이 작품은 국립 보건원의 난청 및 기타 통신 장애의 신경 질환 및 뇌졸중의 국립 연구소 (NINDS) 및 국립 연구소 (NIDCD), (NIH)의 교내 프로그램에 의해 지원되었다. 우리는 NINDS EM 시설과 지원 NIDCD 고급 이미징 코어 (코드 번호 ZIC DC 000081-03)를 감사합니다.

관리 및 동물의 처리는 NIH 동물 관리 및 사용위원회의 가이드 라인에 따라했다. 12 시간 빛 : 출생 후 하루 (P) 양측 우수한 둔덕을 통해 1-1.2 % CTB를 주입 15-21 흰쥐는 12 일 유지 하였다 어두운주기를. 1. 망막 조직 고정 <ol><li> 해부 현미경, 아주 좋은 팁, ​​가위, 셀룰로오스 필터 종이, 플라스틱 피펫과 현미경 슬라이드 2 집게 다음 재료 및 도구를 조립합니다. </li><li> 2.0 ml의 할로 탄 (흡입 마취제)와 밀폐 된 챔버에 쥐를 마취. 발가락 핀치, 또는 깜박임 반사의 부족 후 후방 발의 철회의 부족 :이 방법으로 적절한 마취를 결정합니다. 그런 다음 단두대 즉시 목을 베다. pH가 7.4에서 0.1 M 인산 완충액 (PB)에 4 % 파라 포름 알데히드를 포함하는 유리 접시에 홍채 가위 쌍의 장소 눈을 제거합니다. </li><li> 해부 현미경을 사용하면, 옥수수를 제거안구의 전면을 차단하여 개. 집게와 내부 망막 표면에서 렌즈와 유리체를 제거합니다. </li><li> 망막이 아이 컵에서 분리 될 때까지 두 집게로 공막 껍질. </li><li> 면도기 200 μm의 두께 스트립, pH의 변화 고정 대상 - (100)에 즉시 망막을 잘라. </li><li> 실온에서 20 분 - 10의 pH 10.5에서 30 분 다음에 4 % 파라 포름 알데히드를 더한 0.01 %의 글루 타르 알데히드 - 20 pH가 6.0에서 0.1 M PB 4 % 파라 포름 알데히드의 망막 스트립을 수정합니다. </li><li> (4 ℃에서의 pH 7.4), 0.15 mM의 CaCl2를 가진 PB 여러 회 세척 한 후에 0.1에서 (30 %의 O / N 후, 10 %, 20 %, 30 %로 각각 60 분) M 글리세롤 망막 스트립 cryoprotect 대체를 동결하기 전에 PB. </li></ol> 2. 동결 대체 참고 :이 동결 대체 방법은 이전에 게시 된 프로토콜 (19, 20)에서 수정됩니다. 또한, 악기 (장갑을 착용) 매우 추운 것을 매우 중요하다; 영형악기를 터치하면 therwise, 조직을 부분적으로 해동 수 있습니다. 이 단계의 모든 AFS 챔버 내에서 수행되고 악기는 챔버의 가장자리 위의 이동이 허용되지 않습니다. 유사하게, AFS에 사용 된 모든 화학 물질의 적절한 냉각이 필요하다. <ol><li> - 플 런지 동결 EM의 냉동 보존기구 (CPC)에서 -184 ° C에서 액체 프로판의 망막 스트립. 좋은 브러시를 사용하여, (브러시를 사용하여 별도의 버퍼를 심지) 급락로드의 끝에 가서 금속 스텁에 부착 된 양면 테이프의 작은 조각에 샘플을 배치합니다. </li><li> 액체 프로판으로 봉 런지 약 5 초 동안 거기에 유지하고, 액체 질소로 채워진다 작은 이송 챔버를 이용하여 자동 동결 교체기구 (AFS)으로 전송 ( &quot;LN 2 크라이 이송 컨테이너를 냉각 &#39;). </li><li> AFS에 냉동 샘플 및 기기 (집게와 메스)를 배치 한 후, 그 자체에 대한 실에서 악기를 냉각veral 분 조직을 터치하거나 작은 이송 챔버에 몇 초 동안 기기의 팁을 배치하여 이들을 냉각하기 전에 (액체 질소 충전 () LN 2 크라이 이송 컨테이너를 냉각 &#39;). </li><li> AFS에서하면 벌금 메스를 사용하여 스텁에서 샘플 테이프를 제거합니다. 