This work was supported by NIH R01 EY020533, WSU Startup Fund, and RPB grants.

윤리 문 : 동물 과목을 포함하는 절차는 웨인 주립 대학의 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었다. 실험 솔루션 1. 준비 <ol><li> 실제 실험 전에 해부 솔루션을 일일 1 주를 준비합니다. 때문에 낮은 온도 (16)에서 강력한 완충 능력 망막 박리에 대한 HEPES 완충 용액을 사용합니다. (mm 단위)으로는 다음과 모든 화학 물질을 혼합 : 115의 NaCl, 2.5의 KCl, 2.5 CaCl2를, 1.0의 MgCl 2, 10 HEPES, 28 포도당을. NaOH로 pH 7.4까지를 조정합니다. 일주에 냉장고까지의 솔루션을 유지합니다. </li><li> 기록 액 망막 17 제제 용으로 설계된 인공 뇌척수액 (ACSF)이다 에임스 &#39;매체이다. 실험의 날에 의해, 튜브에 복귀 돌연변이 분말 (500 ml의 용액 4.4 g)을 달아. 또한,의 NaHCO3 (500 ml의 솔루션을위한 0.95 g)을 달아 및 혼합MES &#39;분말. </li><li> 실험의 날에 의해 패치 클램프 레코딩을위한 세포 내 피펫 솔루션을 준비합니다. (mm 단위)으로는 다음과 모든 화학 물질을 혼합 : 111 K 글루코 네이트, 1.0 CaCl2를, 10 HEPES, 1.1 EGTA, 10의 NaCl, 1.0의 MgCl 2, 5 ATP-㎎, 1.0 GTP - 나. KOH으로 pH를 7.2로 조정합니다. 기존의 주사​​기 필터 피펫 솔루션을 필터링합니다. ~ 500 μL 씩을 확인하고 냉장고 나 냉동고에 보관합니다. </li></ol> 실험의 날 2. 준비 <ol><li> O / N 어두운 적응 어두운 공간에서 - (남, 8 주 오래 된 4, C57BL / 6J 또는 유사한 배경) 밤은 실험 전에, 새장에 마우스를 놓습니다. </li><li> 해부를위한 준비 : <ol><li> 적어도 10 분 동안 산소, 유리 비커에 - (200 ml의 100) 얼음에 배치, 거품 솔루션을 해부 입력합니다. </li><li> 망막 준비 저장 어둠 상자의 산소를 시작합니다. </li><li> 재에 대한 그리스 레일 플라스틱 커버 슬립을 준비tinal 슬라이스 제제는 35mm 플라스틱 접시에 이들 각각을 놓고. </li><li> 헬기 (조직 슬라이서)에 새로운 면도날을 연결합니다. </li><li> 두 반으로 여과막을 잘라. 이것은 망막 슬라이스입니다. </li><li> 해부 현미경 근처 해부 도구 및 전송 피펫을 맞 춥니 다. 각 도구를 쉽게 어두운 액세스 할 수 있도록 작업 영역은 잘 조직되어야한다. </li></ol></li><li> 패치 클램프 레코딩을위한 준비 : <ol><li> 에임스 &#39;가루와 증류수의 NaHCO3 (에임스 4.4 g 및 NaHCO3을 0.95g / 500㎖의 DDH 2 O)를 용해. 혼합 산소 버블 에임스 중형 (95 % O 2 및 5 % CO 2) 적어도 30 분 동안 기록 온도 레벨로 가열 (~ 35 ° C). 아직 버블 링 동안의 NaHCO3와 7.4 pH를 조정합니다. </li><li> 해부하는 동안 내부 피펫 솔루션을 녹여. 해동 후 세포 내 염색 0.01 %의 sulforhodamine의 B를 추가합니다. </li></ol></li></ol> 3. 망막 해부 <ol><li> 어두운 방에서, 이산화탄소를 이용하여 양측 기흉 마우스를 안락사. 2 - 후 이산화탄소를 사용하여 3 분간, 마우스가 의식을 잃게 될 것이다. </li><li> 마우스가 더 이상 꼬리 핀치에 응답 할 때, 신속하게 눈을 명백히하다 없으며 플라스틱 접시에 차가운 해부 솔루션에 배치합니다. 동물의 죽음을 확인하기 위해 양측 기흉을 수행합니다. 빨리 어둠 상자에 접시에 눈을 저장합니다. 어둠의 절차를 수행하기 위해 적외선 뷰어를 사용합니다. 