우리는 방법의 수립시 도움이 토론을위한 프레 Heckmann 감사합니다. 또한, 우리는 비디오를 제작 지원을위한 기술 지원 및 프랭크 슈베르트에 대한 클라우디아 서머 감사합니다. 01EO1002와 예나 대학 병원의 임상 연구를위한 학제​​ 간 센터 (IZKF) : 작품은 연방 교육 연구부 (BMBF), 독일, FKZ에 의해 지원되었다.

다음 프로토콜에 언급 된 모든 실험 절차는 튀 링겐 주 당국 (링거 Landesamt FÜR Verbraucherschutz, Reg.-Nr. 02-044/12)에 의해 승인되었다. 1. 실험을위한 준비 <ol><li> 흡입 전극의 제작 <ol><li> 예를 들어, 표준 패치 전극 마이크로 피펫 풀러와 표준 붕규산 유리 모세관을 사용하여 마이크로 피펫을 잡아 당깁니다. </li><li> 브레이크 다이아몬드 파일을 사용하여 (등쪽 뿌리의 직경보다 약간 큰) 0.5 mm의 직경을 팁을 전극. </li><li> 열 광택이 표준 실험실 토치 안으로 빨려 때 등의 루트를 해가되지 않도록 끝이 적당 할 것이다. </li><li> 부압이인가 될 수있는 주사기에 연결된 전극 홀더에 유리 필라멘트를 탑재. </li></ol></li><li> 솔루션의 제조 <ol><li> 마우스의 경우 주입 마취 : 825 ml의 케타민에게 10 %를 혼합, 0.24 ml의 자일 라진 2 %차 5 ㎖의 0.9 % 식염수. 용액 10 μL / g의 체중 (BW)는 (IP)을 복강 내 주사한다. </li><li> 인공 뇌척수 (ACSF) :;의 KCl 3, KH 2 PO 4 1.25, 망초 • H 2 O 2, 탄산 수소 나트륨 (25), 염화칼슘 2, D-NaCl을 134 : 두 번 증류수 (mm 단위) 다음과 같은 염을 희석 10 포도당. 0.003 %의 최종 농도로 H 2 O 2를 추가. 항상 신선하게 준비된 솔루션을 사용합니다. </li></ol></li><li> 녹화 설정의 준비 (그림 2) <ol><li> 데이터를 디지털화하기위한 PC 인터페이스에 앰프를 연결합니다. </li><li> PC 하드 드라이브의 사각 펄스 자극 및 데이터 수집을 트리거하는 PC 기반 프로그램 또는 아날로그 타이머를 사용합니다. </li><li> 자극과 녹음으로 표준 유리 전극 홀더에 연결 염화물로 silverwires를 사용합니다. </li><li> stere 주위에 각각 자극, 기록 및 기준 전극에 대한 세 가지 조종 조립마우스에 대한 otactic 프레임, 모든 전극이 나중에 준비 척수에 액세스 할 수 있도록. </li><li> 부정적인 압력을 적용하는 사과로 전극 홀더에 호스와 주사기를 연결합니다. </li></ol></li></ol> 2. 동물 실험 및 녹음 과정에 대한 동물 준비에 대한 일반적인 설명 <ol><li> 각 기관의 동물 관리 및 사용위원회의 지침에 따라 마우스를 사용하는 모든 실험을 수행합니다. 동물의 고통을 최소화 할 것을 보장 깊은 마취하에 모든 수술과 녹음을 수행합니다. </li><li> (; 상기와 같이 제조 된 용액 10 μL / g의 BW 각각 125mg과 케타민과 자일 라진의 G의 BW 당 8mg의) 케타민 / 자일 라진의 IP 주입하여 동물을 마취. 장기 녹화 중에 필요한 경우 추가 주사는 IP 또는 IM을 할 수있다 주 : 0.05 ~ 0.1의 반복 임 주사를 허벅지 위쪽에있는 케타민 / 자일 라진 용액 ㎖를 적합한 것으로 입증최대 3 시간 동안 깊은 마취에서 동물을 유지합니다. </li><li> 마취 동안 건조를 방지하기 위해 눈에 수의사 연고를 사용합니다. </li><li> 정위 프레임에서 동물의 머리를 고정하고 실험 기간 동안 동물의 체온을 조절하는 직장​​ 프로브 및 휘어진 루프 가열 패드를 사용한다. 고정대에있는 척수의 고정이 필요하지 않습니다. </li><li> 준비를 시작하기 전에, 마우스의 발가락 사이 eyeblink 반사 및 경련에 의해 마취의 깊이를 확인합니다. 