이 연구는 UT 사우스 웨스턴 시작 기금 (SK)에 의해 지원되었다.

<ol>
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저자의 아무도는 경쟁의 이익 또는 충돌하는 이해 관계가 없습니다.

이 프로토콜은 뇌 조각의 뉴런에서 전체 셀 패치 클램프 실험을 수행하기위한 기본 절차를 설명합니다. 그러나,이 기술의 복잡성 전위 감도 완전이 문서에서 설명 될 수 없다. 여기, 우리는 가장 기본적인 단계를 묘사하고 성공적이고 엄격한 전체 세포 녹음을 달성하기위한 제어해야합니다 중요한 매개 변수를 강조하기 위해 노력했다. 더 이론적 인 학습, 많은 책과 기사는 뇌 조각 3,21-24과</su...

패치 클램프 기술은, 1970 년대 후반에 개발 1,2-되었습니다 전기 생리 기술은 실시간 조직에서 단일 또는 다중 이온 채널의 기능을 연구하기위한 기본 툴이다. 달성 될 수있는 다른 패치 구성 중에서, 전체 셀 패치 클램프 기록은 뉴런의 주요부의 전기적 성질의 조사를 허용한다. 고전적으로,이 기술은 시험 관내에서 두 뇌 조각 갓 해리 뉴런 또는 세포 배양 모델 (3)에 행해진 다. 뇌 조각 뉴런에서 수행하는 경우,이 기술은 여러 가지 장점을 제공한다. 특히, (I) 신경 세포는 다소 상대적으로 보존 된 뇌 회로에 기록되고, 세포 배양 용 제제에 비해 3 생리 관련이있는 환경을 제공한다. 이것은 급성 pharmacolog 임의의 유형에 의해 유발되는 세포 및 분자 사건을 초기에 포착, 또는 실시간으로 모니터링 할 수있게iCal의 조작 - 생체 내 조건의 전형적인 사용하여 달성 될 수없는 시간 해상도; 시각적으로 뇌 조각에 뇌 영역을 식별에 (II) 기능은 모두가 형광 마커를 표현 할 때 뇌 영역은 공부에 대한 특정 신경 세포에 대한 높은 지역 특이성 3 수 있습니다; (ⅲ) (세포 내 레코딩을위한 날카로운 마이크로 피펫으로 막을 천공 대조적으로) 원형질막의 상당 부분을 열어 세포의 세포 내 공간에 접근 4. 차례로,이 내부 용액을 구성하는 특정 이온의 함량 또는 농도가 너무 분자 표적을 수정할 또는 셀룰러 메커니즘이 상이한 조건 하에서 조사 될 수 있습니다. 예를 들어, 전체 셀 구성, 특정 약학 제 (예 : 길항제) 설정시 한 기록 마이크로 피펫 (패치 피펫) 용액에 직접 세포질 내로 확산 및 putat에 역할을 추가 할 수 있다는이웃하는 셀들에 상기 타겟의 기능을 변경하지 않고 세포 내 표적 필자. 또한, 마이크로 피펫 날카로운 기록 비교 패치 클램프 전극의 선단에 큰 개구 낮은 저항, 낮은 경쟁중인 잡음 및 셀 (4)의 내부로 더 따라서 전기적 접속을 제공한다. 그러나, 피펫 팁에 큰 구멍이 셀 투석으로 이어질 및 연구 5,6에서있는 생명 현상의 발현에 중요 할 수있다 세포 내 분자 기계함으로써 손실 할 수 있습니다. 이 경우, 날카로운 전극 녹음이 더 적합 할 수있다. 녹음이 유형시켜 세포 내 공간과 내부 피펫 용액의 이온 교환을 최대한 방지 전체 셀 녹음에 사용되는 것보다 훨씬 작은 기공을 가진 마이크로 피펫을 필요로한다. (급성 또는 만성) 경험의 모든 형태를 포함 남용 11,1의 약물에 노출 7-10 학습2 등 응력 13,14은 특정 뇌 영역에서의 신경 기능의 다양한 측면을 변경할 수있다. 이러한 변화는 종종 (일 시간)을 개발하기 위해 시간을 필요로하기 때문에, 특정 경험을받은 동물의 뇌 조각에서 전체 셀 녹음 연구자들은 이러한 변화를 식별 할 수 있습니다. 