Напряжение чувствительные краски Запись с аксонов, дендритов и дендритных шипиков отдельных нейронов в срезах мозга
Summary
Методом визуализации для мониторинга изменений мембранного потенциала с суб-микронных пространственных и суб-миллисекунды временное разрешение описано. Методика, основанная на лазерном возбуждении напряжение чувствительных красителей, позволяет измерять сигналы в аксонов и аксонов залогов, терминал дендритных ветвей и отдельных дендритных шипиков.
Abstract
Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique – optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) – characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.
Protocol
Representative Results
Discussion
В этой статье описывается напряжения чувствительных красителей метод записи для мониторинга электрической активности отдельных нейронов с суб-микронных и суб-миллисекунды пространственно-временного разрешения. Лазерное возбуждение при почти оптимальной длины волны (относительно ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Мы благодарны нашим сотрудникам Кнут Холтофф, Артур Konnerth и Марко Canepari, которые участвовали в начальном развитии этой техники, а также Лесли М. Лоев за любезно предоставленные красителей. Поддерживается NSF гранта IOS-0817969, NIH гранты NS068407 и M136043 и Кавли Институт неврологии Йельского университета.
Materials
| Name of the component | Company | Catalogue number | Comments (optional) |
| Setup components | |||
| Upright Microscope | Olympus Inc. | BX51WI | With three camera ports |
| Motorized Movable Stage | Siskiyou | MXOPi.2 | |
| Epi-fluorescence Condenser for Olympus BX51 | TILL Photonics | 0000-560-11659 | |
| Upright Microscope | Carl Zeiss, LLC | AxioExaminer D1 | With three camera ports |
| Motorized Top Plate | Scientifica Limited | MMBP | |
| Epi-fluorescence Condenser for Zeiss AxioExaminer | TILL Photonics | ||
| Data Acquisition Camera | RedShirtImaging LLC | NeuroCCD-SM | High speed, low read noise |
| CCD for IR-DIC | Dage-MTI | IR-1000 | |
| Spinning-Disc Confocal Scanner | Yokogawa | CSU-10 | |
| High Spatial Resolution CCD on Confocal Scanner | PCO AG | PixelFly | 1392×1024 pixels |
| DPSS CW Laser (532 Nm) | CNI Optoelectronics Tech. Co., Ltd | MLL-III-532 400mW | Excitation light source |
| Multi-Mode Fiber Launcher | Siskiyou | SM-CFT | |
| Light Guide | TILL Photonics | 0000-515-11524 | |
| Shutter | Vincent Associates | LS6 | |
| Vibration Isolation Table | Minus k Technology | MK26 | |
| Specific reagents | |||
| Di-2-ANEPEQ (JPW 1114) | Life Technologies | D-6923 | Voltage sensitive dye |
| Crym-EGFP Mouse Line | GENSAT (MMRRC) | STOCK Tg(Crym-EGFP)GF82Gsat/Mmcd | Sparsely expressing EGFP in Layer 5 cortical neurons |
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