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. Author manuscript; available in PMC: 2020 Sep 1.
Published in final edited form as: Brain Res. 2019 May 15;1718:242–251. doi: 10.1016/j.brainres.2019.05.019

Glucocorticoid receptor expression in the stress-limbic circuitry is differentially affected by prenatal alcohol exposure and adolescent stress

Charlis Raineki 1,*, Erin J Morgan 1, Linda Ellis 1, Joanne Weinberg 1
PMCID: PMC6579044  NIHMSID: NIHMS1530520  PMID: 31102593

Abstract

The dense expression of glucocorticoid receptors (GR) within the amygdala, medial prefrontal cortex (mPFC) and paraventricular nucleus of hypothalamus (PVN) mediates many aspect of emotional and stress regulation. Importantly, both prenatal alcohol exposure (PAE) and adolescent stress are known to induce emotional and stress dysregulation. Little is known, however, about how PAE and/or adolescent stress may alter the expression of GR in the amygdala, mPFC, and PVN. To fill this gap, we exposed PAE and control adolescent male and female rats to chronic mild stress (CMS) and assessed GR mRNA expression in the amygdala, mPFC, and PVN immediately following stress or in adulthood. We found that the effects of PAE on GR expression were more prevalent in the amygdala, while effects of adolescent stress on GR expression were more prevalent in the mPFC. Moreover, PAE effects in the amygdala were more pronounced during adolescence and adolescent stress effects in the mPFC were more pronounced in adulthood. GR expression in the PVN was affected by both PAE and adolescent stress. Finally, PAE and/or adolescent stress effects were distinct between males and females. Together, these results suggest that PAE and adolescent CMS induce dynamic alterations in GR expression in the amygdala, mPFC, and PVN, which manifest differently depending on the brain area, age, and sex of the animal. Additionally, these data indicate that PAE-induced hyperresponsiveness to stress and increased vulnerability to mental health problems may be mediated by different neural mechanisms depending on the sex and age of the animal.

Keywords: prenatal alcohol exposure, adolescent stress, glucocorticoid receptor, amygdala, medial prefrontal cortex, rat

1. Introduction

Clinical and preclinical studies indicate that the bidirectional interconnections between the amygdala and medial prefrontal cortex (mPFC) support fundamental aspects of emotional and stress regulation as well as cognitive processing (Cardinal et al., 2002; Gee, 2016; Myers et al., 2012; Tottenham, 2015; Yan et al., 2017; Yizhar and Klavir, 2018). Despite its critical role, the amygdala-PFC circuitry does not structurally and functionally mature until late adolescence/young adulthood (Cressman et al., 2010; Cunningham et al., 2002; Gabard-Durnam et al., 2014; Gee et al., 2013; Lebel et al., 2012; Perlman and Pelphrey, 2011; Swartz et al., 2014; Verwer et al., 1996; Vink et al., 2014). Importantly, rodent studies indicate that amygdala projections to the mPFC emerge earlier than mPFC projections to the amygdala (Bouwmeester et al., 2002a,b), presumably due to the earlier development of the amygdala relative to the mPFC. Indeed, amygdala development peaks during the first two postnatal weeks (Bayer, 1980; Berdel et al., 1997; Berdel and Morys, 2000; Bouwmeester et al., 2002a,b; Cunningham et al., 2002; Ryan et al., 2016; Thompson et al., 2008), while mPFC development is more protracted and extends until late adolescence/young adulthood (van Eden and Uylings, 1985a,b). Consequently, any insults that disturb the typical maturational process of the amygdala and/or mPFC may have negative consequences for the establishment of appropriate connections between these two areas that could lead to emotional and stress dysregulation as well as altered cognitive processing.

The paraventricular nucleus of the hypothalamus (PVN) is a critical part of the stress limbic circuitry, and works closely in conjunction with the amygdala and mPFC in emotional and stress regulation. More specifically, the medial parvocellular dorsal division of the PVN (mpdPVN) contains a set of neurons that integrates all direct and indirect inputs from the amygdala and mPFC (Myers et al., 2012; Ulrich-Lai and Herman, 2009). In rodents, PVN development is well underway during the second half of gestation, with the PVN achieving secretory capability by embryonic day18 (Karim and Sloper, 1980; Shimada and Nakamura, 1973). Similar to the amygdala and mPFC, any insults that disturb the typical maturational process of the PVN could disrupt this critical stress-limbic circuitry and lead to emotional and stress dysregulation.

