Authors would like to thank Peter Mombaerts for the generous gift of OR-GFP mice; Anne Lefranc and the CSGA animal facility for excellent animal care. Funding was provided by CNRS through an ATIP and ATIP Plus grants, by Conseil R&#233;gional de Bourgogne (FABER and PARI grants), by Universit&#233; de Bourgogne (BQR program).

이 프로토콜은 Université 드 부르고뉴의 동물 보호 지침을 다음과 Université 드 부르고뉴 윤리위원회에 의해 승인되었다. 1. 동물 <ol><li> 잭슨 연구소 가능한 유전자 조작 또는-IRES-tauGFP 마우스를 사용합니다. 이러한 마우스는 후각 기관 (19)의 축삭 타겟팅 및 개발을 분석하기 위해 닥터 피터 Mombaerts &#39;실험실에서 개발되었다. 예를 들어, MOR23-IRES-tauGFP 라인, 재고 번호 006643는 공식 변형 이름 B6 곰, 129P2- Olfr16 tm2Mom / MomJ을; 유사하게, SR1-IRES-tauGFP 라인은 재고 번호 6717의 공식 이름 B6 곰, 129P2- Olfr124 tm1Mom / MomJ을. </li><li> 동물의 연령에 대해서는 : 프로토콜의 더 나은 결과를 위해, 생후 2 4 주 사이에 동물을 사용한다. 이 연령 그룹에서 해부 쉽게 (부드러운 뼈, 탄력있는 후각 상피 세포)와 수지상 노브는 양방향입니다입니다gger 오래된 동물에 비교합니다. </li></ol> 전극 및 솔루션 2. 준비 <ol><li> 자극 피펫의 경우 : 구입 피펫을 자극 prepulled. 그렇지 않으면, 수동으로 준비합니다. <ol><li> 화염을 사용하여, 선단에서 약 1cm의 6 1mm 유리 피펫을 구부리. 구멍에 의해 강화 1.5 CM 열 수축 관에이 여섯 피펫 플러스 바로 7 번째를 삽입합니다. </li><li> 열은 배럴 함께 접착을 유지하기 위해 수축 배관. 배럴의 다른 말단에 추가로 열 수축 튜브를 연결합니다. 멀티 배럴 풀러와 함께이 자극 피펫을 당깁니다. </li><li> 굽​​ 팁을 강화하기 위해 구멍 주위에 약간의 흰색 액체 접착제를 추가합니다. 건조 O를 /의 N하자. </li></ol></li><li> 일반 링어의 세포 외 액의 1 또는 2 L 준비 (MM에서 : 염화나트륨 (124)의 KCl 3, 황산 1.3, CaCl2를 2, NaHCO3를 26의 NaH 2 PO 4 1.25, 포도당 (15), pH가 7.6, 305 mOsm). 4 ° C에서 유지사용할 때까지. </li><li> (MM)에 세포 원액 준비 : KCl을 70, KOH (53), 메탄 설 폰산 (30), EGTA 5, HEPES (10), 수 크로스 (70); pH가 7.2 (KOH), 310 mOsm. 사용까지 4 ℃에서 보관하십시오. 몇 주에 대 한 좋은. </li><li> 실험 전에 (마지막 순간) 즉흥적 니 스타틴과 세포 내 기록 솔루션을 준비합니다. <ol><li> DMSO의 50 μl를 추가, 니 스타틴의 3 mg의 무게; 완전히 희석 될 때까지 소용돌이 20 초 후 2 ~ 3 분을 초음파 처리. </li><li> 세포 내 원액 5ml에 DMSO-니 스타틴 용액 20 μl를 추가합니다. 소용돌이 20 초 후 3 분을 초음파 처리. 4 ° C에서이 솔루션을 유지하고 직사광선으로부터 보호, 니 스타틴은 빛에 민감한입니다. 이 용액을 몇 시간 동안 사용할 수있다. 매일 교체합니다. </li><li> 후각 상피 준비가 현미경되면, 전극을 채우기 위해 불꽃 긴 노란색 팁 1 ML의 주사기에이 솔루션의 일부를 가지고; 직사광선으로부터 보호합니다. RT에서, 니 스타틴 용액 일단안정되지; 매 시간마다 주사기 또는 얼음에 보관 ㎖로 1 솔루션을 교체합니다. </li></ol></li><li> 내부 니 스타틴 솔루션을 15 ~ 20 MΩ의 저항과 긴 목과 작은 팁 (~ 2 μm의)를 얻기 위해 풀러와 기록 전극을 당깁니다. </li><li> 흄 후드 아래에 DMSO 0.5 M에서 취제 솔루션을 준비; 나누어지는 및 사용할 때까지 -20 ° C에서 보관하십시오. 원하는 농도까지 링거액에 부 취제를 희석. 