Onderzoek van Rapid Dopamine Dynamics met Fast scan cyclische voltammetrie Tijdens intra-orale Tastant Administration in Awake Rats

The overall goal of this procedure is to measure subsecond dopamine responses to oral taste dense. This is accomplished by first creating oral catheters, reference electrodes and carbon fiber electrodes needed for the surgical procedures and subsequent recording via fast scan cyclic V telemetry. The second step is to surgically implant the oral catheters, the intracranial electrode, and the guide cannula for the carbon fiber electrode.

One week later, the carbon fiber micro electrode can be lowered into the nucleus accumbens to record changes of extracellular dopamine release in response to intraoral delivery of tastes. The final step after recording is to calibrate the electrode and to use principle component analysis to isolate data that contains information about dopamine. Ultimately, the combined use of intraoral taste and delivery and fast scan cyclic vol telemetry is used to show changes in sub-second dopamine responses to taten.

There are two main advantages of this combined technique over existing methods. First, fast IC Photometry has a high temporal resolution and second intraoral delivery of taten minimizes the movement the rat needs to make to obtain the tatin and allows us to better isolate dopamine responses to the tatin. Generally, individuals new to this method will struggle with fabrication of the oral catheter.

However, constructing a good and paid oral catheter will ensure a snug fit of the catheter between the cheek and the upper molars, and increase the likelihood of a successful experiment. To prepare the recording electrode first aspirate a seven micron diameter T six 50 carbon fiber into a bora silicate glass capillary with an inner diameter of 0.4 millimeters. Next, position the capillary in a vertical electrode polar initially pull with a heater set to 55.0 and the magnet off, then adjust as needed.

After pulling the pipette under a microscope, cut the exposed carbon fiber so it extends 75 to 100 microns beyond the glass. The fiber in the glass must be tightly sealed, otherwise discard the preparation. Next, strip the insulation from half of a three inch 30 gauge wire, apply heat shrink to secure the electrode to the micro manipulator.

Then apply a thin layer of silver paint to the wire while the paint is still wet. Insert the wire into the open end of the electrode and attach it to the carbon fiber therein. Verify this contact under a microscope.

Take the electrode in the micro manipulator and secure it with heat shrink. Place the prepared carbon fiber electrode into IPA for at least 10 minutes to clean the electrode surface and to increase subsequent sensitivity to dopamine. After removing the electrode from IPA, rinse the carbon fiber with tap water to remove the IPA.

Next, prepare the reference electrode from a 13 millimeter silver wire with a six millimeter silver, silver chloride mesh with a 0.4 millimeter diameter, cut off the non meshed portion of the wire and solder it to a gold pin. Then apply epoxy to the solder. Then dip the mesh portion into a copolymer acid five times.

Let it dry for 30 minutes in air between dips after the last dip. Let it air dry overnight. The next day.

Bake the mesh portion of the wire at 120 degrees Celsius for an hour. To make the oral catheter first cut. Polyethylene 100 tubing into six centimeter segments.

Then make a small flat disc at one of the tube segments by melting the end and pressing it onto a flat cool surface at the center of the disc. Use an 18 gauge needle to create a hole. Attach the other end of the tube segment to the blunt end of an 18 gauge needle.

Then using a hole puncher makes several six millimeter diameter, 3.18 millimeter thick pieces of polytetrafluoroethylene. Then at the center of these discs, create a hole, thus making the discs into washers. Cut the washer into a trapezoid shape to help lodge them in during molar implantation.

To complete the catheter, push a needle attached to PE tubing completely through the washer, and slide the washer flush to the tubing. The following protocol takes about 75 minutes to complete. Begin with a rat prepared for surgery and open its mouth using a cotton swab.

Then push the needle on a cannula into the tissue between the cheek and first upper molar so the needle exits just behind and medial to the ears. Then move up the cannula and secure the washer to the molars to steady the catheter onto the exposed needle. Slide on a washer and bring it tight to the incision.

Secure this washer into position. Trim the tubing so two to four centimeters remain exposed above the rat's head. Then using the same technique, put a catheter into the other side of the mouth.

Following this, put the rat into a stereotaxic frame and drill three holes for the cannulas and electrodes to make recordings in the nucleus accumbens core position. The guide cannula 1.2 millimeters anterior and 1.4 millimeters. Lateral torema.

Lower the cannula apparatus until the plastic base is flushed with the skull. Put the reference electrode in the hemisphere contralateral to the guide cannula. Lower the reference about two millimeters below the dura.

Then position the stimulating electrode, 5.2 millimeters posterior and 1.0 millimeters lateral torema. To target the ventral tegmental area. Lower the stimulating electrode 8.0 millimeters below dura.

Then secure the components with screws and cement and provide postoperative care. Before conducting an experiment, always verify the patency of the oral cannula by infusing 200 microliters of water and watch if the rat tastes the water clear the recording site by removing the dummy cannula, adding two drops of 50%heparin to it and then gently repositioning it. The dummy must not be within two millimeters of the recording site.

Now attach and stabilize the head stage to the stimulator implanted in the VTA and then lower the recording electrode into the nucleus accumbens core. Now following the text protocol, produce a training dataset. Always wait at least two minutes, but up to four to five minutes between stimulations to allow for vesicular reloading And must.

Most critical step in the experiment is obtaining a high quality electrode. If the background is a characteristic rounded shape on the rising phase is a good electrode. If the rounded shape is small or closely matches the applied waveform, then try a different electrode.

If the background is right where shifted, apply an offset shift to the applied waveform in a positive direction in 0.1 volt increments. Once a training set is generated at the recording site, collect data while infusing 200 microliter boluses of taten every one to three minutes for 25 infusions. Then wait at least 30 minutes and infuse the next taste in the same manner.

FSCV combined with intraoral catheter implantation was used to examine how sucrose an up AIT of taten modulates phasic dopamine release in the nucleus accumbens core prior to taten infusion electrical stimulation indicated by the red bar of the VTA produced robust increases in phasic dopamine release in the nucleus accumbens core after the training set was acquired 6.5 second infusions of 200 microliters of sucrose indicated by the red bar produced in elevation in current indicated by the white arrow. This occurred at the oxidation potential of dopamine, which is indicated by the white dash line, transforming the data into a concentration versus time plot. After PCA verified that the increased current in response to the taste is the result of an increase in dopamine concentration.

Another tatin menthol was infused during FSCV recordings in the nucleus accumbens core increased dopamine concentration was also observed after infusion of menthol Once mastered, the combined intraoral and cannulation surgery can be performed in 75 minutes. This technique can be combined with other forms of behavior such as a competitive and aversive Pavlovian and opera conditioning in order to answer questions about subsecond dopamine signaling during these processes.