이 작품은 CPF에 과학 연구에 대한 네덜란드기구 (NWO)의 VIDI 인증 번호의 H64.09.016에 의해 재정되었다. CPF는 그녀의 중요한 의견과 최종 원고에 대한 수정 박사 Silvina A. Fratantoni에 감사합니다. GMRDL / 네 ..는 네덜란드의 기술 재단 NWO의 일부입니다 STW (프로젝트 12151 및 11350), 어느 부분적 경제부에 의해 자금 지원에 의해 지원된다. 우리는 도움과 지원을 위해 니콘 인스트루먼트의 유럽 BV의 캐서린 키츠 피터 Drent 감사합니다. HX 보조금 과학 연구에 대한 네덜란드기구 (부여 820.02.006)에 의해 지원되었다 네덜란드 왕립 예술 과학 아카데미 (부여 11CDP10) 및 WT에 의해 지원되었다.

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저자가 공개하는 게 없다.

이 문서에서는 SIM을 사용하여 체외에서 배양 된 쥐의 기본 해마 신경 세포에서 이미지 돌기 쪽을 작동 프로토콜을 설명한다. 차 해마 신경 세포 배양법 Kaech와 뱅커 (18)에 의해 설명 된 방법 일본어의 적응이다. 브루 등 19에 의해 설명 된 주된 차이점은, Neurobasal/B27 배양 astroglial 피더 문화의 요구를 제거 가능 매체, 및 아교 세포의 증식을 억제하면서 신경의 생존을 촉...

수지상 척추 신경 막의 작은 돌기이다. 이 특징적인 구조는 일반적으로 단일 시냅스로부터 입력을 수신하도록 전문이 뉴런 사이의 물리적 접촉 면적을 나타내고있다. 대부분의 기능적 성숙 돌기 쪽은 구형 팁,라고 머리, 그리고 돌기 축에 머리를 연결하는 얇은 목으로 구성되어 있습니다. 그러나 등뼈가 정적되지 않고 적극적으로 이동하고 심지어 성인의 뇌 2에 지속적으로 형태를 변경합니다. 시간이 2 주 기간 내에서 후반 배아 또는 출생 초기 시간에서 파생 된 쥐 차 해마 신경 세포 배양은 척추와 같은 구조 3 초 filipodia 진화 수많은 막 돌기 복잡한 돌기 아버을 개발한다. 이러한 동적 동작과 다른 특성에 기초하여, 돌기 쪽은 메모리 저장 및 시냅스 전달의 4,5위한 해부 기판을 제공하는 것으로 생각된다. 일을 감안할 때돌기 척추의 크기와 모양이 시냅스 기능에, 정확하게 자신의 치수를 측정하는 것이 중요하다 것을 전자 중요한 역할. 등뼈의 길이는 약 200 내지 2000 나노 미터에서 변화 좀처럼 공 초점 레이저 주사 형광 현미경에 의해 식별 될 수있다. 그러나 길이보다 다른 척추의 크기는 일반적으로 이론적으로 200 나노 미터 6 주위 회절에 의해 고정 된 종래의 광 시스템의 해상도, 아래에. 이러한 해결 능력은 척추의 목 및 머리의 폭과 미세한 세부 사항을, 이미징에 충분하다. 많은 일이 문제를 해결하기 위해 전념하고있다 많은 비교적 새로운 슈퍼 해상도 현미경 기술은 상당한 진전을 제공하고 있습니다. 특히, 광 시야, 비 촛점 현미경 7-10에서 측방 구조화 조명 현미경 (SIM)을 사용하여 임의의 발광색을 폐기하지 않고 고전 넘어선 해상도를 달성 할 수있다. 비 리네아와 함께이 기술을 사용R 현미경 기법, 그것은 제한 요소 (11)에 의해 광학 현미경의 측 방향 해상도를 개선하기 위해 이론적으로 가능하다. 그러나, 대부분의 실험 상황에서, SIM은이 하나의 요인에 의한 해상도 한계를 넘어 설 수있다. 이러한 자극 방출 고갈 (STED) 현미경 (12)와 사진 활성화 현지화 현미경 (PALM) 12와 같은 다른 슈퍼 해상도 광학 현미경 기술은 돌기 쪽의 영상에 적용되었습니다. 같은 PALM 등 현지화 기반의 방법은 최고 해상도를 달성하기 위해 RAW 이미지의 매우 큰 숫자를 필요로하기 때문에 속도가 제한됩니다. 한편, STED 높은 촬상 속도를 달성 할 수있는 비교적 낮은 광자 카운트 및 SIM (13)에 대한 경우가 아닐 수도 보이는 작은 필드에서 비록. 이 문서의 목적은 체외에서 배양 한 쥐의 기본 해마 신경 세포에서 이미지 돌기 쪽을 작동하는 프로토콜을 제공하는 것입니다 </EM&gt; SIM을 사용하여. 설립, 개발, 형질 전환 쥐의 기본 해마 신경 세포 배양의 면역 조직 화학 염색과 영상을 샘플링하기 위해 최선을 다하고 늦은 단계로 구성된 최초의 하나 :이 프로토콜은 서로 구분되는 두 단계로 구성됩니다.

