 사용합니다. </li><li> 펜치로, 거미 막, 신경 조직의 표면을 덮는 얇은 조직을 제거합니다. 신경 조직에 대한 피아 교인과 혈관을 유지. 준비 산소를 위해 주사기에 저장된 ACSF를 사용합니다. </li><li> 지느러미와 준비가 아래로 향하게 조심스럽게 자리에 준비를 잡고 가위로 신경과 결합 조직을 절단하여 뇌간과 척수를 가져 놓습니다. 등의 방법도 가능합니다. 가능한 한 오랫동안 뿌리 신경을 유지합니다. 뇌간과 척수를 분리하기 위해 뼈를 제거합니다. 준비 산소를 위해 주사기에 저장된 ACSF를 사용합니다. </li><li> 소뇌를 제거하고 메스를 절편으로 주동이의 구조를 나머지. 준비 산소를하기 위해 바늘에 저장된 ACSF를 사용합니다. </li><li> 선택적 experimen에 따라탈 디자인은 열등한 소뇌 동맥 (22)에 관상 부 전방에 의해 메스와 뇌교를 제거합니다. 전체 뇌교를 유지하는 것은 쥐의 리듬을 느리게하고 완벽하게 쥐를 억제하는 것에주의. 뇌교의 꼬리 부분을 포함 할 준비가 C4 루트에서 안정적인 주파수의 호흡과 같은 활동을 표시합니다. </li></ol> 3. 녹음 <ol><li> 기록 챔버, 복부 얼굴까지의 준비를 놓습니다. 척수와 뇌간의 주동이 - 대부분의 가장 낮은 부분에 핀 준비를 수정합니다. </li><li> 부분적 ACSF으로 전극을 채우기 위해, 주사기 피스톤 당겨서 - (225 μm의 150 유리 팁 직경)의 바늘 전극에 연결된 주사기를 이용하여, 전극의 함몰을 유도한다. </li><li> 미세 조작기를 사용하여 조심스럽게 네 번째 복부 뿌리 중 하나에 가까운 전극을 배치합니다. 기타 복부 뿌리는 호흡과 같은 활동 (예를 들어 제시 할 수있다.G., XII 뇌신경, C1 복부 루트). </li><li> 부드럽게 신경 어린 뿌리를 흡인하기 위하여 피스톤을 잡아 당겨 주사기를 통해 전극 탱크 내 우울증을 유도한다. 조심스럽게 척수에 적용하기 위해 전극을 이동합니다. 주의 : 이상적으로 어린 뿌리의 크기가, 전극 개구의 크기와 일치하고, 따라서 전극의 내부 및 외부 사이의 시일 구획을 생성한다. 차동 증폭기는 일반적으로 이러한 기록을 위해 사용되기 때문에, 전극의 내부 챔버와 기록 사이에 개구 된 신호의 품질을 감소시키고 어려운 배경 잡음을 제거 할 수있다. </li><li> 녹화를 시작합니다. </li><li> 정상 산소의 조건 하에서 제조 제조 리듬을 기록 (즉, ACSF carbogen 버블은 95 % O 2를, 5 % CO 2) 적어도 20 분의 제조 기준 파라미터를 결정하기 위해. </li><li> 에 carbogen - 버블 ACSF에서 관류 전환자극 ACSF (즉, 95 % N 2와 5 %의 저산소증 자극에 대한 CO 2와 버블) 15 분. 실험 프로토콜의 따라 노출 시간을 변경합니다. 기록과 동물 시트에 노출 지속 시간을 기록한다. ACSF 병 및 튜브의 외부로의 가스의 확산을 피하기 위해 가능한 챔버 사이에 스테인레스 스틸 튜브를 사용한다. </li><li> 복구 기록을 위해 적어도 15 분 동안 표준 carbogen - 버블 ACSF로 다시 관류를 전환합니다. 기록과 동물의 데이터 시트에서이를 기록한다. </li><li> 녹음을 종료합니다. </li></ol> 4. 