Name: stripe assay to study the attractive or repulsive activity of a protein substrate using dissociated hippocampal neurons
Uploaded: 2016-06-19T14:00:00.000+00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons at JoVE.com

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (23700412, 25122707 and 26670090 to S.Y.).

동물 주제와 관련된 절차는 의학의 하마 마츠 대학에서 기관 애니멀 케어 및 사용위원회에 의해 승인되었습니다. 행렬의 1. 준비 <ol><li> 전자 레인지에 5 분 동안 핫 플레이트에 4-8 실리콘 매트릭스를 삶아 그들을 층류 (줄무늬면이 위로)에서 1 시간 동안 완전히 건조 할 수 있습니다. 참고 : 다음 절차는 층류에서 수행되어야한다. </li><li> 압축 공기를 불어 또는 행렬의 스트라이프 측면에서 먼지를 제거하기 위해 투명 접착 테이프를 사용합니다. 깨끗한 스트라이프면을 유지 그래서 단단히 플레이트 표면에 부착 할 수 있습니다. </li><li> 조심스럽게 손가락 (; 스트라이프면이 아래 접시 당 하나의 매트릭스)으로 눌러 6 cm 플라스틱 요리 상에 매트릭스를 배치합니다. 매트릭스와 요리 사이에 기포가 들어 가지 않도록. 배양 접시의 바닥면에 줄무늬의 위치를​​ 표시한다. </li><li> 매트릭스 첨부 실패하면, 단계 1.2에서 반복합니다. </li></ol>신경 문화 2. 스트라이프 생성 및 라미닌 코팅 <ol><li> 형광 표지 된 재조합 단백질 준비은 FC-태그 단백질 (10 ~ 50 μg의 / ㎖) 및 형광 염료 -을 혼합하여 (25 μl를 주입 [단계 2.2] 100 ㎕의 배치 방법 [단계 2.2.1]에 대한 각 각) 공액 항 - 인간 항체의 Fc (1 비율 3 : 30-150 μg의 / ㎖) 인산염 완충 식염수 (PBS)이다. 그 후, RT에서 30 분 동안 혼합물을 배양한다. </li><li> 22-G 주사기를 이용하여, 매트릭스의 측면에 작은 구멍을 통해 이전 단계 (2.1)에서 제조 한 재조합 단백질의 25 μl를 주입 (도 1A, 1C에서의 화살표 및 1E). <ol><li> 대안 적으로, 매트릭스의 상단 슬릿으로 재조합 단백질 100 μl를 넣고 있도록 작은 구멍으로부터 액의 흡 약 절반 (도 1A, 1C 및 1E의 화살표), 재조합 단백질은 스트라이프 영역에서 유지된다. </li></ol></li><li> 30 마일의 접시를 품어세포 배양 인큐베이터에서 37 ℃에서 N. </li><li> 행렬의 측면에있는 작은 구멍에서 상단 슬릿 (그림 1B) 및 흡인에 PBS 300 μl를 놓습니다 (그림 1A, C 및 E의 화살표) 부착 재조합 단백질을 제거합니다. 단백질은 건조하기 때문에, 완전히 PBS를 대기음하지 마십시오. 두 번이 단계를 반복합니다. (총 3 회) </li><li> 다른 코팅을 만드는 전체 스트라이프 영역을 덮도록, FC (10 내지 50 μg의 / ㎖)를 조심스럽게 조절 단백질 ~ 100 μL를 배치 즉시 매트릭스를 제거. 그 후, 세포 배양 인큐베이터에서 37 ° C에서 30 분 동안 요리를 배양한다. </li><li> PBS에서 ~로 접시면 20 μg의 / ㎖ 라미닌 100 μl를 PBS로 3 회 코트를 세척 한 후. 주 : 라미닌 응고를 방지하는 얼음에 해동되어야한다. </li><li> 세포 배양 인큐베이터에서 37 ° C에서 1 시간 동안 접시를 부화. </li><li> PBS와 함께 접시 표면을 세 번 씻고 문화 메디칼 추가음. 사용할 때까지 37 ° C에서 5 % CO 2 배양기에서 배양 배지와 코팅 접시를 놓습니다. </li></ol> E15.5 마우스 배아 3. 