우리는 무대 위로 배양기의 설계 및 제조하는 동안 자신의 기술 전문 지식과 훌륭한 기술을 공헌에 대한 도널드 헛슨 감사하고 싶습니다. 우리는 그녀의 뛰어난 기술 지원에도 Ayda Dashtaei에게 감사하고 있습니다. 모든 작품은 Neurosciences 학술 진흥 재단에 의해 지원되었다.

<ol>
	<li>Ligon, L. A., Steward, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+of+mitochondria+in+the+axons+and+dendrites+of+cultured+hippocampal+neurons.">Movement of mitochondria in the axons and dendrites of cultured hippocampal neurons.</a> J Comp Neurol. 427, 340-350 (2000).</li><li>Miller, K. E., Sheetz, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+evidence+for+coherent+low+velocity+axonal+transport+of+mitochondria.">Direct evidence for coherent low velocity axonal transport of mitochondria.</a> J Cell Biol. 173, 373-381 (2006).</li><li>Macaskill, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miro1+is+a+calcium+sensor+for+glutamate+receptor-dependent+localization+of+mitochondria+at+synapses.">Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses.</a> Neuron. 61, 541-555 (2009).</li><li>Morris, R. L., Hollenbeck, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axonal+transport+of+mitochondria+along+microtubules+and+F-actin+in+living+vertebrate+neurons.">Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons.</a> J Cell Biol. 131, 1315-1326 (1995).</li><li>Rintoul, G. L., Filiano, A. J., Brocard, J. B., Kress, G. J., Reynolds, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+decreases+mitochondrial+size+and+movement+in+primary+forebrain+neurons.">Glutamate decreases mitochondrial size and movement in primary forebrain neurons.</a> J Neurosci. 23, 7881-7888 (2003).</li><li>Crawley, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in neuroscience. , (1999).</li><li>Fedoroff, S., Richardson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protocols for neural cell culture. , (2001).</li><li>Chen, S., Owens, G. C., Crossin, K. L., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+stimulates+mitochondrial+transport+in+hippocampal+neurons.">Serotonin stimulates mitochondrial transport in hippocampal neurons.</a> Mol Cell Neurosci. 36, 472-483 (2007).</li><li>Chen, S., Owens, G. C., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+inhibits+mitochondrial+motility+in+hippocampal+neurons.">Dopamine inhibits mitochondrial motility in hippocampal neurons.</a> PLoS One. 3, e2804-e2804 (2008).</li><li>Chen, S., Owens, G. C., Makarenkova, H., Edelman, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HDAC6+regulates+mitochondrial+transport+in+hippocampal+neurons.">HDAC6 regulates mitochondrial transport in hippocampal neurons.</a> PLoS One. , e10848-e10848 (2010).</li><li>Curran, M. A., Kaiser, S. M., Achacoso, P. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transduction+of+nondividing+cells+by+optimized+feline+immunodeficiency+virus+vectors.">Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.</a> Mol Ther. 1, 31-38 (2000).</li></ol>

관심 없음 충돌 선언하지 않습니다.

lentivirus - 중재 감염 교양 뉴런에서 mitochondria를 타겟으로하는 형광 단백질의 표현과 연장 기간이 살 세포의 이미징을 허용 저렴한 실험실 지은 무대 위로 배양기를 채용, 우리는 mitochondrial 운동 neuromodulatory 신호 사이의 링크를 조사할 수있다 , 세로토닌 (5 - HT), 도파민 (DA) 및 아세틸콜린 (ACH) 등. 신경 기능의 핵심입니다 - 우리의 연구는 처음으로, 이러한 5 - HT 및 DA 같은 신경 전달 물질에 의해 뉴런 - 변조된의 활동의 변화에​​ mitochondrial 거래를 링크, 신호 경로를 명료하게하다하는 데 도움이 있습니다. 우리는 대상 단백질 형광의 사용이 더 생리학 관련 중요한 염료를 사용 가능 훨씬 짧은 기간이 아닌 수 있습니다 오랜 기간 동안 교양 뉴런을 살아가 표시 mitochondria의 관찰을 허용 것을 발견했습니다. 또한, 형광 단백질 신호의 강도가 photobleaching 또는 phototoxicity의 가능성을 최소화, 우리는 이미지 수집 중에 노출 시간 간단하게 할 수 있습니다. 마지막으로, 간단하고 저렴한 무대 위로 인큐베이터 미디어의 증발을 최소화하면서, 주위 온도, 습도 및 CO 2 수준을 유지, 우리도 시간 또는 일 동안 뉴런을 생활에 mitochondrial 운동을 수행 할 수 있습니다. 라이브 뉴런에서 mitochondria의 장기적인 관찰을위한 무대 위로 배양기를 조작하고자하는 연구자를 제공, 우리 디자인의 정확한 세부 사항을 수행하지 않아도되는 미디어의 증발을 방지하기 위해 (예를 들어, 가스 투과 막을 사용하는 재료의 성질 )과 (예, 온도 및 습도 제어, osmolarity의 산도, 유지 보수 버퍼링) 적용 원칙은 일반적으로이 프로토콜에서 설명하는 것과 일치합니다.

