우리는 해부 비디오 편집과 지원을위한 절차와 로드니 맥페일을 (생물 과학학과, 퍼듀 대학교) 촬영을 위해 라이언 마네리을 (오이스터캐쳐 프로덕션, LLC) 감사드립니다. 수터 실험실 연구는 DMS에 퍼듀 대학에서 NIH (R01 NS049233)와 빈들리 Bioscience 센터에서 기금에 의해 지원됩니다

솔루션 <ul><li> L15 - ASW의 세포 배양 매체 (일리터) <ul><li> L15 가루 1 봉지 </li><li> H 2 O ultrapure 800 ML을 추가 </li><li> NaCl 400 MM </li><li> MgSO 4 27 MM </li><li> MgCl 2 28 MM </li><li> L - 글루타민 4 MM </li><li> Gentamicin 50 μg / ML </li><li> HEPES 5 MM </li><li> 산도 7.9로 조정 </li><li> (precipitates 경우 스톱) 드롭 CaCl 2 9.3 MM을 들러 추가 </li><li> 1리터에 H 2 O ultrapure 추가 </li><li> osmometer (증기압, Wescor # 5520)와 osmolarity (950-1000 mmol / kg)를 확인 </li><li> 긍정적인 압력 여과 장치 (Millipore의 SVGV010RS 필터)를 사용하여 필터 0.22 μm의 </li><li> 최대 1 개월 4 ° C에 보관 </li></ul></li><li> 폴리 - L - 라이신은 (70-150 KD) 200 살균 H 2 O ultrapure에 μg / ML 주식 솔루션은 -20 O C에서 보관 </li><li> 0.5 M MgCl 2 용액 </li></ul> 1 일 : Aplysia 복부 신경절의 해부 효소 분리 솔루션의 준비 <ol><li> Eppendorf 튜브에 : Dispase 효소 (LS02111 워싱턴, 고양이)의 90-10 MG를 달다 </li><li> L15 - ASW 문화 매체 900 μl 및 살균 H 2 O ultrapure 100 μl 추가 </li><li> 실온 (RT)에서 혼합 및 장소 </li></ol> 복부 신경절의 해부 <ol><li> 0.5 M MgCl 2 솔루션으로 50 ML의 주사기를 채우십시오. </li><li> 탱크에서 한 동물을, 동물의 inking 방어 반응을 피하기 위해 부드럽게 처리할 수 있습니다. </li><li> 왼쪽으로 오른쪽, 꼬리쪽으로 절개 보드의 측면에 Aplysia (스티로폼 보드), 주동이의 옆에 놓습니다. </li><li> 그냥 머리 뒤에 동물에 바늘을 삽입하고 뱃속에 모든 MgCl 2 솔루션을 주입. </li><li> 부드럽게 뱃속 내내 MgCl 2 솔루션을 해산하기 위해 동물을 문질러 완전히 anaesthetized하는 동물을위한 몇 분 동안 기다립니다. </li><li> 이 바늘로 해부 보드 동물의 머리와 꼬리를 핀. </li><li> 측면에 큰 열기를 만들기 위해 머리와 꼬리를 향해 큰 가위로 피부와 근육의 계층을 통해 잘라내기, 피부를 리프트에 집게를 사용하십시오. </li><li> 브러시와 같은 난소에서 복부 신경절을 찾아보십시오. 복부 신경절 앞에서 1cm에 대한 연결 신경 잡아 집게를 사용하여, 포셉 보관 위치 앞에와 복부 신경절 뒤에 해부 가위로 신경을 절단. </li><li> 22 Dispase 솔루션 및 15-16 H에 대한 부화에 플레이스 신경절 ° C 온도 제어 물을 욕조를 사용합니다. </li></ol> 일 2 : Aplysia 가방 세포의 도금 폴리 - 라이신 - 코팅 coverslips 준비 <ol><li> 흐름 벤치에서, 4 ML L15 - ASW 매체 각을 작성, 신경절의 해부를 위해 요리를 작업을 피해 3 35mm 배양 접시를 준비합니다. 