이 작품은 NIH 그랜트 NS065183 및 러트 거스 로버트 우드 존슨 의과 대학에서 창업 자금을 지원합니다.

1. 에어컨 미디어 및 성상 세포 신경 세포 공 배양에 쥐 성상 세포 문화를 준비합니다. <ol><li> (BSS 레시피 표 1 참조) 해부 버퍼를 준비하고 4 ° C에서 보관 사용 준비가 될 때까지. </li><li> 500 ㎖ 비이커에 이소 플루 란과 신생아 쥐 새끼 (P0-P2)를 마취. </li><li> 강아지가 움직 인 경우, 70 % 에탄올로 스프레이 목을 베다. </li><li> 뇌를 제거합니다. <ol><li> 뒤몽 # 5 포셉 한 쌍의 단단히 머리를 잡아, 피부와 두개골을 통해 중간 선 절개를 만들기 위해 미세 가위를 사용합니다. </li><li> 측면에 두개골을 반영하여 뇌를 노출, 차가운 BSS를 포함하는 접시에 두뇌를 제거합니다. </li></ol></li><li> 해부 범위에서 간뇌와 뇌간에서 대뇌 반구를 분리합니다. 조심스럽게 집게 일쌍과 조직을 안정화하고 부드럽게 포셉의 다른 쌍의 수막을 떼어 모든 수막을 제거합니다. </li><li> 모든 반구를 수집 IN 개의 60mm 접시. 작은 가위, 다진 조직과 같은 미세 가능한 한 사용. 그런 다음 50 ML 원뿔 튜브에 다진 조직을 전송합니다. </li><li> 1.5 1 % DNA 분해 효소 I ㎖ 및 1.5 ㎖의 2.5 % 트립신과 두뇌에 BSS (15ML의 총 부피)의 12 ML을 추가합니다. 37 ℃에서 15 분 동안 진탕 수조에서 부화 좋은 혼합을 보장하기 위해, 튜브는 손으로 5 분마다 소용돌이된다. </li><li> 새로운 50 ML 원뿔 관에 70 μm의 셀 스트레이너를 통해 뜨는을 전송합니다. 이 상층에 3 ㎖의 FBS를 추가합니다. </li><li> 나머지 조각, 13.5 BSS ㎖ 및 1.5 ㎖의 2.5 % 트립신을 추가하고 또 다른 15 분 동안 진탕 수조에서 배양한다. </li><li> 셀 스트레이너를 통해 나머지 뜨는을 전송하고 단계 1.8에서 뜨는 결합. </li><li> 펠릿 세포 5 분 1,000 RPM에서 원심 분리기. </li><li> 아교 미디어 5 ㎖에 재현 탁 펠렛. 상청액 나머지 세포 펠렛을 다시 원심 분리 할 수​​있다. </li><li> 혈구를 사용하여 세포를 계산합니다. </li><li> 플레이트 CEL1 × 10 7 150cm 당 2 플라스크의 밀도로 LS. </li><li> 하루 도금 후 신선한 아교 미디어에 미디어를 변경합니다. 그 후, 아교 미디어 일주일에 두 번 세포를 공급. </li><li> 세포가&gt; 80 % 컨 플루 (약 십일 도금 후)에 도달했을 때, 90 % 말 혈청과 10 % DMSO에서 세포를 동결 액체 질소에 주식을 계속. 세포는 2 바이알 당 X 10 6 동결됩니다. 약 5 병 각 플라스크에서 고정 할 수 있습니다. </li><li> 대략 10-14 일 해마 문화를 설정하기 전에, glial 세포는 냉동 주식에서 도금된다. 우리는 일반적으로 해마 신경 세포의 90 커버 슬립 (물론 당 세 개의 15mm 라운드 커버 슬립)의 성장을 지원하기에 충분하다 다섯 6 - 웰 플레이트를, 접시. 우리는 또한 형질에 대한 조절 N2.1 매체에 사용되는 glial 세포의 추가 8 ~ 10 60mm의 요리를 접시. 하루는 신경 세포의 배양하기 전에, 세포 NB27 미디어가 공급되어야한다. 성상 세포는 이상적 일에서&gt; 90 % 합류해야한다지점입니다. 우리는 크게 동결하면 astroglial 문화에서 미세 아교 세포의 수를 줄일 것을 찾을 수 있습니다. 