In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.
This method can help answer key questions in the neuroscience research field about how signal transduction substrate transport and a waste clearance occur in the brain extracellular spaces. The main advantage of this technique is that it allows the sampling and quantification of large extracellular molecules in the Interstitial Fluid or ISF of awake freely-moving animals. After confirming a lack of response to toe pinch, remove the hair from the skull of an anesthetized mouse and use the ear bars and a nose clamp to securely the fix animal onto an adapter.
Apply ointment to the animal's eyes and place the adapter onto the stereotaxic apparatus. Use a scalpel to make a skin incision sagittally over the skull and attach a drill onto the manipulator of the stereotaxic frame. Lower the drill until it gently touches lambda and reset the DV coordinate of the drill to zero.
Then, move the drill to bregma for resetting of the AP and ML coordinates to zero. Move the drill from bregma to the vertical coordinate. Lower the drill to the skull and set the DV coordinate to zero again.
After repeating the procedure for a third coordinate, drill a burr hole carefully at the target coordinate, followed by a second hole on the contralateral side of the parietal bone. Insert a bone screw into the second hole and place the locking piece from a 1.5 milliliter microcentrifuge tube lid onto the skull so that the burr holes are within the circle. Next, place a guide cannula on the shorter arm of the stereotaxic adapter and fasten the cannula with a cap nut.
Set the longer arm of the stereotaxic adapter on the electrode clamp and attach the arm on the manipulator of the stereotaxic apparatus. Rotate the DV stereotaxic assembly on the manipulator arm by 12 degrees and move the guide cannula to the burr hole. Then, slowly lower the guide cannula 1.2 millimeters into the brain.
Add dental cement to the crown until the metal part of the guide cannula, the bone screw, and any exposed skull are covered and secured and allow the cement to dry. After 12 to 20 minutes, remove the stereotaxic adapter from the electrode clamp, remove the cap nut, and replace the stereotaxic adapter with a dummy probe. Then, refasten the cap nut, release the mouse from the stereotaxic apparatus, and house the mouse alone in an individual cage.
Before setting up the microdialysis, fill a disposable one milliliter syringe with distilled water and use a viton tube to connect the syringe to the outlet of a microdialysis probe. Manually cover the vent holes and gently depress the syringe plunger to infuse the probe with water. Confirm that water appears in the probe inlet and that there is leakage onto the surface of the microdialysis membrane.
To activate the probe, submerge the probe membranes in 70-100%ethanol for two seconds, followed by a second distilled water infusion. Attach a connection needle to one inlet and one outlet line and load a disposable three milliliter syringe with freshly prepared perfusion buffer. Connect a syringe equipped with a blunt-end needle to the inlet end of the tubing and use a syringe pump to fill the entire tubing with perfusion buffer.
When the tube is full, replace the connection needle between the inlet and outlet lines with the activated microdialysis probe and the cap nut and mount a roller tube into the outlet tubing on a roller pump. Start the syringe pump at 10 microliters per minute and the roller pump at 9.5-9.8 microliters per minute. Now place the collar around the neck of the anesthetized guide cannula implanted animal and remove the cap nut and dummy probe.
Slowly insert the microdialysis probe through the guide cannula and fasten the cap nut. Then, place the mouse in a cage connected to a free-moving system and tether the mouse with the collar. After at least one hour, sequentially stop the roller pump and the syringe pump, restarting the pumps with the syringe pump set to 20%faster than the roller pump and place the free end of the outlet tubing on a refrigerated fraction collector to collect the brain ISF.
When the appropriate experimental volume has been obtained, remove the probe and handle the mouse recovery as just demonstrated. Consistent with previous observations, when 50 micromolar Picrotoxin is administered via reverse microdialysis as demonstrated in awake C57B6J mice, an increase in interstitial endogenous tau levels is observed compared to levels measured in animals treated with a vehicle control. In addition to drug administrations, microdialysis can be combined with other in vivo methods like EEG recording or optogenetics to answer additional questions about how neuronal activity influences substrate concentration in ISF.