반사 신경은 폐지되어야한다. </li><li> 메스를 사용하여 클리어 연산 필드를 얻을 요추 영역을 낮추는 상부 흉추 레벨로부터 척수 상기 정중선을 따라 피부를 연다. 피부 절개를 만들기 위해 가위를 사용하지 마십시오. 조심스럽게 하부 조직으로부터 피부를 느슨하게. 다음 단계는 0.9 % 식염수에 적신 상처를 계속하는 동안. </li><li> 메스와 가위를 사용하여 힘줄과 가슴 수준으로 요추에서 척추의 양쪽에 결합 조직을 잘라. </리&gt; <li> 작은 니퍼를 사용하여 척추 주위의 극돌기 및 결합 조직의 나머지 부분을 제거합니다. </li><li> 조심스럽게 니퍼 해부 현미경 요추 수준 (L4/L5)에서 시작하는 척추 균열. 척추와 척추 사이의 공간에 니퍼의 끝을 밀어과 떨어져 뼈 조각을 들어 올립니다. 경질을 해와 척수에 압력을 피하지 마십시오. 모두의 성공을 위해 중요하다. 전체 절차를 수행하는 동안 적신 척수를 유지합니다. 중반 흉부 수준으로 진행합니다. </li></ol> 3. 지느러미 뿌리와 DRP 기록의 분리 (그림 2) <ol><li> 조심스럽게 구부러진 끝으로가는 바늘 (30 G)를 사용하여 경막을 엽니 다. 지금부터 습에 ACSF를 사용합니다. </li><li> 게이지를 사용하여 가능한 한 등의 뿌리를 분리하고 가능한 한 말단으로 인접한 두 뿌리를 잘라. 격렬한 분리하는 동안 뿌리에 당기는 실험의 성공에 영향을 미친다. </li><li> 할 수있는 흡입 전극을 아래로 내립니다뿌리를 rsal. 액체 등의 뿌리에서 빨아 수 있기 때문에 가능한 한 척추에 많은 ACSF를 추가합니다. </li><li> 주사기를 통해 부정적인 압력을 적용하여 하나의 유리 피펫에 하나의 지느러미 루트의 단면을 빨아. 필요한 경우, 미세 바늘을 사용하여 전극의 구멍 앞에 지느러미 루트를 이동합니다. </li><li> 지느러미 루트 흡입 전극에서 건조에있는 경우 피펫 충분히 ACSF 가득 될 때까지주의 깊게 빨고있는 동안, 그 팁을 몇 가지 ACSF를 추가합니다. </li><li> 등의 루트를 흡입 한 후, 척수에서 전극의 끝 부분을 올립니다. 주의하는 피펫 팁 및 척수 단락의 기록 / 자극 전극 사이에 &quot;물 다리&quot;.에게 </li><li> 반복 동측 인접 루트에 대한 3.3-3.6 단계를 반복합니다. </li><li> 등의 뿌리에 최대한 가깝게 기준 전극을 배치하고 ACSF을 적용하여 촉촉하게 유지합니다. </li><li> 녹화 설정을 구축함으로써, 하나의 루트가 이미 자극을위한 선택,기록에 대한 다른. 두 번째 지느러미 루트에서 기록이 현재 클램프 모드에서 수행하는 동안 전압을 단계적으로 증가한다. 하나는 DRP를 나타내는 천천히, 오래 지속 상향 편향에 의해 다음에 짧은 하향 편향을 알 수 있습니다. </li><li> (- 0.1 Hz에서 30 스윕 / 후근, 0.3 Hz에서 3 kHz에서 필터를 적어도 20) 수준을 supramaximal 여러 스윕을 기록하는 자극의 전압을 조정합니다. </li><li> DRP의 시간에 따른 합산에 대한 정보를 얻기 위해 세 가지 자극 (100 Hz에서)의 짧은 기차를 기록합니다. </li><li> 추가 반대측 기록에 대한 기록 및 자극 사이트를 전환합니다. </li><li> 동물 절차를 생존하는 것은 아니다. 반면 여전히 깊은 마취 (케타민 / 자일 라진은, 위 참조 2.2 단계) 잘린 마지막 촬영 후 동물을 희생. </li></ol> 4. 데이터 분석 <ol><li> 분석 프로그램에 데이터를 전송 (예 : 시그마 플롯, 이고르 프로, 또는 MATLAB). </li><li> 평균 추적의 FR을 계산톰 20-30 스윕. </li><li> 추가 분석을 위해 (그림 3), (DRP 피크 진폭 기준에서) 전압 편향의 최대 진폭을 가지고. </li><li> 후속 전위의 시간 종속 합산의 측정치를 얻기 위해 세 개의 펄스 및 단일 펄스 후에 DRP 피크 진폭의 비율을 계산한다. </li></ol>

