Anxiety disorders can cause significant biological and psychological changes for an individual as it affects their ability to distinguish cues considered threatening vs. non-threatening. For most individuals, fear is considered an adaptive physiological and behavioral response causing a shift in homeostasis in response to seemingly harmful situations. However, anxiety disorders, a much more diffused state of stress, prevent the individual from being able to identify cues to discern whether the situation requires the shift in homeostasis. The human body’s physiological responses to stressors such as such as increase in blood pressure, trembling, irritability, muscle tension, sleep disturbance and others (American Psychological Association 2016) can lead to further complications in even healthy individuals such as diagnoses of psychiatric illnesses like PTSD, anxiety disorder, panic disorder and others. If these physiological responses are prolonged over extended periods of time with frequent occurrences for a biologically sensitive individual such as a pregnant female, the mechanisms involved in these reactions can flare to higher degrees and expose the fetus to excess levels of stress hormones such cortisol (or corticosterone in rodents), corticotropin releasing hormone, arginine vasopressin, and adrenocorticotropic releasing hormones amongst others (Soreq, H., Friedman, A. Kaufer D. 2010; Vallée, 1997). As a result, these events can cause disturbances in normal neuronal function and lead to an inability to cope with stress as observed in individuals diagnosed with anxiety disorders. These neurological insults in the womb can be a cause for grave concern due to the fact that prenatal and postnatal periods in an individual’s life involve a great deal of neurological development with rapid development of neuronal connections. This means that the neurological insults occurring could have long-term effects on the fetus (Weinstock, 2008).
At present, a large part of the studies that have been conducted on this topic have used a variety of models that aim to elucidate biological mechanisms that underlie the problem. The primary subject used for these studies have been rodents such as mice and rats. Rodent models are very easy to manipulate and thus can be incredibly useful in revealing detailed accounts of what is occurring at a molecular level. These experiments use mechanisms such as restraining, presenting the animals with unpredictable noise, or electric shocks, inducing hypoxia, maternal deprivation, immobilizing the animals and various other methods have been used (Weinstock, 2008; Chung, S., Son, G.H., Park, S.H. et al. 2005; Henry, C., Kabbaj, M., Simon, H. et al. 1994). These methods lead the animal to believe that it is facing a life-threatening situation which causes it to activate its central and peripheral nervous system in response, an ideal method for instituting and testing for prenatal stress (Chung, S., Son, G.H., Park, S.H. et al. 2005; Henry, C., Kabbaj, M., Simon, H. et al. 1994). Inducing hypoxia is a common cause of perinatal distress, morbidity and mortality and therefore creates an emotionally exhausting experience for the animal (Soreq, H., Friedman, A. Kaufer D. 2010). Similarly, maternal deprivation involves removing the animal from the mother leading to a lack of maternal care. This can cause the perinatal animals to suffer from hunger and thirst and therefore causing anxiety for the animals. During pregnancy, stressors like these can significantly impact the bodily functions of the mother such as an observed rise in hormone secretion. In one study, rats were exposed to chronic levels of stress daily throughout the pregnancy to identify changes in the levels of corticosterone (CORT) and the longevity of this elevation of CORT levels (Takahashi, L.K., Turner, J.G. and Kalin, N.H. 1998). CORT hormones are an important aspect in the body’s stress response regulated through the Hypothalamic-Pituitary-Adrenal axis (HPA axis). The HPA axis involves the different regions of the body to initiate release of stress hormones such as cortisol or corticosterone in response to stressors. This sequence of events is activated by the sympathetic nervous system (Know your brain: HPA axis, 2014). Both rapid activation and deactivation of the HPA is necessary for the organism to respond to stress appropriately. The study by Takahashi and colleagues, revealed that inducing chronic stress to the mother, increased the level and duration of CORT concentration levels in both the mother and the fetus as compared to the control group. Under normal conditions, CORT released into maternal blood gets inhibited by CORT binding proteins. This, thereby limiting the influence of CORT on the fetus and the mother (Takahashi, L.K., Turner, J.G. and Kalin, N.H. 1998). However, chronic stress results in a reduction of the effect and sensitivity of corticosterone binding protein (CORT-bp) on plasma levels of CORT (Takahashi, L.K., Turner, J.G. and Kalin, N.H. 1998). Thus, if the mother is being chronically exposed to stress, then the fetus is also indirectly being exposed to CORT for a long period of time since the CORT-bp aren’t effectively inhibiting the plasma levels of CORT. In humans, placental corticotropin releasing hormone (CRH) production rises rapidly, during the gestation period, to levels that are observed in adults under physiological stress (Sandman, C.A., Glynn, L., Schetter, C.D. et al. 2006). Plasma levels of CRH, like CORT in rodents, are normally inhibited by CRH binding proteins to low levels (Soreq, H., Friedman, A. Kaufer D. 2010). However, in humans the levels of CRH-bp present are reduced close to term. This allows CRH levels to rise rapidly, the presence of which can initiate labor (Perkins, A.V., Linton, E.A., Eben F. et al. 1995). As can be predicted, preterm labor limits the amount of neurological development that can happen in utero for the fetus and can therefore effectively increase the likelihood of developing psychological conditions such as anxiety disorders (Cosmi, E.V., Luzi, G., Gori, F. and Chiodi, A. 1990; Myers, R.E. 1975).
