우리는 BAC GLT1 eGFP 마우스와 GLT1 항체를 제공하기 위해 박사 제프리 Rothstein 감사드립니다, 가치의 핵심 시설을 제공하는, 신경 과학 연구 Tufts 센터 (PI, 롭 잭슨 NIH P30 NS047243), 새로운 교수 채용 기금 (NIH P30 5P30NS069254-02 , Tufts 신경 과학학과 PI, 필 Hay​​don).

1. 마이크로 유체 문화 회의소의 조립 (MCP) <ol><li> MCP (그림 1)은 세포 4 종류 compartmented 문화 용으로 설계된 개방형 챔버 있습니다. 그것은 일반적으로 중앙 채널 (직경 3 μm)를 통해 연결된 2 개의 구획이 있습니다. 바닥이 유리로 요리 MCP 조립 문화와 이후의 이미징 분석을 준비하기위한 필요합니다. </li><li> 첫째, Polyornithine (시그마 - 알드리치, 1 MG / ML)와 코트 멸균 유리 바닥 요리 나트륨 테트라 보레이트 (시그마 - 알드리치, 10 밀리미터 산도 8.5)에 용해하고 37 ° C.에서 하룻밤 incubated되었습니다 </li><li> 다음 날, 코팅 유리 바닥 요리는 ddH 2 O로 세 번 씻어되었으며, 멸균 배기함에 넣고 건조. 유리 바닥 요리는 더 laminin (시그마 - 알드리치, 1 MG / ML)로 코팅 1 시간 동안 UV 빛 아래에서 건조했다. 코팅 요리 사용할 수 있습니다 또는 사용 할 때까지 -20 ° C에 저장할 수 있습니다. 이러한 코팅공동 문화에 도금 뉴런과 astrocytes에 필요한 재. </li><li> 문화 플랫폼을 조립하기 위해 마이크로 유체 문화 플랫폼은 방수 밀봉을 형성하기 위해 아래 쪽 중앙 연결 채널을 시청할 수있는 코팅 유리 바닥 요리의 상단에 배치했다. 일반 신경이나 astrocyte 문화 매체는 조립 MCP로 양쪽 (그림 1)에 (셀 도금 지역에서) 추가하고 37 ° C에서 2~4시간에 대한 incubated, MCP와 유리 사이에 누수가 없는지 확인하기 위해 한 바닥 요리. 우리는 누수가없는 보장하기 위해 도금 세포하기 전에 중간에서 테스트했습니다. 유리 바닥 요리에 MCP의 어셈블리가 신선하게 사용하기 전에 준비해야합니다. </li></ol> 2. 조립 MCP에 Neuronal Axons의 Neuronal 문화 유도의 작성 <ol><li> 대뇌 피질의 neuronal 세포 문화는 신선한 배아 14-16일 된 마우스 뇌에서 준비되었다. 뉴런 문화 매체는 neurobasal 매체 2% B27 neurobasa으로 구성되어 있습니다난 보충, 100x GlutaMAX의 1 %, 그리고 1퍼센트 페니실린 - streptomysin를 추가하여 글루타민이 음. 마우스 뇌의 해부 후, meninges는 해부 현미경으로 피질에서 제거되었습니다. 조직은 다음 트립신에 노출 면적을 증가시키기 위해 작은 조직 블록을 만들기 위해 면도날과 다진했다. 조직 블록은 37 ° C의 물을 욕조에 10 분 동안 (시그마 - 알드리치, 0.05 % 트립신) trypsinized, 그리고 불타는 광택 파스퇴르 피펫으로 분쇄하여 부드럽게 한 다음 dissociated했다. Dissociated 세포는 일반 neuronal 세포 현탁액를 수집하기 위해 70 μm의 스트레이너를 통해 필터링되었습니다. </li><li> 뉴런이 세포 보존 지역 (그림 1)에 첨부 할 수 있도록 뉴런은 (2-3 × 10 6 / ML, 150 μl) 조립 MCP의 오른쪽 (그림 1)에서 셀 도금 부분에 도금되었습니다. 신선하게 조리 된 뉴런은 regula에 5% 태아 소 혈청 (Hyclone)로 보완되어 신경 도금 매체를 도금했다R 뉴런 문화 매체입니다. 세포 보존 영역에 첨부 만 뉴런이 중앙 채널을 통해 조립 MCP의 다른 측면에 axons 성장을 할 수 있기 때문에 충분한 뉴런이 연결된 수 있도록하기 위해 셀 도금 지역의 뉴런의 판 고밀도 중요합니다 세포 보존 지역. neuronal axons의 높은 밀도의 대표 이미지가 그림 2A에 표시됩니다. </li><li> 다음 날에서 신경 도금 매체는 일반 뉴런 문화 매체로 대체되었습니다. 