H3 - Brain Stimulation: Flank Marking
Objectives:
To discover how a complex behavior can be triggered by minute application of specific chemicals to discrete brain regions.
To learn about an animal model of Obsessive Compulsive Disorder
To observe how drugs used to treat Obsessive Compulsive Disorder can modify the behavior in this animal model.
Introduction:
Throughout this course we have been using the rat to study behavior. It is important to note that the choice of species to examine can be influenced by a wide variety of factors, one of which is the ability of the species to display the behavior of interest. Sometimes you may wish to examine a behavior which is not ubiquitous in the animal kingdom. One example is the social communication behavior of flank marking in hamsters (Ferris et al., 1984). The neural circuitry of this complex behavior has been well defined (Bamshad and Albers, 1996). This model has found promise as a simple screening tool for Obsessive Compulsive Disorder (OCD). Specifically, antidepressants used to treat OCD inhibit flank marking, while antidepressants that are not indicated for use in OCD have no effect on flank marking (Ferris et al., 2001).
Procedure:
Preparation: Male hamsters will need to have a cannula implanted aimed at the medial preoptic area of the hypothalamus. See Appendix B+C for specifics on the cannulation surgery. The coordinates for this are based on Ferris et al. (2001). The guide cannula should be implanted on an 8 degree declination from vertical. This helps you hit midline structures without damaging the sagittal sinus which sits along midline between the two cerebral hemispheres. The incisor bar should be leveled with interaural line (i.e., set to zero). The coordinates are 1.1 mm anterior to the bregma, 1.8 mm lateral to the midsagittal suture, and 6.5 mm below the dura. An injector cannula that extends 1mm beyond the end of the guide cannula should be used. For the two week prior to testing hamsters should be given daily injections of either 2mg of Clomipramine (Sigma-Aldrich, St. Louis, MO, Catalogue #C7291) or saline vehicle. Randomly assign each hamster to drug or vehicle control, and track this, as they will need the same injection each day. Prepare the drug with to a concentration of 20mg/ml sterile saline, and give 0.1ml to each hamster. Your TA will coordinate this procedure and may make up a duty roster requiring your assistance. Prepare a solution of Arginine Vasopressin (AVP, Sigma Aldrich, St Louis, MO, USA; catalogue number V9879) in saline. The final concentration should be 100µM, or 100µmol in 1000ml. If you have 1mg of AVP, this is equivalent to about 1µmol of AVP, so you would need to dissolve this in 10ml of saline, which is not practical. Instead, make a concentrated stock solution of 1mg/ml (or 1ml of a 1mM solution). This will give you a stock solution 10x stronger than is needed. Mix a small amount of this stock at a ratio of 1:9 with sterile saline to give the working solution for injection. For example, mix 100µl of the stock solution with 900 µl of saline to give 1ml of 100µM AVP in saline.
Test: Prepare a test cage. Connecting the injection cannula to some polyethylene tubing. Fill the tubing with distilled water. Attach the tubing to a 1 µl Hamilton syringe. Lay the assembly flat and then pull back the plunger to the 0.1 µl mark. This will produce an air bubble inside the injector that will keep your drug from mixing with the distiller water filling the PE tubing. Then place the injector into the drug solution or saline vehicle and pull back to the 1 µl mark. Remove the tip from the solution, and watch the tip while pressing the plunger to the 0.8 µl mark. Confirm that a bubble of solution appears. Failure to see this would indicate an incomplete seal in the assembly, or an obstruction in the assembly. If it is confirmed, then you are ready to inject. If this is not confirmed, flush the line with distilled water and load the assembly again. Gently restrain the rat and unscrew the dummy cannula. Insert the injector all the way. Slowly inject 0.2µl the solution over the course of about 1 minute (press the plunger down to the 0.6 µl mark, there will be residual drug in the injector, which prevents the air bubble from being injected). Allow an extra minute for the solution to disperse in the cerebrospinal fluid before withdrawing the injector. Withdraw the injector and replace the dummy cannula. Place the hamster in the testing chamber. Count the number of flank-marking behaviors you observe over the next 30 minutes. At the end of the testing session, return your hamster to its homecage. Report your observations and treatment group to your TA, who will tabulate the results for analysis.
Variations:
Other drugs could be examined. Desipramine, an SNRI is not effective, while SSRIs, such as fluoxetine, are effective at suppressing flank marking.
A 2x2design could also be used where on the test day animals are given either AVP or a vehicle control injection.
Follow-up: In any surgical intervention, histological confirmation of the effectiveness to the surgery is important. Discuss with your TA opportunities for learning about histological procedures (perfusion, tissue slicing and staining, microscopy) that may be available. For this study, perfusion with formalin, tissue sectioning and a nissl stain such as cresyl violet would be appropriate histological procedures.
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