자극 피펫을 채 웁니다. </li></ol> 후각 상피 3. 준비 주 : OR-IRES-tauGFP 마우스는 OR 프로모터의 제어하에 tauGFP 표현. 이 마우스에서 관심의 또는를 표현하는 모든 신경 세포는 GFP로 표시되어 있습니다. 이 프로토콜은 모든 영역에서 발현 논리합하도록 구성된다. 그러나, 해부 및 녹음 등의 영역에서 표현 관찰 보고서에 대한 쉽다. <ol><li> 자일 라진 및 케타민 (150 ㎎ / ㎏ 및 10 ㎎ / ㎏ BOD의 혼합 주사하여 동물을 마취Y 무게, 각각). 참수는 젊은 쥐에 대한 이상 마우스에 대한 제대로 관리 설치류 단두대와 날카로운 가위로 수행 할 수 있습니다. <ol><li> 사용 링 해부 가위는 등쪽 피부를 통해 종 방향 내측 절개를합니다. 그것을 떨어져 당겨 피부를 제거합니다. 가위를 사용하여 턱 관절에서 아래턱을 잘라. 치아 관상 컷 병렬로 위 앞니를 제거합니다. </li><li> 눈 뒤에 머리의 관상 컷을 확인하고 머리의 앞쪽 부분을 유지. 범위에서 해부를위한 얼음처럼 차가운 링거 용액에 담근다. </li></ol></li><li> 범위에서 해부 : 복부 측면에서, 위 턱 / 이빨을 따라 길이 절단을합니다. 비강 배측-외측부 다음, 종 지느러미 뼈를 잘라. 대부분의 뼈와 미각을 제거; 실온에서 산소 벨소리에 부착 된 격벽과 상피 세포를 전송합니다. </li><li> 최종 절개의 경우 : 마우스 오른쪽 버튼으로 녹화를 시작하기 전에세션은, 집게로 기본 격막에서 상피를 벗겨. 집게와의 접착 성이 강한 격막의 앞쪽 부분에 두 4-5mm의 가위 컷 (사용 microvannas 가위)와 상피를 분리합니다. <ol><li> 조심스럽게 중격 상피에 그 등의 연결을 따라 밖으로 절단하여 골코 (vomeronasal) 기관을 제거합니다. 위로 향하게 점액층으로 기록 챔버로 이동 상피; 하프와 챔버에 평면을 유지. </li></ol></li><li> 형광 광학 및 민감한 카메라가 장착 된 수직 현미경 실을 설치합니다. 40X 물 침지 목표 (개구 0.8) 및 확대 렌즈에 의해 달성 여분의 2 배 또는 4 배 확대를 통해 높은 배율에서 컴퓨터 화면의 준비를 시각화. 실온에서 산소 벨소리 (1-2 ml / 분)와 함께 지속적으로 관류. </li></ol> 4. 녹음 세션 <ol><li> 형광 셀에 대한 검색 : 480에서 준비를 자극530-550 nm의 범위에서 발광 용 나노 EGFP; 형광의 밝은 분야에서 확실하게 볼 수 있습니다 하나 수지상 노브를 대상으로. </li><li> 니 스타틴과 세포 내 솔루션 전극을 채우기; 부드럽게 전극에 눌러 거품을 제거합니다. </li><li> , 전극 홀더에 전극을 삽입 피펫에 긍정적 인 압력을 적용; 저항은 15 ~ 20 MΩ해야한다. </li><li> 셀에 가까운 전극을 가져와; 저항 ~ 40 MΩ에 도달하면, 긍정적 인 압력을 해제하고 기가 시일에 도달하려면 약간의 부정적인 압력을 적용합니다. </li><li> 시일에 도달하면, 약 -75 MV의 막 전위를 설정; </li><li> 셀이 열리면 자극 프로토콜 약리 치료 진행. 단일 자극 취제, 기록 자발 활동 200-500 밀리에 대한 응답을 측정하기 위해 500 msec의 자극과 최대 10 초 13 대 세포의 반응을 측정한다. 약물 치료의 경우에서 약리 에이전트를 관류원하는 농도 (20). </li></ol> 5. 데이터 분석 <ol><li> 그 다음으로 취제 자극 유도 전류를 분석 (밀리 초에서 최대 진폭의 90 %에 도달하는데 필요한 시간)의 최대 진폭, 상승 시간, 전체 전류 (PAS의 곡선 아래 영역), 시간을 50 % (개시 및 밀리에서 최대 진폭의 50 %의 응답 시간 사이의 오프셋). <ol><li> 그리는 힐 방정식을 이용하여 투여 량 반응 곡선에 맞는 서로 다른 농도 (최대 진폭에 도달) 피크 전달 전류를 사용 : I = 아이 맥스 / (1 ​​+ (K 1/2 / C) N) I는 피크 전류를 나타내고, , 최대 반응의 절반에 도달되었을 때의 K1 / 2 농도, C 취제의 농도이고; n은 힐 (Hill) 계수를 포화 농도에서 최대 반응을 IMAX. </li></ol></li><li> 최대 탈분극의 현재 클램프자는 tota을 막 전위의 기록을 분석L 전위 (mVsec에서 곡선 아래 면적)를 유발; 30 초 ~ 1 분 녹음을 통해 기록 자발적인 급상승 활동; 전류 (5 ~ 10 PA)의 주입에 의해 유도 스파이크를 통해 기록 흥분. </li></ol>