기본적으로 (모든 경우) 신경 세포의 기능 (예를 들면, 리간드 - 활성화 된 이온 채널 전압 관문 이온 채널, 신경 전달 물질 트랜스 포터)함으로써 뇌 회로 활동 및 동작 환경에 의해 변경 될 수있다 (환경 의존성 참여 요소 많은 소성) 10,15-17. 신경 수준에서 뇌 회로의 활동이 시냅스 사이의 지속적인 상호 작용 (예를 들면, 글루타메이트 전송) 및 고유 세포의 흥분 요인 (예를 들어, axosomato-수지상 이온 채널에서 나온다 : 칼륨, K +, 나트륨, 나트륨 + 칼슘, 칼슘 2 + ). 도 ë을 사용하여 특정 조건에서전자 셀 패치 클램프 전기 생리학 기술, 시냅스 대 고유 흥분의 변화에​​서 특히 발생하는 신호 변경은 분리 될 수있다. 대부분의 경우, 시냅스 흥분성 전체 셀 전압 - 클램프 기술을 이용하여 평가된다. 본 촬영 모드는 α 아미노 -3- 히드 록시 -5- 메틸 -4- isoxazolepropionic 산 수용체에 의해 매개되는 예를 들어, 이온 전류 [의 측정 (허용 AMPA 수용체)와 신경 세포막을 통한 N 메틸 -D- 아스파르트 산 수용체 (NMDA 수용체)] 세트 전압의 막전위 채. 여기에서, 실험자는 세슘을 포함하는 내부 마이크로 피펫 솔루션 (고사 +), K + 채널의 폭 넓은 차단제 (키 고유 흥분 요인)를 사용합니다. 전체 셀 구성의 확립되면, 세포 내 공간에서 고사 +의 확산은 K + 채널을 차단하고, 이에 상대적으로 효율적인 공간 클램프 및 사전을 모두 허용다른 측정치들에 내재 흥분성 요인의 영향 벤트. 불규칙한 모양의 세포 (예를 들어, 신경 세포), 그리고 18, 19 아버 광대하고 복잡한 수지상 특히 신경을 녹음 할 때 공간 클램프의 문제, 즉, 전압 클램프에 어려움 전체 셀은 발생한다. 체세포 전압 클램프 저조한 제어 뉴런의 수지상 트리 전압 때문에, 연구중인 수지상 전기 신호의 다양한 양태는 수지상의 거리에 비례하여 변형된다. (인공 뇌 - 척수액, ACSF) 세포 외 용액에 용해 된 이러한 피크로 톡신 (감마 - 아미노 산, GABA 수용체 길항제) 또는 kynurenic 산 (글루타메이트 수용체의 광범위한 차단제)와 같은 약물 학적 도구와 결합하여,이 기술은 글루타메이트를 측정 할 수 있도록 수용체 -와 GABA 각기 전류를 R은 중재. 대조적으로, 극한의 흥분은 일반적으로 전류 - 클램프 기록 모드에서 평가된다.전압 클램프 기록 반대로, 본 촬영 모드는 신경 세포막에 흐르는 이온 전류에 의한 막 전위의 변화를 측정 할 수있다. 뉴런 나 +와 K + 채널 모두를 필요로 활동 전위를 생성하기 위해 일반적으로 고유 흥분의 변화는 능력의 변화를 통해 평가된다. 현재 클램프 녹음을 수행 할 때 따라서, 가능한 Micropipette는 K + 대신 고사 +를 포함하는 내부 용액으로 채워집니다. 글루타메이트와 GABA에게 ACSF에 용해 된 수용체 매개 전류 차단되는 약물과 결합하여,이 실험 설계 시냅스 흥분성의 전위 변화에 의해 오염되지 않고 신경 소성 본질적인 요인 (예를 들면, K는 + 채널)의 기여를 측정 할 수 있도록 요인. 이 문서에서는 t 기본 필요한 절차 단계를 설명합니다O (I) 건강한 뇌 조각을 제조; (ⅱ) 전체 셀 구성을 달성하고, (ⅲ) 극한 흥분성 시냅스 및 평가하는 기본 파라미터를 모니터링한다.

