이 작품은 NIH 그랜트 EY17606 (APS)에 의해 지원되었다.

1. 준비 전극 <ol><li> 1M NaCl의 모두를 수영하고 둘 사이 15V 운전 ~ 20 S.에 대한 1 Hz에서 사인 파를 사용하여 녹음과 참조 전극을 준비 </li><li> micropipette 풀러 (서터 인스 트루먼 트의 P - 97을)를 사용하여 기록 전극을 만듭니다. 패치 - pipettes은 1.2 mm의 외부 직경 0.69 mm (셔터 인 스트 루먼트, 고양이 # B120 - 69 - 10)의 내경과 borosilicate 유리를 사용하십시오. 전극의 끝부분을 통해 일련의 저항을 측정합니다. MΩ ~ 12 세포 기관에 대한 MΩ ~ 5 ~ 직경 5 μm의 사용 세포 기관에 대한 ~ 직경 20 μm의 사용, 피펫 팁 직경을 선택 대상 세포의 크기에 따라. </li><li> 지상 / 참조 전극을 준비합니다. 1M NaCl에서, 주사기를 사용하면 (CAT # A7002 시그마) 3.5 % 아가르를 포함 1 ㎜ NaCl과 작은 섹션 폴리에틸렌 튜브 (Intramedic, PE205)를 입력합니다. 자세 / AgCl 기준 전극을 통해이 한천 다리를 놓습니다. </li></ol> 2. 준비 솔루션 <ol><li> 외부 목욕 솔루션, 에임스 &#39;비카보네이트 (1 패)를 포함하는 미디어 : 에임 스의 1 병&#39;중간 (시그마, 고양이 # A1420), NaHCO 3 1.9 g 페니실린 - 스트렙토 마이신 7 ML (시그마, 고양이 # P0781) 세균 방지 성장. ~ 280 mOsm에 osmolarity를​​ 조정합니다. </li><li> 솔루션을 깔끔히, 에임 &#39;산도 버퍼 HEPES (1 패)를 포함하는 미디어 : 에임 스의 1 병&#39;매체, NaCl의 0.887 g, 2.38 g HEPES의 페니실린 - 스트렙토 마이신 7 ML. 1M NaOH 및 / 또는 1M HCL을 사용하여 ~ 7.4으로 산도를 조정합니다. ~ 280 mOsm에 osmolarity를​​ 조정합니다. </li><li> 칼륨 - Aspartate (K - ASP) 피펫 내부 솔루션 (MM)을 : 125 K - Aspartate은 10 KCl, 10 HEPES, 5 NMG - HEDTA, 0.5 CaCl 2, 1 ATP - MG, 0.2 GTP - MG, 산도가 조정됩니다 와 ~ 7.3 NMG는 - 아, 그리고 osmolarity는 280 mOsm ~하도록 조정됩니다. </li><li> 망막을 포함한 낮은 겔화 온도 아가로 오스 (무게로 5 %), 아가르의 7.5 G (시그마, 고양이 # A7002)를 안정화에 대한 아가르 역전은 DH 2 O. 150 ML에 녹아 </li></ol> 3. 실험의 날 <ol><li> 해동 ~ K - ASP 내부 솔루션과 냉동고에서 원하는 형광단 1.5 ML. 경험적으로 결정되어야하는 적절한 농도에 형광단을 희석. </li><li> 물 목욕 (30-32 ° C)에 가벼운 꽉 스토리지 컨테이너와 다음 장소로 NaHCO 3 (~ 200 ML)이있는 에임스 &#39;미디어를 붓고 5 %의 CO 2 / 95% O 2 가스를 평형 </li><li> 얼음 HEPES, 장소를 포함하는 에임스 &#39;미디어 ~ 110 ML과 병을 채우기 및 100 % O 2 평형. </li><li> 패러데이 케이지 위에 온수 저수지에서 NaHCO 3가 들어있는 나머지 에임스 &#39;미디어를 삽입하고, 5 % CO 2 / 95% O 2 기체와 평형. Parafilm는 솔루션 위의 공간에 가스를 트랩하는 데 사용할 수 있습니다. </li><li> HEPES와 ~ 115 ° C 아가로 오스를 녹기에서 열 솔루션 에임스 &#39;미디어의 25 ML한다 (CAT # A0701 시그마) 0.75 g을 추가하여 낮은 겔화 온도 아가로 오스 (3 %)를 준비합니다. 물을 욕조에서 거품없이 명확하고, 저장하는 솔루션까지 저어 (~ 42 ° C). </li><li> K - ASP 내부 솔루션과 함께 녹음 피펫을 채울 및 팁 저항을 확인하십시오. </li></ol> 4. 실험 시작 <ol><li> 마우스를 안락사 신속하게 곡선 가위를 사용하여 눈을 제거합니다. 적외선 고글은 시각 색소의 표백 방지하기 위해 모든 절차를 사용해야합니다. </li><li> 포셉로 시선을 누른 상태 압연, 그리고 얇은 이중 단면 블레이드를 사용을 방지하기 위해 필터 종이에 눈을 장소, 각막에 슬릿합니다. </li><li> 곧 눈알이 구멍이므로, 에임스 &#39;미디어 가득한 그릇에 사람들이 시선을 전송합니다. </li><li> 진입 점으로 각막에 슬릿을 사용하여, 작은 가위는 렌즈와 유리를 제거할 수있는 후 각막의 나머지 부분을 버려야하는 데 사용됩니다. 