Brain and Behavior Animal Lab 5
Recently, we radio frequency-lesioned the hippocampus of one group of rats and sham-lesioned the other. We then compared their behavior to those of two unoperated rats to see if we could determine which ones were which.
Whatever we find, our findings will be uninterpretable. This is because we have no way of knowing if a) the lesion generator worked, i.e. is there in fact any lesion at all, b) did we “hit” the hippocampus and if so, what percentage of it was destroyed, and c) was any other structure damaged? These questions must be answered in order to properly interpret the data.
Often, valuable information can be obtained by comparing the behavioral differences of rats which fall into each of these categories. Of course, to be able to make such comparisons, you need several rats in each category.
The next step... to analyze coronal sections of brains from hippocampal lesioned rats to determine the extent and placement of each lesion.
This technique is similar to the embalming process in humans. First, a saline solution is pumped into the rat’s circulatory system to replace the blood. Why? A section of brain that has traces of blood in and around it will obscure the lesion (i.e. the lesion will be poorly visualized). Second, a solution of 10% buffered neutral formalin is pumped into the circulatory system, replacing the saline. This solution hardens and preserves the brain, which is then stored for a minimum of 48 hours in the same formalin solution to await the next step.
CAUTION: We will be using some very toxic chemicals... If you want to assist with the perfusion and/or brain extraction, you MUST wear gloves and a mask. I will supply them.
NOTE: Witnessing a perfusion for the first (or even the 100th time) can be disturbing for some people. If it’s a simple matter of being squeemish, watch from a distance and let me know if you are “not feeling well”. If it’s a matter of concern for the welfare of the rat, it might help to keep several points in mind.
First, perfusions are not done to conscious (or anything close to conscious rats). We will wait until respiration has ceased before we begin the procedure. Even so, it is possible (and even desirable) that the rat’s heart will still be beating. This does not mean that the rat has any chance of regaining consciousness. Many physiological processes take a while to stop, even after the animal is “technically dead”.
Second, keep in mind that scientists who perform the kinds of experiments that require these procedures do so with a firm belief that they will increase our knowledge of the brain, which in turn will benefit both humans and animals. Of course, you may agree with this statement and know that this kind of career is not for you... and there’s nothing wrong with that. You are also free to believe that no animal research should ever be performed. Although I would hope to convince you otherwise, I respect this opinion and hope that you do the same. These issues aside, the only pain felt by a rat during a perfusion is the initial prick of the needle from the injection.
This refers to a set of procedures; using a microtome to slice the brain into thin (usually coronal) sections, mounting brain section onto slides, staining these sections to view cell bodies and/or fibers, and finally analyzing each section to determine the extent and placement of the lesion.
These are two perfusions: one is good, the other is not. Can you guess which is which?
The freezing microtome
MEET IN SCA 210 at 3:00