통계 분석 <ol><li> 통합 된 신호로부터, (버스트 / 분으로 표현) 분당 버스트들의 개수와 주파수, 기준선 및 (MV 표현됨) 버스트의 피크 사이의 차이와 같은 진폭으로부터 통화 시간, 버스트 지속 시간을 계산 (S 표현됨) 버스트의 끝부터 시작하여 아래 CUR 지역 버스트 영역(MV 표현 · S) 통합 신호 (그림 3)에 버스트했습니다. </li><li> 각 조건에 대한 기록의 마지막 5 분의 평균과 주파수, 진폭, 버스트 기간 및 버스트 영역 정상 산소에서 (베이스 라인), 저산소 (자극)과 후 저산소 (포스트 자극) 복구 조건을 계산합니다. 기록 챔버 내에서 관류 ACSF 균질화 스위칭 사이에 지연이 있기 때문에, 각 상태의 제 1 부분을 분석하지 않는다. </li><li> 해당 기록 용 기준 값의 백분율로 자극 후 (즉, 저산소증) 및 후 자극 (즉, 포스트 - 저산소) 복구 값을 표현한다. SD ± 수단으로서 결과를 표현 </li></ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+five+agents/methods+for+anesthesia+of+neonatal+rats.">Evaluation of five agents/methods for anesthesia of neonatal rats.</a> Lab Anim Sci. 47, 386-395 (1997).</li><li>Formenti, A., Zocchi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Error+signals+as+powerful+stimuli+for+the+operant+conditioning-like+process+of+the+fictive+respiratory+output+in+a+brainstem-spinal+cord+preparation+from+rats.">Error signals as powerful stimuli for the operant conditioning-like process of the fictive respiratory output in a brainstem-spinal cord preparation from rats.</a> Behav Brain Res. 272, 8-15 (2014).</li><li>Bierman, A. M., Tankersley, C. G., Wilson, C. G., Chavez-Valdez, R., Gauda, E. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythmic+bursting+of+pre-+and+post-inspiratory+neurones+during+central+apnoea+in+mature+mice.">Rhythmic bursting of pre- and post-inspiratory neurones during central apnoea in mature mice.</a> J Physiol. 502 (Pt 3), 623-639 (1997).</li><li>Morin-Surun, M. P., Boudinot, E., Kato, F., Foutz, A. 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The authors have no competing financial interests or conflicts of interests to disclose.