참고 : 또는 이소 플루 란, 참수, 또는 자궁 경부 전위는 안락사에 사용할 수 있습니다. 안락사의 방법은 국립 보건 연구소 (NIH) 및 / 또는 기관 동물 관리 및 사용위원회의 지침을 따라야합니다. </li><li> 어두운 방에서, 해부 현미경 및 적외선 조명을 조정합니다. 현미경, 10cm 플라스틱 접시 w로 그 접시에서 눈을 전송냉각 해부 솔루션 i 번째. 지속적으로 튜브를 통해 거품 산소는 플라스틱 접시의 바닥에 배치. 너무 부족하지 않도록 유량을 조절하지만 절개를 방해 충분하지 있는지 확인합니다. </li><li> 각막과 렌즈 제거 : <ol><li> 마이크로 수술 칼 각막의 상단에 절개를합니다. 안구의 다른 쪽 끝에서 시신경을 잡고 이동하는 현상을 방지 할 수 있습니다. 수술 가위를 사용하여 절단을 확장합니다. 주위 몇 공막과 각막을 잘라. </li><li> 미세 집게로 렌즈를 잡고 천천히 렌즈를 빼냅니다. 유리체가 렌즈를 분리하기에 너무 끈적 경우 가위로 절단 유리체. </li><li> 눈 컵이 이루어지면, 부드럽게 작은 전송 피펫과 아이 컵에 차가운, 산소 해부 솔루션을 붓는다. 참고 : 절개가 각막을 한 후 안구가 빠르게 움 추러 들게합니다. 그것은이 프로시 변형 눈으로 눈의 형상을 유지하는 것이 중요시저는 망막이 손상되어 망막 박리의 원인이됩니다. </li></ol></li><li> 망막의 지느러미와 복부 측면을 확인 : 참고 : 녹색 및 자외선 (UV)의 불균일 한 분포가 특정 망막 영역을 식별, 18 콘 때문에 빛 응답 레코딩을위한 중요합니다. <ol><li> 시신경 유두 근처에 전달하는 망막 아이 컵을 가로 질러가는 라인을 확인합니다. 이 라인은 주로 시신경의 복부 측면을 가로 질러 통과; 다른 19 복부 동안 시신경 포함 측은 등쪽이다. 적외선 뷰어에서 명확 랜드 마크를 참조하십시오. </li><li> 실험의 목적에 따라, 불필요한 측을 잘라. 이것은 아이 컵으로부터 망막을 분리 후 유용 할 것이다. </li></ol></li><li> 유리체 제거 : <ol><li> 선택적으로, 15 분 동안 히알루로니다 제 (0.5 ㎎ / ㎖)와 눈 컵을 배양한다. </li><li> 여분의 미세 집게로 유리체를 제거합니다. 유리체는 주로바닥과 눈 컵의 바깥 쪽 가장자리에 연결합니다. 조심스럽게 만지거나 망막을 파고하지 않고 제거합니다. 유리체가 단단히 아이 컵에 부착 할 수 있듯이, 눈 컵에서 망막 박리를 방지하기 위해 조심스럽게 제거합니다. 더 긴장 느낌되지 않을 때까지 당겨 계속합니다. 주 :이 단계는 마우스를 해부 망막 특히 중요하다; 그러나, 이러한 쥐 또는 도마뱀 같은 다른 종의 망막에 대한 문제가 아니다. </li></ol></li><li> 눈 컵에서 망막을 분리합니다. 공막을 잡고 부드럽게 집게의 뒷면을 사용하여 망막을 벗겨. 참고 : 망막 준비를 전체 마운트는 분리 된 망막에서 할 수있다. 가장자리에서 3-4 컷을 확인하고 망막을 평평하게, 또는 여러 조각으로 망막을 잘라. </li><li> 망막 트림 및 망막의 불필요한 절반을 버리고 (단계 3.5). 같은 플라스틱 접시에 두 조각으로 남아있는 망막을 잘라. 망막의 각 부분의 경우, 수 있도록 가장자리를 잘라망막 슬래브 및 접는 자리를 떼어. </li><li> 큰 전송 피펫 (~ 2 ㎖)를 사용하여 유리 접시에 망막 슬래브를 전송합니다. 작은 피펫으로 용액을 과량 빨아 슬래브를 평탄화 여과지를 사용하고 빠르게 조직 위에 여과막의 조각을 배치. <ol><li> 작은 전송 피펫을 사용하여 필터 멤브레인 솔루션을 해부 한 방울을 배치합니다. 이 솔루션은 막 퍼져 때까지 ~ 15 초를 기다립니다. 이 시간 동안, 망막 조직 여과막 고집한다. </li><li> 아래의 필터 주위에 더 차가운 해부 솔루션을 붓는다. 여과막과 망막 조직이 뜬다. 집게로 필터 멤브레인을 잡고 슬라이스 챔버에 배치합니다. 조직을 통해 솔루션을 해부 붓고, 어두운 상자에 준비를 저장합니다. 참고 : 여기에 설명 된 절차는 매우 중요하다. 