NINDS, 미국 심장 협회, 노화 연구에 대한 미국 연방, 천 년 3 월에 의해 투자

1. 준비 시작하기 전에, 필요한 수술 도구, 해부, 문화 미디어, 그리고 칠일 늙은 쥐 또는 마우스 새끼를 조립. 프로토콜은 마우스 및 쥐 준비에 대해 동일합니다. (I) 재료 및 장비 <ul><li> 운영 가위 (직선 길이 6 &quot;, 고양이 # RS6818, Roboz, 게이 더 스버그, MD) </li><li> 마이크로 가위, 해부 (길이 4 &#39;, &quot;고양이 # RS5910합니다. Roboz, 게이 더 스버그, MD)는 </li><li> 카본 스틸 더몬트 족집게 (CAT # RS - 5045.Roboz, 게이 더 스버그, MD) </li><li> 수정 유리 &quot;전송&quot;pipettes (삭제 파스퇴르 피펫의 아래쪽과 가장자리 smoothed) </li><li> Aclar 플라스틱 필름은 광장에서 (하니웰, # 812071, 포츠빌, PA) &quot;X 2&quot;2 컷 </li><li> 3에 의한 스폰지 4 (켄달, 고양이 # 3157, 맨스필드, MA) 커버 </li><li> 30mm Millicell 막 삽입 (0.4 μm의, Millipore, 베드 포드, MA) </li><li> 양날 면도날 (맥일웨인 조직 헬기, Vibratome 회사, 세인트 루이스, MO)와 Mcllwain 조직 헬기 </li><li> CCD 카메라 및 이미지 분석 소프트웨어와 함께 바뀌 현미경 </li></ul> (2) 해부 매체 (DM, 산도 7.3에서 1리터) <table border="0"><tbody><tr><td> 행크의 균형 소금 </td><td> 1 병에 (95.2 g/1L) (시그마 : H2387) </td></tr><tr><td> NHCO 3 (4.2 ㎜) </td><td> 350 MG </td></tr><tr><td> HEPES (10 ㎜) </td><td> 2.83 g </td></tr><tr><td> 포도당 (33.3 ㎜) </td><td> 6.0 g </td></tr><tr><td> 페니실린은 / 스트렙토 마이신 (100x) </td><td> 10 ML </td></tr><tr><td> BSA (0.3 %) </td><td> 300 MG </td></tr><tr><td> MgSO 4 7H 2 O </td><td> 1.44 g </td></tr></tbody></table> (3) 문화 매체 (400 ML) <table border="0"><tbody><tr><td> 멤 (Invitrogen 11575-032) </td><td> 200 ML </td></tr><tr><td> *** 행크의 대차 소금 (Invitrogen 24020-117) </td><td> 100 ML </td></tr><tr><td> 호스 혈청 (Invitrogen 26050-070) </td><td> 100 ML </td></tr><tr><td> HEPES 버퍼 (1 M, Invitrogen 15630-080) </td><td> 5 ML </td></tr><tr><td> 페니실린은 / 스트렙토 마이신 (100x) </td><td> 4 ML </td></tr><tr><td colspan="2"> *** 문화 매체를하기 전에 500 ML 행크의 밸런스 솔트 12.8 g의 포도당을 추가 </td></tr></tbody></table> (4) 동물 칠일 늙은 쥐 또는 마우스 출생 후의 새끼, 실험마다 일반적으로 하나의 쓰레기. 2. 문화 당일 <ol><li> 모든 해부 도구와 Aclar 영화는 시작하기 전에 30 분 70 % ETOH에 배어 있어야합니다. 절개 후드, 블레이드 및 조직 헬기의 표면은 에탄올로 소독해야합니다. </li><li> (3-4) 여러 여섯 잘 조직 문화 번호판을 준비, 잘 당 1 ML 문화 매체를 추가하고 각 우물에 Millipore 투과 막를 삽입합니다. 37 장소는 6 - 잘 접시 ° 보육 조각을 전송할 준비까지. </li><li> DM 얼음에 장소 몇 가지 (4-​​6) 60mm 플레이트를 준비합니다. </li><li> 신속하게 가위로 목을 베다와 70% ETOH에서 수영을하고, 신속하게 두개골의 saggital 절개로 머리를 노출. 조심스럽게 추위 DM에 즉시 두뇌과 장소를 제거합니다. 두 반구로 그것을 분리 후 2-3 초 동안 표지 스폰지의 각 반구 내측 면을 아래로 놓습니다. </li><li> 부드럽게 조직 헬기에 Aclar 영화 광장과 장소에 반구를 리프트와 장소, 350 μm의 섹션에 coronally 머리를 잘라. 완료되면, 부드럽게 감기 DM에서 sectioned 북반구와 잠수함과 Alcar 광장을. </li><li> 해부 현미경을 사용하여 hippocampal 섹션을 구분합니다. 밀고 들어, 꼬리 해마에 주동이의 스패닝 5 섹션이 있어야하고, 마우스 되려면 3-4 섹션이 있어야합니다. </li><li> 37 ° 조직 문화의 인큐베이터에서 막 삽입과 함께 6 자 번호판을 가져가라. 유리 전송 피펫을 사용하여 투과 세포막에 개별적으로 hippocampal 조각 전송합니다. 너무 DM은 멤브레인에 출시되면, 파스퇴르 피펫과 DM 초과를 제거합니다. 각 막 위에 다섯개의 hippocampal 조각 플레이스. 조건 당 적어도 3 웰스, 15 조각 허용합니다. </li><li> 독성 실험을 시작하기 전에 12-14일에 대한 5 %의 CO humidified 인큐베이터에서 37 °에있는 문화 번호판을 품어. 문화 매체는 일주일에 두 번 변경할 수 있습니다. </li></ol> 3. 슬라이스 문화 Hippocampal의 연결 부상을 유도하고 Quantifying <ol><li> 문화 12~14일 후, 문화 미디어에 5 μg / ML의 propidium 요오드화물의 (PI)를 추가합니다. 이 단계는 시각화와 기저 세포 사망의 부량 수 있습니다. </li><li> 그 다음날, PI를 계량각 hippocampal 슬라이스의 CA1의 subregion (및 / 또는 CA3 또는 이가있는 원하는 경우)의 형광 및 취득은 기저 세포 죽음 (시간라고도 = 0 시간, 또는 T 0) 대표 PI 형광 측정을 의미합니다. 이미지 수집 및 부량 위해, 우리는 OpenLab 소프트웨어 (순발력, 영국),하지만 다른 이미지 인수를 사용하여 분석 소프트웨어는 형광 강도를 계량하는 데 사용할 수 있습니다. </li><li> 즉시 T 0 PI 형광의 이미지 취득 후, 선택의 방법 (우리의 연결 독성 2,5를 유도 한 시간 10 μm의 NMDA, 산소 - 포도당 박탈, 또는 LPS를 사용한)으로의 연결을 부상을 유발. 독성 자극 후, 5 μg / ML PI를 포함하는 새로운 매체와 매체를 교체하고 야간 조각 유지합니다. </li><li> 일 3, PI 24 시간의 실험 조건의 형광 또는 T 24을 의미 계량. </li><li> 이 이미지 수집 후, 즉시 매체를 변경 및 10 μm의 NMDA 및 5 μg / ML PI와 최대한의 연결을 죽음을 달성하기 위해 밤새 품어 슬라이스를 추가합니다. </li><li> 일 4, T 최대로 정의 최대한의 연결을 죽음의 PI의 형광을 의미 계량. 다음과 같이 %의 세포 죽음을 계산 : (T 24 - T 0) / (T 최대 - T 0). </li></ol> 4. 대표 결과 <img src="/files/ftp_upload/2106/2106fig1.jpg" alt="그림 1" /> 그림 1. hippocampal와 후속 실험 부상 및 이미지 수집의 세대 (A) 타임 라인. (B) hippocampal basally 조각 (T 0), 10 μm의 NMDA (T 24)에 1 시간 노출 다음 24 시간과 최대한의 연결을 부상 (T 최대)을 구하는 NMDA 추가로 24 시간 또는 10 μm의 이후. 대표 이미지

