이 작품은 NINDS와 S.Ge.로 미국 심장 협회가 후원했다 이 접근법은 원래 설립하고 존스 홉킨스 대학에서 박사 Hongjun 노래의 연구실에있는 저자에 의해 개발되었습니다. 우리는 그의지도와 지원을 위해 박사 Hongjun 노래 감사하고 싶습니다.

1. 바이러스 사출 높은 titer 공학 레트로 바이러스 (1 × 10 9 단위 / ML) retroviral 벡터의 공동 transfection에 의해 생산 및 바이러스 뜨는의 ultracentrifugation 다음 HEK293T 세포로 VSVG입니다. 소스 및 생산 방법에 대한, 우수한 조브 데모 (3)을 참조하십시오. 참고 : 젊은 성인 (4-6 주를 넘어서는 안 됨) 여성 C57Bl / 6 생쥐 (찰스 리버)가 표준 조건 하에서 보관되어 있습니다. 모든 절차는 스토니 브룩 대학 IACUC 승인 프로토콜에 따라 실험실 동물 관리 및 사용을위한 국립 연구위원회의 안내를 따르십시오. <ol><li> 얼음 동물 당 냉동 레트로 바이러스의 해동의 2ul. </li><li> 마취시키다 (100 μg의 마취제 + g 체중 당 10 μg의 xylazine)하고 stereotaxic 프레임 (Steolting)에 동물을 탑재합니다. 머리에 머리를 제거하고 70 % 에탄올로 피부를 닦아냅니다. </li><li> 두개골을 노출 다음과 같은 지점에서 치과 드릴 (0.6mm 드릴 비트)를 사용하여 네 얕은 구멍을 드릴 : <ul><li> anterioposterior bregma에서 = -2 mm, 측면 = ± 1.6 mm, 복부 = 2.5 mm; anterioposterior bregma에서 = -3 mm, 측면 = ± 2.6 mm, 복부 = 3.2 mm. </li><li> anterioposterior bregma에서 = -2 mm, 측면 = ± 1.6 mm, 복부 = 2.5 mm; anterioposterior bregma에서 = -3 mm, 측면 = ± 2.6 mm, 복부 = 3.2 mm. </li></ul></li><li> 1μl 해밀턴, 평면 팁 주사기를 탑재하고 이가있는 이랑에 0.25 μl / 분 속도로 사이트마다 0.5 μl 역 바이러스를 주입. 천천히 유체 역류를 방지하기 위해 주사기를 철수하기 전에 각 주입 후 (복부 깊이에 대한 위의 좌표를 참조하십시오.) 일시 정지 2 분. 주사 사이에, 간략하게 멸균 PBS로 주사기 팁 및 혈액 성분을 제거하는 작은 주걱의 외부 표면을 씻어. </li><li> 무균 수술 봉합 재료와 상처 (타입 P - 1; 크기 6-0)을 닫고 사출 후 원하는 시간 단계까지 표준 주택 조건으로 동물을 반환합니다. </li></ol> 2. 슬라이스 준비 성인 생쥐에서 고품질의 급성 조각을 얻으려면, 우리는 (아래 참조) 슬라이스 준비를위한 수정 가공 솔루션을 사용합니다. <ol><li> 0-4 ° 95%와 얼음과 거품에 C O 2 -5 % 적어도 30 분 동안 CO 2 사전 진정 절단 솔루션입니다. </li><li> 마취시키다 및 transcardially 얼음처럼 차가운 산소 포화 절삭 솔루션 동물 perfuse. </li><li> 신속하게 필터 종이 감기, 산소 절단 솔루션으로 사전 침수와 배양 - 접시에 머리를 제거합니다. 