저자는이 작업을 실행하는 동안 도움이 토론의 Harata 실험실의 구성원을 감사드립니다. 이 작품은 미국 심장 협회 (American Heart Association), 근긴장 의학 연구 재단, 에드워드 말린 크로, 주니어 재단, 국립 과학 재단 (National Science Foundation), 그리고 NCH에 화이트 홀 재단의 교부금에 의해 투자되었다

저자는 더 경쟁 재정적 이익을 선언하지 않습니다.

우리는 자연과 소형 시냅스 활동을 유발하고, destaining 단계에서 이미징에 대한 응답으로 시냅스 소포를 염색하고 destaining을위한 프로토콜을 설명했다. 기존 프로토콜 이외에, 우리는 미니어처 시냅스 활성에 근거 FM의 destaining 관찰의 새로운 프로토콜을 포함했다. 이러한 프로토콜을 사용하여, 우리는 이전에 운동 장애 근긴장의 마우스 모델에서 배양 된 신경 세포의 이상을 확인했다. 높은 활성 ...

시냅스 소포의 기능은 시냅스 전달의 중요한 결정 요인이다. 그들은 시냅스 세포막 (세포 외 유출)와 융합 할 때이 소포는 신경 전달 물질을 방출, 그들은 세포막 (엔도 사이토 시스)에서 재생과 신경 전달 물질로 다시로드 된 후 릴리스의 또 다른 사이클에 대한 준비가되어 있습니다. 의 역학과 시냅스 소포 재활용을 기본 메커니즘에 대한 연구는 크게 스티 FM 염료 (1)의 도입에 의해 가속화되었다. 긍정적으로 (그림 1A, 그림 1B의 FM1-43 stereoview에서 여러 염료) 친수성 머리 그룹과 소수성 꼬리가 청구 한이 양친 매성 분자는 가역적으로 입력을 침투하지 않고 지질 세포막을 종료 할 수 있습니다. FM 염료의 그룹들이 방출하는 빛의 범위에 영향을 미치는 유사한 기능을 공유 할 수 있습니다. 예를 들어, FM2-10, FM1-43, 및 FM1-84는 두 개의 고리 형 화합물과 슈 사이에 하나의 이중 결합이w 녹색 방출. 그들 사이의 차이는 소수성을 결정하고, 따라서 멤브레인으로부터 출구의 비율 (departitioning) 소수성 꼬리의 길이이다. FM5-95 및 FM4-64의 경우, 세 개의 이중 결합 사이 클릭 화합물을 연결, 그들은 적색 발광을 보여줍니다. 이 염료는 친수성 ​​부분에 대한 차이가 있습니다. 그들은 인해 친수성 ​​환경 소수성 환경 상대 양자 수율 증가, 생체막에 삽입 될 때 모든 FM 염료에서, 형광 강도가 증가한다. 따라서 FM 강도의 변화는 막 회전율의 변화를 나타냅니다. 다른 색 (발광 스펙트럼)과 hydrophobicities는 FM이 시냅스 소포 재활용의 다양한 연구 도구 염료합니다. 이러한 기능을 바탕으로, FM 염료는 대부분 시냅스 소포 재활용 분석을 다음과 같은 방식 (그림 2)에 따라 사용됩니다. 신경그들은 엔도 시토 시스 (염색)을 통해 형태로의는 시냅스 소포 (의 SV)에 채택되기에 가능, FM 염료를 포함하는 세포 외 용액에 목욕을합니다. 염료는 염료가없는 세포 외 용액을인가함으로써 세척하고, 이것은 즉 만 활발히 진행 엔도 시토 시스는 염료 (도 2 하단)으로로드 된 시냅스 소포의 클러스터를 포함 할 기능적 신경 말단을 보여준다. 이후의 세포 외 유출은 세포 외 공간으로 FM 염료의 손실과 형광의 수반하는 손실로 연결 (destaining; 인해 친수성 ​​환경에 departitioning 멀리 세포 외 유출의 사이트에서 확산 둘 다). 따라서 FM의 형광 강도의 변화는 시냅스 소포 엑소와 엔도 사이토 시스의 지표입니다. FM 염료는 다양한 생물과 준비 2,3에있는 시냅스 소포를 얼룩 destain하는 데 사용되었습니다. 예를 들면 포유류 neur 포함ONAL 문화 4-9, 포유류의 뇌 조각 10, 11, 신경 근육 접합부 (12, 13), 망막 바이폴라 뉴런 14, 15, 및 16 달팽이관의 유모 세포. 일반적으로 실험, 염색 및 destaining 모두 광범위 뉴런 (유발 활동을) 자극에 의​​해 트리거됩니다. 외부 자극 (자연과 소형 시냅스 활동) 9,17-19의 부재에서 재활용을 가지고 최근에, 그러나, 약한 자극에 대한 응답으로 시냅스 소포 재활용도 분석하고있다. 자연과 소형 시냅스 활동은 활동 전위의 자연 발화 (그림 3)을 포함하는 이전에, 외부 자극의 부재에서 발생하는 것과 같이 정의된다. 이러한 약한 시냅스 활동은 다양한 자극에 의​​해 트리거보다 FM 신호의 작은 변화와 관련되어있다. 측정을 요구하는 FM의 fluoresc의 변화줬어 강도는 정확하게 시냅스 소포 세포 외 유출 또는 엔도 시토 시스하지만 강도 인공적하지 않은 변경 사항을 반영합니다. 이슈의 원인 중 하나는 FM 염료에 의한 세포막의 비특이적 염색의 존재이다. 이 구성 요소의 점진적 유실이 잘못 시냅스 활동에 기인한다 측정 된 형광 강도의 점진적인 변화로 이어질 것입니다. 이 계수는 (프로토콜 참조) 적절한 방법에 의해 감소​​ 될 수있다. 이슈의 가장 주목할만한 원인은 시냅스 소포 내에 유지 FM 염료의 광표백입니다. FM 강도 광표백 관련된 변화를 측정하는 생물 (시냅스)의 변화에​​ 비해 작아야한다. 민감한 카메라의 최근의 개발은, 예를 들면 전자 승산 전하 결합 소자 (EMCCD) 카메라가 가능한 노광 시간을 단축하고 형광체를 여기 시키는데 사용되는 빛의 강도를 약화 photobleaching에 의해 최소화 할 수있다. 이슈의 또 다른 원인은 드리프트의 iN 광학 현미경의 초점 수준. 촬상 세션 동안 포커스 드리프트는 기계적 또는 열 효과에 의해 야기 될 수 있고, 잘못 측정 된 형광 강도의 변화로 이어질 것이다. 여기에서 우리는 가능한 특히, 소형 시냅스 활성, 약한 또는 전혀 자극의 컨텍스트에서 시냅스 소포 재활용을 분석하는 FM 염료를 사용할 수 있도록 프로토콜 및 장비를 설명한다. 우리는 배양 된 쥐의 해마 신경 세포를 사용하고 destaining 단계 이미징, 유발과 자발적인 시냅스 이벤트 기간 동안 소포의 염색과 destaining의 예를 보여줍니다. 또한 인하여 시냅스 활동에 대한 FM 염료 손실의 부재에서, FM 염료 광표백의 정도를 평가하는 방법을 설명한다.