Alcohol is a teratogen that disrupts fetal brain development and leads to a wide range of cognitive, neurobehavioral, and physiological deficits (Drew and Kane, 2014; Hellemans et al., 2010a; Riley et al., 2011; Valenzuela et al., 2012; Weinberg et al., 2008). The amygdala, the mPFC, and the PVN are among the many brain regions that are negatively affected by prenatal alcohol exposure (PAE). Children exposed to alcohol in utero show long-lasting structural and functional alterations in the amygdala (Donald et al., 2016; Nardelli et al., 2011; Zhou et al., 2018) and PFC (Fryer et al., 2007; Gross et al., 2018; Malisza et al., 2005; O’Hare et al., 2009; Sowell et al., 2007; Wozniak et al., 2013; Zhou et al., 2011). Animal models have extended these findings, demonstrating that PAE induces deleterious structural and functional effects in the amygdala, mPFC, and PVN (Burke et al., 2009; Cullen et al., 2013; Kozanian et al., 2018; Lam et al., 2018a,b; Raineki et al., 2014, 2016; Weinberg et al., 2008; Wilcoxon et al., 2005). Not surprisingly, functions mediated by these areas are impaired in individuals that are exposed to alcohol during pregnancy. Particularly, individuals exposed to alcohol prenatally are hyperresponsive to wide range of stressful stimuli (Jacobson et al., 1999; Lee and Rivier, 1996; Weinberg et al., 2008), and many go on to develop mental health problems including anxiety, depression, and substance use disorders at a rate disproportionate to that of the general population (Famy et al., 1998; O’Connor et al., 2002; Pei et al., 2011). Importantly, animal models have demonstrated that stress exposure can modulate the link between PAE-related alterations in the brain and increased depressive-and anxiety-like behaviors (Cullen et al., 2013; Hellemans et al., 2008, 2010a,b; Lam et al., 2018a,b; Raineki et al., 2014, 2016; Wilcoxon et al., 2005).

Similar to PAE, exposure to stressors during critical developmental periods can disturb the typical maturational process of the amygdala, mPFC and PVN (Fujioka et al., 1999; Tottenham and Galván, 2016; Wulsin et al., 2016). This is particularly relevant for individuals exposed to alcohol during gestation as, besides being hyperresponsive to stress, they are, in general, at a higher risk of exposure to stressful environments throughout the lifespan (O’Connor and Paley, 2006; Streissguth et al., 2004). The negative effects of stress on brain development are heightened during adolescence, as this period is characterized by higher activity of the stress systems in response to both acute and chronic stressors (Doremus-Fitzwater et al., 2009; Hollis et al., 2013; McCormick and Mathews, 2010; Romeo et al., 2004, 2006; Tottenham and Galván, 2016). Studies in both the clinical and preclinical literature provide evidence that exposure to stress during adolescence can result in structural and functional changes in the amygdala, mPFC, and PVN (Márquez et al., 2013; Raineki et al., 2016; Romeo, 2017; Swartz et al., 2015; Wulsin et al., 2016), which may exacerbate the increased vulnerability to mental health problems observed during adolescence (Andersen and Teicher, 2008; Costello et al., 2002; Paus et al., 2008).

Consistent with the critical roles of the amygdala, mPFC, and PVN in regulating the stress response, there is dense expression of the glucocorticoid receptors (GR) in these areas (Cintra et al., 1994; Honkaniemi et al., 1992; Morimoto et al., 1996; Wang et al., 2014). Notably, both PAE and adolescent stress alter GR expression. For example, male and female PAE rats show decreased amygdala GR expression in adulthood (Wilcoxon et al., 2005). In adolescent mice, PAE reduces levels of GR in the nuclear – but not cytosolic – fraction of mPFC neurons (Allan et al., 2014). Our data indicate that PAE reduces GR expression in the infralimbic region (a subregion of the mPFC) of PAE rats (Lam et al., 2018a). The effects of PAE on GR expression in the PVN have not been specifically assessed. Furthermore, analyses of PAE effects on GR in the hypothalamus are not conclusive, as a previous study indicated that PAE increases GR levels in male, but not female rats (Redei et al., 1993), while other studies have indicated no effect of PAE on hypothalamic GR (Halasz et al., 1993; Kim et al., 1999b).

Studies evaluating the effects of adolescent chronic stress on GR expression have focused their attention primarily on the hippocampus (Isgor et al., 2004; Raineki et al., 2018; Sterlemann et al., 2008). The few studies that have assessed amygdala GR expression following adolescent chronic stress indicate that the effects are not straightforward, as amygdala GR expression is reduced in the short term (Xu et al., 2017), but increased in the long term (Papilloud et al., 2018). In the PVN, the effects of adolescent chronic stress have only been investigated in female rats and the results indicate that stress reduced GR mRNA expression in the PVN of adult females (Wulsin et al., 2016).

To fill this gap in the literature, here we evaluate the short-and long-term effects of adolescent stress on GR mRNA expression in the amygdala, mPFC and PVN of control and PAE animals. We used a well-established rat model of PAE in which pregnant rats received either liquid ethanol diet or control diet throughout pregnancy. After weaning, male and female PAE and control animals were exposed either to adolescent chronic mild stress (CMS; PN31–41 for females, PN 37–47 for males) or left undisturbed (non-CMS). Half the animals were tested in the open field and forced swim test (FST) immediately following CMS exposure (short-term effects of CMS), and the remaining animals were tested on these same tasks in adulthood (long-term effects of CMS). Brains were collected 30-min after the FST and processed for GR mRNA by in situ hybridization. Behavioral data were published previously (Raineki et al., 2016). Here, we assessed GR mRNA expression in the amygdala (basolateral, central, medial, and cortical nuclei), the mPFC [anterior cingulate (ACC), prelimbic (PrL), and infralimbic (IL) cortices], and the mpdPVN (Figure 1). As structural and functional maturation of the amygdala occurs earlier than the mPFC, we hypothesize that PAE will have more marked effects on amygdala GR expression while adolescent stress will have more marked effects on mPFC GR expression. Moreover, because PVN development is ongoing during the period of alcohol exposure and because the PVN is an integral part of the stress response, we hypothesize that both PAE and adolescent stress will affect GR expression in the PVN.

