Drosophila melanogaster의 연결 근육질 교차로 1) 준비 (NMJ) <ol><li> 표준 Drosophila 호수 (130 MM NaCl, 36 MM의 자당, 5 MM KCl, 2 MM CaCl 2, 2 MM MgCl 2, 5 MM Hepes, 산도 7.3 4 준비합니다. </li><li> 식염 (1.1)의 준비를 해부하다. Drosophila의 애벌레는 Sylgard 접시에 지느러미 측면까지 pined이며 지느러미쪽에 세로 sectioned이며, 내장은 제거됩니다. 준비 나서 뻗어와 pined 있습니다. 여러 복부 근육 그런 다음 사용할 수 있습니다. </li></ol> 2) 자극과 FM 염색법 <ol><li> 표백에서 FM 염료를 보호하기 위해, 낮은 조명 조건에있는 모든 다음 단계를 수행하는 것이 좋습니다입니다. </li><li> 신경의 화학적 자극에 대한 높은 칼륨 버퍼가 사용됩니다. KCl 90 MM 및 FM1 -​​ 43 염색 10 μm의를 포함하는 Drosophila 호수 (1.1 참조) 준비합니다. 조명으로부터 보호 솔루션을 유지. </li><li> 실온에서 1 분 이전에 준비를 버퍼로 준비 목욕탕. </li><li> 세포외 FM1 -​​ 43 염료를 제거하는 표준 Drosophila 호수 (1.1)로 씻으십시오. 고정으로 급속하게 진행합니다. </li></ol> 3) 고정 <ol><li> 실온에서 45 분간 인산염 버퍼 호수 (PBS)에 2.5 %의 글루 타 알데히드와 함께 준비 목욕탕. 형태학의 특징을 잘 보존 들어, 글루 타 알데히드는 paraformaldehyde로 선호합니다. </li><li> PBS로 한번 씻어 후 NH 4 Club 호텔의 100 밀리미터에서 15 분 잠수 준비를 두십시오. 이 단계는 남아있는 글루 타 알데히드 정착액의 무료 알데히드 그룹을 담금질하기 위해 수행됩니다. </li><li> 정상 PBS로 NH 4 Club 호텔 솔루션을 씻으십시오. </li></ol> 4) Photoconversion <ol><li> 4 30 분 NMJ 준비를 품어 ° PBS에서 C는 diaminobenzidine (DAB)의 1.5 MG ML - 1을 포함. </li><li> 형광 현미경 샘플을 위치. 상대적으로 낮은 배율 물 침지 목표 (20x 0.5 NA)를 사용하여 형광 신호를 찾아 중점을두고 있습니다. </li><li> FM의 염료가 (그림 1C) 완전히 표백 때까지 최대 램프 강도와 샘플을 조명. photoconversion가 발생하면 제어하려면, FM 염료 표백 단계 이전과 이후 전송 조명 아래의 예제에서 확인하는 것이 좋습니다. photoconversion이 이루어지는 경우, 어두운 갈색 침전물은 (그림 1C) 예정입니다. 조명 시간은 준비에 DAB 보급에 따라 또한 조명의 강도와에 따라 변수이다. 대부분의 준비를 위해 20x 목표를 사용하는 경우 ~ 30-45분의 조명 시간이 최적으로 발견했습니다. 짧은 조명 기간은 높은 배율의 목표 (60x)에 사용됩니다. </li><li> 조명을 위해 우리는 백 흩어진 빛의 광선을 수집하고, 따라서 총 강도를 증가 돌려 거울을 포함하는 램프 주택, 수은 램프를 사용하는 것을 선호합니다. </li></ol> 5) EM에 대한 샘플을 처리 <ol><li> Osmication <ol class="lalpha"><li> 물이 나무 볼륨에 오스뮴 tetroxide (샘플 당 약 300 μl) 중 하나 볼륨있는 솔루션을 준비합니다. 오스뮴의 tetroxide를 사용하는 것은 장갑 알겠 보호 장비를 사용하여 후드 아래에 이루어져야합니다. 작업 </li><li> 실온 (후드 아래)에서 1 시간 오스뮴의 tetroxide 솔루션의 준비를 품어. </li><li> PBS (4-5 회 5 분)와 함께 광범위하게 씻고 깨끗한 유리 플라스크에 각각의 샘플을 전송합니다. </li></ol></li><li> 탈수 <ol class="lalpha"><li> 30, 50, 70 90, 95 및 에탄올의 100 %를 포함하는 별도의 솔루션을 준비합니다. </li><li> 5 분 각각의 샘플을 30 % 에탄올 1 ML을 추가합니다. </li><li> 5 분 각 샘플의 50 % 에탄올 1 ML을 추가합니다. </li><li> 5 분 각각의 샘플을 70 % 에탄올 1 ML을 추가합니다. </li><li> 5 분 각각의 샘플을 90 % 에탄올 1 ML을 추가합니다. </li><li> (배를 반복) 5 분 동안 각각의 샘플을 95 % 에탄올 1 ML을 추가합니다. </li><li> (배를 반복) 5 분 동안 각 샘플에 100 % 에탄올 1 ML을 추가합니다. </li></ol></li><li> 퍼가기 및 추가 처리 <ol class="lalpha"><li> 에탄올 중 하나 볼륨 Epon 수지 1 볼륨을 섞는다. 연속 회전 (2~4시간)에서 준비에 추가합니다. </li><li> 오픈 flasks 100 % Epon에 준비를 놓고, 나머지 에탄올이 증발하도록 (4-6 시간). </li><li> 36 시간 동안 60 ° C에서 금형의 준비를 놓습니다. </li><li> 전자 현미경 처리. 표준 ultramicrotomy 절차를 사용하여 50-80 nm의 얇은 부분의 준비를 처리합니다. </li></ol></li><li> 전자 현미경 이미징 <ol class="lalpha"><li> 준비는 종래의 EM 절차를 사용하여 몇 군데 있습니다. </li></ol></li></ol> 6) 대표 결과 예상 결과는 그림 1C 2에 요약되어 있습니다. 형제의 형성에 조명 절차 결과형광 및 전송 영상 모두에서 표시됩니다 wn의 DAB의 침전물. 조명 중에 첫 번째 사건은 FM 염색의 얼룩과 관련된 형광의 실종이다. 알데히드 정착액에 의해 유도된 배경 형광은 반대로, 실험을 통해 확인할 수 있습니다. 표백은 조명의 시작 후 ~ 10~20분에서 일반적으로 이루어진다. 일반적으로 더 photoconversion 제품이 시점에서 관찰 수 없습니다. 조명은 계속되어야하고, ~ 10 분 후에 준비가 그것은 동시에 자리를하지 않는 이유 DAB 석출 및 FM 염료는 표백 불분명 DAB 축적으로 인해 갈색 그늘 (그림 1C IV)를 설정하는 것이며 그것은 비교적 큰 수 있습니다 산화 DAB의 금액은 집합과 침전하기 전에 축적해야하고, 따라서이 반응은 표백 과정을보다 느린입니다. DAB의 석출 가능성이 불완전으로이 단계에서는, 그러나, 준비, 전자 현미경 처리를위한 준비가되지 않습니다. 우리는 그 동안 준비 (presynaptic 신경 터미널) 완료 변환을 나타내는 어두운 갈색 (또는 검은색) 색상, 가정, 5-10분 이상 기다려야하는 것을 선호합니다. 준비의 일반적인 표면의 약간의 전환 (예를 들어, 신경근육학 교차점에있는 근육 섬유의)은이 단계에서 볼 수 있습니다. 그것은 가능성이 높습니다 autofluorescence (및 / 또는 정착액 형광)에 의해 유도된 DAB의 전환과 관련, 그리고 그것은 프로 시저에 해로운하지 않습니다. 준비 그러면 전자 현미경에 대한 처리이며, 어두운 (레이블) vesicles의 존재를 확인하여야한다. 우리가 5,6하기 전에 보여주는 것처럼, 이러한 vesicles가 아닌 표시된 것들보다 훨씬 밀도 있고, 따라서 쉽게 (그림 2B) 구분합니다. 잘 vesicles의 다른 유형을 구별의 기회를 증가하려면 섹션에 대한 대비를 향상 포스트 얼룩은 (NO uranyl 아세테이트 또는 리드의 구연 산염의 얼룩) 수행되지 않을 것입니다, 오스뮴의 얼룩은 대부분의 세포 요소를 관찰하기에 충분하고, 같이하지 않습니다 DAB의 침전물로 어두운. <img src="/files/ftp_upload/1790/1790fig1.jpg" alt="그림 1" /> 그림 1 : FM 염료하고 photoconversion을 위해 사용합니다. (A) 자극하에 FM 염료를 사용하여 vesicles를로드하고 distaining에 대한 고전적인 실험의 단계를 나타냅니다. (I)는 버퍼를 포함하는 FM 염료에 샘플을 품어. (2) FM 색소의 존재에 소포의 융합을 유도하기 위해 (전기 또는 화학적으로) 자극. (3) 자극 후 subsequence의 endocytosis가 세포 버퍼에있는 사람들 FM 색소 몇 가지 vesicles을로드합니다. (IV) 버퍼와 함께 세탁하여 세포 FM 염료를 교체하십시오. (V) 새로운 자극이 exocytosis에 따라 자신의 FM 염료를 공개로드 vesicles을 유발합니다. (B) 도식의 머리, 다리와 FM1 - 43 염료의 꼬리를 보여주는. Drosophila NMJ에 photoconversion의 실험 (C) 예. (I) 외부 염료 분자와 고정을 세척 FM1 - 43와 NMJ를 로딩 후 신경 단말기의 형광은 종래의 epifluorescence 현미경 (63x)로 볼 수 있습니다. (II - III) 지속적인 조명 아래 염료가 완전히 표백됩니다. (4) 조명이 계속되면 검은 색깔은 diaminobenzidine의 침전으로 인해 나타납니다. 스케일 바는 10 μm의를 나타냅니다. <img src="/files/ftp_upload/1790/1790fig2.jpg" alt="그림 2" /> 그림 2 : EM 그들의 photoconverted 샘플 예제 (A) 이미지 시냅스 vesicles를 포함하는 신경 터미널을 보여줍니다,하지만이 photoconverted되며 이것은 충분한 조명이나 조직에서 가난한 diaminobenzidine의 침투를 가지고 있지으로 인해 발생할 수 있습니다.. (B) FM photoconversion 겪은 여러 어두운 (가득) vesicles와 시냅틱 부통. 더 vesicles 또는 organelles는 구별할 수없는 전반적인 어두운 조명 터미널에서 결과의 (C) 초과. 스케일 바는 200 나노미터를 나타냅니다.

