Edited by: Nuno Sousa, University of Minho, Portugal
Reviewed by: Cesar Venero, National University of Distance Education, Spain; Osborne F. Almeida, University of Minho, Portugal
*Correspondence: Maria Toledo-Rodriguez, School of Biomedical Sciences, Medical School, Queen's Medical Centre, Nottingham NG7 2UH, UK. e-mail:
This is an open-access article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and other Frontiers conditions are complied with.
Adolescence is a period of major physical, hormonal, and psychological change. It is also characterized by a significant increase in the incidence of psychopathologies and this increase is gender-specific. Likewise, stress during adolescence is associated with the development of psychiatric disorders later in life. Previously, using a rat model of psychogenic stress (exposure to predator odor followed by placement on an elevated platform) during the pre-pubertal period (postnatal days 28–30), we reported sex-specific effects on auditory and contextual fear conditioning. Here, we study the short-term impact of psychogenic stress before and during puberty (postnatal days 28–42) on behavior (novelty seeking, risk taking, anxiety, and depression) and hypothalamus–pituitary–adrenocortical (HPA) axis activation during late adolescence (postnatal days 45–51). Peri-pubertal stress decreased anxiety-like behavior and increased risk taking and novelty seeking behaviors during late adolescence (measured with the elevated plus maze, open field and exposure to novel object tests and intake of chocopop pellets before or immediate after stress). Finally neither depressive-like behavior (measured at the forced-swim test) nor HPA response to stress (blood corticosterone and glucose) were affected by peri-pubertal stress. Nevertheless, when controlling for the basal anxiety of the mothers, animals exposed to peri-pubertal stress showed a significant decrease in corticosterone levels immediate after an acute stressor. The results from this study suggest that exposure to mild stressors during the peri-pubertal period induces a broad spectrum of behavioral changes in late adolescence, which may exacerbate the independence-building behaviors naturally happening during this transitional period (increase in curiosity, sensation-seeking, and risk-taking behaviors).
Adolescence is a critical developmental period characterized by change. Despite its gradual onset and offset there are major physical, hormonal, and behavioral differences between adolescence and childhood or adulthood. Adolescence is characterized by major developmental changes in the brain and the hypothalamic–pituitary–adrenal axis (HPA axis). The HPA axis matures before the brain pathways that regulate emotion, cognition, and learning (such as the prefrontal cortex, hippocampus, amygdala, and ventral striatum; Giedd et al.,
This major biological transition is hypothesized to render the adolescent more vulnerable to stress and the development of psychopathologies. Stressful experiences during childhood and adolescence have been associated with the development of psychiatric disorders later in life (Kessler and Magee,
In the rat, adolescence is considered to last from either postnatal days 21–59 (Tirelli et al.,
Type of stressor | ||
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Social | McCormick et al. ( |
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Physical | Tsoory and Richter-Levin ( |
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Pre-puberty | Avital and Richter-Levin ( |
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Puberty | McCormick et al. ( |
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Post-puberty | Laroche et al. ( |
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Entire adolescence | Kabbaj et al. ( |
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Behavior | Avital and Richter-Levin ( |
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Endocrine response | Barha et al. ( |
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Gene expression | Tsoory et al. ( |
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Neuronal and brain morphology | Isgor et al. ( |
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Cell proliferation in the dentate gyrus | Barha et al. ( |
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Drug sensitization | McCormick et al. ( |
|
Ito et al. ( |
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Toledo-Rodriguez and Sandi ( |
Until now most studies focused on the long-term effects of peri-adolescent stress. They investigated the impact on adult behavior, endocrine responses, gene expression, neuronal, and brain morphology, cell proliferation in the dentate gyrus and drug sensitization (Table
Subjects were the offspring of rats purchased from Charles River Laboratories, France. Fifteen male and 18 female Wistar Han rats from different liters were weaned at postnatal day 21. To avoid litter effects rats from different litters were mixed, placing equivalent numbers of animals from each litter in the stress and control groups. Rats were housed in same sex cages (three or four per cage) in standard plastic cages on a 12-h light-dark cycle (light on at 7:00 AM). Food and water were available
The stress protocol used was a longer version of the one previously developed in our laboratory (Toledo-Rodriguez and Sandi,
After the stress period all rats (stressed and control) were handled during 2 min daily for 2 days before testing their spontaneous locomotor activity and reactivity to novelty in an activity box (at 45 days of age). The activity box consisted in a rectangular arena (of 37 cm × 57 cm) divided in nine zones of identical size. The test was started by placing the animals in the center of the arena and consisted in two blocks of 10 and 5 min. During the first 10 min the animal's reactivity to a novel location was measured. Afterward a novel object (dark glass bottle) was placed at the center of the arena and the reactivity of the animal toward the object was measured. The locomotor activity was monitored by a video camera, mounted on the ceiling and a computerized tracking system (Ethovision 1.90, Noldus IT, Wageningen, The Netherlands) recorded the total distance moved, speed, percentage of time spent in each zone, and latency to enter the center. An observer (blind to the animal's experimental condition) scored the animal's sniffing and rearing onto the object. The floor of the arena and the object were washed after each test with 0.1% acetic acid solution to remove odors left by previous subjects. The test was performed simultaneously to all the animals in the cage by using three or four adjacent arenas.