<table border="1"><tbody><tr><th> 양 </th><th> 설명 (및 그림 1에 위치) </th></tr><tr><td> 1 </td><td> 조직 문화 인큐베이터 (H) </td></tr><tr><td> 1 </td><td> 내열성 플라스틱 인클로저 (delrin 또는 유사한) (A) </td></tr><tr><td> 1 </td><td> 35mm GBM의 뚜껑 플라스틱 프레임 (배양 접시에 막을 affixing 용) (M) </td></tr><tr><td> 1-2 </td><td> 선형 피트 클리어 PFTE (테플론) 막 재료 </td></tr><tr><td> 4 </td><td> 브레스 엄지 나사 </td></tr><tr><td> 2 </td><td> 소형 10kOhm 히트싱크 저항 (무대 위로 보육 인클로저 내부 가열 요소로 사용) </td></tr><tr><td> 1 </td><td> 변압기 (저항기에 9V 전류 공급) </td></tr><tr><td> 1 </td><td> 테라리움 온도 컨트롤러와 프로브 (변압기를 통해 방열판의 저항에 전력 조절 온도 조절) </td></tr><tr><td> 1 </td><td> 2000ml Erlenmeyer 플라스크 (호스의 진동 억제하기 전에 무대 위로 보육에 대한 머플러) (K) </td></tr><tr><td> 1 </td><td> 1 / 8 &quot;sorbothane 시트 (무대 상단 인큐베이터의베이스 개스킷 재료) </td></tr><tr><td> 1 </td><td> 밀폐된 ABS (또는 유사한) 플라스틱 박스 (수족관 펌프 주택) (J) </td></tr><tr><td> 1 </td><td> 수족관 펌프 (I) </td></tr><tr><td> 1 </td><td> 소형 컴퓨터 냉각 팬 </td></tr><tr><td> 1 </td><td> 냉각 팬 플라스틱 인클로저 (L) </td></tr><tr><td> 1 </td><td> 컴퓨터 냉각 팬 9V 변압기 </td></tr><tr><td> 20-30피트 </td><td> Nalgene이나 실리콘 호스 (조직 문화와 무대 위로 incubators 사이의 연결, 수분 트랩) (F, G, N) </td></tr><tr><td> 2 </td><td> 플라스틱 stopcocks (무대 위로 보육 전후의 공기 흐름을 열고 닫습니다) (O) </td></tr><tr><td> 4 </td><td> 가시 황동 호스 커넥터 (호스 연결하기 / 무대 위로 보육 및 수족관 펌프 인클로저에서) </td></tr><tr><td> 2 </td><td> 브래스 경감 님이 빠른 couplers (호스 연결하기 / 무대 위로 인큐베이터에서) </td></tr><tr><td> 2 </td><td> 브래스 경감 님이 빠른 couplers (호스 연결하기 / 무대 위로 인큐베이터에서) </td></tr></tbody></table> 표 1. 무대 위로 보육 부품 : <table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 폴리 - D - 라이신 </td><td> 시그마 - 알드리치 </td><td> P7280 - 5MG </td></tr><tr><td> laminin </td><td> 로체 응용 과학 </td><td> 11243217001 </td></tr><tr><td> 35mm 유리 바닥 요리 </td><td> MatTek </td><td> P35GC - 0 - 14 - C </td></tr><tr><td> DMEM </td><td> 생활 기술 </td><td> 10,567 </td></tr><tr><td> B27 </td><td> 생활 기술 </td><td> 17504-044 </td></tr><tr><td> Glutamax </td><td> 생활 기술 </td><td> 35,050 </td></tr><tr><td> 지방질이 풍부한 BSA </td><td> 생활 기술 </td><td> 11020-021 </td></tr><tr><td> L - 아스파라긴 </td><td> 시그마 - 알드리치 </td><td> P0380 - 100G </td></tr><tr><td> L - 프롤린 </td><td> 시그마 - 알드리치 </td><td> A8381 - 100G </td></tr><tr><td> 비타민 B - 12 </td><td> 시그마 - 알드리치 </td><td> V2876 - 100MG </td></tr><tr><td> 5 - HT </td><td> 시그마 - 알드리치 </td><td> H9523 - 25MG </td></tr><tr><td> 8 - OH - DPAT </td><td> 시그마 - 알드리치 </td><td> H8520 - 25MG </td></tr><tr><td> 도파민 </td><td> 시그마 - 알드리치 </td><td> H8502 - 5G </td></tr><tr><td> Bromocriptine </td><td> 시그마 - 알드리치 </td><td> B2134 - 25MG </td></tr><tr><td> SKF38393 </td><td> 시그마 - 알드리치 </td><td> D047 - 100MG </td></tr></tbody></table>