변경 매체는 조심스럽게 챔버의 세포 도금 지역에서 매체를 aspirating하여 수행하지만, 조립 MCP의 세포 보존 지역에서, 셀 보존 지역과 중앙 연결 채널에 기포를 방지하기 위해 인치되었습니다 기포가 심각한 MCP의 중심 채널로 축삭의 가지와 이후의 항목을 억제합니다. Glial 세포 파생 neurotrophic 요인 (GDNF, 10-20 NG / ML)도 다른 (왼쪽) 측면에 추가되었습니다조립 MCP (그림 2A)의 중앙 채널을 통해 neuronal 측면과 십자가에서 축삭의 가지 (5)의 유도를 촉진하기 위해 같은 날 챔버의. GDNF없이 자연 neurite의 파생물의 공정한 금액도 조립 MCP의 중심 채널을 통해 neuronal 측면과 십자가에서 관찰된다. 중앙 채널에 입력 Axons은 종종 2-3일 도금 후 관찰됩니다. axons는 중앙 채널에 입력되면, 그들은 보통 하루 내에 다른 쪽을 입력합니다. Axons은 다른 쪽을 입력 한 후 최소 1 주일 동안 정상적으로 그대로입니다. </li></ol> 3. Compartmentalized 공동 문화 시스템을 구축하기 위해 MCP에 대한 양식 Astrocytes의 추가 기본 astrocyte 문화는 P1-3 마우스 새끼 뇌에서 준비되었다. 뇌 분리 절차는 위에서 설명한 neuronal 세포 분리 절차와 비슷합니다. Astrocytes 먼저 사전에 한 10cm 요리로 도금 된Polyornithine (시그마 - 알드리치, 1 MG / ML)로 코팅. astrocyte 문화 매체는 (DMEM, 10 % 태아 소 혈청, 1 % 페니실린 - streptomysin) 파편을 제거하려면 다음 두 일 동안 매일 변경되었습니다. 그 후, 매체는 매 3 일마다 교체되었습니다. <ol><li> Astrocytes는 90 %의 합류가 7 일 후에 monolayer를 형성하고 있습니다. 합류 astrocytes는 trypsinized되었으며 axons가에 대한 입력하려고합니다 또는 왼쪽의 체결했을 때 다시 중지 astrocytes (1x10 6 / ML) 150 μl은 조립 MCP의 왼쪽의 셀 도금 지역으로 다시 도금했다 MCP 조립. 뉴런이 도금 한 이후 Astrocytes는 일반적으로 4-5일 도금했다. GDNF 먼저 MCP의 왼쪽에 astrocytes의 재 도금하기 전에 삭제되었습니다. 재 도금 astrocytes는 같은 GFAP의 immunostaining (그림 1)에 의해 도시 된 바와 같이, 셀 고정 영역에 첨부되었습니다. 챔버의 양쪽 사이에 매체의 최소한의 흐름 (형광 D에 의해 결정이전에 4,6를 그림과 같이 예)는 MCP에서 발견되었다. </li><li> Astrocytes는 90 %의 합류가 7 일 후에 monolayer를 형성하고 있습니다. 합류 astrocytes는 axons가에 대한 입력 또는 왼쪽에 입력 한 경우 조립 MCP의 왼쪽의 셀 도금 지역으로 다시 도금 된 trypsinized하고 다시 중지 astrocytes (1 × 10 6 / ML) 150 μl되었습니다 조립 MCP의 측면. 뉴런이 도금 한 이후 Astrocytes는 일반적으로 4-5일 도금했다. GDNF 먼저 MCP의 왼쪽에 astrocytes의 재 도금하기 전에 삭제되었습니다. 재 도금 astrocytes는 같은 GFAP의 immunostaining (그림 1)에 의해 도시 된 바와 같이, 셀 고정 영역에 첨부되었습니다. 이전에 4,6를 그림과 같이 챔버의 양쪽 사이에 매체의 최소한의 흐름 (형광 염료에 의해 ​​결정), MCP에서 발견되었다. </li><li> astrocytes의 재 도금 후, 조립 MCP의 왼쪽을 입력 axons 직접 astr과 상호 작용직접 axonal 연락처 또는 수용성 요소의 릴리스에 의해 ocytes. astroglial 플라즈마 막 glutamate의 전송 GLT1와 neuronal βIII-tubulin의 Immunostaining은 그림 2D-F에 도시 된 바와 같이, axons과 astrocytes 사이의 상호 작용을 시각화하기 위해 수행되었다. </li></ol>

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관심 없음 충돌이 선언 없습니다.

