저자는 마우스 배아 5시 슬라이스 문화를 수행할 수있는 아이디어의 원본 소스를 인정합니다. 우리는 촬영하는 동안 우리의 심부름꾼 역할에 대한 관대한 과학 지원과 안나 Degen에 대한 요아킴 커쉬을 인정하고 싶습니다. 그리고 하이 델베르크 (우수 클러스터 셀룰러 네트워크)의 대학이 작품은 독일 연구 재단 (Sonderforschungsbereich 488, Teilprojekt B7/B9 도이치 Forschungsgemeinschaft)에 의해 재정 지원되었다.

1 부 : 깔끔히 및 culturing 준비. <ol><li> 매체 (DMEM, 25 % 1X HBSS, 25 % 태아 소 혈청, 0.5 % 포도당, 1 ㎜의 글루타민, 2.5 MM HEPES, 산도 7.3) 및 3 - cm Millicell - CM 0.4 - μm의 문화를 깔끔히 10 - cm 조직 문화 플레이트 준비 멤브레인 삽입하고 37 인큐베이터에 보관 ° C와 5% CO 2. </li><li> 그것이 약 37 숙박 있도록 전자렌지에 PBS에 4% 낮은 용융 점 아가로 오스를 가열하고 가열 접시에 보관 ° C. </li><li> PBS (140 MM NaCl, 2.7 KCl MM 10 MM 나 2 HPO 4, 1.8mM KH 2 PO 4) 얼음에 장소 10 cm 세균 배양 접시를 채우십시오. </li><li> 마이크로톰 냉각 장치를 설정하거나 버퍼 트레이 및 냉각 요소가 사전에 냉각 그것에 냉장고에 저장됩니다 보장합니다. </li></ol> 2 부 : 엠브료 포함. <ol><li> 자궁에서 배아를 해부하다하여 GFP 발현을 확인하기 위해 거꾸로 형광 현미경으로 그들을 검사합니다. </li><li> 거꾸로 10 cm 페트리 접시와 동양 그들 플레이스 배아 과도한 PBS를 제거하는 왓먼 종이 스트립을 사용합니다. </li><li> 이 자리에서 문제를 해결하기 위해 배아에서 아가로 오스를 적용합니다. 아가로 오스는 고체화하자. </li><li> 면도날로 절단하여 배아를 둘러싼 지역을 제한한다. </li><li> 그 반대편에 포함된 배아를 회전합니다. </li><li> 배아가 완전히 내장된 수 있도록 배아 조직에 대한 추가 아가로 오스를 적용합니다. </li><li> Loctite 406, &quot;Krazy의 접착제&quot;와 유사한 특수 접착제를 사용하여 vibratome 척에 깨끗한 가장자리와 마운트와 아가로 오스 블록을 준비합니다. </li></ol> 파트 3 : 프로 시저를 칼자국. <ol><li> precooled 버퍼 트레이 및 냉각 요소를 설정합니다. </li><li> 붙어 조직과 척을 넣고 1X HBSS를 (CA 2 + - MG 2 + 무료 HBSS, 10 MM의 HEPES 버퍼 산도 7.3, 500 U / ML 페니실린 / 스트렙토 마이신)까지 커버 추가합니다. </li><li> 삽입 및 precleaned (70 % 에탄올) 마이크로톰 블레이드를 수정. </li><li> 350을 준비 - 450 μm의의 조각들을 조직 문화 접시에 단축 유리 파스퇴르 pipettes를 사용하여 전송 얼음이 있었죠. </li><li> 포셉 한 켤레를 사용하여주의 깊게 각 슬라이스 문화 세포막을 Millicell로 전송에서 아가로 오스를 제거합니다. 약 4 조각은 문화 매체 6 ML 가득한 10 cm 조직 문화 판에서 한 막에서 교양 수 있습니다. </li><li> 37 조각 품어 ° C와 5% CO 2 (문화 매체 : DMEM, 25 % 1X HBSS, 25 % 태아 소 혈청, 0.5 % 포도당, 1mM 글루타민, 2.5 MM HEPES, pH를 7.3). 한 시간 경과 영상 시리즈를 수행하는 경우 한 매체 상수의 볼륨을 유지합니다. 짧은 시간 프레임에 대한 시간 초과 이미징 시리즈의 경우에는 그것이 같은 매체를 떠나 충분하다. 더 이상 문화 기간 변경에 대한 이후 12~20시간 것이 좋습니다. </li></ol> 파트 4 : 현미경에서 이미징 척추 신경 가지. <ol><li> 직립 형광 현미경으로 슬라이스와 Millicell 문화 세포막을 포함, 10 cm 조직 문화 플레이트를 삽입 이미지 척수 신경. </li><li> 다음 이미징 시점 동안 올바르게 위치에 현미경의 무대에서 Millicell 문화 점막의 방향을 라벨. </li><li> 사용하는 이미지 척추 신경 가지 4X (숫자 조리개 [NA] 0.1), 10X (NA 0.3), 또는 20x (NA 0.5) 목표. </li></ol> 그림 1은 4X와 10X 목표와 문화를 20 시간 동안 척추 신경 가지를 묘사한 영상 시리즈를 보여줍니다. <img src="/files/ftp_upload/2309/2309fig1.jpg" alt="그림 1" /> homozygous tauGPF 배아의 가로 통에 척수 신경 가지의 그림 1 이미징 시리즈. * 복부 척수, 화살표 = DRG의 = 모터 뉴런. 슬라이스의 지느러미 (D) - 복부 (V) 축에 표시됩니다. Scalebars : 200 μm의.

