Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

The overall goal of this experiment is to demonstrate how to perform acute in vivo electrophysiological recordings of local field potentials and multi-unit activity from the cortico-basal ganglia loop of anesthetized rats. This method can help answer key questions in the neuroscience field such as how pathological oscillations contribute to the complex pathophysiology in neuropsychiatric diseases. The main advantage of this technique is that it can be used to record extracellular potentials from various cortical and subcortical targets of the brain simultaneously.

The implications of this technique extend toward therapy of different neuropsychiatric diseases because local field potentials can be potentially used as a biomarker for treatments such as closed-loop stimulation. Though this method can provide insight into the pathophysiology of the basal ganglia, it can also be applied to other brain areas. To begin this procedure, prepare a five centimeter length of silver wire with a diameter of 200 micrometers and remove any coating if necessary.

Next, hold the wire tip downwards in a flame until the tip starts to melt and forms a ball with a diameter of one millimeter. Then cut off the electrode to a total length of 15 millimeters from the beginning of the ball-shaped tip to the wire end. Solder a precision connector to the end of the wire which fits the electrophysiological recording system.

Then cover the soldering point from the wire end to the connector with conductive silver varnish to improve conductivity. After the conductive varnish has dried, cover the soldering point with a one to three millimeter heat shrinking tube. Carefully flatten the ball-shaped tip to half the thickness using a watchmaker's hammer.

Subsequently, put on examination gloves and use a lint-free cleaning cloth with 100%ethanol to remove any dirt and grease. Then place the electrodes in a 15 milliliter centrifuge tube or a cell culture tube and fill it up with household chlorine bleach until the ball-shaped tip is fully covered. After 23 minutes, take the electrodes out and flush them generously with distilled water.

The electrode surface should appear homogeneously dark purple in the successful application of silver chloride. Following that, leave the electrodes to dry in air. Take the electrodes with fine tweezers after they have been fully dried.

Then use a fine paintbrush to apply liquid electrical insulation onto the electrodes. Start from the wire directly behind the electrode tip and cover everything up to heat shrinking tube. Afterward, let the insulation dry for at least two hours.

In this step, loosely fix the first pair of electrodes to the acrylic block of the holder with pieces of adhesive tape. The electrodes should protrude from the acrylic block by approximately 12 millimeters. Next, carefully fix the second bipolar electrode next to the first electrode.

For targeting the structures of the hyper direct pathway, the distance between the electrode pairs should be two millimeters. Under the microscope, adjust the second bipolar electrode by sliding it carefully to a position in which the most ventral tip is approximately 200 micrometers recessed compared to the first electrode. Press on the adhesive tape and then secure the electrodes with the holder's metal clamp.

After anesthetizing the animal, shave its head to achieve a clean surgical field. Fix the animal in the stereotaxic frame and then disinfect the incision site and the surrounding area with appropriate surgical disinfectant. Subsequently, perform a two centimeter long incision on the scalp in the sagittal direction with a scalpel.

Use the scalpel to slightly scrape off the skull aponeurosis and disinfect the skull with 3%hydrogen peroxide. Then use cotton buds soaked in 3%hydrogen peroxide to remove any remaining tissue. Next, adjust the incisor bar until the top of the skull is leveled which means the bregma and lambda as stereotaxic reference points are in the same plane.

Under the microscope, use a standard stereotaxic rat alignment tool to calibrate the designated tip to the bregma and adjust the incisor bar until the designated points for the bregma and lambda on the tool touch the skull at the same time. Using a stereotaxic holder with cannula, calibrate it to the bregma and then calculate the position of all drill holes on the skull. Then mark the positions of the holes to be drilled either by carefully scratching the skull or by using a surgical color marker.

Then using a micro drill, carefully drill all the holes with a diameter of about one millimeter. For the STN and SNr, drill a common hole approximately two millimeters by three millimeters. Take one fine cannula and bend the tip to form two hooks.

Then use the hooks to remove any debris from the drill holes and carefully cut and remove the dura mater in the common STN/SNr hole. After that, flush the drill holes with physiological saline. Apply a drop of physiological saline every 15 minutes to the drill holes to prevent the brain and dura from drying out.

Then take a micro drill and matching stainless steel micro screw, drill a hole and screw in a micro screw between the drill holes of the reference epidural electrodes above the cerebellum. Next, guide the electrode tips with fine tweezers and slide them directly below the skull bone into the drill holes. Fix all epidural electrodes with two components stencil acrylic.

Make sure not to cover the bregma point nor affect the common STN/SNr hole. Then insert the prepared holder with the tungsten micro wire electrodes into the stereotaxic frame. Calibrate the most ventral electrode to the bregma which is intended to target the STN.

Adjust to the calculated position above the common STN/SNr hole and lower the electrodes down to the brain. Make sure that the tungsten micro wire electrodes go inside the brain smoothly. In this procedure, set up the recording software such as band-pass filter and amplify the raw data signal.

Then use an online LFP and spike filter with appropriate settings. For all filters, use a Butterworth-type filter. Set up a spike threshold for online spike sorting.

Most recording software allows for the setting up of a spike threshold which is an amplitude value above the threshold that a signal is marked as a spike by the software. Next, slowly lower the tungsten micro wire electrodes to one millimeter above the STN and wait for the signal to stabilize if necessary. For the electrophysiological mapping, advance the electrodes ventrally in the steps of 100 micrometers.

At each step, evaluate the firing pattern, firing rate and shape of spikes by comparing those with the typical examples given in figure two of the accompanying manuscript. Once the electrodes are in the desired structures, set up online filtering and spike sorting and then start the recording of the data. Here is a representative 600-second LFP recording of the primary motor cortex.

Time periods with high frequency low amplitude activity corresponding to the activated state and the time periods with the slower rhythm and higher amplitude corresponding to the slow wave activity state can be differentiated. And this is the corresponding time frequency plot over an interval of 600 seconds illustrating the zero to 20 Hertz relative power of the LFPs. Once mastered, this technique can be done in two hours if it's performed properly.

After watching this video, you should have a good understanding of how to perform extracellular in vivo electrophysiological recordings from the cortico-basal ganglia loop of anesthetized rats.