우리는 미르의 사진이 영화와 M. Nicolescu의 초기 단계에 기여 K. Mikic을 인정합니다. C. 후버는 Universtitätsklinikum 튀빙겐의 행운 프로그램에 의한 Universtitätsklinikum 튀빙겐 IZKF, A. 앨윈 프렘 아난드의 친교에 의해 지원되었다.

1. 에서 비켜 Electroporation에 대한 요구 사항 <ol><li> 벡터 구조를 준비 : 미르의 센스와 안티센스 프라이머, Ambion의 지시 후 설계 소둔 및 ApaI로 결찰 / 효소 EcoRI는이 실험에서 pSilencer ™의 U6.1 벡터를 소화. 성공적으로 변환 후 생성 된 클론 중 하나가 성장하고,이 실험에서 pSilencer ™ 플라스미드 Quiagen 미디 준비 키트를 사용하여 알칼리 용해 방법으로 분리 하였다. </li><li> 전극 : 전극을 구입하실 수 있습니다 또는 쉽게 13를 만들 수. 우리는 백금 와이어의 자체 제작 전극 (Φ = 직경 0.2, 0.25, 0.5 mm)를 사용하거나 경우에 따라 더 작은 텅스텐 와이어 전극 (Φ = 0.1 mm) 8 우리가 electroporate하려는 지역에 따라 다릅니다. 조직의 뜨거운을 방지하기 위해, 우리는 조직 전극 얇은에 가까운 값으로 사용하십시오. 중뇌의 넓은 일렉트로 위해, 우리는 황동 / 스테인레스 스틸 관에 납땜 0.5 mm 백금 와이어를 사용샤프트 3-5 ㎜의 거리 (도 1)와 전극 홀더에 고정된다. 특정 영역을 electroporate하기 위해, 우리는 0.25 mm 백금 와이어 양극 및 0.2 mm 백금 와이어 또는 1 mm 이하의 대략적인 거리와 음극 0.1 mm의 텅스텐을 사용합니다. </li><li> 솔루션 : 다음과 같은 솔루션을 준비합니다 : <ol><li> 멸균 치킨 벨소리, pH가 7.5 (9.0 g 염화나트륨, 0.42 g의 KCl, 0.24 g의 염화칼슘, 1 L의 증류수에 용해). </li><li> PBS (8g의 NaCl, 0.2 g의 KCl, 0.24 g의 KH 2 PO 4, 1.44 g의 2 나 HPO 4, 1리터 증류수 H 2 O에 용해). </li><li> 빠른 그린 : 원액 10 ㎎ / ㎖ 증류수 H 2 O </li><li> 천천히 건조 잉크 (이하 셸락 및 배아에 대한, 따라서 적은 중독). 주 : 칸토-더 유일한 및 애들러 (14)의 설명에 따라 광섬유 램프에 연결된 청색 이색 성 필터는 콘트라스트를 향상시키고, 잉크를 주입 할 필요성을 제거 할 수있다. </li></ol></li></ol><img src="/files/ftp_upload/50024/50024fig1.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/50024/50024fig1highres.jpg"/> 그림 1. 전극과 전극 홀더의 개략도. (A, B, C) ​​백금 와이어는 스테인레스 스틸 관형 샤프트의 일측에 납땜 하였다. 구멍 따라서 그 핀 플러그 (A) 빨강과 파랑 색의 전선이 핀 플러그에 연결된 안으로 들어갈 수있는 관 샤프트의 다른 측면 드릴되었다. 