<ol>
	<li>Stoppin, L., Buchs, P. A., Muller, D. <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+method+for+organotypic+cultures+of+nervous+tissue.">A simple method for organotypic cultures of nervous tissue.</a> J Neurosci Methods. 37 (2), 173-173 (1991).</li><li>McCullough, L., Wu, L., Haughey, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+Function+of+the+PGE2+EP2+Receptor+in+Cerebral+Ischemia.">Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia.</a> J Neurosci. 24 (1), 257-257 (2004).</li><li>Czapiga, M., Colton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+microglia+in+organotypic+slice+cultures.">Function of microglia in organotypic slice cultures.</a> J Neurosci Res. 56, 644-644 (1999).</li><li>Newell, D. W., Barth, A., Papermaster, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+and+non-glutamate+receptor+mediated+toxicity+caused+by+oxygen+and+glucose+deprivation+in+organotypic+hippocampal+cultures.">Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures.</a> J Neurosci. 15, 7702-7702 (1995).</li><li>Wu, L., Wang, Q., Liang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divergent+Effects+of+prostaglandin+recetor+signaling+on+neuronal+survival.">Divergent Effects of prostaglandin recetor signaling on neuronal survival.</a> Neuroscience letter. 421, 253-253 (2007).</li><li>Hailer, N. P., Jarhult, J. D., Nitsch, R., R, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resting+microglial+cells+in+vitro:+Analysis+of+morphology+and+adhesion+molecule+expression+in+organotypic+hippocampal+slice+cultures.">Resting microglial cells in vitro: Analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures.</a> Glia. 18, 319-319 (1996).</li></ol>

