This work was funded by the Deutsche Forschungsgemeinschaft DFG (Exc257, SFB958).

참고 : 모든 동물의 절차는 동물의 부당한 고통을 피하는 독일어 동물 보호 법률에 맞게 샤리 테 대학 병원 예나에서 처리되었다. 1. 준비 솔루션 및 해부 직장 <ol><li> 솔루션 참고 : 상피의 해부를 시작하기 전에 다음과 같은 솔루션을 준비합니다. <ol><li> 해부 절차를위한 솔루션 : <ol><li> 140 mM의 염화나트륨, 5 mM의 KCl을 10 mM의 HEPES, 2 mM의 CaCl2를, 1 ㎜의 MgCl 2, 10 mM의 포도당 농도의 링거액 (PH 7.4)을 준비한다. </li><li> - / - 2.68 mM의 KCl을, 1.47 mM의 KH 2 PO 4, 136 mM의 NaCl을, 8.1 mM의 나 2 HPO 4의 농도의 용액 (PH 7.4) PBS를 준비합니다. </li><li> - / - 0.2 mM의 CaCl2를, 4 % 수 크로스, 4 % 파라 포름 알데하이드 PBS 1X의 농도 정착액 용액 (PH 7.2)을 준비한다. 정착액 솔루션 자당 크라이을 향상단면 및 것은 픽업 저온부의를. -20 ° C에서 보관하십시오. </li></ol></li><li> 염색 절차를위한 솔루션 <ol><li> 1X PBS의 농도 PBS + / + 솔루션을 준비 - / -, 0.48 mM의의 MgCl 2, 0.9 mM의 염화칼슘 2. </li><li> 1X PBS + / + 0.1 % 트리톤 X-100의 농도 PBST + / + 솔루션을 준비합니다. </li><li> 1X PBST + / + 1 % 젤라틴의 농도 차단 솔루션을 준비합니다. </li></ol></li></ol></li><li> 직장 <ol><li> 해부 직장를 들어, 밝은 조명과 해부 현미경뿐만 아니라 액체 차단제 펜을 사용합니다. </li><li> 링거액, 접착제 유리 슬라이드 가득 페트리 접시를 준비합니다. </li><li> 다음과 같은 수술 도구를 얻 수술 가위 날카로운 하나는 무딘 팁, 스트레이트 팁 모양 봄 가위, 미세 곡선 팁 모양과 면도날 두 집게 한 쌍의 (로 <strong&gt; 그림 1A). </li></ol></li></ol> 비중격 2. 준비 <ol><li> 음식과 물과 적절한 주 / 야간주기에 정기적으로 액세스 할 수있는 승인 케이지 하우스 동물. </li><li> 후면 발 반사 신경을 테스트하여 100 % 이소 플루 란과 모니터 진통제로 적셔 닫힌 소켓 포함 거즈에 마취를 수행합니다. 자궁 경부 전위가 비강에 혈액을 일으킬 수 있기 때문에, 마취 된 쥐 목을 벨. </li><li> 전체 두개골과 코의 뼈를 노출 피부를 제거하고 철저하게 종이 타월을 사용하여 남아있는 혈액과 조직을 닦아. 아래턱과 앞니를 제거합니다. </li><li> 상악 (측 방향)에서 격벽 (내측)의 한 쪽을 분리하기 위해 봉합선 1 ~ 2 mm 거리에 평행 한 양측 코의 등을 절개 뼈. 하나의 컷으로 코를 분할합니다. 뼈의 잔해 비갑개가 여전히 격벽에 부착하는 경우,주의 깊게을 완전히 제거 격막을 노출을 터치하지 않고. </li><li> 격막과 코 뼈 사이에 격막의 등의 측면을 따라 집게의 미세 곡선 팁을 밀어 지느러미 코 뼈를 제거합니다. 뼈에 약간의 힘을 주어 그것을 밀어 제거합니다. </li><li> 각각의 공간에 두 중격 조직에 대한 액세스를 제공하기 위해 조심스럽게 머리의 다른 쪽의 턱뼈를 제거합니다. 오래된 동물 (&gt; 28 P)를 준비 할 때, 후각 전구를 덮고 정면 뼈의 끝 부분을 제거합니다. </li><li> 그 약간 노란색 (그림 1B)에 의해 후각 상피를 식별합니다. 경계 호흡기 상피은 흰색과 해부 현미경으로 볼 수있는 운동성 섬모의 움직임을 보여줍니다. 