Summary

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

Published: August 24, 2012
doi:

Summary

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

Abstract

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 β-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.

Protocol

1. Приготовление раствора и в агарозном геле 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…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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

Materials

Name Company Cat. N°
Agar Sigma A1296
Fura-red/AM Invitrogen F-3021
Pluronic F-127 Sigma P2443
Dimethyl sulfoxide Fisher Scientific BP231
Vibrating-Blade Microtome Hyrax V 50 Zeiss 9770170
Cooling Device CU 65 for Microtome Hyrax V 50 Zeiss 9920120
O2/CO2 Incubator, CB210-UL Binder 0019389
Super glue, Loctite 406TM Henckel 142580
Double spatulas, spoon shape Bochem 3182
Microspoon spatulas, spoon shape Bochem 3344
Spring Scissors, Moria-Vannas-Wolff – 7mm Blades Fine Science Tools 15370-52
Spring Scissors, Vannas – 3mm Blades Fine Science Tools 15000-00
Wagner Scissors Fine Science Tools 14071-12
Medical Forceps, Dumont 7b Fine Science Tools 11270-20
Large Rectangular Open Bath Chamber (RC-27) Warner Instruments 64-0238
Confocal Microscope BioRad Radiance 2100 Zeiss n.a.

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Cite This Article
Schauer, C., Leinders-Zufall, T. Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices. J. Vis. Exp. (66), e4213, doi:10.3791/4213 (2012).

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