미세 아교 세포가 astroglial 문화에 존재하는 경우 증가 된 신경 독성이 관찰되기 때문에, 우리는 항상 신경 세포와 공 배양 냉동 주식에서 준비 astroglial 문화를 사용합니다. </li></ol> 2. 신경 세포 문화 <ol><li> 를 MilliQ 물 2 배 첫 번째 린스로 청소 유리 커버 슬립. 그 후, 2 시간의 전체에 걸쳐 적어도 5 회 24 시간 동안 진한 질산에 침지하고을 MilliQ 물 세정된다. coverslips는 6 시간 동안 225 ° C의 오븐에 굽고 건조 및 살균된다. </li><li> 전송 멸균 60mm 요리에 커버 슬립. 신경 세포 공 배양시 glial 세포에서 분리 유지하기 위해 각 coverslip에의 외부 가장자리 근처에 멸균 파라핀의 네 점을 놓습니다. </li><li> 37 ° C 배양기에서 하룻밤 0.1 M 붕산 버퍼 / ㎖ 폴리-L-라이신 1 MG와 코트 커버 슬립. </li><li> 다음 날, 멸균 m에 두 번 coverslips를 씻어적어도 15 분마다 illiQ 물. 그들은 다음 37 ° C 배양기에서 도금 미디어에서 하룻밤 배양된다. </li><li> 기흉 다음에 이소 플루 란 마취에 의해 임신 한 쥐 (E18)를 안락사, 그리고 잘린 배아 쥐를 안락사. 머리를 제거하고 상기 한 바와 같이 대뇌 반구를 해부하다. </li><li> 대뇌 반구에서 해마를 해부하다. 15 ML 원뿔 튜브에 모든 해마를 놓고 15 분 동안 37 ℃에서 0.25 % 트립신 (0.5 ml의 2.5 % 트립신, 4.5 ml의 BSS)과 소화. </li><li> 소화 해마는 5 분마다 BSS 배에 씻어서. 이들은이어서 유리 파스퇴르 피펫으로 분쇄하고, 세포 밀도는 혈구 계를 사용하여 결정된다. 뉴런이어서 60mm 디쉬 당 2 × 105 세포의 밀도로 시딩 하였다. </li><li> 대략 2-4 시간 도금 후, 전송이 신경 교세포으로 아래를 향하도록, astroglial 세포를 포함하는 요리에 연결된 뉴런의 coverslips. DIV3에서에 시토신 아라 비노 시드를 추가5 μM의 최종 농도에서 세포 glia의 증식을 정지합니다. </li></ol> 3. 칼슘 형질 <ol><li> glia의 형질 전날의 조건 N2.1 미디어. </li><li> 형질의 날, 아교 오프 에어컨 N2.1 미디어를 가지고 새로운 요리로 전송할 수 있습니다. 전송 에어컨 N2.1 미디어에 뉴런의 coverslips. 10 ~ 30 분 동안 인큐베이터에서 평형을 보자. </li><li> 멸균 튜브의 한 세트로, DNA의 1-4 μg, 100 μL의 총 볼륨에 대한 2 M 염화칼슘 12.5 μL, 멸균 H 2 O를 결합. 튜브의 두 번째 세트에 2 배 HBS 100 μl를 추가합니다. </li><li> 몇 초마다 볼 텍싱, 염화칼슘 / DNA 혼합물을 포함하는 튜브에 한번에 2X HBS의 ⅛ 체적 (12.5 μL)을 추가한다. 튜브가 15 분 동안 앉아 수 있습니다. </li><li> 15 분 배양 후, 세포에 형질 감염 혼합물 적가. 1.5 시간 동안 배양한다. 모래 같은 침전물 층이 볼 수 있어야합니다10X 목표를 사용하여 현미경. </li><li> 배양 후, 따뜻한 HBS 세척 버퍼로 두 번 커버 슬립을 씻어. </li><li> , 아교와 창작 요리로 커버 슬립을 반환 0.5 mm의 최종 농도 kynurenic 산을 추가합니다. </li><li> 형질 전환 된 뉴런의 이미지를 생성하거나 이미 다음 날 같은 면역 세포에 처리 할 수​​ 있습니다. 신경 세포는 형질 전환 후 3 주까지 살아남을 수 있습니다. </li></ol>

