Various models of postnatal early-life stress have explored the influence of later life environmental insults on metabolic function, each of which seem to elicit different outcomes and sex-specific effects (see **Table 2**). Work in our lab demonstrated HFD fed male rats previously exposed to maternal separation, have marked elevations in plasma insulin, and decreased total white adipose tissue mass, independent of body weight vs. HFD controls (131, 132). In agreement, maternal deprivation induced early adulthood hyperinsulinemia and impairments in insulin sensitivity, measured through HOMA-IR, in male offspring fed with an HFD relative to HFD controls (130). Similar metabolic changes are observed in maternal deprivation exposed female offspring, with HFD shown to cause early adulthood hyperinsulinemia, at PND35 compared to PND102 in control rats consuming an HFD (130). Further, maternally deprived female rats consuming a high-fat sucrose diet had a trend for decreased fat depot mass vs. control (121). Less work has examined the metabolic profile arising subsequent to earlylife stress induced by LN material. A recent study explored for the first time the metabolic profile in Wistar female rats; showing reduced body weight at weaning, and reduced food intake, suggesting altered energy utilization and storage. Interestingly, these pups had exaggerated HPA axis activity with delayed clearance of corticosterone from the circulation, and taken together these data further suggest an early-life stress-induced interaction between the HPA axis and metabolic profile (133) (see **Table 2**).

Few studies have explored biological characteristics of peripheral tissues following early-life stress. Recently, maternally deprived female rats were shown to have significantly reduced brown adipose tissue β3-adrenergic receptor mRNA expression and increased white adipose tissue prohibitin mRNA relative to control, with no change in UCP-1 (136). The authors concluded these results may facilitate adipose tissue proliferation later in life. An altered response to nutritional challenge following early-life stress is not only observed in high caloric fed states. Maternally separated rats consuming an omega-3 deficient diet demonstrated marked elevations in plasma insulin and impaired insulin sensitivity, as assessed by HOMA-IR, relative to control animals consuming the same diet (134). Functional studies performed by our lab have demonstrated attenuated adulthood outcomes of early-life stress when siblings are provided with a positive environment of voluntary running wheel exercise (see **Figure 2**). Male rats exposed to maternal separation and weaned onto a standard chow diet demonstrated a hyper-responsive corticosterone response to novel restraint stress, which was dampened with exercise and HFD. Impaired changes in metabolic parameters of insulin and diet-induced obesity were also attenuated with exercise, reversing maternal separation-induced hyperinsulinemia and increased body weight relative to HFD controls and stressed sedentary counterparts (132).

A common aspect across early-life stress models centers around glucocorticoid exposure and HPA axis activation during the stresshypo-responsive period, a stage of neonatal resilience to mild stressors suggested to likely trigger corticosterone secretion in adult life (84). Synthetic glucocorticoid administration attempts to replicate stress models, as this elicits an HPA axis response during the early postnatal period in rodents (41, 138). This also allows for controlled dose administration and with necessary caution can improve understanding of the impact of glucocorticoid exposure during these critical periods. Early-life corticosterone administration reduced adult female rat body weight and decreased fat depot mass relative to control (139). Studies of glucocorticoid administration highlight the marked influence of combining stress hormones and additional metabolic insults. In young male Sprague-Dawley rats, glucocorticoid administration alone did not alter insulin sensitivity, assessed by HOMA-IR, or glucose disposal following an OGTT (140). Conversely, consumption of a palatable HFD in combination with glucocorticoid exposure is known to induce a marked increase in fasting plasma glucose and insulin, with impaired glucose clearance following OGTT (140, 141). Further investigation is required to determine whether the unaltered or improved insulin sensitivity with chow consumption would prove deleterious in the long term (as shown in **Figure 2**).

#### **EARLY-LIFE STRESS AND PROGRAMING OF PERIPHERAL TISSUES**

#### **MECHANISMS UNDERLYING EARLY-LIFE STRESS-INDUCED METABOLIC DEFICITS: ROLE OF THE HPA AXIS, GLUCOCORTICOID, AND TISSUE 11**β**-HSD1**

To uncover the mechanisms underlying early-life stress-induced metabolic derangements, it is first essential to understand the action of glucocorticoids at different concentrations on glucose and insulin homeostasis and lipid metabolism. At pharmacological doses, glucocorticoids act as potent anti-inflammatory agents but high levels of circulating glucocorticoids result in metabolic derangements including increased visceral adiposity, dyslipidemia (increased levels of triglycerides), increased non-esterified fatty acids (NEFA) (142–144), and impaired glucose and insulin tolerance (145–147). In contrast to the anabolic actions of insulin, glucocorticoids are predominantly catabolic, decreasing glucose utilization and insulin sensitivity with both human and animal data revealing insulin intolerance with excess exposure (145, 148).

Glucocorticoids exert tissue-specific metabolic effects, directly targeting tissues for insulin metabolism, and they regulate skeletal muscle, liver, and adipocyte insulin signaling (149). Glucocorticoids alter glucose and protein metabolism; increased levels of

(Continued)

#### **Table 2 | Continued**

glucocorticoids induced during stress increase protein degradation, which results in the generation of amino acids that serve as precursor for glucose synthesis in the liver. In addition, excess glucocorticoids inhibit glucose uptake into muscle by inhibiting translocation of glucose transporter-4 (150–152). Chronically increased circulating or tissue glucocorticoid levels may also lead to insulin resistance, hypertriglyceridemia, and hepatic steatosis (see **Figure 1**). Circulating glucocorticoid concentrations are tightly controlled by activation of the HPA axis, however, tissue-specific availability is regulated by multiple means including glucocorticoid receptor expression, receptor affinity, and alterations in glucocorticoid metabolism and clearance.

Mechanisms regulating intracellular glucocorticoid concentrations are critical to understand the impact of stress on energy metabolism including energy expenditure, storage, and utilization. Intracellular levels of glucocorticoids are influenced by 11β-HSD1 with the type-1 isoform predominantly expressed in the liver (153) and to a lesser degree in adipose and skeletal muscle (154). Evidence shows that tissue glucocorticoid levels are regulated by 11-beta hydroxysteroid dehydrogenase (11β-HSD1) in target tissues (155) as 11β-HSD1 converts inactive cortisone to biologically active cortisol (156, 157). The liver is a major site of glucocorticoid metabolism where 11β-HSD1 regulates the access of glucocorticoid to the glucocorticoid receptor, leading to glucocorticoid metabolism which,is regulated by 5-alpha/beta reductase levels (155, 158). Fat, liver, and muscle-specific increases in 11β-HSD1 are known to increase the risk for metabolic disorders such as insulin resistance, hyperglycemia, and hyperlipidemia (159, 160). Tissue glucocorticoid amplifies the action of insulin to promote lipogenesis within hepatocytes (161). 11β-HSD1 in the liver increases glucocorticoid action in liver to stimulate gluconeogenesis and inhibit beta-oxidation of fat, thus promoting lipid synthesis (162–164). A very recent study demonstrated reduced 5-alpha reductase was associated with fatty liver (165). Thus liver-specific glucocorticoid synthesis and clearance regulated by 11β-HSD1 and 5-alpha reductase appear to affect hepatic lipid accumulation. Interestingly, animal studies demonstrate a link between hepatic glucocorticoid metabolism with regulation of HPA axis activity and lipid synthesis in the liver. For example, transgenic overexpression of 11β-HSD1 in liver of null mice normalized the exaggerated HPA axis activity in response to stress, and led to fatty liver (166, 167). On the other hand, hepatic deletion of 11β-HSD1 led to hyperactivity of the HPA axis (168). These studies suggest liver 11β-HSD1 greatly contributes to amplify circulating glucocorticoid levels, and thus likely mediates the negative feedback activity to dampen HPA axis activity, a concept previously proposed by Chapman et al. (169). Several studies have demonstrated a blunted activity of the HPA axis in response to novel stress following high energy diets such as high sugar or HFD either after adulthood chronic stress or chronic stress exposure during early life (131, 132, 170). As these studies did not report measures of liver 11β-HSD1, it is not clear whether under-activity of the HPA axis following early-life stress and postnatal high energy diet is modulated by liver 11β-HSD1 levels.

