This article represents a detailed description of how to use a photodiode array and voltage-sensitive dyes to record action potentials from large numbers of neurons simultaneously. Compared to calcium imaging of neural activity, which is indirect, the voltage-sensitive dyes we use record action potentials directly similar to sharp electrode recordings, but across many neurons simultaneously. Our optical imaging workflow can be adapted to rapidly visualize network activity with single neuron and single action potential resolution in a variety of non-transgenic invertebrate and even vertebrate systems.
Additionally, demonstrating the procedures in our protocol will be Evan Hill from my laboratory. Begin by anesthetizing a 40 gram Aplysia californica animal and pinning it ventral side up in a waxed-lined dissection dish. Using dissection scissors, make a two to three centimeter midline incision along the most anterior extent of the foot and pin down the flaps of the foot on either side of the incision to reveal part of the central nervous system and buccal mass.
Use forceps and dissection scissors to carefully dissect away the buccal mass, revealing the cerebral ganglia, and sever the nerves innervating the animal's body to excise the central nervous system, leaving a long length of the nerve to be stimulated. Use minutian pins to position the central nervous system in a saline-filled elastomer-lined dish and perfuse the dish with saline passed through a Peltier cooling device to maintain the preparation temperature at 15 to 16 degrees Celsius. Use the forceps and microdissection scissors to remove excess connective tissue from the nerves and dissect away a superficial portion of the sheath on the ganglion or ganglia to be imaged.
Then dip the cleaned central nervous system in a solution of 0.5%glutaraldehyde in saline for 20 seconds before returning the central nervous system to the saline-perfused elastomer-lined dish to rinse away any excess glutaraldehyde. To prepare a working solution of voltage-sensitive dye, add 500 microliters of saline to a previously prepared solid aliquot of RH 155 to achieve a final dye concentration of 0.3 milligrams per milliliter. And use a handheld microdispenser to load 200 microliters of solution into a piece of polyethylene tubing with a similar diameter to that of the ganglion to be stained.
Using a micromanipulator, carefully place the end of the tube over the target ganglion, lowering the tube until it forms a snug seal over the ganglion. Turn the microdispenser applicator knob every five minutes for an hour to force more dye onto the ganglion, checking the sample at 30 minutes to confirm that adequate staining is occurring. After staining, keeping the lights dimmed, place an appropriate buffer material to the left and right of where the preparation will be placed in the saline-containing imaging chamber, and then immerse the central nervous system in the chamber.
Press a suitably-sized piece of a coverslip over the tissue and press firmly on the coverslip to flatten the tissue without damaging the neurons. Then place the imaging chamber under a dissecting microscope. If stimulating a nerve to elicit a fictive motor program, carefully melt a segment of PE100 polyethylene tubing over a flame while gently pulling both ends of the tubing segment and cut the resulting taper at the desired point.
Next, attach a length of thick-walled flexible polymer tubing to the back end of a polyethylene suction electrode. Then use a mouth section to create negative pressure and draw a small volume of saline through the tapered end of the suction electrode. Draw the end of the nerve to be stimulated into the electrode under the microscope and confirm that the saline in the electrode lacks bubbles that could interrupt the electrical conduction.
For imaging of the ganglion, move the chamber to the imaging rig and initiate saline perfusion through the recording chamber. Place a temperature probe near the preparation and set the temperature as appropriate for the species being imaged. Place one chlorided silver wire down the suction electrode, making sure that it contacts the saline in the electrode and place a silver silver chloride wire into the bath saline near the suction electrode.
Lower the water immersion lens into the saline and close the base diaphragm. Raise or lower the sub-stage condenser and adjust the focus until the edges of the diaphragm are in sharp focus, creating Kohler illumination. Focus on the region of the preparation to be imaged and acquire an image of the ganglion of interest with the parfocal digital camera.
Set the control panel gain switch to 1X and click the resting light intensity button to check the average resting light intensity of the diodes. Adjust the voltage level sent from a stimulator to the LED lamp power supply and continue checking the average resting light intensity level until it is in the desired range. Then set the control panel gain switch to 100X for the recording and double-check that the spring or air table is floating.
With the lights still dimmed, set the file duration, pathway, and name in the imaging software and click take data to acquire files up to the capacity of the computer's available RAM. Take care to remain still during the recording as small vibrations can introduce large artifacts into the optical recording data. To view the data immediately after the acquisition, use the superimposed function to superimpose the data collected by all 464 of the diodes over the ganglion image and click any of the diodes represented in the software to expand the recorded data on a separate trace screen.
To achieve an exact alignment of the diodes with respect to the preparation, enter the X, Y, and magnification factors as determined by a pinhole test. To maximize the action potential visibility and improve the neuron yield for subsequent spike sorting, impose a band-pass Butterworth filter with five and 100 Hertz cutoffs to remove both the low and high-frequency noise. To save the filtered optical data as a text file for subsequent analysis, on the page screen, select the TP filter box.
And under the output tab, select save page as ASCII. Then enter an appropriate filename into the dialog box that appears. Skin contact with its sea star predator triggers Tritonia diomedia's escape swim consisting of a rhythmic series of whole body flexions that propel it away to safety.
In this figure, raw and filtered data from 20 diodes recording activity before, during, and after stimulation of pedal nerve three are shown. Immediately after acquisition, the signals measured by all 464 diodes of the recording array can be topographically displayed over an image of the preparation in the imaging software. In this analysis, spike sorting the filtered diode traces with independent component analysis, or ICA, yielded 53 unique neuronal traces, 30 of which are shown here.
A strongly aversive tail stimulus to Aplysia californica elicits repeated waves of a two-part rhythmic escape response consisting of a gallop period of several cycles of head lunges and tail pulls that move the animal quickly forward and a subsequent crawling period. In this representative analysis, one minute into a continuous 20-minute optical recording, pedal nerve nine was stimulated to elicit the gallop-crawl motor program sequence. The probabilistic Gaussian spatial distributions of the signals from all 81 recorded neurons were mapped onto the ganglion.
Dimensionality reduction applied to the full recording revealed that the gallop and crawl phases of the escape program occupied distinct areas and forms different trajectories in the principal component space. When attempting this protocol, important considerations include:optimizing the staining, minimizing vibration, and routing enough light through the preparation to the PDA while minimizing photobleaching of the dye. Examples of post-acquisition analysis that revealed population-level network insights include functional ensemble detection via consensus clustering in unsupervised learning algorithm and dimensionality reduction using principal component analysis.
The combination of our photodiode array-based imaging system and our use of absorbent voltage-sensitive dyes enables the monitoring of dynamical network evolution over extended durations of time.