<ol>
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관심 없음 충돌 선언하지 않습니다.

주의 깊게 지속적으로 성공적인 형질 (10, 11)에 대해 제어 할 필요가 몇 가지 주요 매개 변수가 있습니다. 칼슘 포스페이트 형질 감염에 대한 가장 중요한 매개 변수는 우리 손에 보통 7.10-7.15 사이에서 변화 2X HBS의 pH 값이다. 우리는 산도 미터 사이의 차이를 설명 할 수는 0.05 단위로 pH 값과 주식의 세 가지 배치를하는 것이 좋습니다. 또한, 클론 테크 포유류의 형질 전환 키트는 지속적으로...

차 뉴런들이 postmitotic이며 마이크로 환경 변화에 매우 민감하다 같이 트랜 어려운 세포 유형의 하나이다. 이러한 세포 1 외인성 유전자와 짧은 헤어핀 RNA를 (shRNAs)의 표현 방법의 네 가지 일반적으로 사용되는 유형이있다. 각각은 자신의 장점과 단점이 있습니다. 세포 형질 감염을위한 큐벳에 전송해야 예를 들어, 전기 천공은 일반적 갓 절연 뉴런이 수행된다. 바이러스 감염은 일반적으로 매우 높은 효율 3를 달성하지만, 더 노동 집약적 인 운영과 위험하다 할 수 있습니다. 많은 지질 매개 형질 전환 시약은 신경 세포와 세포 독성의 다른 수준에있는 성공의 학위를 변화와 함께, 상업적으로 이용 가능하다. 인산 칼슘 형질 감염은 뉴런에 외래 유전자를 도입하기위한 편리하고 경제적 인 방법을 나타낸다. 첫번째 방법은 포유 동물 CEL에 아데노 바이러스 DNA를 도입하기 위해 사용 하였다그레이엄과 반 데르 EB를 (1973)에 의해 LS 4. 형질 포스페이트 완충액에서의 재조합 DNA와 염화 칼슘을 혼합함으로써 수행되었다. 이것은 서서히 세포 단층 상에 떨어질 때 DNA / 칼슘 포스페이트의 형성은 세포 표면에 부착, 엔도 사이토 시스에 의해 다루어 최종적 핵 5를 입력되며, 이는 석출물 허용한다. 이 과정은 표적 세포에 도입 된 외래 유전자의 발현을 초래할 것이다. 0.5-5 % 6-8 사이 인산 칼슘 형질 범위의 전형적인 효율성. 그러나, 조심 최적화 실험 프로토콜의 일관된 실행을, 거의 50 %의 형질 감염 효율에 도달 할 수있다. 여기에서 우리는 샌드위치 형식 9 astroglial 세포와 공 배양하는 기본 해마 신경 세포의 칼슘 형질 전환을위한 우리의 상세한 프로토콜을 설명합니다.

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		<tr height="24">
			<td height="24" style="height:24px;width:284px;">Dumont #5 forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11251-20</td>
			<td style="width:269px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Fine scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">14090-09</td>
			<td>straight / sharp 8.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Vannas-Tubinger spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15008-08</td>
			<td>angled / sharp / 8.5 cm / 5 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Standard pattern forceps</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">11000-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Student surgical scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">91401-14</td>
			<td>blunt / 14.5 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Spring scissors</td>
			<td style="width:207px;">Fine Science Tools</td>
			<td style="width:119px;">15006-09</td>
			<td>angled to side / 9 cm / 10 mm cutting edge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Isoflurane</td>
			<td style="width:207px;">Webster Veterinary</td>
			<td style="width:119px;">14043-225-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Poly-L-lysine</td>
			<td>Sigma</td>
			<td style="width:119px;">P2636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM</td>
			<td>Sigma</td>
			<td>M2279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">100 mM Sodium pyruvate</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Glucose</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10x HBSS</td>
			<td>Sigma</td>
			<td>H4385</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">1 M HEPES, pH 7.3</td>
			<td>Gibco/Invitrogen</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GlutaMAX</td>
			<td>Gibco/Invitrogen</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurobasal Media</td>
			<td>Gibco/Invitrogen</td>
			<td>21130-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">B27 Supplement (50x)</td>
			<td>Gibco/Invitrogen</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N2 Supplement</td>
			<td>Gibco/Invitrogen</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ovalbumin</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">A5503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">2.5% Trypsin</td>
			<td>Gibco/Invitrogen</td>
			<td style="width:119px;">15090-046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">DNase I</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">DN-25</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Cytosine arabinoside</td>
			<td style="width:207px;">Calbiochem/EMD Millipore</td>
			<td align="right" style="width:119px;">251010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:284px;">Kynurenic acid</td>
			<td style="width:207px;">Sigma</td>
			<td style="width:119px;">K3375</td>
			<td></td>
		</tr>
	</tbody>
</table>

calcium phosphate transfection of primary hippocampal neurons

인산 칼슘 침전은 배양 된 세포의 형질 전환을위한 편리하고 경제적 인 방법입니다. 최적화로, 일차 뉴런 같은 어려운 형질 세포에이 방법을 사용하는 것이 가능하다. 여기에서 우리는 astroglial 세포와 공 배양 된 해마 신경 세포의 칼슘 형질 전환을위한 우리의 상세한 프로토콜을 설명합니다.

