This work was funded by NIH grants DA013137, DA031604, and HD043680. Partial support was provided by a NIH T32 training grant in Biomedical-Behavioral science.

모든 동물 프로토콜을 검토하고 사우스 캐롤라이나 대학의 동물 관리 및 사용위원회 (보증 번호 : A3049-01)에 의해 승인되었다. 1. 저밀도 배아 신경 문화 <ol><li> 기본 대뇌 피질의 신경 세포 배양을위한 준비 <ol><li> 솔루션 : <ol><li> 10 ml의 보레이트 완충액에 폴리 -L- 리신 5mg을 용해시켜 폴리 -L- 라이신 스톡 용액을 제조 하였다. </li><li> 49 ml의 붕산염 완충액 1 ml의 폴리 - L - 라이신 주식을 희석하여 솔루션을 작동 준비합니다. </li><li> 395 ml의 DH 2 O로 시작하고 모든 재료 (붕사의 1.24 g의 붕산 + 1.9 g)을 추가하여 붕산 완충액, pH가 8.4을 준비합니다. 1 M의 NaOH로 pH를 8.4로 조정합니다. 400 ml의 조정 및 후드 아래에 0.2 μm의 나일론 멤브레인 필터로 필터링 할 수 있습니다. </li><li> 445 ml의 DH 2 O로 시작하는 다른 모든 성분을 추가하여 HBSS 용액의 pH 7.2을 준비합니다 (50 ㎖ 10X HBSS 재고 + 1.2 g HEPES). 1 N 염산, 브리에 의해 7.2의 pH를 조정NG 500 ㎖의 후드 아래에 0.2 ㎛의 나일론 멤브레인 필터로 필터링. </li><li> 10 % 소 태아 혈청 DMEM / F12 : 도금 매체를 준비합니다. 후드 0.2㎛, 나일론 멤브레인 필터로 필터링. </li><li> 전체 성장 매체를 준비 : 50 ㎖로 Neurobasal 매체에, 보충 교재를 추가 (500 μL 루타 (100X) + 500 μL 포도당 항생제 - 안티 곰팡이 솔루션 (100X의 + 1 ML의 B-27 (50X) + 500 μL) + 100 ㎕의 7.5 % 중탄산 나트륨. 후드 0.2㎛, 나일론 멤브레인 필터로 여과. </li></ol></li><li> 이틀 문화 전 : <ol><li> 2 폴리 - L - 라이신 작업 용액 ㎖의, 그리고 후드 O / N에서 유지와 코트 열두 35mm의 유리 바닥 요리. </li></ol></li><li> 문화 전 어느 날 : <ol><li> DDH 2 O와 빈 코팅 요리에서 에이전트와 린스 요리 후드 아래 시간 동안 건조하도록 허용합니다. </li><li> 10 % 소 태아 혈청 (총 용액의 10 %)와 도금 배지 (DMEM은 / F12를 준비한다. 피펫 2ml의 배지 내로각각의 요리는, 37 O를 C, 5 % CO 2에서 O / N을 품어. 참고 : 배양 할 때 플라스틱 문화 요리를 방지하는 것이 중요하다. 유리 바닥 (반전 현미경) 요리 또는 (직립 현미경) 커버 글라스의 사용은 샤프하고 선명한 이미지를 얻을 수 있습니다. 