호흡 활동의 정확한 정량화가 어려울 수 있습니다. 실제로, 호흡 자동 자발적 모두 일 수 함수이며, 즉, 환경, 인체의 요구, 감정 상태와 행동에 따라 변조된다. 이 기술의 장점은 호흡기 명령을 생성 할 책임 신경 요소의 분리이다. 따라서, 뇌간, 척수 - 제제와 혈량 측정법의 전기 생리 학적 기록은 각각 시험 관내 및 생체 내에서 신경 세포 호흡 전체 네트워크를 연구하는 상보적인 기술이?...

호흡 옥시젠 (O 2) 흡수 및 이산화탄소 (CO 2)를 제거 가능하게 뇌에 의해 제어 복잡하고 생명 활동이다. 중앙 호흡기 드라이브는 모두 포유류 1 뇌간에있는 복잡한 네트워크, 양서류 2, 파충류 3, 조류 4 물고기 (5)에 의해 생성된다. 호흡의 연구는 생체 내에서 처리 할 수없는 경우에도, 정확한 역학적 조사 호흡 제어 네트워크에 직접 액세스를 필요로한다. 이를 위해, 아드리안과 Buytendijk는 뇌간 표면 레코드 아가미 환기 5와 연관된 리듬을 생성하는 전극을 배치 감소 금붕어 제제를 개발 하였다. 이 방법은이어서 신생아 설치류에서 사용하기 위해 1984 년 6 Suzue 의해 채택되었다. 이 제제의 출현은 호흡 신경 생물학에서 중요한 발전을 주도하고있다. 그것은 상대적으로 간단하기 때문에,이 기술은 H를 제시감수 리듬 모터 행동과 신생아 설치류 기원의 기본 조사의 넓은 범위에 의무입니다. 이 방법의 전체 목표는 흡기 신경 활동의 상관 관계를 기록하는 것, 호흡기 같은 호흡 리듬 네트워크에 의해 생성 된 가상의 호흡을했다. 이 방법은 모두 야생형 7 및 8 트랜스 제닉 동물의 호흡 변화 또는 약리학에 흡기 응답들을 타겟팅, 연구 목적의 넓은 범위에서 사용할 수있다. 기록 된 신호의 생리 학적 관련성에 관한 실험 구 심성 감각없이 낮은 온도에서 수행되며, ACSF 내 글루코스와 O 2의 농도가 높은 조건 하에서, 문제가 발생되었음을 주어진다. 생체 내 및 시험 관내 조건 사이의 명확한 차이가 있지만 (예., 흡기 버스트의 주파수는) 사실이 존재한다는 남아호흡 네트워크 (6)의 핵심 요소는 가능한 중요 기능 항상성 9,10- 연결된 강력한 리듬을 연구 할 수 있도록. 이러한 기술 개발의 사용 배후의 이론적 근거는 신생아에서 특히, 생체 내에서 거의 액세스 네트워크의 호흡 뇌간 요소에 직접 액세스를 용이하게하는 것이다. 뇌간은 엄격하게 통제 된 조건 아래에 배치되어 기록 된 리듬이 폐 또는 경동맥의 몸에서 말초 구 심성 입력에 의해 변조되지 않은, 연구 중심 호흡 드라이브 자체 11 일에 집중할 수 있습니다. 따라서, 이러한 액세스는 자극을 적용하고 출력 신호를 기록하기 위해 이용된다. 녹음 혈량 측정법 달리 호흡 리듬 체내 모든 구성 요소에 의해 변조된다 (예., 폐 팽만, 주변 화학 센서)를 어렵게 정확한 자극을 적용 할 수있다. 에서ewborn 쥐, 프로토콜은 인공 뇌 - 척수 (ACSF) 유지 고립 뇌간에 네 번째 복부 루트 신호와 절단 된 척수를 기록으로 구성되어 있습니다. 뇌간 - 척수 제제에 의해 생성 된 리듬 흡기 신호 (9)에 연결되어 각각의 느린 버스트들로 구성된다. 7 - 고립 된 뇌간 - 척수 준비 쉽게 4 (P4 P0)에 산후 일 0에서 쥐에서 촬영 가능한된다. 이 방법은 일반적으로 네트워크의 호흡 저산소 반응하고, 또한 탄산 혈증, 산증 또는 약물에 대한 반응을 평가하는 데 사용된다. 급성 저산소증 프로토콜은 여기에 표시됩니다. 이 자극은 ACSF의 O 2의 인출에 의해 얻어진다; 이 방법은 일반적으로 저산소증 욕설 공차 및 응답 성을 평가하는 데 사용된다. 프로토콜은 저산소증 노출 단부 (도 1)까지 12 분부터 제 리듬 우울증을 유도한다. 이 우울증은 반전후 저산소 복구 12시. 실험 설계에 관하여, 그것은 뇌간의 주동이의 부분에있는 뇌교는, 리듬 발생기 (8)에 억제 작용을 가지고주의하는 것이 중요하다. 따라서, 전체 뇌간과 주동이의 척수의 준비는 낮은 리듬을 표시합니다. 기록 용 절연 샘플 뇌교의 포함은 실험 (13)의 목표에 따라 결정된다; 연수 나 네트워크에 뇌교 영향의 연구 결과 (14)를 비교하는 뇌교와없이 녹음을 필요로한다. 또한,이 기술의 이점 중 하나는, 중뇌 및 / 또는 diencephalic 영역 (15, 16)를 포함하는 제제의 입쪽 부분을 연장하는 것이 가능 본토 수질 호흡 네트워크에 이들 영역의 효과를 평가하기 위해 제조하는 가능성이다.

