이 작품은 청각 및 기타 통신 장애에 국립 연구소 (NIDCD) 교내 프로그램에 의해 지원되었다.

1. 신경 세포의 transfection <ol><li> 폴리 - D - 라이신 - 코팅 MatTek에 문화 배아 일 18 일 (E18) 쥐 hippocampal 뉴런은 35 mm 유리 바닥 요리 1. 체외에서 16-18일에서 (DIV), Clontech CalPhos 포유 동물 Transfection 키트를 사용하여 transfect의 뉴런. 첫째, 1.5 ML있는 문화 매체를 교체 <a href="http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/Classical_Media/dmem.html">Dulbecco의 수정된 이글 중간</a> transfection하기 전에 35 - mm 요리 0.5 시간당 (DMEM). 나중에 (단계 1.6) 사용할 수있는 살균 15 ML 튜브에서 원래 문화 매체를 저장합니다. </li><li> 무균 H 2 O (Clontech) 100 μl의 총 볼륨에 12.4 μl 2 M의 칼슘 용액 (Clontech) 10 μg pEGFP - N1 플라스미드 DNA를 섞습니다. </li><li> 100 μl 2 단계 1.2)의 혼합물을 추가 × HBS dropwise vortexing이 동안 × 중간 속도 HBS. </li><li> 혼합물은 20 분 실온에서 앉아 후 DMEM - incubated 뉴런에 1.3 단계)에서 최종 혼합물을 추가하자. </li><li> 1-1.5 시간 동안 37 ° C 배양기로 다시 뉴런 놔. </li><li> 인산 칼슘 함유 매체를 제거 후 DMEM 세 번와 세포를 씻으십시오. 원래 문화 매체와 DMEM 매체 인큐베이터, 교류 문화 요리를 반환하기 전에. </li></ol> 2. 척추에 FRAP 실험 <ol><li> 뉴런은 2~4일 transfection 후 FRAP 실험에 사용됩니다. </li><li> 즉시 (MM)를 NaCl 145, KCl 5, HEPES 10, 포도당 10, 글리신 0.005, CaCl 2 2.6, 및 MgCl가 포함되어 있습니다 미리 예열 Tyrode 솔루션을 추가하여, 35 mm 유리 바닥 요리에서 배지를 교체 이 1.3 (NaOH와 7.4로 조정 PH). </li><li> 자이스 혈구 LSM 710 공촛점 현미경은 FRAP 실험에 사용됩니다. 자이스 혈구 TempModule 시스템은 온도 (37 ° C), 습도 및 작업 시스템의 CO 2 (5 %)을 제어하는 데 사용됩니다. CO 2 탱크가 연결되어 있고 습도를 균형에 사용되는 물 병은, 물로 채워져 있는지 확인합니다. </li><li> 100 × 목표 (αplan - 고차 색지움 100 × / 1.46 오일)와 transfected 성숙 dendrite를 찾습니다. 접시에 transfected 세포가 스파스, 40 원하는 셀 검색 × 목표 (계획 - NEOFLUAR 40 × / 1.3 석유), 다음 100 × 스위치 이미지를 캡처하는 객관적인 경우. </li><li> 5 × 광학 줌 사용 및 이미지 몇 쪽이과 dendrite의 짧은 조각을 위해 256 × 256 픽셀 해상도를. 이미지를 캡처하려면, 0.5 초이 검사를 완료하는 데 걸리는 속도 명목 9 (픽셀 시간이 3.15 μsec 살기)을 사용합니다. 핀홀은 강한 형광을 얻기 위해 2μm로 설정됩니다. 이미지를 복용하면, 전체 이미지를 photobleaching 피하기 위해, 예를 들어 1-5%에 대한 낮은 레이저 전송을 사용하려고합니다. </li><li> 관심 척추를 선택합니다. 우리의 실험에서, 우리는 ~ 1 μm의 자신의 척추 헤드 지름과 버섯 쪽이을 선택합니다. </li><li> FRAP 실험을 수행하려면 488 레이저 라인에서 50 % (15 MW)와 아르곤 레이저의 파워는 30 MW입니다 [표백 후, 표백하기 전에 정격 100 % 레이저 전송에 관심을 10 번의 척추를 5 컨트롤 이미지를 가지고, 약 3-4 MW는 488 레이저 라인]를 통해 현미경에 도달하고 즉시 표백 후 이미지의 시리즈를 캡처합니다. 