This microinjectrode system is designed for drug infusion, electrophysiology, and delivery and retrieval of experimental probes such as microelectrodes and nanosensors. It is optimized for repeated use in awake behaving animals with minor penetration damage to surrounding tissues. The microinjectrode system can be configured for multiple purposes.
The first is a simple arrangement of the cannula for placement of an experimental probe that would otherwise be too fragile to penetrate the dura mater. The second is a microfluidic infusion of a drug either independently or coupled to a cannula containing an experimental probe. The microfluidic components of the system allow for delivery of volumes in the nanoliter scale.
Measure the length of the cannula and the nanosensor probe or microelectrode. Probe must be longer than the cannula by the length that is to protrude from the cannula tip plus approximately one centimeter. Under a magnifier, load the probe into the cannula through the back to protect the tip of the probe.
Insert the cannula containing the probe into the T junction from the bottom ferrule. Place the top flat-end side of the cannula in the middle of the T junction. Avoid blocking the junction via the cannula by retracting it back into the junction and then tighten it.
Attach a tubing to the top of the microelectrode. Back load the microelectrode through the capillary tubing, T junction, cannula, and corresponding ferrules. Cut the microelectrode at the desired length and scrape off the end.
Make sure that the back end of the electrode protrudes less than one centimeter from the back of the capillary tubing and the tip of the electrode protrudes from the cannula at the desired distance on the bottom side. Place the microelectrode terminal into the gold pin and solder the gold pin to the microelectrode terminal. Add epoxy glue between the gold pin and the top ferrule to attach the microelectrode to the ferrule.
After the epoxy is cured, preferably for more than 24 hours, unscrew the top ferrule to make sure that the microelectrode fully retracts inside the cannula. To construct the microfluidic circuit, place a broad board on a stable surface. Place the two three-way valves parallel to the longest sides of the broad board about 12 centimeters apart with one port facing each other.
Use screws to fix the valves to the broad board and cut another 10 centimeters of capillary tubing for the ruler line and place it in between. Use standard ferrules to tighten the tubing to the facing ports of the valves. Cut 10 to 20 centimeters of the capillary tubing and use the standard ferrules and the Luer lock connectors to connect the tubing on the syringe to one of the ports on the input valve.
Cut a small piece of capillary and connect it to the output valve as the flush line. Cut two longer pieces of capillary tubing around 100 centimeters to connect the output valve to the microinjectrode. Connect the drug pump and marker pump to the input valve.
First, make sure the microelectrode experimental probe is retracted in the cannula. To attach a custom-made adapter to the microinjectrode using screws, top load the microinjectrode through the guide tube and secure it to the custom-made micro drive adapter using a pair of screws. Measure the depth of micro drive position at which the microinjectrode protrudes from the guide tube, then retract it one centimeter to prepare for insertion.
For microinfusion experiments, connect the brain line to the unused T junction opening of the microinjectrode. Use a standard ferrule and tighten with the ferrule wrench. Make sure that the top ferrule is tightened as well.
Then, position the micro drive over a beaker. Load chlorhexidine at 20 grams per liter into the one milliliter gas tight syringe and place it in the drug pump. Turn the flow direction of the valves and set a low flow rate of 50 to 200 microliters per minute such that the fluid goes from the drug pump through the input valve to the output valve and out the brain line.
Flush the circuit with chlorhexidine for a minimum of 10 minutes. Repeat the flushing with sterile saline and then with air. Gently apply lint-free wipes at the junctions to help reveal any liquid leaks through the ferrules.
The most critical step is verifying that the assembly of injectrode and microfluidic circuit is leak free. Load the drug in the 500 microliter gas tight syringe, compress the air and then place it in the drug pump. Adjust the flow to 50 microliters per minute and let the liquid travel until a few drops are left in the microinjectrode.
Then, soak the guide tube in chlorhexidine at the concentration of 20 grams per liter for 15 minutes. Turn the direction of the output valve towards the flushing line to displace the marker as the marker pump is advanced until a clear edge of color and oil is observed on the ruler line. Make sure there is always oil between the drug and the color in order to not mix the two water soluble materials and lose the sharp edge between them.
Mark the starting position of this oil dye line. After the necessary experimental setup, retract the microelectrode into the cannula by loosening the top ferrule. Attach the micro drive to the recording chamber and lower the guide tube to penetrate the dura.
Next, lower the microinjectrode to about two millimeters above the recording site located in the brain. Tighten the top ferrule and connect the gold pins to the recording system. Keep advancing the microinjectrode to the target site.
Then, switch the output valve to the brain line. For infusion experiments, use the manual microsyringe pump to move the column of oil by 0.5 centimeters every minute. Once the desired volume has been infused, switch the output valve towards the flushing line.
In this study, injection of a GABA A agonist through the right hemisphere FEF area was performed for reversible inactivation of the frontal eye field while the animal finished a memory-guided saccade task. Polar plot shows the performance of eccentricity for different locations relative to the fixation point. Performance clearly decreased in the left visual hemifield two hours after injection.
Saccade traces for eight peripheral memory locations before and after Muscimol injection in the FEF are shown here. Saccade accuracy in the left visual hemifield decreased after Muscimol injection. Once the setup is complete, the method is very reliable and robust.
However, due to the precipitation of small molecules within the tube and ports, a thorough flushing is required before each use and after each experiment in order to keep the microfluidic free of obstructions and leaks. Although the method was demonstrated in the frontal eye field in a nonhuman primate, the principle can be applied to any other brain region where some combination of electrical stimulation, recording, and drug injection are desired in species of rodent size or larger. Our system has the flexibility to be used for recording either independently or in combination with drug injection and has the ability to precisely place any fragile experimental probe protected from damage through the dura mater and neural tissue with minimal tissue damage due to its small cannula diameter.