<table><tbody><tr><td>Sylgard</td><td>Sigma Aldrich</td><td>761036-5EA</td><td>Use under hood</td></tr><tr><td>NaCl</td><td>Bioshop</td><td>SOD002</td><td></td></tr><tr><td>KCl</td><td>Bioshop</td><td>POC888</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>CCL444</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioshop</td><td>MAG510</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Bioshop</td><td>SOB999</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Bioshop</td><td>SPM306</td><td></td></tr><tr><td>D-glucose</td><td>Bioshop</td><td>GLU501</td><td></td></tr><tr><td>Carbogen</td><td>Linde</td><td>343-02-0006&amp;nbsp;</td><td></td></tr><tr><td>Temperature Controller</td><td>Warner Instruments, Hamden, CT, USA</td><td>TC-324B</td><td></td></tr><tr><td>Suction electrode</td><td>A-M Systems, Everett, WA, USA</td><td>model 573000</td><td></td></tr><tr><td>Differential AC amplifier</td><td>A-M Systems, Everett, WA, USA</td><td>model 1700</td><td></td></tr><tr><td>Moving averager</td><td>CWE, Ardmore, PA, USA</td><td>model MA-821</td><td></td></tr><tr><td>Data acquisition system</td><td>Dataq Instruments, Akron, OH, USA</td><td>model DI-720</td><td></td></tr><tr><td>LabChart software</td><td>ADInstruments, Colorado Springs, CO, USA</td><td></td><td></td></tr><tr><td>Prism sofware</td><td>Graphpad, La Jolla, CA, USA</td><td></td><td></td></tr><tr><td>Dissection chamber</td><td>Plastic box (e.g. petri box) will do</td><td></td><td></td></tr><tr><td>Recording chamber</td><td>Home made</td><td></td><td></td></tr><tr><td>Base</td><td>Kanetec, Bensenville, IL, USA</td><td>MB</td><td></td></tr><tr><td>Micromanipulator</td><td>World Precision Instrument Inc, Sarasota, FL, USA</td><td>KITE-R</td><td></td></tr><tr><td>Base</td><td>Kanetec, Bensenville, IL, USA</td><td>MB</td><td></td></tr><tr><td>Peristaltic pump</td><td>Gilson, Middleton, WI, USA</td><td>MINIPULS 3</td><td></td></tr><tr><td>Faraday Cage</td><td>Home made</td><td></td><td></td></tr><tr><td>Computer</td><td></td><td></td><td></td></tr></tbody></table>

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.

서두에서 언급 한 바와 같이,이 기술의 중요한 장점 중 하나는 뇌간에 직접 액세스 다양한 자극을 적용하는 것이다. 예를 들어, 저산소증 여기에 적용 하였다. 그림 1. AB 두 정상 산소와 저산소 조건, 전체 프로토콜 기록을 표시합니다. 그림 1.CE 즉 정상 산소 조건 (기록 리듬을 표시 ACSF는 95 % O 2 5 %로 버블 26 ° C에서 CO 2). 이전에이 정확한 제제 (1...

Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

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 ...

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Neuroscience

8.5K 조회수.  Laval University. 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.

비디오: Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

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 ...

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신경과학

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

척수 전기 생리학

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

생물학

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In particular, endogenously-generated network activity, which strongly varies as a function of the state of vigilance, significantly modulates neuronal computation. To investigate how different spontaneous cerebral dynamics impact single neurons&#39; integrative properties, we developed a new experimental strategy in the rat consisting in suppressing in vivo all cerebral activity by means of a systemic injection of a high dose of sodium pentobarbital. Cortical activities, continuously monitored by combined electrocorticogram (ECoG) and intracellular recordings are progressively slowed down, leading to a steady isoelectric profile. This extreme brain state, putting the rat into a deep comatose, was carefully monitored by measuring the physiological constants of the animal throughout the experiments. Intracellular recordings allowed us to characterize and compare the integrative properties of the same neuron embedded into physiologically relevant cortical dynamics, such as those encountered in the sleep-wake cycle, and when the brain was fully silent.

Induction of an Isoelectric Brain State to Investigate the Impact of Endogenous Synaptic Activity on Neuronal Excitability In Vivo

This procedure performs long-lasting in vivo intracellular recordings from single neurons during physiologically relevant cerebral states and after complete abolition of ongoing electrical activities, resulting in an isoelectric brain state. The physiological constants of the animal are carefully monitored during the transition to the artificial comatose condition.