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	<li>Lowe, G., Gold, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+amplification+by+calcium-dependent+chloride+channels+in+olfactory+receptor+cells.">Nonlinear amplification by calcium-dependent chloride channels in olfactory receptor cells.</a> Nature. 366 (6452), 283-286 (1993).</li><li>Ponissery Saidu, S., Dibattista, M., Matthews, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-induced+responses+recorded+from+olfactory+receptor+neurons+using+the+suction+pipette+technique.">Odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique.</a> J Vis Exp. (62), e3862 (2012).</li><li>Moss, R. L., 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=Electrophysiological+and+biochemical+responses+of+mouse+vomeronasal+receptor+cells+to+urine-derived+compounds:+possible+mechanism+of+action.">Electrophysiological and biochemical responses of mouse vomeronasal receptor cells to urine-derived compounds: possible mechanism of action.</a> Chem Senses. 23 (4), 483-489 (1998).</li><li>Kaur, A., Dey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+cell+calcium+imaging+of+dissociated+vomeronasal+neurons.">Live cell calcium imaging of dissociated vomeronasal neurons.</a> Methods Mol Biol. 1068, 189-200 (2013).</li><li>Ma, M., Chen, 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=Electrophysiological+characterization+of+rat+and+mouse+olfactory+receptor+neurons+from+an+intact+epithelial+preparation.">Electrophysiological characterization of rat and mouse olfactory receptor neurons from an intact epithelial preparation.</a> J Neurosci Methods. 92 (1-2), 31-40 (1999).</li><li>Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+multigene+family+may+encode+odorant+receptors:+a+molecular+basis+for+odor+recognition.">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65 (1), 175-187 (1991).</li><li>Bozza, T., Feinstein, P., Zheng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+receptor+expression+defines+functional+units+in+the+mouse+olfactory+system.">Odorant receptor expression defines functional units in the mouse olfactory system.</a> J Neurosci. 22 (8), 3033-3043 (2002).</li><li>Bozza, T., 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=Mapping+of+class+I+and+class+II+odorant+receptors+to+glomerular+domains+by+two+distinct+types+of+olfactory+sensory+neurons+in+the+mouse.">Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse.</a> Neuron. 61 (2), 220-233 (2009).</li><li>Vassalli, A., Rothman, A., Feinstein, P., Zapotocky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minigenes+impart+odorant+receptor-specific+axon+guidance+in+the+olfactory+bulb.">Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb.</a> Neuron. 35 (4), 681-696 (2002).</li><li>Feinstein, P., Bozza, T., Rodriguez, I., Vassalli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+guidance+of+mouse+olfactory+sensory+neurons+by+odorant+receptors+and+the+beta2+adrenergic+receptor.">Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor.</a> Cell. 117 (6), 833-846 (2004).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+ligands+for+odorant,+vomeronasal+and+taste+receptors.">Genes and ligands for odorant, vomeronasal and taste receptors.</a> Nat Rev Neurosci. 5 (4), 263-278 (2004).</li><li>Grosmaitre, X., 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=SR1,+a+mouse+odorant+receptor+with+an+unusually+broad+response+profile.">SR1, a mouse odorant receptor with an unusually broad response profile.</a> J Neurosci. 29 (46), 14545-14552 (2009).</li><li>Grosmaitre, X., Vassalli, A., Mombaerts, P., Shepherd, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+responses+of+olfactory+sensory+neurons+expressing+the+odorant+receptor+MOR23:+a+patch+clamp+analysis+in+gene-targeted+mice.">Odorant responses of olfactory sensory neurons expressing the odorant receptor MOR23: a patch clamp analysis in gene-targeted mice.</a> Proc Natl Acad Sci U S A. 103 (6), 1970-1975 (2006).