플라스틱 바닥 접시에 비해 유리 바닥 접시 형광 현미경 동안 원치 않는 색상이나 눈부심을 방지 할 수 있습니다. </li></ol></li></ol></li><li> 세포 배양 프로토콜 : <ol><li> HBSS 버퍼 (100 ~ 150 ㎖), 냉장 넣어 100mm 배양 접시, 집게, 가위, 50 ㎖ 튜브 및 후드 UV 빛 아래 15 ml의 튜브. </li><li> 문화 실의 외부 후드 5 % 세보의 치명적인 흡입 임신 쥐를 안락사. </li><li> 70 %의 EtOH 텐트 복부 cavity.Open 태반을 노출 가위를 이용하여 자궁 중간 꼬리로부터 절단하면서 집게를 사용하여 복부의 피부를 소독. </li><li> 8-10 E18 태아를 제거합니다. 놀이터HBSS 솔루션을 포함하는 배양 접시에 8-10 태아를 CE. </li><li> 집게로 태아의 머리 뒤쪽을 잡고, 다음 몸에서 머리를 절단 가위를 사용합니다. HBSS의 5-7 ml를 가득 다른 배양 접시에 넣습니다. 후드에 접시를 전송합니다. </li><li> 차가운 HBSS와 두 개의 추가 100mm 페트리 접시를 입력합니다. </li><li> 껍질 옆으로 두개골, 그리고 HBSS로 가득 찬 새로운 배양 접시에 머리를 특종. </li><li> 다음에 차가운 HBSS로 새로운 배양 접시에 두뇌를 전송, 뇌에서 날카로운 곡선 집게, 별도의 소뇌와 뇌간을 사용합니다. </li><li> 곡선 집게와 반구를 분리, 고정 핀셋을 사용합니다. 수막을 제거; 표시 15 ML의 원심 분리기 튜브에 정면 피질 및 전송 조각을 격리 할 것. </li><li> 신선한 2 ml의 HBSS로 15 ML 튜브를 입력하고 각 15 ML 튜브에 20 μL 트립신 EDTA (0.5 % 트립신 및 5.3 mM의 EDTA.와 10 배 농축 혼합물)를 추가합니다. </li><li> RT에서 10 ~ 15 분을 품어. 부드럽게 관에게 몇 분마다 소용돌이, 정착 허용하지 않습니다. </li><li> AF터 10 ~ 15 분 사용 유리 피펫 오래된 HBSS를 제거하고 새로운 HBSS로 두 번 씻어합니다. </li><li> (2 ml의 HBSS 1 ㎎ / ㎖, 2 mg을 트립신 억제제) 트립신 억제제에 빠져, 잘 혼합하고 5 분 앉아 보자. 신선한 HBSS로 두 번 씻으십시오. </li><li> 다시 보정 피펫 매우 균질해질 때까지 서서히 감소 팁 직경 멸균 팁을 사용 bubbles.Triturate을 피하기 위해 매우 천천히 씹다 뇌 피스 고무 전구 10-15 번 유리 피펫을 사용. </li><li> 전송 원하는 농도에서 세포를 해리에 코팅 된 배양 접시 (50 셀 / mm2 인) 준비된 사전. 37 O C, 5 % CO2에서 O / N을 품어. </li><li> 24 시간 후, 갓 만든 완전한 성장 혈청 무료로 Neurobasal 매체 DMEM / F12 매체를 교체합니다. </li><li> 실험 전에 적어도 12~14일 대해 5 % CO 2 / 95 %의 실내 공기의 가습 인큐베이터에서 항상 세포를 유지한다. 신선한 배지로 Neurobasal 이전 매체의 약 50 %를 교체하는 모든 5-6일 세포를 보완. </li></ol></li></ol> 2. 