The overall goal of this methodology is to simultaneously record co-localized electroencephalography and local field potential in the anesthetized rat. To begin this procedure, record the rat's weight on a laboratory scale. Anesthetize the rat in an isoflurane chamber.
Then place it onto a stereotaxic holder, with a paper towel underneath its body and rest its teeth on the bite bar. Next administer isoflurane continuously via a hard, plastic nose cone mounted onto the nose clamp. Connect the cone to a small animal isoflurane anesthetic system.
After that, insert a thermostatic heating pad underneath the paper towel upon which the rat is resting and then secure the rat's head with two ear bars. Monitor the body temperature using a rectal thermometer. Now, shave the top of the animal's head.
Then, apply ophthalmic ointment to the eyes to prevent corneal drying. Before exposing the cranium, apply lidocaine drops to the scalp and massage it gently into the skin. After that, make a midline incision of approximately two to three centimeters on the scalp using a scalpel to expose the skull.
Carefully separate the temporalis muscle, contralateral to the whisker pad, to be stimulated from the skull using a jacket scaler and a pair of serrated, curved dissecting forceps. Clean the skull with cotton swabs whenever necessary. Using a braided, silk, non-absorbable suture tie the separated muscle to the scalp with a tight knot and then tie the suture securely to the stereotaxic frame.
Next, use the stereotaxic coordinates to locate the barrel cortex, which is two point five millimeters caudal to bregma and six millimeters lateral to midline. Then draw a dot at the location of the somatosensory cortex using a fine tip, permanent marker. Drill a hole of diameter smaller than two millimeters into the skull.
Take care not to drill into the dura. Thin the bottom of the hole to translucency. To prevent the skull from overheating during drilling, apply sterile saline to the work area every 10 to 15 seconds.
Then use a 27 gauge needle to pierce the dura in order to allow the insertion of a microelectrode. Following that, transfer the stereotaxic frame with the rat to a faraday cage mounted on top of a vibration isolation work station. Attach an oximeter sensor clamp, connected to an oximeter control unit, to the rat's hind paw to monitor the physiological parameters continuously.
After that, replace the hard, plastic nose cone and the nose clamp with a micro flex breather fitted with a transparent soft nose cone to allow easy whisker stimulation to one side of the whisker pad without compromising the isoflurane administration. Next, insert two, stainless steel stimulating electrodes to the whisker pad exposed by the cutout on the nose cone. Following that, connect the stimulating electrodes to an isolated current stimulator.
Subsequently, lift the skin of the midline of the neck with forceps and make a one to two centimeter incision with scissors, ready for the placement of reference electrodes. In this procedure, clean and dry the skull surrounding the burr hole using a cotton swab. Carefully apply the conductive EEG paste on a flat side of an EEG spider electrode.
Leave a small hole clear of EEG paste on the spider electrode to allow a multilamellar microelectrode to pass through the hole without contacting the paste and the spider electrode. Align the spider electrode with the burr hole in the skull with the EEG paste facing the skull. Carefully press the spider electrode onto the skull, making firm contact with the skull via the EEG paste.
Remove any paste obscuring the burr hole, using a needle on a syringe and remove excessive EEG paste beyond the perifia of the spider electrode so that the contact between the spider electrode and the skull is spacial constrained to the size of the electrode. Then, smear EEG paste onto the reference electrode for the the EEG and place it securely inside the incision at the back of the rat's neck. Next, connect the EEG electrodes to the preamplifier via a passive signal splitter for low impedance signals.
At this stage, test the resistance of the EEG probe to make sure it is below five kilo ohms. If not, add more EEG paste and make sure the spider electrode makes a good contact with the skull. Subsequently, mount a micromanipulator arm on the stereotaxic frame.
Connect a linear 16 channel microelectrode to a 16 channel acute head stage, clipped securely onto the micromanipulator arm. Then, smear EEG paste onto the reference electrode for the microelectrode and then place it securely inside the incision, next to the reference electrode for the EEG. Adjust the angle of the micromanipulator arm so that the microelectrode is perpendicular to the cortical surface.
Now, lower the microelectrode under a microscope, so that the tip of the microelectrode is aimed at the tiny opening at the bottom of the burr hole until the upper most electrode just penetrates the cortical surface. Care must be taken to avoid forcing the microelectrode onto the surface of the dura as this would break the electrode. Insert the microelectrode to the cortical surface at a depth of 1, 500 micrometers.
Microadjust the depth by apply a train of stimulas to the whisker pad and observing the 16 channel evoked LFP on a PC monitor. Carefully turn the z-axis knob on the micromanipulator until the highest amplitude of the invoked LFP occurs as this coincides with the layer four in the cortex. This figure shows that the ERP, recorded by the EEG probe is an order of magnitude smaller than the LFP recorded by the microelectrode in the supragranular layer of the barrel cortex.
The temporal profile of the ERP is similar to that of the LFP in the supragranular layer when normalized to the negative peek and super imposed. However, peek latencies of ERP are longer than the corresponding peek latencies in LFP. On the other hand, the temporal profile of ERP is markedly different from that of the granular layer LFP.
Importantly, they are not mirror images of each other with granular LFP dominated by a single negative peek. Whereas, ERP consists primarily of two peeks with opposite polarity. Finally, EEG signals collected around a burr hole in the skull are not significantly different from EEG recordings from an intact skull.
Once mastered, this technique can be done in one hour if it is performed properly. After it's development, this technique paved the way for researchers in the field of EEG and computational neuroscience to explore the neurogenesis of sensory evoked potentials. Thus, providing constraints for the mathematical modeling of ERPs.
After watching this video, you should have a good understanding of how to record co-localized EEG and LFP concurrently.