Figure 1.

Figure 1.

Representative images of GR mRNA in-situ hybridization in the (A) mPFC, (B) mpdPVN, and (C) amygdala.

2. Results

2.1. Amygdala

In adolescent males, PAE increased GR mRNA expression in the central amygdala independently of CMS exposure [Figure 2E; significant main effect of prenatal treatment for central amygdala (F(1,28)=4.31, p=0.047)]. However, neither PAE nor CMS affected GR mRNA expression in the other amygdala nuclei (Figure 2A,I,M). In adolescent females, PAE decreased GR mRNA expression in the basolateral and medial amygdala independently of CMS exposure [Figure 2B,J; significant main effect of prenatal treatment for basolateral (F(1,27)=8.74, p=0.006) and medial (F(1,28)=4.34, p=0.046) amygdala nuclei]. Moreover, CMS increased GR mRNA expression in the central amygdala of control but not PAE females. [Figure 2F; significant interaction between prenatal treatment and CMS for central amygdala (F(1,28)=5.44, p=0.027)]. Neither PAE nor CMS affected GR mRNA expression in the cortical amygdala of adolescent females (Figure 2N).

Figure 2.

Figure 2.

Short-and long-term effects of adolescent CMS on amygdala GR mRNA expression in control and PAE rats. Bars represent the mean ± SEM (mean gray value) of GR mRNA expression in the basolateral (A-D), central (E-H), medial (I-L), and cortical (M-P) amygdala nuclei. † indicates a significant main effect of prenatal treatment, where all PAE animals are different from control animals; * indicates that control CMS is different from all other groups; for C, # indicates that PAE non-CMS is different from control non-CMS based on a priori comparisons; for G and O, # indicates that PAE non-CMS is different from PAE CMS based on a priori comparisons (n = 6–10 for all groups).

In adulthood, PAE non-CMS males showed increased GR mRNA expression in the basolateral amygdala compared to their control counterparts [Figure 2C; a priori analysis for basolateral amygdala comparing PAE non-CMS to control non-CMS (p=0.03)]. Moreover, CMS decreased GR mRNA expression in the central and cortical amygdala nuclei of PAE males, while no changes were observed in the medial amygdala [Figure 2G,K,O; a priori analysis for central (p=0.036) and cortical (p=0.01) amygdala nuclei comparing PAE non-CMS to PAE CMS]. In adult females, neither PAE nor adolescent CMS affected GR mRNA expression in any of the amygdala nuclei analyzed (Figure 2D,H,L,P).

2.2. Medial Prefrontal Cortex

In adolescent males, PAE decreased GR mRNA expression in the IL independently of CMS exposure [Figure 3I; significant main effect of prenatal treatment for IL (F(1,28)=9.92, p=0.002)]. By contrast, CMS increased GR mRNA expression in the PrL of control but not PAE males, while no changes were observed in the ACC [Figure 3A,E; significant interaction between prenatal treatment and CMS for PrL (F(1,28)=4.97, p=0.033)]. In adolescent females, neither PAE nor CMS affected GR mRNA expression in any mPFC subregion (Figure 3B,F,J).

Figure 3.

Figure 3.

Short-and long-term effects of adolescent CMS on mPFC GR mRNA expression in control and PAE rats. Bars represent the mean ± SEM (mean gray value) of GR mRNA expression in the anterior cingulate (A-D), prelimbic (E-H), and infralimbic (I-L) cortices. † indicates a significant main effect of prenatal treatment, where all PAE animals are different from control animals; § indicates a significant main effect of CMS exposure, where all animals exposed to CMS are different from animals not exposed to CMS; * indicates that control CMS is different from all other groups; for H, # indicates that control CMS and PAE non-CMS are different from control non-CMS based on a priori comparisons; for L, # indicates that PAE non-CMS is different from control non-CMS based on a priori comparisons; for H, ^ indicates that PAE CMS is different from PAE non-CMS based on a priori comparisons (n = 6–10 for all groups).

In adult males, adolescent CMS increased GR mRNA expression in all mPFC subregions independently of prenatal treatment [Figure 3C,G,K; significant main effect of CMS for ACC (F(1,30)=7.09, p=0.01), PrL (F(1,30)=7.17, p=0.01), and IL (F(1,30)=4.87, p=0.035)]. In adult females, PAE non-CMS animals showed increased GR mRNA expression in the PrL and IL compared to their control counterparts [Figure 3H,L; a priori analysis for PrL (p=0.05) and IL (p=0.042) comparing PAE non-CMS to control non-CMS]. Moreover, adolescent CMS decreased GR mRNA expression only in the IL of PAE females [Figure 3L; a priori analysis for IL comparing PAE non-CMS to PAE CMS (p=0.05)]. Conversely, adolescent CMS increased GR mRNA expression only in the PrL of control females [Figure 3H; a priori analysis for PrL comparing control non-CMS to control CMS (p=0.046)]. Neither PAE nor CMS affected GR mRNA expression in the ACC of adult females (Figure 3D).