미세 스프링 가위로 후각 및 호흡기 상피 세포 사이의 국경을 따라 잘라. </li><li> 상처를 확장 및 보습 뼈 뼈에 복부 연결에서 격막을 분리합니다. 그런 다음 격막과 OS가 벌집의 사상 판의 경계에 따라 절단전자. 격벽은 이제 완전히 머리에 모든 연결에서 격리됩니다. 도 1C에 도시 된 절단 위치를 사용한다. </li></ol> 면역 염색의 후각 상피의 3. 분리 <ol><li> 링거액 가득 배양 접시에 접착제 유리 슬라이드를 놓습니다. 유리의 절반은 링거액 (그림 1A)에 놓 이도록, 페트리 접시의 가장자리에 배치합니다. </li><li> 조심스럽게 페트리 접시에 후각 상피 사골 뼈를 전송하는 집게를 사용합니다. 포셉의 팁을 잡아없이 들어 올립니다. </li><li> 한 집게와 사골 뼈의 수직 판을 잡고 조심스럽게 격벽의 한쪽에서 상피를 제거하기 위해 두 번째를 사용합니다. 상피를 벗겨 수직 플레이트와 상피 세포 사이의 곡선 끝을 밀어 넣습니다. </li><li> 상피가 링거 soluti에 뒤집 경우, 섬모 측을 식별 할 항상 확인에. 상피가 뒤집 경우 나중에 모양체 측을 거의 식별 할 수있다. 대부분, 상피 세포 내부의 섬모 표면과 롤업. </li><li> 도 1C에 도시 된 절단 위치와 등록 된 조직 형상을 비교한다. 국경에서 위치에서 상피 세포를 잡고 유리 슬라이드에 당깁니다. 조직의 병변을 방지하기 위해 집게와 섬모면을 만지지 마십시오. </li><li> 격막의 전원을 켜고 다른 측면에서 상피 절차를 반복합니다. </li><li> 종이 타월 후각 상피의 두 조각 주위에 유리 슬라이드를 건조 및 액체 차단제 펜으로 조직을 둘러싸. </li><li> 실온에서 10 분 동안 150 ㎕의 정착 용액으로 조직을 수정합니다. 섬모의 안정성과 무결성에 관한 시험 항체의 다양한 짧은 고정 시간을 사용합니다. 이 10 분 섬모 내에서 고정 된, 그러나 아마 전체 상피 조직된다. 따라서, S는 즉시 후속 현미경으로 시각화절차 이닝. 그러나, 정착 시간은 개별 항체 다를 수 있습니다. </li></ol> 4. 염색 프로토콜 참고 : 후각 감각 신경 세포의 섬모 구조를 유지하기 위해 매우 조심스럽게 조직을 처리합니다. 피펫 팅 솔루션을 제거합니다. 기계적인 힘이 미세 섬모를 중단 할 수로, 상피 세포에 직접 솔루션을 떨어 뜨리지 마십시오. 달리 명시하지 않을 경우 실온에서 모든 단계를 수행합니다. <ol><li> 정착액 솔루션을 제거하고 PBS + / +와 함께하는 과정을 2 회 반복한다. </li><li> 차단 솔루션 적어도 1 시간 동안 상피 세포를 품어. </li><li> 어떤 침전물을 제거하기 위해 최고 속도로 5 분 (본 연구에 사용 된 항체 (200) 1) 원심 분리기 차단 솔루션에 항체를 용해하여 일차 항체 솔루션을 준비합니다. </li><li> 4 ° CO / N에 습기 챔버에서 항체 용액으로 상피 세포를 품어. </li><li> PBS + / + 5 분간 씻을 상피 적어도 3 회. </li><li>최고 속도에서 5 분 동안 원심 분리 : 용액 (500 1) 차단에 항체를 용해 차 항체 솔루션을 준비합니다. </li><li> 어두운 챔버에서 1 시간 동안 차 항체 용액으로 상피 세포를 품어. </li><li> PBS + / + 5 분간 씻을 상피 적어도 3 회. </li><li> 면도날이나 종이 타월로 액체 차단제를 제거합니다. 5 초 동안 증류수로 상피 세포를 씻으십시오. </li><li> antifade 장착 시약의 조직을 보존합니다. 주 : 일 이내에 신속하게 배경 형광 증가; 늦어도 2 일 이내에 설치 매체에 삽입 한 후 현미경 분석 따라서 좋습니다. 특별한주의가 올바른 초점면을 검색 할 때이 심각하게 준비를 손상시킬 수 있기 때문에, 조직을 짜내하지 않도록주의해야합니다. 때문에 초점이 맞지 형광에, 공 초점 현미경으로 추가 조사를하는 것이 좋습니다. </li></ol>