배양 해마 신경 세포 <ol><li> 자궁 경부 전위를 통해 어머니를 안락사 세 E15.5 배아를 분리. </li><li> sagittally 가위로 머리의 중간 선에 피부와 두개골을 잘라 작은 스푼을 사용하여 뇌를 제거합니다. 행크의 균형 소금 솔루션 (HBSS) 3 ㎖를 포함하는 60 mm 페트리 접시에 조심스럽게 뇌를 전송합니다. </li><li> 메스를 사용하여, 피질, 해마 및 선조체 (그림 2A)를 포함하여 반구를 잘라. 그리고, 신중 수막을 제거하여 해마 영역을 해부하고, 얼음 (도 2B-C)에서, HBSS 용액 2 ㎖를 함유하는 15 mL의 원심 분리 튜브에 해부 해마 조직을 옮긴다. 8 요리 뉴런을 배양하기위한 충분한 튜브 3 배아에서 6 해마를 수집합니다. </li><li> 수소를 교체트립신 / EDTA 용액 (2 mL) 및 수조에서 15 분 동안 37 ° C에서 튜브를 부화와 BSS 용액. 대기음 HBSS 원심 분리하지 않고 튜브를 기울여 HBSS 솔루션을 가만히 따르다하지 않습니다 있습니다. </li><li> 소 태아 혈청 (FBS) 500 μL를 첨가함으로써 트립신의 활성을 중화. </li><li> 5 분 동안 100 XG에서 혼합물을 원심 분리기. 상층 액을 제거하고 배지 2 ㎖로 펠렛을 씻는다. 다시 한 번이 단계를 반복합니다. </li><li> 피펫은 큰 구멍이 1,000 ㎕의 팁을 사용하여 배지에서 상하 해마 10 배 조직을 해리 (팁을 잘라). </li><li> 단일 세포 현탁액을 얻기 위해 아래 일반 (포경) 1,000 μL 팁으로 변경하고 해리 조직을 위로 피펫합니다. </li><li> 80 ~ 100 μm의 메쉬 셀 스트레이너를 통해 전달하여 단일 세포를 분리합니다. </li><li> 5 분 동안 150 XG에서 필터링 된 단일 세포 현탁액을 원심 분리기. </li><li> 흡입하여 상층 액을 제거하고 펠를 재현 탁등 배지 2 ㎖에 (뉴런). </li><li> 밀도와 생존을 결정 트리 판 블루 염색에 혈구 세포를 사용하여 계산. stripes.The 현탁액의 각 세트는 신중 전체 스트라이프 영역을 커버하는 배치해야합니다 위해 다음, 배양액 150 ㎕를 10,000 신경 세포를 접시. </li></ol> 안티 Tau1 항체를 이용하여 세포 기관 및 면역 형광 염색법에 의한 축삭 4. 시각화 <ol><li> 배양 24 시간 후, 부착 세포를 방해하지 않고 매체를 흡인하고, 예열 된 4 % 파라 포름 알데히드 (PFA) / 4 % 슈 크로스에서 15 분 동안 RT에서 / PBS로 뉴런을 고정한다. </li><li> PBS로 플레이트 표면을 3 회 반복한다. 원하는 경우, 필요한 항체의 양을 감소시키기 위해 스트라이프 영역 주위에 원을 그리 발수 펜을 사용한다. </li><li> 5 분 동안 0.3 % 트리톤 X-100 / PBS로 뉴런 Permeabilize 하시려면. </li><li> 3 % 당나귀 혈청과 0.1 % 트리톤 X-100을 포함 차단 솔루션 뉴런을 품어4 ° C에서 1 % 당나귀 혈청 / 0.1 % 트리톤 X-100 / PBS O / N : 안티 Tau1 항체 (200 일)로 배양 한 다음 30 분. </li><li> PBS로 플레이트 표면을 3 회 반복한다. 1 % 소 혈청 알부민 (BSA) /0.1% 트리톤 X-100 / PBS 30 분에 형광 접합 된 항 - 마우스 IgG 항체와 함께 인큐베이션 뉴런. </li><li> PBS로 플레이트 표면을 3 회 반복한다. antifade 용액 50 μl를 사용하여 18mm X 18mm 커버 유리와 스트라이프 영역을 커버. 이미징을 방해 공기 방울을 피하십시오. </li><li> GFP 및 RFP, 1,000 밀리 초 노출 시간 (그림 3)에 대한 예를 들어, 해당 시스템을 사용하여 형광 현미경, 10 배 대물 렌즈, 필터 세트와 형광 이미지를 얻습니다. </li></ol>