MCP 기반의 뉴런과 astrocytes 공동 문화 시스템은 axons는 중앙 채널을 통과 허용하고 astroglial 세포와 상호 작용에 의해 astroglia 신호 경로에 대한 자세한 뉴런의 절개를 할 수 있습니다. 이 공동 문화 시스템은 편리하게 기존의 신경 세포와 astrocyte 문화 절차를 설정할 수 있습니다. 우리는 또한 astrocytes의 축삭에 의존 GLT1 프로모터 활성화를 입증 eGFP 기반 기자를 고용하여 공동 문화 시스템의 실제 응?...

<table border="1"><tbody><tr><td> 태아 소 혈청 </td><td> Hyclone </td><td> SH30070.03 </td><td> 뉴런의 cutlure 매체에 신경을 도금 용 </td></tr><tr><td> 태아 소 혈청 </td><td> 시그마 - 알드리치 </td><td> F4135 </td><td> astrocyte 문화 매체에 대한 </td></tr><tr><td> Glial 파생 된 신경 요소 </td><td> R &amp; D 시스템 </td><td> 212-GD </td><td> 챔버의 신경 세포 측에 10-20 NG / ML을 적용 </td></tr><tr><td> Dulbecco 수정 독수리 중간 높은 포도당 </td><td> 시그마 - 알드리치 </td><td> 11,995 </td><td></td></tr><tr><td> 70mm 셀 스트레이너 </td><td> BD 팔콘 </td><td> 352350 </td><td></td></tr><tr><td> 멸균 유리 바닥 요리 </td><td> MatTek 공사 </td><td></td><td></td></tr><tr><td> 마이크로 유체 문화 플랫폼 </td><td> Xona Microfluidics LLC </td><td> SND150 </td><td></td></tr><tr><td> 문화 판 6 우물 </TD&gt; <td> Cellstar </td><td> 657 160 </td><td></td></tr><tr><td></td><td></td><td></td><td> 뉴런 문화 매체 <ul><li> Neurobasal 매체 </li><li> 2퍼센트 B27 Neurobasal 보충 </li><li> 1퍼센트 100x GlutaMAX을 추가로 약 2 밀리미터 glutamate </li><li> 1퍼센트 페니실린 - streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> 도금 세포에 대한 신경 세포의 배양액 <ul><li> Neurobasal 매체 </li><li> 2퍼센트 B27 Neurobasal 보충 </li><li> 1퍼센트 100x GlutaMAX을 추가로 약 2 밀리미터 glutamate </li><li> 1퍼센트 페니실린 - streptomysin </li><li> 5% 태아 소 혈청 SH30070.03 </li></ul></td></tr><tr><td></td><td></td><td></td><td> Astrocyte 문화 매체 <ul><li> Dulbecco 수정 EAgle은 중간 높은 포도당 </li><li> 10% 태아 소 혈청 F4135 </li><li> 1퍼센트 페니실린 - streptomysin </li></ul></td></tr><tr><td></td><td></td><td></td><td> 마이크로 유체 문화 플랫폼 기반의 neuronal 축삭과 glia 공동 문화 시스템에서 사용되는 표 1. 자료. </td></tr></tbody></table>

imaging analysis of neuron to glia interaction in microfluidic culture platform (mcp)-based neuronal axon and glia co-culture system

