우리는 기술 지원을 Lihong 음에 감사하고 있습니다. NVD는 NARSAD 젊은 연구자 상을받는 사람이며, 또한 NIH (5 K99 MH095825-02)에서 보조금에 의해 지원됩니다. Fishell 실험실에서 연구는 건강의 국립 연구소, 정신 건강 (5 R01 MH095147-02, 5 R01 MH071679-09)의 국립 연구소, 신경 질환 및 뇌졸중 (5 R01 NS081297-02의 국립 연구소에서 지원 한 P01 NS074972 -01A1)와 시몬스 재단.

모든 동물은 의학의 NYU 학교의 기관 동물 관리 및 사용위원회의 규정 및 지침에 따라 처리 하였다. 마우스 균주 코닉 의해 제공 암컷 스위스 웹스터 마우스는이 실험에 사용 하였다. 특히 표면층의 interneurons를 타겟팅하기 위하여, E15.5 태아 사용 하였다. 주 : 표준 클로닝 기술을 사용하여 생성 된 본 작업 (Dlx5 / 6.eGFP 플라스미드 3 μg / μL)에 사용 된 플라스미드. eGFP는 cDNA를 Dlx5 / 6 PMIN-polyA 플라스미드에 클로닝 하였다. 이 플라스미드를 요청 (demarn02@nyumc.org)에 따라 사용할 수 있습니다. 미세 주입 피펫의 1 준비 <ol><li> 860에 끌어 당기는 열 # 2를 설정합니다. </li><li> 놓고 필라멘트 근처 유리 모세관을 고정합니다. 피펫의 외경 (OD)이 약 30 μm의 있는지 확인합니다. </li><li> &quot;풀&quot;버튼을 눌러. </li><li> 조심스럽게 캐 필러를 제거합니다. 하단 부분은 바늘이다. 상단 부분을 폐기하십시오. </li></ol> 2 마취 절차 <ol><li> 이소 플루 란 수용 실을 준비합니다. 참고 : 아이소 플루 란으로 인해 자사의 빠른 동작과 부작용의 낮은 발생 빈도를 선호하는 마취제입니다. 펜 토바 비탈 다른 옵션입니다; 그러나,이 약의 허용 오차 변수 이소 플루 란보다 높은 사망률의 원인이됩니다. </li><li> 0.5 L / 분 O이 4-5 %의 이소 플루 란의 흐름을 설정합니다. </li><li> 헤드 어댑터의 밸브가 닫혀있는 반면, 챔버의 밸브가 열려 있는지 확인합니다. </li><li> 챔버에 임신 한 마우스를 놓습니다. </li><li> 마우스를 마취의 영향 아래에 갈 수 있습니다. 이 약 2 ~ 3 분 소요됩니다. 호흡 속도를 확인합니다. 마우스가과 호흡 시작하면, 이소 플루 란의 복용량을 낮 춥니 다. </li><li> 마우스를 마취 한 후, 즉시 챔버에서 제거합니다. 가열 패드에 복부 측면을 놓고 전수술을 통해 이소 플루 란을 공급하기 위해 계속 마스크를 머리를 nsert. 헤드 어댑터 조각을 연결하는 튜브에 챔버에서 이소 플루 란의 흐름을 전환해야합니다. 각각의 눈에서 눈 윤활유 한 방울을 넣고 눈을 닫습니다. </li><li> 저체온증을 최소화하기 위해 36 ° C 정도에 전기 패드를 설정합니다. </li><li> 뒷발과 꼬리를 집어 발과 꼬리 반사의 부재를 확인합니다. </li></ol> 3 외과 <ol><li> 70 % 에탄올로 복부를 덮는 피부를 정화. </li><li> 위쪽으로 피부를 당겨 집게를 사용합니다. 각 수술 후 세제와 물을 모든 기기를 세척하고, 72 ° CO / N 살균. </li><li> 유선의 세 번째와 네 번째 쌍 사이의 중간에 시작 정중선을 따라 수직 작은 절개 (약 2 ㎝)를 확인. 미세 수술 가위를 사용합니다. 복부 벽을 손상하지 않도록주의하십시오. </li><li> 조심스럽게 복막으로부터 피부를 구분합니다. </li><li> 동맥을 시각화및 복벽에 정맥. 복강을 노출 (선박을 피) 복막을 통해 다른 2cm 수직 절개를합니다. </li><li> 동맥을 클램핑 피하는 양쪽에있는 피부와 복부 벽을 클램프. 주 : 동맥이 실수로 구멍이있는 경우, 자궁 경부 전위 또는 이소 플루 란 과다 복용을 수행하여 동물을 즉시 희생. </li><li> 37 ° C로 멸균 PBS를 따뜻하게 사전. 