<ol><li>Firestein, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+the+olfactory+system+makes+sense+of+scents%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">How the olfactory system makes sense of scents.</a> Nature. 413 (6852), 211-218 (2001).</li>
<li>Buck, L., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+multigene+family+may+encode+odorant+receptors%3A+a+molecular+basis+for+odor+recognition%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A novel multigene family may encode odorant receptors: a molecular basis for odor recognition.</a> Cell. 65 (1), 175-187 (1991).</li>
<li>Wong, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Disruption+of+the+type+III+adenylyl+cyclase+gene+leads+to+peripheral+and+behavioral+anosmia+in+transgenic+mice%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Disruption of the type III adenylyl cyclase gene leads to peripheral and behavioral anosmia in transgenic mice.</a> Neuron. 27 (3), 487-497 (2000).</li>
<li>Belluscio, L., Gold, G. H., Nemes, A., Axel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mice+deficient+in+G%28olf%29+are+anosmic%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Mice deficient in G(olf) are anosmic.</a> Neuron. 20 (1), 69-81 (1998).</li>
<li>Brunet, L. J., Gold, G. H., Ngai, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((General+anosmia+caused+by+a+targeted+disruption+of+the+mouse+olfactory+cyclic+nucleotide-gated+cation+channel%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">General anosmia caused by a targeted disruption of the mouse olfactory cyclic nucleotide-gated cation channel.</a> Neuron. 17 (4), 681-693 (1996).</li>
<li>Reisert, J., Lai, J., Yau, K. W., Bradley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+the+excitatory+Cl-+response+in+mouse+olfactory+receptor+neurons%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Mechanism of the excitatory Cl- response in mouse olfactory receptor neurons.</a> Neuron. 45 (4), 553-561 (2005).</li>
<li>Hengl, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+components+of+signal+amplification+in+olfactory+sensory+cilia%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Molecular components of signal amplification in olfactory sensory cilia.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (13), 6052-6057 (2010).</li>
<li>Smith, D. W., Thach, S., Marshall, E. L., Mendoza, M. G., Kleene, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mice+lacking+NKCC1+have+normal+olfactory+sensitivity%5BTitle%5D)+AND+%22Physiolog.%2C+%26+Behavior%22%5BJournal%5D)">Mice lacking NKCC1 have normal olfactory sensitivity.</a> Physiolog., & Behavior. 93 (1-2), 44-49 (2008).</li>
<li>Menco, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructural+aspects+of+olfactory+signaling%5BTitle%5D)+AND+%22Chemical+Senses%22%5BJournal%5D)">Ultrastructural aspects of olfactory signaling.</a> Chemical Senses. 22 (3), 295-311 (1997).</li>
<li>Serizawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+neuronal+identity+code+for+the+odorant+receptor-specific+and+activity-dependent+axon+sorting%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting.</a> Cell. 127 (5), 1057-1069 (2006).</li>
<li>Baumgart, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scaffolding+by+MUPP1+regulates+odorant-mediated+signaling+in+olfactory+sensory+neurons%5BTitle%5D)+AND+%22Journal+Of+Cell+Science%22%5BJournal%5D)">Scaffolding by MUPP1 regulates odorant-mediated signaling in olfactory sensory neurons.</a> Journal Of Cell Science. 127 (11), 2518-2527 (2014).</li>
<li>Strotmann, J., Wanner, I., Krieger, J., Raming, K., Breer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+odorant+receptors+in+spatially+restricted+subsets+of+chemosensory+neurones%5BTitle%5D)+AND+%22Neuroreport%22%5BJournal%5D)">Expression of odorant receptors in spatially restricted subsets of chemosensory neurones.</a> Neuroreport. 3 (12), 1053-1056 (1992).</li>
<li>Jenkins, P. M., McEwen, D. P., Martens, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Olfactory+cilia%3A+linking+sensory+cilia+function+and+human+disease%5BTitle%5D)+AND+%22Chemical+Senses%22%5BJournal%5D)">Olfactory cilia: linking sensory cilia function and human disease.</a> Chemical Senses. 34 (5), 451-464 (2009).</li>
<li>Tadenev, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+Bardet-Biedl+syndrome+protein-8+%28BBS8%29+perturbs+olfactory+function%2C+protein+localization%2C+and+axon+targeting%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Loss of Bardet-Biedl syndrome protein-8 (BBS8) perturbs olfactory function, protein localization, and axon targeting.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (25), 10320-10325 (2011).</li>
</ol>

