Name: imaging calcium responses in gfp-tagged neurons of hypothalamic mouse brain slices
Uploaded: 2012-08-24T13:00:00
Description: Watch this Scientific Journal Video about Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices at JoVE.com

Мы благодарим наших коллег, которые принимали участие в работе приведены здесь. Эта работа была поддержана грантами от Deutsche Forschungsgemeinschaft (SFB 894), «Интегративная анализа обоняния&quot; DFG Schwerpunktprogramm 1392 и Фондом Фольксваген (TLZ). TLZ является профессор Лихтенберг от Volkswagen Foundation.

1. Приготовление раствора и в агарозном геле <ol><li> Подготовка внеклеточной решение в соответствии с таблицей с двойным дистиллированной воды. РН будет ~ 7,3 через 10 мин аэрации с карбогена (95% O 2/5% CO 2), осмолярность 300 мосм 8. Если выше осмолярность не требуется, его можно ре...

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Главный вопрос в неврологии, чтобы понять, как мозг обрабатывает социальную информацию. Основным источником информации, необходимой для социального признания кодируется обонятельный или феромонов сигналов. Обнаружение этих сигналов популяций нейронов в носу и признание сигналы в мо...

There was a typo in the abstract of <a href="http://www.jove.com/video/4213">Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices</a>.

Erratum: Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

<table border="1"><tbody><tr><td> Имя </td><td> Компания </td><td> Кат. N ° </td></tr><tr><td> Агар </td><td> Сигма </td><td> A1296 </td></tr><tr><td> Fura-red/AM </td><td> Invitrogen </td><td> F-3021 </td></tr><tr><td> Pluronic F-127 </td><td> Сигма </td><td> P2443 </td></tr><tr><td> Диметилсульфоксид </td><td> Fisher Scientific </td><td> BP231 </td></tr><tr><td> Вибрационный-Blade Микротом Hyrax V 50 </td><td> Zeiss </td><td> 9770170 </td></tr><tr><td> Охлаждающее устройство CU 65 для Микротом Hyrax V 50 </td><td> Zeiss </td><td> 9920120 </td></tr><tr><td> O 2 / CO 2 инкубаторе, CB210-UL </td><td> Связующее вещество </td><td> 0019389 </td></tr><tr><td> Супер-клей, Loctite 406TM </td><td> Хенкель </td><td> 142580 </td></tr><tr><td> Двухместный шпатели, ложки форму </td><td>Bochem </td><td> 3182 </td></tr><tr><td> Microspoon шпатели, ложки форму </td><td> Bochem </td><td> 3344 </td></tr><tr><td> Весна Ножницы, Мория-Vannas-Вольфа - 7 мм Лезвия </td><td> Средства изобразительных наук </td><td> 15370-52 </td></tr><tr><td> Весна Ножницы, Vannas - 3 мм Лезвия </td><td> Средства изобразительных наук </td><td> 15000-00 </td></tr><tr><td> Вагнер ножницы </td><td> Средства изобразительных наук </td><td> 14071-12 </td></tr><tr><td> Медицинские щипцы, Дюмон 7b </td><td> Средства изобразительных наук </td><td> 11270-20 </td></tr><tr><td> Большие прямоугольные ванны с открытой камерой (RC-27) </td><td> Warner Instruments </td><td> 64-0238 </td></tr><tr><td> Конфокальный микроскоп Сияние BioRad 2100 </td><td> Zeiss </td><td> не доступно </td></tr></tbody></table>

imaging calcium responses in gfp-tagged neurons of hypothalamic mouse brain slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

Watch this Scientific Journal Video about Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices at JoVE.com

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

В этом протоколе, мы обновляем последние достижения в области обработки изображений Ca<sup&gt; 2 +</sup&gt; Сигналы GFP с метками нейронов в мозговой ткани срезов при использовании красного флуоресцентного Са<sup&gt; 2 +</sup&gt; Индикатор красителя.

Изображениями кальция Ответы на GFP с метками нейроны гипоталамуса фрагменты мозга мыши

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

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Neuroscience

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Двойной Записи Соматические от гонадотропин-рилизинг гормона (ГнРГ) Нейроны, идентифицируемый зеленый флуоресцентный белок (GFP) в гипоталамус Ломтики

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Кальций изображений запаха, вызвали ответы на Drosophila Усиков лепестка

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The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

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Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using ...

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