망막은 세안 컵에 부착된 상태, 32에서 어둠의 세안 컵을 저장 ° C를 5 % CO 2 / 95% O 2 equilibrated 에임스 &#39;미디어에주의하십시오. </li></ol> 5. 깔끔히 <ol><li> eyecups 중 하나를 Hemisect 및 망막 안료 상피에서 망막을 떼어 놓다. 조직이 평평 거짓말 수 있도록 망막의 가장자리를 제거합니다. </li><li> 녹인 한천과 함께 35mm 페트리 접시를 채우기 위해 라텍스 전구와 작은 유리 파스퇴르 피펫을 사용하고 일관성을 테스트하는 한천을 통해 전체 포셉를 끕니다. </li><li> 당신은 한천이 응고 볼 시작할 수 있습니다 경우 : <ol class="lalpha"><li> 한천의 상단에 망막을 전송합니다. </li><li> 망막 주위의 여분의 솔루션을 흡수 Kimwipe의 뒤틀린 조각을 사용합니다. </li><li> 장소 2-3 망막에 직접 아가르의 방울 신속하게 주위 벽을 형성하기 위해 망막을 통해 플라스틱 실린더를 놓으십시오. 한천 이내에 망막을 중심으로하면서 그런 다음 플라스틱 실린더의 측면 아래 추가 녹인 한천 드립. </li><li> 페트리 접시 T를 전송냉각 얼음 물 목욕 O. </li></ol></li><li> ~ 45 초 얼음 물을 욕조에서 조직을 제거하고 한천은 망막에서 멀리 측면에서 추진, 플라스틱 실린더 밖으로 블록 돌출하는 플런저를 사용하면. </li><li> 망막을 포함 한천의 작은 블록을 잘라 표본 디스크에있는 한천 역전에 대한 장소에 블록을 superglue하는 일방적인 면도날을 사용합니다. </li><li> 진동 마이크로톰 트레이에 표본 디스크를 전송하고 망막과 블록이 완전히 물 속에 잠긴 수 있도록 트레이에 얼음 차가운 에임스 HEPES 잔. </li><li> ~ 200 μm의의 두께 망막 조각은 최대 블레이드 진동 속도로 잘라 앞으로 블레이드 속도는 각각의 슬라이스에있는 망막을 통해 줄일 4-5초 걸립니다 이러한 설정할 수 있습니다. </li><li> 한 번에 하나씩 각 사용 가능한 슬라이스 하나를 제거하는 11 호 메스 블레이드를 사용하여 </li><li> 망막을 포함한 좋은 조각은 기록 챔버에 배치하고 그들을 건너 나일론 스레드와 슬라이스 앵커와 함께 아래 가중치 있습니다. 나일론 스레드는 레코딩을위한 가리지 망막을 떠나, 망막을 둘러싼 한천을 누르십시오. </li><li> 똑바로 현미경의 무대에 녹화 실로 전송, 커튼을 닫고 적외선에 민감한 카메라에 슬라이스를 볼 수있는 적외선 광원을 설정합니다. </li></ol> 6. 녹음 <ol><li> 일단 좋은 망막 슬라이스는 (그대로 photoreceptors하고 적절한 절단 각도) 속도로 망막을 perfusing 시작 확인된 35 37 가열 가열 에임스 &#39;매체와 이상 5 ML / 분 ° C와 5 % CO 2 equilibrated / 95% O 2. </li><li> 일부 표면 손상이 깔끔히 처리로 인해 뚜렷한 수 있습니다. 세포의이 일반적으로 얇은 레이어가 부러 팁있는 피펫을 사용하여 제거하고, 부드럽게 죽은 세포의 시체를 멀리 빠는 수 있습니다. 이것은 일반적으로 살 세포를 아래에 공개합니다. 그의 위치에 대한 관심의 지층에 위치하고 건강한 세포 기관을 식별합니다. </li><li> KAsp 내부 솔루션 micropipette을 입력하고 피펫의 팁을 통해 긍정적인 (외부) 압력을 적용하고, 아래 micromanipulator를 사용하여 세포의 수준으로 기록 챔버에 피펫의 팁을 낮은합니다. </li><li> 그것은 세포막을 보조개 이러한 피펫 팁을 이동하고, 높은 저항 인감 수 있도록 부드러운 부정 (안으로) 압력을 적용합니다. 전체 셀 모드는 세포에 침입하기 위해 전극에 부정적인 압력을 적용하여 얻을 수 있습니다. </li><li> LED를 사용하여 망막 조직을 자극하면 다음 빛에 반응을 보여주고 있습니다. </li><li> 녹음이 피펫 내부 솔루션에 형광단에서 형광을 evoking에 의해 만들어진있는 세포 모습을 사진에 담아보세요. </li></ol> 7. 대표 결과 <img src="/files/ftp_upload/2107/2107fig1.jpg" alt="그림 1" /> 그림 1. 여기는 어두운 적응 증가 강도의 빛을 깜박하는 망막 신경 세포 것이 표시됩니다의 가능성에 불을 - evoked. <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="그림 2" /> 그림 2. 세포가 형광단, 루시퍼 노란색으로 가득했습니다 망막 슬라이스에서 형광 사진. Evoked 형광이 패치 레코딩이되었다하는 세포의 형태를 보여줍니다.