반대쪽 끝에서 와이어가 electroporator의 전기 소켓 작업 바나나 커넥터에 장착되었다. (A, C) 백금 와이어는 90 °의 천사 굽혀했다. 손톱 광택의 층은 굴곡 위의 샤프트입니다. 2. 직접에서 비켜 Electroporation에 전 준비 <ol><li> 다음과 같은 자료를 준비 <ol><li> 닭 벨소리 1시 6분와 잉크를 희석, Gibc에서 항생제 항진균 솔루션 (100 배를 추가) 1:100 오. </li><li> 벡터 솔루션을 준비 : H 2 O의 특정 DNA 1 ~ 2 ㎍ / ㎕의 빠른 그린 (1:20)을 수행 보충. </li><li> 70 % 에탄올로 집게, 가위, 텅스텐 바늘 전극을 소독. </li><li> 붕규산 유리 모세관 (: 100mm X 0.9 mm 외경 길이)에서 핵산 주입 마이크로 피펫을 잡아 당깁니다. 우리는 표 1에 나타낸 설정을 간단한 풀러 PUL-1 (WPI, 베를린)를 사용한다. </li><li> 표 2에 나타낸 바와 같이 electroporator의 매개 변수를 설정. </li></ol></li></ol><table border="1" ><tbody><tr><td fo:width="1in"> 매개 변수 </td><td fo:width="1in"> 값 </td></tr><tr><td> 열 </td><td> (350) </td></tr><tr><td> 당기세요 </td><td> (100) </td></tr><tr><td> 속도 </tD&gt; <td> (50) </td></tr><tr><td> 지연 </td><td> (200) </td></tr></tbody></table> 표 1. PUL-1에 대한 설정 <table border="1"><tbody><tr><td fo:width="1.5in"> 매개 변수 </td><td fo:width="1.5in"> 값 </td></tr><tr><td> 주파수 </td><td> 50 Hz에서 </td></tr><tr><td> 지연 </td><td> 20-50 밀리 초 * </td></tr><tr><td> 폭 </td><td> 20 밀리 초 </td></tr><tr><td> 전압 </td><td> 8-25 V의 * </td></tr><tr><td> 펄스 </td><td> 3 - 5 회 </td></tr><tr><td colspan="2"> * 전극의 직경과의 거리에 따라. 하십시오매개 변수 중 하나를 변경할 때 고려한다 그것을. </td></tr></tbody></table> 표 2. 펄스 자극에 대한 설정 <ol start="2"><li> 일렉트로에 대해 병아리 배아를 준비 <ol><li> 햄버거 &amp; 해밀턴 (HH) 사이의 배아를 얻기 위해 39 시간 동안 65 %의 습도와 37 ° C에서 자신의 긴 측면에 수정 된 닭고기 달걀을 품어 15 ~ 11 스테이지. 배아는 난자의 최상위 부분에 정착, 그래서 당신은 연필 선으로 표시해야합니다. </li><li> 70 % 에탄올로 계란을 소독. </li><li> 공기 구멍이 있어야 할 곳에, 더 넓은 측면에서 계란을 관통. </li><li> 흰자 1 ~ 2 ML를 제거합니다. 