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

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 Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

기본 교양 Hippocampal 신경에 덴스 코어 Vesicles의 라이브 영상

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

연구

생물학

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

살아있는 마우스에 있는 등도 해마 CA1의 세로 2 광자 화상 진찰

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

행동분석학

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 ...

신경과학

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

정확 하 게 지역화 하 고 반복적인 뇌 조각 동봉 된 시스템에서의 장기적인 문화 중 간헐적인 이미징 수정된 롤러 튜브 메서드

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Craniotomy Procedure for Visualizing Neuronal Activities in Hippocampus of Behaving Mice

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit functions in systems neuroscience. However, high absorbance and scattering of light in mammalian tissue limit intravital imaging mostly to superficial brain regions, leaving deep-brain areas, such as the hippocampus, out of reach for optical microscopy. In this video, we show the preparation and implantation of the custom-made imaging window to enable chronic in vivo imaging of the dorsal hippocampal CA1 region in head-fixed behaving mice. The custom-made window is supplemented with an infusion cannula that allows targeted delivery of viral vectors and drugs to the imaging area. By combining this preparation with wide-field imaging, we performed a long-term recording of neuronal activity using a fluorescent calcium indicator from large subsets of neurons in behaving mice over several weeks. We also demonstrated the applicability of this preparation for voltage imaging with single-spike resolution. High-performance genetically encoded indicators of neuronal activity and scientific CMOS cameras allowed the recurrent visualization of subcellular morphological details of single neurons at high temporal resolution. We also discuss the advantages and potential limitations of the described method and its compatibility with other imaging techniques.

This article demonstrates the preparation of a custom-made imaging window supplemented with infusion cannula and its implantation onto the CA1 region of the hippocampus in mice.

마우스를 면도하는 해마에서 신경 활동을 시각화하기위한 두개골 절제술 절차

Imaging neuronal activities at single-cell resolution in awake behaving animals is a very powerful approach for the investigation of neural circuit ...

Live-imaging of Mitochondrial System in Cultured Astrocytes

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and function in astrocytes are implicated in cognition. This article describes the method for time-lapse imaging of astrocyte cultures equipped with a mitochondrial biosensor: MitoTimer. MitoTimer is a powerful and unique tool to assess mitochondrial dynamics, mobility, morphology, biogenesis, and redox state. Here, the different procedures for culture, image acquisitions, and subsequent mitochondrial analysis are presented.

This article describes the method for mitochondrial time-lapse imaging of astrocyte cultures equipped with MitoTimer biosensor and the resulting multiparametric analysis of mitochondrial dynamics, mobility, morphology, biogenesis, redox state, and turnover.

배양 된 성상 세포에서 미토콘드리아 시스템의 라이브 이미징

While much attention has been given to mitochondrial alterations at the neuronal level, recent evidence demonstrates that mitochondrial dynamics and ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

비율 표시기를 사용하여 1 차 신경에 있는 미토콘드리아 글루타티온 Redox 상태의 살아있는 화상 진찰

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal excitability. Recent studies have revealed the differential gene expression and functional heterogeneity of microglia in different brain regions. The unique functions of the hippocampal neural network in learning and memory may be associated with the active roles of microglia in synapse remodeling. However, inflammatory responses induced by surgical procedures have been problematic in the two-photon microscopic analysis of hippocampal microglia. Here, a method is presented that enables the chronic observation of microglia in all layers of the hippocampal CA1 through an imaging window. This method allows the analysis of morphological changes in microglial processes for more than 1 month. Long-term and high-resolution imaging of the resting microglia requires minimally invasive surgical procedures, appropriate objective lens selection, and optimized imaging techniques. The transient inflammatory response of hippocampal microglia may prevent imaging immediately after surgery, but the microglia restore their quiescent morphology within a few weeks. Furthermore, imaging neurons simultaneously with microglia allows us to analyze the interactions of multiple cell types in the hippocampus. This technique may provide essential information about microglial function in the hippocampus.

In Vivo Chronic Two-Photon Imaging of Microglia in the Mouse Hippocampus

This paper describes a method for chronic in vivo observation of the resting microglia in the mouse hippocampal CA1 using precisely controlled surgery and two-photon microscopy.

생체 내 마우스 해마에서 미세아교세포의 만성 이광자 이미징

Microglia, the only immune cells resident in the brain, actively participate in neural circuit maintenance by modifying synapses and neuronal ...

<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.

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

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