glia 상호 작용에 적절한 신경 세포는 중추 신경계 (CNS)의 생리적 기능에 중요합니다. 이 양방향 통신이 sophisticatedly 뉴런과 glia 1,2 사이에 특정 신호 경로에 의해 중재됩니다. 이러한 신호 경로의 식별 및 특성은 glia 상호 작용 형태의 CNS의 생리에 얼마나 신경 세포의 이해에 필수적입니다. 이전 뉴런과 glia 혼합 문화가 널리 뉴런과 glia 사이의 신호 경로를 테스트 및 특성화에 활용되었습니다. 우리가 생체 도구에서 이러한 준비 및 기타에서 배운 것을 그러나, 뉴런과 glia 사이의 상호 신호가 자주 뉴런 (즉, 축삭, dendrite, 또는 소마) 3 내의 특정 구획에서 오류가 발생 것을 제안했습니다. 이것은 중요한 neuronal 구획의 분리 할 수​​있는 새로운 문화 시스템을 개발 할 수 있으며 특별히 glia과 신경 사이의 상호 작용을 검사NAL axons / 수석. 또한, 기존의 혼합 문화 시스템은 가용성 요인과 뉴런과 glia 사이에 직접 멤브레인 연락처 신호를 구별 할 수 없습니다. 또한, 종래의 공동 문화 시스템의 뉴런과 glial 세포의 큰 수량은 하나의 축삭과 glial 세포 사이의 상호 작용을 관찰하는 데 필요한 해상도를 부족합니다. 본 연구에서는, 우리는 마이크로 유체 문화 플랫폼 (MCP)의 사용과 소설 축삭과 glia 공동 문화 시스템을 설명합니다. 이 공동 문화 시스템에서 뉴런과 glial 세포는 여러 중앙 채널을 통해 연결된 2 개의 별도의 방에서 배양되어 있습니다. 이 마이크로 유체 문화 플랫폼에서 만 neuronal 프로세스 (특히 axons) 중앙 채널을 통해 glial 측면을 입력 할 수 있습니다. 강력한 형광 단백질 라벨과 함께,이 시스템은, / axonal 수지상와 glial 상호 작용 사이의 경로를 신호를 직접 검사 할 수 있습니다neuronal 터미널에서 glia, glia로 인한 수용체 인신 매매 및 glia로 인한 축삭의 성장에의 축삭로 인한 전사 조절. 챔버의 좁은 직경은 크게 axons / 수석과 glial 표면 사이의 직접 막 단백질 상호 작용의 프로빙 촉진, glial 챔버에 신경 강화 매체의 흐름을 금지합니다.

astrocytes의 축삭 유도 GLT1 프로모터 활성화의 시간 경과 영상 분석 compartmented 신경 세포와 astrocyte 공동 문화 시스템은 neuronal 프로세스, 특히 axons은 선택적으로 astrocytes와 상호 작용 할 수 있습니다. 조립 MCP의 축삭과 astrocyte (또는 다른 glial 세포) 공동 문화의 성공적인 설립 후, 축삭-glia 상호 작용의 다른 유형은 다음과 같은 연구 될 수있다; astroglial 유전자 프로모터 활성화, astr...

Watch this Scientific Journal Video about Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System at JoVE.com

Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

이 연구는 소설 neuronal 축삭과 (천체) glia 공동 문화 플랫폼을 설정하는 절차를 설명합니다. 이 공동 문화 시스템에서 하나의 축삭 (및 단일 glial 세포) 사이의 직접 상호 작용 조작은 glial 신호에 대한 상호 신경 세포의 기계론의 분석을 수 있도록 가능한된다.

마이크로 유체 문화 플랫폼에 Glia 상호 작용 (MCP) 기반 Neuronal 축삭과 Glia 공동 문화 시스템에 신경의 영상 분석

Proper  neuron to glia interaction is critical to physiological function of the central  nervous system (CNS). This bidirectional communication is ...

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Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform (MCP)-based Neuronal Axon and Glia Co-culture System

14.9K 조회수.  Tufts University. 이 연구는 소설 neuronal 축삭과 (천체) glia 공동 문화 플랫폼을 설정하는 절차를 설명합니다. 이 공동 문화 시스템에서 하나의 축삭 (및 단일 glial 세포) 사이의 직접 상호 작용 조작은 glial 신호에 대한 상호 신경 세포의 기계론의 분석을 수 있도록 가능한된다.

비디오: Imaging Analysis of Neuron to Glia Interaction in Microfluidic Culture Platform MCP-based Neuronal Axon and Glia Co-culture System

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

연구

신경과학

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

미세 유체 공동 문화 장치를 사용하여 치아 세균 신경 분포를 개발 분석

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

결합 된 방법론을 활용하여 축삭 분기 및 신경 세포 형태 형성 동안 플라즈마 멤브레인 배달의 역할을 정의합니다

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly controlled formation and maintenance of complex neuronal networks are not completely understood thus far. The open questions concerning neurons in health and disease are diverse and reaching from understanding the basic development to investigating human related pathologies, e.g.,&#160;Alzheimer&#39;s disease and&#160;Schizophrenia. The most detailed analysis of neurons can be performed in vitro. However, neurons are demanding cells and need the additional support of astrocytes for their long-term survival. This cellular heterogeneity is in conflict with the aim to dissect the analysis of neurons and astrocytes. We present here a cell-culture assay that allows for the long-term cocultivation of pure primary neurons and astrocytes, which share the same chemically defined medium, while being physically separated. In this setup, the cultures survive for up to four weeks and the assay is suitable for a diversity of investigations concerning neuron-glia interaction.