이 달걀 껍질에서 배아를 분리합니다. </li><li> 첼로 테이프 배아에 떨어지는 포탄의 파편을 피하기 위해 달걀 껍질. 연필 선 주위에 달걀 껍질에 작은 창을 잘라 가위를 사용합니다. </li><li> 잉크의 비트를 주입배아를 시각화하는 지역 opaca 아래. </li><li> 핵산 주입 및 전기 충격에 대한 배아의 더 나은 접근성을 달성하기 위해 날카롭게 텅스텐 바늘로 난황 막에 상처를 확인합니다. </li></ol></li></ol> 3. 일렉트로 및 창업 보육 <ol><li> 배아에 따뜻한 링거 용액 몇 방울을 추가합니다. 이것은, 배아를 낮출 과열을 방지, 조직에 전극과 나와 두 전극 사이의 전류를위한 매체를 제공하는 것이다. </li><li> 중뇌 수준에서 신경 튜브의 루멘에 벡터 솔루션을 주입한다. 당신의 DNA 발현 벡터가 더 리포터 유전자 (예 : EGFP)를 포함하지 않으면 DNA 용액에 약간 낮은 농축 리포터 유전자 벡터 (1 ㎍ / μL 미르 0.9 ㎍ / ㎕의 리포터 유전자 벡터와 벡터를 표현)를 추가한다. 이것은 당신이 형질 세포 나중에 8을 인식 할 수 있도록합니다. 우리는 이리저리 소위 pCAX 벡터, 친절한 선물을 사용M C. 크럴 13. </li><li> 중뇌의 다양한 형질의 경우, 중뇌의 높이에서 왼쪽 전극과 태아의 권리를 놓습니다. 전극 간 거리는 3 ~ 5 ㎜로한다. 다른 설정은 15V, 20 밀리 초 펄스와 HH 10 50 밀리 초 지연이다. 약간의 중뇌 dorso-배쪽 (DV) 축을 따라 두 전극의 위치를​​ 변경하는 것은 셀의보다 넓은 형질 DV 축 형질을 보장​​한다. </li><li> 복부 형질의 경우, 노른자 막에서 머리를 올리고 복부 중뇌 아래의 양극을 배치하는 몇 가지 벨소리를 추가합니다. 머리는 신경 튜브의 뜨거운을 피하기 위해 노른자와 배아를 분리하는 막 아래에 전극을 밀어 상승하지 않습니다. 음극은 중뇌 dorso-측면으로 반대 양극 위에 배치해야합니다. 신경 튜브를 만지지 마십시오. 우리는 심장 개발에 어려움을 피하기 위해 중뇌 왼편 복부 절반 형질. 전극 사이의 거리는약 1-0.5 mm, 나머지 설정하는 10V, 20 밀리 폭 50 밀리 초 지연입니다. </li><li> 그것을 아래로 냉각 공기 방울을 제거하는 배아의 위에 생리 식염수의 또 다른 드롭을 적용합니다. </li><li> 테이프로 계란을 밀봉하고 적어도 4 시간 동안 배양, 벡터 표현에 필요한 최소한의 시간. </li></ol> 4. 닭 배아의 고정 <ol><li> 알에서 배아를 제거합니다. </li><li> 실체 현미경 및 형광 실체 현미경으로 형질 감염의 정도를 평가 면도 배아. </li><li> 4 ℃에서 4 % PFA 밤을 통해 배아를 수정 형질 지역의 변화를 분석하기 위해 (현장 하이브리드, 면역 조직 화학 염색에) 구획 및 / 또는 염색을 진행합니다. </li></ol>