<ol>
	<li>Betz, W. J., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+analysis+of+synaptic+vesicle+recycling+at+the+frog+neuromuscular+junction.">Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.</a> Science. 255, 200-203 (1992).</li><li>Henkel, A. W., L&#252;bke, J., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM1-43+dye+ultrastructural+localization+in+and+release+from+frog+motor+nerve+terminals.">FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.</a> Proc Natl Acad Sci USA. 93, 1918-1923 (1996).</li><li>Sandell, J. H., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconversion+of+some+fluorescent+markers+to+a+diaminobenzidine+product.">Photoconversion of some fluorescent markers to a diaminobenzidine product.</a> J Histochem Cytochem. 36, 555-559 (1988).</li><li>Kuromi, H., Kidokoro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+optically+determined+size+of+exo/endo+cycling+vesicle+pool+correlates+with+the+quantal+content+at+the+neuromuscular+junction+of+Drosophila+larvae.">The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.</a> J Neurosci. 19, 1557-1565 (1999).</li><li>Denker, A., Kr&#246;hnert, K., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+synaptic+vesicle+pool+localization+in+the+Drosophila+neuromuscular+junction.">Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction.</a> J Physiol (Lond). 587, 2919-2926 (2009).</li><li>Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+organization+of+the+readily+releasable+pool+of+synaptic+vesicles.">The structural organization of the readily releasable pool of synaptic vesicles.</a> Science. 303, 2037-2039 (2004).</li><li>Darcy, K. J., Staras, K., Collinson, L. M., Goda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+sharing+of+recycling+synaptic+vesicles+between+presynaptic+boutons.">Constitutive sharing of recycling synaptic vesicles between presynaptic boutons.</a> Nat Neurosci. 9, 315-321 (2006).</li><li>Harata, N., Ryan, T. A., Smith, S. J., Buchanan, J., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+recycling+synaptic+vesicles+in+hippocampal+neurons+by+FM1-43+photoconversion.">Visualizing recycling synaptic vesicles in hippocampal neurons by FM1-43 photoconversion.</a> Proc Natl Acad Sci USA. 98, 12748-12753 (2001).</li><li>Lange, R. P. J. d. e., de Roos, A. D. G., Borst, J. G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+vesicle+recycling+in+the+rat+calyx+of+Held.">Two modes of vesicle recycling in the rat calyx of Held.</a> J Neurosci. 23, 10164-10173 (2003).</li><li>Richards, D. A., Guatimosim, C., Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pools+at+the+frog+neuromuscular+junction.">Synaptic vesicle pools at the frog neuromuscular junction.</a> Neuron. 39, 529-541 (2003).</li></ol>