Thus, liver 11β-HSD1-HPA axis is a potential pathway in earlylife stress-mediated metabolic disturbances, particularly insulin sensitivity, glucose metabolism and lipid synthesis, and mobilization as outlined in **Box 1**. This hypothesis, however, needs systematic examination in the future. Early-life stress, modeled through prenatal dexamethasone treatment, has been shown to upregulate 11β-HSD1 in peripheral tissues such as liver, pancreas, and subcutaneous fat in rat offspring at 4 months with persistent increases at 1 year of age (171). Interestingly, it was previously shown that early-life stress alters the expression of liver 5-alpha reductase mRNA (172, 173). Thus, this suggests a programing effect of early-life stress on tissue 11β-HSD1 expression, glucocorticoid metabolism, and glucocorticoid signaling. There has been limited evidence regarding early postnatal stress effects on 11β-HSD1 expression and glucocorticoid metabolism of peripheral tissues, which needs to be explored in future studies (**Figure 1**).

We propose that early-life stress may alter availability of tissue glucocorticoids and glucocorticoid signaling. Specifically, we propose that during the maladaptation period (see **Figure 2**), early-life stress enhances availability of tissue glucocorticoids via increases in liver 11β-HSD1. The tissue glucocorticoid-induced insulin resistance may involve glucocorticoids altering insulin signaling via increasing phosphorylation of PKB/akt, which stimulates insulin secretion, that is, glucocorticoids work synergistically with insulin which increases adipocyte lipolysis and liver lipogenesis (see **Figure 1**). Another possibility is that early-life stress impairs glucocorticoid signaling involving glucocorticoid– glucocorticoid receptor binding and phosphorylation of the glucocorticoid receptor. Impairment in glucocorticoid signaling leads to alteration in glucocorticoid targeted genes that regulate hepatic glucose production, and hepatic lipogenesis including peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1-alpha), phosphoenolpyruvate carboxykinase

#### **Box 1 Outstanding research questions/proposals**.

(PEPCK), glucose 6-phosphate (G6P), and diacylglycerol acyltransferase (DGAT). Genes known to mediate hepatic lipid accumulation such as PGC1-alpha and adipose DGAT1 were altered in rat pups from dams that had been subjected to prenatal stress (174). PGC1-alpha plays an essential role in fatty acid oxidation while increased DGAT1 in adipose tissue increases lipogenesis. DGAT1 transgenic mice fed with an HFD demonstrated a 300% increase in liver triglycerides suggesting a redistribution of the fat from adipose tissue to liver via re-esterification of fatty acid with glycerol (175). Another study showed that prenatal stress induced through manipulation of the availability of food, that is either by limiting intake, or exposing to a high energy food during pregnancy led to fatty liver in pups relative to controls (176).

In conclusion, the role of liver 11β-HSD1 in regulating HPA axis activity, and whether it is modulated by stress early in life, warrants investigation. We propose that early-life stress induces

changes in glucocorticoid metabolism and signaling, likely mediating the metabolic consequences reported. The question of how these affect glucocorticoid-induced insulin dependent processes, including hyperinsulinemia and lipid metabolism following an early-life stress exposure will be addressed in the following section.

#### **PROPOSAL OF HOW ELS MAY INCREASE RISK OF INSULIN RESISTANCE, AND HYPERGLYCEMIA**

It has been known for decades that stress early in life in both humans and animals can affect HPA axis activity in later life (131, 132, 177–179), however, the effects of early-life stress on hypothalamic neuropeptides involved in feeding are less well known, with only a few studies exploring hypothalamic neuropeptides following maternal separation or deprivation (131, 180). Feeding regulation, which is tightly regulated by orexigenic and anorexigenic hypothalamic neuropeptides, is also influenced by HPA axis activity and circulating glucocorticoid concentrations (181, 182). Glucocorticoids stimulate feeding responses by increasing the release of neuropeptide Y (NPY) and inhibiting that of corticotrophin releasing hormone in the hypothalamus however the orexigenic effect of glucocorticoids may be counteracted by leptin (183).

Mela and colleagues explored the effect of 24 h of maternal deprivation on postnatal day 9 in rats on hypothalamic feeding neuropeptides measured at 14 weeks of age in rats fed with chow or HFD (130). No significant differences in orexigenic (NPY, agoutirelated peptide (AgRP) and anorexigenic (pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript) neuropeptides were observed between the chow and HFD fed rats. An interesting finding in this study was that while HFD did not alter these neuropeptides in control females, rats that experienced maternal deprivation had significantly decreased hypothalamic NPY and AgRP mRNA expression and a significant increase in hypothalamic pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript relative to their chow counterparts. The increased caloric intake by HFD fed rats relative to their chow counterparts was similar between the maternally deprived and control rats. But an increased plasma leptin concentration in the maternally deprived HFD fed female rats suggests a programing effect of maternal deprivation which dysregulated feeding homeostasis. Whether this may be linked to either the HPA axis activity or tissue-specific availability of glucocorticoids needs to be explored in the future. The significantly reduced fasting triglycerides and lack of change in fasting insulin by HFD feeding is puzzling, and these may be related to either the strain or the diet used. Thus, use of appropriate controls and dietary conditions are critical to enable exploration of the underlying mechanisms linking combined early-life stress and high-fat feeding with later metabolic risk. Overall, limited studies have directly investigated whether early-life stress affects hypothalamic neuropeptides and inflammation.

The increased availability of glucocorticoids discussed above may play a role in altering tissue insulin signaling by increasing the phosphorylation of PKB/akt. Animals exposed to early-life stress may be resilient to the increased availability of tissue glucocorticoids and thus subsequently show a dampened secretion of insulin. While there are no systematic studies on early-life stress and metabolic consequences, there is evidence in rodents showing early-life stress does not affect basal metabolic hormones if animals consume chow, including non-fasted plasma insulin and leptin (131, 132). Another study demonstrated improved insulin sensitivity following early-life stress when measured during adulthood (170). Taken together, we propose that early-life stress may lead to resilience, and thus the organism potentially may adapt to any changes in the postnatal environment, such as exposure to high energy diet, or later stressors through enhanced negative feedback sensitivity of the HPA axis activity and reprograming of the peripheral tissue sensitivity to glucocorticoid exposure (see **Figure 2**). However, if an organism exposed to early-life stress is chronically exposed to these negative postnatal environments (high energy diet and later stressors), the enhanced negative feedback sensitivity maybe dysregulated, resulting in perturbed HPA axis activity, leading to a phase known as maladaptation (see **Figure 2**). Indeed there is evidence that such maladaptations can influence glucose/insulin homeostasis, resulting in the manifestation of metabolic disorders including insulin resistance, hyperglycemia, hyperlipidemia, as suggested in **Figure 2**.