</li><li>Lam, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant+responsiveness+of+embryonic+mouse+olfactory+sensory+neurons+expressing+the+odorant+receptors+S1+or+MOR23.">Odorant responsiveness of embryonic mouse olfactory sensory neurons expressing the odorant receptors S1 or MOR23.</a> Eur J Neurosci. 38 (2), 2210-2217 (2013).</li><li>Zhang, J., Huang, G., Dewan, A., Feinstein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+stimulus+specificity+and+glomerular+position+in+the+mouse+olfactory+system.">Uncoupling stimulus specificity and glomerular position in the mouse olfactory system.</a> Mol Cell Neurosci. 51 (3-4), 79-88 (2012).</li><li>Zhang, J., Pacifico, R., Cawley, D., Feinstein, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasensitive+detection+of+amines+by+a+trace+amine-associated+receptor.">Ultrasensitive detection of amines by a trace amine-associated receptor.</a> J Neurosci. 33 (7), 3228-3239 (2013).</li><li>Lee, A. C., Tian, H., Grosmaitre, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+patterns+of+odorant+receptors+and+response+properties+of+olfactory+sensory+neurons+in+aged+mice.">Expression patterns of odorant receptors and response properties of olfactory sensory neurons in aged mice.</a> Chem Senses. 34 (8), 695-703 (2009).</li><li>Cadiou, H., 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=Postnatal+odorant+exposure+induces+peripheral+olfactory+plasticity+at+the+cellular+level.">Postnatal odorant exposure induces peripheral olfactory plasticity at the cellular level.</a> J Neurosci. 34 (14), 4857-4870 (2014).</li><li>Mombaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+wiring+in+the+mouse+olfactory+system.">Axonal wiring in the mouse olfactory system.</a> Annu Rev Cell Dev Biol. 22, 713-737 (2006).</li><li>Grosmaitre, X., Santarelli, L. C., Tan, J., Luo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+functions+of+mammalian+olfactory+sensory+neurons+as+odor+detectors+and+mechanical+sensors.">Dual functions of mammalian olfactory sensory neurons as odor detectors and mechanical sensors.</a> Nat Neurosci. 10 (3), 348-354 (2007).</li><li>Duchamp-Viret, P., Chaput, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odor+response+properties+of+rat+olfactory+receptor+neurons.">Odor response properties of rat olfactory receptor neurons.</a> Science. 284 (5423), 2171-2174 (1999).</li><li>Heydel, J. M., 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=Odorant-binding+proteins+and+xenobiotic+metabolizing+enzymes:+implications+in+olfactory+perireceptor+events.">Odorant-binding proteins and xenobiotic metabolizing enzymes: implications in olfactory perireceptor events.</a> Anat Rec (Hoboken). 296 (9), 1333-1345 (2013).</li><li>Pelosi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perireceptor+events+in+olfaction).">Perireceptor events in olfaction).</a> J Neurobiol. 30 (1), 3-19 (1996).</li><li>Spehr, M., Wetzel, C. H., Hatt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3-phosphoinositides+modulate+cyclic+nucleotide+signaling+in+olfactory+receptor+neurons.">3-phosphoinositides modulate cyclic nucleotide signaling in olfactory receptor neurons.</a> Neuron. 33, 731-739 (2002).</li><li>Chen, S., Lane, A. P., Bock, R., Leinders-Zufall, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+adenylyl+cyclase+inhibits+olfactory+generator+currents+induced+by+'IP(3)-odors.">Blocking adenylyl cyclase inhibits olfactory generator currents induced by 'IP(3)-odors.</a> J Neurophysiol. 84 (1), 575-580 (2000).</li><li>Savigner, A., 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=Modulation+of+spontaneous+and+odorant-evoked+activity+of+rat+olfactory+sensory+neurons+by+two+anorectic+peptides,+insulin+and+leptin.">Modulation of spontaneous and odorant-evoked activity of rat olfactory sensory neurons by two anorectic peptides, insulin and leptin.</a> J Neurophysiol. 101 (6), 2898-2906 (2009).</li><li>Ukhanov, K., Brunert, D., Corey, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphoinositide+3-kinase-dependent+antagonism+in+mammalian+olfactory+receptor+neurons.">Phosphoinositide 3-kinase-dependent antagonism in mammalian olfactory receptor neurons.</a> J Neurosci. 31 (1), 273-280 (2011).</li></ol>