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. e., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

관심 없음 충돌 선언하지 않습니다.

척추 망막에서 슬라이스 녹음 수십 년 1,2에 대해되었습니다, 그리고 망막 회로는 입사 광을 인코딩하는 방법에 깊은 이해를 제공하는 매우 성공적으로되었습니다. 진동 마이크로톰와 절삭 오히려 직접보다 면도날로의 장점은 망막 조각이 표면에 덜 손상을 초래한다는 것입니다. 흡입 피펫으로 그것은 단지 세포 유형의 대부분 5,6을 확인했습니다 망막 회로, 그들의 연결을 유지 ?...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>B120-69-10</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Intramedic</td>
<td>PE205</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7002</td>
<td>&nbsp;</td>
<tr>
<td>Low gelling temp agarose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0701</td>
<td>&nbsp;</td>
<tr>
<td>Ames' Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P0781</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

우리의 시각적 경험은 우리의 망막 photoreceptors의 시각 색소가 빛 에너지의 광자를 흡수하여 세포막의 순환 - 염기 - 문이 채널의 폐쇄로 이어질 세포 사건의 폭포를 시작하면 시작됩니다. 멤브레인 전위의 결과로 변경 막대와 원뿔 시냅스 터미널 모두에서 신경 전달 물질 방출의 양을 감소에 차례로 연결됩니다. photoreceptor 막 잠재력에 가벼운 evoked 변화가 망막에 하류 활동으로 연결 방법을 측정하기 위해 과학자들은 수십 년 1,2에 대한 망막 슬라이스 준비에서 electrophysiological 음반을 만들었습니다. 과거에는 이러한 조각은 거름종이에 첨부되어 망막 조직에 면도날로 수동으로 절단되었습니다;과 함께 최근 몇 년 동안 깔끔히의 또 다른 방법은 망막 조직이 낮은 겔화 온도 한천 배지에 포함된 의하여 개발되었으며 냉각 솔루션에 슬라이스 마이크로톰 3,4 진동. 이 준비가 덜 표면 손상이 매우 강력한 빛을 evoked 반응과 함께 망막 조각을 생산하고 있습니다. 여기 우리는이 절차는 시각 색소를 표백되지 않도록 적외선에 따라 할 수있는 방법을 문서.

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

여기 electrophysiological 레코딩을위한 마우스 망막의 어두운 적응 조각을 생성하는 절차를 설명합니다.

어두운 적응 슬라이스 준비에 마우스 망막 뉴런에서 패치 클램프 레코딩

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

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14.3K 조회수.  University of Southern California. 여기 electrophysiological 레코딩을위한 마우스 망막의 어두운 적응 조각을 생성하는 절차를 설명합니다.

비디오: Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

모터 신경 세포의 이점 패치 클램프 레코딩과 Zebrafish의 대상 골격근

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

연구

신경과학

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  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<sub&gt; 1</sub&gt; F<sub&gt; O</subSubmitochondrial 소포 막 패치 클램프 녹음을 공연&gt; ATPase의 소포의 준비와 기술

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

양서류 망막 슬라이스 준비 광수와 2 차 신경 세포에서 동시에 전체 세포 녹음

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

의 감각 뉴런의 분리<em&gt; 군소 californica</em&gt; 글루타메이트 전류의 패치 클램프 녹음을위한

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

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 ...

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

녹화 패치 클램프 기술을 사용하여 어두운 적응 마우스 망막 슬라이스 준비에 신경 세포의 시냅스 응답 빛-유발

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

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 ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

흑색질 도파민 뉴런의 Somatodendritic 도메인에서 세포 내 패치 클램프 녹음

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...