The authors have nothing to disclose.

The en face preparation technique described in this protocol provides a powerful tool for the detailed analysis of the olfactory system. So far, most studies characterizing the localization of signaling proteins use immunostainings of cryosections. They present a good overview of the olfactory epithelium, and protein expression in distinct cell types or regions can be easily identified. However, expression in olfactory cilia is sometimes hard to detect. Even if ciliary localization is obvious, cryosections offer...

비강의 마우스 후각 상피 6-10000000 바이폴라 후각 감각 신경 (1)을 포함한다. 각각의 후각 신경 세포는 표현 1,200 후각 수용체 유전자 중 하나를 선택합니다. 냄새 물질의 검출은 4 OLF 후각 특정 G 단백질 Gα를 통해 아데 닐 레이트 사이 클라 유형 III (ACIII) 3를 활성화 후각 수용체 2에 결합 부 취제에 의해 시작됩니다. 사이 클릭 아데노신 모노 포스페이트 (cAMP)의 상승의 결과가 열립니다 염기 - 게이트 비 선택적 양이온 채널은 칼슘과 나트륨 +, 이후 칼슘의 유입으로 이어지는, (CNG) 2+ 유입 칼슘 활성화 (CL)의 개방에 이르게주기 - 채널 5, 6. 외부 CL 결과 - 플럭스는 높은 세포 (CL)에 의해 촉진된다 - 나 + / K + / CL을 통해 가능성 흡수, - - 공동 수송 NKCC1, 농도는 정상 (CL)에 의해 유지CL - / HCO3 - 교환 SLC4A1, 그리고 아마도 추가로 아직 확인되는 수송 6-8. 바이폴라 후각 신경 세포는 하나의 분지 후각 망울에 직접 투사 축삭과 상피의 표면에 연장하고 전문 구획, 수지상 노브로 끝나는 수상 돌기가있다. 최대 μm의 50 ~ 60까지의 길이에 도달 할 수 있습니다이 노브, 10 ~ 30 섬모,에서, 상피면 (9)을 덮고있는 점액으로 발산. 표준 신호 전달 캐스케이드의 단백질은 주로 이러한 섬모의 막 지역화됩니다. 상피 세포의 증가 된 감각 표면 취기를 감지하는 능력을 증폭한다. 인해 감각 뉴런의 밀도, 인접 수지상 노브로부터 연장 섬모 섞이는. 상피 세포의 표면 상에, 후각 수용체를 발현하는 다른 유형의 다른 뉴런의 섬모의 임의의 혼합물이 섞여서 결과. 검출 및 세포감각 신경의 일부에만 존재하는 섬모 단백질의 울라 할당은 저온부에서 어렵다. 저온부는 섬모의 평균 길이보다 일반적으로 더 얇은 때문에 또한, 섬모 따라 단백질의 정확한 정위 간신히 가능하다. 후각 신경 세포에서 지금까지 규명하지 않은 막 단백질의 섬모 현지화의 조사를 가능하게하기 위해, 우리는 섬모의 단백질 현지화의 상세한 분석을 허용하는 페이싱 준비 기술을 최적화. 요약하면, 마우스들을 희생하고 헤드는 중앙선 근처에 분할된다. 비갑개, 코, 그리고 정면 뼈 격막을 노출 제거됩니다. 안감 상피의 후각 부분 격벽은 비강에 모든 연결을 절단하여 느슨하게된다. 링거액 가득 페트리 접시에 격막을 넣은 후, 상피 코팅 된 유리 슬라이드로 전송 싶게 벗겨. 짧은 fixat 다음취급 깨지기 조직의 손상을 피하기 위해 가능한 한 완만 한 경우, 이온 단계는, 면역 염색 절차를 행할 수있다. 우리는 고전 저온부 및 설명 페이싱 제제 후각 섬모 두 가지 막 단백질의 염색을 비교하여 해상도를 달성 보여준다.