2.3. Paraventricular Nucleus of Hypothalamus

In adolescent males and females and adult males, neither PAE nor adolescent CMS affected GR mRNA expression in the mpdPVN (Figure 4AC). However, in adult females, PAE decreased GR mRNA expression in the mpdPVN independently of CMS exposure. Moreover, adolescent CMS decreased GR mRNA expression in the mpdPVN independently of prenatal treatment [Figure 4D; significant main effects of prenatal treatment (F(1,27)=4.00, p=0.05) and CMS (F(1,27)=7.47, p=0.01) for mpdPVN].

Figure 4.

Figure 4.

Short-and long-term effects of adolescent CMS on mpdPVN GR mRNA expression in control and PAE rats. Bars represent the mean ± SEM (mean integrated density) of GR mRNA expression in mpdPVN. † indicates a significant main effect of prenatal treatment, where all PAE animals are different from control animals; § indicates a significant main effect of CMS exposure, where all animals exposed to CMS are different from animals not exposed to CMS (n = 5–10 for all groups).

3. Discussion

Our data support our hypothesis and demonstrate that the amygdala is more sensitive to the effects of PAE, the mPFC is more sensitive to the effects of adolescent stress, and the PVN is sensitive to both PAE and adolescent stress. Interestingly, PAE effects on GR mRNA expression in the amygdala were more prominent during adolescence, as PAE females showed reduced GR expression in the basolateral and medial amygdala nuclei while PAE males showed increased GR expression in the central amygdala nuclei during adolescence but not in adulthood. The one PAE effect in adulthood was an increase in GR expression in the basolateral amygdala nuclei. In contrast to the PAE effects in the amygdala, the effects of adolescent stress on the mPFC were more prominent in adulthood. Adolescent stress increased GR mRNA expression in all mPFC subregions of adult males independently of prenatal treatment, and increased GR expression in the PrL of adult females. The only short-term effect of adolescent stress was an increase in GR expression in the PrL of adolescent males. Besides confirming our hypothesis of differential vulnerability of the amygdala and mPFC to PAE and adolescent stress, the current data also revealed that PAE effects on the amygdala are more pronounced during adolescence and that adolescent stress effects on the mPFC are more pronounced in adulthood. Finally, we found that both PAE and adolescent stress affected GR mRNA expression in the PVN. PAE decreased GR expression in the mdpPVN of adult females independent of stress while adolescent stress decreased GR expression in the mdpPVN of adult females independent of prenatal treatment.

The dense expression of GR within the amygdala, mPFC, and PVN is essential for emotional and stress regulation (Cardinal et al., 2002; Cintra et al., 1994; Gee, 2016; Honkaniemi et al., 1992; Morimoto et al., 1996; Tottenham, 2015; Wang et al., 2014; Yan et a., 2017). To this end, the observed alterations in GR expression following PAE and/or adolescent stress could be associated with the altered stress response and increased vulnerability to emotional dysregulation observed in these animals (Cullen et al., 2013; Hellemans et al., 2008, 2010a,b; Lam et al., 2018a,b; Raineki et al., 2016; Rouzer et al., 2017; Wieczorek et al., 2015; Wilcoxon et al., 2005). Of note, the current data evaluate the effect of PAE and adolescent stress on GR mRNA expression in response to an acute stressor – exposure to the FST. It has been shown that while acute exposure to forced swim stress in adulthood can alter the expression of GR mRNA, effects are dependent on sex and area of the brain being analyzed. Specifically, forced swim stress increases GR mRNA in the hypothalamus and mPFC of males; however, in females GR mRNA does not change in the hypothalamus and decreases in the mPFC (Karandrea et al., 2002).

3.1. Amygdala GR mRNA expression during adolescence is modulated by PAE

Our ontogenetic approach revealed that PAE effects on GR mRNA expression are more prominent in the amygdala and perhaps more transient, as differential GR expression is apparent during adolescence but declines by adulthood. We have previously demonstrated a similar transient pattern of PAE effects on stress-related receptor expression in the hippocampus (Raineki et al., 2018). Indeed, PAE resulted in widespread alterations in GR, mineralocorticoid (MR) and type 1 CRH (CRHR1) receptor mRNA expression in the dorsal and/or ventral hippocampus during adolescence but not in adulthood (Raineki et al., 2018). Altered expression of these stress-related receptors in key areas regulating stress and emotion during adolescence suggests that this developmental period may represent a possible window of vulnerability for individuals prenatally exposed to alcohol. Assessments of PAE-induced behavioral alterations in several previous studies by our group and others support this notion. Our evaluation of depressive-like behavior across the lifespan in PAE animals indicates that PAE males exhibit depressive-like behavior in the FST in adolescence, but not in adulthood (Lam et al., 2018b; Raineki et al., 2016). Additionally, moderate and acute alcohol exposure on gestational day 12 results in anxiety-like behaviors during adolescence but not in adulthood (Rouzer et al., 2017). However, PAE effects on neurobehavioral development are not as straightforward: our previous analysis of anxiety-like behavior in the open field indicates that adolescent PAE males and females are hyperactive and spend more time in the center of the field (Raineki et al., 2016). As rodents typically stay primarily in the periphery and avoid the center (Gould et al., 2009), this behavior in adolescent PAE animals represents an inappropriate behavioral response. However, classical features of anxiety-like behavior were observed in adult PAE females, as they spent less time in the center of the open field compared to controls (Raineki et al., 2016).

