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There is a significant body of research that indicates exposure to adversity early in life can be a risk factor for dysregulation of homeostasis, including alterations
in neuroendocrine function and sleep. This experiment used the Rhesus Macaque, Macaca mulatta, as a model organism to examine the long-term effects of
perinatal maternal separation on hypothalamic-pituitary-adrenal (HPA) axis function and sleep, when the animals were adolescents. We examined the effects of
maternal separation on: (1) the acute stress response of the HPA axis, (2) the diurnal rhythm of basal HPA axis activity, and (3) another circadian system
bidirectionally related to HPA axis function: sleep/wake patters of activity. Our study used two groups of monkeys. The first group of control subjects did not
experience any maternal separation. The second group of experimental subjects experienced repeated, intermittent, maternal separations for varying periods
of time (either 0.5, 3 or 6 h) on thirty-six randomized occasions between the ages of three and six months old. In order to examine the effects of maternal
separation on the acute stress response, subjects from both groups were transported out of their respective home cages and brought to a novel room. The
handling procedures associated with the transportation of the subjects and the new room itself acted as stressors for the subjects. The subjects were brought
to the new room repeatedly for four weeks in order to habituate them to the experience. It was predicted that both groups would become habituated to the room
with time and their stress response, as measured by the increase in plasma cortisol and ACTH levels from baseline, would decrease with time. However, the
maternally separated subjects showed sensitization to experience as their stress response increased with repeated exposure to the room. The control animals
showed no significant change in their stress response, suggesting that we need to extend the habituation period. In the second part of the study we examined
the effects of maternal separation on basal HPA axis activity, by measuring the diurnal rhythm of cortisol release in saliva samples, in parallel to the circadian
rhythms of sleep/wake activity. It was predicted that the maternally separated subjects would exhibit both a flattened circadian rhythm and as an abnormal sleep
pattern, as compared to the control subjects. Although we found that maternally separated subjects had slightly sharper cortisol rhythm, we detected sleep
disturbances in this group, including a shorter sleep period, more activity during the night, and earlier wake time.
Early life chronic stress can lead to host psychiatric and physiological complications. In particular, early stress can permanently alter neuroendocrine
development, function, and regulation. This project used Rhesus Macaques, Macaca mulatta, to study the effects of maternal separation on the function
of the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis is of particular interest because it plays a key role in maintaining homeostasis. The HPA
axis is activated during stressful situations, causing the release of glucocorticoids, highly catabolic steroid hormones that provoke physiological changes
such as increased blood glucose levels, increased blood pressure and heart rate, decreased reproductive function, and inhibition of the inflammatory response.
These are changes that happen in parallel to the activation of the sympathetic system, causing the reaction commonly referred to as the “fight or flight response”
to stress and can be elicited by any perceived or real threat. The threat is processed by different regions of the brain and is in turn integrated by the neurons of
the paraventricular nucleus (PVN) of the hypothalamus. These neurons in turn release the 41-amino-acid neuropeptide corticotropin releasing hormone (CRH)
into the hypophysial portal circulation. The release of CRH activates the synthesis of precursor protein proopiomelanocortin (POMC) in the anterior pituitary, as
well as the release of a peptide cleaved from POMC, adrenocorticotrophic hormone (ACTH), into the blood stream. ACTH in turn stimulates the release of
glucocorticoids (for example, cortisol in humans and macaques) from the adrenal cortex to the blood stream, where it causes a number of physiological changes
throughout the body. This stress-response is activated over the normal, circadian rhythm of cortisol release. In the normal diurnal rhythm of primates, cortisol levels
rise in the early morning and peak around waking time, then lower for the rest of the day, to reach a trough during sleep, in diurnal species (the opposite is true in
nocturnal animals, such as the rat). It is believed that the rise of cortisol in the morning is directly related to daytime wakefulness and there is strong evidence for
the mutual regulation between the HPA axis activity and sleep cycle of individuals. Early life chronic stress, provoked by the repetitive breaking of the mother-infant
bond, can cause problems in both 1) the stress response of HPA axis and 2) in the circadian rhythm of the axis.