<table><tbody><tr><td>Spring scissors</td><td></td><td></td><td>straight tip, multiple suppliers</td></tr><tr><td>Surgical scissors</td><td></td><td></td><td>sharp and blunt end, multiple suppliers</td></tr><tr><td>Fine forceps</td><td></td><td></td><td>curved tips, Dumont #7, multiple suppliers</td></tr><tr><td>Razor blade</td><td></td><td></td><td>extra thin, multiple suppliers</td></tr><tr><td>Binocular with illumination</td><td></td><td></td><td>multiple suppliers, Stemi 2000-C, Zeiss</td></tr><tr><td>Petri dish</td><td></td><td></td><td>multiple suppliers</td></tr><tr><td>Liquid-blocker pen</td><td>Science Services</td><td>N71310</td><td></td></tr><tr><td>Polysine coated slides</td><td>Thermo Scientific</td><td>J2800AMNZ</td><td></td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>TCS SPE</td><td></td></tr><tr><td>primary antibody Goat anti-Kirrel2</td><td>R&amp;amp;D Systems</td><td>AF2930</td><td>1:200</td></tr><tr><td>primary antibody Rabbit anti-mOR-EG</td><td>Baumgart et al., 2014</td><td></td><td>1:200</td></tr><tr><td>secondary antibodies</td><td>Life Technologies</td><td>A21206, A11057</td><td>1:500</td></tr><tr><td>Mounting medium, ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>441244</td><td>toxic, work under fume hood</td></tr></tbody></table>

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.

얼굴 제제 EN 후각 상피 그 지역화 저온부 분석 후 불분명 단백질의 상세한 조사를 허용 감각 뉴런의 섬모 단백질의 위치 파악을 조사하는데 사용될 수있다. 이 문제는 IRRE 같은 단백질 2 (Kirrel2)의 킨에 대한 염색의 경우에 예시 할 수있다. (Neph3도 불림)는 Kirrel2 homophilic 접착 단백질로서 세포막 단백질의 기능과 면역 글로불린 (IG) 슈퍼 패밀리의 일원이다. 이것은 시스템 후각 축삭 - 유도에?...

Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

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

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 ...

whole-mount-labeling-of-cilia-in-the-main-olfactory-system-of-mice

Research

JoVE Journal

Neuroscience

11.1K 조회수.  Universitaetsklinikum Jena. 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.

