Name: in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity
Uploaded: 2017-02-15T13:00:00.000+00:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

우리는 그들의 우수한 기술 지원 및 편집을위한 LA 바이스에 R. 구티에레스와 A. 모랄레스에 감사하고 있습니다. CGB는 스페인 Ministerio 드 Ciencia 전자 Innovación (MICINN), FPI-BES-2012-056011에 의해 투자된다. 이 작품은 M. Navarrete은에 BBVA 재단과 SAF2014-58598-JIN (MINECO)에서 연구비와 라몬 Areces 재단과 보조금 SAF2014-52119-R과 (MINECO에서) BFU2014-55738 - REDT에에서 보조금에 의해 투자되었다 M. 니에 토.

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저자는 관심의 충돌을 선언합니다.

이 프로토콜은 연결과 흥분을 분석하기 위해 C75BL / 6 마우스의 체성 감각 피질의 뉴런 레이블을하는 방법에 대해 자세히 설명합니다. 기존의 방법과 관련하여, 이러한 신경 세포 축삭 당 분기의 수, 정확한 지형 및 해부학 적 위치로 연결성 판별 측면을 가시화. 전극의 위치를 변경함으로써, 예에 cingulate 피질과 같은 다른 신경 세포 집단을 대상 (전극 뇌와 같은 각도를 유지하지만, 자극의 방향 변...

수지상 및 축삭 구조의 개발은 대뇌 피질에서 비롯한 신경계의 조절 회로의 중요한면이다. 또한 다양한 신경 세포 개체군의 선택 배선 동안 중요한 역할을한다. 최근 보고서 다수 연결 이외에 뉴런의 분자 다양성 소성 높은 특정 모드의 획득에 의해 반사되고, 그 보였다. 그러나, 개발 중에 별개 아형 신경의 흥분성 연결성 결정 메커니즘뿐만 아니라 배위 학위 여전히 잘못 2 1 이해된다. 생체 loss- 및 기능 획득하는 발현 유전자의 특정 레벨과 상기 회로의 개발에 대한 영향 사이의 관계에 대한 연구를 허용 분석한다. 자궁의 전기에서 (IUE)는 광범위하게 연구하는 데 사용하는 기술입니다특정 신경 인구에 대한 관심의 유전자의 기능과는 연결의 전체 패턴을 연구합니다. 그러나, 살아있는 생쥐의 대뇌 피질 층의 축삭과 수상 돌기의 형태 학적 특성을 결정하기 위해서는 띄엄 띄엄 뉴런 레이블을하는 것이 필수적이다. IUE와 결합 Cre 호텔 재조합 시스템은 식별 된 피질 박편의 개별 셀에 의해 방사 돌기를 해결하기 위해 충분히 낮은 밀도 뉴런 저밀도 인구를 표시하는데 사용될 수있다. 이 방법은 전기 천공 뇌 (도 1)의 적절한 수의 정량적 분석 후 데이터를 획득 피질 뉴런 당 충분한 수의 라벨. 이 원고는 연결의 미세 분석을위한 방법을 제시한다. 또한 별도의 실험에서, 분석과 유사한 전략을 제공, 녹색 형광 단백질 (GFP)에 전류 클램프 기록을 수행함으로써 신경 세포의 전기적 특성은 급성 대뇌 피질의 조각에서 세포 -electroporated. 이 프로tocols는 손실 함수의 게인이 IUE 동안 추가 플라스미드 도입 된 뉴런과 같은 다목적 및 WT 형질 전환 동물의 신경 세포의 흥분성 연결성 연구에 적용 할 수 있으며. 이 프로토콜은 배아 일 (E) 15.5에서 마우스의 전기를 설명하지만,이 기술은 E9.5 3 및 출생 후의 일 (P)이 4 사이의 나이에 수행 할 수 있습니다. 초기 단계에서 전기는 신경 세포와 시상의 전구체와 피질의 깊은 층, 이후 단계 전기 마크보다 표면 층 (예를 들어, E15.5 IUE 목표 층 II-III 뉴런)를 대상으로하지만. 요약하면, 단일 셀 형태 학적 분석과 함께 전기 생리학 IUE의 조합 신경계 뉴런의 엄청난 구조적 및 기능적 다양성의 기초가되는 분자 메카니즘을 해명하는 유용한 도구이다.

