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

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

태아 쥐의 Hippocampal 신경 세포의 분리 및 문화

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) 1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis 10. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) 3, 5-7, 9, 11. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic 3, 5-9, 11. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.
Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency 12. Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls 12. The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions 13. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions 9.
In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.

A rapid approach to investigate interactions and effects on molecular mechanisms related to the presence of antibodies in an intracellular environment is described. The method involves transfection of antibodies into live cells using a non-covalent complex formation based on a lipid formulation. The technique is adaptable to immortalized cell lines and primary cells.

질병 Pathogenesis를 공부하는 도구로 뉴런에 항체 Transfection

Antibodies provide the ability to gain novel insight into various events taking place in living systems.  The ability to produce highly specific ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

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 ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

꼬마 선 충 을 행동에 개별 뉴런의 비율 칼슘 이미징

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...