Работа в лаборатории Hammarlund финансируется за счет: NIH гранты R01 NS066082-01 и T32GM007223, Бекман Фонда, и Эллисон Медицинский фонд.

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Нет конфликта интересов объявлены.

Различные лазерные системы были использованы, чтобы сократить нейритов в C. Элеганс, и несколько исследований изучили их работы подробно 3,7,10,11. Лазерной Micropoint используется в нашей протокол под ключ системы, которая проста в установке и обслуживании, и доступен по низкой цене для исследователей, по сравнению с Ti-Sapphire лазерной системы. По сравнению с Ti-сапфир система, однако, лазерная Micropoint, как ожидается, привести к повреждению большей площади, которые могут быть невыгодными для некоторых приложений. Если Ti-сапфир системы желательно, отличный протокол о создании такой системы доступна 12. Текущего протокола могут быть выполнены на различных нейронах C. Элеганс, однако, отметим, что различия в регенеративной способности между различными типами нейронов, как ожидается 13. Кроме того, различные фоны трансгенных может повлиять регенеративной успеха. Хотя процент регенерации нейронов ГАМК достаточно последовательно между различными трансгенных линий маркера, мы уже отмечали общее небольшое увеличение регенерации в juIs76 14 против 15 oxIs12 червей. Различия между маркерами сенсорным нейронам также были описаны 2. Мы предпочитаем, чтобы обездвижить черви с микрошарики, а не в качестве анестетиков бисером приведет к более быстрому и более последовательной иммобилизации 16. Это также выгодно, как мы можем наблюдать регенерации без каких-либо возможных смешанных эффектов, связанных с наркозом 17. Альтернативные анестезии без метод иммобилизации является использование микрожидкостных устройств. Использование микрофлюидики для axotomy была подробно описана 17-22. Мы считаем, что с последовательным использования, лазерный функции лучше всего, когда Кумарин 440 в красителем ячейки меняется один раз в неделю после процедуры, описанной в руководстве Micropoint. При необходимости ослабления слайдер может использоваться для увеличения мощности, но это может быть признаком старого красителя или лазерной смещение. Кроме того, краситель клетка имеет ограниченный срок службы, и в конечном итоге должны быть восстановлены или заменены (см. Поиск и устранение неисправностей). Это может быть трудно маневрировать целевой аксонов под перекрестье, которые отмечают лазерным фокусом, особенно если животное не полностью парализована. Мы считаем, что руководство этапе не является оптимальным для этой цели, хотя это, безусловно, полезная. Джойстика контролируемой моторизованных этапе является более точным, и мы находим, что при использовании программного обеспечения, поддерживающего изображение перетаскиванием с помощью мыши, чтобы переместить моторизованных этапе, тем лучше. Nikon элементов обеспечивает эту возможность и описывается в данном протоколе, но бесплатный пакет микроменеджер, а также другого программного обеспечения обработки изображений, могут иметь схожую функциональность. Другой способ тонкой таргетинг для перемещения лазерного фокуса, а не животное. Гальванометра пучка рулевой механизм доступен как дополнение к Micropoint лазера, если такой подход является предпочтительным. Кроме того его применение к изучению нейронных регенерации, лазер может быть использован для удалять другие типы клеток, такие как кожа, мышцы, или специализированные клетки, или нарушить конкретных нейронных синапсов 23-27. Более того, пути, которые регулируют дегенерацию нейронов, которая сопровождает травмы или болезни может быть исследован с этой системой. Таким образом, использование импульсных лазеров будет продолжаться, чтобы пролить свет на генетические факторы и клеточные биологические изменения, которые способствуют регенерации нейронов и других соответствующих процессов. Устранение неполадок: Описанные здесь некоторые общие проблемы и связанные с ними решения. <ol type="a"><li> Лазерная резка не должным образом. Это, вероятно, потому что либо лазер не в фокусе (см. раздел 2), или красителя в краситель клетка должна быть заменена (см. Micropoint руководства). Кроме того, краситель ячейки, возможно, должны быть заменены или восстановлены. </li><li> Черви переходят на агарозном площадку. Как правило, это признак того, что площадка агарозном слишком влажная. Мы считаем, что колодки необходимо будет оставить в течение приблизительно 30 секунд до червей помещают на них, чтобы обойти эту проблему. Вы можете также попробовать использовать меньшего размера (0,05 мкм) микрошарики. </li><li> Восстановление червей трудно и неэффективно. Это может быть вызвано неправильным удалением покровное или сухой площадке агарозы. Если подушка слишком сухая, можно заметить, что аксоны развивать бисером внешний вид. В некоторых случаях это происходит потому, что черви не были размещены на площадке достаточно скоро после ее изготовления. Кроме того, старые запасы раствора агарозы может быть выше, чем 3%-ной концентрации после многократного плавления. </li></ol>