<table border="0" cellpadding="0" cellspacing="0" width="689">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Isolated pulse stimulus generator</td>
			<td style="width:183px;">A.M.P.I</td>
			<td style="width:139px;">Master-8</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolation unit (ISO-Flex)</td>
			<td>A.M.P.I</td>
			<td>ISO-Flex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer controlled Amplifier&#160;</td>
			<td>Molecular Devices</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital Acquisition system</td>
			<td>Molecular Devices</td>
			<td>Digidata 1500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Olympus</td>
			<td>BX-51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MPC-200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chamber and in-line Heater</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome Slicer</td>
			<td>Leica&#160;</td>
			<td>VT1000 S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging Camera</td>
			<td>Q Imaging</td>
			<td>QIClick-F-M-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narishige pipette puller PC-10</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass capillaries</td>
			<td>WPI</td>
			<td>TW150F-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slice hold-down (harp)</td>
			<td>Warner Instruments</td>
			<td>64-0255</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slice Chamber</td>
			<td>Warner Instruments</td>
			<td>RC-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonmetallic syringe needle</td>
			<td>World Precision Instruments</td>
			<td>MF28G67-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters</td>
			<td>Nalgene</td>
			<td>176-0045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glue Gun</td>
			<td style="width:183px;">Home Depot</td>
			<td style="width:139px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gas dispersion tube</td>
			<td>Ace Glass Inc.</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Decapitation scissors</td>
			<td>Home Depot</td>
			<td>100649198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #3</td>
			<td>World Precision Instruments</td>
			<td>500236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Small straight sharp tips scissors</td>
			<td>World Precision Instruments</td>
			<td>14218</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vessel canulation forceps&#160;</td>
			<td>World Precision Instruments</td>
			<td>500453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curved hemostatic forceps</td>
			<td>World Precision Instruments</td>
			<td>501288</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Economy Tweezers #3</td>
			<td>World Precision Instruments</td>
			<td>501976-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spatula</td>
			<td>Fisher Scientific</td>
			<td>14357Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scooping spatula</td>
			<td>Fisher Scientific</td>
			<td>14-357Q</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-747B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper</td>
			<td>Lab Depot</td>
			<td>CFP1-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;"></td>
			<td style="width:183px;"></td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Cs-Gluconate internal solution (pH 7.2&#8211;7.3, 280&#8211;290 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-gluconic acid 50%&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>G1951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cesium-OH (CsOH) 50%&#160;</td>
			<td>Sigma Aldrich/various</td>
			<td>232041</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl, 2.8 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, 20 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA, 0.4 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tetraethylammonium-Cl, 5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>T2265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2GTP, 0.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgATP, 2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">K-Gluconate internal solution (pH 7.2&#8211;7.3, 280&#8211;290 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K D-gluconate, 120 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G4500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl, 20 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, 10 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA, 0.2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>E4378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich/various</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2GTP, 0.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgATP, 2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Standard artificial cerebrospinal fluid (ACSF, osmolarity &#8776; 300-310 mOsm)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl, 2.5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl, 119 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4-H20, 1 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaHCO3, 26.2 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose, 11 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4-7H2O, 1.3 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2-2H20, 2.5 mM</td>
			<td>Sigma Aldrich/various</td>
			<td>C3881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:183px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">Additional compounds used for solutions preparation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KOH</td>
			<td>various</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kynurenic acid</td>
			<td>Sigma Aldrich/various</td>
			<td style="width:139px;">K3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-cell patch-clamp recordings in brain slices

전체 셀 패치 클램프 기록은 신경 세포의 상당한 부분의 전기적 특성의 조사를 허용하는 전기 생리 기술이다. 이러한 구성에서, 마이크로 피펫은 이전에 사용 된 세포 날카로운 전극 기록 방법보다 더욱 정확한 이온 전류 측정 전류 누설을 방지함으로써 제공 세포막에 밀착된다. 고전적으로, 전체 셀 기록 세포 배양 모델 해리 신경 뇌 조각 뉴런 제제를 포함하는 다양한 형태의 신경 세포에서 수행하고, 그대로 마취 또는 웨이크 동물 일 수있다. 요약하면,이 기술은 대단히 흥분 세포의 수동 및 능동 생물 물리학 적 특성의 이해에 기여하고있다. 이 기술의 주요 장점은 조작 (예, 약물은 실험자 유도 가소성)는 특정한 신경 기능 또는 C를 변경할 수있는 방법의 특정 정보를 제공하는 것이다실시간 hannels. 또한 세포막의 중요한 개구 내부 피펫 용액 자유롭게 도입하는 약물, 예를 들어 작용제 또는 특정 세포 단백질의 길항제를위한 수단을 제공하며 인접 셀의 기능을 변경하지 않고 이러한 대상을 조작하는 세포질 내로 확산 할 수있다. 전체 셀 기록에 초점이 문서는 뇌 조각의 뉴런에 생리 학적으로 관련 맥락에서, 즉 상대적으로 잘 보존 된 뇌 회로에 신경을 기록하는 장점을 가지고 준비를 시행 하였다. 특히, 적절한 약리학과 결합 될 때, 이러한 기술은 학습, 약물 남용에 대한 노출, 스트레스 등의 경험 중 어느 방식에 따른 구체적인 neuroadaptations 발생을 식별 할 수있는 강력한 도구이다. 요약하면, 뇌 조각에서 전체 셀 패치 클램프 녹음은 생체 준비를 오래 지속 변화를 측정 할 수있는 수단을 제공신경 기능에 해당 그대로 깨어 동물에서 개발했습니다.

