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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+the+olfactory+system+makes+sense+of+scents.">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/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>Wong, S. 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=Disruption+of+the+type+III+adenylyl+cyclase+gene+leads+to+peripheral+and+behavioral+anosmia+in+transgenic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+deficient+in+G(olf)+are+anosmic.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+anosmia+caused+by+a+targeted+disruption+of+the+mouse+olfactory+cyclic+nucleotide-gated+cation+channel.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+excitatory+Cl-+response+in+mouse+olfactory+receptor+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+components+of+signal+amplification+in+olfactory+sensory+cilia.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+lacking+NKCC1+have+normal+olfactory+sensitivity.">Mice lacking NKCC1 have normal olfactory sensitivity.</a> Physiolog., &amp; Behavior. 93 (1-2), 44-49 (2008).</li><li>Menco, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+aspects+of+olfactory+signaling.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neuronal+identity+code+for+the+odorant+receptor-specific+and+activity-dependent+axon+sorting.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaffolding+by+MUPP1+regulates+odorant-mediated+signaling+in+olfactory+sensory+neurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+odorant+receptors+in+spatially+restricted+subsets+of+chemosensory+neurones.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+cilia:+linking+sensory+cilia+function+and+human+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Bardet-Biedl+syndrome+protein-8+(BBS8)+perturbs+olfactory+function,+protein+localization,+and+axon+targeting.">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 border="0" cellpadding="0" cellspacing="0" width="629">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spring scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">straight tip, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Surgical scissors</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">sharp and blunt end, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fine forceps</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">curved tips, Dumont #7, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Razor blade</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">extra thin, multiple suppliers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Binocular with illumination</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers, Stemi 2000-C, Zeiss</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">multiple suppliers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Liquid-blocker pen</td>
			<td style="width:127px;">Science Services</td>
			<td style="width:119px;">N71310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polysine coated slides</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td style="width:119px;">J2800AMNZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Confocal microscope</td>
			<td style="width:127px;">Leica Microsystems</td>
			<td style="width:119px;">TCS SPE</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Goat anti-Kirrel2</td>
			<td style="width:127px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2930</td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">primary antibody Rabbit anti-mOR-EG</td>
			<td style="width:127px;">Baumgart et al., 2014</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">secondary antibodies</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">A21206, A11057</td>
			<td style="width:167px;">1:500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Mounting medium, ProLong Gold antifade reagent</td>
			<td style="width:127px;">Life Technologies</td>
			<td style="width:119px;">P36930</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Paraformaldehyde</td>
			<td style="width:127px;">Sigma</td>
			<td align="right" style="width:119px;">441244</td>
			<td style="width:167px;">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

11.0K 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

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

전체 마운트의 배아 신경 계통의 시각화 Drosophila 태아

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

연구

신경과학

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

강한 감각 뉴런의 속눈썹에 갇힌 화합물의 플래시 광분해

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

마우스 배아 척수의 전체 마운트 준비 분기 Axonal 공부 DRG 뉴런의 DiI - 라벨링

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

의 중추 신경계에 단일 세포의 라벨<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

혈관을 조사하기 위해 신생아 마우스 망막의 전체 마운트 면역 형광 염색<em&gt; 생체 내</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

전신 탈염 신경 근육 정션 분석을위한 트랜스 대 복부 근육의 해부

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

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 ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

마우스 뇌의 좌우 심실의 뇌실막 섬모의 라이브 영상

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

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 ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

키메라를 구축의 HSV-중재 형질 전환 유전자의 발현은 마우스의 GPCR Heteromers의 행동 기능을 연구하는

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...