Melissa Wellum
 Effects of Trimethyltin (TMT) Induced Hippocampal Lesions on the Acquisition of Spatial Reference and Working Memory in CF1 and C57/BL/6J Mice
Background Research Question Method
Results Implications Relevant Links

Background

This study examined the effects of trimethyltin (TMT) induced hippocampal lesions on spatial reference and working memory in two strains of mice via the Morris water maze (Morris, Garrud, Rawlins, & O’Keefe, 1982).  C57BL/6J and CF1 mice were randomly divided into two groups of each strain.  One group of each strain received a saline injection and the other group of each strain received 3 mg/kg TMT injected intraperitoneally.  All of the C57BL/6J mice that were injected with TMT died.  In the reference task, both the CF1 TMT and saline mice were significantly impaired compared to the saline C57BL/6J group.  In the working memory task, only the CF1 saline mice were significantly impaired compared to saline C57BL/6J mice.  The lack of significance found between the CF1 saline and TMT mice on both tasks may reflect a recovery from the effect of TMT.  These data also show that C57BL/6J mice may have a more accurate spatial reference and working memory.  This may be a result of their well developed CA3 and CA4 hippocampal regions.  This may further implicate these areas in reference and working memory.  Further research may look at the brain physiology of the C57BL/6J mice in more detail and compare it to that of the CF1 mice through histological examination.
 
 

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Research Question

Does TMT affect spatial working and reference memory in CF1 and C57/BL/6J mice as assessed by the Morris water maze?  If so, does it have more of an affect on memory in C57/BL/6J mice, which have a more highly developed CA3 hippocampal region?

 
 
 
 


 
 

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Method

Subjects
A total of 21 male C57 mice and ten adult male CF1 mice participated in this experiment.  They were kept on a 12-hour light/dark cycle and housed in separate cages with food and water available to them ad libitum.  The 21 C57 mice were randomly assigned to two groups, one of ten (control) and one of 11 (experimental).  The ten CF1 mice were randomly assigned to two groups of five, one control group and one experimental group.  The control groups received a saline injection, while the experimental groups received a 3 mg/kg injection of TMT.  The mean weight of the mice was 30g at time of injection.  All mice were cared for in a manner consistent with the ethical guidelines of the American Psychological Association.

Toxicology
There was a random selection of mice for the control or the TMT group.  The three mg/kg TMT or .25 ml of saline was injected intraperitoneally.  Three mg/kg was used because higher doses are often lethal in mice, while lower doses are usually not sufficient in lesioning the hippocampus (Earley et al, 1992).  The mice were monitored for a period of seven days post-TMT so they could recover from the initial effects of the TMT.

Materials
The Morris water maze (Morris et al, 1982) was used to assess spatial memory.  The maze consists of a submerged platform within a pool of water (Morris, et al, 1982).  It has a diameter and a height of three feet.  The pool was entirely black fiberglass.  The wooden platform was also black and was submerged 1.5 cm below the surface of the water.  The pool was divided into four quadrants with white paint and the platform was located in the center of quadrant four.  The start position for the reference memory task was in the center of quadrant one, marked by an X in white paint.  For the working memory task, the start position was an X painted in the center of quadrant three.  A large lamp and a video camera were visible from within the pool and remained in the same positions throughout the entire experiment.  The data were measured using a stopwatch and a digital video camera that was positioned on top of a counter adjacent to the pool.

Procedure
Prior to testing 11 of the C57s and five of the CF1 mice were randomly selected and injected with 3 mg/kg TMT.  The remaining mice of each strain were injected with .25 ml of saline.  After injection, the mice were given one full week to recover from the initial effects of the drug.
The mice were placed in the pool and had to locate the platform to escape.  They were unable to see the platform, and therefore had to rely on memory of distal cues to locate it.  Resting on the platform provided the motivation, and therefore the mice did not need food- or water-deprivation or an extended training period. 
For the reference memory task, the mice were placed in quadrant one at start position one and allowed to swim freely for 60 seconds or until they found the platform.  If the mouse was unable to find the platform within that time period, they were placed there.  After finding the platform or being placed there, each mouse rested on it for 30 seconds.  There were 22 trials over a period of 11 consecutive days.  Each mouse was tested twice each day, once in the morning and once in the afternoon, at approximately the same times.
In the working memory task, each mouse was placed in the pool at start position two in quadrant three, everything else (e.g., distal cues, water level, position of the platform, etc.) remained the same as in the reference memory task.  The mice were again allowed to swim freely for 60 seconds and if they were unable to locate the platform within that time period, they were placed there.  All of the mice rested on the platform for 30 seconds at the end of each trial.  The working memory task was completed in one day, with three trials.  Two consecutive trials were run in the morning, and the third occurred that afternoon.
The mice were measured for how long it took to find the platform in each trial of both the reference and working memory tasks.  The mean score for each mouse across trials for each task was then computed.  A score of 60 seconds was automatically given for that trial if a mouse did not find the platform within the cut-off period.
 