그것은 보장하기 위해 신속하게 다음 단계를 수행하는 것이 중요하다 필터 (MEM)에 망막 조직 스틱파괴 현상과 건조하지 않습니다. </li></ol></li><li> 헬기를 사용하여 조각으로 망막 조직을 잘라. 약 200 μm의 두께는 패치 클램프 연구 가능하다. </li><li> 슬라이스 챔버 그리스 레일 플라스틱 커버 슬립을 놓습니다. 플라스틱 커버 슬립에 그리스 레일 위에 망막 슬라이스를 전송합니다. 횡단면 볼 수 있도록 슬라이스를 90 ° 회전합니다. 다음, 커버 슬립에 내려 여과막을 눌러 그리스 여과지의 측면을 커버. <ol><li> 슬라이스가 플라스틱 커버 슬립 상에 고정화 된 후, 커버 슬립을 잡아 35mm 플라스틱 접시에 옮긴다. 망막 준비 위에 차가운 해부 솔루션을 붓고 준비 잠수함. 지속적으로 산소해야 어두운 상자에 각각의 접시를 저장합니다. 주 : 전체 절차는 여과막이 변형되지 않도록 조심스럽게 수행되어야한다. 그렇지 않으면, 슬라이스 망막 용이 여과막으로부터 분리한다. </li></ol></li></ol><ol><li> 준비 기록 : <ol><li> 에임스 &#39;매체와 총리는 관류 튜브. 튜브를 채울 때 모든 거품이 통과하도록 허용합니다. </li><li> 풀러와 패치 클램프 녹음 피펫을 확인합니다. 피펫 팁이 채워 때까지 1 분 동안 피펫의 뒷면에있는 피펫 솔루션, 딥 피펫을 작성합니다. 다음에, 마이크로 피펫을 이용하여 필러의 피펫 ~ 1 / 3의 채우기. 촉촉한 피펫 상자에 각각의 펫을 저장합니다. </li><li> 패치 클램프 녹음을위한 장비를 켜십시오; 컴퓨터, 앰프, CCD 카메라와 현미경을 포함. </li></ol></li><li> 실내 조명을 차단합니다. 적외선 뷰어를 사용하여 현미경 스테이지 챔버 상 망막 슬라이스 준비를 놓습니다. 이 고정화 된 후, 연속 관류를 시작한다. 33-37 ° C에서 재관류 온도를 설정합니다. </li><li> CCD 카메라로 슬라이스 표면을 볼 수 있습니다. 대상 위치에 초점 위치를 목표 CELL 유형이 존재합니다. 패치 클램프 녹음을위한 건강한 보이는 셀 선택 (즉, 그것의 자연스러운 표면과 좋은 세포 모양이 있어야합니다). </li><li> 피펫 홀더에 기록 피펫을 놓습니다. 슬라이스 준비에 피펫을 사전. 그것이 슬라이스 제제 (~ 2mm 위)에 가까운 경우에는, 현미경 피펫 팁을 찾기. 피펫 팁은 현미경으로 볼되면, 타겟 셀을 향해 끝이 아래로 이동. </li><li> 앰프를 설정합니다. (제로) 0 MV / 0 펜실베니아 피펫을 조정하고 10 Hz에서 ~에 ~ 5 MV의 연속 전기 펄스를 시작합니다. 피펫 저항을 확인; 이상적인 저항은 대부분의 망막 신경 세포에 대한 5 ~ 10 MΩ입니다. </li><li> 끝에서 불고 시작합니다. 마우스 피스를 사용하거나 피펫은 목욕 솔루션으로 떨어지면 긍정적 인 압력을 적용, 주사기를 사용합니다. 끝이 표적 세포의 표면에있는 동안 연속으로 내부 솔루션을 날려. <ol><li> 양압 차에 작은 딤플을하면전자의 세포 표면, 약간의 팁을 발전 불고 중지합니다. 증가 할 전망 피펫 저항을 확인합니다. 그림 계속 증가하면두고 그것을&gt; 1 GΩ (기가 시일)에 도달 할 때까지 저항을 모니터링한다. 저항이 자발적으로 증가하지 않는 경우가 될 때까지 기가 시일 부드럽게 부압을 적용한다. </li></ol></li><li> 기가 시일이 달성 된 후, MV를 -70 잡고 잠재력을 변경합니다. 그런 다음, 간헐적으로 피펫 팁 내부의 막을 파열 부정적인 압력을 적용합니다. 전체 셀 구성이 이루어지면, 피펫 저항이 500 MΩ 1 GΩ 및 용량 성 전류가 볼 사이 일 수있다. 때로는 자발적인 시냅스 후 전류를 관찰 할 수있다. </li><li> -80에서 +40 MV에 IV 관계를 기록한다. 전압 게이트 된 채널의 종류는 세포 종류에 따라 활성화된다. </li><li> 기록 시냅스 전류 또는 전압을 빛-유발. </li></ol>

