Авторы хотели бы выразить признательность Александру Опи для технической помощи и поддержки, Джеймс Бартон за предоставление видео повествование, и Рамона Flatz и Joellen Суини за появление в сопроводительном видео. Эта работа была поддержана MJ Мердок Благотворительный фонд (грант № 2012205 на JED).

1 Стерео микроскоп Система Примечание: Хотя этот протокол легко адаптируется практически к любой стерео микроскопа системе, связанной с цифровой камеры с возможностью приобретения видео, информация представлена ​​на системы, используемой в нашей лаборатории (Таблица материалов / оборудования). <ol><li> Приобретать цифрового видео с использованием тринокулярный стерео микроскоп, соединенный с коммерческой цифровой камеры. </li><li> Для того, чтобы пара коммерческую цифровую камеру тринокулярной порту стереомикроскопом, удалить ½x C-Mount из фотоэлемента порту стереомикроскопом и заменить его на 1X C-Mount. </li><li> Установите цифровой камеры соединитель (43 мм резьба) к 1X C-Mount. </li><li> Mount две понижающие кольца, 58 мм до 48 мм, и 48 мм до 43 мм, в камеру соединителя сделать мост между цифровыми камерами муфты к переходником объектив для цифровой камеры. </li><li> Установите цифровую камеру к комплекту адаптера объектива. </li><li> Приобретать видео с увеличением микроскопа и оптического зума цифровой набор камеры для комбинированной увеличении примерно 12X (30 кадров в сек, 640 х 480 пикселей). Примечание: увеличение из стереомикроскопа должны быть компенсированы в соответствии с вновь преобразованного 1X C-Mount от тринокулярной порта. </li></ol> 2 изображений Drosophila третьего возраста личинки <ol><li> Лента маркером в черный стадии пластины стереомикроскопом соединен с цифровой камеры так, чтобы сторона маркера крышки занимает примерно ⅓ до ¼ вертикальной поле зрения наблюдается в ЖК-мониторе камеры. С помощью маркера вершины, как на стадии личинок, чтобы выполнить изображений, потому что они приходят в ассортименте цветов, которые могут быть использованы для цветового кода и дифференциации генотипов личинок изображаемого. </li><li> Демаркацию поле зрения наблюдается в цифровом ЖК-мониторе камеры на поверхности маркера сверху с тонкой точкемаркер. </li><li> Выберите третьего возраста личинки к изображению. Критерии для выбора третьего личинок Instar был Длина тела, появление от источника питания во время личиночной фазе жизненного цикла, наличием переднего и заднего дыхальца, и структура нижнечелюстных крючков аппарата рта 11. Убедитесь, личинка чист путем промывания его большим количеством воды. </li><li> Освещать постоянный этап маркер верхнюю сверху света от системы волоконно-оптической освещения. Отрегулируйте угол падающего света, чтобы обеспечить оптимальное освещение. </li><li> Фокус микроскоп на краю постоянного маркера сверху. Начните приобретения цифрового видео. </li><li> Поместите личинку на стороне маркера крышкой примерно 75 ° от вертикальной оси, в ​​непосредственной близости от поля зрения, с передней части личинки, обращенной к полю зрения (Рисунок 1). Примечание: Размещение личинки на стороне маркера крышки позволяет камере записывать движения гоэлектронной личинка с боковой перспективе. Это помогает держать личинку влажной водой, чтобы они не падают на сторону маркера крышкой. Необходимо проявлять осторожность, однако, чтобы не использовать слишком много воды, как чрезмерное количество будет придерживаться личинки, как это ползает по полю. </li><li> Аккуратно нащупывать и личинку с небольшим кисти, чтобы принудить его ползать по полю зрения. Будьте терпеливы, как личинки редко сотрудничают и часто должны быть возвращены в исходную точку много раз, прежде чем они ползают прямо по полю. </li><li> Запись примерно 10-15 мин непрерывной цифровой видеозаписи и культуры и удалить все ненужные кадры после приобретения с цифровым программным обеспечением для редактирования видео. </li></ol> 3 изображений для взрослых Drosophila <ol><li> Поместите одного взрослого Drosophila в одноразовом 1,5 мл спектроскопического полистирола кювете. Примечание: CO 2 обезболивание взрослого дрозофилы непосредственно перед BehavПротокол ioral анализ может поставить под угрозу результаты 12. Рекомендуется взрослых Drosophila быть предоставлен период 24-часовом, чтобы оправиться от CO 2 обезболивания перед выполнением в поведенческой тесте 13. </li><li> Вставьте конец кюветы с небольшим ватным тампоном. Убедитесь, ватный тампон упакован достаточно плотно, чтобы занять большую крышку пространство и ограничивает муху с уменьшением объема отсека кюветы. </li><li> Поместите кювету на белом стадии пластины стереомикроскопом и правильно выровнять снижение громкости отсек кюветы с полем зрения наблюдается в цифровом ЖК-мониторе камеры. </li><li> Illuminate кювету сверху света от системы волоконно-оптического освещения. Отрегулируйте угол падающего света, чтобы обеспечить оптимальное освещение. </li><li> Фокус микроскопа и начать получать цифровые видео. </li><li> Запись примерно 30-45 мин непрерывной цифровой видеозаписи и культуры и удалить все лишнеекадры после приобретения с цифровым программным обеспечением для редактирования видео. </li></ol>