소뇌로를 차단하고 (표시) 사출 좌표 최소 1mm의 앞쪽에 장착 표면 슬라이스. 접착제는 두뇌에 vibrotome 무대와 추위와 산소 절단 솔루션으로 가득 절삭 챔버로를 탑재합니다. </li><li> 코로나 또는 수평 조각 (300 - 350μm)을 준비하고 룸 온도 ACSF을 포함하는 잠복기 챔버에 큰 구멍 피펫으로 그들을 전송는 95 % O 2 / 5% CO 2와 함께 부풀어 오른 모양이었다. </li><li> 30~60분 32 ° C에서 슬라이스, 지속 버블링를 품어. 녹음 기간 동안 실내 온도에 챔버를 반환합니다. </li></ol> 3. Electrophysiological 실험 <ol><li> 34 ° C. - 조각이 지속적으로 32 ° C에서 산소 포화 ACSF로 perfused있는 기록 챔버로 전송됩니다 </li><li> 바이폴라 텅스텐 자극 전극은 낮은 배율 현미경의 목표 (10X)를 사용하여 이가있는 이랑의 분자 층에 배치됩니다. </li><li> 하위 세분화된 영역 / 세분화된 세포 계층의 신생아 DGCs는 GFP 또는 dTomato의 표현에 의해 식별됩니다. 전체 셀 patchclamp 녹음은 높은 배율 (40X)에서 신생아 뉴런에서 수행됩니다. </li><li> 기록된 세포의 소성 특성 전류 클램프 모드에서 전류의 일련의 주입에 의해 얻을 수 있습니다. </li><li> 전기 자극은 레코딩 소프트웨어에 의해 제어되는 자극의 아이 솔 레이터를 통해 자극 전극으로 전달하고, 기록하는 신생아 DGCs에서 postsynaptic 전류를 evoked있다. </li></ol> 4. 데이터 분석 evoked postsynaptic 응답 Amplitudes은 성인에서 태어난 뉴런의 다른 발달 단계에서 분석하고 있습니다. 5. 대표 결과 위의 프로토콜을 사용하여 그림 1과 비슷한 신생아 이가있는 이랑의 뉴런에 GFP 표현이 일반적입니다. 신생아 뉴런 쉽게 시각이며, 모두 dendrites과 axons가 견고하게 표시됩니다. 얇은 DGC axons 작은 쪽이 표현은 바이러스의 품질과 titer, 사용되는 모터와 생체내 표현의 길이에 따라 다릅니다. 우리 손에, 신생아 세포 하위 세포 organelles는 사출 다음과 같은 몇 일 이내 볼 수 있으며, 척추 표현은 개발의 초기 단계에서 추적할 수 있습니다. W다른 시간 단계에서 신생아 뉴런에서 구멍 세포 레코딩은 성숙 (그림 2B) 동안 기존의 신경 회로에 고유한 신생아 셀 속성, 예를 들어 행동 잠재력 (그림 2A),뿐만 아니라 시냅스 통합 과정의 연구를 수 있습니다. <img src="/files/ftp_upload/2548/2548fig1.jpg" alt="그림 1" /> 성인 마우스의 수평 이가있는 이랑 섹션에서 신생아 뉴런의 그림 1은 2 광자 공촛점 이미지. 그린 세포가 21dpi 아르 (일 게시물 주입) GFP - 표현 pUX - GFP의 레트로 바이러스와 라벨 신생아 DGCs합니다. <img src="/files/ftp_upload/2548/2548fig2.jpg" alt="그림 2" /> 그림 2) 액션 가능성 21 신생아 DGC의 속성을 발사 DPI. B)이 대표는 21 DPI에서 신생아 DGC에 기록된 흥분성의 postsynaptic 전류 (EPSCs)를 evoked.