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The authors have nothing to disclose.

이 프로토콜은 E15.5 마우스의 해마에서 재조합 단백질과 해리 신경 세포를 사용하는 스트라이프 분석에 대해 설명합니다. 이 분석은 스트라이프 패턴으로 배치 관심 재조합 단백질에 신경 세포의 반발 매력, 또는 중립적 인 반응을 효율적으로 관찰 할 수 있습니다. 이 프로토콜의 주요 이점은 특별한 행렬 진공 장치를 필요로하는 기존의 방법에 비해 단백질이 직접 플라스틱 접시의 표면에 인쇄?...

축삭지도는 새로 형성된 신경 세포는 신경계의 1, 2의 개발 기간 동안 자신의 목표에 축삭를 전송하는 과정입니다. 개발 축삭의 성장 원추라는 자신의 끝에 높은 운동성 구조를 실시하고 있습니다. 성장 콘 축삭의 경로를 탐색하는 세포 외 신호를 감지한다. 안내 등의 슬릿, semaphorin 같은 분자 및 ephrins는, 유치 또는 축삭 1,3,4에 적합한 수용체와 공동 수용체와의 상호 작용에 따라 축삭을 물리 칠 수 있습니다. 활성화 된 수용체는 축삭 성장 콘의 움직임에 대한 골격 조직에 영향을 미치는 성장 콘에 신호를 전송합니다. 다양한 방법이 유인 및 반발 성 분자의 작용을 평가하기 위해 개발되어왔다. 화학 - 유인 및 방충제는 그라데이션 농도 성장 / 문화 매체에 투여 될 수있다 (예., 던의 챔버 또는 μ-슬라이드) 마이크로-P에 의해 고농도의 자리에 5, 6,ipette (예., 전환 분석) 7 또는 목욕 응용 프로그램 (예., 성장 원뿔 붕괴 분석) 8,9에 의해 균일 한 농도. 다른 방법은 화학 - 유인 또는 반발은 기판 10-12으로서 판 표면에 도포되어있는 스트라이프 분석 또는 마이크로 콘택트 프린팅 (μCP)를 포함한다. Thestripe 분석은 원래 병아리 레티노 - tectal 시스템 (13)의 지형 매핑을 분석하기 위해 1987 년 본회퍼와 동료에 의해 ​​개발되었다. 원래 방법은 스트라이프와 메쉬 행렬을 사용하여 폴리 카보네이트 nucleopore 막에 코트 단백질에 진공 시스템이 필요합니다. 이상에서, 재조합 단백질을 직접적으로 좁은 슬릿 실리콘 매트릭스 (14, 15)를 사용하여, 스트라이프 패턴으로 배양 용기의 표면에 인쇄했다. 최근 여러 연구 그룹이 성공적으로 축삭지도 분자 활동 16-21의 분석이 스트라이프 분석을 적용했습니다. <p claSS = &quot;jove_content은&quot;&gt; 여기, 우리는 해리 해마 신경 세포에 대한 축삭지도 분자의 인력이나 반발력을 측정하는 스트라이프 분석에 대한 자세한 프로토콜을 제시한다. 특히,이 방법은 최소 갖춘 실험실 설정에서 적용될 수있다. 이 분석 들어, 형광 표지 된 기판의 제어 된 단백질의 교번 스트라이프는 90 ㎛의 슬릿 실리콘 매트릭스를 이용한 플라스틱 접시에 생성되고 라미닌 코팅. 우리의 데모에서는, E15.5 마우스의 해마 신경 세포가 피브로넥틴와 류신이 풍부한 막 횡단 단백질 2 (FLRT2)과의 Fc 단백질 (21)를 제어의 재조합 ectodomain의 줄무늬 교대에 배양 하였다 해리. 배양 24 시간 후, 축삭 신경의 세포체는 모두 강하게 FLRT2 줄무늬에서 반발되었다. 항 Tau1 항체로 염색 FLRT2 반발이 강한 것을 나타내는 ~ 뉴런의 90 %가 FLRT2-FC에 ~ 10 %에 비해 된 Fc 피복 영역에 분포 된 것으로 나타났다해마의 뉴런 (21)에 대한 기능.