등전위 뇌 상태의 유도는 신경 흥분에 내생 시냅스 활동의 영향을 조사하기<em&gt; 생체</em

The way neurons process information depends both on their intrinsic membrane properties and on the dynamics of the afferent synaptic network. In ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

새로운 방법으로 자유롭게 움직이는 신생아 쥐의 뇌파 기록

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

심각한<em&gt; In Vivo</em&gt; 마취 된 래트에서의 Hyperdirect 경로로부터의 국소 장 전위 및 다중 단위 활성의 전기 생리 학적 기록

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls body functions and internal organ rhythms when animals are exposed to emotional challenges and changes in their living environments. In general experiments, signals from different organs, such as the brain and the heart, are recorded by independent recording systems that require multiple recording devices and different procedures for processing the data files. This study describes a new method that can simultaneously monitor electrical biosignals, including tens of local field potentials in multiple brain regions, electrocardiograms that represent the cardiac rhythm, electromyograms that represent awake/sleep-related muscle contraction, and breathing signals, in a freely moving rat. The recording configuration of this method is based on a conventional micro-drive array for cortical local field potential recordings in which tens of electrodes are accommodated, and the signals obtained from these electrodes are integrated into a single electrical board mounted on the animal's head. Here, this recording system was improved so that signals from the peripheral organs are also transferred to an electrical interface board. In a single surgery, electrodes are first separately implanted into the appropriate body parts and the target brain areas. The open ends of all of these electrodes are then soldered to individual channels of the electrical board above the animal's head so that all of the signals can be integrated into the single electrical board. Connecting this board to a recording device allows for the collection of all of the signals into a single device, which reduces experimental costs and simplifies data processing, because all data can be handled in the same data file. This technique will aid the understanding of the neurophysiological correlates of the associations between central and peripheral organs.

This study introduces a method for the simultaneous recording of local field potentials in the brain, electrocardiograms, electromyograms, and breathing signals of a freely moving rat. This technique, which reduces experimental costs and simplifies data analysis, will contribute to the understanding of the interactions between the brain and peripheral organs.

대뇌 피 질의 로컬 필드 전위, 심전도, 치십시오, 그리고 자유롭게 움직이는 쥐에서 리듬 호흡의 동시 녹음

Monitoring the physiological dynamics of the brain and peripheral tissues is necessary for addressing a number of questions about how the brain controls ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

리듬-액티브의 준비 체 외 신생아 설치류 Brainstem-척수와 얇은 슬라이스

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Data Acquisition and Analysis In Brainstem Evoked Response Audiometry In Mice

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as visually evoked potentials (VEPs) or somatosensory evoked potentials (SEPs), the auditory evoked potentials (AEPs) are triggered by the repetitive presentation of identical stimuli, the electroencephalographic (EEG) response of which is subsequently averaged resulting in distinct positive (p) and negative (n) deflections. In humans, both the amplitude and the latency of individual peaks can be used to characterize alterations in synchronization and conduction velocity in the underlying neuronal circuitries. Importantly, AEPs are also applied in basic and preclinical science to identify and characterize the auditory function in pharmacological and genetic animal models. Even more, animal models in combination with pharmacological testing are utilized to investigate for potential benefits in the treatment of sensorineural hearing loss (e.g., age- or noise-induced hearing deficits). Here we provide a detailed and integrative description of how to record auditory brainstem-evoked responses (ABRs) in mice using click and tone-burst application. A specific focus of this protocol is on pre-experimental animal housing, anesthesia, ABR recording, ABR filtering processes, automated wavelet-based amplitude growth function analysis, and latency detection.

Brainstem evoked response audiometry is an important tool in clinical neurophysiology. Nowadays, brainstem evoked response audiometry is also applied in the basic science and preclinical studies involving both pharmacological and genetic animal models. Here we provide a detailed description of how auditory brainstem responses can be successfully recorded and analyzed in mice.

마우스에서 뇌간 에서 데이터 수집 및 분석 음성 응답 청력 측정

Brainstem evoked response audiometry (BERA) is of central relevance in the clinical neurophysiology. As other evoked potential (EP) techniques, such as ...