<ol>
	<li>Werblin, F. S., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+retina+of+the+mudpuppy,+Necturus+maculosus+II.+Intracellular+recording.">Organization of the retina of the mudpuppy, Necturus maculosus II. Intracellular recording.</a> J Neurophysiol. 32 (3), 339-355 (1969).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annu Rev Physiol. 46, 455-472 (1984).</li><li>Lukasiewicz, P., Werblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+slowly+inactivating+potassium+current+truncates+spike+activity+in+ganglion+cells+of+the+tiger+salamander+retina.">A slowly inactivating potassium current truncates spike activity in ganglion cells of the tiger salamander retina.</a> J Neurosci. 8 (12), 4470-4481 (1988).</li><li>Kaneko, A., Tachibana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+L-glutamate+on+the+anomalous+rectifier+potassium+current+in+horizontal+cells+of+Carassius+auratus+retina.">Effects of L-glutamate on the anomalous rectifier potassium current in horizontal cells of Carassius auratus retina.</a> J Physiol. 358, 169-182 (1985).</li><li>Kamermans, M., Werblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA-mediated+positive+autofeedback+loop+controls+horizontal+cell+kinetics+in+tiger+salamander+retina.">GABA-mediated positive autofeedback loop controls horizontal cell kinetics in tiger salamander retina.</a> J Neurosci. 12 (7), 2451-2463 (1992).</li><li>Cook, P. B., McReynolds, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+inhibition+in+the+inner+retina+is+important+for+spatial+tuning+of+ganglion+cells.">Lateral inhibition in the inner retina is important for spatial tuning of ganglion cells.</a> Nat Neurosci. 1 (8), 714-719 (1998).</li><li>Pang, J. J., Gao, F., Wu , J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-evoked+current+responses+in+rod+bipolar+cells,+cone+depolarizing+bipolar+cells+and+AII+amacrine+cells+in+dark-adapted+mouse+retina.">Light-evoked current responses in rod bipolar cells, cone depolarizing bipolar cells and AII amacrine cells in dark-adapted mouse retina.</a> J Physiol. 558 (Pt. 558 (Pt 3), 897-912 (2004).</li><li>Euler, T., 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=Light-evoked+responses+of+bipolar+cells+in+a+mammalian+retina).">Light-evoked responses of bipolar cells in a mammalian retina).</a> J Neurophysiol. 83 (4), 1817-1829 (2000).</li><li>Berntson, A., Taylor , W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+characteristics+and+receptive+field+widths+of+on-bipolar+cells+in+the+mouse+retina.">Response characteristics and receptive field widths of on-bipolar cells in the mouse retina.</a> J Physiol. 524 Pt. 524 (Pt 3), 879-889 (2000).</li><li>Sterling, P., Smith, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+for+a+binary+synapse.">Design for a binary synapse.</a> Neuron. 41 (3), 313-315 (2004).</li><li>Borghuis, B. G., Marvin, J. S., Looger, L. L., Demb, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+nonlinear+glutamate+release+dynamics+at+bipolar+cell+synapses+in+the+mouse+retina.">Two-photon imaging of nonlinear glutamate release dynamics at bipolar cell synapses in the mouse retina.</a> J Neurosci. 33 (27), 10972-10985 (2013).</li><li>Dowling, J. E., Sidman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inherited+retinal+dystrophy+in+the+rat.">Inherited retinal dystrophy in the rat.</a> J Cell Biol. 14, 73-109 (1962).</li><li>Eggers, E. D., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA(A),+GABA(C)+and+glycine+receptor-mediated+inhibition+differentially+affects+light-evoked+signalling+from+mouse+retinal+rod+bipolar+cells.">GABA(A), GABA(C) and glycine receptor-mediated inhibition differentially affects light-evoked signalling from mouse retinal rod bipolar cells.</a> J Physiol. 572 (Pt 1), 215-225 (2006).</li><li>Ichinose, T., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mode+of+retinal+presynaptic+inhibition+switches+with+light+intensity).">The mode of retinal presynaptic inhibition switches with light intensity).</a> J Neurosci. 32 (13), 4360-4371 (2012).</li><li>Ichinose, T., Fyk-Kolodziej, B., Cohn, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+ON+cone+bipolar+cell+subtypes+in+temporal+coding+in+the+mouse+retina.">Roles of ON cone bipolar cell subtypes in temporal coding in the mouse retina.</a> J Neurosci. 34 (26), 8761-8771 (2014).</li><li>Baicu, S. C., Taylor, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-base+buffering+in+organ+preservation+solutions+as+a+function+of+temperature:+new+parameters+for+comparing+buffer+capacity+and+efficiency.">Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency.</a> Cryobiology. 45 (1), 33-48 (2002).</li><li>Ames, A., Nesbett, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+retina+as+an+experimental+model+of+the+central+nervous+system.">In vitro retina as an experimental model of the central nervous system.</a> J Neurochem. 37 (4), 867-877 (1981).</li><li>Haverkamp, S., 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=The+primordial,+blue-cone+color+system+of+the+mouse+retina.">The primordial, blue-cone color system of the mouse retina.</a> J Neurosci. 25 (22), 5438-5445 (2005).</li><li>Wei, W., Elstrott, J., Feller, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+targeted+recording+of+GFP-expressing+neurons+for+light+responses+and+live-cell+imaging+in+the+mouse+retina.">Two-photon targeted recording of GFP-expressing neurons for light responses and live-cell imaging in the mouse retina.</a> Nat Protoc. 5 (7), 1347-1352 (2010).</li><li>Farre, C. G., M Bruggemann, A., Fertig, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channel+screening+-+automated+patch+clamp+on+the+rise.">Ion channel screening - automated patch clamp on the rise.</a> Drug discovery today. Technologies. 5 (1), e1-e34 (2008).</li></ol>