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Physiol. 529 Pt 2, 445-460 (2000).</li><li>Geis, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Stiff-Person+Syndrome+IgG+Induces+Anxious+Behavior+in+Rats.">Human Stiff-Person Syndrome IgG Induces Anxious Behavior in Rats.</a> PLoS One. 6, e16775 (2011).</li><li>Martinez-Gomez, J., Lopez-Garcia, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+pharmacological+characterisation+of+ascending+anterolateral+axons+in+the+in+vitro+mouse+spinal+cord.">Electrophysiological and pharmacological characterisation of ascending anterolateral axons in the in vitro mouse spinal cord.</a> J. Neurosci. Methods. 146, 84-90 (2005).</li></ol>

저자는 더 경쟁 재정적 이익을 선언하지 않습니다.

생체 내에서 신경 세포의 활동과 시냅스 전위의 추가 및 세포 내 전기 생리학 기록은 중추 신경계 신경 세포의 기능과 병태 생리를 조사하는 예술 기술의 상태입니다. 척추 통합 운동 기능, 예를 들면 사지 운동과 복합 감각 인식에 중요합니다. 시냅스 억제 감각 입력에 적절한 대응을 보장이 계산 과정에서 하나의 중요한 메커니즘이다. 아이오와 구 심성 섬유에 GABA 성 시냅스가 PAD?...

시냅스 억제는 척수에서 가장 강력한 억제 메커니즘 중 하나입니다. 그것은 시냅스 막 잠재력과 운동 신경원 1-3의 흥분을 변경하지 않고 monosynaptically 흥분 운동 신경원의 흥분성 시냅스 후 전위 (EPSPS)을 억제한다. 감각 시냅스 섬유에 GABA 성 AXO - 축삭의 시냅스에 의해 유도 된 기본 심성 탈분극 (PAD)는 기본 메커니즘 4-7 (도 Figure1a 참조). 이 시냅스는 GABA A-와 B-GABA 수용체 (GABA R과 GABA B R)를 포함하고있다. GABA R 활동으로 인해 로컬 이온 분포에 PAD를 이끌어 클로라이드 컨덕턴스의 증가에 이르게한다. 이 탈분극 블록은 축삭 터미널에 활동 전위의 전파와는 감소 칼슘으로 이어지는 그들의 힘을 감소 + - 유입 및 송신기 방출의 감소. GABA B 수용체의 활성화는 않습니다 없음t는 PAD에 기여하지만, 이에 연접 억제를 강화하는 칼슘 2 + 유입의 감소로 연결됩니다. GABA R의 활성화는 단기간의 억제에 관여하는 것으로 보이지만, GABA B R은 장기 변조 8-10에 참여하고 있습니다. PAD와 연접 억제의 주요 부분을 차지하는 GABA에 더하여, 다른 송신기 시스템은 또한 변조 할 수 있으며이기구 (11, 12)에 기여한다. 시냅스 억제의 병리학 적 변화는, 결함이있는 GABA 성 전송 (17)에 의해 매개 모터 hyperexcitability와 주변 염증 및 신경 병증 성 통증 (13, 14)뿐만 아니라, 이상 중앙 통증 처리 (15), 척수 손상 16, 중추 신경계 질환을 예를 들어 여러 가지 질병 상태에 중요한 것 같습니다 18. 따라서 시냅스 억제를 추정하는 것은 생체 내에서 척수 수준에서 병리학 실험 조건을 조사 할 가치가있다 </em&gt;. PAD는 척수 연접 억제의 직접 측정을 제공하는 양 실시 전위를 일으킨다. 그 잠재력은 후근 전위 (DRP)라고 인접 등의 뿌리 (7)의 자극 후 척수 등쪽 뿌리에서 측정 할 수있다. DRP의 첫 번째 측정은 고양이와 개구리 19에보고 된 집중적으로 1970 년대 초 3,4,20,21에서 에클 스, 슈미트, 그리고 다른 사람에 의해 고양이에서 공부했다. 고양이 (22)와 쥐 23 DRP의 생체 내 녹음이 널리 사용되었지만, 마우스의 측정은 거의 독점적으로 생체 고립 척수 준비 15,24에서 수행되었다. 여기서, 우리는 본래 유기체 시냅스 억제 직접적인 측정을 허용 생체 내에서 마취 된 쥐에서 DRP를 기록하는 방법을 설명한다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass tubing (inner diameter 1.16 mm)</td>
			<td>Science Products (Hofheim, Germany)</td>
			<td>GB200F-10</td>
			<td>Other glass tubing might also be suitable</td>
		</tr>
		<tr>
			<td>Superfusion solution (sterile, 0,9% NaCl)</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>3570350</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Germany)</td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine)</td>
			<td>Bayer Animal Health GmbH (Leverkusen, Germany)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin 10%</td>
			<td>Medistar GmbH (Ascheberg, Germany)</td>
			<td>KETAMIN 10%</td>
			<td></td>
		</tr>
		<tr>
			<td>30G micro needle/ Sterican</td>
			<td>Braun Melsungen AG&#160;</td>
			<td>4656300</td>
			<td></td>
		</tr>
		<tr>
			<td>(Melsungen, Geramny)</td>
		</tr>
		<tr>
			<td>Salts for aCSF</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>Diverse</td>
			<td></td>
		</tr>
		<tr>
			<td>S88 Dual Output Square Pulse</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>S88X</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
		</tr>
		<tr>
			<td>SIU5 RF Transformer Isolation Unit</td>
			<td>Grass Technologies (Warwick, USA)</td>
			<td>SIU-V</td>
			<td></td>
		</tr>
		<tr>
			<td>InstruTECH LIH 8+8</td>
			<td>HEKA (Lambrecht, Deutschland)</td>
			<td>LIH 8+8 + Patchmaster software</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition&#160;</td>
		</tr>
		<tr>
			<td>Universal amplifier</td>
			<td>npi (Tamm, Deutschland)</td>
			<td>ELC-03X</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>P-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Olympus (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Sutter Instruments (Novato, USA)</td>
			<td>MPC-200/MPC-325</td>
			<td>Mechanical micromanipulators also possible</td>
		</tr>
		<tr>
			<td>Homeothermic Blanket System</td>
			<td>Stoelting (Wood Dale, USA)</td>
			<td>50300V</td>
			<td></td>
		</tr>
		<tr>
			<td>Intra-/extracellular recording electrode holder</td>
			<td>Harvard Apparatus (Holliston, USA)</td>
			<td>641227</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring spinal presynaptic inhibition in mice by dorsal root potential recording in vivo

