Мониторинг изменений в внутриклеточной концентрации кальция и синаптической эффективности в Моллюск<em> Aplysia</em
Summary
Мы демонстрируем, как изменения во внутриклеточной концентрации свободного кальция и синаптической эффективности могут быть одновременно контролировать в ганглии подготовки<em> Aplysia</em>. Мы изображений внутриклеточного кальция с помощью флуоресцентного красителя, кальций Orange, и вызывать и контролировать синаптической передачи с острыми (внутриклеточные) электроды.
Abstract
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.
Protocol
Discussion
Мы продемонстрируем методы, которые могут быть использованы для одновременного контроля внутриклеточной концентрации кальция и оценить эффективность синаптической передачи. Эти методы полезны для определения того, как различные формы краткосрочного пластичности опосредовано.
<p …Divulgations
The authors have nothing to disclose.
Acknowledgements
PHS Грант (MH51393) поддержали эту работу. Некоторые из Aplysia которые мы используем, предоставляемых Национальным ресурсов для Aplysia из университета Майами по гранту RR10294 из Национального центра исследовательских ресурсов, NIH.
Materials
| 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.
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