For many years, scientists have understood some of the effects a lack of serotonin has on human behavior. A serotonin deficiency has been credited with numerous uni-polar neurological disorders. However, the total effect that a deficiency of serotonin has on human social behaviors is not completely known. The first part of this experiment was aimed at analyzing what those effects are. Using TPEX wild-type mice with normal levels of serotonin and 5HT-Phypo mice with decreased levels of serotonin, I tested the differences in social behaviors between the two genotypes. Preliminary research showed that a lower level of serotonin resulted in increased levels of aggression, which was seen in the increased number of fights instigated by the 5HT-Phypo mice. To test the validity of these findings I paired unfamiliar wild-type mice with each other, unfamiliar 5HT-Phypo mice with each other, and I also paired wild-type mice with 5HT-Phypo mice. This way I could see how those with normal levels of serotonin and those with diminished levels of serotonin interact with those of their same genotype and those of different genotypes. The experiment used is called a “dynamic affiliation test”. In a dynamic affiliation test I place each test mouse in a clean viewing cage and record the mice on video for three hours and ten minutes. Once the videos are recorded I can score the mice based on a number of social behavior variables. The different variables of importance for this study are allo-sniffing, sleeping together, sleeping alone, and fighting. Allo-sniffing is a control variable as it shows evidence of normal mouse interaction. Sleeping together is a positive affiliation variable. Last, sleeping alone and fighting are negative affiliation variables used to observe the level of aggression in the mice. These variables are scored the first ten minutes of every hour and the last ten minutes of the recording. The rest of the 3 hour, 10 minutes recording is scored solely for fight count. This is used to see the overall aggression that accompanies each genotype.
My hypothesis is that the lower level of serotonin will effectively increase the aggressive behavior in the mice. I am looking to see if there is sufficient evidence to support this. At the same time, I have noticed that the 5HT-Phypo mice also show a lack of friendliness to go along with the increased aggression. I will look to the positive affiliation tests to see if there is significant evidence of this effect as well. I hypothesize that lower serotonin levels lower positive affiliation.
The second part of my experiment concerns treatment of low serotonin. For years, depression, borderline personality disorder, and many other uni-polar disorders have been treated with selective serotonin re-uptake inhibitors (SSRIs). The results have been unsatisfactory, to say the least. I started looking to see if there was a more effective way to reverse the effects of low serotonin. I re-ran the experiment having treated the mice with Prozac or Prozac with a 5HT-P booster. In this way I could find whether Prozac alone is effective, or whether a combination of Prozac and 5HT-P is more effective. At the same time, if none of the two treatment methods show signs of effectiveness, this would be evidence that serotonin deficiency cannot be reversed as symptoms are not just the result of genotype, but may be the result of environmental factors that effected the maturation within each specific genotype. I hypothesized that the combination of the two drugs would have the greatest effect on reversing the signs of serotonin deficiency.
Data Analysis
In order to complete hypothesis testing, I reorganized the data, comparing the mean fight counts and mean sleeping together occurrence counts for my three comparisons. When analyzing the difference between the two genotypes before treatment, the null hypothesis is that there is no difference between the mean fight counts and mean sleeping together occurrence counts for the two genotypes. The alternative hypothesis is that there is a difference between the mean fight counts and mean sleeping together occurrence counts for the two genotypes, in that the 5HT-P mice have, on average, more fights than the wild-type (Wt) mice. When analyzing the difference between the 5HT-P mice before and after treatment, the null hypothesis is that there is no difference between the mean fight counts and mean sleeping together occurrence counts for the two treatment stages. The alternative hypothesis is that there is a difference between the mean fight counts and mean sleeping together occurrence counts for the two treatment stages, in that the treated 5HT-P mice have, on average, fewer fights than the untreated 5HT-P mice. When analyzing the difference between the Wt/5HT-P mouse pairs before and after treatment, the null hypothesis is that there is no difference between the mean fight counts and mean sleeping together occurrence counts for the two treatment stages. The alternative hypothesis is that there is a difference between the mean fight counts and mean sleeping together occurrence counts for the two treatment stages, in that the treated mouse pairs have, on average, fewer fights than the untreated mouse pairs. All conditions necessary for hypothesis testing are assumed to apply to the data sets in question. Although there are only 9 or 7 mouse pairs per category, these pairs were drawn from nearly normal populations of mice with engineered genotypes by means of random experimentation through double blind scoring techniques throughout the experiment. For more reliable results, an experiment with a larger population of engineered mice, meeting the conditions for hypothesis testing, should be run in future behavioral analyses. A significance level of .05 is used in all analyses. For each comparison, I will use a t-test to test for sufficient evidence to support the alternative hypotheses.
