The aim of this study was to investigate whether the effects of maternal serotonin transporter expression and prenatal stress combine in such a manner to produce autistic-like behaviors in offspring. Our specific hypothesis was that prenatally stressed offspring of 5-HTT +/− dams would display decreased social interaction in comparison to control offspring of WT dams. Our results seem to support our hypothesis. Prenatally stressed offspring of 5-HTT +/− dams appeared to display social deficits despite having no observed increase in anxious behavior. The neurological exam battery indicated no major developmental abnormalities resulting from our experimental manipulations in the studied mice.
The behavioral battery revealed significant differences between prenatally stressed offspring of 5-HTT +/− dams and control offspring of WT dams. Further study will be needed to fully understand how prenatal stress and maternal genotype interact. During the 3-chamber social approach task comparing interaction with a novel object and stranger mouse, the offspring of prenatally stressed 5-HTT +/− dams spent significantly less time with the stranger mouse than the offspring of WT control dams, suggesting decreased preference for social interaction. Offspring of WT control dams spent significantly more time with both the stranger mouse and the object than prenatally stressed offspring of 5-HTT +/− dams, further indicating that the prenatally stressed offspring of 5-HTT +/− dams have decreased social interaction and social interest. The prenatally stressed offspring of 5-HTT +/− dams appeared to display decreased levels of social novelty seeking compared to control offspring of WT dams, as assessed in the trial comparing interaction with a novel stranger and a familiar stranger, although this comparison did not withstand Bonferroni correction. The prenatally stressed offspring of 5-HTT +/− dams also preferred to stay in the center chamber, and not interact with either mouse, as compared to the other groups, further suggesting a decreased preference for social novelty seeking. Decreased total time spent with the novel stranger as compared to the familiar stranger can be interpreted as decreased social novelty seeking as well as decreased recognition of social cues (Crawley, 2004
), and less time spent in the chamber with a novel mouse in comparison to a novel object is indicative of decreased sociability (Moy et al., 2004
). In our study, presence of either prenatal stress or maternal 5-HTT +/−genotype alone was sufficient to result in an increase in time spent in the central chamber in the novel stranger versus familiar stranger experiment, since the non-stressed offspring of WT dams spent less time in the central chamber than all other groups. In the novel stranger versus novel object experiment, increased time in the central chamber suggests a decreased preference for novelty in general, since it would result from the combined effects of less approach for both the object and the mouse. Decreased novelty seeking is also part of the increased insistence on sameness that is observed in autism clinically (APA, 2000
). The initial trial in which mice explored all of the empty chambers equally suggests that these findings are not due to the mice having a general decrease in exploratory behavior.
It is important to note that in any task assessing levels of social interest, interaction or exploration in the mouse, difficulties arise in attempting to interpret the from data in mice as applicable in humans. In individuals with ASD, not only the amount of social interaction is decreased, which is assessed in this task, the quality of interaction is also decreased. While this task assesses the amount of social interaction between the experimental mouse and the stranger mouse, it does not assess the quality or content of the interactions. Further, interactions between two sex-matched mice can be very different than those of two sex-matched humans. For example, one would expect mice to demonstrate more aggressive, territorial behavior toward each other than would be expected in humans. Therefore, while this task is useful in that it helps shed light on deficits in social interaction, we acknowledge that further social testing is necessary to truly understand the nature of the interactions of these proposed autistic-like mice.
Results from the ultrasonic vocalization measure show a decrease in communication of the prenatally stressed offspring of 5-HTT +/− dams in early development in comparison to control offspring of WT dams, as seen in an interaction of maternal genotype and prenatal stress. Decreased communication is a cardinal sign of ASD. As main effects for prenatal stress or maternal genotype did not reach significance alone, the significant interaction between maternal genotype x prenatal stress further supports our hypothesis that a combined effect between maternal genotype and prenatal stress produces behavioral differences in the prenatally offspring of 5-HTT +/− dams. Overall, the USV results indicate a basic disruption of communication, but further investigation is necessary to better characterize these findings. It is important to note that the decrease in communication was in the range of 20 – 40 kHz, and not calls in the range of 40 – 100 kHz. These findings demonstrate a change in the communicative phenotype, but the social role of these vocalizations is not yet fully understood (Hahn and Lavooy, 2005
). Furthermore, while it is generally well agreed upon that social calls are in a frequency range above 40 kHz, the role of vocalizations at any frequency is still in need of further understanding. Therefore, more research is needed to understand the social and communicative relevance of the USV effects detected in our study. As a heating pad was not used as part of the USV procedure, it is possible that the vocalizations observed could be due to a reaction to the cold environment. However, length of time isolated from the nest and dam was minimized to lessen the effects of temperature, and mice in all experimental groups were subjected to the same conditions.