김 물티슈 촉촉한 PBS와 클램프를 커버. </li><li> PBS와 노출 복강 보습. 수술을 수행하는 동안이 단계를 여러 번 반복합니다. </li><li> 라운드 집게를 사용하여 부드럽게 떨어져 공간에 배아 체인 (E15.5)을 당깁니다. 물티슈의 체인 휴식을 보자. 첫번째 자궁의 절반을 노출함으로써 시작합니다. 그 안에 배아 전기 천공되면, 내부를 돌아 오게 한 후 하반기에 작동합니다. </li></ol> 4 일렉트로 <ol><li> 배아를 조작하는 라운드 집게를 계속 사용합니다. </li><li> 바이올렛측면 심실을 sualize. 중간 선 (그림 1b)을 따라 실행 측면 심실. </li><li> 빠른 그린 (주식을 10 % / w V) 추가 1:20 혼합물에 대한 DNA 용액에 주입하는 동안 시각화 할 수 있습니다. 증류수 표준 맥시 프렙 정제 프로토콜로부터 얻어진 펠릿을 용해시킴으로써 DNA 용액을 준비한다. </li><li> 빠른 그린으로 DNA 용액 (Dlx5 / 6-eGFP는, 3 μg / μL) 1 ㎖를 주입하는 플런저 미리 뽑아 마이크로 피펫을 사용합니다. 피펫을 넣기 전에 바늘의 팁 단부에 어깨의 선두로부터 6mm 길이를 남겨 미세 forcep # 5 (듀)를 마이크로 피펫의 선단을 잘라. 라운드 핀셋으로 태아의 머리를 잡아. 중간 선 근처에있는 뇌실 목표로하는 45 ° 각도로 피펫 뇌를 입력합니다. 바늘이 뇌실을 관통하는 경우가 DNA를 주입 플런저를 사용할 때, 천천히 피펫을 철회. 심실이 녹색해야 할 때솔루션을 작성하기 시작합니다. 이 시점에서, 피펫을 움직이는 멈추고 DNA 1 ㎖를 주입. 조심스럽게 피펫을 철회. </li><li> 950 밀리 초 간격으로 50 밀리의 오 45V 펄스 CUY21의 electroporator을 설정합니다. </li><li> E15.5 배아에 대한 5mm 패들 전극을 사용합니다. 효율적인 전류 전송을 보장하기 위해 PBS에 전극을 담근다. </li><li> DNA 솔루션을 포함하는 심실에 긍정적 인 패들과 태아의 머리 주위에 전극 패를 놓습니다. 약간 신경절 eminences을 통해 복부 전류를 만들 수있는 음의 일의 대한 긍정적 인 패를 내립니다. 이 조작은 피라미드 세포에서 이소 식을 최소화 할 수 있습니다. </li><li> 전극을 배치 한 후, 한 번 풋 페달을 눌러 펄스를 제공한다. </li><li> 전극을 제거하고 깨끗하게 닦아주십시오. 반복 각 배아와 4.6-4.9 단계를 반복합니다. </li><li> 모든 배아를 electroporating 후 복강에 PBS 3 ㎖를 부어 복강에 반환. </li><li>제 봉합사 곡선 바늘과 5-0 실크 봉합사 후 피부 복벽. 상처에 리도카인 (4 %)을 적용합니다. 진통제 아스피린 내 복막의 120 ㎎ / ㎏을 관리 할 수​​ 있습니다. 주 : 전체 절차는 20 분 이하로 수행되어야한다. 그것은 초보자는 짧은 수술 시간을 유지하기 위해 몇 배아를 electroporate하는 것이 좋습니다. 장시간 수술 배아의 생존율을 감소한다. </li><li> 마취 튜브에서 동물을 제거하고 종이에 가열 패드에 복구를 닦아 수 있습니다. </li><li> 케이지에 동물을 돌려 37 ° C에서 가열 패드에 그것을 유지하고 건강 상태를 모니터링 할 수 있습니다. 동물은 일반적으로 수술을 잘 견딜 그들이 마취 튜브에서 제거 된 직후 서서히 이동하기 시작합니다. 과도한 출혈이나 혼수가 관찰되는 경우, IACUC는 자궁 경부 전위 또는 마취 과다 복용으로 안락사 절차를 승인 수행하여 즉시 동물을 희생. </li><li> 비바에 케이지를 반환rium. 여성은 분만 자연적으로 줄 것이다. </li></ol>

<ol>
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저자가 공개하는 게 없다.