<ol>
	<li>Zhao, C., Deng, W., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functional+implications+of+adult+neurogenesis.">Mechanisms and functional implications of adult neurogenesis.</a> Cell. 132, 645-660 (2008).</li><li>Ge, S., Goh, E. L., Sailor, K. A., Kitabatake, Y., Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABA+regulates+synaptic+integration+of+newly+generated+neurons+in+the+adult+brain.">GABA regulates synaptic integration of newly generated neurons in the adult brain.</a> Nature. 439, 589-593 (2006).</li><li>Gavrilescu, L. C., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+replication-defective+retrovirus+by+transient+transfection+of+293T+cells.">Production of replication-defective retrovirus by transient transfection of 293T cells.</a> J Vis Exp. , (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

성인 포유류의 두뇌의 해마에서 지속 neurogenesis은 성숙한 두뇌에의 연결을 개발 및 통합을 공부하는 독특한 실험 모델 시스템을 제공합니다. 여기에 제시 프로토콜 retroviral 레이블과 성인 생쥐의 두뇌에서 신생아 이가있는 과립 뉴런의 통합을 연구 electrophysiological 방법을 결합한 제품입니다. 실험 성공되도록하기 위해, 우리는 절차의 중요한 단계 중에 다음 사항을 권장합니다 : 원치 않는 감염 및 염증을 방지하려면, 모든 도구는 수술 전에 (autoclaving, 살균기 모든 종류의, 또는 70 % 에탄올에 의해) 소독해야합니다. 주사하기 전에 주사기 바늘의 팁을 씻어 살균 PBS를 사용합니다. 바이러스 주입의 경우, 동물은 잘 stereotaxic 장치에 장착해야해야하며 불필요한 움직임의 출처는 주입하는 동안 최소화해야합니다. 머리가 잘 고정하고 기업 있는지 확인하고, 사전에 계산 사출 좌표 수준으로 bregma과 람다에 마우스의 코를 위치를 조정합니다. 객실 온도에 노출을 최소화하기 위해 신속하게 주사기로 바이러스를 추출 - retroviruses 특히 온도를 구분합니다. 압력 손상을 최소화하기 위해 권장되는 속도로 천천히 바이러스를 주사. 권장 평면 밀고 주사기 (대 일반 beveled 팁)을 사용하면 균일하게 분산 이가있는 이랑 감염 영역을 보장합니다. 조각을 준비하기 전에 솔루션을 절단하고 ACSF을 잠복기는 산소가 솔루션을 포화 수 있도록 최소 30 분 95% O 2 -5 %의 CO 2와 함께 부풀어 오른해야합니다. 동물은 빠르게 perfused 및 조직 최고의 슬라이스 품질 추위, 산소 포화 용액에 유지되어야합니다. postsynaptic 응답이있을 때까지 녹화, 서서히 자극 강도를 향상시킬 수 있습니다. 필요한 이가있는 이랑의 분자 층 내에서 자극 전극의 위치를​​ 변경합니다. 요약, 권장되는 접근 방법은 적극적으로, 성숙한 회로에 초기 통합하는 동안 서로 다른 특성과 성인에서 태어난 뉴런 가능한 기능 역할을 탐구하는 방법을 제공합니다.

<ul><li> 110 콜린 염화물, 2.5 KCl, 1.3 KH 2 PO 4, 25.0 NaHCO 3, 0.5 CaCl 2, 7 MgCl 2, 10 덱스 트로 오스, 1.3 나트륨 아스코 브, 0.6 나트륨 pyruvate, 5.0 kynurenic 산성 : 솔루션 (MM)을 커팅. </li><li> 인공 세리 브로 - 척수액 (ACSF, MM의) : 125.0 NaCl, NaHCO 3 25.0, 2.5 KCl, 2 CaCl 2, 1.3 KH 2 PO 4, 1.3 MgCl 2, 1.3 나트륨 아스코 브, 0.6 나트륨 pyruvate, 10 덱스 트로 오스. </li><li> 전체 셀 patchclamp위한 내부 솔루션 (MM)을 : 120 글루 콘 산 칼륨, 15 KCl, 4 MgCl 2, 0.1 EGTA, 10.0 HEPES, 4 MgATP, 0.3 나 3 GTP, 7 phosphocreatine (산도 7.4, 300 mOsm). </li></ul>