The authors have nothing to disclose.

Good recordings can only be achieved with good retinal preparations and well-designed patch clamp setups. Although all the steps described above are important, the discussion highlights some critical steps both for the dissection and recordings. 
For dissection, two things are especially important: cooling and oxygenation. After enucleating the eye, quickly remove the front part of the eye in a dissecting chamber with oxygen-bubbled, cooled dissecting solution, and pour cold solution into the eye-cup. When the retinal tissue is in the dissecting chamber, cold solution should be poured on the tissue every 10 min. The tissue should be sliced quickly without pulling or poking the retinal tissue during the dissection, which could easily damage its structure. Using good dissecting tools (sharp forceps and scissors) is also crucial. A chopper is equally as effective as a vibratome because it allows for speedy maneuvering. Extra-fine forceps should be used to remove vitreous matter as efficiently as possible because residual vitreous damages the retinal structure when slicing the tissue.
Beginners tend to damage the retina when removing the lens. The retina can detach from the eye-cup, or can even be accidentally removed with the lens. To avoid this issue, the sclera can be cut around the Ora Serrata which is located close to the extra-ocular muscle attachment point. An eye-cup with a wide opening makes it easier to remove the lens. Also, it is important not to deform the eye when removing the front portion. Another critical point is fixing the retinal slice onto a coverslip. This can be facilitated by practicing rotating the filter paper without distorting it to prevent the retinal slice from coming off during the experiment. 
A sturdy, well-built rig is crucial for patch clamp recordings. It must be free of electric noise, vibration, and pipette drifting. In addition, light response can be recorded in dark conditions to prevent desensitization. Our cage is made with a metal frame and aluminum panels with black plastic drapes that act as an electric shield while also completely blocking light from entering the cage; computer monitor illumination will not affect the retinal preparation. Use a remote controller for the pipette manipulator and close the cage while setting up the patch clamp configuration, which also avoid illuminating the preparation.
Although we have been tried to simplify the procedures involved, patch clamp study is still a difficult experiment that requires extensive knowledge and training. An automated patch clamp has been developed by some groups20; this system is applicable to dissociated cells and might be an efficient technique for pharmacological screenings. 
In summary, patch clamp recording in dark-adapted retinal slice preparations is a useful technique to investigate visual information processing. Compared to other methods, patch clamp offers better temporal and spatial resolution. Additional technical innovations will make the patch clamp method more user-friendly in the near future.

The retina is one of the unique parts of the nervous system. As an accessible part of the brain, its synaptic architecture has been well characterized. In addition, the functions of this neural network can be examined with a physiological stimulus: light. If the retinal tissue is isolated in a dark room with appropriate procedures, neurons in the tissue will respond to light. This preparation has been used to study visual signal processing and elucidate various synaptic mechanisms and neural network functions, as well as disease mechanisms. 
Light responses in retinal neurons have been recorded for decades. Early studies used sharp electrodes to make intracellular recordings from mudpuppy retinal neurons1. In the 1980s, the patch clamp technique was invented2, and soon became a popular method among vision researchers3,4. Single cell recordings from lower vertebrates, including mudpuppy and fish retinal neurons, were popular methods that contributed to the elucidation of visual signal processing mechanisms5,6. 
After genetic mutation techniques were developed, the mouse retina became a more popular model for vision researchers7-9. The mammalian retina is more attractive than that of lower vertebrates because it is evolutionarily closer to the human retina, and there is an opportunity to use disease models. However, mouse retinal cells are small and fragile10, and making retinal preparations and conducting patch clamp recordings in a dark room is challenging. As technology has improved, diverse approaches have become available to study visual signaling mechanisms such as imaging studies11 and the electroretinogram (ERG)12. Nevertheless, single cell recording with the patch clamp method is still important because it is highly temporally and spatially sensitive compared to other methods. Therefore, we have continuously conducted patch clamp recordings and improved our methods to investigate visual signal processing in mouse retinal slice preparations13-15.
In this video tutorial, the protocols are presented with important tips. Good recordings can only be achieved with good preparation. Practicing animal dissection and building a sturdy patch clamp rig will enable most researchers to achieve successful recordings.