The present data also demonstrate that GR mRNA expression in the amygdala and mPFC of male and female rats was differentially affected by PAE. PAE reduced GR expression in the basolateral and medial amygdala of adolescent females and increased GR expression in the PrL and IL subregions of adult females. Conversely, PAE increased GR expression in the central amygdala and decreased GR expression in the IL of adolescent males, but increased GR expression in the basolateral amygdala of adult males. These differential PAE-related effects on GR expression in the amygdala-mPFC circuit between males and females suggest that the altered stress response and increased vulnerability to emotional dysregulation may be mediated by different underlying mechanisms in males and females (Cullen et al., 2013; Hellemans et al., 2008, 2010a,b; Lam et al., 2018a,b; Raineki et al., 2016; Rouzer et al., 2017; Wieczorek et al., 2015; Wilcoxon et al., 2005).

Decreases in amygdala GR induced by early-life adversity have been shown to mediate the reduction in anxiety-like behaviors and overall increase in risk-taking behavior (Arnett et al., 2015). This adversity-related behavioral profile is similar to what we previously observed in PAE animals during early adolescence (Raineki et al., 2016). Adolescent PAE animals were hyperactive and spent more time in the center of the open field, a behavioral profile that might reflect a pathological increase in novelty-seeking and risk-taking behaviors. Accordingly, we suggest that, similar to early-life adversity (Arnett et al., 2015), the reduction in GR mRNA expression observed in the basolateral and medial amygdala nuclei of PAE adolescent females may mediate their increased risk-taking behaviors (Raineki et al., 2016).

The clinical and preclinical literature have consistently provided evidence demonstrating that PAE individuals exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness to a wide range of stressful stimuli (Jacobson et al., 1999; Lee and Rivier, 1996; Nelson et al., 1986; Taylor et al., 1982; Weinberg et al., 2008). Activation of GR in the amygdala, including the central nuclei, has been implicated in mediating the positive feedback loop that potentiates the activity of the HPA axis (Myers et al., 2012). Our data indicate that PAE increased GR mRNA expression in the central amygdala of adolescent males, which potentially contributes to the stress hyperresponsivity observed following PAE.

Similar to the amygdala, activation of GR in the IL subregion of the mPFC stimulates the HPA axis (Myers et al., 2012). Specifically, projections from the IL target the central nucleus of the amygdala (Hurley et al., 1991; Vertes 2004), and this pathway appears to transmit glucocorticoid feedforward information that activates the HPA axis. Nevertheless, our data indicate that PAE reduced GR expression in the IL of adolescent males, a paradoxical effect that suggests either that IL GR during adolescence is not a critical component mediating PAE-related stress hyperresponsivity or that the reduction may be a compensatory response to counterbalance stress hyperresponsivity.

3.2. mPFC GR mRNA expression in adulthood is modulated by adolescent stress

Our data demonstrate that the effects adolescent stress on GR mRNA expression are more prominent in the mPFC and mdpPVN than the amygdala, and that these effects are observed primarily in adulthood. Taken together, these results suggest a possible incubation period for some of the effects of adolescent stress on GR expression. This interpretation is supported by previous work showing that the effects of adolescent chronic stress on the neurocircuitry that underlies stress and emotional regulation can manifest differently depending on the timing of assessment. Indeed, some effects are only observed immediately after stress, some effects only manifest long after the termination of stress, and some effects are enduring and can be observed any time following stress exposure (Bourke and Neigh, 2011; Leussis and Andersen, 2008; Leussis et al., 2008; Raineki et al., 2016, 2018; Romeo, 2017; Tsoory et al., 2008).

The few studies that have evaluated how adolescent stress may affect the structure of the mPFC found that adolescent stress induces dendritic atrophy in adolescent male and female rats (Eiland et al., 2012). Moreover, examination of synaptic plasticity markers, such as spinophilin and synaptophysin reveals that adolescent stress produces persistent decreases in synaptic plasticity in several subregions of the mPFC (Leussis and Andersen, 2008; Leussis et al., 2008). More is known about how adolescent stress affects the long-term function of the mPFC. It has been shown that adolescent stress does not change basal or stress-induced (resident-intruder test) neural activity of the mPFC in adulthood (Márquez et al., 2013). Despite having no effect on neural activity, exposure to adolescent stress promoted long-term alterations in the mPFC serotonergic and GABAergic systems (Márquez et al., 2013; Tzanoulinou et al., 2016). The current data extend these findings by demonstrating that exposure to adolescent stress alters glucocorticoid function in the mPFC. Indeed, adolescent stress increased GR mRNA expression in all subregions of the mPFC of adult males and in the PrL of adult females. Together, these data suggest that adolescence may represent a developmental window of vulnerability for the mPFC, as exposure to stress during this period promoted long-term alterations in the function of several neuroregulatory systems (current data; Márquez et al., 2013; Tzanoulinou et al., 2016). It is not unreasonable to speculate that stress-induced changes in serotonin, GABA, and glucocorticoid function in the mPFC during adolescence may mediate the behavioral dysregulation observed following stress. Indeed, adolescent stress exposure has been shown to induce higher rates of aggression, impair attention, and disrupt emotional regulation (Márquez et al., 2013; Raineki et al., 2016; Tzanoulinou et al., 2016).