During periods of maladaptation, increased tissue glucocorticoids and circulating corticosterone will impair the insulin signaling pathway, leading to hypersecretion of insulin; a condition leading to insulin resistance (see **Figure 2**). This will mediate tissue specific effects, that is, increased adipose lipolysis through beta-oxidation which releases free fatty acids into the circulation and re-esterification in the liver to promote lipogenesis. In addition, increased tissue glucocorticoid levels via insulin stimulation increases hepatic glucose production. Prenatal stress has been shown to increase hepatic PEPCK mRNA and this was enhanced with high energy feeding (174). There are no data thus far on the impact of early postnatal stress on hepatic glucose production, which is an important measure to be considered for future studies to improve understanding of the link between postnatal stress and risk of pre- or diabetes. In addition, increased glucocorticoid-induced hyperinsulinemia also alters the muscle glucose utilization via affecting the glucose transporter gene and expression, including the GLUT4-transporter.

#### **CONCLUSION AND FUTURE DIRECTIONS**

Despite significant progress in the field of early life programing and metabolic disease risk many challenges exist. Human studies are vital in providing evidence of the association between earlylife adversity and disease incidence but data must be interpreted with necessary caution. A majority of human evidence is based on parental or offspring self-report raising the possibility of confounding due to issues with information recall, lack of accuracy, and the potential for bias.

Future studies in humans should seek to better quantify stress exposure during the early-life period and at the time of assessment to improve knowledge of how different stress types may alter disease risk. Given both environment and genetic predisposition determine health outcome, studies should not only consider stressor experience during early life but also control for socioeconomic status, food and education availability, ethnicity, and lifestyle factors such as nutritional status, smoking, and physical activity throughout their lifetime.

According to the hypothesis explored throughout this review, programed adaptation during early life occurs in an attempt to adapt to the predicted later life environment and hence even seemingly trivial variations in stressors during these periods may vary the observed outcomes. Inconsistency in findings across experiments could be due to the marked differences in study design. Procedural variations in maternal separation have been reviewed elsewhere (177). In the literature, there is no consistent procedure, rather multiple experimental conditions fall under the broad term of maternal separation. Thus, duration, and age at separation, temperature in which pups are separated, whether pups are isolated from litter mates during separation and whether pups are removed or remain in their home cage have all been shown to influence behavioral outcomes and brain function. Animal models must consider the influence of maternal care on long term outcomes, which is a major aspect the novel model of limiting nesting material attempts to explore. Work exploring prenatal or gestational stress exposure could benefit from cross-fostering to better delineate effects of maternal stress and the influence of received postnatal care. Offspring of low care dams that were cross-fostered by high-licking high arched back nursing dams were resilient in terms of the decline in hippocampal synaptogenesis and spatial learning seen in offspring reared by low care dams (184).

Animal research should ultimately aim to improve public health outcomes. To ensure this, analysis of behavioral, physiological, and molecular parameters is required. The current literature lacks assessment of whole body insulin sensitivity measures or assessment of β-cell structural changes – key factors that influence metabolic outcome. Models need to reflect the human condition upon which they are based, which brings the need for valid controls. Given the complex heterogeneity of both the stress system and metabolic disease the phenotype of experimental animals would ideally be comprehensively assessed, rather than examining single factors. Species and strain differences must be considered and these animals must be valid models for the environmental conditions that the study aims to investigate.

Investigation of the environmental influence provides us with an opportunity to better identify factors determining vulnerability and resilience to early-life experience. A better understanding of factors driving the association between genetic predisposition with both early and adult environment will help with the identification of targets for intervention with the hope of minimizing disease incidence. As known, high energy feeding impairs glucose and insulin tolerance and affects lipid metabolism, thus chronic HFD could serve as a good model to study the altered effects of early-life stress on metabolism. Our lab and others have shown early evidence that HFD or altered nutrition lead to altered insulin levels in early-life stressed animals relative to control (131,132,134, 170). Together these studies appear to suggest that both short term diet and long term diet exaggerate plasma insulin. Despite this, a single marker or circulating hormone levels limit ability to draw conclusions of how early-life stress may exert negative impact on insulin sensitivity and overall metabolic risk. Future studies need to adopt a mechanistic approach, examining animals using appropriate metabolic tests that will provide answers to the outstanding questions outlined in **Box 1**. An alternate model of early-life stress, variable foraging demand, demonstrated impaired insulin

resistance in non-human primates as measured by hyperglycemicinsulin clamp (185). Functional studies in rodent early-life stress models, such as glucose or insulin tolerance tests, or preferably glucose clamp, would be useful to explore this further.

In conclusion, the mechanisms whereby adverse early-life events accelerate metabolic deficits have received little attention to date. When combined with a sub-optimal subsequent environment (e.g., poor diet, stress, physical inactivity) early-life stress may exacerbate the risk of metabolic disease. One potential mechanism underlying early-life stress-induced metabolic deficits is the interaction between the HPA axis and liver 11β-HSD1. Positive later environments may modulate the negative impact of early-life stress not only on behavioral outcomes, but also on metabolism. Given the burgeoning issues of metabolic and mental health disorders, the question of how early-life stress impacts subsequent disease risk warrants further investigation.

#### **REFERENCES**

adults born preterm at very low birth weight. *PLoS One* (2012) **7**:e32430. doi:10.1371/journal.pone.0032430

diet-induced obesity in rats. *PLoS One* (2010) **5**:e8779. doi:10.1371/journal. pone.0008779

**Conflict of Interest Statement:** 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.

*Received: 31 December 2013; accepted: 28 April 2014; published online: 13 May 2014. Citation: Maniam J, Antoniadis C and Morris MJ (2014) Early-life stress, HPA axis adaptation, and mechanisms contributing to later health outcomes. Front. Endocrinol. 5:73. doi: 10.3389/fendo.2014.00073*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2014 Maniam, Antoniadis and Morris. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited andthatthe original publication inthis journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Neurocircuitry underlying stress and emotional regulation in animals prenatally exposed to alcohol and subjected to chronic mild stress in adulthood

#### **Charlis Raineki <sup>1</sup> , Kim G. C. Hellemans 1,2,Tamara Bodnar <sup>1</sup> , Katie M. Lavigne3,4, Linda Ellis <sup>1</sup> , Todd S.Woodward3,4 and JoanneWeinberg<sup>1</sup>\***

<sup>1</sup> Department of Cellular and Physiological Sciences, The University of British Columbia, Vancouver, BC, Canada

<sup>2</sup> Department of Neuroscience, Carleton University, Ottawa, ON, Canada

<sup>3</sup> Department of Psychiatry, The University of British Columbia, Vancouver, BC, Canada

<sup>4</sup> BC Mental Health and Addictions Research Institute (BCMHARI), Vancouver, BC, Canada

#### **Edited by:**

Nikolaos P. Daskalakis, Icahn School of Medicine at Mount Sinai, USA

#### **Reviewed by:**

Gábor B. Makara, Hungarian Academy of Sciences, Hungary Ishwar Parhar, Monash University, Malaysia

#### **\*Correspondence:**

Joanne Weinberg, Department of Cellular and Physiological Sciences, The University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada e-mail<EMAIL_ADDRESS>
Individuals exposed to alcohol during gestation show higher rates of psychopathologies. The hyperresponsivity to stress induced by prenatal alcohol exposure (PAE) may be related to this increased rate of psychopathologies, especially because this population is more likely to be exposed to stressful environments throughout life. However, alcohol-induced changes in the overlapping neurocircuitries that underlie stress and the expression of psychopathologies are not fully understood. Here, we performed a comprehensive analysis of the neural activity within central areas known to play key roles in both emotional and stress regulation. Adult male and female offspring from PAE, pair-fed, and ad libitum-fed control conditions were exposed to chronic mild stress (CMS). Following CMS, the neural activity (c-fos mRNA) of the amygdala, ventral hippocampal formation, medial prefrontal cortex (mPFC), and paraventricular nucleus of hypothalamus (PVN) was assessed in response to an acute stress (elevated plus maze). Our results demonstrate that, overall, PAE decreased neural activity within the amygdala and hippocampal formation in males and increased neural activity within the amygdala and mPFC in females. CMS reduced neural activity within the mPFC and PVN in PAE males, but reduced activity in all areas analyzed in control males. By contrast, CMS reduced neural activity in the mPFC in PAE females and had no effects in control females. Furthermore, the constrained principal component analysis revealed that these patterns of neural activity resulted in differential activation of the functional neural networks in males compared to females, indicating sexually dimorphic effects of PAE and CMS. Importantly, the altered networks of brain activation in PAE animals may underlie the hyperresponsivity to stress and increased psychopathologies observed among individuals prenatally exposed to alcohol.