시냅스 억제는 척수에서 가장 강력한 억제 메커니즘 중 하나입니다. 기본 생리 학적 메커니즘은 GABA 성 AXO - 축삭의 시냅스 (기본 심성 탈분극)에 의해 매개 차 구 심성 섬유의 탈분극입니다. 기본 심성 탈분극의 강도는 후근 (후근 전위, DRP)에 볼륨 실시 전위의 기록에 의해 측정 할 수있다. 시냅스 억제의 병리학 적 변화는 특정 두통 상태의 비정상적인 중앙 처리 및 모터 hyperexcitability 일부 질환에 중요하다. 여기, 우리는 생쥐에서 생체 내에서 기록 DRP하는 방법을 설명합니다. 마취 된 동물 흡입 전극을 사용하여 기록 절차의 척수 등쪽 뿌리의 제조는 설명합니다. 이 방법은 GABA 성 DRP를 측정함으로써 살아있는 마우스에서 척추 시냅스 억제를 추정 할 수 있습니다. 형질 전환 마우스 모델과 함께, DRP 녹화 할 수있다 SE질병 관련 척추의 병태 생리를 조사 할 수있는 강력한 도구로 RVE는. 생체 내 기록은 예를 들어, 동시 녹음 또는 supraspinal 네트워크 및 말초 신경의 자극에 의해 DRP의 유도 조작의 가능성 생체 고립 척수 준비에 비해 몇 가지 장점이 있습니다.

전형적인 DRP 흔적은도 3에 나타낸다. 눈에 띄는 자극 이슈는 일반적으로 짧은 아래로 편향옵니다. 그 후 DRP 나타내는 느리고 오래 상향 편향은, 명확히 구별이다. 녹음의 하위 집합으로, 지느러미 루트 반사 신경 DRP의 상단에 작은 스파이크로 볼 수 있습니다. 자극의 전압이 과도한 경우 정상적인 야생형 생쥐에서 지느러미 루트 반사 신경이 가장 자주 나타납니다. 지느러미 루트 반사...