3.3. PVN GR mRNA expression in adulthood is modulated by both PAE and adolescent stress

While PAE-induced hyperresponsiveness to stress has been reported in both males and females, sexually dimorphic effects are often observed. Indeed, some studies evaluating the responses of PAE male and female rodents to acute stressors, including swim stress, show heightened corticosterone and/or ACTH responses in females, but not in males (Halasz et al., 1993; Kelly et al., 1991; Lee and Rivier, 1996; Nelson et al., 1986; Taylor et al., 1981, 1982; Weinberg, 1988, 1992; Weinberg et al., 2008). By contrast, in studies using more prolonged and/or repeated stressors, HPA hyperresponsiveness may be observed in PAE males or in both male and female offspring (Kim et al., 1999a; Lee et al., 2000; Taylor et al., 1982; Weinberg, 1992; Weinberg et al., 1996). The current data demonstrate that PAE differentially reduced GR mRNA expression in the mpdPVN only in adult females. As GR within the PVN is a critical regulator of HPA negative feedback (Myers et al., 2012), the reduction in PVN GR expression observed in PAE female, may play a critical role in the increased vulnerability to hyperresponsiveness to acute stressors and impaired negative feedback observed in PAE females.

Similar to PAE, our results indicate that exposure to adolescent stress also reduced mpdPVN GR mRNA expression only in adult females, data that corroborates previous research showing that adolescent chronic stress reduces PVN GR expression in adult females (Wulsin et al., 2016). Exposure to adolescent stress has been shown to induce sex-specific effects in several domains including stress and emotional regulation, cognitive function, and neuroplasticity (Barha et al., 2011; McCormick et al., 2008; Raineki et al., 2016, 2018; Toledo-Rodriguez et al., 2012; Toledo-Rodriguez and Sandi, 2007, 2011). Moreover, some reports suggest that females exposed to adolescent stress show greater and longer-lasting changes in emotional-related behavior compared to males (Bourke and Neigh, 2011; Raineki et al., 2016). Indeed, our previous results indicate that adult females, but not males, exposed to adolescent stress show anxiety-like behavior, as they spent less time in the center of the open field compared to controls (Raineki et al., 2016). Together, these data suggests that females may be more vulnerable than males to the long-term neurobehavioral effects of adolescent stress.

3.4. Unique responses of PAE animals to adolescent stress

A goal of the current study was to evaluate if PAE animals are more vulnerable than controls to the effects of adolescent stress. Overall, our data revealed two patterns in which PAE animals responded to adolescent stress differently from controls. First, PAE animals failed to respond to adolescent stress as compared to control animals. That is, immediately following exposure to adolescent stress, control but not PAE females showed increased GR expression in the central amygdala and control but not PAE males showed increased GR expression in the PrL. This lack of response to adolescent stress in PAE animals may suggest that the mechanisms necessary for a short-term adaptation to chronic stressors may be altered in PAE animals (Allan et al., 2014; Caldwell et al., 2014; McCormick and Mathews, 2010; Romeo, 2017; Romeo et al., 2004, 2006). Second, PAE animals were more susceptible to adolescent stress, as PAE but not control animals showed evidence of adolescent stress effects in adulthood. Adult PAE males exposed to adolescent stress showed reduced GR expression in the central and cortical amygdala nuclei and adult PAE females exposed to adolescent stress showed reduced GR expression in the IL. This increased susceptibility to adolescent stress may further exacerbate the PAE-induced HPA axis dysregulation (Weinberg et al., 2008). These findings could have critical implications for individuals exposed to alcohol in utero, as the clinical literature has shown that individuals prenatally exposed to alcohol are at a higher risk of encountering a more stressful environment throughout the lifespan, including adolescence (O’Connor and Paley, 2006; Streissguth et al., 1991, 2004).

3.5. Conclusions

Here we investigate how exposure to stress during adolescence alters GR mRNA expression in the amygdala, mPFC, and PVN, potentially contributing to the stress hyperresponsivity and increased vulnerability to mental health problems observed following PAE. Our data demonstrated that PAE effects on GR expression were more pervasive in the amygdala and adolescent stress effects on GR expression were more pervasive in the mPFC, while both PAE and adolescent stress affected GR expression in the PVN. Moreover, PAE effects on amygdala GR expression were more pronounced during adolescence, while the adolescent stress effects in the mPFC were more pronounced in adulthood. Finally, males and females were differentially affected by PAE and/or adolescent stress, underscoring the importance of including both sexes. Together, these results suggests that PAE and adolescent CMS induce dynamic alterations in GR expression in the amygdala, mPFC, and PVN, and that these alterations manifest differently depending on brain area, age, and sex of the animal. These results further suggest that the PAE-induced hyperresponsiveness to stress and increased vulnerability to mental health problems may be mediated by different neural mechanisms depending on the sex and age of the animal.

4. Experimental Procedure

4.1. Animals and Breeding

Male and female Sprague-Dawley rats were obtained from Charles River Laboratories (St. Constant, Canada). Rats were pair-housed by sex and maintained at a constant temperature (21 ± 1°C) and on a 12 h light-dark cycle (lights on at 0700 h) with ad libitum access to water and standard lab chow (Harlan, Canada). After a 10-day acclimation period, male and female pairs were placed together for breeding. Vaginal smears were taken each morning, and the presence of sperm was used as an indicator of pregnancy (gestation day 1; G1). All experiments were performed in accordance with National Institutes of Health (NIH) guidelines for the care and use of laboratory animals, Canadian Council on Animal Care guidelines, and were approved by the University of British Columbia Animal Care Committee.