studying the integration of adult-born neurons

Neurogenesis은 측면 뇌실의 하위 심실 구역 (SVZ) 및 생활 전반에 걸쳐 hippocampal 이가있는 이랑의 하위 세분화된 영역 (SGZ)에 성인 포유류의 두뇌에서 발생합니다. 이전 보고서는 성인 hippocampal neurogenesis가 간질, 정신 분열증, 우울증과 불안 (1) 등 다양한 뇌 장애와 관련된 것으로 나타났습니다. 정상 및 비정상적 성인 태어난 뉴런 통합의 과정을 해독하는 것은 이러한 질병의 병인에 빛을 창고 및 새로운 치료법의 개발을 알려 수 있습니다. SGZ 성인 neurogenesis은 운명의 사양, 마이 그 레이션, 시냅스 통합 및 성숙의 단계를 포함하여, 배아 및 사후 산후의 연결을 개발을 거울. 그러나, 전체 통합은 장기, 6 주 기간 동안 발생합니다. 성인 출생 SGZ 이가있는 과립 세포 (DGCs)에 초기 시냅스 입력을 14 일 (2)에서 glutamatergic 입력 다음, GABAergic이다. 이가있는 이랑에 성인에서 태어난 뉴런의 회로 형성을 조절하는 특정 요소는 현재 알 수 있습니다. 우리 연구실이 proliferating 세포에 형광 단백질과 가상 규제 유전자를 전달하기 위해 몰로니 murine 백혈병 바이러스에 따라 복제 - 결함 retroviral 벡터를 사용합니다. 이 바이러스 기술은 세포 생년월일, 혈통 형태, 그리고 synaptogenesis 분석에 대해 높은 특이성과 해상도를 제공합니다. 전형적인 실험은 종종 독특한 라벨, transgene, 그리고 생체내에서 원하는 개발 프로세스의 단일 세포 분석을위한 발기인 요소가 포함된 두 개 또는 세 바이러스를 고용합니다. 다음 프로토콜은 하나의 녹색 (GFP) 또는 빨간색 (dTomato) 형광 단백질 레트로 바이러스 및 패치 - 클램프 전기 생리학을 사용하여 기능성 신생아 신경 통합을 분석하는 방법을 설명합니다.

Watch this Scientific Journal Video about Studying the Integration of Adult-born Neurons at JoVE.com

Studying the Integration of Adult-born Neurons

성인 동물 신생아 이가있는 과립 세포의 통합을 연구하는 방법이 설명되어 있습니다. 이 기술은 생체내 기능 통합 확인할 수 electrophysiological 녹음 뒤에 신생아 뉴런을 라벨 설계 레트로 바이러스를 사용합니다.

성인에서 태어난 뉴런의 통합을 공부

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

studying-the-integration-of-adult-born-neurons

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JoVE Journal

Neuroscience

13.8K 조회수.  State University of New York at Stony Brook. 성인 동물 신생아 이가있는 과립 세포의 통합을 연구하는 방법이 설명되어 있습니다. 이 기술은 생체내 기능 통합 확인할 수 electrophysiological 녹음 뒤에 신생아 뉴런을 라벨 설계 레트로 바이러스를 사용합니다.

