Day 3

Microinjections

Today we learned how to prepare and deliver microinjections to the rats’ PBN.  There is a very elaborate process that goes into prepping.  First, the microinjectors are sterilized and flushed.  They are very fragile and expensive, so it is important to be careful.  The plunger must be pulled out slowly and should not exceed the 1.0μL mark (0.9 μL is a good amount).  The microinjector is filled with ethanol and then injects the ethanol into the waste beaker 3-5 times.  It is then filled with water and injects the water into the waste beaker 6-10 times.  This ensures that there is not leftover ethanol injecting into the rats’ brains.  The microinjectors are inserted comfortably into the injector machines (the right and left microinjector should be inserted into the proper side).  A syringe is then filled with water and releases the water into the tube several times.  There should be no air bubbles.  The syringe is then removed, and the microinjector is inserted into the tube.  Again, there should be no air bubbles.  Clay can be used to hold the tubes when they are not in use.  When one of the two drugs (treatment: CDP control: aCSF) is ready to be injected into the rat’s PBN, an air bubble is formed in the tube before the specified drug is sucked up into the tube of both the right and left side.  During this time, I have removed the rat’s dummies and will paint the left dummy tip with nail polish to differentiate it from the right.  The injectors on the tip of the tubes are inserted into the correct cannula.  The injector machine inserts 0.2μL of the drug into the rat’s PBN per minute.  After the drug has been injected into the PBN, we wait two minutes while the injector sits into the cannula.  Then we remove the injector and screw back in the dummies.  The rats are placed in an assigned cage in the testing room, are given a specific flavor of liquid to drink, and their licks are recorded in a 60 minute time period.  We also replaced their cages with a new one for when they complete the experiment.  We tested the water restricted rats using an ABBA within subjects design.  The tubes were cleaned again after the second microinjection and then at the end of the experiment.  The cages in the testing room were wiped using an antibacterial wipe and dried using a paper towel after each trial.  Notes are made for each rat.

Difficulties

There were three difficulties.  One was having enough skill and good eyesight to place the dummies back into the cannulae of squirming rats.  The second was that one rat did not lick the baby bottle at all.  This was one of the same rats that did not lick the bottle during training.  A third difficulty was that one rat had scratched and irritated his head, so he had to be run in a separate trial by himself after he was cleaned up.