<ol>
	<li>Tucker, K. L., Meyer, M., Barde, Y. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotrophins+are+required+for+nerve+growth+during+development.">Neurotrophins are required for nerve growth during development.</a> Nat Neurosci. 4, 29-37 (2001).</li><li>Brachmann, I., Jakubick, V. C., Shaked, M., Unsicker, K., Tucker, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+slice+culture+system+for+the+imaging+of+nerve+development+in+embryonic+mouse.">A simple slice culture system for the imaging of nerve development in embryonic mouse.</a> Dev Dyn. 236, 3514-3523 (2007).</li><li>Collingridge, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+brain+slice+preparation:+a+tribute+to+the+pioneer+Henry+McIlwain.">The brain slice preparation: a tribute to the pioneer Henry McIlwain.</a> Journal of neuroscience methods. 59, 5-9 (1995).</li><li>Katz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+circuitry+of+identified+projection+neurons+in+cat+visual+cortex+brain+slices.">Local circuitry of identified projection neurons in cat visual cortex brain slices.</a> J Neurosci. 7, 1223-1249 (1987).</li><li>Hotary, K. B., Landmesser, L. T., Tosney, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+slices.">Embryo slices.</a> Methods Cell Biol. 51, 109-124 (1996).</li></ol>

중간 회임 마우스 배아의 배아 슬라이스 문화 (E10 - E12)를 준비하는 방법의 광범위한 비교에서, 우리는 vibratome가 문제없이 문화의 전반적인 생존과 재현성 모두와 관련하여 가장 신뢰할 수있는 결과를 생산하는 것을 관찰했습니다 신경 가지 패턴. 반대로, 조각이 맥일웨인 조직 헬기 3 완전히 inviable 것으로 판명을 사용하여 준비 하였다. 우리는 원래 배아의 전체 쓰레기가 동시에 400 μm의 ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>HBSS 10x</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>14180</td>
<td>&nbsp;</td>
<tr>
<td>Dissection tools</td>
<td>&nbsp;</td>
<td>FST</td>
<td>various</td>
<td>&nbsp;</td>
<tr>
<td>L.M.P. agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-022</td>
<td>&nbsp;</td>
<tr>
<td>Whatmann paper</td>
<td>&nbsp;</td>
<td>Whatman Int.</td>
<td>3030917</td>
<td>&nbsp;</td>
<tr>
<td>Shortened firepolished pipettes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>41966</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>10270-106</td>
<td>&nbsp;</td>
<tr>
<td>Pen Strep</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>L-glutamine 100x</td>
<td>&nbsp;</td>
<td>GIBCO</td>
<td>25030</td>
<td>&nbsp;</td>
<tr>
<td>Vibratome</td>
<td>&nbsp;</td>
<td>Microm International GmbH</td>
<td>HM 650 V</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>BX61WI</td>
<td>&nbsp;</td>
<tr>
<td>analySIS</td>
<td>&nbsp;</td>
<td>Soft Imaging System</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Millicell-CM inserts</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>PICMORG 50</td>
<td>&nbsp;</td>
<tr>
<td>10 cm culture plates</td>
<td>&nbsp;</td>
<td>Greiner Bio-One</td>
<td>633171</td>
<td>&nbsp;</td>
<tr>
<td>LOCTITE 406</td>
<td>&nbsp;</td>
<td>Henkel</td>
<td>142580</td>
<td>&nbsp;</td>
<tr>
<td>Razor blades</td>
<td>&nbsp;</td>
<td>Thermo Fisher</td>
<td>none</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>SMZ800</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>9105.2</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G7021</td>
<td>&nbsp;</td>
<tr>
<td>x4 objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>PL series</td>
<td>&nbsp;</td>
<tr>
<td>x10 objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>UPLFL &#x2013;PH series</td>
<td>&nbsp;</td>
<tr>
<td>Filter</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>U-MNIBA2</td>
<td>&nbsp;</td>
<tr>
<td>CCD camera</td>
<td>&nbsp;</td>
<td>Soft Imaging System</td>
<td>SIS F-View II</td>
<td>&nbsp;</td>
<tr>
<td>Equipment for heated chamber</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>CTI-Controller 3700 and incubator S #11531171</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