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Авторы заявили, что не существует конкурирующие интересы.

Сила Дрозофилы &#39;ы в качестве модельной системы для изучения функции нервной системы значительной мере обусловлено сходимости мощных генетических инструментов доступных и широкого круга надежных поведенческих анализов развитой. Здесь мы представляем простой и широко доступн?...

Общая плодовой мушки дрозофилы является мощным экспериментальная модель системы для изучения функции нервной системы 1-3. Эволюционная сохранение структуры и функции нервной системы с человека, а также простота генетических манипуляций и огромный массив генетических инструментов делает Drosophila премьерный организм для моделирования человеческого нейродегенеративных заболеваний 4. Генные мутации, вызывающие дисфункции нервной системы часто приводит к жизнеспособным мутант личинок и взрослых дрозофилы с нарушением двигательной. Фенотипы наблюдаемые в нервной системы дефектных мутантов включают снижение темпов передвижения, аномальным координации и спастических движений у взрослых, а также дефицит в перистальтического сокращения стене мускулатуры и частичный паралич личинок. Эти фенотипы были использованы в разработке высокопроизводительных генетических экранов и опорно-двигательного анализов мутантных личинок 5, 6 и 7-10 взрослых дрозофилы направлена ​​на количественное определение нарушения локомоции и идентификации генов, необходимых для функции нервной системы. В то время как эти подходы являются чрезвычайно полезными для количественного личинок и взрослых локомотивных поведения, они не в состоянии передать качественную информацию о каждом конкретном девиантным поведением. Например, в то время как мутант третьего личинки возрастной стадии могут проявлять измененных параметров передвижения, в поведенческой анализа, это может быть непонятно, является ли это результатом изменений в художественной перистальтических сокращений в течение цикла ползать, общее отсутствие координации, или частичным параличом задней тела стена мускулатура. Здесь мы опишем простой и широко доступного технику микроскопии для получения высококачественного цифрового видео Drosophila взрослого и личинок фенотипов локомотивных от боковой перспективе. Цифровое видео приобрела у боковой перспективе позволяет прямое наблюдение и анализ тонких различий в Локомотиве поведения от более информативное ориентации бокового обзора.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Trinocular Stereozoom Microscope</td>
			<td>Olympus Corporation</td>
			<td>SZ6145TR</td>
			<td>&#189; C-mount was removed and replaced with 1x C-mount</td>
		</tr>
		<tr>
			<td>1X C-mount</td>
			<td>Leeds Precision Instruments</td>
			<td>LSZ-1XCMT2</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital Camera Coupler (43mm thread)</td>
			<td>Qioptiq Imaging Solutions</td>
			<td>25-70-10-02</td>
			<td></td>
		</tr>
		<tr>
			<td>58mm to 48mm Step Down Ring</td>
			<td>B&#38;H Video</td>
			<td>GBSDR5848</td>
			<td></td>
		</tr>
		<tr>
			<td>48mm to 43mm Step Down Ring</td>
			<td>B&#38;H Video</td>
			<td>GBSDR4843</td>
			<td></td>
		</tr>
		<tr>
			<td>Lensmate Adapter Kit for Canon G10</td>
			<td>LensMateOnline.com</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Canon PowerShot G10 Digital Camera</td>
			<td>Canon U.S.A., Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml Spectroscopic PolystereneCuvette</td>
			<td>Denville Scientific</td>
			<td>U8650-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

acquisition of high-quality digital video of drosophila larval and adult behaviors from a lateral perspective

