Optogenetic Activation of Intrinsic Cardiac Autonomic Neurons in Excised Perfused Mouse Hearts

We present a valuable experimental approach to investigate the kinetics of sudden intrinsic autonomic neuron activation in perfused hearts and the interaction between cardiac catecholaminergic and cholinergic activity. Perfused mouse hearts can be very sensitive to changes in the environment, such as temperature, oxygenation, and perfusate concentration. They require closer monitoring than larger animal models.

Additionally, the micro-LED can take some trial and error at first to get consistent results. We are working to understand what happens in the heart when the sympathetic and parasympathetic systems are activated at the same time. What is new is that we are studying that topic using optogenetics to photostimulate the cardiac ganglia and neurons within the heart itself.

The micro-LED shown here is low-cost and relatively simple to replicate. Due to its size, the micro LED is maneuverable and can target areas of the heart more precisely than larger light sources. To begin, under a dissecting microscope in a well-ventilated area, solder the stripped ends of two insulated copper wires to the contact points of a 465-nanometer micro-LED.

Connect the micro-LED to a power source, and turn it on to test the soldering. Cut the bottom one centimeter of a 200-microliter filtered pipette tip. Push out the filter using a small-diameter rod.

Insert the micro-LED with attached wires into the pipette tip so the LED is flush with the end of the tip. Replace the filter at the top of the pipette tip to secure the LED and wires. Then superglue the edges of the LED to the pipette tip.

Allow the superglue to dry. To prepare silicone elastomer, mix the base and curing agent until the solution becomes uniform. Remove any bubbles from the mixture using a vacuum chamber.

Next, take a 0.5-milliliter centrifuge tube and score the sides to facilitate LED removal. Tape the outside of the centrifuge tube to prevent leaking. Pour approximately 0.2 milliliters of the silicone elastomer into the tube.

Place the micro-LED pipette tip into the tube, ensuring at least one millimeter of space between the LED and the bottom of the tube. Place the centrifuge tube micro-LED upright in a 50 degree Celsius oven for eight hours or overnight. Once the elastomer is hardened, remove the LED from the tube.

Once cured, trim any excess elastomer from the LED tip with a precision utility knife leaving no more than one millimeter. To begin, add 175 milliliters of Krebs-Henseleit or KH solution to the perfusion system. Place a 10-micrometer membrane filter in the Langendorff perfusion system.

Then start circulation. Turn on the water baths and set them to maintain the perfusate temperature at 37 degrees Celsius. To calibrate the flowmeter, halt the flow in the perfusion system using a stopcock.

Then press the zero button on the flowmeter to perform the calibration. Open the LabChart data acquisition software. Configure the software to include 12 channels.

Set up channels for heart bath temperature, aortic perfusate temperature, ECG leads one to three for additional computed leads, heart rate calculation, flow rate, and a function generator output to track LED pulses. Next, use the channel designated for heart rate to calculate heart rate. Activate the cyclic measurements feature, setting it to detect mouse ECG on lead one.

Use LabChart's Cardiac Access extension to compute leads three aVR, aVL, and aVF based on leads one and two. After anesthetizing and euthanizing the mouse, hold the xiphoid process with a pair of forceps, and cut into the abdominal cavity using surgical scissors. Carefully cut through the diaphragm to open the thoracic cavity.

Then cut through the ribs to expose the heart and lungs. Gently grab the lungs, and excise the heart and lungs. Place the heart into a dish containing heparinized KH solution.

Remove the lungs and any large fat deposits. Under a dissecting microscope, set to 2x magnification. Transfer the cleaned heart to a second dish of heparinized KH solution.

Locate the aorta and slide it over the cannula using fine forceps. Secure the heart to the cannula with a 4-0 silk suture. Next, flush the cannula with a bolus of heparinized KH to remove blood from the coronary vessels.

Connect the cannulated heart to the perfusion system and place it in the PDMS dish filled with perfusate. Place ECG needle electrodes into the PDMS dish according to Einthoven's triangle. Rotate the heart so the left atrium is accessible.

Use micro self-opening scissors to create a one-millimeter incision in the left atrium. Insert a one-millimeter diameter tube into the incision to allow trapped perfusate in the left ventricle to drain. Next, rotate the heart so the right atrium is facing upward and the sinoatrial node is accessible for illumination.

Adjust the ECG electrodes as needed by moving them closer to the heart to improve the signal-to-noise ratio. For optogenetic activation, connect the micro-LED device to a function generator and configure it to produce pulse waves with a frequency of 10 hertz, a pulse width of 30 milliseconds, and an amplitude of 10 volts peak to peak. Place the micro-LED gently on the sinoatrial node.

Then turn on the function generator and observe changes in heart rate from photostimulation. Immediate changes in heart rate indicate effective activation. Turn off the function generator.

Allow the heart rate to return to pre-activation levels. If optogenetic activation results in a heart rate change of less than 100 BPM, reposition the micro-LED to illuminate the neurons in the right atrium. Optogenetic stimulation of ChAT neurons reduced heart rate by over 100 BPM during light stimulation without norepinephrine sustaining the reduction throughout stimulation.

With 2, 000 nanomoles of norepinephrine, the heart rate dropped by 40 BPM and began recovering before the light was turned off. Optogenetic suppression of heart rate by ChAT neuron photostimulation was less effective in overcoming heart rate increases induced by high norepinephrine doses, resulting in shorter suppression times and smaller decreases in heart rate.