The authors declare that they have no competing financial interest.

올바르게 건강한 OSNs의 특성을 모니터링이 프로토콜의 능력은 제제의 품질에 크게 의존한다. 따라서, 해부 단계는 매우 중요하다. 첫째는 품질 (PH, 삼투압), 산소 및 온도 (빙냉하지만 냉동 생략) 해부 배지에 주목하는 것이 중요하다. 둘째, 해부 도구 상피의 조작 등의 손상을 피하기 위해 가능한 한 제한해야한다. 마지막으로, 가능한 최대 OSN 인구를 액세스하기 위해 가능한 한 평탄 제제를 얻는 ?...

후각 감각 뉴런 (OSN)는 후각 인식의 첫 번째 단계를 나타냅니다. 설치류의 비강에 선을 긋고있는 후각 상피에 위치한, 그들은 뇌에 자신의 축삭을 통해 전송 활동 전위에 냄새 물질의 화학적 정보를 변환. 더 후각 부호화 메커니즘을 이해하기 위해서는 OSNs의 전달 및 막 특성을 특성화하는 것이 필요하다. 최근까지, 포유 동물 OSNs의 성질을 특성화하기 위해 사용되는 기법은 대부분 해리 OSNs 1-4에서 수행 하였다. 해리 과정은 자신의 환경에서 OSNs을 확보하기 위해 다양한 기계 및 화학 물질 (즉, 효소) 프로세스를 사용합니다. 이러한 과정은 녹화 가능 세포의 낮은 번호를 유도한다. 이러한 낮은 수는 GFP 표지 된 세포의 경우에 훨씬 더 중요 할 수있다. 해리도 및 OSNs surviv 중을 상승시킬 수 후각 상피의 다른 셀 간의 로컬 세포 간 상호 작용을 제거알과 OSNs &#39;속성의 변조. 해리 과정을 생략하기 위해, 제제는 그대로 5 개발되었다. 각 OSN은 큰 multigene 가족 (6)에서 선택 (OR) 하나의 후각 수용체를 표현한다. 마우스의 주요 후각 상피 세포에서 발현 1,000 관찰 보고서는 ~가 있습니다. 인해 야생형 동물 OR의 다수에, 기회는 동일한 발현 OSNs 기록 OR 매우 낮은한다. 이러한 한계를 극복하기 위해, 유전자 표적으로 마우스를 사용할 수있는 식별 또는 형광 단백질 7-9로 표시되어 표현하는 모든 OSNs. 이 표시 OSNs는 해리 준비 앞에서 언급 한 단점과 7,10,11에 기능 분석을 수행 하였다. 유전자 표지 된 마우스의 상피 손상 5- 따라서 이러한 문제를 회피. 이것은 VI에서에 가까운 환경에서 정확하게 정의 논리합을 표현 OSNs의 활동의 모니터링을 허용가능한 한 VO. 게다가, OSNs의 패치 클램프 녹음도 막 특성, 전달 경로의 약리학, 리간드 / 또는 상호 작용의 정확한 분석을 할 수 있습니다. 이러한 모든 항목은 거의 세포 외 녹음을 사용하여 분석 할 수 없습니다. 우리 취제 수용체 SR1 및 MOR23 12,13 발현 OSNs의 응답을 모니터링하기 위해이 기술을 사용했다. 기술의 실현 가능성은 뉴런 15,16 표현 OSNs 14 발현 MOR23에 다른 그룹뿐만 아니라 다른 논리합에 의해 확인 하였다. OSNs의 정의 인구의 모니터링 (17) 노화, 이러한 개발 (14)와 같은 많은 다른 상황에서 그 특성의 분석으로 이어질 수, 부 취제 유도 소성 (18), 15 코딩 냄새에 후각 수용체의 서열 변화의 역할. 이 프로토콜은, 따라서 멤브레인 레벨에서 정의 OSNs의 기능적 특성을 모니터링하는 강력한 도구를 제공한다.