Дрозофилы является мощным экспериментальная модель системы для изучения функции нервной системы. Генные мутации, которые вызывают дисфункцию нервной системы часто производят жизнеспособный личинок и взрослых, которые имеют передвижения, дефектные фенотипы, которые трудно адекватно описать с текстом или полностью представляют с одной фотографического изображения. Текущие режимы научных публикаций, однако, поддерживает представление цифрового видео СМИ как дополнительного материала, чтобы сопровождать рукопись. Здесь мы опишем простой и широко доступного технику микроскопии для получения высококачественного цифрового видео как дрозофилы личинок и взрослых фенотипов с боковой перспективе. Видео из личинок и взрослых передвижения от побочного зрения выгодно, потому что это позволяет наблюдать и анализировать тонких различий и вариаций в аберрантных поведения локомотивных. Мы успешно использовали технику для визуализации и количественной aberranт ползет поведения в третий личинок возрастной стадии, в дополнение к взрослым мутантных фенотипов и поведения в том числе ухода.

Мы успешно использовали эту технику, чтобы приобретать и количественной оценки личинок поведенческий фенотип, связанный с потерей функции гена stathmin (рисунок 2) 14. Ген stathmin кодирует микротрубочек регуляторный белок, которая разделяет димеров тубулина из пулов р...

Watch this Scientific Journal Video about Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective at JoVE.com

Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Здесь мы опишем простой и широко доступного технику микроскопии на приобретение высококачественного цифрового видео Drosophila взрослого и личинок мутантных фенотипов с боковой перспективе.

Приобретение высококачественного цифрового видео из<em&gt; Drosophila</em&gt; Личинки и взрослые особи поведения от боковой перспективе

Drosophila melanogaster is a powerful experimental model system for studying the function of the nervous system. Gene mutations that cause dysfunction of ...

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Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

8.7K Views.  Willamette University.  Здесь мы опишем простой и широко доступного технику микроскопии на приобретение высококачественного цифрового видео Drosophila взрослого и личинок мутантных фенотипов с боковой перспективе. 

Video: Acquisition of High-Quality Digital Video of Drosophila Larval and Adult Behaviors from a Lateral Perspective

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of its convenient genetics and fully sequenced genome. Additionally, the larval nervous system is an ideal model system to study mechanisms of axonal transport as the larval segmental nerves contain bundles of axons with their cell bodies located within the brain and their nerve terminals ending along the length of the body. Here we describe the procedure for visualization of synaptic vesicle proteins within larval segmental nerves. If done correctly, all components of the nervous system, along with associated tissues such as muscles and NMJs, remain intact, undamaged, and ready to be visualized. 3rd instar larvae carrying various mutations are dissected, fixed, incubated with synaptic vesicle antibodies, visualized and compared to wild type larvae. This procedure can be adapted for several different synaptic or neuronal antibodies and changes in the distribution of a variety of proteins can be easily observed within larval segmental nerves.

Visualization of Larval Segmental Nerves in 3rd Instar Drosophila Larval Preparations

Drosophila melanogaster larvae provide an ideal model system to investigate the mechanisms of axonal transport within larval segmental nerves. Using this procedure, 3rd instar larvae carrying various mutations can be compared to wild type larvae.

Визуализация личинок Сегментные Нервы у 3 Й Инстар Drosophila Подготовка личинок

Drosophila melanogaster is emerging as a powerful model system for studying the development and function of the nervous system, particularly because of ...

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

Карт и применение Enhancer-ловушки Выражение Flippase в личинок и взрослых Drosophila ЦНС

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Вскрытие и иммуногистохимического личинок, куколок и взрослых<em&gt; Drosophila</em&gt; Сетчатки

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Functional Analysis of the Larval Feeding Circuit in Drosophila

The serotonergic feeding circuit in Drosophila melanogaster larvae can be used to investigate neuronal substrates of critical importance during the development of the circuit. Using the functional output of the circuit, feeding, changes in the neuronal architecture of the stomatogastric system can be visualized. Feeding behavior can be recorded by observing the rate of retraction of the mouth hooks, which receive innervation from the brain. Locomotor behavior is used as a physiological control for feeding, since larvae use their mouth hooks to traverse across an agar substrate. Changes in feeding behavior can be correlated with the axonal architecture of the neurites innervating the gut. Using immunohistochemistry it is possible to visualize and quantitate these changes. Improper handling of the larvae during behavior paradigms can alter data as they are very sensitive to manipulations. Proper imaging of the neurite architecture innervating the gut is critical for precise quantitation of number and size of varicosities as well as the extent of branch nodes. Analysis of most circuits allow only for visualization of neurite architecture or behavioral effects; however, this model allows one to correlate the functional output of the circuit with the impairments in neuronal architecture.