**Keywords: prenatal alcohol exposure, chronic mild stress, amygdala, hippocampus, prefrontal cortex, paraventricular nucleus of hypothalamus, c-fos, constrained principal component analysis**

#### **INTRODUCTION**

Fetal development is a dynamic process strongly influenced by the quality of the environment in which it occurs (1, 2). Adverse intrauterine environments can negatively alter the developmental trajectory, resulting in physical and mental health problems later in life (1, 3). In mammals, the mother plays a critical role in fetal development, not only by supplying nutrients and oxygen via the placenta, but also by modulating the environmental stimuli to which the fetus is exposed (4). Clinical and experimental studies have clearly demonstrated that alcohol consumed during pregnancy has pervasive and long-lasting negative effects on fetal development (5–9). Indeed, prenatal alcohol exposure (PAE) is strongly associated with a wide range of neural, behavioral, hormonal, and cognitive deficits in humans, non-human primates, and rodents (5–9). In addition, the rates of psychopathologies (e.g., anxiety, depression, other mood disorders, and substance use disorders)

among individuals prenatally exposed to alcohol are disproportionately higher when compared to unexposed individuals (10– 13). Unfortunately, little is known about the underlying neural processes that support this high incidence of psychopathologies following PAE.

Brain areas implicated in psychopathologies overlap to a large extent with areas that mediate responses to stress. The amygdala, hippocampus, and medial prefrontal cortex (mPFC) are intrinsically involved in the modulation of hypothalamic–pituitary– adrenal (HPA) axis activity (14, 15), and are also associated with several mental health disorders (16, 17). Abnormal function of any one of these highly interconnected areas may result in abnormal responses to stress and/or the emergence of psychopathologies. This is especially relevant for individuals exposed to alcohol during gestation, as neuroimaging studies have demonstrated structural and functional alterations in brain regions

involved in stress regulation, such as the hippocampus and PFC (18–21).

Not surprisingly, a striking deficit induced by PAE involves changes in how affected individuals process and/or respond to a stressful stimulus or situation. The human and animal literature clearly demonstrates that alcohol consumption during pregnancy results in offspring that are hyperresponsive to a variety of stressors (5, 9, 22–24). For example, children exposed to alcohol *in utero* show higher salivary cortisol levels following exposure to stressors such as blood draw and the still-face procedure when compared to unexposed counterparts (22, 23). Additionally, higher basal cortisol levels were also observed in children exposed to alcohol prenatally (23, 24). In parallel with these studies, rats prenatally exposed to alcohol show increased HPA activation and/or a delay in return to basal levels as compared to controls in response to a wide range of stressful stimuli including footshock, restraint, immune challenge, and exposure to novel environments (25–30). In addition, HPA dysregulation is observed not only following stress, but also under basal conditions, even in the face of similar basal hormone levels. Dysregulation is evident at multiple levels of the axis, and appears to reflect changes in both HPA drive and feedback regulation and/or in the balance between drive and feedback. Taken together, these findings suggest that for individuals prenatally exposed to alcohol, stressors occurring over the life span may be acting on an already dysregulated or sensitized stress neurocircuitry. Thus, stress hyperresponsivity and HPA dysregulation may be a crucial factor mediating the increased vulnerability of these individuals to develop later psychopathologies (5, 31, 32). An aggravating factor to this already unfavorable situation is that individuals prenatally exposed to alcohol are,in general, at a higher risk of encountering a more stressful environment throughout the lifespan (12, 13, 33).

Using a rodent model of PAE, our laboratory has been successful in demonstrating that the combination of PAE and chronic unpredictable stress in adulthood increases anxiety- and depressive-like behaviors in a sexually dimorphic manner (5, 31, 32). However, the neurocircuitry underlying both stress dysregulation and anxiety- and depressive-like behaviors of individuals prenatally exposed to alcohol is still not fully understood. In the present study, adult male and female rats that were prenatally exposed to alcohol underwent a 10-day chronic mild stress (CMS) regimen in adulthood, which was followed by a comprehensive analysis of the neural activity within brain areas known to play key roles in stress and emotional regulation. Specifically, we assessed *c-fos* mRNA expression as a measurement of neural activity in the medial parvocellular dorsal division of the paraventricular nucleus of hypothalamus (mpdPVN), ventral hippocampal formation (CA1, CA3, DG, and ventral subiculum), the amygdala (central, cortical, lateral, basal, and medial nuclei), and the mPFC [anterior cigulate cortex (ACC), prelimbic (PrL), and infralimbic (IL)] in response to an acute stressor – exposure to the elevated plus maze (34, 35). In order to identify networks of coordinated activity in these brain regions rather than simply to identify whether activity differed between experimental groups for each individual brain area, we employed constrained principal component analysis (CPCA) in addition to a traditional univariate analysis (ANOVAs followed by *post hoc* group comparisons). CPCA

is a multivariate technique that combines multivariate multiple regression and principal component analysis (PCA) into a unified framework (36–41). In the current study, CPCA allowed for the identification of brain networks that were specifically related to our experimental conditions (i.e., PAE and CMS). Thus, we were able to identify the networks of brain regions associated with differences in neural activity between animals that were prenatally exposed to alcohol, with or without CMS exposure in adulthood, and their respective controls. Below, we present results from a traditional univariate technique (ANOVA) as well as CPCA to demonstrate how this multivariate technique can facilitate interpretation when group differences are observed in a variety of distinct, but interconnected, brain regions.

#### **MATERIALS AND METHODS**

#### **ANIMALS AND BREEDING**

Female Sprague-Dawley rats were obtained from Charles River Laboratories (St. Constant, QC, Canada) and male Sprague-Dawley rats were obtained from the UBC Animal Care Centre. 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 6 a.m.) with *ad libitum* access to water and standard laboratory chow (Jamieson's Pet Food Distributors Ltd., Canada). After a 10-day acclimation period, male and female pairs were placed together suspended in stainless steel cages with mesh front and floor (25 cm × 18 cm × 18 cm). Day 1 of gestation (G1) was indicated by the presence of a vaginal plug on the wax paper beneath the breeding cages,which were checked daily. All experiments were performed in accordance with National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and the Canadian Council on Animal Care guidelines and were approved by The University of British Columbia Animal Care Committee.