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우리는 공개 아무것도 없어.

이 비디오는 병아리 중뇌의 특정 영역의 neuroepithelial 세포에 플라스미드를 형질 할 수있는 효과적인 방법을 보여줍니다. 낮은 전압의 직사각형 전기 펄스는 6,16을 OVO에 병아리 신경 튜브의 세포에 DNA를 도입 할 수 있습니다. 그러나, DNA의 타겟팅 정확성은 종종 비교적 큰 전극 (Φ = 0.5 mm)을 통해 상승 넓은 전기장에 의해 방해된다. 우리는 모모 세 등. 8의 지침에 따라 ?...

유전자 발현에 대한 추가 플레이어와 같은 작은 비 코딩 RNA를 인식 게놈 프로그래밍 / 유전자 조절에 새로운 복잡성을 시작했다. 비 코딩 RNA를 다른 종은 작은 비 코딩 RNA를 1-4 포함하는 신경 세포의 기능적인 중요성이있다. 예를 들어, 마이크로 RNA (miRNA의 미르 나)는 두뇌 개발 5 별개의 변화 발현 프로파일을 보여줍니다. 개발하는 동안 유전자 발현과 침묵의 시간 및 공간 제어를위한 유일한 기회는 병아리 태아의 비켜 일렉트로에 제공 대상. 이 동영상은 일렉트로 6-10 OVO에서 사용하는 병아리 중뇌의 특정 영역에 미르의 이소성 표현을 수행하는 여러 단계를 보여줍니다. 세포에있는이 작은 비 코딩 RNA를의 긴 지속 효과를 보장하기 위해, 미르의 DNA 시퀀스는 모노 - 또는 바이 cistronic 벡터에 클로닝 하였다. 비켜 일렉트로의 경우, 미르는 V를 포함엑터는 달걀 껍질에 작은 창을 한 후 배아를 노출하여 중뇌의 신경 튜브에 주입된다. 중뇌 작은 플러스 (양극)과 음극 (캐소드)의 특정 영역을 형질 백금 전극을 특정 위치에 배치됩니다. 복측 중뇌 형질 들어, 애​​노드는 좌 복부 중뇌 및 전류를인가하기 전에 중뇌의 우측 절반 상기 캐소드 밑에 배치된다. 달걀 껍질에 구멍을 테이프로 닫히고 배아는 한 어떤 분석을 위해 필요에 따라 배양된다. 이 방법은 원래 무라 마츠 등. (6)에 의해 설명 된 특정 지역의 형질 전환을위한 모모 세 등. 8로 향상되었다. <img src="/files/ftp_upload/50024/50024schematic1.jpg" fo:content-width-="6in" fo:src="/files/ftp_upload/50024/50024schematic1highres.jpg"/> 도식 개요. <ol><li> 계란의 배아는 일에 작은 창을 절단하여 노출전자 껍질. </li><li> 용해 벡터 (들)는 마이크로 모세관을 사용하여 중뇌 주입된다. </li><li> 두 개의 전극 - 병렬 또는 배아에서와 위에 배치는 - 펄스 전기장을 생성합니다. </li><li> 전기장은 일시적으로 양극 (11, 12)에 매력을 부정 청구 DNA (또는 RNA)에 의해 세포에 진입을 용이하게 세포막에 구멍을 만듭니다. </li></ol>

<table border="1">
<tbody>
 <tr>
 <td>Name </td>
 <td>Company</td>
 <td></td>
 <td>Model</td>
 </tr>
 <tr>
 <td>Borosillicate glass capillaries </td>
 <td>Hartenstein</td>
 <td></td>
 <td>Model: 0.9 mm</td>
 </tr>
 <tr>
 <td>Microcapillary puller</td>
 <td>WPI, Berlin</td>
 <td></td>
 <td>Model: Pul1-E</td>
 </tr>
 <tr>
 <td>Electroporator</td>
 <td>Intracel</td>
 <td></td>
 <td>Model: TSSIC</td>
 </tr>
 <tr>
 <td>Stereomicroscope - fluorescence </td>
 <td>LEICA</td>
 <td></td>
 <td>Model: MZFLIII</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Stemi</td>
 </tr>
 <tr>
 <td>Camera and software</td>
 <td>Zeiss</td>
 <td></td>
 <td>Model: Axiocam MRc/ Axiovision Re. 4.8</td>
 </tr>
 </tbody>
</table>

in ovo expression of microrna in ventral chick midbrain

비 코딩 RNA를 유전자 발현을 조절하는데 추가 플레이어. 특정 지역의 OVO 일렉트로의 대상은 자궁외 마이크로 RNA 발현의 공간 및 시간 제어를위한 독특한 도구를 제공합니다. 그러나, 복부 중뇌와 같은 복부 뇌 구조는 모든 조작에 도달 오히려 어렵습니다. 여기, 우리는 얇은 백금 전극을 사용하여 복부 중뇌에 miRNA의를 electroporate하는 효율적인 방법을 보여줍니다. 이 방법은 중뇌 및 생체 연구를위한 유용한 도구의 특정 영역을 감염 될 수있는 신뢰할 수있는 방법을 제공합니다.

미르로 형질 중뇌 영역의 범위는 미르 발현 벡터 (그림 2)와 함께 주입 기자 벡터에서 GFP의 표현을 통해 볼 수 있습니다. 0.5 mm의 직경을 갖는 백금 전극을 사용하고 중뇌 후뇌, 때때로 간뇌 (도 2A와 B)를 포함하여 DV-및 AP-축을 따라 넓은 영역의 형질 전환에 일반적 중뇌 리드 AP 축에 평행하게 배치. 넓은 전계의 전극 결과의 크기는 뇌 영역 따라서 형질 넓은 영역에 적용. </p...