<table><tbody><tr><td>vibration table with Faraday cage</td><td>TMC</td><td>63-500 SERIES</td><td>required : isolates the recording system from vibrations induced by the environment (movements of experimenter, vibrations of equipment such as fans for cooling computers, etc); can also be purchased with a Faraday cage, or equipped by a custom made Faraday cage; this cage is recommended to avoid electric noise from the environment</td></tr><tr><td>optics</td><td></td><td></td><td></td></tr><tr><td>microscope</td><td>Olympus</td><td>BX51WI</td><td>upright microcope equipped with epifluorescence; fixed or moving stage depending on the user&#039;s preference</td></tr><tr><td>objectives</td><td>Olympus</td><td>LUMPLFL40XW</td><td>at least 2 objectives required: a 4X or 10X for coarse approach to the cell; and a 40X immersion long distance example Olympus LUMPLFL40XW / IR /0,8 / WD:3.3 MM</td></tr><tr><td>magnifier</td><td>Olympus</td><td>U-TVCAC</td><td>ABSOLUTELY REQUIRED: placed in the light path between the objective and the camera; allows to magnify the image on the screen in order to reach precisely the knob with the recording electrode</td></tr><tr><td>camera</td><td>Olympus</td><td>DP72</td><td>a good camera is required to see the neurons in fluorescence as well as in bright field; the controlling software is simple and allows to take pictures and do live camera image to monitor the approach of the electrode to the cell. An ultrasensitive camera is not necessary</td></tr><tr><td>filters</td><td>Olympus/Chroma</td><td></td><td>depending on the fluorescent protein used in the mice; example for GFP: excitation : BP460-490: emission: HQ530/50m</td></tr><tr><td>amplifier</td><td>HEKA</td><td>EPC10 USB</td><td>monitors the currents flowing through the recording electrode and also controls the puffing by sending a TTL signal to the spritzer; the EPC10 setup is controled by computer</td></tr><tr><td>software</td><td>HEKA</td><td>Patchmaster</td><td>controls the amplifier during the experiment</td></tr><tr><td>micromanipulator</td><td>Sutter</td><td>MP225</td><td>precision micromanipulator, allows precise movements down to 1/10th of a micrometer; this model is very stable; avoid hydraulic manipulators that may drift</td></tr><tr><td>electrode puller</td><td>Sutter</td><td>P97</td><td>with a FT345-B wide trough filament;&amp;nbsp; to prepare recording pipets of about 2&amp;micro;m diameter with a long tip to reach the cells; the resistance should be 15 to 20Mohm with perforated patch clamp solution</td></tr><tr><td>glass</td><td>Sutter</td><td>BF120-69-10</td><td>in our recording conditions, this glass is ideal for recording pipets</td></tr><tr><td>recording chamber</td><td>Warner Instruments</td><td>RC-26G</td><td>a chamber is needed to set the preparation under the microscope. To maintain the preparation in the center of the chamber, a net/anchor should be used.</td></tr><tr><td>stimulation</td><td></td><td></td><td></td></tr><tr><td>glass</td><td>WPI</td><td>TW100F-4</td><td>attached in groups of 7, these pipettes are used to prepare prepulled stimulating pipettes</td></tr><tr><td>multibarrel puller</td><td>MDI</td><td>PMP-107-Z</td><td>by association of pull and twist, this puller allows us to prepare puffing electrodes with 7 barrels</td></tr><tr><td>precision pressure injector&amp;nbsp;</td><td>Toohey Company</td><td>P/N T25-1-900 Single Channel&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>this precision pressure injector&amp;nbsp; controls the pressure ejected in the multibarrel puller; it is controlled manually or by the amplifier by a 5V&amp;nbsp; TTLs</td></tr><tr><td>micromanipulator</td><td>Narishige</td><td>YOU-1</td><td>a coarse manipulator is enough to bring the puffing electrode close to the recording site</td></tr><tr><td>tubings</td><td>N/A</td><td></td><td>tygon tubing to bring the pressure from the puffer to the puffing pipette</td></tr><tr><td>solutions/perfusion/chemicals</td><td></td><td></td><td></td></tr><tr><td>vacuum pump</td><td>gardner denver</td><td>300 series</td><td>a vibrating membrane pump is more quiet and efficient than other types of pumps</td></tr><tr><td>perfusion system</td><td>N/A</td><td>N/A</td><td>gravity perfusion system with polyethlylen tubing to bring in and out the external solution from the recording chamber</td></tr><tr><td>nystatin</td><td>Sigma-Aldrich</td><td>N3503</td><td>mandatory to perpare internal solution for perforated patch clamp</td></tr><tr><td>DIMETHYL SULFOXIDE</td><td>Sigma-Aldrich</td><td>D5879</td><td>used to disolve nystatin for internal solution for perforated patch&amp;nbsp;</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S9625</td><td>extracellular solution</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P4504</td><td>intracellular/extracellular solution</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td>extracellular solution</td></tr><tr><td>Sodium phosphate monobasic monohydrate (NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S9638</td><td>extracellular solution</td></tr><tr><td>Magnesium sulfate heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt; 7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Sigma-Aldrich</td><td>63140</td><td>extracellular solution</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td>extracellular solution</td></tr><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S6297</td><td>extracellular solution</td></tr><tr><td>EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&amp;prime;,N&amp;prime;-tetraacetic acid)</td><td>Sigma-Aldrich</td><td>E3889</td><td>internal solution</td></tr><tr><td>Potassium hydroxyde</td><td>Sigma-Aldrich</td><td>P1767</td><td>internal solution</td></tr><tr><td>Methyl Sulfoxide</td><td>Sigma-Aldrich</td><td>W387509</td><td>intracellular solution</td></tr><tr><td>Hepes-Na</td><td>Sigma-Aldrich</td><td>H7006</td><td>intracellular solution</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td>intracellular solution</td></tr></tbody></table>

perforated patch-clamp recording of mouse olfactory sensory neurons in intact neuroepithelium: functional analysis of neurons expressing an identified odorant receptor