소화의 15.5 시간 후 작업 요리 중 하나 dispase 솔루션에서 복부 신경절을 전송합니다. </li><li> 포셉 취할 사용하면 산성 - 청소 # 1.5 coverslips (22x22 mm)는 에탄올에 저장된, 알코올을 제거하고 알콜 램프를 통해 간단히 그들을 불타는하여 coverslips을 소독. 문화 요리에 coverslips를 삽입하기 전에 그들이 잠시 더위 좀 식히자. </li><li> 알콜 램프에 비해 노란색 팁 (불꽃은 간단히 플라스틱의 용융을 피하기 위해를!) 굴곡하여 곡선 도금 팁을 준비합니다. </li><li> 무균 H 2 O ultrapure 200 μg / ML 주식 솔루션을 diluting 20 μg / ML - 폴리 리신 솔루션의 적절한 금액을 준비합니다. </li><li> 20 μg / ML - 폴리 리신과 RT에서 20 분에 대해 품어 500 μl 각 coverslip을 커버. </li><li> 500 μl 무균 H 2 O ultrapure 각 각 coverslip 3-5 번 씻으십시오. </li><li> coverslips 물을 제거, L15 - ASW 문화 매체 4 ML 각 요리를 입력합니다. </li></ol> 가방 세포의 분리 <ol><li> 해부 현미경에서 이전에 95 %의 에탄올 (그림 1 라인 1) 청소 이분의 일 사용 해부 가위와 집게의 복부 신경절를 잘라. </li><li> 가방 전지 클러스터 hemiganglion 세포 (그림 1 2 호선) 사이 컷 신경절, 그리고 가방 세포의 반대편에 남아있는 신경 확장 (그림 1 라인 3) 버려야 중 절반에. 한 번에 신경절의 반은 작업, 첫 번째 클러스터의 세포가 도금되는 동안 접시에있는 다른 반쪽을 두십시오. </li><li> 이 포셉 (또는 한 포셉 및 해부 가위), 가방 전지 클러스터를 둘러싼 결합 조직 칼집을 별도로 추진하여 릴리스 가방 셀 클러스터를 사용합니다. 필요한 경우 해부 가위 들고 도망 결합 조직을 잘라. 이 과정에서 당기 너무 많은 가방 셀 클러스터를 만지지 않도록 조심하십시오. </li><li> 대부분 결합 조직의 제거되면 B를 전송벤트 노란 팁으로 새 작업을 그릇에 AG 전지 클러스터는 이전 준비. 가방 전지 클러스터가 노란색 팁 안에 갇혀 또는 공기 / 미디어 인터페이스에 붙어있을 수 있도록하지 않도록주의하십시오. </li><li> 개인 가방 세포를 제거하는 노란색 팁으로 클러스터를 씹다. 팁에서 퇴원 클러스터를 pipetting하여이 작업을 수행합니다. 낮은 전단 병력과 함께 시작하여 필요에 따라 힘을 향상시킬 수 있습니다. 항상 너무 많은 세포를 죽이지 않고 뉴런을 제거하는 데 필요한 최저 힘을 사용합니다. </li><li> 3-5 건강한 가방 세포를 수집하고 쓰임새 라이신 - 코팅 coverslip있는 요리로 전송할 수 있습니다. 죽은 세포 (그들은 중앙에 검은 / 투명) 및 결합 조직의 파편을 채취하지 마십시오. </li><li> 6 단계를 반복합니다. 각 coverslip의 중심에 15-25 살 뉴런에 대한 곳. 죽은 세포이나 먼지가 잘으로 전송하는 경우, 그것을 수확하거나 건강한 세포에서 멀리 불어하여 ​​원치 않는 물질을 제거합니다. 이 과정은 두 클러스터에 대한 2-3시간 걸립니다. </li><li> 일단 (원치 않는 진동을 피하기 위해 해제 흐름 벤치 송풍기 유지) 세포를 첨부할 수 있도록 RT 적어도 2 시간 흐름 벤치에서 문화 요리를 보관 마쳤다. </li><li> ° C 하룻밤, 또는 추가를 사용하기 전까지 14에서 인큐베이터에 요리를 놓습니다. 