Watch this Scientific Journal Video about In ovo Expression of MicroRNA in Ventral Chick Midbrain at JoVE.com

In ovo Expression of MicroRNA in Ventral Chick Midbrain

이소성 표현은 두뇌 발달의 마이크로 RNA의 역할을 명료하게 한 기술이다. 그러나, 비켜 일렉트로에 사용하여 특정 영역을 대상으로하는 것은 도전이다. 여기, 우리는 효율적으로 선택적으로 electroporate 복부와 등쪽 중뇌 영역에 표시됩니다.

복부 병아리 중뇌에있는 마이크로 RNA의 오보, 식

Non-coding RNAs are additional players in regulating gene expression. Targeted in ovo electroporation of specific areas provides a unique tool for spatial ...

in-ovo-expression-of-microrna-in-ventral-chick-midbrain

Research

JoVE Journal

Neuroscience

In ovo Expression of MicroRNA in Ventral Chick Midbrain

10.3K 조회수.  University of Tübingen.  이소성 표현은 두뇌 발달의 마이크로 RNA의 역할을 명료하게 한 기술이다. 그러나, 비켜 일렉트로에 사용하여 특정 영역을 대상으로하는 것은 도전이다. 여기, 우리는 효율적으로 선택적으로 electroporate 복부와 등쪽 중뇌 영역에 표시됩니다. 

비디오: In ovo Expression of MicroRNA in Ventral Chick Midbrain

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 ...

연구

신경과학

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

개발 Neuroepithelium의 세포 문제의 고해상도 라이브 영상

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain dopaminergic neurons is implicated in Parkinson's disease, Schizophrenia, depression, and dementia1-5. Thus, studying the regulation of midbrain dopaminergic neuron differentiation could not only provide important insight into mechanisms regulating midbrain development and neural progenitor fate specification, but also help develop new therapeutic strategies for treating a variety of human neurological disorders.
Dopaminergic neurons differentiate from neural progenitors lining the ventricular zone of embryonic ventral midbrain. The development of neural progenitors is controlled by gene expression programs6,7. Here we report techniques utilizing electroporation to express genes specifically in the midbrain of Hamburger Hamilton (HH) stage 11 (thirteen somites, 42 hours) chick embryos8,9. The external development of chick embryos allows for convenient experimental manipulations at specific embryonic stages, with the effects determined at later developmental time points10-13. Chick embryonic neural tubes earlier than HH stage 13 (nineteen somites, 48 hours) consist of multipotent neural progenitors that are capable of differentiating into distinct cell types of the nervous system. The pCAG vector, which contains both a CMV promoter and a chick &beta;-actin enhancer, allows for robust expression of Flag or other epitope-tagged constructs in embryonic chick neural tubes14. In this report, we emphasize special measures to achieve regionally restricted gene expression in embryonic midbrain dopaminergic neuron progenitors, including how to inject DNA constructs specifically into the embryonic midbrain region and how to pinpoint electroporation with small custom-made electrodes. Analyzing chick midbrain at later stages provides an excellent in vivo system for plasmid vector-mediated gain-of-function and loss-of-function studies of midbrain development. Modification of the experimental system may extend the assay to other parts of the nervous system for performing fate mapping analysis and for investigating the regulation of gene expression.

In ovo Electroporation in Chick Midbrain for Studying Gene Function in Dopaminergic Neuron Development

To assess the function and the regulation of genes during the development of midbrain dopaminergic neurons, we describe a method that involves in ovo electroporation of plasmid DNA constructs into embryonic chick ventral midbrain dopaminergic neuron progenitors. This technique can be used to achieve efficient expression of genes of interest to study different aspects of midbrain development and dopaminergic neuron differentiation.