In order to produce early chronic stress by repetitive disruption of the mother-infant bond, experimental subjects were separated from their mothers beginning at 3
months postpartum (infancy). In the experiment, the mothers were removed from the social groups for variable lengths of time – either for 0.5, 3, or 6 hr. A control
group did not have any maternal separation. The experimental animals experienced a total of 36 separations over a 90-day period (from 3-6 months of age). Both
the duration and timing of the separations were randomized.
Part 1: Response to acute stressors
Our goal was to use 3 maternally-separated monkeys and 2 controls that are currently 5 years old to expose them to the Social Intruder Stress Test, which has
been demonstrated to evoke physiological and behavioral responses in macaques and other nonhuman primates. Before that, though, the animals needed to be
trained for awake blood sampling and habituated to the room and procedures.
Training, Habituation and Blood Sample Collection: First, the animals were trained to voluntarily present their legs for awake blood sampling from their saphenous
vein, via positive reinforcement. Once they were successfully trained, each subject was brought trough a trial habituation process for 4 weeks in order to familiarize
them with the pre-testing procedures (boxing out of their homecage, handling, and novel room/cage). The subject was transported to a testing room and placed in
a testing cage, and a baseline 3-ml blood sample (0 min) was taken from the saphenous vein. The subjects were then left alone in the novel room for 30 min.
Another 3-ml blood sample was taken after this 30 min period to test for HPA axis stress responses.
Blood Processing and Assays: Once collected, blood samples were placed in pre-chilled polypropylene tubes with EDTA and centrifuged at 1000 x g for 10 min
at 4°C. Plasma was then aliquoted and stored at -80°C until assayed. Plasma cortisol concentrations was assayed by radioimmunoassay (R.I.A.) and ACTH levels
by radioimmunometric assay (I.R.M.A.), using commercially available kits (cortisol: Diagnostic Systems Laboratories, Webster, TX; ACTH: DiaSorin Corporation,
Stillwater, MN).
Part 2: Diurnal Cortisol Rhythms and Activity-Based Sleep Monitoring (Actigraphy)
Saliva Sample Collection: In order to examine the effect of maternal separation on the diurnal rhythm of basal HPA axis activity, a pilot study using 2 maternally
-separated monkeys and 5 control monkey was performed. Saliva samples were taken at 6:45 AM, 7:15 AM, and 7:45 AM in order to investigate cortisol
awakening responses, and again at 2 PM to follow the daytime decrease in cortisol levels. The subjects had been trained to chew on dental cotton that had been
soaked in Kool Aid solution and then dried, following previously published protocols for children and monkeys.
Saliva Processing and Assays: The cotton was then placed in salivette commercial tubes, immediately stored on ice and subsequently centrifuged at 3000 rpm for
15 min at 4 °C. The extracted saliva samples were then aliquoted and stored at -80°C until assayed. The salivary cortisol was also assayed by R.I.A. (DiaSorin
Corporation, Stillwater, MN).
Activity-Based Sleep Monitoring (Actigraphy): Sleep data was obtained using the actigraphic monitors (“Actiwatch,” Mini-Mitter, Sunriver, OR) to correlate with
daytime rhythms of salivary cortisol. The actigraphic monitors were used to collect data over 3 days with a sampling epoch of .25 min. The monitors were housed
in specially designed primate collars. The sleep data was analyzed using the Sleepwatch Software Program (Mini-Mitter) that applies an algorithm to extract the
following information: sleep onset, sleep offset, average sleep time, sleep efficiency, number of sleep and wake bouts and, the amount of time moving during sleep.