Twenty-four hours after the activity box and reactivity to novelty tests, anxiety-like behavior of stressed and control rats was evaluated using the elevated plus maze (EPM) test. The EPM consisted of two opposing open arms (45 cm × 10 cm) and two closed arms (45 cm × 10 cm × 50 cm) that extended from a central platform (10 cm × 10 cm) elevated 65 cm above the floor. Rats were placed individually on the central platform facing a closed arm and were allowed to freely explore the maze for 5 min. The behavior of each rat was monitored using a video camera and the movement of the rats automatically registered and analyzed with a computerized tracking system (Ethovision 1.90, Noldus IT, The Netherlands). Entry into an arm was defined as entry of all four paws into the arm. Total distance moved, speed, time spent in the open and closed arms, number of times the animal entered each type of arm, latency before entering an open arm and number of defecations were measured. An observer (blind to the animal's experimental condition) scored the animal's grooming, stretching, and rearing behaviors as well as the exploration outside the maze (head-dipping). The floor of the EPM was washed after each testing with 0.1% acetic acid solution to remove odors left by previous subjects.
The parents of the experimental animals used in this study were also assessed for their anxiety-like behavior in the EPM test before breeding took place.
On postnatal days 45–49, animals were habituated to eat Chocopop flakes (Kellogg's, Switzerland) by feeding four flakes per animal in their homecages daily. Afterward two test of food intake were performed:
In all cases the number of flakes eaten by the animal were measured. The floor and walls of the arena were washed after each testing with 0.1% EtOH solution to remove odors left by previous subjects. The test was performed simultaneously to all the animals in the cage by using three or four adjacent arenas.
To evaluate depression-like behavior animals underwent forced-swim test as previously described (Toledo-Rodriguez and Sandi,
On postnatal day 51 rats were placed individually in cylinders (25 cm diameter) for 30 min. Immediately after, rats were decapitated and their trunk blood collected in heparinized tubes. Plasma corticosterone and glucose levels were measured using an enzyme immunoassay kit (Correlate-EIA from Assay Designs Inc., USA) and (RTU BioMerieux ref
All data from the adolescent animals was analyzed using two-way ANOVA. When the sex × treatment interaction was significant, Student's
First, we studied whether peri-pubertal stress affected the spontaneous locomotor activity of adolescent animals. Two days after the end of the stress period (i.e., 45 days of age) the basal locomotor activity and exploratory behavior of stressed and control animals were measured. Stressed animals showed significantly longer latency to reach the center of the arena [
Next we examined whether peri-pubertal stress influenced the reactivity to explore a novel object. Stress during adolescence had a significant effect on the exploratory behavior of the animals. Stressed animals showed significant lower latency to approach the object [
On postnatal day 46, animals underwent the EMP test to measure their anxiety-like levels. Two-way ANOVA revealed that stress during adolescence had a significant effect on anxiety-like behavior in the adolescent rat. Stressed animals: (a) spent more time in the open arms [
On postnatal day 50 peri-pubertal stressed and control animals underwent forced-swim test (training took place 24 h before). Exposure to peri-pubertal stress did not affect swimming behavior as revealed by two-way ANOVA performed on the percentage of time spent floating [treatment effect,
In this test, performed on postnatal day 50, each rat was allowed 2 min to eat 10 chocopop pellets. Statistical analysis showed significant treatment effect with stressed animals eating significantly more chocopop pellets [
On postnatal day 51, animals were exposed to a novel environment for 30 min and immediate afterward blood samples were taken to assess plasma corticosterone and glucose levels. Two-way ANOVA analysis revealed that while peri-pubertal stress did not have any effect on glucose levels [
Finally, we evaluated whether the behavior and reactivity to stress of the adolescent rats was influence by the parental anxiety-like traits. For this purpose, we repeated the previous analyses including the covariate “basal anxiety of either the mother of the father before breeding” (measured as the time spent in the open arms of the EPM; see
ANCOVA analysis revealed that the covariate “basal anxiety of the mothers before breeding” was significantly related only to the offspring's latency to approach the object [
The covariate “basal anxiety of the fathers before breeding” was marginally but not significantly related to the offspring's blood corticosterone levels [
The present study investigated the behavioral and hormonal effects of psychogenic stress (exposure to predator odor and placement on an elevated platform) during the peri-pubertal period in adolescent male and female rats. Our findings indicate that peri-pubertal stress reduces anxiety-like behaviors without affecting depressive-like behavior in late adolescence. Stressed animals exhibited increases in: (a) risk-taking behaviors (measured as time spent in the open arms of the EPM, time spent head-dipping, and number of entries to the EPM open arms) and (b) novelty seeking behaviors (measured as latency to reach a novel object, distance walked at the EPM, frequency, and time spent sniffing the object and number of chocopop pellets eaten under basal or stress conditions). Yet, stressed animals did not differ from controls in: (a) time spent floating during the forced-swim test or (b) blood corticosterone and glucose levels immediate after an acute stress.