<table border="0" cellpadding="0" cellspacing="0" width="1767">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Pulse generator</td>
			<td>AMPI</td>
			<td>Master-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolated stimulator</td>
			<td>Digitimer</td>
			<td>DS3</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>This is used for assessing the cell morphology at low magnification.</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TiE</td>
			<td>This is used for high-resolution fluorescence and transmitted light imaging, with minimal focus drift.</td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Nikon</td>
			<td></td>
			<td>Water-immersion lens is recommended. Oil-immersion lens is usable unless an imaged structure is deep from the coverslip surface (e.g. &#62;10 &#956;m).</td>
		</tr>
		<tr>
			<td>Filter cube</td>
			<td>Nikon</td>
			<td>77032509</td>
			<td>490/20-nm ex, 510-nm dclp, 520-nm-LP em for FM1-43</td>
		</tr>
		<tr>
			<td>Filter cube</td>
			<td>Nikon</td>
			<td>77032809</td>
			<td>490/20-nm ex, 510-nm dclp, 650-nm-LP em for FM4-64.&#160;</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor Technology</td>
			<td>iXon EM+ DU-860</td>
			<td>This EMCCD camera is used for high-sensitivity detection of fluorescence.</td>
		</tr>
		<tr>
			<td>Liquid recirculating chiller</td>
			<td>Solid State Cooling Systems</td>
			<td>Oasis 160</td>
			<td>This is used for continuously perfusing the camera with chilled water for maintaining a temperature of -80&#176;C, and thereby reducing noise.</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>CoolLED-Custom Interconnect</td>
			<td>490 nm&#160;</td>
			<td>This light source is used for rapid on/off control of fluorescence excitation.</td>
		</tr>
		<tr>
			<td>Image acquisition software</td>
			<td>Andor Technology</td>
			<td>Solis</td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging chamber</td>
			<td>Warner Instruments</td>
			<td>RC-21BRFS</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast perfusion system</td>
			<td>Warner Instruments</td>
			<td>SF-77B</td>
			<td></td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris Bioscience</td>
			<td>1045</td>
			<td></td>
		</tr>
		<tr>
			<td>D,L-AP5</td>
			<td>Tocris Bioscience</td>
			<td>0106</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris Bioscience</td>
			<td>1069</td>
			<td>Caution: toxic reagent. Handle with care.</td>
		</tr>
		<tr>
			<td>FM1-43</td>
			<td>Invitrogen</td>
			<td>T35356</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde-fixable FM1-43 (FM1-43FX)</td>
			<td>Invitrogen</td>
			<td>F35355</td>
			<td></td>
		</tr>
		<tr>
			<td>FM4-64</td>
			<td>Invitrogen</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>Aldehyde-fixable FM4-64 (FM4-64FX)</td>
			<td>Invitrogen</td>
			<td>F34653</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma-Aldrich</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#8217; balanced salt</td>
			<td>Sigma-Aldrich</td>
			<td>H2387</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium</td>
			<td>Invitrogen</td>
			<td>51200-038</td>
			<td>This solution does not contain phenol red that will interfere with fluorescence imaging.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Caution: toxic reagent. Handle with care.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
	</tbody>
</table>