본 실험의 경우, 이미지는 표백 후 15 초 동안 매 일초를 점령하고 있습니다. 시간 간격은 다른 타겟팅 단백질 및 다른 실험 설계 (그림 1)에 따라 조정되어야합니다. </li><li> 이미지를 저장합니다. </li></ol> 3. 데이터 분석 <ol><li> ImageJ 소프트웨어와 이미지를 엽니다. </li><li> 관심 척추가 물에 뜨지 않는다 그래야, ImageJ에서 - (→ → &#39;번역&#39;및 / 또는 &#39;엄격한 신체&#39;스택에 조각 정렬 &#39;플러그인&#39;→ &#39;걸어갔다 스택&#39;)은 정렬 도구를 사용하여 이미지의 스택 정렬 다른 단어 - 그것은 이미지에 동일한 위치에 남아 있습니다. </li><li> ImageJ의 &#39;강도 V 타임 모니터&#39;도구 ( &#39;플러그인을 사용하여 관심있는 척추 (F S), transfected지만, 표백하지 않은 영역 (제어), 그리고 시간 경과 이미지에서 배경 영역 (F B)의 상대 형광 강도를 측정 &#39;→&#39;스택 - 걸어갔다 &#39;→&#39;강도 V 타임 모니터 &#39;). 제어 영역은 잘 집중 말초 dendrite의 조각 될 수 있습니다. 배경 지역이 아닌 형광등 지역입니다. </li><li> 전에 (F C0)와 (F C) photobleaching 후 제어 영역의 형광을 비교하여 photobleaching 속도를 (R)을 계산. R = F C / F C0. </li><li> 다음과 같이 대상 척추의 형광 강도 (F)를 정상화 : F = (F S - F B) / R. </li><li> 커브가 한 단계 지수 Graphpad의 프리즘 소프트웨어의 방정식 (그림 2) 또는 인접해있는 대상 척추의 형광 강도에 맞게R 유사한 소프트웨어. </li><li> 모바일 분율 (F M) 다음과 같은 등식으로 고정되어 분수를 (F I) 계산 : F M = F ∞ / F 0, 어디 F ∞ 전체 복구 후 형광 강도이며, F 0 photobleaching 전에 형광 강도입니다. F I = 1 - F M. </li></ol> 4. 대표 결과 : 본 연구에서는, 우리는 성숙한 hippocampal 뉴런에 FRAP 실험을 수행합니다. 18-22 DIV에서 버섯 쪽이 이미 형성됩니다. 우리의 방법을 사용하여 같은 척추와 같은 작은 영역에서 형광 강도의 동적 변경, 녹음 수 있습니다. EGFP의 형광 복구 프로세스를 분석하기 위해, 우리는 표백하기 전에 컨트롤로 5 이미지 다음 즉시 15 초 동안 표백 후 매 일초 한 이미지를 가져가라. 이미지의 해상도가 정량 분석​​을 위해 충분합니다. 태그 형광 단백질의 형광 복구 프로필이 높은 재현성 있습니다. 우리는 또한 간략하게 ImageJ 및 Graphpad의 프리즘 소프트웨어를 사용하여 형광 단백질의 모바일 및 고정되어 분수를 정의하는 방법을 보여줍니다. 우리가 여기에 표시 FRAP 방법 및 분석은 크게 신경 과학, 세포 생물학 연구와 다른 연구에서 사용할 수 있습니다. <img src="/files/ftp_upload/2568/2568fig1.jpg" alt="그림 1" /> 그림 1. 교양 hippocampal 신경 세포에서 척추에 EGFP 형광의 FRAP 측정. 빨간 화살촉은 photobleaching의 시간을 나타냅니다. 사진은 전에 동일한 영역을 (사전) 대표와 photobleaching 0, 1, 5, 10, 15 초 후. 척추, 제어 및 배경의 지역은 문자 각각 S, C와 B와 함께 표시됩니다. 뉴런은 실험 기간 동안 37 ° C에서 유지되었다. 스케일 바, 1 μm의. <img src="/files/ftp_upload/2568/2568fig2.jpg" alt="그림 2" /> 그림 2. 15 초 동안 EGFP 형광의 FRAP 곡선. 녹색 라인은 원래의 곡선을 보여주는, 빨간색 선은 표준 곡선을 보여줍니다. 곡선에있는 점들은 FRAP 매 일초을 보여줍니다. 곡선은 한 단계 지수 방정식으로 장착되었습니다. photobleaching 전에 평균 형광 100 %로 계산되었다. 본 실험에서는 모바일 분수 (MF)는 94%이고 고정되어 분수 (IF) 6 %입니다.