<table><tbody><tr><td>15 ml centrifuge tube</td><td>Violamo&amp;nbsp;</td><td>1-3500-01</td><td></td></tr><tr><td>4% Paraformaldehyde (PFA)</td><td>Nacalai</td><td>01954-85</td><td></td></tr><tr><td>Alexa Fluor 488 Goat anti-human IgG antibody</td><td>Thermo Scientific</td><td>A11013</td><td></td></tr><tr><td>Alexa Fluor 594 Donkey anti-mouse IgG antibody</td><td>Thermo Scientific</td><td>A-21203</td><td>Dilution 1/500</td></tr><tr><td>Anti-Tau1 antibody</td><td>Chemicon</td><td>MAB3420</td><td>Dilution 1/200</td></tr><tr><td>Antifade</td><td>Thermo Scientific</td><td>P7481</td><td>Alternative mounting media may be used</td></tr><tr><td>B27 supplement</td><td>Thermo Scientific</td><td>17504-044</td><td>Dilution 1/50</td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>01-2030-2</td><td></td></tr><tr><td>Cell strainer 100 um</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Centrifugation machine</td><td>Kubota</td><td>2410</td><td></td></tr><tr><td>Cover glass 18mmx18mm</td><td>Matsunami</td><td>18x18 mm No. 1</td><td></td></tr><tr><td>DAKO pen</td><td>DAKO</td><td>S2002</td><td>Alternative water-repellent pen may be used</td></tr><tr><td>Disposable scalpel</td><td>Feather</td><td>2975#11</td><td></td></tr><tr><td>FBS</td><td>Thermo Scientific</td><td>10437-028</td><td></td></tr><tr><td>Fluorecent microscope</td><td>Nikon</td><td>E600</td><td></td></tr><tr><td>Forceps No. 5</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>GlutaMAX</td><td>Thermo Scientific</td><td>35050-061</td><td>Dilution 1/100</td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>805N</td><td>22 gauge, 50 uL</td></tr><tr><td>HBSS</td><td>Thermo Scientific</td><td>14170-112</td><td></td></tr><tr><td>Human IgG, Fc Fragment</td><td>Jackson</td><td>009-000-008</td><td></td></tr><tr><td>Laminin</td><td>Thermo Scientific</td><td>23017-015</td><td></td></tr><tr><td>Neurobasal</td><td>Thermo Scientific</td><td>21103-049</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson</td><td>017-000-121</td><td></td></tr><tr><td>PBS</td><td>Nacalai</td><td>14249-24</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Scientific</td><td>15070-063</td><td>Dilution 1/100</td></tr><tr><td>Plastic culture dish, 60 mm</td><td>Thermo Scientific</td><td>150288</td><td></td></tr><tr><td>Silicone Matrices</td><td></td><td></td><td>Available and purchasable from Prof. Martin Bastmeyer (bastmeyer@kit.edu)</td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZ61</td><td></td></tr><tr><td>Tip, 1000 uL</td><td>Watson</td><td>125-1000S</td><td></td></tr><tr><td>Transparent sticky tape</td><td>Tesa</td><td>57315</td><td>Alternative sticky tape may be used</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Trypan blue, 0.4%</td><td>Bio-Rad</td><td>145-0013</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Thermo Scientific</td><td>25300-054</td><td></td></tr><tr><td>Culture medium</td><td></td><td></td><td>Neurobasal supplemented with B27, GlutaMAX and Penicillin-Streptomycin.</td></tr></tbody></table>