성장 원뿔은 40-10시간에서 개발 있습니다. </li></ol><img src="/files/ftp_upload/old/662_2008_2_10_0/image001.jpg" alt="그림 1. Aplysia californica 복부 신경절의 개략도. 가방 전지 클러스터를 얻기 위해 상처 어디에 컬러 라인 나타냅니다." /> 그림 1. Aplysia californica 복부 신경절의 개략도. 가방 전지 클러스터를 얻기 위해 상처 어디에 컬러 라인 나타냅니다. 3 일 : Aplysia 가방 세포의 라이브 영상을위한 챔버 설정 이미지 챔버 조립 <ol><li> 라이브 셀 이미징 실험을위한 가방 세포를 사용하기 전에, 낮은 전력 현미경에서 세포를 검사합니다. 세포가 건강하게 성장 콘을 개발하지 않은 경우, 1-2 시간 동안 RT에서 신선한 매체와 휴가를 제공합니다. </li><li> 신선한 L15 - ASW 매체와 번호판을 공급하기 위해 진공 라인 오래된 매체의 절반 (~ 2ml)를 제거하고 새로운 2 ML L15 - ASW의 배양 접시 당 매체를 추가하십시오. 이 coverslip에서 세포를 제거하므로 완전히 한꺼번에 접시에서 모든 매체를 제거하지 않는 것이 중요합니다. </li><li> 가방 세포 이미징을 위해, 우리는 샌드위치와 같은 영상 챔버의 두 coverslips 두 플라스틱 스페이서를 조립. 샌드위치의 맨 coverslip는 중간 및 시약을 쉽게 교환할 수 있도록 짧은 컷입니다. 조립하는 동안 연결된 가방 세포와 coverslips는 dislodging에서 세포를 방지하기 위해 항상 L15 - ASW 매체에 빠져들해야합니다. </li><li> 챔버를 준비하려면, 잘라 ~ 2mm를 다이아몬드 펜 (유리 커터)와 통치자와 # 1 coverslips 22x22 mm의 오른쪽에서. </li><li> 1 ML의 주사기를 사용하여 짧은 coverslip의 양면 높은 진공 그리스를 적용하고, 각 측면에 플라스틱 스페이서를 연결합니다. 두 스페이서의 상단에 진공 그리스를 적용합니다. </li><li> coverglass에 가방 세포를 포함하는 페트리 접시에 coverslip와 스페이서 조립, 장소 반전. 정렬 부드럽게이 Q - 팁 리버스 엔드와 하단 coverslip 맨 coverslip을 막대기 아래로 누르십시오. </li><li> 포셉을 사용하여 부드럽게 밖으로 누출 매체없이는 배양 접시의 샌드위치 조립을 리프트. 필요한 경우, 두 coverslips를 정렬하고, Kimwipe로 초과 매체를 닦아. 현미경 슬라이드 홀더의 하단 부분에 샌드위치를​​ 장소와 클립을 사용하여주의 덮개를 부착합니다. </li><li> Q - 팁을 샌드위치의 맨 위에 측면의 중심을 깨끗이 유리 청소기 ( &#39;스파클&#39;은 유리 청소기로 사용됩니다)에 dabbed. 신속 리크 라인을 피하기 위해 두 번째 건조 Q - 팁과 유리 청소기를 건조. 하단 coverslip에 대해 동일한 작업을 수행. 리크 라인 무료 깨끗한 coverslips는 고품질의 영상을 위해 중요합니다. </li></ol> 현미경에 가방 세포 Aplysia 가방 세포의 연결을 문화가 저밀도에 있기 때문에 귀하의 현미경 설정은 세포 사이를 전환할 촉진 무대 위치 기능을 가지고있다면, 그것은 최고입니다. 가방 세포는 쉽게 이미징 실험을하는 동안 무대에서 몇 시간 동안 RT에 보관하실 수 있습니다. 증발을 방지하기 위해 정기적으로 매체를 교환.