또한, 질소 가스의 유량 제어를 유지 TF 이러한 과정에서 오픈 (TF는 &#39;LN이 증발기 용 조정기 제어&#39;라고 기재 함). </li><li> (AFS를 설정하기 전에, IE) 평면 매립 홀더에 고정 된 조직을 배치하기 전에, 투명 플라스틱 시트 얇은 원형 절단 각 웰의 바닥 라인하도록 홀더의 하부에 배치. 완료 할 때 중합 시험편 블록 비교적 쉽게 제거 할 수있다. 참고 : 이전에, 우리는 두 번 라이 헤르 + 젤라틴 캡슐 (Petralia와 Wenthold, 1999 참조) 사용하는 평면 삽입 홀더에 다른 방법을 사용하고, &lt;SUP&gt; 20. </li><li> = 4 ° C / 시간 (11.3 시간), T2에 의해 32 시간, S1은 = 증가 온도 T1 = -90 ° C ~ -45 ° C ~ 50 시간, S2에 대한 : AFS (악기 용어를 사용)에서 다음 자동 시퀀스를 사용하여 = 5 ° C / 시간 (9 시간), (총 = 142.3 시간) 40 시간 동안 T3 = 0 ° C로 증가 온도. 이전에 Timed 시퀀스를 시작하기 (-90 ° C에서) AFS에서 샘플을 배치하는 4 시간은 2 - 걸릴 수 있습니다. </li><li> AFS에서&gt; 32 시간 동안 -90 ℃에서 메탄올 1.5 % 우라 닐 아세테이트에 얼어 붙은 섹션을 담가. 평면 포함 알루미늄 홀더에 일곱 우물의 각 조직 (일반적으로) 두 가지를 놓고 (세 AFS에 맞게). <ol><li> 우물에서 우라 닐 아세테이트 / 메탄올로 조직 + 테이프를 놓고는 테이프에서 조직을 제거 너무 어려운 경우, 직전에 침투 (예 : Lowicryl HM20)를 시작 포함 매체에, 나중에 테이프를 제거합니다. </li></ol></li><li> 이어서, 온도를 단계적으로 증가 시간 당 -45 ° C (+ 4 ℃로; 자동 순서대로). </li><li> 각 플랫 포함 홀더에서 기존의 용액을 제거하기 위해 뾰족한 플라스틱 피펫을 사용하여, 다음 예비 냉각 신선한 메탄올을 추가하는 또 다른 표준 플라스틱 피펫을 사용하여, 미리 냉각 된 메탄올의 샘플을 세 번 씻으십시오. </li><li> 1, 2 :이어서, 동일한 방법을 사용하여 예 1의 매체 (HM20은 / 메탄올 매립 낮은 온도 매립 수지 점진적 샘플 침투 2 시간 동안 순수 수지이어서 2 시간 동안 한 각 후 수지를 변경할 등) O / N을 유지합니다. </li><li> 단지 웰의 상단에 도달하는 수지의 레벨을 조정 다음날 다시 수지를 변경한다. </li><li> 샘플을 중합 (-45 ° C 자동 시퀀스에서 0 ° C로, 시간 당 + 5 ° C) 40 시간 동안 UV 빛. </li><li> 그런 다음 AFS에서 샘플을 제거합니다. 일반적으로 샘플 블록은 여전히​​ 색상에 약간의 핑크색을 보여줍니다. RT O에서 화학 물질 흄 후드에서 형광 빛에 가까운 샘플을, 배치 / N 이상 그들이 때까지 앱귀를 완전히 취소합니다. </li></ol> 3. Postembedding EM Immunogold 면역 세포 화학 참고 : 23,24,25 설명 된대로 Postembedding의 면역 세포가 수행된다. <ol><li> 1 % 톨루이딘 블루와 1 ㎛을 Ultramicrotome와 부분, 얼룩 부분을 잘라 및 단면 방향을 위해 그들을 검사; 절편의 최적의 횡단면을 달성하기 위해 조직 블록의 방향. </li><li> 을 Ultramicrotome 70 nm 두께의 얇은 부분을 잘라 Formvar - 탄소 코팅 니켈 슬롯 그리드에 그들을 수집합니다. </li><li> 워시는 TBS 버퍼 세 번 (TBS, 0.05 M 트리스 완충액, 0.7 %의 NaCl, pH를 7.6) 세척 한 다음 증류수 H 2 O에 한 번 메쉬. </li><li> 하나 