#### **PRENATAL ALCOHOL EXPOSURE**

On G1, females were single-housed and randomly assigned to one of the three prenatal treatment groups: PAE, Pair-Fed (PF), or *ad libitum*-fed Control (C). Dams in the PAE group were offered *ad libitum* liquid ethanol diet with 36% ethanol-derived calories (Dyets, Inc., Bethlehem, PA, USA). This diet is formulated to provide adequate nutrition to pregnant rats regardless of ethanol intake (42, 43). PF dams were offered a liquid control diet with maltose–dextrin isocalorically substituted for ethanol, in an amount matched to the consumption of a PAE partner according to gestation day (gram/kilogram body weight/day of gestation). The control dams were offered *ad libitum* access to standard laboratory chow (Jamieson's Pet Food Distributors Ltd.). All animals were provided with fresh diet daily within 1 h of lights off to prevent a shift in corticosterone circadian rhythms, which occurs in animals that are on a restricted feeding schedule, such as the PF dams (44, 45). 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 and weighing, which occurred on G1, G7, G14, and G21. On the day of birth [postnatal day 1 (PN1)], litters were weighed and culled to 10 pups with an attempt to preserve an equal number of males and females per litter. Dams and pups were weighed

on PN1, PN8, PN15, and PN22 [dam and pup body weight data were published in Ref. (31)]. On PN22, pups were weaned and group-housed by litter and sex.

#### **CHRONIC MILD STRESS**

On PN40, one male and one female rat from each litter were randomly assigned to either the CMS or non-CMS condition and were pair-housed with another animal of the same sex, prenatal treatment, and stress condition (CMS or non-CMS). In adulthood (PN60–90), CMS animals were subjected to 10 consecutive days of randomized stressors. Animals were exposed to two different stressors each day: the first at a randomized time in the morning (between 8:00 and 12:00 h) and the second at a randomized time in the afternoon (between 13:00 and 16:00 h), with a minimum of 2 h between stressors. On day 1 of CMS, all animals were weighed, handled, and moved to new colony rooms in a neighboring building. CMS and non-CMS animals were housed in separate colony rooms so that non-CMS animals were not exposed to the disturbance of either moving CMS animals between stressors, or stress odors and vocalizations produced by CMS animals. CMS exposure occurred in a room separate from the colony room. The order and type of stressor were randomized, but all animals received the same number of exposures to each stressor over the 10-day period. Stressors included: (1) platform: 5 min exposure to an elevated Plexiglass platform (20 cm × 20 cm) mounted on a 90-cm high post; (2) cage tilt: the home cage was tilted at a 30° angle for 2 h; (3) novel cage: exposure to a novel, small (18 cm × 25 cm × 15 cm), and opaque cage without food and water for 1 h; (4) soiled cage: exposure to the soiled cage from another sex-matched pair of animals for 1 h; (5) restraint: restraint in PVC tubes (15 cm × 6 cm for females and 19 cm × 7 cm for males) for 30 min; (6) social isolation: overnight isolation in hanging wire mesh cage (20 cm × 23 cm × 18 cm) without food and water for 12 h; (7) white noise: exposure to white noise (40 dB; Lafayette Instruments model no. 15800) for 2 h; and (8) tail nick: a cut was made 1 mm from the tip of the tail for blood collection from the tail vein [blood sample data was published in Ref. (31)]. Non-CMS animals remained undisturbed other than routine husbandry during this same period.

#### **ACUTE STRESS (ELEVATED PLUS MAZE EXPOSURE) AND BRAIN COLLECTION**

The day after the end of CMS (day 11), all animals from both CMS and non-CMS conditions were habituated to the behavioral testing room for 10 min. The following day (day 12), animals were exposed to an elevated plus maze for 5 min in a dimly lit room during the light phase (09:00 and 12:00 h) of the light– dark cycle [elevated plus maze data were published in Ref. (31)]. Exposure to the elevated plus maze is a stressor known to produce an increase in corticosterone levels (35). The elevated plus maze consisted of two closed arms (69 cm × 10.5 cm) and two open arms (69 cm × 10.5 cm) with a central platform (diameter 35 cm). Closed arms had walls of darkened Plexiglass 20 cm high along their length. Open arms had a 2-cm-high lip along the edges of the arms. Immediately following the elevated plus maze exposure, animals were individually housed and left undisturbed in a quiet holding room for 30 min. Animals were then decapitated, and brains were removed, frozen on dry ice, and stored at −80°C.

#### **NEURAL ASSESSMENT OF C-FOS mRNA BY IN SITU HYBRIDIZATION Probes and labeling**

The ribonucleotide probe was prepared using a rat *c-fos* 2116 bp template provided by Dr. Victor Viau (Department Cellular and Physiological Sciences, The University of British Columbia, Canada). Probes were labeled with <sup>35</sup>S-UTP (Amersham Biosciences, NJ, USA) using Polymerase T<sup>7</sup> and Promega Riboprobe System (Promega Corporation, Madison, WI, USA). All probes were purified using Micro Bio-Spin 30 Columns (Bio-Rad, CA, USA). One molar of DTT was added to prevent oxidation.

#### **In situ hybridization**

Brains were sectioned coronally (20µm) using a cryostat (−16°C) and stored at −80°C. Thawed sections were fixed in formalin for 30 min and then pre-hybridized as follows: 1× PBS twice for 10 min each, proteinase K (100µg/L; at 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 twice for 10 min each, dehydration by a graded series of ethanol, chloroform for 5 min, and finally 100% ethanol before being air dried. Hybridization buffer (75% formamide, 3× SSC, 1× Denhardt's solution, 200µg/mL yeast tRNA, 50 mM sodium phosphate buffer (pH 7.4), 10% dextran sulfate, and 10 mM DTT) was applied (1 × 10<sup>6</sup> cpm/slide) and covered with HybriSlips (Sigma-Aldrich, ON, Canada). Sections were incubated overnight at 55°C in chambers humidified with 75% formamide. HybriSlips were removed and the slides were rinsed as follows: 2× SSC twice for 20 min, 2× SSC for 30 min, 50µg/L RNAse A solution (at 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 (at 60°C) for 60 min, and 0.1× SSC for 5 min. Sections were dehydrated by a graded series of ethanol and air dried overnight.

Kodak BioMax autoradiography film was exposed to hybridized slides of the ventral hippocampus for 28 days, and then developed using Kodak GBX developer and fixer. For all other areas, the hybridized slides were dipped in Kodak NTB2 autoradiography emulsion (diluted 50:50 with distilled water) and exposed in desiccated sealed, light tight boxes (4°C) for 49 days for mPFC, 70 days for mpdPVN, and 92 days for the amygdala. Slides were developed using Kodak D19 developer and standard Kodak fixer, counterstained with Toluidine Blue, and coverslipped with Permount (Fisher Scientific Ltd.). For all brain areas analyzed,*n* = 4–5 with the exception of the ventral hippocampal formation (CA1, CA3, DG, and ventral subiculum) where *n* = 3 for PF non-CMS males.

#### **Densitometric analysis**

The autoradiographic films for the ventral hippocampal formation were scanned and analyzed with Scion Image <IP_ADDRESS> (National Institutes of Health, USA). The left and right DG, CA1, and CA3, and ventral subiculum were traced freehand according to a stereotaxic rat brain atlas (46) in two sections per animal to determine mean gray density levels. Background was measured from the corpus callosum, and corrected gray levels were obtained by subtracting the background level from each of the four measurements. Left and right levels in each measured area were averaged together for analysis. For the emulsion dipped slides (amygdala, mPFC, and

mpdPVN), *in situ* signals were visualized with a Q-imaging monochrome 12-bit camera attached to a Zeiss Axioskop 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 10.3 software (National Institutes of Health, USA).

