This work was supported by the National Health and Medical Research Council [570930]. Imaging was carried out at the Bosch Institute Advanced Microscopy Facility. Former members of the lab, whose work is cited, are thanked for their contributions to developing these methods.

<ol>
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The authors declare they have no competing financial interests.

여기에 설명 된 프로토콜은 신뢰성있게 측정하고 정상 노화와 질병 상태를 포함한 조건의 범위에 걸쳐 NMJ의 특성의 변화를 정량화 할 수있게했다. EN에 대해 기재된 방법 NMJ 이미지 연구자 사전 및 시냅스 전문 분야의 오버랩 영역과 연접 / 배향의 면적과 비교 할 수 직면한다. 사전 및 시냅스 단백질 횡단 섹션을 사용하여 광 제 2 프로토콜의 상대 강도를 비교하기 위해 바람직하다. 세 번째 프로?...

신경 근육 접합 (NMJ)는 신경계와 골격 근육 사이의 통신을 매개하는 중요한 릴레이 시냅스이다. 그것은 모든 자발적인 운동이 필요합니다. 형광 현미경 긴 NMJ 1-3 마우스 유전자의 효과를 연구하기 위해 또는 설치류 4-11 NMJs시 연령,식이 요법, 운동 및 질환의 효과를 비교하기 위해 사용되었다. 이러한 연구는 NMJ의 생리학 및 병리학 소개 훨씬 가르친하지만 다양한 매개 변수 (예, ACHR 영역 종판 영역, 경계 길이, 분열 인덱스)가 종종 어렵게 이들 연구의 결과를 비교할 수 있도록보고. 전임상 연구는 특히 질병 (12)의 설치류 모델 연구에서, 재현성을 입증 할 수 있으려면 증가 기대가있다. 여기에 설명 된 프로토콜은, 생리 학적 및 병태 생리 학적 발달 CH 조사 일련의 연구를 통해 정제시켰다NMJ에 ANGES. 이러한 연구는 마우스 모터 종판에서 시냅스 전문화 영역과 시냅스 전문화 13-15 시냅스 내의 단백질의 패킹의 상대 밀도의 측정을 필요로한다. 이 방법의 유용성은 중증 근무력증 안티 사향 마우스 모델에서 최근 연구에 의해 예시된다. 안티 사향 양성 중증에서의 IgG 매일 ​​주사는 성인 마우스로 환자들을 2 주 (16) 내에서 약이 될 인한 근무력증. 근육 (신경 터미널에서) synaptophysin에 대한 이중 표지 된 섹션과 시냅스 AChRs의 공 초점 최대 프로젝션 이미지가 주 변화로 ACHR 염색 분야의 진보적 인 감소를 한 것으로 밝혀졌습니다. 감소의 중요한 비율은 시냅스 전위, 시냅스 전달 및 근육 약화 (17, 18)의 실패의 진폭에 비교 하락을 설명하기에 충분했다. 질적으로 비슷한 연구 결과는 다른 연구 그룹에 의해보고되었다10,19. NMJ 동일한 측정 방법은 사람이 마우스 모델 (20, 21)에 근무력증 안티 사향 중증의 치료 세 약물의 영향을 평가하기 위해 사용되어왔다. 정주의 노화는 신경 근육 연결의 손실을 초래할 수 있습니다. 마우스는 나이로 진행하면서 여기에 설명 된 프로토콜은 모터 종판에서 신경 터미널 시납의 영역에서 연령 관련 감소를 계시했다. 같은 방법은 자발적인 운동이 크게 다른 그룹 4로 이전의 연구와 일치하는 시냅스 전 신경 말단 영역 (22)의 감소를 방지 할 수있는 것으로 나타났습니다. 신경 근육 연결의 손실은 근 위축성 측삭 경화증 9,23의 SOD1G93A 마우스 모델에서 발생합니다. 상기 언급 된 연구들은 건강 상태의 다양한 NMJ에서 전후 시냅스 전문 하나의 영역에서 감소를 초래할 수 있음을 입증한다. 이것은 손상된 시냅스 재미가 발생할 수 있습니다ction의는 신경 근육 연결의 완전한 손실을 예고하거나. 세 가지 프로토콜은 지역과 시냅스 전문의 밀도의 정량을 허용하는 설명되어 있습니다. 제 1 프로토콜의 목적은 형광 현미경을 이용하여, 전후 및 포유 동물의 시냅스 전문 NMJs 그들의 배향의 영역의 실제적이고 재현 가능한 측정 값을 제공하는 것이다. 이차원 최대 투영 공 초점 이미지 및 NIH ImageJ에 함께 이미지 분석 시납 염색 (시냅스 소포), 시냅스 AChRs 시냅스 오버랩 영역의 면적의 변화를 감지하는 데 사용된다. 시냅스 전문 영역을 식별하는 데 사용되는 시각 정보를 최대화하기 위해 공 초점 촬상 파라미터 (이득 및 오프셋 레벨)이 각각 NMJ 최적화되어있다. 신경 근육 장애는 시냅스 ACHR 및 / 또는 기타 시냅스 단백질의 밀도의 변화로 발생할 수 있습니다. 제 2 프로토콜은 시냅스 단백질 등의 상대 밀도의 변화를 검출하도록 적용될 수있다사향, rapsyn, dystroglycan, 인산화의 Src 키나제 인산화 ACHR 18,21있다. 중증 근무력증에서 시냅스 막 내 ACHR의 감소 밀도는 시냅스 실패와 근육 약화의 직접적인 원인이다. 세 번째 프로토콜은 시냅스 막 (14, 15) 내에서 인접 AChRs의 근접의 변화를 평가하기 위해 형광 공명 에너지 전달 (FRET) 방법을 설명합니다. 이 방법은 형광-α-bungarotoxin (BGT)으로 표시 이웃 AChRs 사이의 에너지 전달을 검출한다. 형광 기증자 및 수용체 프로브는 10nm 이하 떨어져있는 경우 FRET가 발생합니다. 이것은 직접 시냅스 전위의 진폭에 관련 될 수있다 ACHR 포장의 압박감에서 (초 현미경) 변경을 표시 할 수 있습니다. 지난 10 년 동안 정제 된이 세 가지 프로토콜은 일관되고 재현 가능한 방식으로 NMJ 무결성의 보완을 제공합니다. 표준화 된 프로토콜의 사용ND 파라미터 NMJ 포유류 유전자, 환경에 따라 중재 효과 비교를 용이하게한다.