형질의 다른 매개 변수를 최적화하고 신중하게 실험 실험에서 제어하는 경우, 최대 50 %의 형질 전환 효율을 얻을 수있다. 1 DIV4에 GFP 형질 전환 된 뉴런의 필드를 보여줍니다. 필드 (16)가 형​​질 전환 된 가운데 28 뉴런의 전체를 포함한다. 이것은 50 %의 효율을 나타냅니다. 동일한 커버 슬립에 다른 필드의 샘플링은 전체적인 효율 (데이터 미도시) 주변의 50 %이다. astroglia...

Watch this Scientific Journal Video about Calcium Phosphate Transfection of Primary Hippocampal Neurons at JoVE.com

Calcium Phosphate Transfection of Primary Hippocampal Neurons

차 해마 신경 세포의 칼슘 인산염 형질

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

calcium-phosphate-transfection-of-primary-hippocampal-neurons

Research

JoVE Journal

Neuroscience

25.1K 조회수.  Rutgers University.  인산 칼슘 침전은 배양 된 세포의 형질 전환을위한 편리하고 경제적 인 방법입니다. 최적화로, 일차 뉴런 같은 어려운 형질 세포에이 방법을 사용하는 것이 가능하다. 여기에서 우리는 astroglial 세포와 공 배양 된 해마 신경 세포의 칼슘 형질 전환을위한 우리의 상세한 프로토콜을 설명합니다. 

비디오: Calcium Phosphate Transfection of Primary Hippocampal Neurons

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 ...

연구

신경과학

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

구조적 조명 현미경을 사용하여 쥐 차 해마 신경 세포의 돌기 쪽 이미징

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

마우스 도파민 뉴런의 차 문화

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

하는 세포 칼슘의 형광 및 생물 발광 영상<sup&gt; 2 +</sup&gt; 노인 해마 신경 세포에서

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

낮은 밀도 차 해마 신경 세포 문화

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

교양된 Hippocampal 신경에 시 냅 스 소포 Endocytosis를 측정

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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 ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

축 사 초기 세그먼트의 조립을 공부 하는 기본 배양 된 해마 뉴런의 사용

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

배아 해마 뉴런의 냉동 재고를 사용한 쉽고 재현 가능한 저밀도 1차 배양

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the cytoplasm or the release of intracellular Ca2+ stores. Pirt-GCaMP3-based Ca2+ imaging of primary sensory neurons of the dorsal root ganglion (DRG) in mice offers the advantage of simultaneous measurement of a large number of cells. Up to 1,800 neurons can be monitored, allowing neuronal networks and somatosensory processes to be studied as an ensemble in their normal physiological context at a populational level in vivo. The large number of neurons monitored allows the detection of activity patterns that would be challenging to detect using other methods. Stimuli can be applied to the mouse hindpaw, allowing the direct effects of stimuli on the DRG neuron ensemble to be studied. The number of neurons producing Ca2+ transients as well as the amplitude of Ca2+ transients indicates sensitivity to specific sensory modalities. The diameter of neurons provides evidence of activated fiber types (non-noxious mechano vs. noxious pain fibers, A&#946;, A&#948;, and C fibers). Neurons expressing specific receptors can be genetically labeled with td-Tomato and specific Cre recombinases together with Pirt-GCaMP. Therefore, Pirt-GCaMP3 Ca2+ imaging of DRG provides a powerful tool and model for the analysis of specific sensory modalities and neuron subtypes acting as an ensemble at the populational level to study pain, itch, touch, and other somatosensory signals.

In Vivo Calcium Imaging of Neuronal Ensembles in Networks of Primary Sensory Neurons in Intact Dorsal Root Ganglia

This protocol describes the surgical exposure of the dorsal root ganglion (DRG) followed by GCaMP3 (genetically-encoded Ca2+ indicator; Green Fluorescent Protein-Calmodulin-M13 Protein 3) Ca2+ imaging of the neuronal ensembles using Pirt-GCaMP3 mice while applying a variety of stimuli to the ipsilateral hind paw.

생체 내 온전한 등근 신경절에 있는 일차 감각 뉴런 네트워크에서 뉴런 앙상블의 칼슘 이미징

Ca2+ imaging can be used as a proxy for cellular activity, including action potentials and various signaling mechanisms involving Ca2+ entry into the ...