Download Alison Poulson Effects of Maternal Care and the 5

Alison Poulson
Effects of Maternal Care and the 5-HTT Gene on Anxiety in Rodents
Abstract
Depression and anxiety are widespread disabilities. Many risk factors predispose an
individual for depression and anxiety, including polymorphisms in the 5-HTT gene and
childhood parental care. Both of these factors have been modeled in animals. Mice with one
copy of the 5-HTT gene knocked out are more anxious, and mice and rats raised with abnormally
high levels of maternal care are less anxious. This study investigates the interaction between
these two effects in mice to see if 5-HTT +/- mice display more variation in anxiety based on
maternal care than 5-HTT +/+ mice.
Introduction
Depression is a major health concern and one of the leading causes of disability (Murray
and Lopez 1997). Major depressive episodes are triggered by stressful life events, but not all
people experience depression after such events; genetic predispositions to depression and anxiety
and environmental factors both affect an individual's risk of developing depression (Jacobs et al
2006). Many factors, both genetic and environmental, have been identified that predispose for a
major depressive episode. It is clear that children who are neglected or abused are at higher risk
for anxiety and depression later in life (Brown et al 1999). However, studies of allelic variation
in the serotonin transporter gene known as 5-HTT show that this effect can be changed by
genetic factors. Individuals with two copies of the long form show no relationship
between childhood neglect and risk of anxiety and depression, while those with one or two
copies of the short form have variable risk for depression based on parental care (Caspi et al
2003). Both of these factors have been modeled in animals, but the relationship between the two
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has not been examined. This study aims to explore the relationship between 5-HTT allelic
variation and parental care and their effects on adult anxiety in rodents.
The Long-Evans hooded rat is a useful model for maternal care. Females display a range
of behavior in the amount of licking and grooming (LG) and arched-back nursing (ABN) given
to offspring in the first ten days after birth (Champagne et al 2003). This variation allows dams
to be characterized as "high" and "low" LG-ABN mothers. Pups raised by high LG-ABN
mothers were shown to be less fearful in multiple experiments. Corticosterone secretion
increases in response to stress and is a measure of anxiety (Rodgers et al 1999). Pups raised by
high LG-ABN mothers secreted less corticosterone in stressful situations (Liu and Diorio 1997).
Behaviorally, another study found that pups raised by high LG-ABN dams were less fearful
when placed in an unknown environment; they were quicker to eat when placed in a new cage
and spent more time exploring a novel open field (Caldji et al 1998). Cross-fostering showed
that these results were dependent on the mothering style, not the genotype of the birth
mother (Weaver et al. 2004). Similar results were found when mice were raised with a rat "aunt"
in the same cage; increased maternal care from the rat led to decreased anxiety in the pups
(Rosenberg et al 1970).
Maternal care affects gene expression during the development of the brain in the rats, and
presumably in all rodents. Pups raised by high LG-ABN mothers have more GR in their brains
during the first week of development, which inhibits later response to stress by methylating, or
inactivating, parts of the DNA important in the stress response (Kaffman and Meaney 2007). As
a result, throughout their lives these rats display less anxiety.
In humans, the 5-HTT transporter gene affects the response to parental care and the
probability of later depressive episodes (Caspi et al 2003). Similarly, in mice the 5-HTT gene
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affects anxiety levels; mice with one copy of the 5-HTT gene inactivated (5-HTT +/-) are more
anxious (Carola et al 2008). However, the relationship between 5-HTT mutations and parental
care and its effect on anxiety has not been examined in rodents. This study will include 5HTT +/- mice and 5-HTT +/+ controls, raised with high and low LG-ABN rat aunts. As adults,
the mice will be tested for anxiety behaviors, and HPA activation levels will be measured. I
predict that the 5-HTT +/- mice will show greater variation in anxiety based on maternal care,
behaviorally and by corticosterone levels, than the +/+ controls.
Methods
Methods are based on the work of Caldji et al (1998) and Rodgers et al (1999).
Forty female mice will be used for this experiment, raised in groups of five with a mouse
mother and a Long Evans hooded rat “aunt” who recently gave birth. The mice will belong to
four groups: ten 5-HTT +/+ mice raised with high LG-ABN aunts, ten 5-HTT +/- mice raised
with high LG-ABN aunts, ten 5-HTT +/+ mice raised with low LG-ABN aunts, and ten 5-HTT /- mice raised with low LG-ABN aunts. All groups will be raised in identical standard cages.
