Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

Summary

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

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

1. Preparation Anesthetize the animal by injecting 75-100 ml isotonic magnesium chloride solution. The Aplysia we use for imaging are generally 150-200 grams and are obtained from Marinus Scientific. Pin the anesthetized animal to a wax covered dish. Syringe needles work well for this purpose; sterile techniques are not necessary. Using gross forceps and standard scissors make an incision in the animal’s foot and expose the buccal mass. Locate the buccal ganglion. Using spring sci…

Discussion

We demonstrate techniques that can be used to simultaneously monitor the intracellular calcium concentration and evaluate the efficacy of synaptic transmission. These techniques are useful for determining how various forms of short-term plasticity are mediated.

The imaging is carried out with a fluorescence microscope and CCD camera. These equipment requirements are relatively modest when compared to most functional imaging set-ups. The technique is simple and easy to learn. While imaging wit…

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.