특정 리간드로 자극시 후각 감각 뉴런의 생리적 반응 (OSN)를 분석하는 것은 후각 구동 동작과 그 변조의 기초를 이해하는 것이 중요하다. 이러한 코딩 특성은 냄새 분자와 후각 수용체 (OR) 사이의 초기 상호 작용에 크게 의존하는 OSNs로 표현. 명시 적 또는 중요한의 ID, 특이 리간드 스펙트럼. 또는의 리간드를 찾을 수있는 확률은 상피 내에서 무작위로 선택된 OSN 매우 낮은 표현했다. 이러한 과제를 해결하기 위해,이 프로토콜은 전적으로 정의 논리합의 프로모터의 제어하에 형광 단백질 GFP를 발현하는 태깅 된 마우스를 사용한다. OSNs는 이웃 세포가 자신의 성숙과 기능에 영향을 미치는으로, 비강 안감 꽉 및 조직 상피에 있습니다. 여기에서 우리는 OSNs 전자의 속성을 그대로 후각 상피를 분리하고 패치 클램프 녹음을 기록하는 방법을 설명정의 후각 수용체를 xpressing. 프로토콜은 인접한 조직의 영향을 유지하면서 하나 OSN 막 성질을 특성화 할 수있다. 패치 클램프 결과의 분석은 리간드 / 또는 상호 작용, 전달 경로 및 약리학, OSNs &#39;코딩 특성과 막 수준에서 자신의 변조의 정확한 정량을 산출한다.

이 프로토콜의 결과는 절개의 품질에 따라 달라집니다. 이 해부 단계는 짧은 (이하 10 분 ~ 15) 및 (즉, 상피 세포의 손상을 방지하기 위해) 정확해야합니다. 그림 1은 이상적인 준비가 다른 배율 수준에서처럼 보이는 방법을 보여줍니다. 밝은 필드 아래 낮은 배율 (예컨대 세포를지지 OSNs의 손잡이와 같은) 다른 유형의 세포는 구별 (도 1A)이다. 최대 배율 수준에서, 일?...

Watch this Scientific Journal Video about Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant

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

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 ...

perforated-patch-clamp-recording-mouse-olfactory-sensory-neurons

Universidad de Cartagena UDEC

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Neuroscience

26.6K 조회수.  Université de Bourgogne. 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.

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

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

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

흡입 피펫 기법을 사용하여 강한 수용체 뉴런에서 기록 Odorant 유발 응답

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

메뚜기에 신경 활동을 특성화하기 위해 다중 단위 기록 방법 (<em&gt; Schistocerca 아메리카나</em&gt;) 강한 회로

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

마우스의 메인 후각 시스템의 섬모의 전체 마운트 라벨링

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

In-depth Physiological Analysis of Defined Cell Populations in Acute Tissue Slices of the Mouse Vomeronasal Organ

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory neurons (VSNs) express a specific type of G protein-coupled receptor (GPCR) from at least three different chemoreceptor gene families allowing sensitive and specific detection of chemosensory cues. These families comprise the V1r and V2r gene families as well as the formyl peptide receptor (FPR)-related sequence (Fpr-rs) family of putative chemoreceptor genes. In order to understand the physiology of vomeronasal receptor-ligand interactions and downstream signaling, it is essential to identify the biophysical properties inherent to each specific class of VSNs.
The physiological approach described here allows identification and in-depth analysis of a defined population of sensory neurons using a transgenic mouse line (Fpr-rs3-i-Venus). The use of this protocol, however, is not restricted to this specific line and thus can easily be extended to other genetically modified lines or wild type animals.

Here, we describe a physiological approach that allows identification and in-depth analysis of a defined population of sensory neurons in acute coronal tissue slices of the mouse vomeronasal organ using whole-cell patch-clamp recordings.