<table>
	<tbody>
		<tr>
			<td>Fine forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Big forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Big scissor</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt spatula</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope with illumination</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow cell culture hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High-temperature dry-oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator (5% CO2, 37 &#176;C)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon structured illumination microscope setup consisting of:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti research inverted microscope with Perfect Focus System</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon CFI Apo TIRF 100x oil objective lens (N.A. 1.49)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 Coherent Sapphire Lasers (458, 488, 514 and 561 nm exitation wavelength)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SIM Illuminator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Stage Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MCL Nano-Drive piezo controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Intensilight C-HGFIE mercury lamp</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SIM Microscope Enclosure temperature control</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Andor EM-CCD Camera iXon DU897</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PC with Microsoft Windows 7 Home Edition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon&#8217;s NiS Elements 6.14 SIM software package</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon type A immersion oil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy

돌기 쪽은 신경 세포의 수상 돌기에서 나오는 돌출부이며, 뇌의 흥분성 입력의 기본 시냅스 대상을 나타냅니다. 기술의 발전은 신경 세포의 연결 및 시냅스 가소성의 핵심 요소로 이러한 구조를 발견했습니다. 광학 현미경을 사용하여 척추의 형태의 정량 분석​​에 의한 빛의 굴절 고유 제한과 관련된 기술적 인 한계에 대한 근본적인 문제가 남아있다. 돌기 쪽이 용이하게 공 초점 레이저 주사 형광 현미경에 의해 식별 될 수있다. 길이 아닌 다른 척추 치수는 일반적으로 200 ㎚의 광학 현미경의 이론적 인 해상도 한계에 의해 고정 된 기존의 광학 해상도보다 작은 있기 때문에, 척추의 모양과 크기에 미묘한 변화를 측정하는 것은 어렵다. 몇 가지 최근에 개발 된 슈퍼 해상도 기술이 돌기 쪽을 포함하여 200 나노 미터보다 작은 이미지 세포 구조에 사용되어왔다. 티HESE 기술은 고전적인 원방 조작에 기초하기 때문에 시료의 표면을 넘어 기존의 샘플 준비 방법과 이미지를 사용할 수있게됩니다. 여기에 설명 된 기본 해마 신경 세포의 문화에 돌기 쪽의 영상을 초 고해상도 구조 조명 현미경 (SIM)을 적용하는 작업 프로토콜입니다. SIM의 가능한 응용 프로그램은 공 초점 현미경의 사람들과 겹칩니다. 그러나,이 기술은 다양한 적용 가능성을 제시한다. 공 초점 현미경으로 인해 실제 구멍의 사용에 초점 빛을 밖으로 제거의 비용으로 해상도 향상을 달성하면서 SIM은, 높은 효과적인 측면 해상도를 제공합니다. 이 프로토콜에서 기본 뉴런은 DNA 플라스미드는 형광 단백질을 인코딩하고 SIM을 사용하여 몇 군데로 형질 표준 프로토콜을 사용하여 유리 커버 슬립에 배양. 돌기 쪽이 체외에서 16-17일 후, 때 이미지가 있기 때문에 여기에 설명 된 전체 프로토콜은, 약 2 주 소요수지상 개발이 최적이다. 프로토콜을 완료 한 후, 돌기 쪽이 전문 소프트웨어를 사용하여 SIM 이미지 스택의 시리즈에서 3D로 재구성 할 수있다.