<table><tbody><tr><td>pCAG-Cre</td><td>Addgene</td><td>13775</td><td></td></tr><tr><td>pCALNL-GFP</td><td>Addgene</td><td>13770</td><td></td></tr><tr><td>pCAG-DsRed2</td><td>Addgene</td><td>15777</td><td></td></tr><tr><td>pCAG-GFP</td><td>Addgene</td><td>11150</td><td></td></tr><tr><td>Fast Green</td><td>Carl Roth</td><td>301.1</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td></td></tr><tr><td>Carprofen (Rimadyl)</td><td>Pfizer GmbH</td><td>1615 ESP</td><td></td></tr><tr><td>Isoflurane (IsoFlo)</td><td>Abbott (Esteve)</td><td>1385 ESP</td><td></td></tr><tr><td>Ketamine (Imalgene)</td><td>Merial</td><td>2528-ESP</td><td></td></tr><tr><td>Xylazine (Xilagesic)</td><td>Calier</td><td>0682-ESP</td><td></td></tr><tr><td>Povidone Iodine</td><td>Meda</td><td>694109.6</td><td></td></tr><tr><td>Eye Ointment (Lipolac)</td><td>Angelini</td><td>65.277</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS)</td><td>Gibco by Life Technologies</td><td>24020-091</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma -Aldrich</td><td>P4333</td><td></td></tr><tr><td>Scalpel Handle # 3 - 12 cm</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Scalpel Blades # 10</td><td>Fine Science Tools</td><td>10010-00</td><td></td></tr><tr><td>Adson Forceps-Serrated - Straight 12 cm</td><td>Fine Science Tools</td><td>1106-12</td><td></td></tr><tr><td>Hardened Fine Scissors - Straight 11 cm</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Scissors Mezenbaum-Nelson Curved L=14.5 cm</td><td>Teleflex</td><td>PO143281</td><td></td></tr><tr><td>Thin curved tips - Style 7 Dumoxel</td><td>Dumont</td><td>0303-7-PO</td><td></td></tr><tr><td>Dumont #5 Forceps-Inox</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>Mathieu Needle Holder - Serrated</td><td>Fine Science Tools</td><td>12010-14</td><td></td></tr><tr><td>AutoClip Applier</td><td>Braintree scientific, Inc</td><td>ACS&amp;nbsp;APL</td><td></td></tr><tr><td>9 mm AutoClips</td><td>MikRon Precision, Inc.</td><td>205016</td><td></td></tr><tr><td>Sutures - Polysorb 6-0</td><td>Covidien</td><td>UL-101</td><td></td></tr><tr><td>Electric Razor&amp;nbsp;</td><td>Panasonic</td><td>ER 240</td><td></td></tr><tr><td>Borosilicate glass capillaries (100 mm, 1.0/0.58 Outer/Inner diameter)</td><td>World Precision Instrument Inc.</td><td>1B100F-4</td><td></td></tr><tr><td>Aspirator tube assemblies for calibrated microcapillary pipettes</td><td>Sigma -Aldrich</td><td>A5177-5EA</td><td></td></tr><tr><td>Gauze (Aposan)</td><td>Laboratorios Indas, S.A.U.</td><td>C.N. 482232.8</td><td></td></tr><tr><td>Cotton Swabs (Star Cott)</td><td>Albasa</td><td>-</td><td></td></tr><tr><td>Needle 25 G (BD Microlance 3)</td><td>Becton, Dickinson and Company</td><td>300600</td><td></td></tr><tr><td>Sucrose</td><td>Sigma -Aldrich</td><td>S0389</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma -Aldrich</td><td>158127</td><td></td></tr><tr><td>OCT Compound</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>Tissue Culture Dish 100 x 20 mm</td><td>Falcon</td><td>353003</td><td></td></tr><tr><td>GFP Tag Polyclonal Antibody</td><td>Thermo Fisher Scientific</td><td>A-11122</td><td></td></tr><tr><td>Secondary Antibody, Alexa Fluor 488 conjugate</td><td>Thermo Fisher Scientific</td><td>A-11008</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542&amp;nbsp;</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>10270106&amp;nbsp;</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-500ML</td><td></td></tr><tr><td>Electroporator ECM 830&amp;nbsp;</td><td>BTX Harvard Apparatus</td><td>45-0002</td><td></td></tr><tr><td>Platinum electrodes 650P 7 mm</td><td>Nepagene</td><td>CUY650P7</td><td></td></tr><tr><td>Microscope&amp;nbsp;for Fluorescent Imaging - MZ10F</td><td>Leica</td><td>-</td><td></td></tr><tr><td>VIP 3000 Isofluorane Vaporizer</td><td>Matrx</td><td>-</td><td></td></tr><tr><td>TCS-SP5 Laser Scanning System</td><td>Leica</td><td>-</td><td></td></tr><tr><td>Axiovert 200 Microscope</td><td>Zeiss</td><td>-</td><td></td></tr><tr><td>Cryostat - CM 1950</td><td>Leica</td><td>-</td><td></td></tr><tr><td>P-97 Micropette Puller</td><td>Sutter Instrument Company</td><td>P-97</td><td></td></tr><tr><td>Patch clamp analysis softwarw (p-Clamp Clampfit 10.3)</td><td>Molecular Devices</td><td>-</td><td></td></tr><tr><td>Acquisition software (MultiClamp 700B Amplifier)</td><td>Molecular Devices</td><td>DD1440A</td><td></td></tr><tr><td>Motorized Micromanipulator + Rotating Base&amp;nbsp;</td><td>Sutter Instrument</td><td>MP-225</td><td></td></tr><tr><td>Air Table</td><td>Newport</td><td>-</td><td></td></tr><tr><td>Miniature Peristaltic Pumps</td><td>WPI</td><td>-</td><td></td></tr></tbody></table>