온도 용이 실험자에 의해 제어되는 요소는, 이온 채널과 수용체의 생물 리 학적 특성에 영향을 미치는, 이로써 시냅스 전류 (PSC를) (EPSC 및 IPSCs) 스파이크를 유발하는 신경 기능의 파형.도 3 도 4는 신경 세포에 소성 온도의 영향을 각각 유발 EPSCs (eEPSCs)의 기울기를 나타낸다. 소성 패턴 (도 3)는 초과되어 조정 개구 특정 전압 - 게이트 ?...

Watch this Scientific Journal Video about Whole-cell Patch-clamp Recordings in Brain Slices at JoVE.com

Whole-cell Patch-clamp Recordings in Brain Slices

이 프로토콜은 전체 셀 패치 클램프 녹음을 수행하기위한 기본 절차 단계를 설명합니다. 이 기술은 신경 세포의 전기적 동작의 연구를 허용하고, 뇌 조각에서 수행하는 경우, 여전히 상대적으로 잘 보존 된 뇌 회로에 통합되어 신경 세포에서 다양한 신경 기능의 평가를 할 수 있습니다.

뇌 조각에서 전체 셀 패치 클램프 녹음

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

whole-cell-patch-clamp-recordings-in-brain-slices

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

59.3K 조회수.  University of Texas Southwestern Medical Center.  이 프로토콜은 전체 셀 패치 클램프 녹음을 수행하기위한 기본 절차 단계를 설명합니다. 이 기술은 신경 세포의 전기적 동작의 연구를 허용하고, 뇌 조각에서 수행하는 경우, 여전히 상대적으로 잘 보존 된 뇌 회로에 통합되어 신경 세포에서 다양한 신경 기능의 평가를 할 수 있습니다. 

비디오: Whole-cell Patch-clamp Recordings in Brain Slices

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

귀에 세포의 패치 클램프 녹음 절연 Zebrafish에서

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

연구

신경과학

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

적외선 신경 자극의 메커니즘을 조사하는 전체 세포 패치 클램프

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

배아 Zebrafish의 MAUTHNER 세포에서 패치 클램프 녹음

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

A Computer-assisted Multi-electrode Patch-clamp System

The patch-clamp technique is today the most well-established method for recording electrical activity from individual neurons or their subcellular compartments. Nevertheless, achieving stable recordings, even from individual cells, remains a time-consuming procedure of considerable complexity. Automation of many steps in conjunction with efficient information display can greatly assist experimentalists in performing a larger number of recordings with greater reliability and in less time. In order to achieve large-scale recordings we concluded the most efficient approach is not to fully automatize the process but to simplify the experimental steps and reduce the chances of human error while efficiently incorporating the experimenter's experience and visual feedback. With these goals in mind we developed a computer-assisted system which centralizes all the controls necessary for a multi-electrode patch-clamp experiment in a single interface, a commercially available wireless gamepad, while displaying experiment related information and guidance cues on the computer screen. Here we describe the different components of the system which allowed us to reduce the time required for achieving the recording configuration and substantially increase the chances of successfully recording large numbers of neurons simultaneously.

Multi-electrode patch-clamp recordings constitute a complex task. Here we show how, by automating of many of the experimental steps, it is possible to accelerate the process leading to qualitative improvement in performance and number of recordings.