In Vivo Intracellular Recording of Type-Identified Rat Spinal Motoneurons During Trans-Spinal Direct Current Stimulation

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological characteristics in the intact spinal network and holds significant advantages relative to classical in vitro or extracellular recording techniques. An advantage of in vivo intracellular recordings is that this method can be performed on adult animals with a fully mature nervous system, and therefore many observed physiological mechanisms can be translated to practical applications. In this methodological paper, we describe this procedure combined with externally applied constant current stimulation, which mimics polarization processes occurring within spinal neuronal networks. Trans-spinal direct current stimulation (tsDCS) is an innovative method increasingly used as a neuromodulatory intervention in rehabilitation after various neurological injuries as well as in sports. The influence of tsDCS on the nervous system remains poorly understood and the physiological mechanisms behind its actions are largely unknown. The application of the tsDCS simultaneously with intracellular recordings enables us to directly observe changes of motoneuron membrane properties and characteristics of rhythmic firing in response to the polarization of the spinal neuronal network, which is crucial for the understanding of tsDCS actions. Moreover, when the presented protocol includes the identification of the motoneuron with respect to an innervated muscle and its function (flexor versus extensor) as well as the physiological type (fast versus slow) it provides an opportunity to selectively investigate the influence of tsDCS on identified components of spinal circuitry, which seem to be differently affected by polarization. The presented procedure focuses on surgical preparation for intracellular recordings and stimulation with an emphasis on the steps which are necessary to achieve preparation stability and reproducibility of results. The details of the methodology of the anodal or cathodal tsDCS application are discussed while paying attention to practical and safety issues.

This protocol describes in vivo intracellular recording of rat lumbar motoneurons with simultaneous trans-spinal direct current stimulation. The method enables us to measure membrane properties and to record rhythmic firing of motoneurons before, during and after anodal or cathodal polarization of the spinal cord.

트랜스 척추 직접 전류 자극 중 유형 식별 쥐 척추 운동 신경의 생체 내 세포 내 기록

Intracellular recording of spinal motoneurons in vivo provides a &#8220;gold standard&#8221; for determining the cells&#8217; electrophysiological ...

<meta charset="utf8"></head><body>Angled Silicon Multielectrode Recordings for Deep Brainstem Structures in Mice

Acute Single-Unit Multi-Electrode Recordings from the Brainstem of Head-Fixed Mice

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many brain regions. However, recording neuronal activity from deep caudal structures like the brainstem using multi-channel probes remains challenging. A significant concern is finding a trajectory for probe insertion that avoids large blood vessels, such as the superior sagittal venous sinus and the transverse venous sinus. Injuring these large veins can cause extensive bleeding, damage to the underlying brain tissue, and potentially death. This approach describes targeting brainstem structures by coupling anterior coordinates with an angled approach, allowing the recording probe to penetrate the brain below high-risk vascular structures. Compared to a strictly vertical approach, the angled approach maximizes the number of brain regions that can be targeted. Using this strategy, the ventrolateral periaqueductal gray (vlPAG), a brainstem region associated with REM sleep, can be reproducibly and reliably accessed to obtain single-unit, multi-electrode recordings in head-fixed mice before and during sevoflurane anesthesia. The ability to record neuronal activity in the vlPAG and surrounding nuclei with high temporal resolution is a step forward in advancing the understanding of the relationship between REM sleep and anesthesia.

<meta charset="utf8"></head><body>저자 스포트라이트: Investigating Anesthesia-Induced Sleep Pathways and Neuronal Excitability in Mice (생쥐의 마취 유도 수면 경로 및 신경 흥분성 조사)

This protocol describes a method to obtain in vivo, high-density single-neuron recordings from the brainstem of head-fixed mice. This approach is deployed to measure the action potential firing of neurons in the ventrolateral periaqueductal gray - a brainstem region inactive during Rapid Eye Movement (REM) sleep - before and during general anesthesia.

Head-Fixed Mice의 뇌간에서 발생한 급성 단일 단위 다중 전극 기록

Silicon multielectrode-based recordings are increasingly popular for studying neuronal activity at the temporal resolution of action potentials in many ...