형광 라벨링 및 면역 세포 화학 주 : 일차 대뇌 피질 세포 배양의 면역 라벨 1 ㎖가 작동 부피 유리 바닥 35mm 세포 배양 접시에서 행했다. <ol><li> 인산염 완충 식염수 산도 7.4 (PBS)로 두 번 유리 바닥 접시를 씻으십시오. </li><li> 실온에서 15 분 동안 4 % 파라 포름 알데히드와 세포를 수정. </li><li> PBS로 두 번 세포를 씻으 5 분 동안 PBS에서 0.1 % 트리톤 X-100 Permeabilize 하시려면. </li><li> F - 굴지 특정 얼룩 실온에서 20 분 동안 세포 치료, AlexaFluor 488 Phalloidin의 (1시 40분, 1 ml의 PBS에서 Phalloidin의 25 μL). </li><li> PBS로 두 번 세포를 씻어 1 ~ 2 시간 동안 실온에서 10 % 정상 염소 혈청으로 차단합니다. </li><li> 2 % 정상 염소 혈청과 함께 PBS에서 4 O ℃에서 O / N을 부화 : 닭 항 MAP2 클론 항체 (2500 1) 희석 </li><li> 배양 한 다음, 두 번 PBS에 헹군다. </li><li> 이차 항체 (알렉사 R 희석ED 594 - 복합 염소 항 - 닭 IgG를 (1 : 500) 2 % 정상 염소 혈청와 실온에서 2 시간 동안 품어. </li><li> PBS로 헹구고 10 μL / Hoescht 염료의 요리를 추가합니다. </li><li> RT에서 3 분을 품어 PBS로 2 회 씻는다. 참고 : 3 단계 (F - 굴지 puncta의 계산) 이제 표지 세포가 준비. </li><li> coverslipping 제로 antifade 시약 100 ㎕로 세포 샘플을 보존 및 장기 저장을 위해 어둠 속에서 4 ℃로 유지한다. 참고 : Phalloidin의이 antifade coverslipping 시약으로 보존 할 수 일주 형광까지의 어둠 속에서 4 ℃에서 PBS에서 상대적으로 안정적이다. Phalloidin의 확장 된 스토리지 밖으로 확산 시작할 수 있기 때문에, 최적의 이미지는 염색 후 1-3일를 얻을 수있다. </li></ol> 3. F - 굴지 puncta의 카운팅 <ol><li> 형광 현미경의 전원을 켜고 20 × 목표로 전환합니다. 오픈 현미경 소프트웨어, 1280 × 960 화소의 화상 사이즈에 프로그램을 설정하고, 0.11 배 줌에 7 μm의 / 픽셀 이미지 해상도. </li><li> 빨강, 녹색 (495 ㎚) / (613 ㎚) 형광 채널에서 공동 표지 F - 굴지 / MAP2 뉴런의 이미지를 획득. </li><li> 5 그린 (F - 굴지) / 레드 (MAP2) immunolabeled / 블루 (Hoescht) 명확하게 정의 된 수지상 아버 개별 신경 세포의 형광 이미지를 선택합니다. </li><li> 연속 MAP2의 면역와 두 번째 순서 수지상 세그먼트 (길이 범위 25 ~ 75 μm의)에서 F - 굴지 풍부한 구조를 확인합니다. </li><li> 수평 수준으로 이미지의 선택 영역을 돌립니다. 새로운 이미지로 복사 및 붙여 넣기. </li><li> 각 접시에 대한 일관성을 조정하여, 이미지의 배경을 뺀다. </li><li> 밝은 녹색 F - 굴지 puncta의 카운트 수동으로 훈련 된 독립적 인 관찰자에 의해 선택 수지상 세그먼트의 길이를 추적. 스프레드 시트 파일에 데이터를 보냅니다. </li><li> MAP2는 수상 돌기 레이블의 길이 (L)에 의해 총 F - 굴지 라벨 puncta에 (N)을 나누어 밀도를 계산합니다. F-교류의 수와 같은 고속 데​​이터수상 돌기의 10 μm의 당 주석 puncta의 참고 puncta의 수지상 샤프트 염색의 평균 강도보다 최소한 50 %의 피크 강도 F 액틴 형광 (크기 ≤1.5 것이 μm의) 각각의 선택된 수지상 세그먼트에 포함되었다. </li></ol>

