H1 - Brain Stimulation: Feeding
Objectives:
To learn about a surgical model that elicits feeding behavior.
To quantify feeding in rats following activation or sham activation of the lateral hypothalamus.
Introduction:
Feeding is one of the most critical behaviors that any organism performs. This behavior must be tightly regulated so ensure against energy deficits when food is scarce. This must be balanced with being encumbered by energy reserves. In today’s modern society where food is always plentiful, my members of society are encumbered by their energy reserves (i.e., obesity) which has serious implications for health by increasing the risk of developing a variety of diseases. Understanding the neural mechanisms that regulate energy balance is critical if effective treatments for eating disorders are to be developed. The rat is a good model for studying eating behavior (Clifton, 2005).
Procedure:
Preparation: Rats will need to have a cannula implanted aimed at the lateral hypothalamus. See Appendix B+C for specifics on the cannulation surgery. The coordinates for this are based on Hettes et al. (2010). The guide cannula should be implanted 6.1mm anterior to the interaural line, 1.8mm lateral of midline, and 8.2mm ventral to the surface of the skull. The incisor bar should be set to 3.3mm below the center of the ear bars. An injector cannula that extends 1mm beyond the end of the guide cannula should be used. For the week prior to testing, rats should have ad libitum access to a milk-mash diet (Hettes et al., 2010) consisting of powdered Purina rat chow (500 g), sucrose (400 g), and evaporated milk (354 ml). This will prevent neophobia to this diet during testing.
Test: Prepare a solution of RS-AMPA (Tocris Bioscience, Ellisville, MO, USA; catalogue number 0169) in saline. Mix 6.2mg of drug into 1ml of saline (this will give a 33mM solution, or 10nmol in 0.3µl). Warm slightly and vortex to aid getting the drug into solution. Prepare some milk-mash in a dish, and weigh it. Place it in the test chamber (clean empty cage). Load the injector with at least 0.5 µl of either RS-AMPA or saline. Do this by 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 0.2 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 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 0.3µl the solution over the course of about 1 minute (press the plunger down to the 0.5 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 brain tissue before withdrawing the injector. Withdraw the injector and replace the dummy cannula. Place the rat in the testing chamber containing the food-dish of mash. Observe its behavior for 60 minutes. At the end of the test, return the rat to its home cage and weigh the food-dish. Report your weight and injection solution to the TA, who will tabulate the data and supply summary data to the group.
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.
References:
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