기술의 한계 이 기술은 셀 프로세스 셀 자율적 분석이 가능하지만,이 집단 분석에 적합하지 않다. electroporations 뇌 당 electroporation하여보다 천 세포와 매우 드물다. 결과적으로, 기술은 CGE의 interneurons 유래의 유전자 조작으로 인한 행동 결과를 평가하기 위해 사용될 수 없다. electroporations가 E13.5-E14.5 대상 MGE에서 파생 된 하위 유형에서 수행하지만, 효율이...

대뇌 피질의 GABA 성의의 interneurons의 대부분은 중간과 꼬리 신경절 eminences (각각 MGE 및 CGE)을 명명 된이 과도 배아 구조에서 발생 4. Parvalbumin 및 소마토스타틴 (somatostatin) 표현의 interneurons는의​​ interneurons는 CGE에서 발생 표현 Calretinin 반면 MGE 크롬 (Cr), Vasointestinal 펩티드 (VIP) 및 Reelin (재)에서 발생. 이 interneuron 하위 유형은 자신의 생일에 의해 구별 될 수있다. MGE 파생 된 하위 유형은 배아 일 9.5 (E9.5)와 e16.5 5,6 사이에서 태어난다. 반면, CGE 유래의 interneurons는 생산이 E15.5 6 고점으로 E18.5 통해 E12.5에서 태어난다. 늦게 태어난 인구의 유전자 타겟팅은, 그러나, 어려운 남아있다. 쥐의 말초없는 (DLX) 유전자를 독점적으로 개발 복부 전뇌 3 표현됩니다. GABA 성의 선조체의 interneurons 및 투영 아니라 피질 뉴런 피라미드 세포 Dlx1 표현할초기 발달 단계 3에서 2,5, 6 유전자. 실제로, DLX 유전자는 모든 GABA 성 전구 세포의 MGE 및 CGE 뇌실 영역 (SVZ)로 표현된다. 이들 유전자의 발현은 postmitotic 단계 7-9에서 서브 타입을 선택하도록 제한된다. 이전 실험적 증거는 Dlx5 / 6 인핸서 요소가 형질 전환 마우스 모델 2에서 GABA 성 계통의 선택 타겟팅을 허용 것으로 나타났다. 우리는 현상 마우스 뇌에서 에피 솜 발현의 문맥에서 이들 인핸서 요소 중 하나의 사용을 시험 하였다. 우리는 최소한의 발기인 및 bluescript (BS) 백본 플라스미드의 강화 된 녹색 형광 단백질 (eGFP는) (그림 1)과 함께 Dlx5 / 6 증강 요소를 복제 서브. 우리는 선택적으로 CR-, VIP를 대상으로 E15.5에서 자궁 일렉트로에 의해 플라스미드를 도입 재는 3,8,10을 아류. 우리의 기술은 용이 스파 스 일렉트로, 수 있습니다그을음 세포의 형태 학적 특징의 재건입니다. 또한, 피질 GABA 성 뉴런의 유전자 발현의 예외적으로 높은 수준의 기능 연구를 허용한다. 우리는 손실과 여러 야생 유형 및 지배적 인 부정적인 유전자 11를 사용하여 기능 연구의 이득을 실시했다.

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subtype-selective electroporation of cortical interneurons

중추 신경계 (CNS) 성숙의 연구는 신경 세포 집단의 유전자 타겟팅에 의존한다. 그러나, 특정 신경 세포 아형의 관심의 유전자의 발현을 제한하는 작업으로 인해 특정 프로모터 요소의 상대적 부족에 현저 도전 입증되었다. GABA 성의의 interneurons 광범위한 유전 적, 형태 적 다양성을 가진 신경 세포의 인구를 구성한다. 실제로, GABA 성의의 interneurons의 11 개 이상의 서로 다른 하위 유형 마우스 피질 한 것을 특징으로하고있다. 여기에서 우리는 GABA 성 인구의 표적으로 선택에 대한 적응 프로토콜을 제시한다. 우리는 호 메오 박스 (homeobox) 전사의 증강 요소를 사용하여 GABA 성의의 interneurons의 하위 표적으로 선택 달성 자궁 일렉트로 특정 유전자의 발현을 통해 운전 Dlx5 및 Dlx6, 초파리 말초없는 (DLL) 유전자 2,3의 상 동체를, 요인.