Watch this Scientific Journal Video about Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo at JoVE.com

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABA 성 시냅스 억제는 모터와 척수 네트워크에서 감각 신호 통합을위한 중요한 척수에서 강력한 억제 메커니즘입니다. 기초 기본 심성의 탈분극은 후근 전위 (DRP)의 기록에 의해 측정 할 수있다. 여기에서 우리는 생쥐의 DRP의 생체 기록하는 방법을 보여줍니다.

배근 잠재적 기록하여 생쥐의 척수 시냅스 억제를 측정<em&gt; 생체</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

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Neuroscience

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

13.9K 조회수.  Jena University Hospital, Jena, Germany.  GABA 성 시냅스 억제는 모터와 척수 네트워크에서 감각 신호 통합을위한 중요한 척수에서 강력한 억제 메커니즘입니다. 기초 기본 심성의 탈분극은 후근 전위 (DRP)의 기록에 의해 측정 할 수있다. 여기에서 우리는 생쥐의 DRP의 생체 기록하는 방법을 보여줍니다. 

비디오: Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

마우스 망막 신경절 세포 Transfection로 생체내에 Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

연구

신경과학

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

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

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

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

동시<em&gt; 생체</em두 망막의&gt; 기능 테스트로<em&gt; 생체</em&gt; 전위도 시스템

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Polygraphic Recording Procedure for Measuring Sleep in Mice

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the homeodynamics and circuitry of sleep-wake regulation. In many laboratories, a cable-based sleep recording system is used to monitor the EEG and EMG in freely behaving mice in combination with computer software for automatic scoring of the vigilance states on the basis of power spectrum analysis of EEG data. A description of this system is detailed herein. Steel screws are implanted over the frontal cortical area and the parietal area of 1 hemisphere for monitoring EEG signals. In addition, EMG activity is monitored by the bilateral placement of wires in both neck muscles. Non-rapid eye movement (Non-REM; NREM) sleep is characterized by large, slow brain waves with delta activity below 4 Hz in the EEG, whereas a shift from low-frequency delta activity to a rapid low-voltage EEG in the theta range between 6 and 10 Hz can be observed at the transition from NREM to REM sleep. By contrast, wakefulness is identified by low- to moderate-voltage brain waves in the EEG trace and significant EMG activity.

The recording of electroencephalogram (EEG) and electromyogram (EMG) in freely behaving mice is a critical step to correlate behavior and physiology with sleep and wakefulness. The experimental protocol described herein provides a cable-based system for acquiring EEG and EMG recordings in mice.

Recording of the epidural electroencephalogram (EEG) and electromyogram (EMG) in small animals, like mice and rats, has been pivotal to study the ...

Hydraulic Extrusion of the Spinal Cord and Isolation of Dorsal Root Ganglia in Rodents

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may result in damage to the spinal cord caused by the dissection process. Here, we show how the spinal cord can be extruded using hydraulic pressure. Handling time is significantly reduced to only a few minutes, likely decreasing protein damage. The low risk of damage to the spinal cord tissue improves subsequent immunohistochemical analysis. By performing hydraulic spinal cord extrusion instead of traditional laminectomy, the rodents can further be used for DRG isolation, thereby lowering the number of animals and allowing analysis across tissues from the same rodent. We demonstrate a consistent method to identify and isolate the DRGs according to their localization relative to the costae. It is, however, important to adjust this method to the particular animal used, as the number of spinal cord segments, both thoracic and lumbar, may vary according to animal type and strain. In addition, we illustrate further processing examples of the isolated tissues.

Here, we present a protocol for hydraulic extrusion of the spinal cord as well as identification and isolation of specific dorsal root ganglia (DRGs) in the same rodent. Compared to standard spinal cord isolation methods, this method is significantly faster and reduces the risk of tissue damage.

설치류의 등쪽 뿌리 신경절의 척수 및 절연의 유압 압출

Traditionally, the spinal cord is isolated by laminectomy, i.e. by breaking open the spinal vertebrae one at a time. This is both time consuming and may ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

등쪽 뿌리 신경절 사출 및 지느러미 루트 감각 축삭의 재생을위한 모델로 부상 크러시

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Laminotomy for Lumbar Dorsal Root Ganglion Access and Injection in Swine