The monkeys were kept on a 12:12-h light/dark cycle, with the lights in the housing room going on at 7:00 and going off at 19:00. Actigraphy has proved to be an
accurate determinate of sleep patterns in humans and it has been shown to have high correlations with PSG recordings. It has also been used to perform studies
of sleep/wake activity in non-human primates.
Statistical Analysis: Using two way repeated measures ANOVA, the difference in the mean values among the two groups was not statistically significant
(p = 0.581). We did not find a statistically significant effect across the 4 weeks, either (p = 0.509). The group cortisol levels did not depend on the week.
There was not a statistically significant interaction between Group and week (p = 0.715)
Using Two Way Repeated Measures ANOVA, the difference in the mean values between the Control and Maternal Separation groups was not statistically
significant (p = 0.863). The difference in the mean values among the different sample times was statistically significant (p = 0.047). AM salivary cortisol levels
were higher than at 2 PM.
Part 1: HPA response to acute stress
In response to acute stressors, there was no significant change in HPA axis response with repeated habituation, from week 1-4 (p=.715). Nor was there a
significant difference in HPA axis response between the two groups (p= 0.581). Thus, it can be concluded that neither the control nor the experimental animals
are habituating to the testing procedure. In fact, the maternally separated subjects may be showing signs of sensitization to the testing procedures because their
HPA axis response becomes more pronounced with time, yet this result was not statistically significant.
Part 2: Diurnal Cortisol Rhythms and Activity-Based Sleep Monitoring (Actigraphy)
With respect to the diurnal cortisol levels, while there was a significant decline in salivary cortisol levels from morning to afternoon, (p=.047), there was not a
significant difference between the maternally separated and control subjects (p=.836). However, visual inspection of the data suggests that maternally separated
monkeys showed a slightly steeper decline in cortisol rhythm from 6:45 AM to 2:00 PM).
On the other hand, preliminary actigraphy data indicates that maternally separated subjects exhibit sleep deficits, such as less sleep time per night, earlier wake
time and they also seem to spend a greater percentage of their sleep time moving. Thus, the sleep alterations in the maternal separation indicates the need to
look closer into alterations in circadian HPA axis.
There is a significant body of research that indicates exposure to adversity early in life can be lead to many problems later in life. This experiment used the
Rhesus Macaque, a monkey from India, as a model organism to examine the long-term effects of perinatal maternal separation on endocrine function and sleep,
when the animals were adolescents. We examined the effects of maternal separation on: (1) the acute stress response in terms of the release of the horomone
cortisol, (2) the daily wake-sleep cycle of cortisol activity, and (3) the sleep/wake cycle. Our study used two groups of monkeys. The first group of control subjects
did not experience any maternal separation. The second group of experimental subjects experienced maternal separations for varying periods of time (either 0.5,
3 or 6 h) on thirty-six randomized occasions between the ages of three and six months old. In order to examine the effects of maternal separation stress
response, subjects from both groups were transported out of their respective home cages and brought to a new room. The transportation of the subjects
and the new room itself acted as stressors to the subjects. The subjects were brought to the new room repeatedly for four weeks in order to habituate them
to the experience. It was predicted that both groups would become used to the room with time and that their stress response, as measured by the increase
in plasma cortisol and ACTH levels from baseline, would decrease with time. However, the maternally separated subjects became more stressed in response
. The control animals showed no significant change in their stress response, suggesting that we need to extend the habituation period. In the second part of
the study we examined the effects of maternal separation on daily, normal cortisol levels by measuring the amount of cortisol in saliva samples, in parallel to
the rhythms of sleep/wake activity. It was predicted that the maternally separated subjects would exhibit both a flattened circadian rhythm and as an abnormal
sleep pattern, as compared to the control subjects. Although we found that maternally separated subjects had slightly sharper cortisol rhythm, we detected
sleep disturbances in this group, including a shorter sleep period, more activity during the night, and earlier wake time.
Behavioral observation, radioimmunoassays, actigraphy
Rhesus Macaque, maternal separation, stress, HPA axis, cortisol
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