stripe assay to study the attractive or repulsive activity of a protein substrate using dissociated hippocampal neurons

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to navigate axonal growth. Netrin, slit, semaphorin, and ephrins are known guidance molecules that can attract or repel axons upon binding to receptors and co-receptors on the axon. The activated receptors initiate various signaling molecules in the growth cone that alter the structure and movement of the neuron. Here, we describe the detailed protocol for a stripe assay to assess the ability of a guidance molecule to attract or repel neurons. In this method, dissociated hippocampal neurons from E15.5 mice are cultured on laminin-coated dishes processed with alternating stripes of ectodomain of fibronectin and leucine-rich transmembrane protein-2 (FLRT2) and control immunoglobulin G (IgG) fragment crystallizable region (Fc) protein. Both axons and cell bodies were strongly repelled from the FLRT2-coated stripe regions after 24 h of culture. Immunostaining with tau1 showed that ~90% of the neurons were distributed on the Fc-coated stripes compared to the FLRT2-Fc-coated stripes (~10%). This result indicates that FLRT2 has a strong repulsive effect on these neurons. This powerful method is applicable not only for primary cultured neurons but also for a variety of other cells, such as neuroblasts.

E15.5 마우스 해리 해마 뉴런 도금의 형광 제어을 FC (도 3A-C) 또는 비 - 표지 된 Fc 제어 교대 FLRT2-FC (도 3d-F) 라벨 줄무늬 24 시간 동안 배양 하였다. 두 경우 모두, 뉴런은 집계했고, 번들로 자신의 축삭을 확장했다. 컨트롤을 된 Fc / 된 Fc 줄무늬가 신경이 형광 표지하고 비 표지 된 줄무늬 균일하게 분포하고, 이들은 임의의 방향 (도 3A-C)에서</str...

Watch this Scientific Journal Video about Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons at JoVE.com

Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons.

스트라이프 분석은 해리 해마 신경 세포를 사용하여 단백질 기판의 매력적인 또는 혐오감 활동을 연구하는

Growing axons develop a highly motile structure at their tip, termed the growth cone. The growth cone contacts extracellular environmental cues to ...

stripe-assay-to-study-attractive-or-repulsive-activity-protein

Research

JoVE Journal

Neuroscience

10.6K 조회수.  Hamamatsu University School of Medicine. Axon guidance molecules regulate neuronal migration and targeted growth-cone navigation. We present a powerful method, the stripe assay, to assess the ability of guidance molecules to attract or repulse neurons. In this protocol, we demonstrate the stripe assay by showing FLRT2's ability to repel cultured hippocampal neurons. 

비디오: Stripe Assay to Study the Attractive or Repulsive Activity of a Protein Substrate Using Dissociated Hippocampal Neurons

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 ...

연구

신경과학

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 ...