At first glance our results may seem paradoxical when compared with previous studies reporting an increase of anxiety-like behaviors in rats exposed to stress during adolescence (Avital et al.,
The increase in risk-taking behaviors in animals that underwent stress during adolescence seems to be restricted to the adolescent period. In fact, many of the above cited studies reported that the decrease in anxiety-like behaviors following adolescent stress disappeared when animals were tested in adulthood, both for males (Peleg-Raibstein and Feldon,
What could be the implications of the increase in novelty seeking and risk-taking behaviors resulting from exposure to stress during adolescence? Early life stress has been hypothesized to lead to an increase in vulnerability to develop psychopathologies later in life (Kessler and Magee,
At this stage, it is not possible to indicate what is the predictive value of the stress-induced changes in behavioral reactivity for maladaptive behavior later in life. Further studies are required in order to determine how these alterations relate to behavioral responses in adulthood.
The use of males and female rats in our study enabled us to investigate whether the effects of peri-pubertal stress were sex specific. We found a significant interaction between treatment and sex factors in some parameters of the open field and novel object tests, but not in the standard variables from these tests (such as those related to visits to the center of the arena or to the exploration of the novel object). Interestingly, there was a significant sex difference in the behavior at the EPM with females showing increased risk-taking behaviors. So far there are only a handful of studies investigating the effects of stress during adolescence on behavior at the EPM in males and females. The results from these studies are not consistent. Exposure to chronic social stress during adolescence was shown to result in only females displaying higher risk-taking behaviors at the EPM, when tested in late adolescence (McCormick et al.,
In addition, we found that the basal anxiety of the mothers was significantly related to the offspring's blood corticosterone levels after acute stress. Future experiments should address the molecular and/or behavioral mechanisms underlying this link between maternal anxiety and the offspring's HPA reactivity after adolescent stress.
One should be cautious when comparing the results of the present study with human adolescence, since brain maturation in rodents and humans follows different developmental time patterns. For example, while the rodent hippocampus continues to develop well into adolescence (Meyer et al.,
It should be noted that a shortcoming in the present study is the fact that only one measure of the HPA axis and glucose responses to acute stress was performed, with basal levels not being included. Given that corticosterone (McCormick et al.,
Adolescence, and in particular the peri-pubertal period, is a critical developmental period when major changes take place in the brain (i.e., synapse pruning, fiber sprouting, and myelination; Giedd et al.,
Juvenile stress has been reported to delay the maturational changes in expression of GABAa receptor subunits in the amygdala (Jacobson-Pick et al.,
In conclusion, this set of experiments shows that exposure to stress during the peri-pubertal period results in an increase of novelty seeking and risk-taking behavior in adolescent male and female rats. These data suggest that peri-pubertal stress exacerbates the independence-building behavior characteristic of this transitional period. This behavioral change may reflect alterations in the plastic maturational changes undergone by brain regions regulating emotionality and stress responsiveness during this developmental stage.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We would like to thank Coralie Siegmund and Clara Rosetti for their invaluable experimental assistance, Dr. M. Isabel Cordero and Dr. Cristina Marquez for her help with the behavioral analyses and Prof. Charles Marsden for his useful comments on the manuscript. This work was supported by the Roche Research Foundation, and grants from the EU 7th Framework Program (MemStick), the Swiss National Science Foundation (310000-120791; Sinergia CRSIK0-122697 and CRSIK0-122691).