By measuring anxiety-like behaviors, we demonstrated that decreases in social interactions witnessed here are not likely to be an indirect result of greater anxiety. With the elevated-plus maze, stressed mice had significantly higher open arm ratio than control mice, indicating lower levels of anxiety. Thus, according to this task, the decrease in social interaction described above is not likely due to an increase in anxiety. However, stressed offspring spent less time in the inner region during the open field task, suggesting they were more anxious than control offspring. For this task a significant interaction between genotype and stress was observed, driven by reduced open field exploration by the prenatally stressed WT-dam group. It is possible that this is reflected in the first part of the social interaction task, in which male mice of WT dams subjected to prenatal stress show no preference for social interaction versus an inanimate object. However, in both tasks which measure anxiety, offspring of 5-HTT +/− dams that were prenatally stressed displayed low to normal levels of anxiety-like behaviors. While these initial findings require further study to definitively determine the role of maternal 5-HTT expression and prenatal stress on anxiety-like behaviors in adult offspring, they support the notion that any detected social interaction effects observed in the offspring of stressed 5-HTT +/− dams are not likely due to increased overall anxiety.
As no significant differences were observed in maternal care behaviors or in offspring behavior across groups that were cross-fostered, the counterbalancing issues between groups are unlikely to confound our results. Additionally, the lack of any noticeable effect on behavior for cross-fostered groups would also suggest against the possibility of introducing confounding factors as a result of the cross-fostering procedure.
This study generally supports our hypothesis that a combined effect of maternal genotype and prenatal stress produces autistic-like deficits in social interaction in prenatally stressed offspring of 5-HTT +/− dams compared to control offspring of WT dams. These findings may relate to some of the findings in the current literature on 5-HTT polymorphisms in patients with ASD (Cook et al., 1997
; Brune et al., 2006
; Losh et al., 2008
; Freitag, 2007
). While some studies have found effects of 5-HTT polymorphisms (Cook et al., 1997
), others have not (Maestrini et al., 1999
). This may be due to the need for environmental factors to be present in order to augment the effects of the gene. Many of the studies that did not show a relationship between the S allele of the 5-HTT gene and autism predominantly examined families with multiple affected family members (Maestrini et al., 1999
), where an environmental interaction would less likely be important. Studies that have revealed a relationship between the S allele of the 5-HTT gene were not typically exclusive to families with multiple affected family members (Cook et al., 1997
). This implies that while they may serve as risk factors individually, both 5-HTT polymorphisms and prenatal stress could interact to increase ASD risk.
One limitation of this study is the small number of behavioral tasks used. Additional tasks that measure social interaction need to be considered, such as social transmission of food preference, to further support this hypothesis that global social functioning is present in these animals (Wrenn et al., 2003
). Additional communication tasks, anxiety, repetitive and restrictive behaviors amongst others should also be included in future studies to expand upon these initial findings (Moy et al., 2006
). In the future, the time that experimental mice spend near the stranger mouse as well as the novel object will be assessed in order to obtain a more accurate reading of social interaction. Larger numbers of animals assigned to each condition can also help with matching the number of animals per group as well as with further defining how the combined effects of genetics and prenatal stress interact. We did not collect offspring genotype information in the present study. This is an important point which will need to be further examined.
The neurobiological underpinnings of the combined effects of maternal stress and 5-HTT genetics in offspring remains to be fully elucidated. Post-mortem analysis of receptor binding and gross anatomical differences will help support the hypothesis that 5-HTT maternal genotype and prenatal stress combine in effect to produce a phenotype in the offspring related to ASD, while helping to delineate the anatomical basis of the observed behaviors, by comparing brain structure and function to the known human ASD abnormalities. For example, as previous structural observations of autism patients have revealed a significant decrease in the number of Purkinje cells, these cells should be studied in a mouse model of autism as well (Bauman and Kemper, 2005
). Furthermore, abnormal serotonin (5-HT) function is observed in autism (Pardo and Eberhart, 2007
). As receptors for serotonin (5-HT) are present on the Purkinje cells of neonatal rats (Maeshima et al., 2004
), further investigation of serotonergic ligand binding in Purkinje cells is needed to assess whether the changes in this animal model resemble those in humans. Finally, deceased glutamic acid decarboxylase (GAD) mRNA is observed in Purkinje cells in autism (Yip et al., 2007
), and decreased GABA receptor binding is also seen in the hippocampus in autism (Blatt et al., 2001
). Further investigation of the effects of GABA in the cerebellum by performing in situ
hybridization for GAD mRNA in the Purkinje cells is needed to further assess whether the changes in this animal model resemble those in humans.