in utero electroporation approaches to study the excitability of neuronal subpopulations and single-cell connectivity

신경계의 연결은 별개 형태의 엄청난 범위로 구성된다. 이러한 신경 세포의 소집단 다른 기능, 자신의 고유 한 수지상 형태학, 축삭 연결의 특정 패턴과 선택적 발사 응답 중 특징으로한다. 개발하는 동안 차별화의 이러한 측면을 담당하는 분자 및 세포 메커니즘은 여전히 ​​저조한 이해된다. 여기, 우리는 라벨에 대한 결합 된 프로토콜을 설명하고 대뇌 피질의 신경 세포의 구조적 연결과 흥분을 특성화. 에서 자궁 전기 (IUE) 프로토콜의 수정은 뉴런의 스파 스 인구의 표시를 할 수 있습니다. 이것은, 차례로, 개별 돌기 신경 축색 돌기 층류 위치의 정확한 특성 및 형태 계측 학적 분석 축삭의 식별 및 추적 할 수있다. IUE 또한 흥분성의 변화를 조사하기 위하여 사용될 수있다야생형 (WT) 또는 전기 천공 두뇌의 급성 조각에서 전체 셀 녹음과 결합하여 유전자 변형 뉴런. 이 두 기술은 구조적 및 기능적 연결성의 결합에 대한 이해 및 개발 중에 신경 다이버 시티를 제어하는 ​​분자 메커니즘에 기여한다. 이러한 발달 과정은 축삭 배선, 신경 세포의 기능 다양성,인지 장애의 생물학에 중요한 의미를 가지고있다.

상세 및 개발을 통해 신경 세포의 형태 학적 변화를 특성화하기 위해, 띄엄 띄엄 뉴런 레이블을하는 것이 필수적이다. 이 효소를 통합 만 신경 GFP (그림 1A)를 표현할 수 있도록 Cre 호텔 - 재조합 효소 희석 시스템은 뉴런의 스파 스 인구에 대한 관심의 유전자의 발현을 허용한다. 이 전략을 사용하여 레이어 II-III는 대상과 E15.5에서 IUE에 의해 표시된다. CAG-DsRed2?...