<ol>
	<li>Forscher, P., Kaczmarek, L. K., Buchanan, J. A., Stephen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+AMP+induces+changes+in+distribution+and+transport+of+organelles+within+growth+cones+of+Aplysia+bag+cell+neurons.">Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons.</a> J. Neurosci. 7, 3600-3611 (1987).</li><li>Suter, D. M., Errante, L. D., Belotserkovsky, V., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ig+superfamily+cell+adhesion+molecule,+apCAM,+mediates+growth+cone+steering+by+substrate-cytoskeletal+coupling.">The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling.</a> J. Cell. Biol. 141, 227-240 (1998).</li><li>Schaefer, A. W., Kabir, N., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filopodia+and+actin+arcs+guide+the+assembly+and+transport+of+two+populations+of+microtubules+with+unique+dynamic+parameters+in+neuronal+growth+cones.">Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones.</a> J. Cell. Biol. 158, 139-152 (2002).</li><li>Suter, D. M., Schaefer, A. W., Forscher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtubule+dynamics+are+necessary+for+SRC+family+kinase-dependent+growth+cone+steering.">Microtubule dynamics are necessary for SRC family kinase-dependent growth cone steering.</a> Curr. Biol. 14, 1194-1199 (2004).</li></ol>

Aplysia 가방 세포 뉴런은 거의 비의 연결을 세포와 혈청의 연결이없는 세포 배양 시스템을 제공합니다. 이러한 뉴런 중요한 세포 생물 학적 질문 숫자를 해결하기 위해 적합한 매우 큰 성장 원뿔을 형성합니다. 가방 세포 뉴런 쉽게 몇 시간 동안 조작하고 실온에서 몇 군데하실 수 있습니다. 형광등을 사용하면 하나 양적 F - 굴지와 미세 소관의 중합 및 translocation 역학의 다양한 매개 변수를 분석할 수 현미경 (FSM)?...

<table><tbody><tr><td>#1 glass coverslips 22x22 mm</td><td>Tool</td><td>VWR international</td><td>48366067</td><td></td></tr><tr><td>#1.5 glass coverslips 22x22 mm</td><td>Tool</td><td>Corning</td><td>2870-22</td><td></td></tr><tr><td>35 mm Petri dishes</td><td>Tool</td><td>Falcon BD</td><td>353001</td><td></td></tr><tr><td>Filters for medium filtration</td><td>Tool</td><td>EMD Millipore</td><td>SVGV010RS</td><td></td></tr><tr><td>High vacuum grease</td><td>Tool</td><td>Dow Corning</td><td>1597418</td><td></td></tr><tr><td>Osmometer</td><td>Tool</td><td>Wescor</td><td>5520</td><td></td></tr><tr><td>Plastic shims</td><td>Tool</td><td>Small Parts, Inc.</td><td>SHSP-200</td><td></td></tr><tr><td>Calcium chloride</td><td>Reagent</td><td>Fisher Scientific</td><td>C79-500</td><td></td></tr><tr><td>Gentamicin</td><td>Reagent</td><td>Invitrogen</td><td>15750-060</td><td></td></tr><tr><td>HEPES</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H4030</td><td></td></tr><tr><td>L15 medium</td><td>Reagent</td><td>Invitrogen</td><td>41300-039</td><td></td></tr><tr><td>L-glutamine</td><td>Reagent</td><td>Sigma-Aldrich</td><td>G8540</td><td></td></tr><tr><td>Magnesium chloride</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>5958-04</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>6070-12</td><td></td></tr><tr><td>Poly-L-lysine (70-150 kD) </td><td>Reagent</td><td>Sigma-Aldrich</td><td>P6282</td><td></td></tr><tr><td>Sodium chloride</td><td>Reagent</td><td>Mallinckrodt Baker Inc.</td><td>7581</td><td></td></tr><tr><td>Neutral Protease (Dispase)</td><td>Reagent</td><td>Worthington Biochemical</td><td>LS02111</td><td></td></tr></tbody></table>

