The overall goal of this experiment is to examine the neurobiology and behavior underlying anorexia nervosa. This method can help answer key questions in the field of psychiatry related to eating disorders such as what are the neural underpinnings of anorexia nervosa, and what brain circuits are involved. The main advantage of this technique is that it can be used to study some underlying mechanisms of anorexia, which can't be assessed in humans.
Setup an experimental area by choosing a cage rack close to the laptop and wheel hubs. This will limit issues with data transmission. Now activate the connection between the wheel hubs and the software.
First open the wheel management software to ensure both wireless hubs are active. From the software reset the default wheel connections. Open tools and select delete wheels.
Repeat this step five times then close the software and reopen it. Next, provide power to the wheels. Connect the battery pack wires to the wheel hardware and insert the batteries into the wheel base.
Then have a look at the software. The wheelbase should be listed with the ID of one under the appropriate wireless hub. If not, unhook the battery pack, delete the wheel in the software, and try again.
If the wheel is recognized, replace the ID with the appropriate animal's ID.Next, adhere the running wheel's base to the bottom of the cage with duct tape to ensure stability. Do not allow any tape to stick out as mice will chew on exposed tape. Next, place a running wheel disc without a corroded magnetic on the base and rotate the wheel to verify clearance from the cage walls and grating.
Spin the disc and check that each revolution registers properly with the software. Next, use duct tape to secure the food jar to the cage. Position it away from the running wheel and water bottle.
Then place two pieces of chow in the jar, attach full water bottle to the cage, and add bedding to the cage bottom. After all the cages have been prepared in this manner, individually place mice into their appropriate cages. Once all the mice are in their cages, go to the software and click on file, and then start acquisition.
Throughout the experiment start new acquisitions to breakup the trial phases. Now let the mice acclimate to the housing conditions for two full days. During this phase, decide if each mouse is healthy enough to handle the ABA experiment.
On the third day of the experiment, measure out about nine grams chow and record the weight to 1/1000 of a gram. Next, carefully remove the mouse from the cage and weigh it in a large plastic beaker to and nearest tenth of a gram. Do this with minimal stress to the mouse.
With the mouse out of the cage, search out and remove all of the food in the cage. Besides removing the food in the jar, look through the bedding for scattered food. Next, if needed, clean the running wheel with 70%isopropanol and paper towels.
Before returning the mouse to its cage, be sure that the isopropanol has dried. Then load the mouse and the nine grams of chow into the cage. On the next day return to the animal facility within 10 minutes of the last visit.
Again load nine grams of chow in a small plastic beaker and record the exact weight. Again gently remove the mouse and weigh it with minimal stress to the mouse. Now remove the food jar out of the cage and investigate it.
Remove any feces with tweezers and use a strainer over the beaker to remove bedding. Crumble the larger pieces of food so they pass through the filter leaving just bedding in the strainer. Then search the cage bedding for food remnants and add them to the beaker.
Then record the mass of the food that remained in the cage. Next, wipe the jar clean and return it to the cage empty. Apply new tape if necessary.
Then load the jar with the prepared nine gram food ration. As before, clean any soiled running wheels and return them to the cages. Reset all the cages in this manner everyday.
On days five, seven, and nine, restart the computer data acquisition in wheel running program. After collecting the baseline data, on day 11 restart the data acquisition and begin the restriction phase. Basically, follow the baseline protocol with a few changes.
First, do not clear the running wheels when changing the food. Second, limit the animals'access to their nine grams food ration to just six hours. The feeding period can be diurnal or nocturnal.
Both choices have merit. After six hours, weigh the food remaining in the jar and cage. At this point, if needed, clean the wheel, but do so quickly so the next cage can be rapidly attended to.
On each and every day of the restriction phase, repeat this procedure. Now it becomes very important to monitor the animals'weight loss. If a mouse drops to 75%of its baseline body weight, remove it from the experiment.
Provide these mice unlimited food from the cage floor and no access to a running wheel. After all the cages are assessed, euthanize the dropped mice unless their recovery is to be studied. Using the described experiment, a group of activity based anorexia mice were assessed along with three control groups.
Following the described procedure, the food was restricted to six diurnal hours. When a mouse was dropped from the study due to excessive weight loss, a randomly selected control mouse was as well. Body weight was reduced on days one through five in both groups exposed to scheduled feeding, and this correlated logically with reduced food intake.
Curiously, weight loss had no effect on wheel running activity. After the behavior study, brains were harvested and frozen for biochemical analysis. One analysis of tissue from the ventral tegmental area showed that the wheel running significantly increased BDNF expression while food restriction increased NCAM1 MRNA expression but did not alter BDNF MRNA expression.
While attempting this procedure, it's important to remember to limit stress on the mice, be consistent in timing your measurements, be patient so you acquire accurate readings, and consistently check on the wheel running software for any technical issues. Following this procedure, other methods like immunohistochemistry and radioligand binding can be performed on mouse brains in order to answer additional questions pertaining to virus function in the anorexia paradigm.