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th><th> Комментарии </th></tr><tr><td> 0,05 мкм бисер полистирола </td><td> Polysciences, Inc </td><td> 08691 </td><td></td></tr><tr><td> 0,10 мкм бисер полистирола </td><td> Polysciences, Inc </td><td> 00876 </td><td></td></tr><tr><td> Агарозном GPG / LE </td><td> Американская Биоаналитическая </td><td> 00972 </td><td> Ультра чистый </td></tr><tr><td> Сокол 14 мл Полистирол с круглым дном трубы </td><td> BD Biosciences </td><td> 352057 </td><td> 17 х 100 мм стиль, nonpyrogenic </td></tr><tr><td> Thermo Scientific Обычная precleaned слайды микроскопом </td><td> Эри ОАО Научно- </td><td> 420-004T </td><td> 3 &quot;х 1&quot; х 1 мм </td></tr><tr><td> Покровные стекла </td><td> VWR </td><td> 48366 205 </td><td> 18 мм х 18 мм Количество 1 1 / 2 </td></tr><tr><td> KIMTECH науки Kimwipes </td><td> Kimberly-Clark </td><td> 34155 </td><td></td></tr><tr><td> BD Falcon 100 х 15 мм Стиль чашки Петри </td><td> BD Biosciences </td><td> 351029 </td><td></td></tr><tr><td> Лента пустой 3/4w х 500L </td><td> TimeMed </td><td> T-512 </td><td></td></tr><tr><td> Погружение нефти </td><td> Nikon </td><td></td><td> Тип А, й = 1,515 </td></tr><tr><td> EG1285 или CZ1200 </td><td> C. Элеганс генетический центр </td><td> http://www.cbs.umn.edu/CGC/strains/ </td><td></td></tr><tr><td> OptiScan II </td><td> ДО Научно </td><td></td><td></td></tr><tr><td> NIS-Elements Ar или Br </td><td> Nikon </td><td></td><td></td></tr><tr><td> Micropoint абляция лазерная система </td><td> Фотонные Научные </td><td></td><td></td></tr><tr><td> Соединения микроскопом </td><td> Nikon </td><td> Затмение 80i </td><td></td></tr><tr><td> Hamamatsu камеры </td><td> Hamamatsu Photonics </td><td> Модель C8484-05G01 </td><td></td></tr><tr><td> Dell Precision T3400 ПК с процессором Intel Core 2 Duo </td><td> Лощина </td><td></td><td></td></tr><tr><td> Windows XP Professional </td><td></td><td> Microsoft </td><td> Версия 2002, Series Pack 3 </td></tr><tr><td> Двойной сухие ванны инкубатор </td><td> Fisher Scientific </td><td></td><td> Аналоговый контроль </td></tr><tr><td> 4X Plan Fluor цель </td><td> Nikon </td><td></td><td></td></tr><tr><td> 100X Plan Apo VC нефти цель </td><td> Nikon </td><td></td><td></td></tr><tr><td> Дюмон # 5 / 45 пинцетов </td><td> Дюмон </td><td> 11251-35 </td><td> Dumoxel стандартные советы, можно также использовать # 7 </td></tr><tr><td> Препаровальная лупа </td><td> Nikon </td><td> SMZ800 </td><td> С NI-150 Высокая интенсивность Просветителя </td></tr></tbody></table>

