A Novel Procedure for Evaluating the Reinforcing Properties of Tastants in Laboratory Rats: Operant Intraoral Self-administration

The overall goal of this procedure is to apply principles of operant intravenous drug self-administration to the investigation of operant self-administration of sweet solutions delivered by intraoral infusion. This is accomplished by first surgically implanting rats with an IO cannula and a head cap. The second step of the procedure is to select the sugar of interest and prepare appropriate concentrations of the solution for self-administration in operant chambers.

The third step of the procedure is to place rats in the operant chambers, attach their IO cannulas to the appropriate tubing, and allow the animals to self-administer IO infusions of the suite solution using different concentrations and different schedules of reinforcement. The final step of the procedure is to remove the rats from the operant chambers and flush their cannulas with water in preparation for the following self-administration session. Ultimately, results can be obtained that show that rats will acquire and maintain self-administration of sweet solutions, and that their behavior is sensitive to changes in the concentration of the solution and the schedule of reinforcement employed through IO self-administration.

The main advantage of this technique over existing methods like drinking a solution from a spout or open responding for solid pellets is that this metal allows to test any concentration and any volume of any water-soluble food additive. More importantly, we can deliver passive interval infusions of control, quantities of the solutions without any action required on the part of the animal. This makes it possible to measure artificial responses of liking using activity tests.

Any studies of relapse plus interval infusions can be used to study priming induced reinstatement as well as potential cross reinstatement between Swiss solutions and drugs of abuse. This method can help answer key questions in the food addiction and obesity fields, such as identifying the neuro substrates responsible for the acquisition, maintenance, and relapse to compulsive sweet seeking, as well as identifying factors involved in diet induced obesity. It'll also be possible to study the genetic and epigenetic epigenetic factors involved in the development of compulsive food seeking.

All experiments are approved by the Animal Care Committee of the University of Guelph and are carried out in accordance with the recommendations of the Canadian Council on Animal Care. To construct the IO cannula, cut a piece of polyethylene plastic tubing, approximately 130 millimeters in length to create a F flanged end for securing in the oral cavity. Use a soldering iron to heat the end of the tube for two to five seconds, and then immediately flatten the heated edge of the tube against a flat surface.

Cut a surgical mesh disc approximately six to seven millimeters in diameter and thread it onto the length of the tube until it rests against the back of the F flanged, end of the cannula. For each IO cannula, cut one additional surgical mesh disc approximately eight millimeters in diameter and three six by six millimeters, squares of an elastic band for at least 24 hours prior to surgery. Sterilize the IO cannula, surgical mesh discs and elastic squares in an antibacterial solution approximately 30 minutes prior to surgery.

Administer a subcutaneous injection of the antibiotic dein and the analgesic meloxicam. After using isof fluorine to induce general anesthesia according to the text protocol and verifying sedation with a toe pinch, use a cotton tipped swab to administer a topical anesthetic along the inner left cheek where the cannula will be implanted. Then inject a local anesthetic such as lidocaine HCL at the incision site for the biopsy needle and the head cap.

Apply lubricating eye ointment to both eyes. Next, shave a 10 by 10 millimeter patch of hair at the base of the neck. Use sterile gauze to swab and cleanse the area with antibacterial soap, followed by 70%alcohol.

And finally, Betadine. Then insert a 15 gauge stainless steel needle at the base of the neck and move it subcutaneously around the ear to the left cheek. Position the needle tip and pass it through the cheek behind the first molar inside the oral cavity.

Pass the IO cannula through the tip of the protruding needle, and then remove the needle, leaving only the cannula Thread one surgical mesh disc followed by three square elastic discs onto the exterior portion of the cannula, and slide them to the exposed skin at the back of the neck to stabilize it. Then use antibacterial oral rinse to flush the IO cannula prior to surgery on the top of the head. Modify a nylon bolt by removing its head, and then carve a two millimeter groove along the length of the thread.

After shaving a 10 by 20 millimeter strip of hair along the midline of the head, use sterile gauze to swab and cleanse the shaved area as shown earlier for the cheek with a surgical scalpel, make a 20 millimeter long incision along the sagittal. Then use a hand drill to make four holes, two on each side of the suture. Finally, use jeweler screws and dental cement to mount the bolt to the skull after surgery.

House the rat in an individual cage and monitor for 24 hours. Refer to the text protocol for additional care and cleaning of the IO cannula for experiments conducted in the operant chamber. Described in the text protocol.

Using reverse osmosis water, make 8%25%and 50%dilutions of 55%high fructose corn syrup or HFCS after weighing the rat and transferring it in its home cage to the testing room. Remove food or bedding that can accumulate in the IO cannula by using a 12 milliliter syringe Fixed with a 16 gauge needle and 50 millimeters of polyethylene tubing to pump air through the cannula in the operant chamber. Use a 20 gauge needle as a shaft to connect the IO cannula to tigon tubing.

Affix the lower portion of the tubing within the groove of the belt, and connect the upper portion to a swivel protected by a swing. Then connect the base of the spring to the thread of the bolt mounted on the head of the rat. Allow the rat to acclimate in the chamber for five minutes and then begin the self-administration session by activating the house light.

Introducing the two retractable levers and illuminating the light stimulus above the active lever for 30 seconds. For the progressive ratio or PR test one and all subsequent PR tests, use the PR schedule described by Richardson and Roberts where the response ratio escalation is equal to five times E raised to the injection number times 0.2 minus five. For acquisition sessions one through 10, use a continuous schedule of reinforcement whereby each press on the active lever results in the delivery of an 80 microliter infusion of the test solution over 2.5 seconds to allow sufficient time for ingestion, impose a timeout period of 27.5 seconds during which the active lever is retracted and the stimulus light is activated.

Do not impose a limit on the number of infusions available within each IO self-administration session. If the rat does not respond to the active lever for 20 consecutive minutes, prime the rat by facilitating it to press once on the active lever. Following self-administration sessions, remove the rat from the operant chamber.

Use two milliliters of water to flush the IO cannula and return the rat to its home cage in the colony room. Follow the feeding schedule found in the text protocol. This figure shows opera responding on the active and inactive levers during self-administration of 8 25 or 50%HFCS for 10 consecutive sessions.

Rats acquire IO self-administration of HFCS within the first three sessions and maintain stable active lever responding for the remainder of acquisition. Concentration dependent differences in active lever responses are also evident. For example, compared to 25%HFCS responding is significantly lower in rats self-administering 50%HFCS.

The rats maintain an intermediate level of operant responding when given 8%HFCS and demonstrate greater individual variability of intake active lever responses and breakpoint, which is defined as the last infusion received due to a lack of responding on the active lever for at least one hour are presented in these graphs respectively. In PR test one, there is a modest frequency of response for all concentrations of HFCS. However, group differences emerge in test two, where higher concentrations elicit more responses and higher break points.

While attempting this procedure, it's important to check on your animals frequently for fluid buildup on the head cap and leaks during the self-administration session. Also, attrition in these experiments should be minimal because the IO cannulation surgery can be performed a second time if the animals were to pull the cannula out or chew it off in between the self-administration sessions. After watching this video, you should have a good understanding on how to modify surgical procedures and open chambers to perform intraoral self-administration experiments in laboratory rats.