Here, we describe a method for in vivo microdialysis to analyze aspartate and glutamate release in the ventral hippocampus of epileptic and non-epileptic rats, in combination with EEG recordings. Extracellular concentrations of aspartate and glutamate may be correlated with the different phases of the disease.
This method can help answer key questions in the neurosciences such as the alterations in neurochemistry during the natural history of neurological diseases. The main advantage of proposed microdialysis EEG technique is that such combined approach allows the pairing of changes in the neurotransmitter release within specific stages of the disease development and progression. Begin by switching on the amplifier positioned outside of the Faraday Cage.
Open the EEG software and start the EEG acquisition and observe the EEG signal produced by unconnected cables. Next, connect the animal to the tethered EEG recording system by holding the animal's head between two stretched fingers of one hand and screwing down the connectors to the electrode pedestal using the other free hand. Set an amplification factor on each channel of the amplifier according to the electrode signal of a single animal so the EEG signal is in scale.
Then let the animal explore the new cage for at least one hour under the direct observation of the researcher. Adjust the cable according to the animal's commodity, and ensure that the cables do not interfere with the animal's movements and lying posture. Finally, check for the correct image framing on the video cameras and then start the video EEG recording.
Begin by preparing the microdialysis probes for the first use according to the manufacturer user's guide and fill them with Ringer's Solution. Then, cut 10 centimeter long pieces of FEP Tubing and connect them to the inlet and outlet cannulas of the probe using the tubing adapters of different colors. Ensure that tubing touches the adapters with no dead space in all connections.
Remove the dummy cannula from its guide using the tweezers by holding the animal's head firmly. Insert the microdialysis probe into the guide cannula and use modeling clay to further firm up the cannula. Next, connect the animal to the tethered EEG recording system.
Then put the animal into the plexiglass cylinder and let it explore the new environment. Follow the awake and freely moving rat movements. Then, connect the inlet of the probe to the 2.5 milliliter syringe with a blunted 22 gauge needle containing Ringer's Solution using the tubing adapters.
Push one milliliter of Ringer's Solution into the probe in 10 seconds by pushing the piston of the 2.5 milliliter syringe continuously. Check for the drop of the liquid appearing on the outlet to signify when the probe is ready for use. Finally, fill up the 2.5 milliliter syringes connected to FEP tubing by tubing adapters with Ringer's Solution and mount them onto the infusion pump.
Start the pump at two microliters per minute and let it run overnight. After verifying via video EEG recordings the absence of seizures in the three hours preceding sample collection, stop the pump carrying the FEP tubing cannulated syringes filled up with Ringer's Solution. Mount another set of 2.5 milliliter syringes connected to FEP tubing with tubing adapters filled up with a modified Ringer's Solution containing 100 millimolar potassium solution.
Start the pump at two microliters per minute and let it run. Check for the absence of air bubbles in the system and ensure that the tubing touches the adapters with no dead space in all connections. Then, check for the drop of the liquid appearing on the outlet to signify when the probe is ready for use.
Next, connect the FEP tubing of syringes filled up with Ringer's Solution to the inlet cannula of the probe in each animal and wait for the appearance of the liquid drop on the tip of the outlet. Connect the outlet of the probe to the FEP tubing which leads to collection in the test tube. Insert the FEP tubing into the closed 0.2 milliliter test tube with a perforated cap and ensure that the tube stays in place by fixing it with a piece of modeling clay.
After running the pump at two microliters per minute for 60 minutes without collecting samples to equilibrate the system, collect five consecutive 30 minute dialysate samples under baseline conditions, and store samples on ice. After 10 minutes, switch the tubing from the syringes containing 100 millimolar potassium Ringer's Solution to normal Ringer's Solution and let the pump run. After collection of the fifth post-equilibration dialysate, collect 20 microliter dialysate fractions every 10 minutes for one hour.
Then collect three additional 30 minute dialysate samples and stop the pump. Lastly, store the samples at minus 80 degrees Celsius after the experiment until HPLC analysis. After the experiment is complete, rinse the used microdialysis probes with distilled water and store them in a vial filled with clean distilled water until next use.
Finally, rinse the entire microdialysis setup with distilled water followed by 70 per cent ethanol. Replace ethanol with air, and store the setup in a sterile environment. These are the representative EEG traces recorded from the hippocampus of control and epileptic rats during the chronic phase of the disease.
No EEG alteration was observed in control rats, whereas a synchronized paroxysmal epileptiform activity was observed about 500 milliseconds before and during the behavioral seizures in epileptic rats. Basal glutamate concentration found in chronic epileptic rats was significantly higher than in control animals. High potassium evoked an additional release of glutamate for about 30 minutes in control rats and for about 60 minutes in chronic epileptic rats.
While attempting this procedure, it is important to remember that animals implanted with the device are prone to lose it over a course of prolonged experiment, so use a light head set and sufficiently long cables to avoid in interfering with animal mobility. Depending on the availability of an appropriate analytical essay, the method may be further used to test different soluble molecules in the brain when employing EEG recording at the same time. After its development, this technique allowed the researchers in the field of epileptology to provide an insight into the alterations in excitatory neurotransmission in the case of a temporal lobe epilepsy.
After watching this video, we trust you will have a better understanding of how to perform in vivo microdialysis together with depth electrode EEG recording in a freely moving epileptic rat.