examination of synaptic vesicle recycling using fm dyes during evoked, spontaneous, and miniature synaptic activities

기능 신경 말단에서 시냅스 소포는 세포 외 유출 및 엔도 시토 시스를 받고있다. 이 시냅스 소포 재활용 효과적으로 막 매출을 공개 스티 FM 염료를 사용하여 분석 할 수 있습니다. FM 염료의 사용을위한 종래의 프로토콜이 자극 (유발) 시냅스 활성 다음 뉴런을 분석을 위해 설계되었다. 최근 프로토콜은 자연 또는 소형 시냅스 이벤트와 같은 약한 시냅스 활동을 함께 FM 신호 분석을위한 유효하게되었다. FM 신호의 이러한 작은 변화의 분석 영상 시스템은 강도의 작은 변화를 감지하기에 충분히 민감하다, 그러나 큰 진폭의 인공적인 변화가 억제되어 있어야합니다. 여기서 우리는, 유발 자발 및 미니어처 시냅스 활동, 예를 들어 배양 된 해마 뉴런을 사용하여 적용 할 수있는 프로토콜을 설명한다. 이것은 중요하다으로서이 프로토콜은 또한, FM 염료의 광표백의 속도를 평가하는 수단을 포함강도의 작은 변화 이미징 유물의 소스.

예를 들어, 우리는 시냅스 소포의 destaining 시간 코스 (그림 4)에 대한 대표적인 결과를 보여줍니다. 배양 된 해마의 신경 세포가 자발적 시냅스 활동 (단계 2.3)를 사용하여 FM4-64로 염색 및 염료 무료 솔루션 (솔루션 2 린스)로 세척 하였다. 영상은 자발적인 활동 (단계 5.3) (연속 라인의 초기 부분, 그림 4A)를 사용하여 초기 destaining 시간의 과정을 보여줍니다. 이것은 120 초 동...

Watch this Scientific Journal Video about Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities at JoVE.com

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous and Miniature Synaptic Activities | Neuroscience | Video

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

우리는 기능적 신경 말단에서 화상 시냅스 소포 재활용 스티 릴 FM 염료의 사용을 설명한다. 이 프로토콜은 없습니다 만 유발하는 것이 아니라 또한 자연과 소형 시냅스 활동에 적용 할 수 있습니다. 프로토콜은 효과적으로 평가할 수 시냅스 이벤트의 다양한 확장한다.

유발, 자발적, 미니어처 시냅틱 활동을하는 동안 FM 염료를 사용하여 시냅틱 소포 재활용의 시험

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

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20.9K 조회수.  University of Iowa Carver College of Medicine.  우리는 기능적 신경 말단에서 화상 시냅스 소포 재활용 스티 릴 FM 염료의 사용을 설명한다. 이 프로토콜은 없습니다 만 유발하는 것이 아니라 또한 자연과 소형 시냅스 활동에 적용 할 수 있습니다. 프로토콜은 효과적으로 평가할 수 시냅스 이벤트의 다양한 확장한다. 

비디오: Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Styryl 염료의 Photoconversion를 사용하여 시냅틱 소포 풀 유학

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

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Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

FM 염료를 사용하여 양식 소뇌 과립 뉴런에서 시냅틱 소포 풀 보충의 정량 분석

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

교양 뉴런의 막 단백질의 측면 보급과 Exocytosis은 형광 복구 및 형광 손실 Photobleaching를 사용하여 평가

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

성상 세포 운송업자 전류의 디컨 볼 루션 분석 글루타민산 정리의 시간 과정을 도출

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

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 ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

급성 Hippocampal 조각에 작은 볼륨 재활용-, 관류-침수 형 챔버 시스템 유지에 시 냅 스가 소성을 기록

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

Vivo에서경작-Murine 이다 빠르고, 간단 하 고 저렴 한 AWESAM 프로토콜을 사용 하 여 같은

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

연 접 기능 그대로 신경 회로 내에 유전자 최저 효과 특성 인공 microRNAs Optogenetics 결합

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

시 냅 스 소포 교양된 신경 연 접 단백질 Overexpressing에 있는 재활용에 대 한 광학 분석 결과

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...