उप सुक्ष्ममापी स्थानिक और उप millisecond अस्थायी समाधान के साथ झिल्ली संभावित परिवर्तनों की निगरानी के लिए एक इमेजिंग तकनीक वर्णित है. तकनीक, वोल्टेज के प्रति संवेदनशील रंगों के लेजर उत्तेजना, axons और collaterals अक्षतंतु टर्मिनल वृक्ष के समान शाखाओं, और व्यक्तिगत वृक्ष के समान spines संकेतों के मापन के आधार पर अनुमति देता है.
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.
इस अनुच्छेद के उप सुक्ष्ममापी और उप millisecond spatiotemporal संकल्प के साथ व्यक्तिगत न्यूरॉन्स की विद्युत गतिविधि की निगरानी के लिए एक डाई वोल्टेज के प्रति संवेदनशील रिकॉर्डिंग विधि का वर्णन करता है. निकट इष्टतम त?…
The authors have nothing to disclose.
हम हमारे Knut Holthoff, आर्थर Konnerth और मार्को Canepari जो कृपया रंजक प्रदान करने के लिए इस तकनीक का प्रारंभिक विकास में के रूप में के रूप में अच्छी तरह से भाग लिया Leslie एम. Loew सहयोगियों के लिए आभारी हैं. NSF IOS 0817969 अनुदान, NIH NS068407 और M136043 अनुदान द्वारा और Kavli येल विश्वविद्यालय में तंत्रिका विज्ञान के लिए संस्थान द्वारा समर्थित है.
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 |