여기에 설명하는 것은 SIM을 사용하여 체외에서 쥐 차 해마 신경 세포의 돌기 쪽 이미징을위한 표준화 작업 프로토콜입니다. 프로토콜 워크 플로우와의 중요한 단계는 그림 1에 나타내었다. 전반적으로, 프로토콜 문화, 개발, 쥐 차 해마 신경 세포 및 면역 조직 화학 염색의 형질을 포함한 샘플 준비의 첫 단계에서 분리 실험적인 작업의 약 2 주가 걸리고, 두 번째 단계 SIM을 사용...

Watch this Scientific Journal Video about Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy at JoVE.com

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

이 문서는 구조적 조명 현미경 (SIM)을 사용하여 체외에서 해마 신경 세포에서 이미지 돌기 쪽을 작동 프로토콜을 설명합니다. SIM을 사용하여 초 고해상도 현미경 개선 된 세부 사항과 함께 개별 돌기 쪽의 영상을 허용, 크게 세 가지 차원 공간에서 빛의 회절 한계를 넘어 이미지 해상도를 제공합니다.

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. ...

imaging-dendritic-spines-rat-primary-hippocampal-neurons-using

Research

JoVE Journal

Neuroscience

15.7K 조회수.  University of Amsterdam.  이 문서는 구조적 조명 현미경 (SIM)을 사용하여 체외에서 해마 신경 세포에서 이미지 돌기 쪽을 작동 프로토콜을 설명합니다. SIM을 사용하여 초 고해상도 현미경 개선 된 세부 사항과 함께 개별 돌기 쪽의 영상을 허용, 크게 세 가지 차원 공간에서 빛의 회절 한계를 넘어 이미지 해상도를 제공합니다. 

비디오: Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection 및 배아 마우스 Hippocampal과 두피 뉴런의 주요 문화

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

연구

신경과학

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

교양 Hippocampal 뉴런의 돌기 쪽이에서 형광 단백질의 태그 Photobleaching 후 형광 복구 (FRAP)

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Axons, 수석 및 뇌 슬라이스의 개별 뉴런의 수지상 쪽의 전압에 민감한 염료 기록

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

차 해마 신경 세포의 칼슘 인산염 형질

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

두 광자<em&gt; 생체</em&gt; 시닝 두개골 준비를 사용하여 마우스 피질 돌기 쪽의 영상

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

마우스 기본 Hypothalamic 뉴런 Deconvolution 현미경을 사용 하 여 매매 GLUT4 단백질의 라이브 이미지

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

E14-E16 스프라그 Dawley 쥐 배아에서 분리된 혼합 된 기본 해마 및 피질 뉴런에서 신경 구의 생성

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the mechanisms of synaptic plasticity in dendritic spines. In this protocol, the detailed 3D structure of the dendrites and dendritic spines is modeled with meshes on the software Blender with CellBlender. The synaptic and extrasynaptic regions are defined on the mesh. Next, the synaptic receptor and synaptic anchor molecules are defined with their diffusion constants. Finally, the chemical reactions between synaptic receptors and synaptic anchors are included and the computational model is solved numerically with the software MCell. This method describes the spatiotemporal path of every single molecule in a 3D geometrical structure. Thus, it is very useful to study the trafficking of synaptic receptors in and out of the dendritic spines during the occurrence of synaptic plasticity. A limitation of this method is that the high number of molecules slows the speed of the simulations. Modeling of dendritic spines with this method allows the study of homosynaptic potentiation and depression within single spines and heterosynaptic plasticity between neighbor dendritic spines.

The protocol develops a three-dimensional (3D) model of a dendritic segment with dendritic spines for modeling synaptic plasticity. The constructed mesh can be used for computational modeling of AMPA receptor trafficking in the long-term synaptic plasticity using the software program Blender with CellBlender and MCell.

시냅스 가소성을 가진 수지상 척추의 3D 모델링

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

Caenorhabditis elegans의 수지상 가시 이미징

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

수지상 척추 연구를 위한 직렬 블록-얼굴 스캐닝 전자 현미경 검사(SBEM)

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...