Авторы хотели бы признать, что эта работа была поддержана естественным наукам и инженерным исследовательского совета Канады (NSERC) гранта.

<ol>
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Авторы не имеют ничего раскрывать.

Этот протокол описывает использование различных видов микроскопии в изображение клеточных делений в течение середине эмбриогенеза. В частности, этот протокол подчеркивает, как изображение разделение нейробластах, клетки, которые могут способствовать эпидермальный морфогенез. Межк?...

В то время как существуют протоколы, описывающие, как разделение изображения клеток в начале C. Элеганс эмбрионов, этот протокол описывает, как разделение изображения клеток в ткани во время середине эмбриогенеза. Одна из основных проблем в визуализации организмов в процессе разработки была их чувствительность к фототоксичности. Тем не менее, расширение доступности к вращающемся диске конфокальной микроскопов или прокатилась микроскопов полевых позволило более широкое применение визуализации. Обе системы используют твердотельные лазеры и рассеянный свет, ограничивая уровни УФ, что организмы подвергаются. Тем не менее, Widefield стенды-прежнему можно использовать для работы с изображениями в естественных условиях, особенно если они оснащены камерами с высокой чувствительностью (например EMCCD), управление диафрагмой и управления освещением (например, светодиодов или регулируемые ламп ртуть). Этот протокол описывает, как использовать либо систему конфокальной основе или системы с широким полем зрения до разделения изображений клеток в развитии С. Элеганс &lt;/ EM&gt; эмбрионы. В качестве примера, мы опишем, как изображение деление нейробластов клеток. Нейробласты может способствовать эпидермальный морфогенез, предоставляя химические или механические сигналы в клетки эпидермиса вышележащих, и обеспечить отличный пример важности межклеточной коммуникации в формировании тканей. Caenorhabditis Элеганс является идеальным модельным организмом для исследований микроскопии, основанные из-за его прозрачности и простой организации ткани 1. Кроме того, С. Элеганс поддается генетических методов и RNAi, и так как многие из его генов у человека гомологи, он может быть использован для идентификации консервативных механизмов для формирования ткани 2-5. В С. Элеганс, формирование эпидермиса происходит в течение середины эмбриогенеза, когда эмбрион имеет&gt; 300 клеток. Эпидермального морфогенез состоит из нескольких основных этапов, в течение которых эмбрион закрытых в слое клеток эпидермиса, что сужают и расширяют превратить EMBRлет от яйцевидной формы в продолговатой формы червя 6. Брюшной корпус описывает один из этих морфогенетических событий, когда брюшные клетки эпидермиса мигрируют в направлении вентральной срединной линии, чтобы покрыть вентральной поверхности эмбриона (рис. 1). Во-первых, две пары передних расположенный передней кромки клеток мигрировать в направлении вентральной средней линии, где они прилипать и плавким предохранителем с их соседей контралатеральной 6. Это сопровождается миграции задних расположен карман клеток, которые образуют клин как форм, создающих вентральной карман 6-7. Механизм, который закрывает карман не очень хорошо понял. Одной из возможностей является то, что supracellular актина структура миозина сократительная связывает карманные клетки вместе в кошелек строки, как мода, подобно ранозаживляющее 8. Интересно, что миграция некоторые из кармана клеток опосредовано специфическими подмножеств, лежащих в основе нейробластов 9 (нейрональных предшественников, которые находятся под epidermiс; фиг.1В). Предыдущие исследования показали, что нейробласты регулировать вентральной миграцию клеток эпидермиса и вентральной корпус. VAB-1 (эфрин рецептор) и VAB-2 (эфрин лиганд) экспрессируются на высоком уровне в нейробластах и облегчить сортировку передних и задних нейробластах друг от друга, и мутации в VAB-1 или VAB-2 вызывают брюшной корпуса фенотипы 10-13. Тем не менее, промоутер спасательные эксперименты показали, что VAB-1 также требуется в вышележащих брюшных клеток эпидермиса и рецепторов для других сигналов наведения секретируемых нейробластах выражаются в вентральной клеток эпидермиса 9. Хотя мутации в любом из этих рецепторов вызывает брюшной фенотипа шкафа, это не ясно, если дефекты возникают из-за проблем в нейробласта позиционирования или из-за отказа вентральных клеток эпидермиса реагировать на сигналы наведения 14. Изменение разделение нейробласты Wiства без сказывается на их способности выделять сигналы наведения могли бы пролить свет на роль нейробластов и их способности обеспечивать механическую вход во эпидермального морфогенеза. Недавно было обнаружено, что ген, деление клеток, ани-1 (anillin) экспрессируется на высоком уровне в нейробластов (рис. 2а) и его истощение вызывает нейробластов деление дефектов. Интересно, что эти эмбрионы отображения брюшной фенотипа шкафа (Fotopoulos, Wernike и Piekny, неопубликованные данные). Anillin требуется для деления клеток, и особенно для цитокинезе, которая описывает процесс, в котором материнская клетка физически делит на две дочерние клетки. Цитокинез движет формированию сократительной кольца актомиозинового, который должен быть под жестким контролем в пространстве и времени, чтобы убедиться, что он правильно подключен с сестринских хроматид сегрегации. Главный регулятор многоклеточных цитокинезе является RhoA (Ро-1 в C. Элеганс), небольшой ГТФ, что являетсяCTIVE в ГТФ-связанной форме. ГЭФ Ect2/ECT-2 активирует RhoA, после чего RhoA-GTP взаимодействует с нижестоящих эффекторов, которые формируют сократительную кольцо и опосредуют его втеканию 15. Anillin является мульти белок домена, который связывается с RhoA через его С-конце и с актином и миозином через его N-конце. Anillin требуется, чтобы стабилизировать положение сократительной кольца в млекопитающих или Drosophila S2 клетках 16. Истощение Anillin вызывает сократительные кольца пройти боковые колебания, и в конечном итоге терпит неудачу цитокинез формирования многоядерных клеткам 17-19. Интересно, что хотя С. Элеганс ани-1 координат актомиозина сократимость в ранних эмбрионов, это не является необходимым для цитокинеза. Однако, как описано выше, ани-1 требуется для нейробластов цитокинеза в течение середины эмбриогенеза (Fotopoulos, Wernike и Piekny, неопубликованные данные). Отказ Цитокинез бы менять количество и положение нейробластах и ​​может повлиять на Locданию химических сигналов наведения, или он может изменить механические свойства ткани. Обе модели выделить неавтономную роль нейробластах для брюшной корпусе, и важность ткани ткани связи во время эмбрионального развития. Это экспериментальный протокол описывает, как разделение изображений клеток во время С. Элеганс середине эмбриогенеза с помощью флуоресцентной микроскопии. Большинство экспериментов по изучению механизмов клеточного деления были выполнены в единичных клеток в культуральные чашки (например. HeLa или S2 клетках) или в ранних эмбрионов с ограниченным числом клеток (например, C. Элеганс один клеток эмбриона, Xenopus, или иглокожих эмбрионы). Тем не менее, важно также изучить деление клеток в тканях, так как есть внешние сигналы, которые могут влиять на выбор времени и размещение разделительной плоскости. Кроме того, клетки могут обеспечить химические или механические сигналы, чтобы влиять на развитие соседних тканейс, и очень важно, чтобы понять, как межклеточное общение помогает тканей с образованием в процессе разработки.