## X Ho.pre.treatment.fight.count Wt.pre.treatment.fight.count
## 1 n 9.00 9.00
## 2 x? 29.88 1.56
## 3 s 22.00 4.39
## 4 SE 2.44 0.48
## Ho.pre.treatment.Sleeping.Together.Occurrence.Count
## 1 9
## 2 0
## 3 0
## 4 0
## Wt.pre.treatment.Sleeping.Together.Occurrence.Count
## 1 9.00
## 2 1.22
## 3 1.93
## 4 0.21
## Response variable: numerical, Explanatory variable: categorical
## Difference between two means
## Summary statistics:
## n_Ho = 9, mean_Ho = 29.89, sd_Ho = 23.34
## n_Wt = 9, mean_Wt = 1.556, sd_Wt = 4.667
## Observed difference between means (Ho-Wt) = 28.33
##
## H0: mu_Ho - mu_Wt = 0
## HA: mu_Ho - mu_Wt > 0
## Standard error = 7.935
## Test statistic: T = 3.571
## Degrees of freedom: 8
## p-value = 0.0036
## Response variable: numerical, Explanatory variable: categorical
## Difference between two means
## Summary statistics:
## n_Ho = 9, mean_Ho = 0, sd_Ho = 0
## n_Wt = 9, mean_Wt = 1.222, sd_Wt = 2.048
## Observed difference between means (Ho-Wt) = -1.222
##
## H0: mu_Ho - mu_Wt = 0
## HA: mu_Ho - mu_Wt < 0
## Standard error = 0.683
## Test statistic: T = -1.79
## Degrees of freedom: 8
## p-value = 0.0556
Comparing the average fight counts for the two genotypes before treatment will show whether or not genotype has any effect on the aggression levels in the mice. The test statistic for the fight count comparison is 3.571 with 8 degrees of freedom. This yields a p-value of 0.0036. Due to the fact that this p-value is far below my significance level, I conclude that there is significant evidence to support the notion that the 5HT-P mice fight more on average than the Wt mice. Comparing the sleeping together occurrence counts for the two genotypes before treatment will show whether or not genotype has any effect on the positive affiliation levels in the mice The test statistic for the sleeping together occurrence count comparison is -1.79 with 8 degrees of freedom. This yields a p-value of 0.0556. Due to the fact that this p-value is not less than my significance level, I cannot conclude that genotype has any bearing on the average number of sleeping together occurrences before 5HT-P treatment.