in vivo laser axotomy in c. elegans

Нейроны общаются с другими клетками с помощью аксонов и дендритов, тонкая мембрана расширений, содержащих пре-и пост-синаптические специализации. Если нейрон поврежден-за травмы или болезни, он может регенерировать. Cell-внутренние и внешние факторы влияют на способность нейронов к регенерации и восстановления функции. В последнее время нематоды C. Элеганс стала отличная модель организма для выявления генов и сигнальных путей, которые влияют на регенерацию нейронов 1-6. Основным способом инициировать регенерацию нейронов в C. Элеганс является лазерная резка-опосредованной или axotomy. Во время axotomy, флуоресцентно меченных нейронов процесс разорвал использованием высоких энергий импульсов. Первоначально нейронные регенерации в C. Элеганс были изучены с помощью усиления фемтосекундных лазерных 5. Однако последующие исследования регенерации показали, что обычный импульсный лазер может быть использован для точного разорвать нейронов в естественных условиях и вызывать аналогичные регенеративный ответ 1,3,7. Мы представляем протокол для выполнения в естественных условиях лазерной axotomy в использовании червя Micropoint импульсного лазера, под ключ системы, которые легко доступны и широко используется для целевых абляция клетки. Мы описываем выравнивания лазера, монтаж червей, резка специфических нейронов, а также оценка последующей регенерации. Система предоставляет возможность сократить большое количество нейронов в нескольких червей в течение одного эксперимента. Таким образом, лазерная axotomy как описывалось выше, эффективной системой для инициирования и анализа процесса регенерации.

Watch this Scientific Journal Video about In vivo Laser Axotomy in C. elegans at JoVE.com

In vivo Laser Axotomy in C. elegans

Протокол сократить нейронов в<em&gt; С. Элеганс</em&gt; С лазерным импульсным Micropoint представлена. Мы описываем создание системы иммобилизации червей и разрыва меченых нейронов. Среди преимуществ относительно недорогие системы и возможность разорвать нейронных процессов или удалять клетки<em&gt; В естественных условиях</em&gt;.

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 ...

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Neuroscience

In vivo Laser Axotomy in C. elegans

18.1K Views.  Yale University School of Medicine. Протокол сократить нейронов в С. Элеганс С лазерным импульсным Micropoint представлена. Мы описываем создание системы иммобилизации червей и разрыва меченых нейронов. Среди преимуществ относительно недорогие системы и возможность разорвать нейронных пр...

Video: In vivo Laser Axotomy in C. elegans

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

Трансфекция Мышь ганглиозных клеток сетчатки при В естественных условиях Электропорация

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Функциональные нейровизуализации с использованием ультразвуковых Гематоэнцефалический Разрушение барьеров и марганца-МРТ

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

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 ...

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, ...

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this protocol focuses on how to image dividing neuroblasts, which are found underneath the epidermal cells and may be important for epidermal morphogenesis. Tissue formation is crucial for metazoan development&#160;and relies on external cues from neighboring tissues. C. elegans is an excellent model organism to study tissue morphogenesis in vivo due to its transparency and simple organization, making its tissues easy to study via microscopy. Ventral enclosure is the process where the ventral surface of the embryo is covered by a single layer of epithelial cells. This event is thought to be facilitated by the underlying neuroblasts, which provide chemical guidance cues to mediate migration of the overlying epithelial cells. However, the neuroblasts are highly proliferative and also may act as a mechanical substrate for the ventral epidermal cells. Studies using this experimental protocol could uncover the importance of intercellular communication during tissue formation, and could be used to reveal the roles of genes involved in cell division within developing tissues.

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

This protocol describes how to image dividing cells within a tissue in Caenorhabditis elegans embryos. While several protocols describe how to image cell division in the early embryo, this protocol describes how to image cell division within a developing tissue during mid late embryogenesis.

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 ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

Лазерная нанохирургии мозжечка Аксоны<em&gt; В Vivo</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

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 ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

Одновременный<em&gt; Экс естественных условиях</em&gt; Функциональное тестирование двух сетчатки по<em&gt; В естественных условиях</em&gt; Система электроретинограммы

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...