비디오: Studying the Integration of Adult-born Neurons

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

utero에서 Electroporation은 두피 뉴런의 하위 집합의 유전자 기능을 학습에 대한 기본의 연결을 문화 뒤에

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

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신경과학

Studying the Neural Basis of Adaptive Locomotor Behavior in Insects

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those leg movements that result from passive forces and interactions with other legs through the common contact with the substrate. If we want to understand, which part of a given movement is produced by nervous system motor output, kinematic analysis of stepping movements, therefore, needs to be complemented with electrophysiological recordings of motor activity. The recording of neuronal or muscular activity in a behaving animal is often limited by the electrophysiological equipment which can constrain the animal in its ability to move with as many degrees of freedom as possible. This can either be avoided by using implantable electrodes and then having the animal move on a long tether (i.e. Clarac et al., 1987; Duch &amp; Pfl&uuml;ger, 1995; B&ouml;hm et al., 1997; Gruhn &amp; Rathmayer, 2002) or by transmitting the data using telemetric devices (Kutsch et al, 1993; Fischer et al., 1996; Tsuchida et al. 2004; Hama et al., 2007; Wang et al., 2008). Both of these elegant methods, which are successfully used in larger arthropods, often prove difficult to apply in smaller walking insects which either easily get entangled in the long tether or are hindered by the weight of the telemetric device and its batteries. In addition, in all these cases, it is still impossible to distinguish between the purely neuronal basis of locomotion and the effects exerted by mechanical coupling between the walking legs through the substrate. One solution for this problem is to conduct the experiments in a tethered animal that is free to walk in place and that is locally suspended, for example over a slippery surface, which effectively removes most ground contact mechanics. This has been used to study escape responses (Camhi and Nolen, 1981; Camhi and Levy, 1988), turning (Tryba and Ritzman, 2000a,b; Gruhn et al., 2009a), backward walking (Graham and Epstein, 1985) or changes in velocity (Gruhn et al., 2009b) and it allows the experimenter easily to combine intra- and extracellular physiology with kinematic analyses (Gruhn et al., 2006).
We use a slippery surface setup to investigate the timing of leg muscles in the behaving stick insect with respect to touch-down and lift-off under different behavioral paradigms such as straight forward and curved walking in intact and reduced preparations.

We describe a method to record motor activity, timed to the electrically recorded tarsal contact signal in a tethered insect, walking on a slippery surface. This is used to study the neural basis of adaptive behavior under reduced influence of mechanical interaction between legs through the substrate.

곤충의 적응 운동력이있는 사람의 행동의 신경 기초를 공부

Studying the neural basis of walking behavior, one often faces the problem that it is hard to separate the neuronally produced stepping output from those ...

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells in the walls of the lateral ventricle give rise to neuroblasts that migrate for several millimeters along the rostral migratory stream (RMS) to reach and incorporate into the olfactory bulb. To study the different steps and the impact of adult-born neuron integration into preexisting olfactory circuits, it is necessary to selectively label and manipulate the activity of this specific population of neurons. The recent development of optogenetic technologies offers the opportunity to use light to precisely activate this specific cohort of neurons without affecting surrounding neurons4,5. Here, we present a series of procedures to virally express Channelrhodopsin2(ChR2)-YFP in a temporally restricted cohort of neuroblasts in the RMS before they reach the olfactory bulb and become adult-born neurons. In addition, we show how to implant and calibrate a miniature LED for chronic in vivo stimulation of ChR2-expressing neurons.

Selective Viral Transduction of Adult-born Olfactory Neurons for Chronic in vivo Optogenetic Stimulation

Adult-born neurons of the olfactory bulb can be optogenetically controlled using Channelrhodopsin2-expressing lentiviral injection in the rostral migratory stream and chronic photostimulation with an implanted miniature LED.

만성에 대한 성인 태어나 강한 뉴런의 선택 바이러스성 전달 생체내에 Optogenetic의 자극

Local interneurons are continuously regenerated in the olfactory bulb of adult rodents1-3. In this process, called adult neurogenesis, neural stem cells ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.

This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.

