Adam Field and Dr. Jeff Edwards, Department of Physiology and Developmental Biology
In April of 2012, IACUC committee approved the methods which we had set forth for the carrying out of the experiment. At this point we began gathering the tools and materials needed for the performing of the experiment. Mice were obtained, and the electrophysiological research begun.
The experiment was set up with three groups for testing, a control group, a stress group, and a stress and exercise group. For each of these groups mice were sacrificed, and the portions of their hippocampi were sectioned coronally for testing. These coronal sections were obtained using a vibratome located within the lab, and placed in artificial cerebrospinal fluid until ready for use. At which time, the technician in the lab took the slices and performed the required protocol. The protocol used in this experiment is a preset program consisting of a series of electrical impulses designed to induce long tern depression (LTD), or in other words, to decrease the connection strength between neurons. After this was done the results were to be compared between the various groups to determine if there was a difference between the groups in the amount of long term depression observed.
Treatment of the mice among the various groups was carried out in the following manner. To the control group, nothing was done until the time in which they were ready for sacrificing. The stress group, however, underwent a stressful protocol in which the mouse was placed in a plastic tube with electrodes connected to the tail. The protocol was then initiated, delivering a single electrical impulse every minute for one hour. After which, the mouse was sacrificed. The stress and exercise group is to undergo the same protocol as the stress group, only that they are also given access to running wheels in their respective cages.
Of the groups tested, we know from previous studies1 that there should be a great difference between the control group and the stress group. The control group is used to show what the normal amounts of LTD should be in a mouse that has not undergone a stressful protocol. The stress group is supposed to show greater amounts of LTD than the control group, and the stress/exercise group is hypothesized to show similar amounts of LTD when compared with the control group.
Thus far, the difficulty in the experimental process has come when trying to get the stress group to show a different response than the control group. Again, it has been demonstrated in numerous papers that acute stress causes an increase in LTD in mice. However, for some reason we have struggled to be able to see results that reflect this. For the last couple months we have tried different things to obtain results consistent with the other studies that have been published on the subject. In the process, we have slowly been able to narrow down possible explanations as to why we were not receiving consistent results.
Around July, when we first noted the inconsistency of the data, we began taking note of anything the stressed and non-stressed mice might be doing behaviorally to throw off the data. One of the things that we noted was that while the stressed mice were in the plastic tube undergoing the protocol, they would gnaw on the rubber casing the covered the end of the tube. After some research, we discovered a an article2 that showed a marked difference in plasticity of mice that were allowed to gnaw on the rubber, and those that were not. So to remedy the issue, we ordered a new plastic tube with a plastic cover on the end that the mice could not reach to gnaw on during the stress protocol.
After some time recording data with the new protocol instruments the data reflected a slight difference, but the difference was not enough to produce the results that we were looking for. Part of the reason for this was that the stress experiments showed great differences in plasticity among themselves. Strangely, these differences did not seem to occur from day to day, but from slice to slice. Experiments performed on slices from the same brain acted differently from each other. This was unexplainable until one of the members of the lab found a research paper3 thatshowed that there were marked differences in LTD as well as LTP (long term potentiation) when performing experiments in the ventral verses the dorsal hippocampus, with greater responses gleaned from the dorsal hippocampus. As the slices were obtained coronally it was difficult to ascertain whether experiments were activating the dorsal region of the hippocampus or the ventral. To resolve this issue, we have recently begun obtaining experimental data from transverse slices rather than coronal slices to be sure that all experiments are activating neurons in the dorsal hippocampus.
Since that time we have collected a few pieces of data that seem to be more consistent with the data from other papers, but it is still difficult to be sure at this point. Once we have established firmly that the stressed group of mice is reacting as they are expected, we will begin work on the stress/exercise group to be able to analyze the difference between the two groups. This project is expected to continue through the beginning of next year when we will finally have obtained all the data necessary to write a report and publish an article with the acquired data.
References
- Gao LC, Wang YT, Lao X, Wang C, Wang FY, Yuan CG. The change of learning and memory ability in the rat
model of depression. In: Journal of Molecular Biology. Feb 2009;42(1):20-6 - Helmreich DL, Tylee D, Christianson JP, Kubala KH, Govindarajan ST, O’Neill WE, Becoats K, Watkins L, Maier SF. Active behavioral coping alters the behavioral but not the endocrine response to stress.
Psychoneuroendocrinology. May 9, 2012 - Maggio N, Segal M. Persistent changes in ability to express long-term potentiation/depression in the rat
hippocampus after juvenile/adult stress.In: Biological Psychiatry.Apr 15, 2011:69(8):748-53