## X Ho.pre.treatment.fight.count Ho.post.treatment.fight.count
## 1 n 9.00 9.00
## 2 x? 29.88 0.66
## 3 s 22.00 1.33
## 4 SE 2.44 0.15
## Ho.pre.treatment.Sleeping.Together.Occurrence.Count
## 1 9
## 2 0
## 3 0
## 4 0
## Ho.post.treatment.Sleeping.Together.Occurrence.Count
## 1 9.00
## 2 1.44
## 3 1.77
## 4 0.20
## Response variable: numerical, Explanatory variable: categorical
## Difference between two means
## Summary statistics:
## n_post = 9, mean_post = 0.6667, sd_post = 1.414
## n_pre = 9, mean_pre = 29.89, sd_pre = 23.34
## Observed difference between means (post-pre) = -29.22
##
## H0: mu_post - mu_pre = 0
## HA: mu_post - mu_pre < 0
## Standard error = 7.795
## Test statistic: T = -3.749
## Degrees of freedom: 8
## p-value = 0.0028
## Response variable: numerical, Explanatory variable: categorical
## Difference between two means
## Summary statistics:
## n_post = 9, mean_post = 1.444, sd_post = 1.878
## n_pre = 9, mean_pre = 0, sd_pre = 0
## Observed difference between means (post-pre) = 1.444
##
## H0: mu_post - mu_pre = 0
## HA: mu_post - mu_pre > 0
## Standard error = 0.626
## Test statistic: T = 2.307
## Degrees of freedom: 8
## p-value = 0.025
Comparing the average fight count for the 5HT-P mice before and after treatment will show whether or not treatment with 5HT-P has any therapeutic effect on the aggression levels. The test statistic for the fight count comparison is -3.749 with 8 degrees of freedom. This yields a p-value of 0.0028. Due to the fact that this p-value is far below my significance level, I conclude that there is significant evidence to support the notion that the 5HT-P mice fight more on average before treatment than after treatment. Comparing the average sleeping together occurrence count for the 5HT-P mice before and after treatment will show whether or not treatment with 5HT-P has any therapeutic effect on the positive affiliation levels. The test statistic for the sleeping together occurrence count comparison is 2.307 with 8 degrees of freedom. This yields a p-value of 0.025. Due to the fact that this p-value is less than my significance level, I can conclude that treatment increases the average number of sleeping together occurrences in 5HT-P mice.
## X X.Ho.Wt.pre.treatment.fight.count X.Ho.Wt.post.treatment.fight.count
## 1 n 7.00 7.00
## 2 x? 9.71 4.29
## 3 s 6.32 10.49
## 4 SE 2.39 3.97
## X.Ho.Wt.pre.treatment.Sleeping.Together.Occurrence.Count
## 1 7
## 2 0
## 3 0
## 4 0
## X.Ho.Wt.post.treatment.Sleeping.Together.Occurrence.Count
## 1 7.00
## 2 2.57
## 3 2.49
## 4 1.58
## Response variable: numerical, Explanatory variable: categorical
## Difference between two means
## Summary statistics:
## n_post = 7, mean_post = 4.286, sd_post = 11.34
## n_pre = 7, mean_pre = 9.714, sd_pre = 6.824
## Observed difference between means (post-pre) = -5.429
##
## H0: mu_post - mu_pre = 0
## HA: mu_post - mu_pre < 0
## Standard error = 5.002
## Test statistic: T = -1.085
## Degrees of freedom: 6
## p-value = 0.1597
## Response variable: numerical, Explanatory variable: categorical
## Difference between two means
## Summary statistics:
## n_post = 7, mean_post = 2.571, sd_post = 2.699
## n_pre = 7, mean_pre = 0, sd_pre = 0
## Observed difference between means (post-pre) = 2.571
##
## H0: mu_post - mu_pre = 0
## HA: mu_post - mu_pre > 0
## Standard error = 1.02
## Test statistic: T = 2.521
## Degrees of freedom: 6
## p-value = 0.0226
Comparing the average fight count for the WT/5HT-P mouse pairs before and after treatment will show whether or not treatment with 5HT-P has any therapeutic effect on the aggression levels in homozygous mice when paired with wild-type mice. The test statistic for the fight count comparison is -1.085 with 6 degrees of freedom. This yields a p-value of 0.1597. Due to the fact that this p-value is not below my significance level, I cannot conclude that there is significant evidence to support the notion that the Wt/5HT-P mouse pairs fight more on average before treatment than they do after treatment. This result is most likely due to the large outlier in the post treatment mouse pairs. Only 1 mouse pair fought in the post treatment group. Should this pair be thrown out there would be evidence supporting the effect 5HTP has on reducing aggression in mouse models. Comparing the average sleeping together occurrence count for the WT/5HT-P mouse pairs before and after treatment will show whether or not treatment with 5HT-P has any therapeutic effect on the positive affiliation levels in homozygous mice when paired with wild-type mice. The test statistic for the sleeping together occurrence count comparison is 2.521 with 6 degrees of freedom. This yields a p-value of 0.0226. Due to the fact that this p-value is less than my significance level, I can conclude that treatment increases the average number of sleeping together occurrences in Wt/5HT-P mouse pairs.