Care from the mouse mother and rat aunt will be carefully recorded for the first 21 days of life.
At weaning, day 22, mice will be placed in individual cages. Testing will begin 100 days after
birth.
Two mice from each group will perform the open field test. Each mouse will be placed
in a novel circular open area two meters in diameter. They will be left for ten minutes and the
amount of time spent in the interior circle, away from the walls, will be recorded. The mice will
then be decapitated and trunk blood collected to analyze for corticosterone.
Two other mice from each group will be deprived of food for 24 hours and then placed in
a novel environment where food is provided. The new environment will be the same size as the
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home cage and have food in a wire mesh container in the center. Latency time before eating and
time spent eating will be measured for each mouse. At the same time, two mice from each group
will be deprived of food for 24 hours and then placed in the home cage with food available;
latency time before eating and time spent eating will be measured to compare with the mice in
the novel environment.
Two more mice from each group will participate in the elevated plus-maze test,
consisting of a central platform where the mouse is placed with two open and two closed arms
opening off it. Mice will be left in the maze for five minutes and the percent of open-arm and
closed-arm entries, as well as the percent of time spent on the central platform, in open arms, and
in closed arms, will be recorded. Immediately after this test, mice will be decapitated and trunk
blood collected for corticosterone analysis.
The final two mice from each group will be decapitated directly from the home cage to
provide a baseline for corticosterone levels. All trunk blood will be analyzed with a commercial
corticosterone assay.
Discussion
This study examines the relationship between maternal care and 5-HTT allelic variation
in mice, as each of these factors has been individually shown to affect the risk of anxiety in
rodents. Presumably, maternal care will have a more marked effect on 5-HTT +/- mice than on
5-HTT +/+ mice.
It is expected that 5-HTT +/+ mice will display less variation in anxiety levels based on
maternal care than +/- mice. The +/+ mice should spend more time in the center of the open
field and in the closed arms of the elevated plus-maze, have less latency before eating in novel
environments, and have lower corticosterone levels in their blood after the tests. There should be
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less variation in the results between those raised with high and low LG-ABN aunts, as compared
to the 5-HTT +/- mice. This would provide evidence that the 5-HTT gene can help prevent adult
anxiety, even with less parental care. Also, 5-HTT +/- mice will have a higher rate of anxiety
with low LG-ABN rat aunts than those with high LG-ABN rat aunts. These results would show
that maternal care reduces anxiety in rodents at high risk due to genetic predispositions. Both the
5-HTT gene and maternal care influence the stress response, and these results would show that
the two can interact to make long-term changes to the organism. The molecular mechanisms by
which DNA methylation as a result of maternal care and the 5-HTT gene interact would warrant
considerable further study; both of these systems are well understood in isolation, but not in
concert.
If the 5-HTT +/+ mice with low LG-ABN rat aunts display the same levels of anxiety as
the 5-HTT +/- mice with low LG-ABN aunts, and higher levels than those with high LG-ABN
aunts, then the effects of maternal care and the GR system display greater effects on the risk for
anxiety than the 5-HTT gene in this model. This would further establish the importance of
maternal care. The stress response could be sufficiently inhibited by DNA methylation due to
maternal care to overcome the risk factors associated with 5-HTT variations. Conversely, if the
5-HTT +/- mice show the same anxiety levels regardless of maternal care, then maternal care
cannot change the effects of 5-HTT allelic variation. This is apparently contrary to current
human research and additional study would be needed to understand it. If one factor is a more
powerful predictor of anxiety than another, this masking effect would need to be explained on a
molecular level. If the pattern is conserved across species, there would need to be a reassessment
of human data on 5-HTT variation and parental care.
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This study could be expanded in multiple directions. Rat aunts are an imperfect model of
parental care; if 5-HTT knockouts could be developed in rat pups, further tests could be done
with low and high LG-ABN mothers. Studying the effects of these systems on depression, in
addition to anxiety, would also more clearly reflect human research and provide additional
understanding. And molecular analysis of the DNA and hormone responses could explain how
the two systems interact. Depression and anxiety are highly complex problems, and the factors
that put an individual at risk are insufficiently understood. This study attempts to link only two
of the common factors in depressive behavior, but it aims to show the complexity of risk factors
involved. To understand complex phenomena like this, we must examine the interactions of risk
factors and learn how seemingly unrelated processes can affect one another and the phenotype.
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