organotypic slice culture of gfp-expressing mouse embryos for real-time imaging of peripheral nerve outgrowth

다양한 목적을 위해, organotypic 조각으로 마우스 배아의 전의 생체내의 재배가 바람직합니다. 예를 들어, 우리는 모두 영화의 innervation에 가능성을 허용, 녹색 형광 단백질 (EGFP)의 향상된 버전이 독점적으로 개발 중심과 주변 신경계 하나의 뉴런에서 표현되는 유전자 변형 마우스 라인 (tauGFP)를 채택 forelimb가와 약리과 유전 기법이이 과정을 조작합니다. 이러한 슬라이스 문화의 성공적인 재배에서 가장 중요한 매개 변수는 조각을 준비하는 방법입니다. 다양한 방법의 광범위한 테스트 후, 우리가 vibratome들이 일상적으로 몇 일 동안 생존하고, 가장 중요한을 보여 문화에 따라서 그러한 배아를 슬라이스하는 최선의 장치는 것을 발견, 시대에 발전 특정 방식. 중반에 태동하고 배아의 경우, 이것은 척수와 지느러미 루트 신경의 주변과 골격 및 근육 조직의 적절한 판단에 자신의 목표에 척수 신경의 정상적인 파생물이 포함되어 있습니다. 슬라이스 준비 후 2 일을 위해 공부를 할 수있는 표준 조직 문화 인큐베이터에서 재배 400 마이크로 미터 조각 -이 작품에서 우리는 300로 (E) E10 E12에 배아 일 전체 배아를 처리하는 방법을 제시. 이 방법의 성공을 위해 중요 각각의 아가로 오스 - 임베디드의 배아를 슬라이스하는 vibratome의 사용입니다. 이 인터페이스 문화 기술에서 발생하는 중간의 작은 볼륨에 배치 Millicell 문화 막 삽입,시 조각의 재배옵니다. 7 배아의 평균 한 쓰레기는 정기적으로 인해 태아의 나이뿐만 아니라, 조각의 두께에 약간 차이가 최소 14 조각 (태아 당 forelimb 영역로부터 2-3 조각)을 생산하고 있습니다. 교양 조각의 80 % 정도는 culturing 기간 2 througout 측정할 수 신경 가지를 보여줍니다. tauGFP 마우스 라인을 사용하는 대표적인 결과가 증명하고 있습니다.

Watch this Scientific Journal Video about Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth at JoVE.com

Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

우리는 주변 신경 가지의 재배와 시간 경과 영상을위한 중간 회임 마우스 배아의 organotypic 조각을 준비하는 방법을 제시한다.

의 Organotypic 슬라이스 문화 GFP - 표현 주변 신경 가지의 실시간 이미징을위한 마우스 배아를

For many purposes, the cultivation of mouse embryos ex vivo as organotypic slices is desirable. For example, we employ a transgenic mouse line (tauGFP) in ...

organotypic-slice-culture-gfp-expressing-mouse-embryos-for-real-time

Research

JoVE Journal

Neuroscience

16.1K 조회수.  University of Heidelberg. 우리는 주변 신경 가지의 재배와 시간 경과 영상을위한 중간 회임 마우스 배아의 organotypic 조각을 준비하는 방법을 제시한다.

비디오: Organotypic Slice Culture of GFP-expressing Mouse Embryos for Real-time Imaging of Peripheral Nerve Outgrowth

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

의 연결을 부상을 검사에 대한 Organotypic Hippocampal 슬라이스 문화 모델

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

연구

신경과학

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotypic Hippocampal 슬라이스 문화

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

출생 후의 두뇌에의 연결을 마이 그 레이션의 고해상도 시간 경과 이미징을위한 Organotypic 슬라이스 분석

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

여러 마우스 Neuroanatomical 자기 공명 영상

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

네크로 텍스의 Organotypic 슬라이스 준비에서 전체 셀 녹음

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of the ...