 

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Results

The purpose of this experiment was to both examine the effects of TMT on spatial reference and working memory, and to compare CF1 and C57 mice on a spatial memory task in which time was the dependant variable.  After injection of the TMT all 11 of the C57 mice died.  Univariate analyses of variance were computed across the three remaining groups (CF1 saline, CF1 TMT, C57 saline).
For the reference task, a significant difference between groups was found (F(2,12) = 5.07, p<.05).  A post-hoc comparison was then performed and the CF1 mice, both control (p=.01) and TMT (p=.036) groups, were found to be significantly impaired compared to the C57s (see figure 1) with means of 44, 40, and 26 respectively.  However, no significant difference was found between the CF1 control and TMT mice (p=.513).
For the working memory task, no significant effect was found for TMT (F(2,12) = 3.21, p>.05), although it was approaching significance.  However, a post-hoc comparison showed that the CF1 saline group (p=.028) was significantly impaired compared to the C57s (see figure 2) with means of 8 and 33, respectively.
These findings suggest that reference memory is not dependent on the areas of the brain affected by TMT because the CF1 control and TMT groups were not significantly different from each other.  However, because the C57s were much faster and more accurate in both acquisition and performance in completing the reference memory task than both of the CF1 groups, it is very possible that the differences are a result of the different brain physiology of the two strains.
Further, because the CF1 control and TMT groups were not significantly different from each other in the working memory task, the areas of the brain destroyed by the TMT may not be necessary for working memory to function properly.  In fact, the CF1 TMT group did slightly better than the CF1 saline group in this task, with means of 24 and 33, respectively.  The C57s once again outperformed both the CF1 saline and TMT groups.  This suggests that the different brain physiology of the C57s may be responsible for more accurate working memory as well.

figure 1


 
 

figure 2

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Implications

Many areas of this study were inconsistent with previous research.  This may be due to possible confounds.  First, because there were only five mice in each group due to the lethality of TMT in the C57s, the sample may not have been large enough to give an accurate representation of the mice's abilities.  Second, the scoring of the mice was subject to experimenter reliability, and a more accurate method would entail using a computer program that would score each mouse in a consistent manner.  Third, as previously mentioned, the TMT mice were injected approximately six months prior to testing and the effects of the drug may have worn off by the time it began.  Fourth, it is possible that the somewhat inconsistent testing patterns caused some fatigue.  For the working memory portion of the experiment, two of the three trials were run consecutively and the third trial was run a few hours later that same day.  Finally, the dose of TMT that was administered to the C57 mice was too high.  These mice may require a much lower dose to produce lesions in the hippocampal area and affect spatial working memory.
As the many confounds may have skewed the results, in the future researchers might attempt to repeat this study using more consistent and reliable techniques.  For instance, a larger sample size should be used so that it can be safely assumed that the results are representative of the strain of mouse being tested.  Also, testing the mice approximately one to two weeks after TMT-injection  will ensure that the effects of the drug have not worn off.  Furthermore, testing should be done in a consistent manner, with a set number of trials each day, dispersed evenly throughout to reduce fatigue effects.  Finally, to avoid lethality while pursuing the effects of TMT on C57 mice, a lower dose should be injected (maybe as low as .5 mg/kg TMT).
Information on the effects of TMT is very important.  TMT is found in many large and vital industries, including plastics, agriculture, paper, and disinfectants (Wenger et al, 1982).  Furthermore, approximately 100 people developed toxic symptoms in the 1950s in France, after treatment with a preparation containing triethyltin, an organic tin closely related to trimethyltin (Wenger et al, 1982). This is what initially spurred interest into the effects of TMT on rats and mice.
Research in this area is also important for understanding what the regions of the brain affected by the TMT are responsible for.  Although, in this study TMT did not appear to negatively affect spatial reference or working memory ability in mice, much previous research has found that TMT does impair working memory in rats (Alessandri et al, 1994; Bushnell & Angell, 1992; Earley et al, 1992; Nation et al, 1984) and mice (Oullette, 1998).  Previous studies have also found increased aggression, hyperirritability, tremor, hyperreactivity, and changes in schedule-controlled behavior in rats as a result of TMT exposure (Earley et al, 1992).  This suggests that the areas of the brain affected by TMT are involved in more than spatial reference and working memory.  Future research should further address what these areas are responsible for.
Overall, the results of this study suggest that C57 mice perform better on the Morris water maze than CF1 mice.  Further, despite previous research findings in the area, these results do not support a link between TMT and spatial-working memory impairment.  Further research with C57 mice and TMT will be essential to a better understanding of spatial memory in humans and animals.
 
 

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