Assessment of Excitatory Neurotransmitter Levels in Chronic Epilepsy-Induced Rats

Take a control rat and a chronic epilepsy-induced rat implanted with a guide cannula and an electroencephalogram, or EEG, electrode in the hippocampus.

Epilepsy induction increases excitatory neurotransmitter release, causing ion influx into postsynaptic cells, which generates continuous excitatory potentials. This leads to synchronized bursts of electrical activity, termed seizures, recorded using the electrode.

Insert a microdialysis probe with a semi-permeable membrane into the cannula.

Tether the rat to the cage of an EEG recording system.

Infuse a physiological buffer into the brain tissue via the probe inlet.

Ensure the absence of seizure and collect extracellular fluid via the probe outlet to assess neurotransmitter levels.

An elevated basal level in the epileptic rat indicates an epilepsy-mediated increase.

Infuse a high potassium buffer and recollect samples. 

The increased potassium prolongs neuronal excitability, triggering continuous neurotransmitter release.

A temporary spike in neurotransmitter levels in both the epileptic and control rats indicates a high potassium-induced increase.

Begin by preparing the microdialysis probes for the first use according to the manufacturer users guide and fill them with ringer's solution. Then, cut 10 centimeters-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 the 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 plexiglas 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 milliliters 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 milliliters 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 milliliters 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 milliliters 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 2 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 milliliters 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 2 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.