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

손상 망막의 특성 Electrophysiological GFP - 표현 세포 집단

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

A Protocol for the Administration of Real-Time fMRI Neurofeedback Training

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control neuroplastic processes and correct abnormal function, or even shift functions from damaged tissue to physiologically healthy brain regions, hold the potential to dramatically improve overall health. Of the current neuroplastic interventions in development, neurofeedback training (NFT) from functional Magnetic Resonance Imaging (fMRI) has the advantages of being completely non-invasive, non-pharmacologic, and spatially localized to target brain regions, as well as having no known side effects. Furthermore, NFT techniques, initially developed using fMRI, can often be translated to exercises that can be performed outside of the scanner without the aid of medical professionals or sophisticated medical equipment. In fMRI NFT, the fMRI signal is measured from specific regions of the brain, processed, and presented to the participant in real-time. Through training, self-directed mental processing techniques, that regulate this signal and its underlying neurophysiologic correlates, are developed. FMRI NFT has been used to train volitional control over a wide range of brain regions with implications for several different cognitive, behavioral, and motor systems. Additionally, fMRI NFT has shown promise in a broad range of applications such as the treatment of neurologic disorders and the augmentation of baseline human performance. In this article, we present an fMRI NFT protocol developed at our institution for modulation of both healthy and abnormal brain function, as well as examples of using the method to target both cognitive and auditory regions of the brain.

The ability to induce and/or control neural plasticity may be critical in future treatments for neurologic disorders and the recovery from brain injury. In this paper, we present a protocol on the use of neurofeedback training with functional magnetic resonance imaging to modulate human brain function.

관리의 실시간 fMRI Neurofeedback 훈련에 대 한 프로토콜

Neurologic disorders are characterized by abnormal cellular-, molecular-, and circuit-level functions in the brain. New methods to induce and control ...

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It has helped to describe the different steps of radial migration and characterize the molecular mechanisms controlling this process. To directly and dynamically analyze migrating neurons they have to be traced over time. This protocol describes a workflow that combines in utero electroporation with organotypic slice culture and time-lapse confocal imaging, which allows for a direct examination and dynamic analysis of radially migrating cortical neurons. Furthermore, detailed characterization of migrating neurons, such as migration speed, speed profiles, as well as radial orientation changes, is possible. The method can easily be adapted to perform functional analyses of genes of interest in radially migrating cortical neurons by loss and gain of function as well as rescue experiments. Time-lapse imaging of migrating neurons is a state-of-the-art technique that once established is a potent tool to study the development of the cerebral cortex in mouse models of neuronal migration disorders.

Time-lapse Confocal Imaging of Migrating Neurons in Organotypic Slice Culture of Embryonic Mouse Brain Using In Utero Electroporation

This protocol provides instructions for direct observation of radially migrating cortical neurons. In utero electroporation, organotypic slice culture, and time-lapse confocal imaging are combined to directly and dynamically study the effects of overexpression or downregulation of genes of interest in migrating neurons and to analyze their differentiation during development.

배아 마우스 두뇌의 organotypic 슬라이스 문화에서 마이 그 레이션 뉴런의 저속 공 촛점 이미징<em&gt; Utero에서</em&gt; 일렉트로 포 레이션

In utero electroporation is a rapid and powerful approach to study the process of radial migration in the cerebral cortex of developing mouse embryos. It ...

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons

Here, we provide a low-cost and reliable method to generate electroporated brain organotypic slice cultures from mouse embryos suitable for confocal microscopy and live-imaging techniques.

전 Utero Electroporation 및 마이그레이션 GABAergic 수의 라이브 이미징 배아 마우스 두뇌의 Organotypic 조각 문화

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate ...

Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, where it is associated with functional deficits. Here, we provide a detailed description of an ex vivo model consisting of mouse organotypic cerebellar slices, which can be maintained in culture for several weeks and further be labeled to visualize myelin.

Here we present a method to prepare organotypic slice cultures from mouse cerebellum and myelin sheath staining by immunohistochemistry suitable for investigating mechanisms of myelination and remyelination in the central nervous system.

준비 및 Immunostaining Myelinating Organotypic 소 뇌 조각 문화

In the nervous system, myelin is a complex membrane structure generated by myelinating glial cells, which ensheathes axons and facilitates fast electrical ...