<ol>
	<li>Zheng, C. Y., Petralia, R. S., Wang, Y. X., Kachar, B., 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=SAP102+is+a+highly+mobile+MAGUK+in+spines.">SAP102 is a highly mobile MAGUK in spines.</a> J Neurosci. 30, 4757-4766 (2010).</li><li>Grati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+turnover+of+stereocilia+membrane+proteins:+evidence+from+the+trafficking+and+mobility+of+plasma+membrane+Ca(2+)-ATPase+2.">Rapid turnover of stereocilia membrane proteins: evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2.</a> J Neurosci. 26, 6386-6395 (2006).</li><li>Liu, L. N., Aartsma, T. J., Thomas, J. C., Zhou, B. C., Zhang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+Analysis+on+Red+Alga+Reveals+the+Fluorescence+Recovery+Is+Ascribed+to+Intrinsic+Photoprocesses+of+Phycobilisomes+than+Large-Scale+Diffusion.">FRAP Analysis on Red Alga Reveals the Fluorescence Recovery Is Ascribed to Intrinsic Photoprocesses of Phycobilisomes than Large-Scale Diffusion.</a> PLoS ONE. 4, e5295-e5295 (2009).</li><li>Wiedenmann, J., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+with+EosFP+and+other+photoactivatable+marker+proteins+of+the+GFP+family.">Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family.</a> Expert Rev Proteomics. 3, 361-374 (2006).</li><li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li><li>Sprague, B. L., McNally, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+analysis+of+binding:+proper+and+fitting.">FRAP analysis of binding: proper and fitting.</a> Trends Cell Biol. 15, 84-91 (2005).</li></ol>

관심 없음 충돌 선언하지 않습니다.

FRAP 분석은 크게 생체내 및 시험 관내 1-2 연구에 사용되고 있습니다. 이 기술은 일반적으로 이용 GFP <a href="http://en.wikipedia.org/wiki/Fusion_protein" title="융합 단백질">융합 단백질을</a> 그것도 빨간 알가의 융합 단백질 3을 사용할 수 있지만. 이 분석은 민감하고 GFP - 태그 단백질의 이동성을 특징하는 데 사용할 수 있습니다. 의미 FRAP 분석을 생산하기 위해서는 이전과 FRAP 실험 도중 불필요한 photobleaching을 방지하는 것이 중요합니다. 이것을 달성하기 위해 두 가지 방법이 있습니다. 첫째, 검색 및 실험 신경 세포를 관찰하는 과정이 빨리되어야합니다. 특히, 오랫동안 100X 목적으로 뉴런의 관찰 크게 형광 표백제로 닦았을. 둘째, 높은 레이저 전원과 상용 스캔은 종종 photobleaching의 가능성을 높일 수 있습니다. 그러므로, 그것은 통제 지역에서 photobleaching 율을 계산하고 실험 지역에서 FRAP 곡선을 정상화하기 위해 필요합니다. 정규화 방법 (자세한 내용은 단계 3.3-3.5 참조) 프로토콜에 설명되어있다. photobleaching 단계는 또한 좋은 FRAP 결과를 확보 중요합니다. 본 실험에서는, 우리는 100 % 표백 레이저 전송에 관심이 10 배의 척추 있습니다. 이 조건은 표백 고정 준비 배경 수준으로 척추의 형광을하기에 충분합니다. 따라서, 우리는 photobleaching 후 0초에서 배경 같은 번호로 형광 강도를 설정합니다. 첫 번째 스캔의 속도에 따라 형광 복구의 상당량은 이미 관심의 단백질이 높은 휴대 때 감지 수 있습니다. 많은 형광 단백질 프로브는 FRAP과 단백질 역학을 연구하기 위해 개발되었습니다. 보완 접근 photoactivatable GFP (PA - GFP) 또는 photoconvertible 변종을 사용하는, 예를 들어 있습니다. 함께 FRAP 기술과 함께,이 도구는 라이브 셀 이미징 연구 4-6위한 필수가되고있다.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>35 mm glass-bottom dishes </td>
<td>&nbsp;</td>
<td>MatTek</td>
<td>P35GC-1.0-14-C</td>
<td>&nbsp;</td>
<tr>
<td>CalPhos Mammalian Transfection Kit</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>631312</td>
<td>&nbsp;</td>
<tr>
<td>pEGFP-N1 plasmid</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>6085-1</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss confocal microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>LSM 710</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss TempModule system</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>N.A.</td>
<td>&nbsp;</td>
<tr>
<td>ImageJ software</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>N.A.</td>
<td>&nbsp;</td>
<tr>
<td>Graphpad Prism software</td>
<td>&nbsp;</td>
<td>Graphpad software</td>
<td>N.A.</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fluorescence recovery after photobleaching (frap) of fluorescence tagged proteins in dendritic spines of cultured hippocampal neurons