neuromodulation and mitochondrial transport: live imaging in hippocampal neurons over long durations

mitochondrial 교통과의 연결 기능 사이의 관계를 이해하기 위하여, 1-3 연장 기간이 삶의 양식 뉴런의 mitochondrial 행동을 관찰하는 것이 중요합니다. 이것은 cytoskeletal 컴포넌트, organelles, 생활 세포에서 다른 구조 동적 형광 현미경을 통해 시각 다음 표시하고 수있는 중요한 염료와 형광 단백질을 사용하여 지금은 가능합니다. 예를 들어, 배아 닭 교감 신경에 mitochondrial 운동은 중요한 염료 rhodamine 123 4를 사용 특성화했다. 또 다른 연구에서는 mitochondria는 mitochondrially 대상 eYFP 5 transfection하여 쥐 forebrain의 뉴런에서 시각되었습니다. 그러나, 심지어 기본 분 이상의 뉴런, 시간 또는 일의 영상은 문제의 번호를 보여줍니다. 최고 이들 가운데는 다음과 같습니다 긴 영상 세션 동안 같은 온도, 습도, 그리고 산도와 같은 문화 조건 1) 보수, 2) 인수 이미지와 이미지를 분석하는 동안 신호 강도의 정확한 측정의 품질을 모두 보장하는 강력하고 안정적​​인 형광 신호; 3) 이미지 수집 중에 노출 시간을 제한하는 것은 photobleaching을 최소화하고 phototoxicity을 피할 수 있습니다. 여기, 우리는 관찰, 시각화, 높은 시간적 해상도와 최적의 생활 지원 조건 교양 hippocampal 뉴런의 mitochondrial 운동의 분석을 허용하는 프로토콜을 설명합니다. 우리는 좋은 온도 조절 및 대기 가스 흐름을 제공하는 저렴한 무대 위로 배양기를 구축하고, 또한 안정적인 산도와 osmolarity 추청, 미디어 증발의 정도를 제한합니다. 이 인큐베이터는 일정한 습도 수준 및 5~10%의 CO 2 / 공기의 분위기를 제공하는 표준 조직 문화 배양기에, 유입 및 배출구 호스를 통해 연결되어 있습니다. 이 디자인은 반드시 몇 시간 또는 며칠 동안 세포의 생존을 보장하지 훨씬 더 비싼 현미경 incubators에 비용 효율적인 대안을 제공합니다. mitochondria 시각화하기 위해, 우리는 mitochondrion를 타겟으로하는 적색 형광 단백질을 인코딩 lentivirus와 세포를 감염. 이것은 안정적인 크세논 광원의 사용과 함께, 우리는 이미지 수집 중에 노출 시간을 제한할 수 있으며 제외한 모든 photobleaching과 phototoxicity 걸로, 강하고 지속적인 신호를 보장합니다. 무대 상단 인큐베이터의 상단에 두 사출 포트위한 신경 전달 물질 및 기타 시약의 급성 행정부가 mitochondrial 움직임을 조절하실 수 있습니다. organelle 타겟 빨간색 형광 단백질과 우리 무대 - 최고 인큐베이터, 종래의 거꾸로 형광 현미경, CCD 카메라, 크세논 광원의 조합 합계, lentivirus - 중재 표현은 우리가 mitochondrial 운송 시간이 경과 이미지를 얻기위한 수 종래의 중요한 염료 및 오프 - 더 - 수명 지원 시스템을 배포 연구에서 가능한 그 이상 기간이 이상의 살아있는 뉴런.

