Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

The overall aim of this procedure is to measure hippocampal metabolism during behavioral testing. This is accomplished by surgically preparing a well-handled animal with a microdialysis guide cannula. On the day of testing, a microdialysis probe is inserted into the cannula and the PERFUSE eight flow is confirmed.

Next, the probe is allowed to equilibrate while the animal is sitting in a control chamber. Ultimately, appropriate analytical methods, for instance, a glucose oxidase, NADP, N-A-D-P-H reaction and flu metric analysis. If measuring glucose is used to show the level of metabolism and or neurochemical release throughout the testing period, One of the most difficult aspects of this technique is the care needed in handling the microdialysis tubing and also often chew it or tangle the lines if care isn't taken.

So the first time I used hippocampal microdialysis during behavioral testing was doing my graduate work, and we wanted to check whether the existing models bring glucose supply. Were in fact compatible with the known psychological data regarding enhancement of memory by glucose. This methodology allowed us to show that in fact, the existing models of glucose applied to the brain were completely wrong.

Following standard surgical procedures, prepare the animal, expose the skull and drill holes for cannula placement. Insert a cannula at predetermined coordinates and then slowly lower it to the target depth. Once correctly positioned, secure the cannula in place with dental cement.

Use a sterile surgical suture to close the wound if needed. Lastly, a style is used to maintain cannular patency. Next, prepare the PERFUSE eight to be used for microdialysis.

It should be noted that accurate fluid composition is essential for optimal results. On the day of testing bovine serum albumin BSA should be added and fully dissolved. After preparation, the PERFUSE eight should be filtered through a 0.2 micron filter.

Next, prepare the treatment that will be administered to the brain, if any, using an aliquot of the prepared A ECF. Here insulin is prepared for delivery into the hippocampus. A fresh microdialysis probe and line should be prepared the day before testing.

Create two separate lines for inflow and outflow. Use PE 50 tubing to connect two one meter long pieces of FEP tubing and ensure that there is minimal dead space between the lines. Tubing should be trimmed as shown to create slanted ends for ease of insertion into the PE 50 connectors.

Connect the inflow tubing to a one milliliter Hamilton syringe filled with sterile filtered deionized water and attach the probe to the other end to allow the animal to move freely while taking measurements. Connect the inflow and outflow lines to a two channel swivel. Next, connect the outflow tubing to the pump and run it at 1.5 microliters per minute until you see deionized water exiting the outflow tube on the probe.

Turn the pump off and connect the other line between the outflow tube and a sample collection tube. Run five milliliters through the tube and place the probe in a vial containing sterile deionized water overnight, ensuring that the probe tip always remains wet. 24 hours before testing, pre-Roe the test subject by removing the dummy stylet and inserting a microdialysis probe that is used only for this purpose.

For 10 minutes afterwards, replace the dummy style it and put the rat back into its cage. Alternatively, use a fresh probe in this stage and leave in place without removal until taking measurements. The following day.

On the day of Microdialysis, fill the Hamilton syringe and scintillation vial with filtered A ECF and allow the pump to equilibrate for one hour. If needed, fill a second Hamilton syringe with prepared treatment and place into the syringe pump. The key to success in this procedure is to remain calm and to handle the animal really extensively.

So remain calm, move slowly and precisely and above all practice. When the equilibration is complete, remove the dummy stylet and gently insert the equilibrated microdialysis probe into the rat's brain via the cannula. Place the rat into a clear plastic box that contains some of the rat's home bedding.

Make sure to counterbalance the tubing. Attach a 1.6 milliliter micro centrifuge tube filled with water to the tubing outside the cage in such a way that gravity keeps the microdialysis lines un kinked but not taught. Allow the probe and rat to equilibrate for two hours.

At the start of this period, confirm the flow of Perfuse eight is unblocked. To do this, collect PERFUSE eight outflow over a defined period and weigh the sample to confirm that the expected volume is exiting the system before collecting samples. Ensure that the correlation between sample dialysis and collection is accurately calculated.

Here we have a 30 microliter volume between the probe and collection point at a rate of 1.5 microliters per minute. There is a 20 minute lag from when the sample exits the brain until it is collected. After the equilibration period, collect at least three samples while the rat is at rest in the home chamber.

To establish a stable baseline of measurements Before behavioral testing, carefully plan out the timing of delivery of your treatment to the brain. So in this setup where we have a volume of 30 microliters between the syringe tip and the probe going into the animal's brain, we change the treatment syringe to the profuse eight treatment solution 20 minutes prior to. We want that treatment to arrive at the hippocampus To change syringes.

Simply disconnect the inflow line from the control perfuse eight syringe and rapidly connect to the treatment perfuse eight syringe. This should take no more than five seconds to avoid interruption to the PERFUSE eight flow. Take care not to introduce air bubbles that may reduce proficiency at the appropriate time period.

After treatment delivery, gently move the rat into the behavioral testing apparatus of choice. Here, spontaneous alternation is measured on a forearm plus shaped maze. Allow the animal to freely explore the maze for 20 minutes.

Record the sequence and timing of arm Aries either using a video recording or by hand to collect samples. During this period, move the outflow tubing to a new collection tube. Every five minutes hold the tubing such that it moves freely.

It should not hinder the rat's movement or move in front of the animal and distracted remain still so that your movements do not impact the rat's behavior. Following testing, remove the rat gently from the maze and return it to the control chamber. Continue microdialysis sample collection for at least four samples to cover the period of recovery from task performance.

After collecting the desired samples, gently remove the probe from the animal's head and place into a storage vial. Return the animal to its home cage and observe closely for any post experimental change in health or behavior. Finally, flush the tubing by switching to a syringe containing a solution of catho to prevent microbe growth.

This example shows a task associated dip in hippocampal ECF glucose. The gray box represents the time the animal spent in the maze. The purple line shows hippocampal glucose in animals with no maze testing, while the red line represents animals performing the forearm spontaneous alternation task.

The orange line shows measurements in animals performing the easier three arm version of this task. The changes in hippocampal, glucose and lactate after administration of insulin are seen here. Note that these changes are seen without any behavioral testing, and hence reflect the impact of insulin alone under baseline conditions.

When performing this procedure, it's really important to remain calm. Rats can pick up on the experiment of stress and they'll become agitated, and that can both make it much more difficult and probably confound your results. This combination of techniques has allowed for accurate neurochemical investigation of behavioral processes.

Similar techniques are now also used in human patients. For example, patients with epilepsy that have electrodes implanted to localize their seizure focus may also have microdialysis cannula implanted to permit metabolic measurements.