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None of the authors have conflicts of interest to declare.

이 프로토콜에서는, 우리는 우리가 개별 뉴런의 수상 돌기를 식별 할 수 있습니다 35mm의 유리 바닥 요리 낮은 밀도에서 쥐의 대뇌 피질의 신경 세포를 배양 설명합니다. 다음으로, 우리는 수지상 변화를 감지 할 Phalloidin의 및 MAP2 염색을 사용합니다. 그 후, 우리는 F - 굴지 puncta의 변화를 정량화하는 전문 소프트웨어를 사용했다. F - 굴지 puncta의 개별 신경 세포의 전체 신경 네...

본 연구의 주된 목적은 신경 돌기 네트워크 시냅스 무결성 측정 (추정)의 신뢰성있는 방법을 개발하는 것이다. 여기에서 우리는 Phalloidin의 염색 전문 (NIS-요소) 소프트웨어를 사용하여 후속 분석을 수상 돌기의 면역 세포 화학 (ICC) 검출의 조합을 사용하여 기본 쥐 배양 된 신경 세포의 F - 굴지 puncta의 정량화를 설명합니다. 레이블 phallotoxins는 크고 작은 필라멘트 (F-액틴)에 대한 유사한 친 화성을 ​​가지고 있지만, 일부 액틴 항체 1과는 달리, 단량체 구형 액틴 (G-액틴)에 결합하지 않는다. Phalloidin의의 결합 특이성 따라서 세포 이미징 동안 최소한의 배경을 제공 무시할 수있다. Phalloidin의는 일반적으로 Phalloidin의에 의해 F - 굴지의 훨씬 더 강렬한 라벨을 허용 형광 현미경에 대한 세포 단백질을 라벨 사용되는 항체보다 훨씬 작다. 따라서, 뉴런에서 F - 굴지 현지화의 상세한 이미지가 될 수 있습니다레이블 Phalloidin의 사용을 통해 얻을. Phalloidin의 신경 세포 수상 돌기의 (F - 굴지) 염색 성숙 쪽이 아닌 가시 시냅스 2 미숙 한 쪽을 포함하여 수지상 구조의 다양한 표현 이산 &quot;핫스팟&quot;또는 밝은 &quot;puncta에&quot;를 생성합니다. 미성숙 등뼈 얇은 filopodia 패치 형태의 일부 형태를 포함하고 spinogenesis 3의 개시를 나타낼 수있다. 미성숙 가시 및 비 - 가시 패치 PSD95 4 부족하다. 뿐만 아니라 등뼈의 후속 변경뿐만 아니라 추가 수지상 구조에 F - 굴지의 납 생산의 변화, 따라서 synaptodendritic 무결성 5-7을 조사하기위한 중요한 도구 Phalloidin의 제작. 일반적으로, Phalloidin의 양성 (F - 굴지) puncta의의 수는 활성 시냅스 사이의 균형 (흥분성과 억제), 굴지 역학과 시냅스 안정성 (8)을 반영한다. 특정 t을 연구하는 중요하지만치료의 대상이 알려지지 않은 경우에 시냅스 (즉, 흥분성 등뼈)의 씨네마 스테이션 먼저 수지상 구조의 다양한 일반 무결성을 추정 할 필요가있다. F - 굴지는 억제 시냅스, synaptopathy을 나타낼 수 F - 굴지 puncta에의 변경 번호를 포함 돌기 쪽과 다른 구조의 주요 구성 요소이기 때문에. 이 synaptopathy는보다 구체적인 변경에 대한 추가 조사를 할 수있다. 여러 시냅스 유형 / 구조를 검출하기위한 우리의 정량화 방법은 다양한 실험적인 치료 다음과 같은 돌기 시냅스 변화 (증가 및 감소)의 전체 추정치를 산출한다.

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quantification of filamentous actin (f-actin) puncta in rat cortical neurons

(F 액틴) 필라멘트 액틴 단백질 spinogenesis 시냅스 가소성 및 시냅스 안정성에 중요한 역할을한다. 수지상 F - 굴지 풍부한 구조의 변화는 시냅스 통합 및 연결에 변경을 제안한다. 여기에서 우리는 F - 굴지 puncta에, 이후의 정량화 기법에 대한 기본 쥐의 대뇌 피질의 뉴런, Phalloidin의 얼룩을 배양하기위한 상세한 프로토콜을 제공합니다. 먼저, E18 쥐 배아 전두엽는, 저밀도 세포 배양에 적어도 12~14일 체외에서 배양 신경 세포 해리된다. 실험 치료 후, 대뇌 피질의 신경 세포가 AlexaFluor 488 Phalloidin의 염색하고 미세 소관 - 관련 단백질 2 (수지상 F - 굴지 puncta의 레이블을) (MAP2을, 신경 세포와 수지상 무결성을 검증하기 위해). 마지막으로, 전문 소프트웨어 분석하고 무작위로 선택된 신경 세포의 수상 돌기를 정량화하는 데 사용됩니다. F는 액틴 풍부한 구조하는 두 번째 순서 수지상 가지 (길이 범위 25 ~ 75 &amp;에 식별# 181; 연속 MAP2의 면역와 m). 여기에 제시된 프로토콜은 실험 치료 이후 수지상 시냅스 구조의 변화를 조사하기위한 유용한 방법이 될 것입니다.