#### **STATISTICAL ANALYSIS**

Data were first analyzed using two-way analysis of variance (ANOVA for the factors of prenatal treatment and CMS exposure) followed by Newman–Keuls *post hoc* tests for each brain region (presented in **Figures 1**–**4**). Differences were considered significant at *p* ≤ 0.05. Further analyses utilized planned comparisons to test the *a priori* hypotheses that: (1) PAE will alter the response to acute stress, i.e., non-CMS PAE animals will show differential neural activity in response to acute stress (exposure to the elevated plus maze) compared to non-CMS control animals; and (2) CMS will differentially alter neural activity in response to acute stress in PAE compared to control animals.

In order to identify networks of brain regions altered by our experimental conditions, we employed CPCA, which is performed in two steps, referred to as the external analysis and the internal analysis. The external analysis consists of multivariate leastsquares multiple regression, which serves to separate the overall variance (i.e.,*c-fos* mRNA activity in the 13 brain regions of interest) into the variance that can (and cannot) be predicted by the experimental conditions (i.e., prenatal treatment and CMS exposure). This first step of CPCA simply separates the predicted and residual scores through multivariate multiple regression, using dummy-coded groupings as the independent variables [1 for group membership, 0 for not; (36)]. This first step produces a matrix of predicted scores that reflects the portion of variance in brain neural activity that is attributable to the experimental conditions (i.e., the predictable variance). A matrix of residual scores is also produced, which reflects the portion of variance in brain neural activity that is not attributable to experimental conditions (i.e., the residual variance). This residual variance was not further analyzed for the present study, as we were interested in brain networks associated with our experimental conditions. The second step in CPCA, the internal analysis, simply consists of a PCA on the predictable variance (i.e., the predicted scores from the multivariate regression in the first step). PCA is a data reduction technique that serves to combine large sets of related variables by reducing them to a smaller number of components (referred to as networks when the dependent variables consist of brain regions) that best explain the variance, while losing as little information as possible. The components that emerge from the PCA on the predicted scores reflect the brain networks that characterize the differences in brain activity between the experimental conditions (i.e., the brain networks that show differing degrees of activity across the different prenatal treatment and CMS groups). All PCA solutions were separately rotated using Varimax with Kaiser normalization, and the number of the components extracted was determined by inspection of scree plots (47, 48). In order to examine specifically how the groups differ in terms of brain activity in each of the resulting networks, correlations were computed between the experimental groups and the component scores from each of the extracted components. A more detailed description of the theory

**FIGURE 1 | Amygdala c-fos mRNA expression in response to the elevated plus maze test in male and female control (C), pair-fed (PF), and prenatal alcohol exposed (PAE) rats, with or without exposure to chronic mild stress (CMS) in adulthood**. Bars represent the integrated density (mean ± SEM) of c-fos mRNA expression in the lateral **(A,F)**, basal **(B,G)**, central **(C,H)**, medial **(D,I)**, and cortical **(E,J)** amygdala nuclei. \*Indicates a significant interaction between prenatal treatment and CMS

exposure where all groups are different from C non-CMS; & indicates a significant main effect of prenatal treatment where the post hoc test shows that PAE is different from C and PF, independent of CMS; <sup>∧</sup> 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 and PAE non-CMS are different from control non-CMS based on a priori comparisons (n = 4–5 for all groups).

and methodology of CPCA can be found in previously published manuscripts (38–41, 49, 50). CPCA was carried out using SPSS.

### **RESULTS**

#### **AMYGDALA**

#### **Males**

*c-fos* mRNA expression within the medial and cortical amygdala nuclei in response to acute stress was significantly reduced in non-CMS PAE and PF compared to non-CMS control males (**Figures 1D,E**). Moreover, while CMS reduced *c-fos* mRNA expression within the medial and cortical amygdala nuclei in control males compared to that in their non-CMS counterparts, *c-fos* mRNA expression of PAE and PF rats was similar under CMS and non-CMS conditions following acute stress {significant interaction between prenatal treatment and CMS exposure for medial [*F*(1,23) = 6.578; *p* < 0.007] and cortical [*F*(1,23) = 4.196; *p* < 0.03] amygdala nuclei}.

Similarly, *c-fos* mRNA expression within the basal amygdala in response to acute stress was significantly reduced in non-CMS PAE compared to non-CMS control males (**Figure 1B**). Moreover,CMS control males showed reduced *c-fos* mRNA expression within the basal nucleus compared to that in their non-CMS counterparts, whereas PAE rats were similar in *c-fos* mRNA expression following acute stress under both CMS and non-CMS conditions {no significant interaction between prenatal treatment and CMS interaction [*F*(1,23) = 3.129; *p* = 0.06]; *a priori* analysis indicated that PAE non-CMS (*p* < 0.005) and control CMS (*p* < 0.02) showed lower *c-fos* mRNA expression than the control non-CMS}.

Neither prenatal treatment nor CMS significantly altered *c-fos* mRNA expression within the central or lateral amygdala nuclei following acute stress {**Figures 1A,C**; no significant interaction between prenatal treatment and CMS exposure for central [*F*(1,22) = 0.803] and lateral [*F*(1,23) = 1.499] amygdala nuclei}.

#### **Females**

Among females, a different pattern of neural activity within the amygdala was observed. PAE, independent of CMS exposure, increased *c-fos* mRNA expression within the central and lateral amygdala nuclei in response to acute stress when compared to that in control and PF animals {**Figures 1F,H**; no significant interaction between prenatal treatment and CMS exposure for central [*F*(1,23) = 0.448] or lateral [*F*(1,23) = 0.387] amygdala nuclei, but significant main effect of prenatal treatment for central [*F*(2,23) = 7.198; *p* < 0.005] and lateral [*F*(2,23) = 4.910; *p* < 0.02] nuclei in response to acute stress}.

In the medial amygdala, CMS reduced *c-fos* mRNA expression in response to acute stress independent of prenatal treatment group {**Figure 1I**; no significant interaction between prenatal treatment and CMS exposure [*F*(1,23) = 0.015], but significant main effect for CMS [*F*(1,23) = 4.176; *p* = 0.05]}.

Neither prenatal treatment nor CMS significantly altered *c-fos* mRNA expression in response to acute stress within the cortical and basal amygdala nuclei {**Figures 1G,J**; no significant interaction between prenatal treatment and CMS exposure for cortical [*F*(1,23) = 0.312] or basal [*F*(1,23) = 0.257] amygdala nuclei}.

**FIGURE 3 | Medial prefrontal cortex c-fos mRNA expression in response to the elevated plus maze test in male and female control (C), pair-fed (PF), and prenatal alcohol exposed (PAE) rats, with or without exposure to chronic mild stress (CMS) in adulthood**. Bars represent the integrated density (mean ± SEM) of c-fos mRNA expression in the ACC **(A,D)**, PrL **(B,E)**, and IL **(C,F)**. \*Indicates a significant interaction between prenatal treatment and CMS exposure where the PAE non-CMS group is different from all other groups; <sup>∧</sup> indicates a significant main effect of CMS exposure (n = 4–5 for all groups).

**nucleus of hypothalamus c-fos mRNA expression in response to the elevated plus maze test in male (A) and female (B) control (C), pair-fed (PF), or prenatal alcohol exposed (PAE) rats, with or without exposure to chronic mild stress (CMS) in adulthood**. Bars represent the integrated density (mean ± SEM) of c-fos mRNA expression in the mpdPVN. ∧ Indicates a significant main effect of CMS exposure (n = 4–5 for all groups).