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			<td height="21" style="height:21px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:91px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:341px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Scanning confocal microscope</td>
			<td style="width:91px;">Leica</td>
			<td style="width:119px;">DM IRE2 with&#160; TCS SP2 system</td>
			<td>Most scanning confocal microscopes should be suitable.&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:91px;">Zeiss</td>
			<td>LSM 510 Meta&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;"></td>
			<td style="width:91px;">Leica</td>
			<td>SPE-II</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;">Alexa555-a-bungarotoxin (red-BGT)</td>
			<td style="width:91px;">Life technologies</td>
			<td style="width:119px;">B35451</td>
			<td>Used for labelling AChRs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Alexa647-&#945;-bungarotoxin (far-red-BGT)</td>
			<td style="width:91px;">Life technologies</td>
			<td>B35450</td>
			<td>Far red fluorescence: barely visible through the eyepiece&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">rabbit anti-synaptophysin</td>
			<td style="width:91px;">Life technologies</td>
			<td>18-0130</td>
			<td>Different batches of primary antibody differ in effective working dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">FITC-anti-rapsyn mab1234</td>
			<td>Milipore</td>
			<td style="width:119px;">FCMAB134F</td>
			<td>Monoclonal antibody conjugated to FITC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">FITC-donkey anti-rabbit IgG</td>
			<td style="width:91px;">Jackson</td>
			<td>711-095-152</td>
			<td>Polyclonal secondary antibodies can vary in quality according to source and batch</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optimal Cutting Temperature compound (O.T.C.)</td>
			<td style="width:91px;">ProSciTech</td>
			<td>IA018</td>
			<td>Cryostat embedding matrix for freezing&#160; muscles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DABCO</td>
			<td>Sigma</td>
			<td align="right">10981</td>
			<td>Mounting medium that slows photobleaching of fluorophors</td>
		</tr>
	</tbody>
</table>

the neuromuscular junction: measuring synapse size, fragmentation and changes in synaptic protein density using confocal fluorescence microscopy

신경 근육 접합 (NMJ)은 포유 동물의 운동 신경이 자발적 근육 수축을 제어되는 큰, 콜린성 릴레이 시냅스이다. NMJ에서 구조 변경 약화, 위축과 근육 섬유의 사망을 초래, 신경 전달 오류가 발생할 수 있습니다. 많은 연구는 유전자 변형 또는 질환 마우스 NMJ의 구조를 변경할 수있는 방법을 연구 하였다. 불행히도, 직접들은 종종 다른 파라미터 분석 방법을 사용하기 때문에 이들 연구에서 조사 결과를 비교하기 어려울 수있다. 세 개의 프로토콜이 설명되어 있습니다. 제는 아세틸 콜린 수용체 (ACHR)의​​ 면적을 측정하는 최대 강도 투영 공 촛점 이미지를 사용하여 단부 판과 상부 시냅스 전 신경 말단의 시냅스 소포 염색이 풍부한 영역에서 시냅스 막 도메인. 두 번째 프로토콜은 시냅스 막 시냅스 단백질에 대한 면역 염색의 상대적 강도를 비교합니다. 세 번째 홍보otocol은 엔드 플레이트에서 시냅스 AChRs의 포장의 변화를 감지하는 형광 공명 에너지 전달 (FRET)를 사용합니다. 프로토콜이 개발 및 일련의 연구를 통해 정제되었다. 품질과 결과의 일관성에 영향을 미치는 요인에 대해 설명하고 규범 적 데이터는 건강한 젊은 성인 마우스의 NMJs 제공됩니다.

