H2 - Brain Stimulation: Drinking
Objectives:
To learn about a surgical model that elicits drinking behavior.
To quantify drinking in rats following intracerebroventricular injection of a dipsogenic substance.
Introduction:
Fluid homeostasis is carefully controlled in animals. Obligatory losses of body water due to respiration and urination require regular consumption of water. The brain anticipates needs and responds to losses in a homeostatic manner. The brain also regulates the tonicity of body water to maintain the correct osmotic pressure across membranes, and to maintain an appropriate blood pressure. Osmoreceptors in the brain and baroreceptors in the heart provide the brain with feedback for that drinking behavior and kidney function can maintain body water volume and tonicity in the optimal range (Rowland, 2005).
Procedure:
Preparation: Rats will need to have a cannula implanted aimed at the lateral ventricle. See Appendix B+C for specifics on the cannulation surgery. The coordinates for this are based on those of Harland et al., (1988). The guide cannula should be implanted 0.8 mm posterior to bregma, 1.5 mm lateral to the midline, and 3.2 mm ventral to the surface of the skull. The incisor bar should be set so that bregma and lambda have the same dorsoventral level. An injector cannula that extends 1mm beyond the end of the guide cannula should be used.
Test: Prepare a solution of Angiotensin II (Sigma Aldrich, St Louis, MO, USA; catalogue number A9525) in saline. The final concentration is 100µg/ml. Since 100µg is difficult to measure, weight out a small amount and mix it at a concentration of 1mg/ml in saline. 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 to give 1ml of 100µg/ml Angiotensin II in saline. Prepare a test cage. Fill a water bottle, and weigh it. Carefully place it on its holder, avoiding any leakage. This may take practice. Connecting the injection cannula to some polyethylene tubing. Fill the tubing with distilled water. Attach the tubing to a 10 µl Hamilton syringe. Lay the assembly flat and then pull back the plunger to the 0.5 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 10 µl mark. Remove the tip from the solution, and watch the tip while pressing the plunger to the 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 5µl the solution over the course of about 1 minute (press the plunger down to the 3 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 rat in the testing chamber containing a drinking bottle. Time latency until it begins to drink. Continue to observe its behavior for 30 minutes. At the end of the time, return the rat to its home cage. Carefully remove the water bottle, avoiding any leakage, and weight it again to calculate how much the rat drank. Report your values 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, injection of 5 µl of a 1% aqueous solution of Evans blue dye and examining the brain for staining of the ventricular spaces, tissue sectioning and a nissl stain such as cresyl violet would be appropriate histological procedures.
Variations:
A 2x2 design could be used where the saline vs. Angiotensin II treated rats are given a choice or 2 solutions (water vs. hypotonic saline; water vs. glucose solution)
References:
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