Intracellular calcium has been implied as an important part of several mechanisms of synaptic plasticity. These ideas can be tested by simultaneously monitoring changes in calcium and alterations in synaptic efficacy. Here we demonstrate how this is accomplished by combining calcium imaging with intracellular recording.
Our experiments are conducted with a ganglion of the mollusk Aplysia Cahora. This preparation has a number of advantages such as large identified neurons, the low temperatures that are natural for apia, slow signals and facilitate imaging, but the general techniques we demonstrate are easily transferred to other systems. Hello, I'm Colin Evans in the laboratory of Elizabeth Cropper in the Fishburg Department of Neuroscience and the Freedman Brain Institute at the Mount Sinai School of Medicine in New York City.
Today, we are going to demonstrate simultaneous imaging of changes in intracellular calcium concentration and synaptic efficacy in the buccal ganglion of the mollusk Aplysia cahora. To detect intracellular calcium, we will inject a pre-synaptic neuron with the cell IMP permeant calcium indicator dye calcium orange. The preparation is then moved under microscope and pre and postsynaptic neurons are impaled with sharp electrodes.
Repeated presynaptic stimulation results in an enhanced postsynaptic response visible as increasing postsynaptic potential or PSP amplitude. Measuring the fluorescent signal of the calcium indicator dye will allow us to detect simultaneous changes in presynaptic intracellular calcium. To begin anesthetize an adult animal weighing about 150 grams by injecting 75 mils of isotonic magnesium chloride solution, pin the anesthetized animal to a wax covered dish, then using gross forceps and scissors, make an incision in the animal's foot and expose the buccal mass.
The buccal ganglion lies on the ventral side of the buccal mass and consists of two connected hemi ganglia. It contains several hundred neurons that together with neurons in other ganglia contribute to the generation and control of the feeding behavior of the animal. Carefully free the ganglion by cutting all buckled nerves with fine scissors and remove it from the animal.
Then pin the freed ganglion to the base of a sil guard coated dish filled with artificial seawater. The ganglion is wrapped in a sheath of connective tissue, which has to be removed to expose the individual neurons. Use verifying forceps, iris scissors, and extreme care to cut away this sheath.
To avoid damaging neurons, stabilize the de sheath ganglion with about 15 minute pins as any movement will be problematic during imaging. To prepare sharp electrodes for intracellular recording and dye injection, pull capillary tubing with a micro electrode puller. We use thin walled filament boa silicate glass with an outer diameter of one millimeter.
The recording electrodes should have a resistance of about 10 mega when filled with three molar potassium acetate, and so we adjust the puller accordingly to fill the D electrodes. Dip the back of the pulled electrode into a 10 millimolar solution of the calcium orange dye capillary action along the glass filament inside the electrode will draw dye into the tip, then backfill the electrode with 200 millimolar potassium chloride to ensure good electrical contact. We use a custom built Bela to improve the electrode properties and lower the recording electrode resistance to about five mega.
The electrode is held at a 45 degree angle into a stream of an aluminum oxide suspension. Mixing sodium chloride into the suspension and connecting an ome meter allows monitoring of the electrode resistance. During the beveling process, we transfer the dish containing the deceived buckle ganglia to an electrophysiology rig and locate the neurons of interest by impaling them with electrodes and verifying their identity.
To prepare neuron for calcium imaging, we first insert a dye filled electrode into the cell soma and load the dye electro theoretically with 30 minutes of hyperpolarizing, 15 nano amp current pulses. As the D calcium orange enters the cell, the SOR will acquire a faint pink color. We then carefully retract the recording electrodes and leave the preparation sitting for at least 30 minutes to allow the dye to diffuse into the neuron's fine processes.
Next, transfer the dish with the preparation to a fluorescent microscope. We use a 10 times NA 0.3 water immersion lens on an upright fixed stage microscope, which focuses by moving the lens and not the stage. This makes it easier to mount manipulators.
For the recording electrodes select an appropriate filter block. A SI three filter block will provide the correct excitation and emission filters for calcium orange, adjust the camera parameters and the illumination intensity with neutral density filters and the field aperture. More light will result in less noise, but can potentially bleach the preparation.
The cool snap CCD camera can capture a 500 by 300 pixel field at a frame rate of around 30 frames per second, and a good signal to noise ratio. Minimize the illumination of the fil neurons by keeping the light sources shutter closed whenever possible. In the imaging software, select regions of interest or R ois.
These regions define where the software will take intensity measurements. We generally image primary, secondary, and tertiary branches of the presynaptic neuron. Place an additional ROI next to the difi neuron to obtain a background value that is later subtracted from all other data points.
Intracellular recording electrodes in the pre and post synaptic neurons are used to induce and monitor synaptic transmission. You can ensure synchronization between electrophysiological and imaging data acquisition if you use the camera's frame out trigger signal to start the data acquisition software. Another way to ensure synchronization is to mount an LED inside the camera port.
This LED is briefly turned on at the beginning of the recording session, and thereby provides a synchronization mark. During a typical experiment, you trigger action potentials in the presynaptic neuron by injection of brief current pulses. In this example, we show a burst of spikes and corresponding increases in the calcium signal to test specific hypotheses.
The calcium signal can be manipulated and effects on synaptic transmission determined. For example, drugs such as the calcium chelator EGTA can be presynaptically injected or applied through the perfusion system. Data are analyzed by exporting them from the imaging software into a text file and then into the software that was used to acquire the electrophysiological data, which in our case is spike two by Cambridge Electronic Design.
We use a custom written script to plot the ROI intensity values and corresponding changes in membrane potential quantify changes in the calcium signal by subtracting the background signal obtained from the background ROI and calculating the relative change with the equation shown. F zero is the fluorescence just before stimulation, and F is the fluorescence during stimulation. Here we show results of an experiment in which we imaged changes in calcium fluorescence in the identified neuron B 21.
The region we imaged is indicated in A, in B one, we induced a burst of spikes in B 21, which induced postsynaptic potentials in B eight and a change in the calcium signal. B two shows the effect of the injection of the calcium chelator. EGTA spikes in B 21 no longer induce widespread increases in calcium fluorescence and the post synaptic potential amplitude is decreased.
In conclusion, we have demonstrated techniques that can be used to simultaneously monitor the intracellular calcium concentration and evaluate the efficacy of synaptic transmission. These techniques require only modest equipment compared to most functional imaging and are easy and quick to learn.