FRAP는 GFP - 태그 단백질의 이동성을 계량하는 데 사용되었습니다. 강한 여기 레이저를 사용하여 GFP - 태그 단백질의 형광은 관심 지역에 표백됩니다. 지역의 외부에서 표백하지 않은 GFP - 태그 단백질 관심의 영역으로 diffuses 때 지역의 형광은 복구합니다. 단백질의 이동성은 다음 형광 복구 속도를 측정하여 분석합니다. 이 기술은 단백질 이동성과 회전율 속도를 특성화하는 데 사용할 수 있습니다. 본 연구에서는, 우리는 교양 hippocampal 뉴런의 (녹색 형광 단백질 강화) EGFP 벡터를 표현한다. 자이스 혈구 710 공촛점 현미경을 사용하여, 우리는 하나의 척추에 GFP 단백질의 형광 신호를 photobleach 다음 photobleaching 후 형광 회복을 기록 시간 경과 이미지를 가져가라. 마지막으로, 우리는 ImageJ와 Graphpad의 소프트웨어를 사용하여 영상 데이터를 분석하여, 쪽이에서 GFP의 모바일 및 고정되어 분수의 비율을 예상하고있다. 이 FRAP 프로토콜은뿐만 아니라 데이터를 분석하는 방법으로 기본 FRAP 실험을 수행하는 방법을 보여줍니다.

Watch this Scientific Journal Video about Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons at JoVE.com

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP는 녹색 형광 단백질 (GFP) 태그 교양 세포에있는 단백질의 이동성을 계량하는 데 사용되었습니다. 우리는 photobleaching 후 형광 복구 비율을 분석하여 GFP의 모바일 / 고정되어 분수를 조사. 본 연구에서는, FRAP는 hippocampal 뉴런의 쪽이에서 수행되었다.

교양 Hippocampal 뉴런의 돌기 쪽이에서 형광 단백질의 태그 Photobleaching 후 형광 복구 (FRAP)

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

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JoVE Journal

Neuroscience

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

54.1K 조회수.  National Institutes of Health, Bethesda. FRAP는 녹색 형광 단백질 (GFP) 태그 교양 세포에있는 단백질의 이동성을 계량하는 데 사용되었습니다. 우리는 photobleaching 후 형광 복구 비율을 분석하여 GFP의 모바일 / 고정되어 분수를 조사. 본 연구에서는, FRAP는 hippocampal 뉴런의 쪽이에서 수행되었다.

비디오: Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

교양 CNS의 뉴런의 돌기 스파인 형태론 분석

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

연구

신경과학

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

교양 공감 뉴런의 돌기 성장을 유도

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

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 ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Axons, 수석 및 뇌 슬라이스의 개별 뉴런의 수지상 쪽의 전압에 민감한 염료 기록

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

이미징은 기본 양식 마우스 뉴런의 플라즈마 막에 수용체 삽입을 pHluorin 태그

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Immunoprecipitation 및 아실-비오틴 거래소 (아베)에 의한 양식 Hippocampal 뉴런의 단백질 Palmitoylation의 감지

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

라이브 배양 된 신경 세포의 항체 수유 후 세포 표면과 내면 단백질의 차등 라벨링

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

구조적 조명 현미경을 사용하여 쥐 차 해마 신경 세포의 돌기 쪽 이미징

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

하는 세포 칼슘의 형광 및 생물 발광 영상<sup&gt; 2 +</sup&gt; 노인 해마 신경 세포에서

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

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.