NMDAR 소단위 (항 - 토끼 GluN2A 1:50 항체 : 염소 항 - CTB를 함유하는 혼합물을 20 μL의 방울 (3000 1)의 다음, 30 분 동안 TBS 5 % BSA 20 μL 떨어진다 그리드 부화 및 , GluN2B 1:30)을, 또는 항 - 토끼 GluA2 / 3 (1:30)을2 % BSA와 TBS-트리톤 (TBST, 0.01 % 트리톤 X-100, 산도 7.6) 및 0.02 M NaN이 3 O / RT에서 N. </li><li> 5 분 TBS (산도 8.2) 다음 세 가지 별도의 10 TBS 방울 (20 μL) (PH 7.6), 10, 20 분에 세척 그리드. </li><li> TBST에 18 나노 금 입자에 결합 된 10 나노 금 입자와 당나귀 방지 염소 IgG에 (1:20)을 수행 결합 당나귀 항 - 토끼 IgG를 (1:20)를 포함하는 혼합물의 방울에 2 시간 (20 μL)에 대한 그리드를 품어 2 % BSA 및 0.02 M NaN의 3 (PH 8.2). </li><li> 초순수에 다음, 5, 5, 10 분 동안 TBS의 20 μl의 방울 (PH 7.6)에 그리드를 세척 한 다음 건조. </li><li> 어두운 조건 하에서 각각 8 및 5 분 동안 증류하여 H 2 O 5 % 우라 닐 아세테이트 및 0.3 %의 납 시트르산 Counterstain과 그리드. </li><li> 트리플 - 표지 실험, 그리드 부화 O / 항 - 염소 CTB의 혼합물 20 μL 방울 (1 : 3000)과 함께 RT에서 N, 항 - 마우스 PSD-95 (1 : 100), 및 항 - 토끼 GluN2A (1 : 50). 그런 다음 2를 2 시간 동안 그리드를 품다2 % BSA 및 0.02 M NaN이 3 TBST에 18, 10, 5 nm의 금 입자에 결합 된 IgG를 혼합 (PH 8.2) 0 μL 방울. 세척 사이에 항체 배양 후으로 대조 이중 라벨과 같은 절차를 유지합니다. </li><li> 컨트롤 이차 항체 단독인가 된 수행 하였다. </li><li> EM에보기 그리드 및 디지털화 된 이미지. 이 24 필요한 경우에만 밝기 및 대비를위한 어도비 포토샵 6.0에서 최종 수치를 처리합니다. </li></ol> 4. 정량 <ol><li> 수동 8,000X 확대에 구멍이나 균열이없는 내부 얼기 층 (IPL)의 선택 영역은, 무작위 25000 배율에서 IPL의 전체 깊이를 합성 사진. </li><li> 그들은) 역행 수송 CTB 신호 (큰 금 입자)가 포함되어, b)을 나타낸다 잘 정의 막은, 갈라진 틈 및 시냅스 밀도, C)는 적어도 두개의 작은 금 부분을 포함 할 때 원뿔 바이폴라 댓구에서 RGC 수지상 식별PSD 내의 icles 또는 extrasynaptic 세포막을 따라 하나 이상의 작은 금 입자. </li><li> 정량 분석​​을 위해 상기 기준에 따라, 55 RGC 수지상 - (45)를 수집한다. </li><li> PSD 및 NIH ImageJ에 소프트웨어 개별 RGC 덴 드라이트의 extrasynaptic 세포막의 길이를 측정한다. 같은 막 10 nm의 금 입자 내의 카운트 (7)의 평균 두께에 기초하여, 막 - 관련 - 세포막 26 9 내지. </li><li> 선형 마이크​​로 미터 (금색 / μm의) 당 금 입자의 수​​가 입자 밀도를 계산한다. 각 금 입자의 중심 (시냅스 위치) PSD의 중간 또는 (extrasynaptic 위치) PSD의 가장자리 사이의 거리를 측정한다. </li><li> 소프트웨어에 의해 통계 분석을 수행합니다. 수단을 비교하는 두 꼬리 t-테스트를​​ 사용합니다. 의미 P &lt;0.05 결론 지었다. 달리 명시하지 않으면, 리포트 값으로 ± SEM (18)를 의미한다. </li></ol>