본 발명의 방법에서, 우리는 첫 번째 문화 쥐 우리가 개별 뉴런의 수상 돌기를 식별 할 수 있습니다 35mm의 유리 바닥 요리 낮은 밀도에서 대뇌 피질의 뉴런. 도 1에있어서, 미분 간섭 대비 (DIC) 이미지 일 4, 6, 10, 시험관 내에서 14, 21 및 27에서 태아 래트 피질 뉴런 개발 형태 변화를 나타낸다. 길이와 수상 돌기의 수는 배양 된 쥐 주 신경 세포의 성숙에 따?...

Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons at JoVE.com

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

쥐 두피 뉴런의 사상 액틴 (F - 굴지) puncta의 정량

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

quantification-filamentous-actin-f-actin-puncta-rat-cortical

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JoVE Journal

Neuroscience

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

12.0K 조회수.  University of South Carolina. Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

비디오: Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

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

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Nucleofection 및 배아 마우스 Hippocampal과 두피 뉴런의 주요 문화

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

Permeabilized의 연결을 성장 콘스에서 Rhodamine - 굴지와 F - 굴지 이던가 엔드 레이블

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of &Delta;&Psi;m renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine &Delta;&Psi;m and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. 
Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine &Delta;&psi;m in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess &Delta;&Psi;m or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of &Delta;&Psi;m or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of &Delta;&Psi;m or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in &#x2206;&#x03A8;m and ROS in different cell types and in neurons isolated from other brain regions.

We demonstrate application of the fluorescence indicator, TMRM, in cortical neurons to determine the relative changes in TMRM fluorescence intensity before and after application of a specific stimulus. We also show application of the fluorescence probe H2DCF-DA to assess the relative level of reactive oxygen species in cortical neurons.

라이브 랫 두피 뉴런에서 Mitochondrial 분리막 잠재력과 반응 산소 종의 결정

Mitochondrial membrane potential (&Delta;&Psi;m) is critical for maintaining the physiological function of the respiratory chain to generate ATP.  A ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

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

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

차 쥐 두피 뉴런과 Glia의 Bilaminar의 공동 문화

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

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

두피 뉴런에서 생체 내 두 - 광자의 영상은 경험에 의존 분자 변경

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive functions including decision making, sensory-guided behavior or memory. To understand how such intricate neuronal networks perform such tasks, a crucial step is to determine the function (or electrical activity) of individual cell types within the network, preferentially when the animal is performing a relevant cognitive task. Additionally, it is equally important to determine the anatomical structure of the network and the morphological architecture of the individual neurons to allow reverse engineering the cortical network. Technical breakthroughs available today allow recording cellular activity in awake, behaving animals with the valuable option of post&#160;hoc identifying the recorded neurons. Here, we demonstrate the juxtasomal biocytin labeling technique, which involves recording action potential spiking in the extracellular (or loose-patch) configuration using conventional patch pipettes. The juxtasomal recording configuration is relatively stable and applicable across behavioral conditions, including anesthetized, sedated, awake head-fixed, and even in the freely moving animal. Thus, this method allows linking cell-type specific action potential spiking during animal behavior to reconstruction of the individual neurons and ultimately, the entire cortical microcircuit. In this video manuscript, we show how individual neurons in the juxtasomal configuration can be labeled with biocytin in the urethane-anaesthetized rat for post&#160;hoc identification and morphological reconstruction.

To understand the structure of neuronal networks, functional and morphological characterization of individual neurons is a necessity. Here, we demonstrate juxtasomal biocytin labeling, which allows electrophysiological recordings in the extracellular configuration, yet maintaining the ability to intracellularly label the neuron for post&#160;hoc reconstruction of dendritic and axonal architecture.

Juxtasomal Biocytin 레이블은 개별 두피 뉴런의 구조 - 기능 관계를 연구하는

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

형광 프로브를 이용한 배양 된 뉴런의 엔도 좀과 리소좀 기능의 정량화

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

Vivo에서 신생아 쥐에 있는 대뇌 피 질의 뉴런의 두 광자 영상

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

E14-E16 스프라그 Dawley 쥐 배아에서 분리된 혼합 된 기본 해마 및 피질 뉴런에서 신경 구의 생성

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...