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

Simple and Computer-assisted Olfactory Testing for Mice

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected with aging and is a strong predictor of neurodegenerative diseases. Thus, olfaction testing is used as a non-invasive diagnostic method to detect neurological deficits early on. In order to understand the mechanisms underlying olfactory network susceptibility, olfactory research in rodents has gained momentum in the past decade.
Here, we present a very simple, time efficient and reproducible olfactory testing method of innate odor perception and sensitivity in mice without the need of any prior food or water restriction. The tests are performed in a familiar environment to the mice, require only the scents and a 2 min session of odorant exposure. The analysis is performed, post-hoc, using computer-assisted commands on ImageJ and can be, therefore, carried out from start to end by one researcher.
This protocol does not require any special hardware or setup and is indicated for any laboratory interested in testing olfactory perception and sensitivity.

We present a simple and unbiased olfactory test in mice. With this protocol olfactory discrimination, preference, avoidance and sensitivity to a novel odor as compared to water can be assessed in single behavioral sessions. This method is indicated for a single experimenter and analysis is based on computer-assisted video processing.

간단하고 컴퓨터 지원 마우스에 대한 후각 테스트를

Olfaction is highly conserved among species and is required for reproduction and survival.
In humans, olfaction is also one of the senses that is affected ...

연구

행동분석학

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. Pheromones are volatile or non-volatile short-lived molecules2 secreted and/or contained in biological fluids3,4, such as urine, a liquid known to be a main source of pheromones3. Pheromonal communication is implicated in a variety of key animal modalities such as kin interactions5,6, hierarchical organisations3 and sexual interactions7,8 and are consequently directly correlated with the survival of a given species9,10,11. In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO)10,12, a paired structure located at the base of the nasal cavity, and enclosed in a cartilaginous capsule. Each VNO has a tubular shape with a lumen13,14 allowing the contact with the external chemical world. The sensory neuroepithelium is principally composed of vomeronasal bipolar sensory neurons (VSNs)15. Each VSN extends a single dendrite to the lumen ending in a large dendritic knob bearing up to 100 microvilli implicated in chemical detection16. Numerous subpopulations of VSNs are present. They are differentiated by the chemoreceptor they express and thus possibly by the ligand(s) they recognize17,18. Two main vomeronasal receptor families, V1Rs and V2Rs19,20,21,22, are composed respectively by 24023 and 12024 members and are expressed in separate layers of the neuroepithelium. Olfactory receptors (ORs)25 and formyl peptide receptors (FPRs)26,27 are also expressed in VSNs.
Whether or not these neuronal subpopulations use the same downstream signalling pathway for sensing pheromones is unknown. Despite a major role played by a calcium-permeable channel (TRPC2) present in the microvilli of mature neurons28 TRPC2 independent transduction channels have been suggested6,29. Due to the high number of neuronal subpopulations and the peculiar morphology of the organ, pharmacological and physiological investigations of the signalling elements present in the VNO are complex.
Here, we present an acute tissue slice preparation of the mouse VNO for performing calcium imaging investigations. This physiological approach allows observations, in the natural environment of a living tissue, of general or individual subpopulations of VSNs previously loaded with Fura-2AM, a calcium dye. This method is also convenient for studying any GFP-tagged pheromone receptor and is adaptable for the use of other fluorescent calcium probes. As an example, we use here a VG mouse line30, in which the translation of the pheromone V1rb2 receptor is linked to the expression of GFP by a polycistronic strategy.

In mice, the ability to detect pheromones is principally mediated by the vomeronasal organ (VNO). Here, an acute tissue slice preparation of VNO for performing calcium imaging is described. This physiological approach allows observations of subpopulations and/or individual neurons in a living tissue and is convenient for receptor-ligand identification.