몇 가지 중요한 단계를 고려해야한다 : <ul><li> DAB 보육은 준비 철저한 세척과 담금질 후에 실시하여야한다. 그렇지 않은 반응 글루 타 알데히드는 DAB과 상호 작 용할 수 있으며 (일반적으로 고밀도 전자되지 않은 평면 결정의 형태로)의 강수량을 일으킬 것입니다. 이 강수량이 넘치게 이루어지는 준비는 전자 현미경에 대한 거의 사용할 수 있습니다. </li><li> 조명 시간은 다른 조명 시간을 몇 가?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FM 1-43</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F10317</td>
<td>&nbsp;</td>
<tr>
<td>Epon resin</td>
<td>&nbsp;</td>
<td>Plano</td>
<td>R1030</td>
<td>&nbsp;</td>
<tr>
<td>di-aminobenzidine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5905</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde</td>
<td>&nbsp;</td>
<td>AppliChem</td>
<td>A3166</td>
<td>EM grade</td>
</tr>
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>104186298</td>
<td>&nbsp;</td>
<tr>
<td>Axioskop 2 FS plus</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Objective 20x 0.5 NA</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Dry objective</td>
</tr>
<tr>
<td>100W Hg Lamp</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lamp housing with back mirror</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>1007-980</td>
<td>&nbsp;</td>
<tr>
<td>MRm camera</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>0445-554</td>
<td>Image acquisition</td>
</tr>
<tr>
<td>Ex. Filter (HQ 470/40)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F49-671</td>
<td>&nbsp;</td>
<tr>
<td>Dichroic (495 DCLP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F33-100</td>
<td>&nbsp;</td>
<tr>
<td>Em. Filter (HQ 500 LP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F42-018</td>
<td>&nbsp;</td>
<tr>
<td>EM</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Proscan CCD HSS</td>
<td>&nbsp;</td>
<td>Proscan Electronic Sys.</td>
<td>&nbsp;</td>
<td>1024 x 1024</td>
</tr>		</tbody>
		</table>
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studying synaptic vesicle pools using photoconversion of styryl dyes

