हम प्रदर्शन कैसे intracellular कैल्शियम एकाग्रता और synaptic प्रभावकारिता में परिवर्तन के साथ ही एक नाड़ीग्रन्थि की तैयारी में नजर रखी जा सकती है<em> Aplysia</em>. हम intracellular कैल्शियम छवि एक फ्लोरोसेंट रंजक, कैल्शियम ऑरेंज, और प्रेरित और तेज इलेक्ट्रोड (intracellular) के साथ synaptic प्रसारण पर नजर रखने का उपयोग.
It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 °C) that are natural for Aplysia, preparations are stable for long periods of time.
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily ‘loaded’ into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations.
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.
हम तकनीक है कि करने के लिए एक साथ intracellular कैल्शियम एकाग्रता की निगरानी और synaptic प्रसारण की प्रभावकारिता का मूल्यांकन करने के लिए इस्तेमाल किया जा सकता है प्रदर्शित करता है. इन तकनीकों का निर्धारण कैसे अल्प?…
The authors have nothing to disclose.
एक PHS अनुदान (MH51393) इस काम का समर्थन किया. कुछ का उपयोग हम Aplysia के अनुसंधान संसाधन, एनआईएच के लिए राष्ट्रीय केंद्र से अनुदान RR10294 तहत मियामी के विश्वविद्यालय के Aplysia के लिए राष्ट्रीय संसाधन द्वारा प्रदान की जाती हैं.
Reagent Name | Company | Catalogue Number | Comment |
Calcium Orange | Invitrogen | C-3013 | |
EGTA | Sigma | E-4378 | |
Calcium calibration buffer kit | Invitrogen | C-3008MP | useful for testing the sensitivity and dynamic range of the signal |
Magnesium chloride hexahydrate | Sigma | M0250 | used in 0.33 M solution to anesthetize animal |
Table 1. Reagents used.
Equipment Name | Company | Comment |
FN-1 upright fluorescence microscope | Nikon Instruments | with Narishige ITS-FN1 stage |
NMN-21 manipulators | Narishige | mounted on stage with magnets |
CoolSNAP HQ2 CCD camera | Photometrics | |
NIS elements AR (version 3.22) |
Nikon Instruments | imaging software used to acquire fluorescence data |
10X/0.3w Plan Fluor objective | Nikon Instruments | this water immersion lens has a very long working distance of 3.5 mm |
X-Cite 120 PC metal halide lamp | EXFO | used for fluorescence imaging |
LS-DWL halogen lamp |
Sumica | |
ET-CY3 filter set | Chroma Technology | |
Power 1401 A/D converter | Cambridge Electronic Design | sampling was done at 3 kHz |
Spike II (version 7.07) |
Cambridge Electronic Design | software used to acquire electrophysiology data |
SEC-10 LX amplifier | NPI electronics | used with a 10X headstage |
Model 410 amplifier | Brownlee precision | used to amplify and filter the signal |
WS-4 | minus k Technology | vibration isolation for imaging |
cooling platform | custom made | brass plate through which ice water is pumped at a variable rate |
Table 2. Equipment used.