neuronal cell cultures from aplysia for high-resolution imaging of growth cones

의 연결을 성장 콘은 환경에서지도 신호를 감지하고 적절한 대상 세포쪽으로 방향 움직임에이 정보를 transduce 수 axons의 끝에 높은 운동성이있는 구조입니다. 완벽하게지도 정보가 기본 동적 cytoskeletal 네트워크에 세포 표면에서 전달하는 방법 이해하기 위해서, 하나는 높은 시간적 및 공간적 해상도에서 단백질 역학 라이브 셀 이미징에 적합한 모델 시스템이 필요합니다. 전형적인 척추 성장 콘은 양적 F - 굴지와 미세 소관의 역학을 분석하는 너무 작은 수 있습니다. 군소 Aplysia californica에서 뉴런은 뉴런 척추보다 5-10 배 큰 쉽게 실온에서 보관 수 있으며, 미세 조작 및 biophysical 측정을 매우 강력한 세포 수 있습니다. 그들의 성장 콘은 매우 세포질 지역과 잘 설명 cytoskeletal 시스템을 정의했습니다. 의 연결을 세포 기관은 성장 콘 운동성 및지도를 공부에 대한 프로브의 다양한 microinjected 수 있습니다. 현재의 프로토콜에서는 복부 신경절, 가방 셀 뉴런의 문화와 성장 콘 라이브 셀 이미징에 대한 이미징 챔버를 설정 해부를위한 절차를 설명합니다.

Watch this Scientific Journal Video about Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones at JoVE.com

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Aplysia californica의 뉴런 성장 콘 운동성 및지도 고해상도 이미지를위한 최고의 아르 문화의 큰 성장 콘을 개발. 여기서 우리는 절개 및 Aplysia 가방 셀 뉴런의 도금뿐만 아니라 라이브 셀 이미징을위한 챔버를 설정을위한 프로토콜을 제시한다.

성장 콘스의 고해상도 이미징을위한 Aplysia에서의 연결을 셀 문화

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

neuronal-cell-cultures-from-aplysia-for-high-resolution-imaging

Research

JoVE Journal

Biology

11.4K 조회수.  Purdue University. Aplysia californica의 뉴런 성장 콘 운동성 및지도 고해상도 이미지를위한 최고의 아르 문화의 큰 성장 콘을 개발. 여기서 우리는 절개 및 Aplysia 가방 셀 뉴런의 도금뿐만 아니라 라이브 셀 이미징을위한 챔버를 설정을위한 프로토콜을 제시한다.

비디오: Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Sf9 세포에서 Aplysia 감각 뉴런에서 PKC Translocation의 라이브 영상

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

연구

신경과학

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

아피시아 단일 뉴런의 전기 생리 및 분자 분석을 위한 신경리아 준비

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

생체공학

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

연체 동물에서 세포 내 칼슘 농도와 시냅틱 효능의 모니터링 변경<em&gt; Aplysia</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

에서 신경 Explant의 문화<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

의 준비 Aplysia 감각 - 모터의 연결을 셀 문화

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

생물학

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

의 감각 뉴런의 분리<em&gt; 군소 californica</em&gt; 글루타메이트 전류의 패치 클램프 녹음을위한

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

동물의 신속한 유전자형은 뇌 신경 세포의 차 문화 구축에 이어

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

Rewiring Neuronal Circuits: A New Method for Fast Neurite Extension and Functional Neuronal Connection