관심 없음 충돌 선언하지 않습니다.

실험을 복제하면 재현할하고 일관된 결과를 보장하기 위해 수행하는 두 가지 중요한 포인트가 있습니다. 첫째, 실험 전에 12-14일에 대한 hippocampal 조각의 시대를하게하는 것이 중요합니다. 슬라이스 절연을 통해 상당한 microglial 응답이 있으며, microglia는 체외에서 3,6 일 10 가지를내는 발생 휴식 상태로 돌아와, 일정 기간 동안 활성 상태로 유지됩니다. 둘째, PI 형광 강도가 부상 세포의 수를 4로 비례 의미합니다. 선형 형광 세 시간 지점에서 측정한다 (I) T 0, NMDA 또는 ​​자연의 연결을 죽음의 기저 수준을 측정하는 OGD 자극하기 전에, (2) T 24, 또는 24 시간 NMDA 또는 ​​OGD 또는 다른 자극과 자극 후 그리고 (iii) T 맥스에서의 연결을 죽음의 최대한의 금액을 생산하는 10 μm의 NMDA와 함께 24 시간 야간 치명적인 치료와 조각의 최종 치료를 다음과. / (T 최대 - T 0) - %의 세포 사망은 다음 수식을 (T 0 T 24)를 사용하여 계산됩니다. 하나 크게 해마 및 글루 탐 산염 수용체의 표현 및 유통의 변화의 크기가 꼬리 뇌, 그리고 그 자체로 각각의 슬라이스를 정상화하기 위해 실패 주동이의 나갈 수 있기 때문에이 방법을 사용하여 자체 각각의 슬라이스를 정상화하기 위해 중요한 것은 artifactual 데이터가 발생할 수 있습니다 .

the organotypic hippocampal slice culture model for examining neuronal injury

Organotypic hippocampal 슬라이스 문화의 기본 아키텍처와 해마의 구성이 상대적으로 1 보존되어있는 부상의 연결 메커니즘을 검토에서 체외 방식입니다. organotypic 문화 시스템은 astrocytic의 연결 및 microglial 효과 시험은 가능하지만, 전직 생체내 준비로 주변 염증 세포의 혈액 흐름, 또는 모집의 효과를 해결하지 않습니다. 이를 위해,이 문화 방법은 자주 해마의 피라미드 뉴런에 excitotoxic 및 hypoxic 부상을 확인하는 데 사용됩니다뿐만 아니라 염증 반응을 검사하는 데 사용되었습니다. 여기에 우리는 출생 후의 쥐 두뇌에서 hippocampal 슬라이스 문화를 생성의 연결 부상을 유발하는 유해 자극을 관리하고, 시금 및 hippocampal의 연결을 죽음을 quantifying위한 방법을 설명합니다.

Watch this Scientific Journal Video about The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury at JoVE.com

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

organoptypic hippocampal 슬라이스 문화 모델입니다<em&gt; 체외에서</em&gt; 모델 패러다임의 다양한 부상의 연결을 검사하는 데 사용됩니다. 이 문서에서는, 우리는 슬라이스 문화를 생성하고의 연결 부상을 quantifying위한 방법을 설명합니다.