인간 만능의 줄기 세포에서 Telencephalic Glutamatergic 뉴런의 사양

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

글루타민산 염과 GABA의 빠른 마이크로 이온 도입 : 시냅틱 통합을 조사하는 유용한 도구

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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

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

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

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

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

A Microbiomechanical System for Studying Varicosity Formation and Recovery in Central Neuron Axons

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with neurodegenerative diseases or injuries, but also in the normal brain. Here, we describe a newly-established micromechanical system to rapidly, reliably, and reversibly induce axonal varicosities, allowing us to understand the mechanisms governing varicosity formation and heterogeneous protein composition. This system represents a novel means to evaluate the effects of compression and shear stress on different subcellular compartments of neurons, different from other in vitro systems that mainly focus on the effect of stretching. Importantly, owing to the unique features of our system, we recently made a novel discovery showing that the application of pressurized fluid can rapidly and reversibly induce axonal varicosities through the activation of a transient receptor potential channel. Our biomechanical system can be utilized conveniently in combination with drug perfusion, live cell imaging, calcium imaging, and patch clamp recording. Therefore, this method can be adopted for studying mechanosensitive ion channels, axonal transport regulation, axonal cytoskeleton dynamics, calcium signaling, and morphological changes related to traumatic brain injury.

This protocol describes a physiologically relevant, pressurized fluid approach for rapid and reversible induction of varicosities in neurons.

공부 Varicosity 형성 및 중앙 신경 축 삭에서 복구 하기 위한 Microbiomechanical 시스템

Axonal varicosities are enlarged structures along the shafts of axons with a high degree of heterogeneity. They are present not only in brains with ...

An In Vitro Model for Studying Tau Aggregation Using Lentiviral-mediated Transduction of Human Neurons

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). Although mouse models of tauopathy have provided a valuable resource for investigating the neurotoxic mechanisms of aggregated tau, it is becoming increasingly apparent that, due to interspecies differences in neurophysiology, the mouse brain is unsuitable for modeling the human condition. Advances in cell culture methods have made human neuronal cultures accessible for experimental use in vitro and have aided in the development of neurotherapeutics. However, despite the adaptation of human neuronal cell cultures, in vitro models of human tauopathy are not yet widely available. This protocol describes a cellular model of tau aggregation in which human neurons are transduced with lentiviral-derived vectors that code for pathogenically mutated tau fused to a yellow fluorescent protein (YFP) reporter. Transduced cultures produce tau aggregates that stain positively for thioflavin and display markers of neurotoxicity, such as decreased axonal length and increased lysosomal volume. This procedure may be a useful and cost-effective model for studying human tauopathies.

This protocol details a procedure in which human neuronal cultures are transduced with lentiviral constructs coding for mutant human tau. Transduced cultures display tau aggregates and associated pathologies.

인간 뉴런의 렌 티 바이러스 매개 된 형질 도입을 이용한 타우 응집 연구를 위한 시험관 내 모델

Aberrant aggregation of the protein tau is pathogenically involved in a number of neurodegenerative diseases, including Alzheimer&#8217;s disease (AD). ...

Wireless Electrophysiological Recording of Neurons by Movable Tetrodes in Freely Swimming Fish

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain activity from freely moving fish would advance research on the neural basis of fish behavior considerably. Moreover, precise control of the recording location in the brain is critical to studying coordinated neural activity across regions in fish brain. Here, we present a technique that records wirelessly from the brain of freely swimming fish while controlling for the depth of the recording location. The system is based on a neural logger associated with a novel water-compatible implant that can adjust the recording location by microdrive-controlled tetrodes. The capabilities of the system are illustrated through recordings from the telencephalon of goldfish.

A novel wireless technique for recording extracellular neural signals from the brain of freely swimming goldfish is presented. The recording device is composed of two tetrodes, a microdrive, a neural data logger, and a waterproof case. All parts are custom-made except for the data logger and its connector.

자유롭게 수영 물고기에서 이동 식 Tetrodes에 의해 뉴런의 무선 전기 생리적 기록

The neural mechanisms governing fish behavior remain mostly unknown, although fish constitute the majority of all vertebrates. The ability to record brain ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

1 차 배아 마우스 중뇌 도파민 뉴런에서 사전 형성 된 피브릴 유도 α-시뉴클레인 축적 연구

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...