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive targets for injection of novel therapeutics aimed at treating chronic pain. In small animal models, laminectomy has been used to facilitate DRG injection because it involves surgical removal of the vertebral bone surrounding each DRG. We demonstrate a technique for intraganglionic injection of lumbar DRG in a large animal species, namely, swine. Laminotomy is performed to allow direct access to DRG using standard neurosurgical techniques, instruments, and materials. Compared with more extensive bone removal via laminectomy, we implement laminotomy to conserve spinal anatomy while achieving sufficient DRG access. Intraoperative progress of DRG injection is monitored using a non-toxic dye. Following euthanasia on post-operative day 21, the success of injection is determined by histology for intraganglionic distribution of 4&#39;,6-diamidino-2-phenylindole (DAPI). We inject a biologically inactive solution to demonstrate the protocol. This method could be applied in future preclinical studies to target therapeutic solutions to DRG. Our methodology should facilitate testing the translatability of intraganglionic small animal paradigms in a large animal species. Additionally, this protocol may serve as a key resource for those planning preclinical studies of DRG injection in swine.

We describe a method for laminotomy in swine that provides access to lumbar dorsal root ganglia (DRG) for intraganglionic injection. Injection progress is monitored intraoperatively and histologically confirmed up to 21 days after surgery. This protocol could be used for future preclinical studies involving DRG injection.

Laminotomy 허리 지 루트 신경 절의 액세스 및 돼지에 주입

Dorsal root ganglia (DRG) are anatomically well defined structures that contain all primary sensory neurons below the head. This fact makes DRG attractive ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

성인 마우스 지 루트 신경 절의 Electroporation Vivo에서 조사 포유류 축 삭 재생:

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

Use of In Vivo Single-fiber Recording and Intact Dorsal Root Ganglion with Attached Sciatic Nerve to Examine the Mechanism of Conduction Failure

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for nerve fibers in the central and peripheral nervous systems. This method is particularly applicable to dorsal root ganglia (DRG), which are primary sensory neurons that exhibit a pseudo-unipolar structure of nervous processes. The patterns and features of the action potentials passed along axons are recordable in these neurons. The present study uses in vivo single-fiber recordings to observe the conduction failure of sciatic nerves in complete Freund&#8217;s adjuvant (CFA)-treated rats. As the underlying mechanism cannot be studied using in vivo single-fiber recordings, patch-clamp-recordings of DRG neurons are performed on preparations of intact DRG with the attached sciatic nerve. These recordings reveal a positive correlation between conduction failure and the rising slope of the after-hyperpolarization potential (AHP) of DRG neurons in CFA-treated animals. The protocol for in vivo single fiber-recordings allows the classification of nerve fibers via the measurement of conduction velocity and monitoring of abnormal conditions in nerve fibers in certain diseases. Intact DRG with attached peripheral nerve allows observation of the activity of DRG neurons in most physiological conditions. Conclusively, single-fiber recording combined with electrophysiological recording of intact DRGs is an effective method to examine the role of conduction failure during the analgesic process.

Single-fiber recording is an effective electrophysiological technique that is applicable to the central and peripheral nervous systems. Along with the preparation of intact DRG with the attached sciatic nerve, the mechanism of conduction failure is examined. Both protocols improve the understanding of the peripheral nervous system&#39;s relationship with pain.

전도 실패의 메커니즘을 검사하기 위해 연결된 Sciatic 신경을 가진 생체 내 단일 섬유 기록 및 손상되지 않은 등철 루트 신경절의 사용

Single-fiber recording has been a classical and effective electrophysiological technique over the last few decades because of its specific application for ...

Rapid Isolation of Dorsal Root Ganglion Macrophages

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after peripheral nerve injury. Peripheral monocytic cells, including macrophages, are known to respond to a tissue injury through phagocytosis, antigen presentation, and cytokine release. Emerging evidence has implicated the contribution of dorsal root ganglia macrophages to neuropathic pain development and axonal repair in the context of nerve injury. Rapidly phenotyping (or &#8220;rapid isolation of&#8221;) the response of dorsal root ganglia macrophages in the context of nerve injury is desired to identify the unknown neuroimmune factors. Here we demonstrate how our lab rapidly and effectively isolates macrophages from the dorsal root ganglia using an enzyme-free mechanical dissociation protocol. The samples are kept on ice throughout to limit cellular stress. This protocol is far less time consuming compared to the standard enzymatic protocol and has been routinely used for our Fluorescence-activated Cell Sorting analysis.

Here we present a mechanical dissociation protocol to rapidly isolate macrophages from the dorsal root ganglion for phenotyping and functional analysis.

등갈 뿌리 신경절 대식세포의 신속한 절연

There are growing interests to study the molecular and cellular interactions among immune cells and sensory neurons in the dorsal root ganglia after ...