<table border="0" cellpadding="0" cellspacing="0" width="886">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:145px;">Company</td>
			<td style="width:203px;">Catalog Number</td>
			<td style="width:323px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mice (28-60 days old, male)</td>
			<td style="width:145px;">Jackson laboratory</td>
			<td style="width:203px;">C57BL/6J strain</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ames&#39; medium powder</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:203px;">A1420</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Stereo microscope</td>
			<td style="width:145px;">Nikon</td>
			<td style="width:203px;">SMZ745</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_forceps</td>
			<td style="width:145px;">Dumont</td>
			<td style="width:203px;">#4, #5, #55</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_scissors</td>
			<td style="width:145px;">Roboz</td>
			<td style="width:203px;">RS-5605</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dissecting tool_surgery knife</td>
			<td style="width:145px;">Surgistar</td>
			<td style="width:203px;">7514</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">razor blade (for chopper)</td>
			<td style="width:145px;">EMS</td>
			<td style="width:203px;">71970</td>
			<td>excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chopper</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">infrared viewer</td>
			<td style="width:145px;">Night Owl Optics</td>
			<td style="width:203px;">NOBG1</td>
			<td>It shows bright view.&#160; Focusing small objects is an issue.</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:216px;">infrared pocket scopes</td>
			<td style="width:145px;">B.E. Meyers</td>
			<td style="width:203px;">OWL Gen 3 NV pocketscope</td>
			<td>excellent view</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">puller</td>
			<td style="width:145px;">Sutter</td>
			<td style="width:203px;">P-1000</td>
			<td>excellent.&#160; Make consistent size pipettes.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dark box</td>
			<td style="width:145px;">Pelican</td>
			<td style="width:203px;">dark box</td>
			<td>excellent</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">patch clamp system</td>
			<td style="width:145px;">Scientifica</td>
			<td style="width:203px;">slice scope 2000</td>
			<td style="width:323px;">Excellent setup.&#160; Most key components are included in one package.&#160; Micromanipulators are excellent.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amplifier</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">multiclamp 700B</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">acquiring software</td>
			<td style="width:145px;">Molecular Devices</td>
			<td style="width:203px;">pClamp software</td>
			<td>Excellent and easy control.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:216px;">light source (LED)</td>
			<td style="width:145px;">Cool LED</td>
			<td style="width:203px;">pE-2 4 channel system</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CCD camera</td>
			<td style="width:145px;">Q-imaging</td>
			<td style="width:203px;">Retiga 2000</td>
			<td>Excellent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Faraday cage</td>
			<td style="width:145px;">handmade</td>
			<td style="width:203px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

recording light-evoked postsynaptic responses in neurons in dark-adapted, mouse retinal slice preparations using patch clamp techniques

망막은 시각 체계의 관문이다. 영상 신호 처리 메커니즘을 이해하기 위해 망막 신경 네트워크 기능을 조사. 네트워크의 망막 신경 세포는 다양한 아형 포함한다. 양극성 세포, 신경절 세포 및 무 축삭 세포의 10 개 이상의 아형은 형태 학적 연구에 의해 확인되었다. 망막 뉴런의 여러 아형 모션 및 컬러 같은 시각적 신호의 뚜렷한 기능을 인코딩 및 다중 신경 경로를 형성하는 것으로 생각된다. 그러나, 영상 신호 처리의 각 신경 세포의 기능적 역할은 완전히 이해되지 않습니다. 패치 클램프 방법이 근본적인 문제를 해결하는 데 유용합니다. 여기에, 프로토콜을 제공한다 어두운 적응 조건에서 패치 클램프 녹음을 사용하여 마우스 망막 신경 세포에서 빛 유발 시냅스 응답을 기록합니다. 마우스 눈 O / N 암순응이며, 망막 슬라이스 제제는 적외선 조명을 이용하여 시청자 암실에서 해부. 적외선하지 않습니다마우스의 광 수용체를 활성화하고, 따라서 그들의 빛 응답 성을 유지합니다. 패치 클램프 망막 뉴런의 광 유발 된 응답을 기록하는데 사용된다. 형광 염료는 신경 학적 아형의 특성을 기록하는 동안 분사된다. 이 절차는 마우스 망막에 각각의 신경 세포의 생리 학적 기능을 결정하기 위해 우리가 할 수 있습니다.