플라즈마 막 (exocytosis)와 시냅스 vesicles의 융합은 신경 전달 물질 릴리스와의 연결을 통신에 필요한 단계입니다. 그들이 새로운 엑소과 endocytosis주기 (소포 재활용)을 받아야 때까지 vesicles 그런 다음, 플라즈마 막 (endocytosis)에서 검색 및 신경 터미널 내에 vesicles의 일반 수영장과 함께 그룹화됩니다. 이러한 프로세스는 이러한 전자 현미경, 전기 생리학 녹음, amperometry 및 커패시턴스 측정과 같은 기술을 사용하여 다양한 연구하고 있습니다. 중요한 것은 지난 20 년간 동안 찬란 분류 마커의 숫자는 광학 기술들이 재활용 역학 vesicles를 추적할 수있게 나타났다. 가장 일반적으로 사용되는 마커 중 하나는 styryl 또는 FM 염료 1, 구조적, 모든 FM 염료는 친수성 머리와 아로마 링과 하나 이상의 이중 채권 (그림의 1B)을 통해 연결된 lipophilic 꼬리가 포함되어 있습니다. vesicles의 수영장 레이블 클래식 FM 염료의 실험 (전기 또는 높은 K +와 함께) 신경의 자극 동안 염료와 준비 (그림 1Ai)를 목욕으로 구성되어 있습니다. 이것은 최근 endocytosed vesicles (그림 1A I - III)에 소포 재활용 및 염료의 후속 로딩을 유도. 염료, 염료없는 욕조에서 자극의 두 번째 라운드로 vesicles 로딩 후 exocytosis를 통해 FM 릴리스 (그림 1A IV - V), 형광 강도 감소 (destaining)를 모니터링하여 다음 수있는 프로세스를 실행하는 것입니다. FM 염료는 소포 재활용 분야에 크게 공헌했지만, 그것은 종래의 형광 현미경을 사용하여 개별 vesicles의 정확한 지방화이나 형태를 결정하는 것은 불가능합니다. 그런 이유로, 우리는 어떻게 FM 염료가 photoconversion 통해 전자 현미경을 사용하여 endocytic 마커로도 사용될 수 있습니다 여기에 설명합니다. photoconversion 기술은 강한 조명 아래의 반응 산소 종을 생성하는 형광 염료의 속성을 이용. 찬란 분류 준비는 diaminobenzidine (DAB)을 포함하는 솔루션에 빠져들와 조명입니다. 염료 분자에 의해 생성되는 반응 종족은 어두운 모습을 쉽게 전자 현미경 2,3로 구분할 수 안정, 불용성 침전물을 형성 DAB를 산화. DAB에만 형광 분자 (반응 산소 종은 단기 살고 있으므로)의 바로 근처에 산화되기 때문에, 기술은 찬란 표시된 구조는 전자 - 밀도 침전물을 포함 될 것을 보장합니다. 기술은 따라서 적극적으로 organelles을 재활용의 정확한 위치와 형태의 연구를 수 있습니다.

