A Lateralized Odor Learning Model in Neonatal Rats for Dissecting Neural Circuitry Underpinning Memory Formation

This protocol introduces lateralized early odor preference learning in rats using acute single nary occlusions. This is accomplished by first constructing removable polyethylene nose plugs. The second step is to occlude the desired Aries before behavioral training.

Next, prepare the scented bedding and then train the animal in the scented bedding while stroking it with a paintbrush. The final step is to test for lateralized odor preference using a two choice testing apparatus. Ultimately, immunohistochemistry of phosphorylated C amp response element binding protein or P crib is used to demonstrate unilateral silencing of neuronal activity due to single nary seclusion and the reversibility of the method.

The main advantage of this technique of our existing between animal control methods is that we are able to reduce variability and animal numbers through the nature of our intra animal control experimental design before postnatal layer 11, due to lacking of MA mature anterior commes projections, that two olfactory hemispheres are not connected. This allows us to manipulate one hemisphere independently of the other through the single nourish occlusion. The occluded hemispheres serves as an intra animal control.

Although this technique focuses on olfactory learning in neonatal rats, the implications will allow us to answer fundamental questions regarding neural processes during learning and memory, such as synaptic plasticity, metaplastic, and their underpinning molecular mechanisms. The conclusions drawn from studies using this method can be generalized to basic principles of learning and memory and mammals, especially during postnatal critical periods. This will help us to build up a promising background for potential therapeutics for memory loss associated with pathophysiological conditions.

This method is also highly suitable for other olfactory related processes such as odor perception and odor coating. The nose plug is constructed from polyethylene 20 tubing and three zero silk suture thread. First, cut a small piece of polyethylene 22 to approximately 0.8 centimeters.

Then thread the silk suture through the prepared tubing such that there is thread on either side of the section of tubing. On one end of the thread outside of the plug, tie a knot. Pull the section of tubing down over the knot in the thread.

The knot should lodge inside the tubing. Trim both ends of the thread, such that about two millimeters of thread is protruding from one end of the tubing. To begin this procedure, remove the pup from the dam and place it in a secure dish covered with regular bedding.

Using a cotton tip applicator dab a local anesthetic jelly, 2%Xylocaine on the Aries to be occluded. Allow the pup to rest in the dish for about three minutes. Lucin in the pup is the most critical step in this procedure.

Cure should be taken during nose plug insertion to prevent bleeding and tissue damage. Next, pick up the pup and hold it gently but securely in the non-dominant hand with the dominant hand, pick up a nose, plug and dab the same local anesthetic jelly around the tip from which the thread is not protruding. This will act both as an anesthetic for any minor pain associated with plug insertion and a lubricant inside the nes.

Gently insert the nose plug by firmly holding the pup and slowly rotating the plug with very gentle pushes until the plug is fully inserted and only the two millimeter thread is protruding from the Aries. There should be no bleeding from either Aries during this process. Pups with bleeding during nose plug insertion cannot be used for the experiment.

Allow the animal to rest in the dish for five minutes in order to habituate to the plug Before beginning the training to prevent odor contamination, prepare the scented bedding in a fume hood and while wearing new gloves, place 500 milliliters of wood chip bedding into a plastic bag. Use a syringe to draw up 0.3 milliliters of peppermint extract and spray this over the bedding in the plastic bag. Tie the bag shut, shake the bag vigorously and allow the bedding to rest in the bag for five minutes, place the scented bedding in a clear shallow 20 by 20 by five centimeter acrylic training box uncovered in the fume hood for five minutes before use.

Once the bedding is prepared, discard the used gloves. Do not allow the gloves to come in contact with the animals. Place 500 milliliters of unscented bedding in another clean acrylic training box and ensure that it does not come into contact with as scented bedding or used gloves.

Begin the odor conditioning paradigm by placing the habituated pup on the scented bedding for experimental odor. Plus stroke OS positive pups. Stroke the pup for 30 seconds.

Using a small paintbrush, use rapid circular motions primarily around the hind region of the pup. Stroking induces norepinephrine release to the olfactory bulb and olfactory cortex. This serves as an uncondition stimulus.

Allow the pup to rest for 30 seconds. Repeat the 30 seconds stroking, followed by 30 seconds of rest for a total of 10 minutes. When the 10 pairings of stroking plus odor are complete, remove the pup from the conditioning box.

Remove the nose plug, and return the pup to the dam for control. Odor only OS negative pups. Place the habituated pup on the scented bedding for 10 minutes without any stroking.

After 10 minutes, remove the pup from the conditioning box. Remove the nose plug, and return the pup to the dam. Lateralized odor preference testing is performed at various time points following the final training session.

Testing is carried out in a 30 by 20 by 18 centimeter stainless steel testing chamber that is placed on top of two acrylic training boxes separated by a two centimeter neutral zone. One training box contains scented bedding while the other box contains clean unscented bedding. Prepare one peppermint scented bedding and one unscented bedding as shown earlier, and place each box under opposite sides of the testing chamber.

Two centimeters apart. Place the plastic mesh on the metal grid floor of the testing chamber. Remove the P from the dam and place a firm dab of odorless silicone grease on the Aries that is occluded.

During training, the grease can be reapplied throughout the first testing procedure as needed. Place the pup in the neutral zone of the testing chamber. Allow the pup to explore the chamber for one minute recording how long the pup spent over the two sides of the chamber, IE over peppermint or neutrally scented bedding.

Transfer the pup to a covered plastic holding chamber and allow it to rest for one minute. Repeat the test trials for a total of 10 minutes, switching the initial orientation of the pup in the chamber in order to control for direction preferences. Immediately following testing, wipe away the grease from the Aries.

Insert a polyethylene nose plug into the opposite Aries, following the same RY seclusion procedure demonstrated earlier, and allow the animal to rest for 10 minutes. Subsequently, lateralized odor preference testing is performed on the pup.Again. When the testing is done, remove the plug and return the pup to the dam.

Single narrow seclusion during odor plus stroking training leads to a lateralized odor memory that is confined to the spared NRIs. When pups are tested for an odor preference with the same nary secluded as during training, they show a preference for the conditioned odor. This was not observed when testing was done with the opposite nary secluded.

In contrast, odor only control groups do not show odor preference regardless of which nease is occluded. During testing, the lateralized odor training results in lateralized activation of the olfactory system during odor exposure, immunohistochemistry of the olfactory bulb or OB and anterior piriform cortex or a PC showed significantly less phosphorylated Creb in the occluded hemisphere following odor exposure compared to the contralateral spared hemisphere. Nissel staining demonstrates comparable cell bodies in the mitral cell layer of the OB and in the pyramidal cell layer of the PC of both hemispheres.

24 hours following the removal of the nose plug pea crab expression Following odor exposure in mitral cells of the OB and the pyramidal cells in the PC are comparable between the occluded and spared hemispheres. This indicates that the effect of a single trial nary seclusion is transient and reversible and does not result in visible long-term neuronal damage that could affect odor perception and neuronal activation to odors during testing. Once mastered NA occlusion can be done in less than five minutes if it is performed properly.

While attempting this procedure, it's important to be extremely careful when plugging the nostril. Any excessive force or improper technique can cause bleeding and therefore damage to the nostril Following this procedure. Other methods like ex vivo electrophysiology, and molecular techniques can be used to answer additional questions regarding synaptic plasticity.

After watching this video, you should have a good understanding of how to use temporary unilateral narrow occlusions in order to lateralize odor learning and to dissect the neural circuitry underpinning memory formation.