Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat

The overall goal of this surgical implantation of electrodes is to record central and peripheral bioelectric signals simultaneously from a freely moving rat. This method can help answer key questions in the neuroscience field, such as memory, cognition, emotion, and brain-body interactions. The main advantage of this technique is that a single recording device collects all the bioelectrical signals from the wide range of body areas.

We first had the idea for this method when we noticed that muscle and cardiac activities are both represented by electrical signals similar to brain extracellular signals. We therefore considered that all signals can be recorded by a single, multichannel recording device. To begin, prepare a standard micro-drive array for cortical LFP recordings.

Leave at least six metal holes open on an electrode interface board for the signaling channels. Next, make six five-centimeter lengths of Bioflex wire to carry the signals. Peel off about five millimeters of PFTE coating on both ends.

Then, connect one wire to each metal hole in the board using a gold pin. Next, make two five-centimeter lengths of enamel wire, and solder them to the two ground/reference channels on the board. To make the ECG electrodes, cut two 16-centimeter lengths of Bioflex wire and peel off the 0.5 millimeter of coating from one end and 15 millimeters of coating from the other end.

Then, form each long stripped section into a two-millimeter-diameter circle, and secure the loop with solder. To make the EMG electrodes, cut two eight-centimeter lengths of Bioflex wire and peel off about five millimeters of coating from both ends. To make the BR electrodes, cut two six-centimeter lengths of Bioflex wire and peel off about five millimeters of coating off both ends of each wire.

For each of the two BR electrodes, solder one end of each wire to the head of a stainless steel screw. To make the ground/reference electrodes, cut two six-centimeter lengths of enamel wire. For each of the two ground-reference electrodes, solder one end of the wire to a slightly larger screw.

Now sterilize all of the electrodes, and then handle them under sterile conditions. To begin, fix an anesthetized rat on its back on a flat heat pad. Provide buprenorphine as an analgesic, and apply ophthalmic ointment to prevent dryness.

Next, clean the chest and neck with alternating scrubs of 70%ethanol and betadine. Then, drape the animal to leave only the chest area exposed. First, implant the ECG electrodes.

Begin with a two-centimeter incision into the medial chest area. Then, expose the intercostal muscles by separating the chest muscles. Now, suture the rings of the ECG electrodes to the intercostal muscles.

Do this tightly so that the electrodes are very secure and there is a high signal-to-noise ratio. Then, make a one-centimeter incision in the dorsal neck area, and tunnel the ECG electrodes subcutaneously to the incision, leaving a slack of wire under the skin to minimize physical stress at the electrode. Then, suture closed the chest incision.

Next, reposition and secure the animal to attach the EMG electrodes. Insert one end of each electrode subcutaneously about one centimeter into the neck incision and into the neck muscles. Then, suture these electrodes in place.

After the electrode implantations, secure the rat to a stereotaxic device. Then, shave and clean the skull, and start the array implantation with a three-centimeter incision along the midline from the point between the eyes to the neck. Using a high-speed drill, make a pair of circular craniotomies between 0.7 and 1.0 millimeters in diameter above the olfactory bulb, which is 11.0 millimeters anterior and one millimeter bilateral to bregma.

Then, implant the two BR electrodes just deep enough for the tips of the screws to make contact with the brain, which is about two millimeters deep. Use between six and eight full turns of the screw. Next, make another pair of craniotomies above the frontal cortex, 2.7 millimeters anterior and 2.7 millimeters bilateral to bregma.

Into these holes, secure the two ground/reference electrodes so they also just contact the brain about 1.6 millimeters deep. This takes four to five full turns of the screw. Now, plan a large circular craniotomy with a diameter of about 2.0 millimeters above the hippocampus, 3.8 millimeters posterior and 2.5 millimeters bilateral to bregma.

Then, make six to eight 1.0-millimeter holes in the area that will surround the craniotomy. Into each of these holes, implant the anchor screws. Then, make the planned craniotomy between the screws.

Above the large hole, position the integrative micro-drive array such that the cannula tip is just above the large craniotomy. Fill the gap space between the cannula tip and the brain surface with about 100 microliters of two-part epoxy. Over the next five minutes, allow the mixture to change into a transparent gel.

Next, cover the cannula, BR electrodes, ground/reference electrodes, and anchor screws with dental cement. Apply about five millimeters of cement without covering the open ends of BR or ground/reference electrodes. Now, solder all the open ends of the electrodes to where they connect to the board.

Then, cover the bottom part of the integrative micro-drive array and all electrode wires with dental cement. It is important to completely cover the electrode wires so that the rat cannot scratch them out after the implantation. Now, allow the rat to recover.

After regaining sufficient consciousness to maintain sternal recumbency, house it solo without any cage mates, and give it free access to food and water. After the surgery, gradually advance the tetrodes by advancing the screws daily. Once the tetrodes are adjacent to the target brain areas, let them settle for several days, during which signals will stabilize.

Using the described methods, bioelectrical signals from the brain, heart, lungs, and skeletal muscles can be captured simultaneously and correlated. For example, a freely moving rat undergoing foraging behavior provides a dataset with transitions between active and resting states. A power spectrum was computed from a hippocampal LFP trace using wavelet analysis.

The BR signal recorded from the surface area of the olfactory bulb was used to roughly estimate the relative changes in breathing frequencies, such as those that occur during exploratory sniffing behavior. Once mastered, this technique can be done in three hours if it is performed properly. After its development, this technique paved the way for researchers in the field of neuroscience to explore the relationship between the central and peripheral organs with high temporal resolution in rodents.