이미징 페로몬은 마우스 Vomeronasal 급성 조직 슬라이스 준비에 감지

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

신경과학

Imaging Odor-Evoked Activities in the Mouse Olfactory Bulb using Optical Reflectance and Autofluorescence Signals

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. Functional optical imaging techniques are advantageous to visualize the activation of these modules in sensory cortices with high spatial resolution. In this context, endogenous optical signals that arise from molecular mechanisms linked to neuroenergetics are valuable sources of contrast to record spatial maps of sensory stimuli over wide fields in the rodent brain.
Here, we present two techniques based on changes of endogenous optical properties of the brain tissue during activation. First the intrinsic optical signals (IOS) are produced by a local alteration in red light reflectance due to: (i) absorption by changes in blood oxygenation level and blood volume (ii) photon scattering. The use of in vivo IOS to record spatial maps started in the mid 1980's with the observation of optical maps of whisker barrels in the rat and the orientation columns in the cat visual cortex1. IOS imaging of the surface of the rodent main olfactory bulb (OB) in response to odorants was later demonstrated by Larry Katz's group2. The second approach relies on flavoprotein autofluorescence signals (FAS) due to changes in the redox state of these mitochondrial metabolic intermediates. More precisely, the technique is based on the green fluorescence due to oxidized state of flavoproteins when the tissue is excited with blue light. Although such signals were probably among the first fluorescent molecules recorded for the study of brain activity by the pioneer studies of Britton Chances and colleagues3, it was not until recently that they have been used for mapping of brain activation in vivo. FAS imaging was first applied to the somatosensory cortex in rodents in response to hindpaw stimulation by Katsuei Shibuki's group4.
The olfactory system is of central importance for the survival of the vast majority of living species because it allows efficient detection and identification of chemical substances in the environment (food, predators). The OB is the first relay of olfactory information processing in the brain. It receives afferent projections from the olfactory primary sensory neurons that detect volatile odorant molecules. Each sensory neuron expresses only one type of odorant receptor and neurons carrying the same type of receptor send their nerve processes to the same well-defined microregions of &tilde;100&mu;m3 constituted of discrete neuropil, the olfactory glomerulus (Fig. 1). In the last decade, IOS imaging has fostered the functional exploration of the OB5, 6, 7 which has become one of the most studied sensory structures. The mapping of OB activity with FAS imaging has not been performed yet.
Here, we show the successive steps of an efficient protocol for IOS and FAS imaging to map odor-evoked activities in the mouse OB.

This article presents the protocols of intrinsic optical signals and flavoproteins autofluorescence signals imaging to map odor-evoked activities at the surface of the olfactory bulb in mice.

광 반사율과 Autofluorescence 신호를 사용하여 마우스 후각 망울에서 이미징 냄새 Evoked 활동

In the brain, sensory stimulation activates distributed populations of neurons among functional modules which participate to the coding of the stimulus. ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

에있는 후각 수용체 뉴런의 전체 마운트 Immunolabeling<em&gt; 초파리</em&gt; 안테나

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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 Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

전체 마운트 해부 및 성인 마우스 달팽이관의 면역 형광

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

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 Objective and Reproducible Test of Olfactory Learning and Discrimination in Mice

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and parenting. Importantly, mice can be trained to associate novel odors with specific behavioral responses to provide insight into olfactory circuit function. This protocol details the procedure for training mice on a Go/No-Go operant learning task. In this approach, mice are trained on hundreds of automated trials daily for 2&#8211;4 weeks and can then be tested on novel Go/No-Go odor pairs to assess olfactory discrimination, or be used for studies on how odor learning alters the structure or function of the olfactory circuit. Additionally, the mouse olfactory bulb (OB) features ongoing integration of adult-born neurons. Interestingly, olfactory learning increases both the survival and synaptic connections of these adult-born neurons. Therefore, this protocol can be combined with other biochemical, electrophysiological, and imaging techniques to study learning and activity-dependent factors that mediate neuronal survival and plasticity.

Here, we train mice on an associative learning task to test odor discrimination. This protocol also allows for studies on learning-induced structural changes in the brain.

후 각 학습 및 쥐에 있는 차별의 객관적이 고 재현성 시험

Olfaction is the predominant sensory modality in mice and influences many important behaviors, including foraging, predator detection, mating, and ...

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.

<meta charset="utf8"></head><body>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.

Investigation of Activated Mouse Olfactory Sensory Neurons via Combined Immunostaining and in situ Hybridization (결합된 면역염색 및 현장 혼성화를 통한 활성화된 마우스 후각 감각 뉴런 조사)

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