우리는 성숙 뉴런 세포 유형 특정한 타겟팅을 달성하기에 자궁 일렉트로 기법을 적응. CGE 유래의 interneurons에서 eGFP는의 발현을 구동하기 위해, 우리는 Dlx5 / 6 증강 요소를 사용 E15.5에 우리의 주사를 제한, 무대 CGE 유래의 interneurons의 대부분이 발생하는 경우. 우리는 P8에서 분석을 수행하고 P15 (11) (도 1 및도 2). 우리는 E15.5에서 몰 농도에서 CAG-mCherry 및 D...

Watch this Scientific Journal Video about Subtype-selective Electroporation of Cortical Interneurons at JoVE.com

Subtype-selective Electroporation of Cortical Interneurons

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 ...

subtype-selective-electroporation-of-cortical-interneurons

Research

JoVE Journal

Neuroscience

8.7K 조회수.  New York University School of Medicine. 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.

비디오: Subtype-selective Electroporation of Cortical Interneurons

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

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

두피 네트워크 개발에 기능성 칼슘 이미징

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

마우스 두피 Astrocytes의 분리와 문화

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

신생아 Subventricular 지대 Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Morphologically- 및 Neurochemically 확인 된 해마의 interneurons에서 전체 세포 패치 클램프 녹음

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral cortex. We here share our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex using a method developed by Lima and colleagues1. Recordings are performed in mice expressing Channelrhodopsin-2 (ChR2) in specific neuronal subpopulations. Members of the population are identified by their response to a brief flash of blue light. This technique &#8211; termed &#8220;PINP&#8221;, or Photostimulation-assisted Identification of Neuronal Populations &#8211; can be implemented with standard extracellular recording equipment. It can serve as an inexpensive and accessible alternative to calcium imaging or visually-guided patching, for the purpose of targeting extracellular recordings to genetically-identified cells. Here we provide a set of guidelines for optimizing the method in everyday practice. We refined our strategy specifically for targeting parvalbumin-positive (PV+) cells, but have found that it works for other interneuron types as well, such as somatostatin-expressing (SOM+) and calretinin-expressing (CR+) interneurons.

A Guide to In vivo Single-unit Recording from Optogenetically Identified Cortical Inhibitory Interneurons

Here we describe our strategy for obtaining stable, well-isolated single-unit recordings from identified inhibitory interneurons in the anesthetized mouse cortex. Neurons expressing ChR2 are identified by their response to blue light. The method uses standard extracellular recording equipment, and serves as an inexpensive alternative to calcium imaging or visually-guided patching.

에 가이드<em&gt; 생체</emOptogenetically 확인 된 대뇌 피질의 억제의 interneurons에서&gt; 단일 단위 기록

A major challenge in neurophysiology has been to characterize the response properties and function of the numerous inhibitory cell types in the cerebral ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

의 기록 세포 내 전압 응답에 대한 전기 생리 방법<em&gt; 초파리</em&gt; 빛 자극에 광수와의 interneurons<em&gt; 생체</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Visualization of Cortical Modules in Flattened Mammalian Cortices

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and can be delineated by certain histochemical and immunohistochemical methods. In this study, we highlight a method to isolate the cortex from mammalian brains and flatten them to obtain sections parallel to the cortical sheet. We further highlight selected histochemical and immunohistochemical methods to process these flattened tangential sections to visualize cortical modules. In the somatosensory cortex of various mammals, we perform cytochrome oxidase histochemistry to reveal body maps or cortical modules representing different parts of the body of the animal. In the medial entorhinal cortex, an area where grid cells are generated, we utilize immunohistochemical methods to highlight modules of genetically determined neurons which are arranged in a grid-pattern in the cortical sheet across several species. Overall, we provide a framework to isolate and prepare layer-wise flattened cortical sections, and visualize cortical modules using histochemical and immunohistochemical methods in a wide variety of mammalian brains.

This article describes a detailed methodology to obtain flattened tangential sections from mammalian cortices and visualize cortical modules using histochemical and immunohistochemical methods.

병합 된 포유류 외피가 대뇌 피 질의 모듈의 시각화

The cortex of mammalian brains is parcellated into distinct substructures or modules. Cortical modules typically lie parallel to the cortical sheet, and ...