4.2. Prenatal Alcohol Exposure

On G1, females were single-housed and randomly assigned to one of three treatment groups: Prenatal Alcohol Exposure (PAE), pair-feeding, or control. Dams in the PAE group were offered ad libitum liquid ethanol diet with 36% ethanol-derived calories (Weinberg-Keiver High Protein Control Diet #710109, Experimental Diet # 710324, Dyets Inc., Bethlehem, PA). The liquid ethanol diet was introduced gradually over the first 3 days with bottles containing: G1–66% control diet, 34% ethanol diet; G2–34% control diet, 66% ethanol diet; G3-21-100% ethanol diet. This diet is formulated to provide adequate nutrition to pregnant rats regardless of ethanol intake (Lan et al., 2006). Blood alcohol levels were measured as previously reported (Workman et al., 2015) and alcohol-consuming dams showed an average of 118.20 ± 8.11 mg/dl. Pair-fed dams were offered a liquid control diet with maltose-dextrin isocalorically substituted for ethanol, in an amount matched to the consumption of an alcohol-fed partner (g/Kg body weight/day of gestation). The control dams were offered ad libitum access to a pelleted form of the liquid control diet. All animals had ad libitum access to water, and those in the PAE and pair-fed groups were provided with fresh diet daily within 1 h prior to lights off to maintain the normal HPA circadian rhythm (Gallo and Weinberg, 1981; Krieger, 1974). Experimental diets were continued through G21. Beginning on G22, all animals were offered ad libitum access to standard laboratory chow and water, which they received throughout lactation. Pregnant dams were left undisturbed except for cage changing (G1, G7, and G14) and weighing (G1, G7, G14, and G21). On the day of birth (postnatal day 1, PN1) the litters were culled to 12 pups with an attempt to preserve an equal number of males and females per litter. Dams and pups were cage changed and weighed on PN1, PN8, PN15, and PN22. Dam and pup body weight data were published in Raineki et al., 2016. On PN22 pups were weaned and group-housed by litter and sex. Effects of adolescent CMS on GR expression in PAE and ad libitum-fed control offspring were presented in the Results section above, whereas specific pair-feeding effects are analyzed and presented separately in the Supplementary Materials.

4.3. Adolescent Chronic Mild Stress (CMS)

One male and one female from each litter were randomly assigned to either the CMS or the non-CMS condition and were pair-housed with another animal of the same sex and prenatal group. Animals in the CMS condition were subjected to 10 consecutive days of chronic, unpredictable, mild stressors. To account for the sexually dimorphic time of pubertal onset (McCormick and Mathews, 2010; Vetter-O’Hagen and Spear, 2012), males and females were exposed to CMS at ages consistent with puberty onset – PN31–41 for females, PN 37–47 for males. On each CMS day, animals received two different stressors at random times: one in the morning (between 0800 and 1200 h) and one in the afternoon (between 1300 and 1800 h), with a minimum of 2 h between stressors. On day 1 of CMS and the day immediately following the end of CMS, within 2 h of lights on, basal blood samples were obtained from all animals (including those in the non-CMS group) via tail nick. After tail nick, all animals were weighed and placed in a new home cage. Pre-and post-CMS blood sample and body weight data were published in Raineki et al., 2016. Except for blood sampling and routine husbandry, animals in the non-CMS condition were left undisturbed during this period. CMS and non-CMS animals were housed in different colony rooms so that non-CMS animals were not exposed to the disturbance and stress odors of the CMS animals (Mackay-Sim and Laing, 1980). The order and type of stressor was randomized, but all animals received the same number of exposures to each stressor over the 10-day period. Stressors included: 1) Platform: exposure to an elevated Plexiglas platform (20 × 20 cm) mounted on 90 cm high post for 10 min; 2) Cage tilt: the home cage was tilted at a 30° angle for 2 h; 3) Novel cage: exposure to a novel cage without food and water, with a small amount of novel bedding for 1 h; 4) Soiled cage: exposure to a soiled cage of another animal of the same sex for 1 h; 5) Restraint: restraint in PVC tubes (tube size varied to ensure proper restraint/immobility of each animal) for 30 min; 6) Social isolation without food and water: overnight isolation in a smaller cage (28 × 17 cm with 12.5 cm high) for 12 h; and 7) Empty water bottle: animals given their empty water bottles for 1 h following the social isolation/food and water deprivation period.

4.4. Behavioral Exposure and Brain Extraction

All animals were tested on the open field before and after CMS exposure. For the post-CMS test, half the animals were tested in adolescence (short-term effects of CMS) and the remaining animals were tested in adulthood (long-term effects of CMS). Following post-CMS open field tests, all animals were exposed to the FST (habituated for 15 min and then tested for 5 min the following day). Pre-and post-CMS behavioral data were published in Raineki et al., 2016. Animals were decapitated 30-min after the end of day 2 FST testing and brains were collected, quickly frozen on dry ice and stored at −80°C.