The Indirect Neuron-astrocyte Coculture Assay: An In Vitro Set-up for the Detailed Investigation of Neuron-glia Interactions

This protocol describes the indirect neuron-astrocyte coculture for compartmentalized analysis of neuron-glia interactions.

간접 신경-성상 세포 공동 배양 분석 일 :<em&gt; 체외</em&gt; 신경 세포 - glia 상호 작용의 상세한 조사를위한 셋업

Proper neuronal development and function is the prerequisite of the developing and the adult brain. However, the mechanisms underlying the highly ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

레이저 유도 된 망막 변성 및 재생 Zebrafish 모델 뮐러 명과 셀 활성화

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

미세 유체 챔버 시스템을 사용하여 모터 뉴런 배양에서 세포기관의 축축한 수송

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague&#8211;Dawley rats. In this method, the neurons and glia were incubated in a 37 &#176;C, 5% CO2 incubator for 5&#8211;7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by &#946;-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

성인 쥐 요로 방광에서 1 차신경및 glia의 격리 그리고 문화

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

뇌 회로의 비 침습적 단절에 대한 표적 신경 부상

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...

Dissection and Isolation of Murine Glia from Multiple Central Nervous System Regions

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine neonates for the isolation of glial subpopulations. The purpose of the procedure is to dissociate microglia, oligodendrocyte progenitor cells (OPCs), and astrocytes from cortical, cerebellar, brainstem, and spinal cord tissue to facilitate further in vitro analysis. The CNS region isolation procedures allow for the determination of regional heterogeneity among glia in multiple cell culture systems. Rapid CNS region isolation is performed, followed by the mechanical removal of meninges to prevent meningeal cell contamination of glia. This protocol combines gentle tissue dissociation and plating on a specified matrix designed to preserve cell integrity and adherence. Isolating mixed glia from multiple CNS regions provides a comprehensive analysis of potentially heterogenous glia while maximizing the use of individual experimental animals. Additionally, following dissociation of regional tissue, mixed glia are further divided into multiple cell types including microglia, OPCs, and astrocytes for use in either single cell type, cell culture plate inserts, or co-culture systems. Overall, the demonstrated techniques provide a comprehensive protocol of broad applicability for careful dissection of four individual CNS regions from murine neonates and includes methods for the isolation of three individual glia cell types to examine regional heterogeneity in any number of in vitro cell culture systems or assays.

Here we present a protocol for in vitro isolation of multiple glial cell populations from a mouse CNS. This method allows for the segregation of regional microglia, oligodendrocyte precursor cells, and astrocytes to study the phenotypes of each in a variety of culture systems.

여러 중추 신경계 영역에서 쥐 아교 세포의 해부 및 분리

The methods presented here demonstrate laboratory procedures for the dissection of four different regions of the central nervous system (CNS) from murine ...

A Cell Culture Model for Studying the Role of Neuron-Glia Interactions in Ischemia

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent neuronal loss. Numerous evidence suggests that the interaction between glial and neuronal cells exert beneficial effects after an ischemic event. Therefore, to explore potential protective mechanisms, it is important to develop models that allow studying neuron-glia interactions in an ischemic environment. Herein we present a simple approach to isolate astrocytes and neurons from the rat embryonic cortex, and that by using specific culture media, allows the establishment of neuron- or astrocyte-enriched cultures or neuron-glia cultures with high yield and reproducibility.
To study the crosstalk between astrocytes and neurons, we propose an approach based on a co-culture system in which neurons cultured in coverslips are maintained in contact with a monolayer of astrocytes plated in multiwell plates. The two cultures are maintained apart by small paraffin spheres. This approach allows the independent manipulation and the application of specific treatments to each cell type, which represents an advantage in many studies.
To simulate what occurs during an ischemic stroke, the cultures are subjected to an oxygen and glucose deprivation protocol. This protocol represents a useful tool to study the role of neuron-glia interactions in ischemic stroke.

Here, we present a simple approach using specific culture media that allows the establishment of neuron- and astrocyte-enriched cultures, or neuron-glia cultures from the embryonic cortex, with high yield and reproducibility.

이혈증에서 신경 신경-Glia 상호 작용의 역할을 공부 하기 위한 세포 배양 모델

Ischemic stroke is a clinical condition characterized by hypoperfusion of brain tissue, leading to oxygen and glucose deprivation, and the consequent ...