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			<td height="21" style="height:21px;width:291px;">Agar</td>
			<td style="width:247px;">BioShop Canada Inc.</td>
			<td style="width:185px;">#AGR001.1</td>
			<td style="width:335px;">For making C. elegans NGM and RNAi plates</td>
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			<td height="21" style="height:21px;">Agar</td>
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			<td>Store powder and dissolved IPTG at -20 &#176;C</td>
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			<td>For optimal results coat microscope slides three times&#160;</td>
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			<td>Store powder at 4 &#176;C and dissolved tetracycline at -20 &#176;C</td>
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			<td>CAS#9005-64-5</td>
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visualizing neuroblast cytokinesis during c. elegans embryogenesis

Этот протокол описывает использование флуоресцентной микроскопии для изображения делящихся клеток в развитии Caenorhabditis Элеганс эмбрионов. В частности, этот протокол фокусируется на том, как изображение деления нейробластов, которые находятся под клеток эпидермиса и могут иметь важное значение для эпидермального морфогенеза. Формирование тканей имеет решающее значение для развития многоклеточных и полагается на внешние сигналы из соседних тканей. С. Элеганс является отличным модельным организмом для изучения тканей морфогенез в естественных условиях из-за его прозрачности и простой организации, что делает его ткани легко изучать с помощью микроскопии. Брюшные корпус является процесс, в котором вентральной эмбриона покрыта одним слоем эпителиальных клеток. Это событие, как считается, способствует основных нейробластах, которые обеспечивают химические сигналы наведения посредничать миграцию вышележащих эпителиальных клеток. Тем не менее, нейробласты весьма пролиферативного а также могут действоватьв качестве субстрата для механической вентральных клеток эпидермиса. Исследования с использованием этого экспериментальный протокол может раскрыть важность межклеточной коммуникации во время формирования ткани, и могут быть использованы для выявления роли генов, участвующих в клеточном делении в развивающихся тканях.

Это экспериментальный протокол описывает, как разделения изображения клеток в С. Элеганс эмбрионов во время середине эмбриогенеза. В частности, он описывает, как изображения нейробластов, которые могут способствовать эпидермальный морфогенез. Эпидермального морфогенез происхо?...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Этот протокол описывает, как изображение делящихся клеток в ткани в Caenorhabditis Элеганс эмбрионов. В то время как несколько протоколов описывают, как разделение изображения клеток в раннем эмбрионе, этот протокол описывает, как изображение деление клетки в развивающемся ткани во время середины позднего эмбриогенеза.

Визуализация нейробласта Цитокинез Во<em&gt; С. Элеганс</em&gt; Эмбриогенеза

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

visualizing-neuroblast-cytokinesis-during-c-elegans-embryogenesis

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Neuroscience

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

11.7K Views.  Concordia University.  Этот протокол описывает, как изображение делящихся клеток в ткани в Caenorhabditis Элеганс эмбрионов. В то время как несколько протоколов описывают, как разделение изображения клеток в раннем эмбрионе, этот протокол описывает, как изображение деление клетки в ...

Video: Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Опробование β-амилоида Токсичность использованием трансгенных С. Элеганс Модель

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

Элеганс C. Положительные обучения бутанон, краткосрочный и долгосрочный Ассоциативные Анализы памяти

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

В естественных условиях лазерной Axotomy на языке C. Элеганс

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Автоматизированная количественная оценка Synaptic флуоресценции в<em&gt; С. Элеганс</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

C. Элеганс отслеживания и поведенческих измерений

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

Молекулярная Считывание Долгосрочные обонятельной адаптации в<em&gt; C. Элеганс</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Гиппокампа Микроинъекция инсулина и<em&gt; В естественных условиях</em&gt; Микродиализ Во время тестирования пространственной памяти

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

В естественных нейронных изображений кальция в C. Элеганс

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

Nucleofection из грызунов нейробласты по изучению нейробласта миграции<em&gt; В пробирке</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

В естественных изображений из Dauer конкретным нейронов ремоделирования в C. Элеганс

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...