Functional Analysis of the Larval Feeding Circuit in Drosophila

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Функциональный анализ из питания личинок округа в Дрозофилы</i

The serotonergic  feeding circuit in Drosophila  melanogaster larvae can be used to investigate neuronal substrates of  critical importance during the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

Живая съемка<em&gt; Дрозофилы</em&gt; Личиночные нейробласты

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

Обонятельные Поведения, испытываемых Computer Отслеживание<em&gt; Drosophila</em&gt; В четырехквадрантном одориметра

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in communication with the epidermal cells they innervate and thus their analysis benefits from in situ visualization of both neuronally and epidermally expressed proteins by immunofluorescence. Traditional methods of preparing larval fillets for immunofluorescence experiments leave intact the muscle tissue that covers most of the body wall, presenting several challenges to imaging neuronal and epidermal proteins. Here we describe a method for removing muscle tissue from Drosophila larval fillets. This protocol enables imaging of proteins that are otherwise obscured by muscle tissue, improves signal to noise ratio, and facilitates the use of super-resolution microscopy to study da neuron development.

Removal of Drosophila Muscle Tissue from Larval Fillets for Immunofluorescence Analysis of Sensory Neurons and Epidermal Cells

Studies of neuronal morphogenesis using Drosophila larval dendritic arborization (da) neurons benefit from in situ visualization of neuronal and epidermal proteins by immunofluorescence. We describe a procedure that improves immunofluorescence analysis of da neurons and surrounding epidermal cells by removing muscle tissue from the larval body wall.

Удаление<em&gt; Drosophila</em&gt; Мышечной ткани из личиночной филе для иммунофлуоресценции анализа сенсорных нейронах и эпидермальных клеток

Drosophila larval dendritic arborization (da) neurons are a popular model for investigating mechanisms of neuronal morphogenesis. Da neurons develop in ...

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.

Фокуса Macropatch записи синаптических токов от дрозофилы личиночной нервно-Джанкшен

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

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a considerable amount of information has been generated regarding ORN responses to odorants, the role of specific ORNs in driving behavioral responses remains poorly understood. Complications in behavior analyses arise due to different volatilities of odorants that activate individual ORNs, multiple ORNs activated by single odorants, and the difficulty in replicating naturally observed temporal variations in olfactory stimuli using conventional odor-delivery methods in the laboratory. Here, we describe a protocol that analyzes Drosophila larval behavior in response to simultaneous optogenetic stimulation of its ORNs. The optogenetic technology used here allows for specificity of ORN activation and precise control of temporal patterns of ORN activation. Corresponding larval movement is tracked, digitally recorded, and analyzed using custom written software. By replacing odor stimuli with light stimuli, this method allows for a more precise control of individual ORN activation in order to study its impact on larval behavior. Our method could be further extended to study the impact of second-order projection neurons (PNs) as well as local neurons (LNs) on larval behavior. This method will thus enable a comprehensive dissection of olfactory circuit function and complement studies on how olfactory neuron activities translate in to behavior responses.

Tracking Drosophila Larval Behavior in Response to Optogenetic Stimulation of Olfactory Neurons

This protocol analyzes navigational behavior of Drosophila larva in response to simultaneous optogenetic stimulation of its olfactory neurons. Light of 630 nm wavelength is used to activate individual olfactory neurons expressing a red-shifted channel rhodopsin. Larval movement is simultaneously tracked, digitally recorded, and analyzed using custom-written software.

Отслеживание дрозофилы личиночной поведение в ответ на Optogenetic стимуляции обонятельных нейронов

The ability of insects to navigate toward odor sources is based on the activities of their first-order olfactory receptor neurons (ORNs). While a ...

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

Метаболический Анализ Drosophila melanogaster Larval и взрослых мозги

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...