Day 16

Ink Microinjections
In order to determine whether the microinjections given to the rats throughout the experiment were isolated to both sides of the parabrachial nucleus, we give rats microinjections of dye.  Whoever is doing histology will then cut the brain into pieces, put them on slides, and examine them under a microscope to see where the dye is located.  Our goal is for the dye to only be in the PBN (bilateral).  Lindy let me give the rat the ink microinjections.  First, they are injected with anesthesia.. Lindy let me wrap the rat for its injection as well, which was a lot more difficult than I expected.  Every time I correctly positioned the rat, he would move.  Sometimes he would even grab the cloth and unfold it when I picked him back up to try again.  The time I finally got him wrapped properly, it was not tight enough and he could still move when the needle was being injected.  Lindy eventually had to go ahead and do it herself.  We then waited for the rat to go unconscious.  I unscrewed its dummies and placed the injectors in the cannulae.  I also set up for microinjections.  The hardest part was getting the tubing onto the microinjector without having air bubbles.  There would be air bubbles where the microinjector inserted into the tube, and  I poked a hole in the tubing a couple times.  It took a few tries to finally get it right.  Once the microinjections were complete, I would hand the rat with its label to Dr. Holstein for perfusions.

Cleaning
Julia and I deep cleaned the testing room.  We took apart the testing boxes and wiped down all the solution that had stuck to the counters and floor.  This is important so the mess does not accumulate over time. 

Day 15

CTA Testing
Today, we began CTA testing with group C and group D.  Before testing them, we had to weigh all the rats.  We could run a maximum of 8 rats at a time.  We first gave each rat saccharine for one hour and then injected them with LiCl directly after.  The LiCl makes the rats feel sick which they then associate with the saccharine they just drank.  The goal is for their lick count to decrease the next day when they are given the saccharine again.  We run each rat and then clean out their cages in between.  After the rats finished with CTA testing, we reattempted microinjections for three rats.  The air bubble only moved in both tubes for one rat so we ran him alone and quit trying the reinjections for the others.  We agitated the cannulae of all other rats.

Day 14

Microinjections

Today, we did microinjections for the last time with group C and group D.  We began with group D1 and had to reinject both rats receiving CDP for the air bubble to move.  All rats today received 0.05M quinine.  While group D1 rats were running, we gave water to group C for 60 minutes.  The rats in group D1 had very few licks, which Dr. Holstein attributed to quinine.  While group D2 was running, I made and labeled jars with subject numbers for group C and group D.  For group D3, we had trouble, again, with the CDP in which both rats had to have more than one microinjection.  Group C and group D will continue with CTA tests.

Day 13

Microinjections

The first set of microinjections went well for group C1.  However, the second rat receiving CDP had problems.  He received three microinjections on the right side, and the air bubble still didn’t move in the tubing.  We decided to run him today anyways and retest him another day as well.  We also had trouble with the rats in group C2 receiving CDP.  The first rat had three microinjections, but thankfully the second rat only needed one microinjection for the air bubble to move.  All rats in group C1 and group C2 received 0.01M quinine.  Group D rats were given 60 minutes of water.  While group C2 was testing, I prepared jars with formalin.  I labeled 17 jars total with the subject number of each rat in group A and group B.  I then filled the jars about 2/3 full with formalin.  We ran group C3 rats, and only had to reinject the first rat receiving CDP once before the air bubble moved.  Rats in Box 1, Box 2, and Box 3 received 0.01M quinine while the rat in Box 4 received 0.03M NaCl.  One rat only had one lick.  Some rats in group A and group B were retested for CTA.  The other rats of group A and group B who successfully experienced taste aversions the previous day were finished testing.  I was able to see the preparation for tissue sampling performed by Dr. Holstein and Dr. Pittman.

Day 12

Microinjections

We ran group D rats today, starting with group D2.  The first two rats’ microinjection ran well, but the second rat receiving CDP had to have a second microinjection on the left side.  When I inserted his left dummy back in his cannula, a little liquid was coming out.  All rats in group D received 0.01M quinine.  I gave 60 minutes of water to group C while group D2 was running.  For group D3, all aCSF microinjections went well.  The first rat receiving CDP had to have two microinjections, and the second rat receiving CDP had to have three microinjections.  I changed out the bedding for the D2 rats while they were running and took out the trash with Alex.  For group D1, all aCSF microinjections went well, and both rats receiving CDP had to have two microinjections on the left side for the air bubble to move in the left tubing. 

CTA Testing

Julia and I prepared four syringes with LiCl to use for any rats that not react aversely to the NaCl.  They would need to be reinjected with the LiCl after drinking the NaCl to establish an aversive relationship.  We encountered some problems when preparing the syringes.  Some needles did not securely fit on the syringes, and we had more trouble today than yesterday getting rid of the air bubbles in the LiCl solution.  It took longer, but we finally managed to properly prepare the four syringes.

Day 11

Microinjections

Today, we are testing rats in group C.  This morning, I came in and prepared the syringes for microinjections.  I cleaned the fragile microinjectors with ethanol and distilled water.  I then filled them with water and attached them to the pump.  I also prepared the tubes for microinjections.  After I vortexed the drugs, we began microinjections for group C2.  All rats received 0.01M quinine (bitter).  All aCSF injections went well.  The first rat receiving CDP had problems with the left side.  It took three microinjections for the air bubble to move on the left side.  The second rat also had trouble, and when I reinserted his dummy into his right cannula, liquid was coming out.  Group C3 rats all received 0.01M quinine except for the rat in Box 4 who received 0.03M NaCl.  Group C1 rats all received 0.01M quinine.  We gave water to group D rats for 60 minutes, and agitated the cannulae of and weighted rats in group A and group B.  I discovered that rats can unscrew their dummy if it is not properly secured.  When agitating one cannula of a rat, his was missing for the second time because he had methodically taken it out.

CTA Testing

Dr. Holstein taught us how to prepare syringes for CTA (conditioned taste aversion) testing.  First, you take the barrel and move the plunger in and out a few times in the LiCl to get rid of air bubbles. Then you hit the barrel against your hand to move the air bubbles to the top.  After they are at the surface, you pull back on the plunger so there is air in the barrel and push the plunger back in until liquid comes out.  You can then screw on the needle tightly, pop off the cap, and squirt the liquid out of the needle.  We prepared 16 syringes with LiCl to inject group A and group B rats with.  The LiCl was injected to the left or right of the midline about an inch above the base of the tail.  It is important to pull back on the plunger when injecting the rats to make sure no blood is coming out.  LiCl will make the rats feel sick, which we hope they will associate with the NaCl they are given to drink (rats in group A and group B had not been given NaCl before, so it is a new taste for them).  Julia and I did not yet experience CTA testing, but will have that opportunity when it is time for the rats in group C and group D.

Day 10

Microinjections

Today was a much simpler day, but we encountered a lot more problems.  Group A and group B are done with microinjections until CTA testing.  We gave microinjections only to group C today.  Of group C3, rats in Box 1 and Box 2 received 0.3M NaCl, and rats in Box 3 and Box 4 received 0.5M NaCl.  Group C1 rats received 0.05M saccharine, and group C2 rats received 0.3M MSG.  For group C3, all aCSF injections went well.  However, the first rat receiving CDP had to be reinjected on both sides.  The second rat receiving CDP was reinjected on the left side twice for the air bubble to move in the left tubing.  Two rats from group C1, one receiving aCSF and one receiving CDP, had to be reinjected for the air bubble to move.  We were behind schedule today due to the many difficulties with the microinjections.  Julia and I agitated the cannulae of the rats in group A and group B.  We also gave the rats in group D water for one hour.