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Here a procedure is described to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved reach over 1.2 mm/h, 30-60 times faster than the in vivo rates of the fastest growing axons from the peripheral nervous system (0.02 to 0.04 mm/h)28 and 10 times faster than previously reported for the same neuronal type at an earlier stage of development4. First, isolated populations of rat hippocampal neurons are grown for 2-3 weeks in microfluidic devices to precisely position the cells, enabling easy micromanipulation and experimental reproducibility. Next, beads coated with poly-D-lysine (PDL) are placed on neurites to form adhesive contacts and pipette micromanipulation is used to move the resulting bead-neurite complex. As the bead is moved, it pulls out a new neurite that can be extended over hundreds of micrometers and functionally connected to a target cell in less than 1 h. This process enables experimental reproducibility and ease of manipulation while bypassing slower chemical strategies to induce neurite growth. Preliminary measurements presented here demonstrate a neuronal growth rate far exceeding physiological ones. Combining these innovations allows for the precise establishment of neuronal networks in culture with an unprecedented degree of control. It is a novel method that opens the door to a plethora of information and insights into signal transmission and communication within the neuronal network as well as being a playground in which to explore the limits of neuronal growth. The potential applications and experiments are widespread with direct implications for therapies that aim to reconnect neuronal circuits after trauma or in neurodegenerative diseases.

This procedure describes how to rapidly initiate, extend and connect neurites organized in microfluidic chambers using poly-D-lysine-coated beads fixed to micropipettes that guide neurite elongation.

재귀 신경 회로의 연구 : 빠른 신경 돌기 확장과 기능적 신경 연결을위한 새로운 방법

Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model system to explore disease mechanisms and carry out high content screening (HCS) to interrogate compound libraries or identify gene mutation phenotypes. However, with human cells the transition from NPC to functional, mature neuron requires several weeks. Synapses typically start to form after 3 weeks of differentiation in monolayer culture, and several neuron-specific proteins, for example the later expressing pan-neuronal marker NeuN, or the layer 5/6 cerebral cortical neuron marker CTIP2, begin to express around 4-5 weeks post-differentiation. This lengthy differentiation time can be incompatible with optimal culture conditions used for small volume, multi-well HCS platforms. Among the many challenges are the need for well-adhered, uniformly distributed cells with minimal cell clustering, and culture procedures that foster long-term viability and functional synapse maturation. One approach is to differentiate neurons in a large volume format, then replate them at a later time point in HCS-compatible multi-wells. Some main challenges when using this replating approach concern reproducibility and cell viability, due to the stressful disruption of the dendritic and axonal network. Here we demonstrate a detailed and reliable procedure for enzymatically resuspending human induced pluripotent stem cell (hiPSC)-derived neurons after their differentiation for 4-8 weeks in a large-volume format, transferring them to 384-well microtiter plates, and culturing them for a further 1-3 weeks with excellent cell survival. This replating of human neurons not only allows the study of synapse assembly and maturation within two weeks from replating, but also enables studies of neurite regeneration and growth cone characteristics. We provide examples of scalable assays for neuritogenesis and synaptogenesis using a 384-well platform.

This protocol describes a detailed procedure for resuspending and culturing human stem cell derived neurons that were previously differentiated from neural progenitors in vitro for multiple weeks. The procedure facilitates imaging-based assays of neurites, synapses, and late-expressing neuronal markers in a format compatible with light microscopy and high-content screening.

신형성 및 시냅스 성숙의 고함량 스크리닝을 위한 인간 다능성 줄기 세포 유래 뉴런의 분화 후 리포팅

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

성장 콘스의 고해상도 이미징을위한 Aplysia에서의 연결을 셀 문화

요약

더 많은 비디오 탐색

이 비디오의 챕터

Sf9 세포에서 Aplysia 감각 뉴런에서 PKC Translocation의 라이브 영상

아피시아 단일 뉴런의 전기 생리 및 분자 분석을 위한 신경리아 준비

연체 동물에서 세포 내 칼슘 농도와 시냅틱 효능의 모니터링 변경

에서 신경 Explant의 문화

의 준비 Aplysia 감각 - 모터의 연결을 셀 문화

의 감각 뉴런의 분리

동물의 신속한 유전자형은 뇌 신경 세포의 차 문화 구축에 이어

재귀 신경 회로의 연구 : 빠른 신경 돌기 확장과 기능적 신경 연결을위한 새로운 방법

신형성 및 시냅스 성숙의 고함량 스크리닝을 위한 인간 다능성 줄기 세포 유래 뉴런의 분화 후 리포팅

축 사 초기 세그먼트의 조립을 공부 하는 기본 배양 된 해마 뉴런의 사용