#### **HIPPOCAMPAL FORMATION**

#### **Males**

*c-fos* mRNA expression within the CA3 subregion of the ventral hippocampal formation in response to acute stress was significantly reduced in CMS PAE compared to non-CMS control males (**Figure 2B**). Moreover, similar to the medial and cortical amygdala, while CMS reduced *c-fos* mRNA expression within the CA3 region in control males compared to their non-CMS counterparts,*c-fos* mRNA expression of PAE and PF rats was similar under CMS and non-CMS conditions in response to acute stress {significant interaction between prenatal treatment and CMS exposure [*F*(1,19) = 3.714; *p* < 0.05]}.

Consistent with these findings, *c-fos* mRNA expression in response to acute stress within the CA1 subregion of the ventral hippocampal formation was significantly reduced in non-CMS PAE compared to non-CMS control males (**Figure 2A**). Moreover, while *c-fos* mRNA expression was reduced within the CA1, DG, and ventral subiculum subregions in CMS control compared to non-CMS control males, *c-fos* mRNA expression of PAE rats was similar under CMS and non-CMS conditions in response to acute stress {**Figures 2A,C,D**; no significant interaction between prenatal treatment and CMS interaction for CA1 [*F*(1,20) = 2.257], DG [*F*(1,20) = 1.472], or ventral subiculum [*F*(1,19) = 1.839]; *a priori* analysis indicates that non-CMS PAE showed a significantly lower *c-fos* mRNA expression within the CA1 (*p* = 0.05) than non-CMS controls, and that CMS controls (*p* < 0.03) showed lower *c-fos* mRNA expression than non-CMS control within CA1, DG, and ventral subiculum}.

#### **Females**

Among females, neither prenatal treatment nor CMS significantly altered *c-fos* mRNA expression in response to acute stress within any subregion of the hippocampal formation {**Figures 2E–H**; no significant interaction between prenatal treatment and CMS exposure for CA1 [*F*(1,21) = 0.193], CA3 [*F*(1,21) = 0.399], DG [*F*(1,21) = 0.130], or ventral subiculum [*F*(1,21) = 0.490]}.

#### **MEDIAL PREFRONTAL CORTEX**

#### **Males**

In all mPFC subregions (ACC, PrL, and IL), CMS reduced *cfos* mRNA expression in response to acute stress independent of prenatal treatment {**Figures 3A–C**; no significant interaction between prenatal treatment and CMS exposure for ACC [*F*(1,22) = 0.299], PrL [*F*(1,22) = 0.474], or IL [*F*(1,22) = 0.421], but significant main effect for CMS for ACC [*F*(1,22) = 7.426; *p* < 0.02], PrL [*F*(1,22) = 8.936; *p* < 0.008], and IL [*F*(1,22) = 5.365; *p* < 0.03]}.

#### **Females**

Similar to findings for the amygdala, *c-fos* mRNA expression in response to acute stress within all areas of the mPFC was significantly increased in non-CMS PAE females compared to non-CMS control and PF females (**Figures 3D–F**). Moreover,while CMS PAE females showed reduced *c-fos* mRNA expression within the ACC, PrL, and IL compared to non-CMS PAE females, *c-fos* mRNA expression of control and PF rats was similar under CMS and non-CMS conditions in response to acute stress {significant interaction between prenatal treatment and CMS exposure for ACC [*F*(1,23) = 4.614; *p* < 0.03], PrL [*F*(1,23) = 5.139; *p* < 0.02], and IL [*F*(1,23) = 6.525; *p* < 0.007]}.

#### **MEDIAL PARVOCELLULAR DORSAL DIVISION OF THE PARAVENTRICULAR NUCLEUS OF HYPOTHALAMUS Males**

In the mpdPVN, CMS reduced *c-fos* mRNA expression in response to acute stress independent of prenatal treatment {**Figure 4A**; no significant interaction between prenatal treatment and CMS exposure [*F*(1,23) = 0.257], but significant main effect for CMS [*F*(1,23) = 15.541; *p* < 0.001]}.

#### Raineki et al. Prenatal alcohol: stress and emotional regulation

#### **Females**

By contrast to males, neither prenatal treatment nor CMS significantly altered *c-fos* mRNA expression among females within mpdPVN in response to acute stress {**Figure 4B**; no significant interaction between prenatal treatment and CMS exposure [*F*(1,23) = 0.783]}.

#### **CONSTRAINED PRINCIPAL COMPONENT ANALYSIS Males**

The experimental conditions (i.e., prenatal treatment and CMS exposure) accounted for 34.17% of the total variance in *c-fos* mRNA expression in males. PCA on this constrained variance (i.e., the predictable variance) revealed a two-component solution; **Table 1** displays the component loadings for each of the 13 brain regions on each component. The first component explained 19.40% of the total variance and 56.78% of the predictable variance and was defined as *Amygdala* + *Hippocampal Formation* because the medial, cortical, and basal amygdala nuclei, and CA3 and CA1 hippocampal regions showed the highest loadings, with lesser contributions from the ventral subiculum, DG, and lateral amygdala (**Figure 5B**). The second component explained 11.26% of the total variance and 32.97% of the predictable variance and was defined as *Prefrontal Cortex* + *Paraventricular Nucleus* because the PrL, mpdPVN, and ACC showed the highest loadings, with lesser contributions from the IL and central amygdala (**Figure 5D**).

Correlations between the subjects' component scores and their experimental condition membership are displayed in **Figure 5A** for the *Amygdala* + *Hippocampal Formation network* and in **Figure 5C** for the *Prefrontal Cortex* + *Paraventricular Nucleus network*. The *Amygdala* + *Hippocampal Formation network* showed a significant positive correlation with the control non-CMS condition (*r* = 0.96, *p* < 0.0001), but a significant negative correlation with the PAE non-CMS condition (*r* = −0.39, *p* < 0.05). Furthermore, although not reaching statistical significance, the *Amygdala* + *Hippocampal Formation network* was also negatively correlated with the control CMS condition. As seen in **Figure 5C**, the *Prefrontal Cortex* + *Paraventricular Nucleus network* showed positive correlations with all non-CMS conditions; however only PAE non-CMS (*r* = 0.79, *p* < 0.0001) reached significance. Conversely, the *Prefrontal Cortex* + *Paraventricular Nucleus network* showed negative correlations with all CMS groups, with PAE (*r* = −0.44, *p* < 0.05) and PF (*r* = −0.45, *p* < 0.05) CMS conditions showing significant correlations.

#### **Females**

The experimental conditions accounted for 25.72% of the total variance in *c-fos* expression in females. PCA on this constrained (i.e., predictable) variance revealed a two-component solution (see **Table 2** for component loadings). The first component explained 12.04% of the total variance and 46.81% of the predictable variance and was defined as *Prefrontal Cortex* because all mPFC subregions (ACC, IL, and PrL) contributed with the highest loadings to this component (**Table 2**; **Figure 6B**). The second component explained 9.14% of the total variance and 35.56% of the predictable variance and was defined as *Amygdala* because the majority of amygdala nuclei (central, lateral, medial, and basal)

Values ≥0.30 are set in bold.

contributed with the highest loadings to this component; however, the IL also contributed to this component (**Table 2**; **Figure 6D**).

Correlations between the subjects' component scores and their experimental condition membership are displayed graphically in **Figure 6A** for the *Prefrontal Cortex network* and in **Figure 6C** for the *Amygdala network*. The *Prefrontal Cortex network* showed a significant positive correlation with the PAE non-CMS condition (*r* = 0.82, *p* < 0.0001). In contrast, the *Prefrontal Cortex network* showed a significant negative correlation with the PAE CMS condition (*r* = −0.64, *p* < 0.0001). Additionally, as seen in **Figure 6C**, the *Amygdala network* showed a significant positive correlation with the PAE group, independent of CMS condition (*r* = 0.56, *p* < 0.01 for PAE non-CMS and *r* = 0.67, *p* < 0.0001 for PAE CMS); however, this network was negatively correlated with the control group, again, independent of CMS condition. Furthermore, the *Amygdala network* showed a significant negative correlation with the PF non-CMS condition (*r* = −0.55, *p* < 0.01).