One of the primary examinations in identifying the effects of prenatal stress, is to observe the changes taking place in the fetal HPA axis. Indeed, rodent models have been subjected to maternal stress to observe these changes. In one study by Koehl and colleagues, maternal stress was induced in rats by restraining them for 45 minutes, three times daily (Koehl, M., Darnaudery, M., Dulluc, J. et al. 1999). This induction altered the circadian rhythms of CORT of the offspring postnatally. The prenatally stressed offspring began secreting higher plasma levels of CORT at the end of the light period as compared to controls (Koehl, M., Darnaudery, M., Dulluc, J. et al. 1999). Thus, the basal HPA axis activity is altered postnatally due to the stress endured prenatally by the offspring. Additionally, disruptions and subjection of distress to pregnant rats through methods such as restraint, electric shocks, or unpredictable noise can cause an increase in the plasma levels and duration of CORT in offspring postnatally when compared to control rats (Koehl, M., Darnaudery, M., Dulluc, J. et al. 1999). This was further confirmed through a study, on rats specifically bred for high anxiety and low anxiety that were subjected to equal levels of prenatal stress (Bosch, O.J., Kromer, S.A. and Neumann, I.D. 2006). High anxiety bred rats showed an increase in the plasma levels and duration of CORT whereas low anxiety bred rats did not (Bosch, O.J., Kromer, S.A. and Neumann, I.D. 2006). Additionally, the presence of CRH mRNA was reduced in the high anxiety bred rats with no effect in low anxiety rats (Bosch, O.J., Kromer, S.A. and Neumann, I.D. 2006). This suggests, that there is a genetic basis for identifying which animal will experience a shift in the functioning of the HPA axis in response to prenatal stress. Pup retrieval and food-seeking behavior are basic survival skills present in wild type rats, and the absence of these behaviors are categorized as symptoms of anxiety disorders (Soreq, H., Friedman, A. Kaufer D. 2010). However, one study observed an impaired ability of prenatally stressed rats to perform this task (Fride, E., Dan, Y., Gavish, M. and Weinstock M. 1985). The prenatally stressed female rats showed the same amount of latency to retrieve their pups as the control rats under normal conditions (Fride, E., Dan, Y., Gavish, M. and Weinstock M. 1985). However, when placed in aversive conditions of cold air through which they had to pass, 50% of prenatally stressed females failed to retrieve their pups as compared to 5% of the control group rats (Fride, E., Dan, Y., Gavish, M. and Weinstock M. 1985). Similarly, after being exposed to mild foot shocks, the food seeking behavior of the prenatally stressed female rats was disrupted as opposed to the control rats who showed no change in behavior (Fride, E., Dan, Y., Gavish, M. and Weinstock M. 1985). The experimental group mice were also slower to learn that the environment posed no threat to their survival as it was observed that the plasma CORT levels of the experimental group remained at an elevated rate despite eight exposures (Fride, E., Dan, Y., Gavish, M. and Weinstock M. 1985). Similar results were observed in another study where the prenatally stressed rats showed a lack of exploration in the elevated plus maze, constant elevation of plasma corticosterone after repeated exposure to an open field, dysregulation of the HPA axis in response to footshock and increased levels of freezing when induced with shock (Fride, E., and Weinstock M. 1988). Additionally, the prenatally stressed rats showed higher levels of corticotropin releasing hormone in the amygdala, which was abolished when they were injected with CRH antagonist (Cratty, M.S., Ward, He.E., Johnson, E.A. et al. 1995). The CRH antagonist also suppressed the anxiogenic behavior of failing to recognize and distinguish the environment as threatening and non-threatening, as previously observed in the experimental rats (Cratty, M.S., Ward, He.E., Johnson, E.A. et al. 1995). These studies demonstrate a postnatal effect on the anxiogenic behavior of prenatally stressed mice through the biological alterations that occurred in utero. Thus, prenatal stress has a long-lasting impact on the stress mechanisms of the offspring, which may be prolonged postnatally through other experiences.