Watch this Scientific Journal Video about In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity at JoVE.com

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

이 논문은 단일 세포 수준과 형광 표지 된 뉴런의 흥분성 뉴런의 연결 구조를 설명하는 자궁 전기 (IUE)에서 사용하는 프로토콜을 제공한다. 조직학은 수지상 및 축삭 돌기를 특성화하는 데 사용됩니다. 급성 슬라이스 전체 셀 기록 흥분성을 조사하기 위해 사용된다.

자궁 내 일렉트로는 신경 세포의 부분 집단의 흥분 및 단일 세포의 연결을 연구에 접근

The nervous system is composed of an enormous range of distinct neuronal types. These neuronal subpopulations are characterized by, among other features, ...

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JoVE Journal

Neuroscience

In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

9.6K 조회수.  Centro Nacional de Biotecnología (CNB-CSIC).  이 논문은 단일 세포 수준과 형광 표지 된 뉴런의 흥분성 뉴런의 연결 구조를 설명하는 자궁 전기 (IUE)에서 사용하는 프로토콜을 제공한다. 조직학은 수지상 및 축삭 돌기를 특성화하는 데 사용됩니다. 급성 슬라이스 전체 셀 기록 흥분성을 조사하기 위해 사용된다. 

비디오: In Utero Electroporation Approaches to Study the Excitability of Neuronal Subpopulations and Single-cell Connectivity

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells.
The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab 1, 2. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

In utero Electroporation followed by Primary Neuronal Culture for Studying Gene Function in Subset of Cortical Neurons

In utero electroporation is a valuable method for transfecting neuronal progenitor cells in vivo. Depending upon the placement of the electrodes and the developmental timepoint of electroporation, certain subsets of cortical cells can be targeted. Targeted cells can then be analyzed in vivo or in vitro for effects of genetic alteration.

utero에서 Electroporation은 두피 뉴런의 하위 집합의 유전자 기능을 학습에 대한 기본의 연결을 문화 뒤에

In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect ...

연구

신경과학

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos1,2 that we have successfully adapted3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick5. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described6. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.
To date, in utero electroporation has been most widely used for the analysis of neocortical development1,2,3,4. More recent studies have targeted the embryonic retina7,8,9 and thalamus10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

In utero electroporation allows for rapid gene delivery in a spatially- and temporally-controlled manner in the developing central nervous system (CNS). Here we describe a highly adaptable in utero electroporation protocol that can be used to deliver expression constructs into multiple embryonic CNS domains, including the telencephalon, diencephalon and retina.

사용하여 여러 개의 CNS 준주로 효율적인 유전자 전달 Utero에서 Electroporation

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental ...

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date this tool has been widely used to study the regulation of cellular proliferation, differentiation and neuronal migration especially in the developing cerebral cortex 6-8. Here we detail our protocol to electroporate in utero the cerebral cortex and the hippocampus and provide evidence that this approach can be used to study dendrites and spines in these two cerebral regions.
Visualization and manipulation of neurons in primary cultures have contributed to a better understanding of the processes involved in dendrite, spine and synapse development. However neurons growing in vitro are not exposed to all the physiological cues that can affect dendrite and/or spine formation and maintenance during normal development. Our knowledge of dendrite and spine structures in vivo in wild-type or mutant mice comes mostly from observations using the Golgi-Cox method 9. However, Golgi staining is considered to be unpredictable. Indeed, groups of nerve cells and fiber tracts are labeled randomly, with particular areas often appearing completely stained while adjacent areas are devoid of staining. Recent studies have shown that IUE of fluorescent constructs represents an attractive alternative method to study dendrites, spines as well as synapses in mutant / wild-type mice 10-11 (Figure 1A). Moreover in comparison to the generation of mouse knockouts, IUE represents a rapid approach to perform gain and loss of function studies in specific population of cells during a specific time window. In addition, IUE has been successfully used with inducible gene expression or inducible RNAi approaches to refine the temporal control over the expression of a gene or shRNA 12. These advantages of IUE have thus opened new dimensions to study the effect of gene expression/suppression on dendrites and spines not only in specific cerebral structures (Figure 1B) but also at a specific time point of development (Figure 1C).
Finally, IUE provides a useful tool to identify functional interactions between genes involved in dendrite, spine and/or synapse development. Indeed, in contrast to other gene transfer methods such as virus, it is straightforward to combine multiple RNAi or transgenes in the same population of cells. 
In summary, IUE is a powerful method that has already contributed to the characterization of molecular mechanisms underlying brain function and disease and it should also be useful in the study of dendrites and spines.

Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation

This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.

사용하는 마우스 대뇌 피질과 해마의 시각화 및 Dendrites와 쪽이의 유전자 조작<em&gt; utero에서</em&gt; Electroporation

In utero electroporation (IUE) has become a powerful technique to study the development of different regions of the embryonic nervous system 1-5. To date ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

예 자궁 Electroporation 및 전체 반구의 Explants : 초기 두피 개발 연구 간단한 실험 방법

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

신생아 Pial 표면 에레

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Subtype-selective Electroporation of Cortical Interneurons

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression of genes of interest to specific neuronal subtypes has proven remarkably challenging due to the relative scarcity of specific promoter elements. GABAergic interneurons constitute a neuronal population with extensive genetic and morphological diversity. Indeed, more than 11 different subtypes of GABAergic interneurons have been characterized in the mouse cortex1. Here we present an adapted protocol for selective targeting of GABAergic populations. We achieved subtype selective targeting of GABAergic interneurons by using the enhancer element of the homeobox transcription factors Dlx5 and Dlx6, homologues of the Drosophila distal-less (Dll) gene2,3, to drive the expression of specific genes through in utero electroporation.

This procedure shows how to target interneurons in the developing mouse forebrain by means of in utero electroporation. This technique was particularly efficient to achieve selective gene expression in interneuron subtypes destined to the superficial layers of the cortex.

두피의 interneurons의 하위 유형 선택적 일렉트로

The study of central nervous system (CNS) maturation relies on genetic targeting of neuronal populations. However, the task of restricting the expression ...

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

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

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

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

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

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

Double In Utero Electroporation to Target Temporally and Spatially Separated Cell Populations

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.

Double in utero electroporation allows targeting cell populations that are spatially and temporally separated. This technique is useful to visualize interactions between those cell populations using fluorescent proteins in normal conditions but also after functional experiments to perturb genes of interest.

자궁에서 두 배의 자궁 전기화는 시간적으로 그리고 공간적으로 분리된 세포 인구를 표적으로 합니다

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian ...

In Vivo Targeting of Neural Progenitor Cells in Ferret Neocortex by In Utero Electroporation

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.

Presented here is a protocol to perform genetic manipulation in the embryonic ferret brain using in utero electroporation. This method allows for targeting of neural progenitor cells in the neocortex in vivo.

자궁 에서 뇌포화에 의해 페렛 신피질에서 신경 전구 세포의 생체 포팅

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian ...

Murine In Utero Electroporation

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and maturation of cells during neural development. Electroporation enables the rapid and targeted delivery of material into cells by utilizing electrical pulses to create transient pores in cell membranes. Although electroporation has traditionally been used in in vitro studies, scientific advancements have now broadened its utilization to intact organs, such as those found in mouse embryos developing in utero. This video will introduce the key principles behind in utero electroporation in addition to reviewing the basic surgical techniques required to access developing embryos within a pregnant rodent. Details of the injection and electroporation steps are provided along with important considerations for directing gene delivery to specific brain regions. Finally, neurobiological applications of in utero electroporation are presented, such as investigating how specific genes contribute to neural development and how connections form between developing neurons.

Murine In Utero Electroporation

쥐 In Utero 전기 천공

In utero electroporation is an important technique for studying the molecular mechanisms that guide the proliferation, differentiation, migration, and ...