<ol>
	<li>Copenhagen, D. R., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+endogenous+excitatory+amino+acids+from+turtle+photoreceptors.">Release of endogenous excitatory amino acids from turtle photoreceptors.</a> Nature. 341, 536-539 (1989).</li><li>W&#228;ssle, H., Boycott, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+architecture+of+the+mammalian+retina.">Functional architecture of the mammalian retina.</a> Physiol. Rev. 71, 447-480 (1991).</li><li>Mittman, S., Taylor, W. R., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+activation+of+two+types+of+glutamate+receptor+mediates+excitation+of+salamander+retinal+ganglion+cells.">Concomitant activation of two types of glutamate receptor mediates excitation of salamander retinal ganglion cells.</a> J. Physiol. 428, 175-197 (1990).</li><li>Matsui, K., Hosoi, N., Tachibana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excitatory+synaptic+transmission+in+the+inner+retina:+paired+recordings+of+bipolar+cells+and+neurons+of+the+ganglion+cell+layer.">Excitatory synaptic transmission in the inner retina: paired recordings of bipolar cells and neurons of the ganglion cell layer.</a> J. Neurosci. 18, 4500-4510 (1998).</li><li>Chen, S., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptically+released+glutamate+activates+extrasynaptic+NMDA+receptors+on+cells+in+the+ganglion+cell+layer+of+rat+retina.">Synaptically released glutamate activates extrasynaptic NMDA receptors on cells in the ganglion cell layer of rat retina.</a> J. Neurosci. 22, 2165-2173 (2002).</li><li>Diamond, J. S., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+NMDA+and+non-NMDA+receptors+to+the+light-evoked+input-output+characteristics+of+retinal+ganglion+cells.">The contribution of NMDA and non-NMDA receptors to the light-evoked input-output characteristics of retinal ganglion cells.</a> Neuron. 11, 725-738 (1993).</li><li>Lukasiewicz, P. D., Wilson, J. A., Lawrence, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA-preferring+receptors+mediate+excitatory+synaptic+inputs+to+retinal+ganglion+cells.">AMPA-preferring receptors mediate excitatory synaptic inputs to retinal ganglion cells.</a> J. Neurophysiol. 77, 57-64 (1997).</li><li>Higgs, M. H., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+uptake+limits+synaptic+excitation+of+retinal+ganglion+cells.">Glutamate uptake limits synaptic excitation of retinal ganglion cells.</a> J. Neurosci. 19, 3691-3700 (1999).</li><li>Taylor, W. R., Chen, E., Copenhagen, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+spontaneous+excitatory+synaptic+currents+in+salamander+retinal+ganglion+cells.">Characterization of spontaneous excitatory synaptic currents in salamander retinal ganglion cells.</a> J. Physiol. 486, 207-221 (1995).</li><li>Kennedy, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+of+PDZ+(DHR,+GLGF)+domains.">Origin of PDZ (DHR, GLGF) domains.</a> Trends. Biochem. Sci. 20, 350 (1995).</li><li>Kim, E., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDZ+domain+proteins+of+synapses.">PDZ domain proteins of synapses.</a> Nat. Rev. Neurosci. 5, 771-781 (2004).</li><li>Migaud, 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=Enhanced+long-term+potentiation+and+impaired+learning+in+mice+with+mutant+postsynaptic+density-95+protein.">Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein.</a> Nature. 396, 433-439 (1998).</li><li>Aoki, C., 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=Electron+microscopic+immunocytochemical+detection+of+PSD-95,+PSD-93,+SAP-102,+and+SAP-97+at+postsynaptic,+presynaptic,+and+nonsynaptic+sites+of+adult+and+neonatal+rat+visual+cortex.">Electron microscopic immunocytochemical detection of PSD-95, PSD-93, SAP-102, and SAP-97 at postsynaptic, presynaptic, and nonsynaptic sites of adult and neonatal rat visual cortex.</a> Synapse. 40, 239-257 (2001).</li><li>Davies, C., Tingley, D., Kachar, B., Wenthold, R. J., Petralia, 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=Distribution+of+members+of+the+PSD-95+family+of+MAGUK+proteins+at+the+synaptic+region+of+inner+and+outer+hair+cells+of+the+guinea+pig+cochlea.">Distribution of members of the PSD-95 family of MAGUK proteins at the synaptic region of inner and outer hair cells of the guinea pig cochlea.</a> Synapse. 40, 258-268 (2001).</li><li>Hartveit, E., Brandst&#228;tter, J. H., Sasso&#232;-Pognetto, M., Laurie, D. J., Seeburg, P. H., W&#228;ssle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+developmental+expression+of+the+NMDA+receptor+subunit+NR2A+in+the+mammalian+retina.">Localization and developmental expression of the NMDA receptor subunit NR2A in the mammalian retina.</a> J. Comp. Neurol. 348, 570-582 (1994).</li><li>Fletcher, E. L., Hack, I., Brandst&#228;tter, J. H., W&#228;ssle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+localization+of+NMDA+receptor+subunits+in+the+rat+retina.">Synaptic localization of NMDA receptor subunits in the rat retina.</a> J. Comp. Neurol. 420, 98-112 (2000).</li><li>Pourcho, R. G., Qin, P., Goebel, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+subcellular+distribution+of+NMDA+receptor+subunit+NR2B+in+the+retina.">Cellular and subcellular distribution of NMDA receptor subunit NR2B in the retina.</a> J. Comp. Neurol. 433, 75-85 (2001).</li><li>Ottersen, O. P., Landsend, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+glutamate+receptors+at+the+synapse.">Organization of glutamate receptors at the synapse.</a> Eur. J. Neurosci. 9, 2219-2224 (1997).