컴퓨터를 이용한 다중 전극 패치 클램프 시스템

The patch-clamp technique is today the most  well-established method for recording electrical activity from individual  neurons or their subcellular ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- 및 Neurochemically 확인 된 해마의 interneurons에서 전체 세포 패치 클램프 녹음

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

해마의 Organotypic 조각의 쌍으로 전체 셀 녹음

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

급성 뇌 조각에서 신경 세포의 미세 회로의 전기 생리 및 형태 학적 특성은 페어 패치 클램프 녹음을 사용하여

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate electrical processes in target cells. In this context, ion selectivity of a channelrhodopsin is of particular importance. This article describes the investigation of chloride selectivity for a recently identified anion-conducting channelrhodopsin of Proteomonas sulcata via electrophysiological patch-clamp recordings on HEK293 cells. The experimental procedure for measuring light-gated photocurrents demands a fast switchable &#8211; ideally monochromatic &#8211; light source coupled into the microscope of an otherwise conventional patch-clamp setup. Preparative procedures prior to the experiment are outlined involving preparation of buffered solutions, considerations on liquid junction potentials, seeding and transfection of cells, and pulling of patch pipettes. The actual recording of current-voltage relations to determine the reversal potentials for different chloride concentrations takes place 24 h to 48 h after transfection. Finally, electrophysiological data are analyzed with respect to theoretical considerations of chloride conduction.

This article describes how the ion selectivity of channelrhodopsin is determined with electrophysiological whole-cell patch-clamp recordings using HEK293 cells. Here, the experimental procedure for investigating chloride selectivity of an anion-selective channelrhodopsin is demonstrated. However, the procedure is transferable to other channelrhodopsins of distinct selectivity.

Channelrhodopsins에서 이온 선택성의 전기 생리 학적 결정을위한 전체 셀 패치 - 클램프 기록

Over the past decade, channelrhodopsins became indispensable in neuroscientific research where they are used as tools to non-invasively manipulate ...

Application of Automated Image-guided Patch Clamp for the Study of Neurons in Brain Slices

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a challenging and low-throughput technique due to its complexity and high reliance on user operation and control. This manuscript demonstrates an image-guided automatic patch clamp system for in vitro whole-cell patch clamp experiments in acute brain slices. Our system implements a computer vision-based algorithm to detect fluorescently labeled cells and to target them for fully automatic patching using a micromanipulator and internal pipette pressure control. The entire process is highly automated, with minimal requirements for human intervention. Real-time experimental information, including electrical resistance and internal pipette pressure, are documented electronically for future analysis and for optimization to different cell types. Although our system is described in the context of acute brain slice recordings, it can also be applied to the automated image-guided patch clamp of dissociated neurons, organotypic slice cultures, and other non-neuronal cell types.

This protocol describes how to conduct automatic image-guided patch-clamp experiments using a system recently developed for standard in vitro electrophysiology equipment.

뇌 조각에서 뉴런 연구를위한 자동 이미지 유도 패치 클램프의 적용

Whole-cell patch clamp is the gold-standard method to measure the electrical properties of single cells. However, the in vitro patch clamp remains a ...

Auditory Brainstem Response and Outer Hair Cell Whole-cell Patch Clamp Recording in Postnatal Rats

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to amplify the weak vibration of low-level sound signal. The morphology and electrophysiological property of outer hair cells (OHCs) develop in early postnatal ages. The maturation of outer hair cell may contribute to the development of the auditory system. However, the process of OHCs development is not well studied. This is partly because of the difficulty to measure their function by an electrophysiological approach. With the purpose of developing a simple method to address the above issue, here we describe a step-by-step protocol to study the function of OHCs in acutely dissociated cochlea from postnatal rats. With this method, we can evaluate the cochlear response to pure tone stimuli and examine the expression level and function of the motor protein prestin in OHCs. This method can also be used to investigate the inner hair cells (IHCs).

This protocol describes methods to record the auditory brainstem response from postnatal rat pups. To examine the functional development of outer hair cells, the experimental procedure of whole-cell patch clamp recording in isolated outer hair cells is described step-by-step.

청각 Brainstem 응답 및 출생 후 쥐에 외부 머리 세포 전체 셀 패치 클램프 기록

The outer hair cell is one of the two types of sensory hair cells in the mammalian cochlea. They alter their cell length with the receptor potential to ...