NMJ에서 시냅스 지역 측정 영역의 모든 추정치는 시냅스 전문의 범위를 정의하는 경계의 도면에 의존. 건강한 젊은 성인 근육에 NMJ 이미지 ACHR과 synaptophysin에 염색 (그림 2A와 B) 모두 잘 정의 된 경계를 표시해야합니다. ACHR 및 시납 모두 형광 강도는 모터 엔드 플레이트 (도 5a &#39;및 B&#39;)의-주위염 시냅스와 시냅스 사이의 경...

Watch this Scientific Journal Video about The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy at JoVE.com

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

신경 근육 접합 : 측정 시냅스 크기, 공 초점 형광 현미경을 사용하여 시냅스 단백질 농도의 분열 및 변경

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

the-neuromuscular-junction-measuring-synapse-size-fragmentation

Research

JoVE Journal

Neuroscience

21.4K 조회수.  University of Sydney. The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

비디오: The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

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 ...

연구

신경과학

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

의 활성 영역의 해부 및 이미징 Drosophila 신경근육학 융티온

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

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 ...

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

연체 동물에서 세포 내 칼슘 농도와 시냅틱 효능의 모니터링 변경<em&gt; Aplysia</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

연속 신경 근육 분기점에서 싱글 Schwann 세포를 윤곽을 그리다하는 사진 표백

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development.
Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.

Detection of In Situ Protein-protein Complexes at the Drosophila Larval Neuromuscular Junction Using Proximity Ligation Assay

This protocol demonstrates how Proximity Ligation Assay can be used to detect in situ protein-protein interactions at the Drosophila larval neuromuscular junction. With this technique, Discs large and Hu-li tai shao are shown to form a complex at the postsynaptic region, an association previously identified through co-immunoprecipitation.

검출<em&gt; 현장에서</em상기&gt; 단백질 - 단백질 복합체<em&gt; 초파리</em근접 내고 분석을 사용하기&gt; 애벌레 신경 근육 접합부

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval ...

Loading a Calcium Dye into Frog Nerve Endings Through the Nerve Stump: Calcium Transient Registration in the Frog Neuromuscular Junction

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using calcium-sensitive fluorescent dyes that change their emission intensity or wavelength depending on the concentration of free calcium in the cell. There are several methods used to stain cells with calcium dyes. Most common are the processes of loading the dyes through a micropipette or pre-incubating with the acetoxymethyl ester forms of the dyes. However, these methods are not quite applicable to neuromuscular junctions (NMJs) due to methodological issues that arise. In this article, we present a method for loading a calcium-sensitive dye through the frog nerve stump of the frog nerve into the nerve endings. Since entry of external calcium into nerve terminals and the subsequent binding to the calcium dye occur within the millisecond time-scale, it is necessary to use a fast imaging system to record these interactions. Here, we describe a protocol for recording the calcium transient with a fast CCD camera.

Here, we describe a method for loading a calcium-sensitive dye through the frog nerve stump into the nerve endings. We also present a protocol for the recording and analysis of fast calcium transients in the peripheral nerve endings.

칼슘 염료를 개구부 신경 절편에 삽입하기 : 신경 개구부를 통해 개구리 신경 근육 계통에서 칼슘 과도 등록

One of the most feasible methods of measuring presynaptic calcium levels in presynaptic nerve terminals is optical recording. It is based on using ...

Measuring Neuromuscular Junction Functionality

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is impaired, such as aging and amyotrophic lateral sclerosis (ALS). Here we describe an experimental protocol that can be used to measure NMJ functionality by combining two types of electrical stimulation: direct muscle membrane stimulation and the stimulation through the nerve. The comparison of the muscle response to these two different stimulations can help to define, at the functional level, potential alterations in the NMJ that lead to functional decline in muscle.
Ex vivo preparations are suited to well-controlled studies. Here we describe an intensive protocol to measure several parameters of muscle and NMJ functionality for the soleus-sciatic nerve preparation and for the diaphragm-phrenic nerve preparation. The protocol lasts approximately 60 min and is conducted uninterruptedly by means of a custom-made software that measures the twitch kinetics properties, the force-frequency relationship for both muscle and nerve stimulations, and two parameters specific to NMJ functionality, i.e. neurotransmission failure and intratetanic fatigue. This methodology was used to detect damages in soleus and diaphragm muscle-nerve preparations by using SOD1G93A transgenic mouse, an experimental model of ALS that ubiquitously overexpresses the mutant antioxidant enzyme superoxide dismutase 1 (SOD1).

A functional assessment of the neuromuscular junction (NMJ) can provide essential information on the communication between muscle and nerve. Here we describe a protocol to comprehensively evaluate both the NMJ and muscle functionality using two different muscle-nerve preparations, i.e. soleus-sciatic and diaphragm-phrenic.

Neuromuscular junction (NMJ) functionality plays a pivotal role when studying diseases in which the communication between motor neuron and muscle is ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

시 냅 스 밀도 Hippocampal 설치류 두뇌 분할 영역에서의 평가

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

초파리 애벌레 신경 근육 학 교차로에서 시 냅 스 전류의 초점 Macropatch 녹음

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...