</li><li>Petralia, R. S., Wenthold, R. J., Ariano, 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=Glutamate+receptor+antibodies:+Production+and+immunocytochemistry.">Glutamate receptor antibodies: Production and immunocytochemistry.</a> Receptor Localization: Laboratory Methods and Procedures. , 46-74 (1998).</li><li>Petralia, R. S., Wenthold, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemistry+of+NMDA+receptors.">Immunocytochemistry of NMDA receptors.</a> Methods. Mol. Biol. 128, 73-92 (1999).</li><li>Nusser, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+and+NMDA+receptors:+similarities+and+differences+in+their+synaptic+distribution.">AMPA and NMDA receptors: similarities and differences in their synaptic distribution.</a> Curr. Opin. Neurobiol. 10, 337-341 (2000).</li><li>Qin, P., Pourcho, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+of+AMPA-selective+glutamate+receptor+subunits+in+the+cat+retina:+a+light-+and+electron-microscopic+study.">Localization of AMPA-selective glutamate receptor subunits in the cat retina: a light- and electron-microscopic study.</a> Vis. Neurosci. 16, 169-177 (1999).</li><li>Zhang, J., Wang, H. H., Yang, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+organization+of+GABAergic+amacrine+cells+in+salamander+retina.">Synaptic organization of GABAergic amacrine cells in salamander retina.</a> Visual. Neurosci. 21, 817-825 (2004).</li><li>Zhang, J., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+perisynaptic+and+synaptic+localization+of+NMDA+and+AMPA+receptors+on+ganglion+cells+in+rat+retina.">Distinct perisynaptic and synaptic localization of NMDA and AMPA receptors on ganglion cells in rat retina.</a> J. Comp. Neurol. 498, 810-820 (2006).</li><li>Zhang, J., Diamond, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subunit-+and+Pathway-Specific+Localization+of+NMDA+Receptors+and+Scaffolding+Proteins+at+Ganglion+Cell+Synapses+in+Rat+Retina.">Subunit- and Pathway-Specific Localization of NMDA Receptors and Scaffolding Proteins at Ganglion Cell Synapses in Rat Retina.</a> J. Neurosci. 29, 4274-4286 (2009).</li><li>Peters, A., Palay, S., Webster, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The fine structure of the nervous system: neurons and their supporting cells, 3rd ed. , (1991).</li><li>Baude, A., Nusser, Z., Roberts, J. D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabotropic+glutamate+receptor+(mGluR1)+is+concentrated+at+perisynaptic+membrane+of+neuronal+subpopulations+as+detected+by+immunogold+reaction.">The metabotropic glutamate receptor (mGluR1) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.</a> Neuron. 11, 771-787 (1993).</li><li>Van Lookeren Campagne, M., Oestreicher, A. B., van der Krift, T. P., Gispen, W. H., Verkleij, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freeze-substitution+and+Lowicryl+HM20+embedding+of+fixed+rat+brain:+Suitability+for+immunogold+ultrastructural+localization+of+neural+antigens.">Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: Suitability for immunogold ultrastructural localization of neural antigens.</a> J. Hist. Cyt. 39, 1267-1279 (1991).</li><li>Matsubara, A., Laake, J. H., Davanger, S., Usami, S., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+AMPA+receptor+subunits+at+a+glutamate+synapse:+A+quantitative+immunogold+analysis+of+hair+cell+synapses+in+the+rat+organ+of+Corti.">Organization of AMPA receptor subunits at a glutamate synapse: A quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.</a> J. Neurosci. 16, 4457-4467 (1996).</li><li>Petralia, R. 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=Organization+of+NMDA+receptors+at+extrasynaptic+locations.">Organization of NMDA receptors at extrasynaptic locations.</a> Neuroscience. 167, 68-87 (2010).</li><li>Merighi, A., Polak, J. M., Priestley, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embedding+electron+microscopic+immunocytochemistry.">Post-embedding electron microscopic immunocytochemistry.</a> Electron Microscopic Immunocytochemistry: Principles and Practice. , 51-87 (1992).</li><li>Ottersen, O. P., Takumi, Y., Matsubara, A., Landsend, A. S., Laake, J. H., Usami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+organization+of+a+type+of+peripheral+glutamate+synapse:+the+afferent+synapses+of+hair+cells+in+the+inner+ear.">Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear.</a> Prog. Neurobiol. 54, 127-148 (1998).</li><li>Nusser, Z., Lujan, R., Laube, G., Roberts, J. D., Molnar, E., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+and+pathway+dependence+of+synaptic+AMPA+receptor+number+and+variability+in+the+hippocampus.">Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.</a> Neuron. 21, 545-559 (1998).</li><li>Takumi, Y., Ramirez-Leon, V., Laake, P., Rinvik, E., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+modes+of+expression+of+AMPA+and+NMDA+receptors+in+hippocampal+synapses.">Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.</a> Nat. Neurosci. 2, 618-624 (1999).</li><li>Grimes, W. N., 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=Complex+inhibitory+microcircuitry+regulates+retinal+signaling+near+visual+threshold.">Complex inhibitory microcircuitry regulates retinal signaling near visual threshold.</a> J. Neurophysiol. 114, 341-353 (2015).</li><li>Sassoe-Pognetto, M., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+ionotropic+glutamate+receptors+at+dendrodendritic+synapses+in+the+rat+olfactory+bulb.">Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb.</a> J. Neurosci. 20, 2192-2201 (2000).</li></ol>