Dopaminergic 신경 세포 개발에서 유전자 기능을 유학 칙의 Midbrain에서 비켜 Electroporation에

Dopaminergic neurons located in the ventral midbrain control movement, emotional behavior, and reward mechanisms1-3. The dysfunction of ventral midbrain ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

Substantia Nigra 및 복부 Tegmental 지역의 도파민 규정의 종합적인 프로파일

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

급성 Hippocampal 슬라이스에 Synaptically-evoked Astroglial와 Neuronal 응답의 듀얼 Electrophysiological 레코딩

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

electroporation하여 병아리 배아 척수에서 차동 유전자 발현의 RNA-SEQ 분석

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

성인 마우스의 중간 뇌 도파민 뉴런의 수의 환경 변조

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

RNA 시퀀싱 및 TH-프로모터 중심 eGFP는 표현을 사용하여 중간 뇌 문화에서 생활 도파민 뉴런의 신뢰성 확인

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

An In Ovo Model for Testing Insulin-mimetic Compounds

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and &#946;-cell failure. Various strategies, including the injection of insulin or the usage of insulin-sensitizing drugs, were pursued to treat T2DM or at least reduce the symptoms. In addition, the application of herbal compounds has attracted increasing attention. Thus, it is necessary to find efficient test systems to identify and characterize insulin-mimetic compounds. Here we developed a modified chick embryo model, which enables testing of synthetic compounds and herbal extracts with insulin-mimetic properties. Using a fluorescence microscopy-based primary screen, which quantifies the translocation of Glucose transporter 4 (Glut4) to the plasma membrane, we were able to identify compounds, mainly herbal extracts, which lead to an increase of intracellular glucose concentrations in adipocytes. However, the efficacy of these substances requires further verification in a living organism. Thus, we used an in-ovo approach to identify their blood glucose-reducing properties. The approval by an ethics committee is not needed since the use of chicken embryos during the first two-thirds of embryonic development is not considered an animal experiment. Here, the application of this model is described in detail.

An In Ovo Model for Testing Insulin-mimetic Compounds

In this study, we describe an in-ovo system as a promising tool to test insulin-mimetic compounds in a living organism. The main advantages include adequate throughput rates and acceptable costs, which enable the identification of phytochemicals with insulin-mimetic properties.

Elevated blood glucose levels in type 2 diabetes mellitus (T2DM), a complex and multifactorial metabolic disease, are caused by insulin resistance and ...

Combined In Vivo Anatomical and Functional Tracing of Ventral Tegmental Area Glutamate Terminals in the Hippocampus

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a premise for determining the role of neuron types within a neural circuit, and their significance in information encoding relative to learning. Likewise, the method can be used to test the physiological significance of two or more connected brain regions in awake and anesthetized animals. The current study demonstrates how VTA glutamate neurons modulate the firing rate of putative pyramidal neurons in the CA1 (hippocampus) of anesthetized mice. This protocol employs adeno-associated virus (AAV)-dependent labeling of VTA glutamate neurons for the tracing of VTA presynaptic glutamate terminals in the layers of the hippocampus. Expression of light-controlled opsin (channelrhodopsin; hChR2) and fluorescence protein (eYFP) harbored by the AAV vector permitted anterograde tracing of VTA glutamate terminals, and photostimulation of VTA glutamate neuron cell bodies (in the VTA). High-impedance acute silicon electrodes were positioned in the CA1 to detect multi-unit and single-unit responses to VTA photostimulation in vivo. The results of this study demonstrate the&#160;layer-dependent distribution of presynaptic VTA glutamate terminals in the hippocampus (CA1, CA3, and DG). Also, the photostimulation of VTA glutamate neurons increased the firing and burst rate of putative CA1 pyramidal units in vivo.

The current protocol demonstrates a simple method for tracing of ventral tegmental area (VTA) glutamate projections to the hippocampus. Photostimulation of VTA glutamate neurons was combined with CA1 recording to demonstrate how VTA glutamate terminals modulate CA1 putative pyramidal firing rate in vivo.

해마의 생체 내 해부학 및 기능적 추적

Optogenetic modulation of neuron sub-populations in the brain has allowed researchers to dissect neural circuits in vivo and ex vivo. This provides a ...