Isolation of CA1 Nuclear Enriched Fractions from Hippocampal Slices to Study Activity-dependent Nuclear Import of Synapto-nuclear Messenger Proteins

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular mechanisms of synaptic plasticity. Long-term potentiation (LTP) and long-term depression (LTD) induced in acute hippocampal slices are widely accepted as cellular models of learning and memory. There are numerous studies that use live cell imaging or immunohistochemistry approaches to visualize activity dependent protein dynamics. However these methods rely on the suitability of antibodies for immunocytochemistry or overexpression of fluorescence-tagged proteins in single neurons. Immunoblotting of proteins is an alternative method providing independent confirmation of the findings.&#160;The first limiting factor in preparation of subcellular fractions from individual tetanized hippocampal slices is the low amount of material. Second, the handling procedure is crucial because even very short and minor manipulations of living slices might induce activation of certain signaling cascades. Here we describe an optimized workflow in order to obtain sufficient quantity of nuclear enriched fraction of sufficient purity from the CA1 region of acute hippocampal slices from rat brain. As a representative example we show that the ERK1/2 phosphorylated form of the synapto-nuclear protein messenger Jacob actively translocates to the nucleus upon induction of LTP and can be detected in a nuclear enriched fraction from CA1 neurons.

We provide a detailed protocol for induction of long-term potentiation in the CA1 region of the hippocampus and the subsequent isolation of nuclear enriched fractions from the tetanized area of the slice. This approach can be used to determine activity dependent nuclear protein import in cellular models of learning and memory.

해마 조각에서 CA1 핵 농축 분획의 분리는 Synapto 핵 메신저 단백질의 활동에 의존하는 핵 가져 오기를 공부합니다

Studying activity dependent protein expression, subcellular translocation, or phosphorylation is essential to understand the underlying cellular ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn: Synapse 밀도의 변화를 감지하는 빠르고 편향되지 않은 형광 방법

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

생화학

A High-content Assay for Monitoring AMPA Receptor Trafficking

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different stimuli. The &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, which are responsible for fast excitatory synaptic transmission in neurons, are trafficked to and from the postsynaptic surface to dynamically alter neuronal excitability. AMPA receptor trafficking is essential for synaptic plasticity and can be disrupted in neurological disease. However, prevalent approaches for quantifying receptor trafficking ignore entire receptor pools, are overly time- and labor-intensive, or potentially disrupt normal trafficking mechanisms and therefore complicate the interpretation of resulting data. We present a high-content assay for the quantification of both surface and internal AMPA receptor populations in cultured primary hippocampal neurons using dual fluorescent immunolabeling and a near-infrared fluorescent 96-well microplate scanner. This approach facilitates the rapid screening of bulk internalized and surface receptor densities while minimizing sample material. However, our method has limitations in obtaining single-cell resolution or conducting live cell imaging. Finally, this protocol may be amenable to other receptors and different cell types, provided proper adjustments and optimization.

Neuronal excitability can be modulated through a dynamic process of endo- and exocytosis of excitatory ionotropic glutamate receptors. Described here is an accessible, high-content assay for quantifying surface and internal receptor population pools.

AMPA 모니터링을 위한 하이 콘텐츠 분석 결과 수용 체 매매

Postsynaptic trafficking of receptors to and from the cell surface is an important mechanism by which neurons modulate their responsiveness to different ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

뇌 슬라이스와 기본 세포 배양에서 피라미드 뉴런의 탄도 라벨링

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

설치류 및 인간의 뇌 조직에서 액틴 중합 상태를 평가하기위한 시간 효율적인 형광 분광 법 분석 기반 분석

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (&gt;90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

마우스 뇌 해마 조직의 기계적 및 효소적 해리 결합

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in ...

<meta charset="utf8"></head><body>Live-Cell Imaging을 통한 Primary Hippocampal Neurons의 단백질 시각화

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

<meta charset="utf8"></head><body>저자 스포트라이트: 신경퇴행성 질환에서 병리학적 단백질이 축삭 수송에 미치는 영향 이해

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.