4.5. Neural assessment of GR by in situ hybridization

4.5.1. Probe

Rat GR probe (456bp fragment in pGem4), provided by Dr. James Herman, was transcribed using 35S-UTP (Perkin-Elmer, Waltham, MA) and the Promega Riboprobe System (Promega Corp., Madison, WI) with polymerase T7 antisense probe. GR probe was purified using Micro Bio-Spin 30 Columns (Bio-Rad, CA, USA) and 0.1 M DTT was added to prevent oxidation.

4.5.2. In situ hybridization

Brains were sectioned coronally (20 μm) using a cryostat (−16°C) and stored at −80°C. Thawed sections were fixed in formalin for 30 min and pre-hybridized as follows: 1 × PBS for 10 min twice, proteinase K (0.1μg/L; 37°C) for 9 min, 0.1 M triethanolamine-hydrochloride (TEA) for 10 min, 0.1 M TEA with 0.25% acetic anhydride for 10 min, 2 × SSC for 10 min twice, dehydrated by a graded series of ethanol, chloroform, and 100% ethanol and air-dried. Probe was applied at 1 × 106 cpm/slide in 75% Hybridization buffer (75% formamide, 3 × SSC, 1× Denhardt’s solution, 200 μg/mL yeast tRNA, 50 mM sodium phosphate buffer (pH 7.4), 10% dextran sulphate, 10 mM DTT) and covered with HybriSlips (Sigma-Aldrich, ON). Sections were incubated overnight at 55°C in chambers humidified with 75% formamide. HybriSlips were removed and slides were rinsed as follows: 2 × SSC twice for 20 min, 2 × SSC for 30 min, 50 μg/L RNAse A solution (37°C) for 60 min, 2 × SSC with 0.01 M DTT for 10 min, 1 × SSC for 10 min, 0.5 × SSC with 0.01 M DTT for 10 min, 0.1 × SSC with 0.01 M DTT (60°C) for 60 min, 0.1 × SSC for 5 min. Sections were dehydrated by a graded series of ethanol and air dried overnight. Amygdala and mPFC hybridized slides were exposed to Kodak BioMax MR film, and developed using Kodak GBX developer and fixer. mpdPVN hybridized slides were dipped in Kodak NTB2 autoradiography emulsion (diluted 50:50 with distilled water), exposed in desiccated sealed, light tight boxes (4°C), developed using Kodak D19 developer and standard Kodak fixer, counterstained with Toluidine Blue, and coverslipped with Permount (Fisher Scientific Ltd.).

4.5.3. Densitometric analysis

The autoradiograph films for the amygdala and mPFC were scanned and analyzed with ImageJ 1.48v (National Institutes of Health, USA). The left and right subregions of the amygdala (basolateral, medial, central, and cortical; bregma −2.16 to −3.00) and mPFC (ACC, PrL, and IL; bregma 3.73 to 2.52) were traced free-hand according to a stereotaxic rat brain atlas (Paxinos and Watson, 2005) in two sections per animal to determine mean grey density levels. Corrected grey levels were obtained by subtracting the background level (corpus callosum) from each of the four measurements. For the emulsion-dipped slides (mpdPVN; bregma −1.72 to −1.92), in situ signals were visualized with a Q-imaging monochrome 12-bit camera attached to a Zeiss Axiokop 2 motorized plus microscope. Images were captured under dark field illumination using Northern Elite 6.0v (Empix Imaging Inc., Mississauga, ON, Canada) and analyzed with ImageJ 1.48v software (National Institutes of Health, USA). The left and right mpdPVN were traced by outlining a fixed circle (0.75 in diameter; scale 300 pixels ⁄ inch) in two sections per animal to determine mean integrated density levels. Corrected integrated densities were obtained by subtracting the background level (corpus callosum) from each of the four measurements. For all the areas, the left and right levels in each measured area were averaged together for analysis.

4.6. Statistical analysis

All data are expressed as mean ± SEM and were analyzed by two-way ANOVA (prenatal treatment and adolescent CMS as factors). When significant, ANOVAs were followed by Newman-Keuls post hoc tests. Sex was not used as a factor in the ANOVAs because females and males were exposed to CMS during different ages (PN31–41 and PN 37–47 respectively). Age was also not used as factor because in situ hybridizations for brains collected during adolescence and adulthood were run separately. Further analyses utilized planned comparisons to test the a priori hypothesis that: 1) PAE will alter animals’ GR mRNA expression compared to controls; and 2) CMS will differentially alter animals’ GR mRNA expression. In all cases, differences were considered significant when p≤0.05. Outliers were identified and removed using the Robust regression and Outlier removal (ROUT) method with Q=0.05.

Supplementary Material

1
  • glucocorticoid receptor (GR) expression in the amygdala is altered by prenatal alcohol exposure (PAE)

  • PAE effects on amygdala GR expression were more pronounced during adolescence

  • GR expression in the mPFC is altered by adolescent stress

  • adolescent stress effects on mPFC GR expression were more pronounced in adulthood

Acknowledgments

This research was supported by NIH/NIAAA grants R37 AA007789 and R01 AA022460, Kids Brain Health Network (Canadian Networks of Centers of Excellence) grant 20R64153 to JW. We thank all members of the Weinberg laboratory for their assistance with the animal work. We also thank Parker J. Holman for help in editing the manuscript.

Footnotes

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