The authors have no disclosures.

우리는 성공적으로 정량 후 삽입 immunogold에게 EM을 네 가지 기술을 설명했다 : 1) 짧고 약한 고정, 2) 동결 대체, 3) 포스트 포함 immunogold 염색, 4) 정량. EM의 immunogold는 얇은 조직 섹션에서 특정 단백질의 검출을 할 수 있습니다. 금 입자로 표지 된 항체를 직접 EM을 사용하여 가시화 할 수있다. 막 수용체의 subsynaptic 현지화를 검출하는 강력한 반면, EM의 immunogold 기술적으로 도전, ?...

글루타메이트는 망막 1에서 주요 흥분성 신경 전달 물질이다. 바이폴라 전지 (2)로부터 글루타메이트 시냅스 입력을 수신하는 망막 신경절 세포 (망막 신경절 세포)는, 뇌 영상 정보를 전송 망막의 출력 뉴런이다. 생리 학적 연구는 망막 신경절 세포의 시냅스 여기가 NMDA 수용체 (NMDARs)와 AMPA 수용체 (AMPARs) 3,4,5에 의해 postsynaptically 매개되는 것으로 나타났다. 망막 신경절 세포의 흥분성 시냅스 후 전류 (EPSCs)가 AMPARs 및 NMDARs에 의해 매개되어 있지만 망막 신경절 세포에 3,5,6,7,8, 자연 미니어처 EPSCs (mEPSCs)는 만 AMPARs 매개 구성 요소 4,5,9을 나타낸다. 그러나, 글루타메이트 흡수를 감소시키는 것은 RGC의 수상 돌기에 NMDARs이 흥분성 시냅스의 외부에 위치 될 수 있음을 시사 자발적인 EPSCs 5 NMDAR 구성 요소를 한 것으로 밝혀졌습니다. 막 - 관련 구아닐산 키나제 등 PSD-95 (MAGUKs)이 글루타메이트 수용체 이온 채널을 포함한 클러스터 신경 전달 물질 수용체시냅스 부위에서의도 구별 subsynaptic 10,11,12,13,14 발현 패턴을 나타낸다. 최근 수십 년간, 촛점 면역 사전 매립 전자 현미경 (EM) 면역 위에 막 수용체 발현을 연구하기 위해 사용되었다. 촛점 면역 수용체 발현 넓은 패턴을 보여준다하더라도, 그 해상도가 낮은 것이 불가능 세포 내 위치를 구별하는 데 사용할 수있다. 포유 동물의 망막에 사전 내장 EM 연구는 NMDAR의 서브 유닛이 콘 바이폴라 전지 리본 시냅스 15, 16, 17에서 시냅스 요소 내에 존재 함을 나타냅니다. 이것은 생리 학적 증거 명백 대조적이다. 그러나, 반응 생성물의 확산을 미리 매립 immunoperoxidase 방법에서 공지 된 이슈이다. 따라서,이 방법은 일반적으로 통계적으로 신뢰할 수있는 데이터를 제공하지 않습니다 및 extrasynaptic 막 18,19,20,21 대 시냅스 막에 현지화 사이의 구별을 제외 할 수 있습니다. 에다른 한편으로는, 생리 및 해부학 적 데이터는 망막 신경절 세포 3,5,7,9,22에 AMPARs의 시냅스 현지화과 일치한다. 따라서, 망막 리본 시냅스에서 글루타메이트 수용체와 MAGUKs는 시냅스에뿐만 아니라 perisynaptic 또는 extrasynaptic 막 구획뿐만 아니라 현지화됩니다. 그러나, 망막 리본 시냅스에서 이들의 막 단백질의 고해상도의 정량 분석​​이 여전히 필요하다. 여기서는 콜레라 독소 소단위 B를 사용하여 라벨 (CTB)를 래트 망막 신경절 세포 상 시냅스에서 이들 단백질의 개수 밀도 변동을 추정 하였다 NMDAR 소단위, AMPAR 소단위와 PSD-95 subsynaptic 현지화를 검사 postembedding EM의 immunogold 기술 개발 역행 추적 방법.