Methods:
In this study, 50 Long Evans strain female rats were used. Mice were housed same sex, two per cage with a 12:12 light/dark cycle with food and water provided to them ad libitum.
12 of these rats were pregnant and part of the control group, 12 were pregnant and part of the experimental group, and 12 female rats inbred for high maternal care. The two remaining female rats were used in case of a problem with the experiment such as the premature death of a rat.
During the experiment, in order to induce stress to the 12 experimental pregnant rats, we followed the methods of Koehl and colleagues, and restrained the rats for 45 minutes, 3 times daily throughout their gestation period. To measure the CORT levels, we collected the samples of the CORT plasma levels of the rats of each group (experimental and control) throughout the pregnancy. There was no manipulation of high maternal care rats or control rats during this phase of the experiment.
Once the rats had completed their gestation period and given birth, we performed the elevated plus maze for the newborns from both the control and experimental group of rats. After this we placed the experimental rats with adult female high maternal care rats.
2 weeks postnatally, we perfused the neonatal animals and collected brain sample tissues to analyze the amygdala. The extraction of the amygdala was based on the experiment performed by Cratty and colleagues. The brains were removed, chilled and placed on 0.9% saline for 2 minutes. After this the brains were coronally sectioned in the cryostat for the amygdala at 40 μm. The tissue was then transferred to tubes containing 800 μl Kreb’s Ringer buffer. Tissues of CORT were extracted from the frozen tissues to analyze levels of CORT within the amygdala.
Hypothesized Results:
In order to analyze the results statistically we will use a 3-way ANOVA of treatment x trial x CORT level. Based on the 3-way ANOVA of treatment x trial x CORT level, there will be a significant difference in CORT plasma levels of control vs. experimental rats in the restraining phase with the experimental group having significantly higher levels of CORT as compared to the control group.
We predict that the experimental animals will also show significantly more anxiety-like behavior in the Elevated Plus Maze trials as compared to controls. This will be obvious in their behavior as the experimental rats will show less exploratory behavior by staying in the closed arms of the maze as opposed to the control group which will show more exploratory behavior as they will choose to explore the open arms significantly more.
After being exposed to high maternal care, however, we predict that the experimental rats will have significantly lower levels of CORT as compared to their CORT levels measured during the restraining phase of the experiment. Additionally, amygdala samples will show somewhat higher CORT levels for the experimental group as compared to the controls.
Discussion:
The results will show higher levels of anxiety like behavior and CORT levels in experimental mice as compared to the controls. We predict that this trend will be reduced through exposure to high maternal care rats. However, overall the experimental mice will still have higher levels of CORT even after exposure to high maternal care as compared to the controls.
These results will be in line with some of the previous literature found on similar rodent models. This shows that the subjects that are exposed to high levels of stress in utero have a much greater likelihood of developing anxiety like behavior postnatally. The biological mechanisms that underlie this, while still somewhat unclear, point to connections with the HPA axis and how this physiological stress response triggers the release of these stress hormones that are then catalysts for anxiety like behavior postnatally. However, the predicted decrease of CORT levels after exposure to maternal care shows that environmental stimuli have the ability to reduce neurological insults experienced in the womb through biological and psychological mechanisms that are still unclear.
Some caveats to this study, that can interfere with the results of this experiment could be due to the fact that this study focused on a lot of different variables and how they impact the neonatal animals. Additionally, the restraining phase of the experiment is quite harsh. This could possibly mean that some of the animals who are subjected to this much restraining daily may not be able to handle that much stress and have other unintended physiological/psychological effects. Lastly, the results may not be as clear or significant as we have predicted.
Future studies should look at whether longer periods of maternal care may significantly reduce or abolish the trend of anxiety like behavior. This will confirm and strengthen the idea that environmental stimuli have great influence on the reversal of some of the effects experienced prenatally. Additionally, future studies should manipulate external to determine the resilience of anxiety like behavior at different time periods during gestation. These findings may help elucidate the biological basis and origin of anxiety-like behavior and why it effects an individual’s ability to distinguish cues considered threatening vs. non-threatening.