Watch this Scientific Journal Video about Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations at JoVE.com

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

우리는 오랜 기간이 이상의 형광 현미경을 통해 뉴런을 생활에서 mitochondria의 이미징 수있는 프로토콜을 설명합니다. 연장 기간 동안 이미지가 mitochondrially 대상으로 형광 단백질과 설계 및 우리 연구실에 지어진 저렴한 무대 위로 보육의 이용 lentivirus - 중재 표현을 통해 수행됩니다.

Neuromodulation 및 Mitochondrial 교통 : 긴 기간이 이상 Hippocampal 뉴런의 라이브 영상

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

neuromodulation-mitochondrial-transport-live-imaging-hippocampal

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

15.9K 조회수.  The Neurosciences Institute. 우리는 오랜 기간이 이상의 형광 현미경을 통해 뉴런을 생활에서 mitochondria의 이미징 수있는 프로토콜을 설명합니다. 연장 기간 동안 이미지가 mitochondrially 대상으로 형광 단백질과 설계 및 우리 연구실에 지어진 저렴한 무대 위로 보육의 이용 lentivirus - 중재 표현을 통해 수행됩니다.

비디오: Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

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

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

라이브 랫 두피 뉴런에서 Mitochondrial 분리막 잠재력과 반응 산소 종의 결정

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

혜성 분석을 사용하여 Hippocampal 뉴런의 DNA 손상을 시금

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

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. ...

Real-time Imaging of Axonal Transport of Quantum Dot-labeled BDNF in Primary Neurons

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are increasingly viewed as an early feature of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD) and Huntington&#8217;s disease (HD). As yet unclear is the mechanism(s) by which axonal injury is induced. We reported the development of a novel technique to produce biologically active, monobiotinylated BDNF (mBtBDNF) that can be used to trace axonal transport of BDNF. Quantum dot-labeled BDNF (QD-BDNF) was produced by conjugating quantum dot 655 to mBtBDNF. A microfluidic device was used to isolate axons from neuron cell bodies. Addition of QD-BDNF to the axonal compartment allowed live imaging of BDNF transport in axons. We demonstrated that QD-BDNF moved essentially exclusively retrogradely, with very few pauses, at a moving velocity of around 1.06 &#956;m/sec. This system can be used to investigate mechanisms of disrupted axonal function in AD or HD, as well as other degenerative disorders.

Axonal transport of BDNF, a neurotrophic factor, is critical for the survival and function of several neuronal populations. Some degenerative disorders are marked by disruption of axonal structure and function. We demonstrated the techniques used to examine live trafficking of QD-BDNF in microfluidic chambers using primary neurons.

양자의 축삭 교통 수단의 실시간 이미징 차 뉴런에서 BDNF를 도트 표지

BDNF plays an important role in several facets of neuronal survival, differentiation, and function. Structural and functional deficits in axons are ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

니코틴 유도 칼슘 신호 전달 및 복부 해마 축삭을 따라 신경 전달 물질 방출의 라이브 영상

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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.

교양된 Hippocampal 신경에 시 냅 스 소포 Endocytosis를 측정

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

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

초파리 뉴런 개발시 빠른 수지상 분지 역학의 시간 경과 라이브 이미징 및 정량화

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

유전 경직 성 하반신 마비에서 인간 유도 만능 줄기 세포 유래 뉴런에서 미토콘드리아 수송 및 형태 분석

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...