대표적인 제제는 슬라이스 슬라이스 제제 평면에서 신경절 세포를 나타내는 감광체, 직선 위치에있다.도 1에 도시하지 않고, 여과지로부터 박리된다. 슬라이스가 기울어 진 경우, 제제의 일부만 어려운 패치 클램프 적절한 셀을 식별 할 수있는 초점이다. 레코딩을위한, 일반적으로 빛나는 표면을 갖는 좋은 소마, 심지어 둥근 형상 및 가시적 어두운 플라크를 선택하는 것이 중요하다. 전체 셀 구성이 이루어진 후, 슬라이스는 스텝 - 광 (도 2) 다음에 배경 광에 노출시켰다. 빛 유발 흥분성 시냅스 후 전위 (L-EPSPS)는 (타이밍이 노란색으로 표시된다) 단계 빛에 반응 유발했다. 피크 진폭과 세포 유형 및 하위 유형에 따라 변할 감쇠 시간. 신경절 세포는 밝은 자극 (그림 2 D &amp; E)에 대한 응답으로 활동 전위를 생성합니다. CELL 형과 형태 학적 아형 생리 학적 기록 (오른쪽 그림 2) 후 sulforhodamine 라벨에 의해 밝혀졌다. <img alt="그림 1" src="/files/ftp_upload/52422/52422fig1highres.jpg" width="650" /> 그림 1. 망막 슬라이스 준비. (A) 10 배 목적으로 볼 망막 슬라이스 준비. 망막 슬라이스 준비는 (위) 여과지 (아래)의 조각에 부착했다. (B) 60X 목적으로 볼 망막 슬라이스 준비를 보여주는 네 이미지 편집. 각 셀 층이 명확하게 관찰된다 (: 외부 핵 층, OPL : 외부 망상 층, INL : 내부 핵 층, IPL : ONL 내부 얼기 층, GCL : 신경절 세포 층) <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52422/52422fig1highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 그림 망막 신경 세포 2. 빛 유발 흥분성 시냅스 후 전위 (L-EPSPS). 스텝 빛 L-EPSPS (왼쪽) 유발. 60 % 웨버 대비 - 빛의 강도는 30이었다. 배경 빛 적응 수준은 4 × 10 4 광자 / μm의 2 / 초였다. Sulforhodamine B (오른쪽) 기록 된 신경 세포를 시각화 기록 패치 클램프 동안 주입 하였다. 기록 피펫은 여전히 ON 콘 양극 세포에서 소마 (A) L-EPSPS와 sulforhodamine 염색에 부착했다. (B) OFF 콘 바이폴라 셀. (C) 무 축삭 세포. 신경절 세포 ON (D). 신경절 세포 OFF (E). 언급 한 바와 같이 스케일 바는 2 또는 5 MV를 나타냅니다. 빛 자극이 1 초 동안 적용되었다. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52422/52422fig2highres.jpg" target="_blank">여기를 클릭하십시오</a>이 그림의 더 큰 버전을 볼 수 있습니다.

Watch this Scientific Journal Video about Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques at JoVE.com

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

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. ...

recording-light-evoked-postsynaptic-responses-neurons-dark-adapted

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.4K 조회수.  Wayne State University School of Medicine. 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.

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

Analyzing Responses of Mouse Olfactory Sensory Neurons Using the Air-phase Electroolfactogram Recording

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and other social interactions. The electroolfactogram (EOG) recording is an informative, easy to conduct, and reliable method to assay olfactory function at the level of the olfactory epithelium. Since the 1956 description of the EOG by Ottoson in frogs1, the EOG recording has been applied in many vertebrates including salamanders, rabbits, rats, mice, and humans (reviewed by Scott and Scott-Johnson, 2002, ref. 2). The recent advances in genetic modification in mice have rekindled interest in recording the EOG for physiological characterization of olfactory function in knock-out and knock-in mice. EOG recordings have been successfully applied to demonstrate the central role of olfactory signal transduction components3-8, and more recently to characterize the contribution of certain regulatory mechanisms to OSN responses9-12.
Odorant detection occurs at the surface of the olfactory epithelium on the cilia of OSNs, where a signal transduction cascade leads to opening of ion channels, generating a current that flows into the cilia and depolarizes the membrane13. The EOG is the negative potential recorded extracellularly at the surface of the olfactory epithelium upon odorant stimulation, resulting from a summation of the potential changes caused by individual responsive OSNs in the recording field2. Comparison of the amplitude and kinetics of the EOG thus provide valuable information about how genetic modification and other experimental manipulations influence the molecular signaling underlying the OSN response to odor. 
Here we describe an air-phase EOG recording on a preparation of mouse olfactory turbinates. Briefly, after sacrificing the mouse, the olfactory turbinates are exposed by bisecting the head along the midline and removing the septum. The turbinate preparation is then placed in the recording setup, and a recording electrode is placed at the surface of the olfactory epithelium on one of the medial turbinates. A reference electrode is electrically connected to the tissue through a buffer solution. A continuous stream of humidified air is blown over the surface of the epithelium to keep it moist. The vapor of odorant solutions is puffed into the stream of humidified air to stimulate the epithelium. Responses are recorded and digitized for further analysis.

The electroolfactogram (EOG) recording is an informative, easy-to-conduct, and reliable way of assessing olfactory function at the level of the olfactory epithelium. This protocol describes a recording setup, mouse tissue preparation, data collection, and basic data analysis.