Watch this Scientific Journal Video about Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes at JoVE.com

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

FM의 염료는 시냅스 역학의 이해에 귀중한 도움이되었습니다. FMS는 일반적으로 다른 자극 조건 동안 형광 현미경으로 다음 있습니다. 그러나, 전자 현미경과 함께 FM 염료의 photoconversion는 시냅스 부튼스의 다른 ultrastructure 구성 요소 간의, 서로 다른 시냅스 소포의 수영장의 시각화 수 있습니다.

Styryl 염료의 Photoconversion를 사용하여 시냅틱 소포 풀 유학

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

studying-synaptic-vesicle-pools-using-photoconversion-of-styryl-dyes

Research

JoVE Journal

Neuroscience

11.6K 조회수.  European Neuroscience Institute Göttingen. FM의 염료는 시냅스 역학의 이해에 귀중한 도움이되었습니다. FMS는 일반적으로 다른 자극 조건 동안 형광 현미경으로 다음 있습니다. 그러나, 전자 현미경과 함께 FM 염료의 photoconversion는 시냅스 부튼스의 다른 ultrastructure 구성 요소 간의, 서로 다른 시냅스 소포의 수영장의 시각화 수 있습니다.

비디오: Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

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 ...

연구

신경과학

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

교양 뉴런의 막 단백질의 측면 보급과 Exocytosis은 형광 복구 및 형광 손실 Photobleaching를 사용하여 평가

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

유발, 자발적, 미니어처 시냅틱 활동을하는 동안 FM 염료를 사용하여 시냅틱 소포 재활용의 시험

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

성상 세포 운송업자 전류의 디컨 볼 루션 분석 글루타민산 정리의 시간 과정을 도출

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

교양된 Hippocampal 신경에 시 냅 스 소포 Endocytosis를 측정

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Vivo에서경작-Murine 이다 빠르고, 간단 하 고 저렴 한 AWESAM 프로토콜을 사용 하 여 같은

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

연 접 기능 그대로 신경 회로 내에 유전자 최저 효과 특성 인공 microRNAs Optogenetics 결합

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

시 냅 스 소포 교양된 신경 연 접 단백질 Overexpressing에 있는 재활용에 대 한 광학 분석 결과

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

전기 생리학 전체 셀 패치 클램프를 사용 하 여 시 냅 스 다양성의 평가

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...