Day 1

Experiment Training 
Today was a very eventful day with much to learn.  I first learned how to fill the baby bottles with 0.2 moles of sucrose (about 3/4 full), place the stopper on, and secure it with a rubber band ensuring that the small white rubber disk is not covered so it can properly sit in the holder.  Every morning, I have to log onto the computers so they are ready to run.  We had a set time to "cuddle" with the rats and pet the heads of the ones who have not yet had surgery.  This gets them used to their heads being touched.  For the ones who have had surgery, we were able to practice taking out the dummies and placing them back into the cannulas (it is important not to mix up the right and left dummy).  There are two ways to do this: one way is to hold the rats on your chest where the pocket is and the second is to hold the rat in the crook of your arm.  I found the latter to be best for me because it is easier to see the hole in order to place the dummy back in the cannula.  Today, we ran the rats who have not had surgery in a training session.  This gets them used to the equipment and being transported, so they are not intimidated during the actual test.  Each rat is placed in a separate clear, plastic cage with the sucrose water.  The lights are turned off with only a red lamp to see.  A device records the number of licks by each rat in a 60 minute period.  Rats that had a low number of licks during the time period were rerun.  The bottle weights are recorded before and after each trial.  After the rats are placed back in their original cages, the cages used for the trials are wiped down with sterile wipes and dried.  At the end of the day after all test runs, the computer and lights are turned off and the bottles cleaned.

Surgery
I was also able to watch Dr. Pittman perform a surgery today on a rat.  The rat is first anesthetized with two injections, one to the right and one to the left of the midline about an inch above the base of the tail.  When injecting, it is important to pull up on the plunger and make sure there is no blood before pushing down.  Once the rat is asleep, it is secured in a stereotactic apparatus.  Once everything is in the correct place, it is time to start the surgery.  The skin is cut using a scalpel and the connective tissue is pulled back.  The most tedious part of surgery is to ensure that the rat is in a particular position so the cannula is placed in the correct location.  This should not be rushed because it would be a flawed experiment if the microinjections were not delivered to the parabrachial nucleus (PBN).  The rat's lambda and bregma are used to make sure the rat is correctly positioned.  The distance has to be measured each time the head is moved.  It is important also to pay attention to when the coordinates need to be zeroed.    Based on previous research, the stereotaxic atlas is used to locate the PBN.  Circles are drawn in sharpie.  Small holes are drilled into the rat's skull where screws are inserted as an anchor for the cement.  Dr. Pittman allowed me to drill one of the holes in the skull which was very interesting.  After four screws are fastened, the cannula is inserted into either the left or right hole.  A gel pad is placed over the other hole while the cement secures the cannula in place and hardens (this is a precaution so the other hole does not fill with cement).  Once the cement hardens, the same procedure is repeated on the other side.  During the entire surgery, it is crucial to sterilize all tools and to recheck measurements multiple times.  It can also help to use a microscope.

Tomorrow: I will go into the lab and practice handling the rats and placing the dummies into the cannulas.