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 ...

the-organotypic-hippocampal-slice-culture-model-for-examining

Universidad San Sebastian

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JoVE Journal

Neuroscience

19.0K 조회수.  Stanford University School of Medicine. organoptypic hippocampal 슬라이스 문화 모델입니다 체외에서 모델 패러다임의 다양한 부상의 연결을 검사하는 데 사용됩니다. 이 문서에서는, 우리는 슬라이스 문화를 생성하고의 연결 부상을 quantifying위한 방법을 설명합니다.

비디오: The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

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

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 which the enhanced version of the green fluorescent protein (EGFP) is exclusively expressed in all neurons of the developing central and peripheral nervous system1, allowing the possibility to both film the innervation of the forelimb and to manipulate this process with pharmacological and genetic techniques2. The most critical parameter in the successful cultivation of such slice cultures is the method by which the slices are prepared. After extensive testing of a variety of methods, we have found that a vibratome is the best possible device to slice the embryos such that they routinely result in a culture that demonstrates viability over a period of several days, and most importantly, develops in an age-specific manner. For mid-gestation embryos, this includes the normal outgrowth of spinal nerves from the spinal cord and the dorsal root ganglia to their targets in the periphery and the proper determination of skeletal and muscle tissue. 
In this work, we present a method for processing whole embryos of embryonic day (E) E10 to E12 into 300 - 400 micrometer slices for cultivation in a standard tissue culture incubator, which can be studied for up to two days after slice preparation. Critical for the success of this approach is the use of a vibratome to slice each agarose-embedded embryo. This is followed by the cultivation of the slices upon Millicell culture membrane inserts placed upon a small volume of medium, resulting in an interface culture technique. One litter with an average of 7 embryos routinely produces at least 14 slices (2-3 slices of the forelimb region per embryo), which varies slightly due to the age of the embryos as well as to the thickness of the slices. About 80% of the cultured slices show nerve outgrowth, which can be measured througout the culturing period2. Representative results using the tauGFP mouse line are demonstrated.

We present a method to prepare organotypic slices of mid-gestation mouse embryos for the cultivation and time-lapse imaging of peripheral nerve outgrowth.

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 ...

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 ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

배아 복부 Midbrain의 Organotypic 슬라이스 문화 : Dopaminergic의 연결을 개발 연구하는 시스템 체외에서</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Organotypic 슬라이스 문화의 Purkinje 세포 수지상의 형태학의 분석

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

해마의 Organotypic 조각의 쌍으로 전체 셀 녹음

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

에서 신경 혈관 리모델링의 분석 Entorhino - 해마 Organotypic 슬라이스 문화

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

낮은 밀도 차 해마 신경 세포 문화

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Organotypic Hippocampal 슬라이스 문화 연구 Neuroprotection 및 종양 세포의 침입을 모델로

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Optogenetic Entrainment of Hippocampal Theta Oscillations in Behaving Mice

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools to selectively manipulate brain rhythms. Here we describe an approach combining projection-specific optogenetics with extracellular electrophysiology for high-fidelity control of hippocampal theta oscillations (5-10 Hz) in behaving mice. The specificity of the optogenetic entrainment is achieved by targeting channelrhodopsin-2 (ChR2) to the GABAergic population of medial septal cells, crucially involved in the generation of hippocampal theta oscillations, and a local synchronized activation of a subset of inhibitory septal afferents in the hippocampus. The efficacy of the optogenetic rhythm control is verified by a simultaneous monitoring of the local field potential (LFP) across lamina of the CA1 area and/or of neuronal discharge. Using this readily implementable preparation we show efficacy of various optogenetic stimulation protocols for induction of theta oscillations and for the manipulation of their frequency and regularity. Finally, a combination of the theta rhythm control with projection-specific inhibition addresses the readout of particular aspects of the hippocampal synchronization by efferent regions.

We describe the use of optogenetics and electrophysiological recordings for selective manipulations of hippocampal theta oscillations (5-10 Hz) in behaving mice. The efficacy of the rhythm entrainment is monitored using local field potential. A combination of opto- and pharmacogenetic inhibition addresses the efferent readout of hippocampal synchronization.

Optogenetic 쥐 행동에 Hippocampal 세타 진동의 유입

Extensive data on relationships of neural network oscillations to behavior and organization of neuronal discharge across brain regions call for new tools ...