After completing the statistical analysis of the three different comparisons, evaluating both the negative affiliation and positive affiliation marker variables, several conclusions can be drawn from the data sets. When comparing the untreated wild-type mice with the untreated 5HT-Phypo mice, it is seen that there is a significant increase in the aggression levels, represented by fight counts, in the 5HT-Phypo mice over the wild-type mice. This correlates with previous studies, suggesting that the lower levels of serotonin in 5HT-Phypo mice caused increased levels of aggression. Continued support for this claim of increased levels of aggression due to lowered levels of serotonin, could be derived from larger experiments, including more test subjects from multiple breeders, with 5HT-Phypo mice with varying levels of diminished serotonin. Concerning the analysis of positive affiliation, insufficient evidence was found to support the claim that decreased serotonin levels lead to decreased positive affiliation levels as expressed in sleeping together occurrence counts. This discovery was new to the experiment as witnessed in tandem with other observations. While the counts of occurrence does not result in sufficient evidence, the average duration for these occurrences of sleeping together might yield more profitable results. While one mouse pair might sleep together for three hours, this would be statistically less than the pair that sleeps together fifteen times, though only for a total of ten minutes. Analyzing the duration of sleeping together would most likely show a truer picture of the difference in positive affiliation levels between the two genotypes, wild-type with normal levels of serotonin, and 5HT-Phypo mice with diminished levels of serotonin. Another important factor in experimentation is timing. These experiments all took place during the night phase when the mice are most active as mice are nocturnal creatures. An alternative experiment could test the mice for positive affiliation levels during the late morning hours when the mice are more prone to sleeping for longer periods of time. However, testing the mice by this design strays from any true correlation with the human behaviors the mouse model is used to predict. Testing the mice for specific behaviors when they are most apt to do those desired behaviors, leads to invalid results and inefficient pharmaceuticals down the line. Once the genotypic differences were observed, the test genotype, 5HT-Phypo, was tested for differences in affiliation levels before and after treatment. Hypothesis testing provided sufficient evidence for conclusions stating that 5HT-P significantly decreased the levels of negative affiliation, and increased the levels of positive affiliation in the 5HT-Phypo mice. While testing the wild-type mice for the same responses would have provided nice controls, the results would not have supported or negated any of the claims in question. These results suggest that the 5HT-P in conjunction with Prozac, produces far more satisfactory alterations in behavior than Prozac on its own has achieved in the past. At the same time, these results allow us to conclude that depression does not result from environmental factors, as the genetic factor associated with depression can be reversed, thus resulting in reversed signs of depression. Further support for these results would be gained through further testing with more subjects in different testing environments, with different recorders and scorers. The final comparison, analyzed how the affiliation levels in half wild-type, half 5HT-Phypo mouse pairs change throughout the treatment process. This analysis will show how treatment affects both the wild-type mouse and the 5HT-Phypo mouse when interacting with one another. Although there is insufficient evidence in support of the therapeutic value of 5HT-P, this is most likely due to the extreme outlier in the data. For the sake of having the same number of observations, this value was kept. However, in the future, where a greater number of tests could be completed, such outliers would be overwhelmed by the majority of supporting data. Nevertheless, the mixed pairs did show signs of increased positive affiliation levels in the presence of 5HT-P treatment. Future analysis would be needed to show whether it is the wild-type or the 5HT-Phypo mouse that is most effected by the treatment, and most responsible for the evident levels of both negative and positive affiliation. It is possible that one genotype contributed one affiliation while the other was a strong supplier of the opposite affiliation level. These and certainly many more questions can be answered with further testing. However, the results found in this test suggest the need for changes in pharmaceutical therapy for patients suffering from uni-polar disorders.