<table border="0" cellpadding="0" cellspacing="0" width="761">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Paraformaldehyde</td>
			<td style="width:167px;">EMS</td>
			<td style="width:127px;">15710</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutarldehyde</td>
			<td>EMS</td>
			<td>16019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Sigma</td>
			<td style="width:127px;">7782-85-6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Sigma</td>
			<td>C-8106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>Sigma</td>
			<td>A-7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>Sigma</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaN3</td>
			<td>JT Baker</td>
			<td>V015-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Gibco BRL</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lowicryl HM 20</td>
			<td>Polysciences</td>
			<td>15924-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-Base</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris</td>
			<td>Fisher</td>
			<td>04997-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluN2A</td>
			<td>Millipore</td>
			<td>AB1555P</td>
			<td>Dilution 1/50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluN2B</td>
			<td>Millipore</td>
			<td>AB1557P</td>
			<td>Dilution 1/30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-GluA2/3</td>
			<td>Millipore</td>
			<td>AB1506</td>
			<td>Dilution 1/30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-PSD-95</td>
			<td>Millipore</td>
			<td>MA1&#8211;046</td>
			<td>Dilution 1/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-rabbit IgG-10 nm gold particles</td>
			<td>EMS</td>
			<td>25704</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-mouse IgG-10 nm gold particles</td>
			<td>EMS</td>
			<td>25814</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-mouse IgG-5 nm gold particles</td>
			<td>EMS</td>
			<td>25812</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-goat IgG-18 nm gold particles</td>
			<td>Jackson ImmunoResearch</td>
			<td style="width:127px;">705-215-147</td>
			<td>Dilution 1/20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formvar-Carbon coated nickel-slot grids.</td>
			<td>EMS</td>
			<td>FCF2010-Ni</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uranyl acetate</td>
			<td>EMS</td>
			<td>22400-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>EMS</td>
			<td>67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lead citrate</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica EM AFS</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica EM CPC</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultromicrotome</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JEOL 1200 EM</td>
			<td>JEOL</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">liquid nitrogen&#160;</td>
			<td>Roberts Oxygen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propane</td>
			<td>Roberts Oxygen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CTB</td>
			<td>List Biological Laboratories</td>
			<td>104</td>
			<td>1-1.2%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-CTB</td>
			<td>List Biological Laboratories</td>
			<td>703</td>
			<td>Dilution 1/4000</td>
		</tr>
	</tbody>
</table>

high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

이전 24, 25 설명한 바와 같이 여기에 제시된 결과는 쥐의 망막에서 RGC의 수상 돌기에 현저하게 다른 subsynaptic 현지화 GluA 2/3의 패턴과 NMDARs을 보여줍니다. RGC 수지상 정보에 GluA 2/3 immunogold 입자의 77 %가 가장 중심 시냅스 마찬가지로, PSD (도 1a) 내에 위치 하였다. 그러나 NMDARs은 하나 시냅스 또는 extrasynaptically에 있었다. GluN2A의 immunogold 입자의 83 %가 PSD ?...

Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

망막 리본 시냅스에서 막 수용체의 고해상도 양이 Immunogold 분석

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

high-resolution-quantitative-immunogold-analysis-membrane-receptors

Research

JoVE Journal

Neuroscience

7.4K 조회수.  National Institutes of Health. The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

비디오: High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

여러 스케일에서 디지털의 연결 형태론의 자동 숄 분석

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

연구

신경과학

Postsynaptic Recordings at Afferent Dendrites Contacting Cochlear Inner Hair Cells: Monitoring Multivesicular Release at a Ribbon Synapse

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the postsynaptic activity of a single ribbon synapse 1-4. Ribbon synapses of sensory cells release neurotransmitter continuously, the rate of which is modulated in response to graded changes in IHC membrane potential 5. Ribbon synapses have been shown to operate by multivesicular release, where multiple vesicles can be released simultaneously to evoke excitatory postsynaptic currents (EPSCs) of varying amplitudes 1, 4, 6-11. Neither the role of the presynaptic ribbon, nor the mechanism underlying multivesicular release is currently well understood.
The IHC is innervated by 10-20 auditory nerve fibers, and every fiber contacts the IHC with a unmyelinated single ending to form a single ribbon synapse. The small size of the afferent boutons contacting IHCs (approximately 1 &mu;m in diameter) enables recordings with exceptional temporal resolution to be made. Furthermore, the technique can be adapted to record from both pre- and postsynaptic cells simultaneously, allowing the transfer function at the synapse to be studied directly 2. This method therefore provides a means by which fundamental aspects of neurotransmission can be studied, from multivesicular release to the elusive function of the ribbon in sensory cells.

Whole-cell patch-clamp recordings from auditory nerve fiber dendrites at the inner hair cell ribbon synapse in the mammalian cochlea.

달팽이 내부 세포에 문의 수입 성의 Dendrites에서 Postsynaptic 레코딩 : 리본 시냅스에서 모니터링 Multivesicular 릴리스

The afferent synapse between the inner hair cell (IHC) and the auditory nerve fiber provides an electrophysiologically accessible site for recording the ...

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

망막 안료 상피의 전체 마운트를위한 마우스 아이의 해부

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

인간 Midbrain위한 고해상도 기능성 자기 공명 영상 방법

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

개발 Neuroepithelium의 세포 문제의 고해상도 라이브 영상

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

의 해부, 문화, 및 분석<em&gt; Xenopus laevis</em&gt; 배아 망막 조직

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

High-throughput Analysis of Mammalian Olfactory Receptors: Measurement of Receptor Activation via Luciferase Activity

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors&#8212;an essential step toward a model of odor coding in the mammalian olfactory system.

Olfactory receptor activation patterns encode odor identity, but the lack of published data identifying odorant ligands for mammalian olfactory receptors hinders the development of a comprehensive model of odor coding. This protocol describes a method to facilitate high-throughput identification of olfactory receptor ligands and quantification of receptor activation.

루시 페라 제 활동을 통해 수용체 활성화의 측정 : 포유류의 후각 수용체의 높은 처리량 분석

Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors ...

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

조직 추출물의 고해상도 핵 자기 공명 분광법에 의한 쥐 뇌의 대사 체 분석

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Peering at Brain Polysomes with Atomic Force Microscopy

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, formed by ribosomes, mRNAs, several proteins and non-coding RNAs, represent integrated platforms where translational controls take place. However, while the ribosome has been widely studied, the organization of polysomes is still lacking comprehensive understanding. Thus much effort is required in order to elucidate polysome organization and any novel mechanism of translational control that may be embedded. Atomic force microscopy (AFM) is a type of scanning probe microscopy that allows the acquisition of 3D images at nanoscale resolution. Compared to electron microscopy (EM) techniques, one of the main advantages of AFM is that it can acquire thousands of images both in air and in solution, enabling the sample to be maintained under near physiological conditions without any need for staining and fixing procedures. Here, a detailed protocol for the accurate purification of polysomes from mouse brain and their deposition on mica substrates is described. This protocol enables polysome imaging in air and liquid with AFM and their reconstruction as three-dimensional objects. Complementary to cryo-electron microscopy (cryo-EM), the proposed method can be conveniently used for systematically analyzing polysomes and studying their organization.

While ribosome structure has been extensively characterized, the organization of polysomes is still understudied. To overcome this lack of knowledge, we present here a detailed preparation protocol for accurate imaging of mammalian polysomes by atomic force microscopy (AFM) in air and liquid.

원자 힘 현미경과 뇌 Polysom​​es에서 피어링

The translational machinery, i.e., the polysome or polyribosome, is one of the biggest and most complex cytoplasmic machineries in cells. Polysomes, ...