에어 - 상 Electroolfactogram 기록을 사용하여 마우스 강한 감각 뉴런의 분석 응답

Animals depend on olfaction for many critical behaviors, such as finding food sources, avoiding predators, and identifying conspecifics for mating and ...

연구

신경과학

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

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

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 ...

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 ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

패치 - 클램프 커패시턴스 측정 및 CA 2 +</sup망막 조각의 단일 신경 터미널에서> 영상

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within the same species 1,2. These intraspecies signals, the pheromones, as well as signals from some predators 3, activate the vomeronasal sensory neurons (VSNs) with high levels of specificity and sensitivity 4. At least three distinct families of G-protein coupled receptors, V1R, V2R and FPR 5-14, are expressed in VNO neurons to mediate the detection of the chemosensory cues. To understand how pheromone information is encoded by the VNO, it is critical to analyze the response profiles of individual VSNs to various stimuli and identify the specific receptors that mediate these responses.
The neuroepithelia of VNO are enclosed in a pair of vomer bones. The semi-blind tubular structure of VNO has one open end (the vomeronasal duct) connecting to the nasal cavity. VSNs extend their dendrites to the lumen part of the VNO, where the pheromone cues are in contact with the receptors expressed at the dendritic knobs. The cell bodies of the VSNs form pseudo-stratified layers with V1R and V2R expressed in the apical and basal layers respectively 6-8. Several techniques have been utilized to monitor responses of VSNs to sensory stimuli 4,12,15-19. Among these techniques, acute slice preparation offers several advantages. First, compared to dissociated VSNs 3,17, slice preparations maintain the neurons in their native morphology and the dendrites of the cells stay relatively intact. Second, the cell bodies of the VSNs are easily accessible in coronal slice of the VNO to allow electrophysiology studies and imaging experiments as compared to whole epithelium and whole-mount preparations 12,20. Third, this method can be combined with molecular cloning techniques to allow receptor identification.
Sensory stimulation elicits strong Ca2+ influx in VSNs that is indicative of receptor activation 4,21. We thus develop transgenic mice that express G-CaMP2 in the olfactory sensory neurons, including the VSNs 15,22. The sensitivity and the genetic nature of the probe greatly facilitate Ca2+ imaging experiments. This method has eliminated the dye loading process used in previous studies 4,21. We also employ a ligand delivery system that enables application of various stimuli to the VNO slices. The combination of the two techniques allows us to monitor multiple neurons simultaneously in response to large numbers of stimuli. Finally, we have established a semi-automated analysis pipeline to assist image processing.

The vomeronasal organ (VNO) detects intraspecies chemical signals that convey social and reproductive information. We have performed Ca2+ imaging experiments using transgenic mice expressing G-CaMP2 in VNO tissue. This approach allows us to analyze the complicated response patterns of the vomeronasal neurons to large numbers of pheromone stimuli.

영상의 연결 응답 Vomeronasal 조직의 슬라이스 준비에 유전적으로 인코딩된 칼슘 센서를 표현

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

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 ...

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

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

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

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

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

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 ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

Deafferentated 마우스 망막의 플랫 마운트 준비에 별 모양 무 축삭 세포의 패치 클램프 녹음

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze motor-learning induced plasticity, we trained rats using an accelerated rotor rod task. Rats performed the task 10 times at 30-s intervals for 1 or 2 days. Performance was significantly improved on the training days compared to the first trial. We then prepared acute brain slices of the primary motor cortex (M1) in untrained and trained rats. Current-clamp analysis showed dynamic changes in resting membrane potential, spike threshold, afterhyperpolarization, and membrane resistance in layer II/III pyramidal neurons. Current injection induced many more spikes in 2-day trained rats than in untrained controls.
To analyze contextual-learning induced plasticity, we trained rats using an inhibitory avoidance (IA) task. After experiencing foot-shock in the dark side of a box, the rats learned to avoid it, staying in the lighted side. We prepared acute hippocampal slices from untrained, IA-trained, unpaired, and walk-through rats. Voltage-clamp analysis was used to sequentially record miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) from the same CA1 neuron. We found different mean mEPSC and mIPSC amplitudes in each CA1 neuron, suggesting that each neuron had different postsynaptic strengths at its excitatory and inhibitory synapses. Moreover, compared with untrained controls, IA-trained rats had higher mEPSC and mIPSC amplitudes, with broad diversity. These results suggested that contextual learning creates postsynaptic diversity in both excitatory and inhibitory synapses at each CA1 neuron.
AMPA or GABAA receptors seemed to mediate the postsynaptic currents, since bath treatment with CNQX or bicuculline blocked the mEPSC or mIPSC events, respectively. This technique can be used to study different types of learning in other regions, such as the sensory cortex and amygdala.

The slice patch clamp technique is an effective method for analyzing learning-induced changes in the intrinsic properties and plasticity of excitatory or inhibitory synapses.