마우스 보습 코 기관의 급성 조직 조각에서 정의 된 세포 집단의 심층 생리 학적 분석

In most mammals, the vomeronasal organ (VNO) is a chemosensory structure that detects both hetero- and conspecific social cues. Vomeronasal sensory ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

라이브 셀 측정의 Odorant 수용 체 활성화를 사용 하 여 분석 결과 실시간 캠프

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

후각 감각 축색 돌연변이 분자 식별 코드의 시각화를위한 뒷 전구의 4 배 면역 면역 측정법

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

An Effective Manual Deboning Method To Prepare Intact Mouse Nasal Tissue With Preserved Anatomical Organization

The mammalian nose is a multi-functional organ with intricate internal structures. The nasal cavity is lined with various epithelia such as olfactory, respiratory, and squamous epithelia which differ markedly in anatomical locations, morphology, and functions. In adult mice, the nose is covered with various skull bones, limiting experimental access to internal structures, especially those in the posterior such as the main olfactory epithelium (MOE). Here we describe an effective method for obtaining almost the entire and intact nasal tissues with preserved anatomical organization. Using surgical tools under a dissecting microscope, we sequentially remove the skull bones surrounding the nasal tissue. This procedure can be performed on both paraformaldehyde-fixed and freshly dissected, skinned mouse heads. The entire deboning procedure takes about 20-30 min, which is significantly shorter than the experimental time required for conventional chemical-based decalcification. In addition, we present an easy method to remove air bubbles trapped between turbinates, which is critical for obtaining intact thin horizontal or coronal or sagittal sections from the nasal tissue preparation. Nasal tissue prepared using our method can be used for whole mount observation of the entire epithelia, as well as morphological, immunocytochemical, RNA in situ hybridization, and physiological studies, especially in studies where region-specific examination and comparison are of interest.

보존 해부 조직 본래대로 마우스 비강 조직을 준비하는 효과적인 수동 발골 방법

The mammalian nose is a multi-functional organ with intricate internal structures.  The nasal cavity is lined with various epithelia such as olfactory, ...

생물학

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of available mating partners or threats. In mice, this task is executed primarily by the olfactory system and its underlying subsystems, including the main and accessory olfactory systems. Both have peripheral organs populated by sensory neurons expressing G-protein coupled receptors able to bind chemical cues that reach the nasal cavity. Even though the molecular characteristics of these receptors is well understood, little is known about their cognate specific ligands. The method described here combines in situ hybridization detection of olfactory or vomeronasal receptors with immunodetection of phosphorylated ribosomal protein S6 (pS6) - a marker of neuronal activation. This protocol was devised to identify neurons activated after a single event of exposure to purified or complex chemical stimuli detected by the olfactory organs. Importantly, this technique allows the investigation of neurons triggered in biologically relevant contexts. Ideally, this method should be used to probe the molecular biology of the olfactory system and to study olfactory behaviors.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

This protocol combines in situ hybridization with immunofluorescence to identify olfactory and vomeronasal receptor genes expressed in olfactory sensory neurons after activation by chemical stimuli in the mouse.

Animals rely on chemical communication to convey and perceive relevant environmental information, ranging from assessment of food quality to detection of ...

Perforated Patch-Clamp Recording of Olfactory Sensory Neurons in Mice

Source: Jarriault, D., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52652">Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor.</a> J. Vis. Exp. (2015).
This video demonstrates electrophysiological recording of olfactory sensory neurons (OSNs) in mice using a perforated patch-clamp technique. The olfactory epithelium, containing OSNs with fluorophore-tagged odor receptors, is isolated from a mouse and secured in a recording chamber. A recording pipette is clamped to the dendritic knob of an OSN, and nystatin within the pipette perforates the membrane to form electrical connectivity with the cell. Finally, another pipette is used to deliver an odor stimulant, and the electrophysiological activity of the patched cell is recorded.

생쥐의 후각 감각 뉴런에 대한 천공된 패치 클램프 기록

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

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

흡입 피펫 기법을 사용하여 강한 수용체 뉴런에서 기록 Odorant 유발 응답

메뚜기에 신경 활동을 특성화하기 위해 다중 단위 기록 방법 (

마우스의 메인 후각 시스템의 섬모의 전체 마운트 라벨링

마우스 보습 코 기관의 급성 조직 조각에서 정의 된 세포 집단의 심층 생리 학적 분석

라이브 셀 측정의 Odorant 수용 체 활성화를 사용 하 여 분석 결과 실시간 캠프

후각 감각 축색 돌연변이 분자 식별 코드의 시각화를위한 뒷 전구의 4 배 면역 면역 측정법

보존 해부 조직 본래대로 마우스 비강 조직을 준비하는 효과적인 수동 발골 방법

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization

생쥐의 후각 감각 뉴런에 대한 천공된 패치 클램프 기록