#### **DISCUSSION**

Stress and emotional regulation are dynamic and complex processes achieved by the fine coordination of interconnected brain regions, including, but not limited to, the amygdala, mPFC, hippocampus, and paraventricular nucleus of hypothalamus (PVN) (14–17, 51). The majority of these brain areas have dual functions, playing major roles in both the stress response and emotional regulation. Because of these dual roles and the intrinsic interconnectivity among these brain regions, dysfunction within or among structures in this neurocircuitry could result in dysregulation of the stress response and/or lead to mood and anxiety disorders. The present results indicate that, regardless of chronic stress later in life, PAE produces widespread alterations in neural activity within and between several brain areas of the stress/emotional neurocircuitry analyzed. Notably, male and female rats in the current study were differentially affected by PAE. Overall, PAE decreased neural activity within the amygdala and hippocampal formation in males and increased neural activity

**FIGURE 5 | Bars represent the correlation between the experimental group and the component scores for components 1 (A) and 2 (C) extracted from the CPCA for males**. Schematic figure highlighting the

**Table 2 | Component loadings for the predicted solution in females.**

Values ≥0.30 are set in bold.

within the amygdala and mPFC in females. Exposure to CMS also resulted in a sexually dimorphic effect, as it reduced neural activity within the mPFC and PVN in PAE males, but reduced activity in all areas analyzed in controls. Conversely, CMS reduced neural activity only in the mPFC of PAE females and had no effects in control females. Importantly, these patterns of neural activity were reflected in the differential networks of brain activation for PAE and control male and female rats. Indeed, the CPCA revealed that, contrary to non-CMS PAE males, non-CMS control males showed a strongly active *Amygdala* + *Hippocampal Formation network*. Conversely, non-CMS PAE males showed a strongly active *Prefrontal Cortex* + *Paraventricular Nucleus network*. Among females, non-CMS PAE animals showed strongly active *Prefrontal Cortex* and *Amygdala networks*, which contrasts with the network activity observed in non-CMS controls. Of note, CMS also altered the

networks of brain activation in PAE animals, as the pattern of activity within the *Prefrontal Cortex* + *Paraventricular Nucleus network*

areas that contributes to each neural network **(B,D)**. Images were adapted from Ref. (46), \*\*\*p < 0.0001; \*p < 0.05. C, control; PF, pair-fed; and PAE,

in PAE males and the *Prefrontal Cortex network* in PAE females switched from being active in the non-CMS animals to showing decreased activation in CMS animals. Overall, the changes in the network of brain areas following PAE could be implicated in the hyperresponsivity to stress and increased vulnerability to the development of psychopathologies observed among individuals prenatally exposed to alcohol (5, 9, 11–13, 22, 23).

#### **AMYGDALA**

prenatal alcohol exposed.

Clinical studies have consistently demonstrated that amygdala dysfunction is associated with mood and anxiety disorders (16, 17, 52, 53). These disorders are among the most prevalent mental health problems in children and adults exposed to alcohol during gestation (11–13). Animal models have supported the relationship between amygdala dysfunction and anxiety- and depressive-like behaviors (52–55). Specifically, the basal and lateral amygdala nuclei have been defined as part of the neural circuitry activated in animals exhibiting depressive-like behaviors (56, 57). Additionally, the central, lateral, and basal amygdala nuclei have a large population of neurons that express corticotropin-releasing hormone (CRH) and its receptors (58, 59); hyperfunction of these neurons can result in increased levels of anxiety-like behaviors (60, 61).

The present results indicate sexually dimorphic effects of PAE and/or CMS on neural activity of the amygdala nuclei. PAE females showed an increase in neural activity within the central and lateral amygdala nuclei in response to acute stress, regardless of whether they were exposed to CMS. Increased neural activity in the amygdala is consistent with the increased stress responsiveness as well as anxiety-/depressive-like behavior often seen in PAE females (5, 31, 32). However, vulnerability to anxiety-/depressive-like behavior is often greater in PAE animals following CMS than in their non-CMS counterparts, and under some conditions, non-CMS

PAE animals may not differ from non-CMS controls. We found, for example, that while non-CMS PAE females were similar to non-CMS controls in time spent on the open arms of an elevated plus maze, exposure to CMS significantly reduced frequency of total open arm entries for PAE but not control females (31). These discrepancies in neural activity and behavior suggest that other brain areas besides the amygdala may be modulating anxiety-like behavior among PAE females. In males, PAE and pair-feeding prevented the increase in neural activity within the medial, cortical, and basal amygdala nuclei shown by control non-CMS animals. However, compared to controls, neither PAE nor CMS changed the neural activity of central and lateral amygdala nuclei in response to acute stress. These data are interesting in light of the finding that PAE males exposed to CMS show more robust increases in anxiety-like behavior in the elevated plus maze than PAE females (31), suggesting that the neural mechanisms involved in mediating the anxiety response among males may be dissociable from those in females. This is supported by our finding of networks of brain regions that were differentially associated with PAE and/or CMS in males and females in the current study, as discussed further below.

In addition to its well-characterized role in emotional processing, the amygdala also regulates different aspects of the neuroendocrine stress response (15, 62). In general, the central nucleus of the amygdala regulates the HPA response to systemic stressors, and integrates autonomic responses to psychogenic stressors; the basal, lateral, and the medial amygdala nuclei regulate the HPA response to psychological stressors (62). The increased neural activity within the lateral amygdala of PAE females reported here may, at least partially, underlie the hyperresponsivity to stress observed in these animals (5, 9, 22, 23).

#### **HIPPOCAMPAL FORMATION**

The hippocampal formation is well known for its role in learning and memory, stress, and emotional regulation (63). However, the dorsal and ventral components of the hippocampal formation have different functions: the dorsal hippocampus is primarily involved in cognitive functions while the ventral hippocampus is more essential for stress and emotional regulation (63). For example, lesions to the ventral hippocampus result in decreased anxietylike behaviors (63, 64), whereas increased activity in the ventral hippocampus is positively correlated with anxiety-like behavior (65, 66). Additionally, the hippocampal formation is consistently associated with an overall inhibition of the stress response (14, 15). Indeed, hippocampal lesions result in increased stress responses and, in some cases, increased basal levels of corticosterone (67). Stimulation of the CA3, DG, and ventral subiculum subfields, on the other hand, results in reduced glucocorticoid secretion (68). Interestingly, PAE is known to induce morphological changes that can lead to functional deficits in the hippocampus: studies in the clinical literature reveal reduced hippocampal volume in individuals exposed to alcohol during gestation (20, 21). In rats, PAE results in hypertrophy of mossy fibers (69), reduced numbers of dendritic spines (70), as well as impaired long-term potentiation (71, 72) and adult neurogenesis – primarily in males (73, 74). In agreement with these findings, our results indicate that PAE males show reduced neural activity in the hippocampal formation in response to acute stress when compared to controls. This deficit in neural activity suggests that the inhibitory action of the hippocampal formation may be reduced, which could contribute to the hyperresponsivity to stress, and ultimately to the increase in anxiety-like behaviors observed in PAE animals (31).

Interestingly, PAE results in no apparent changes in basal expression of glucocorticoid and mineralocorticoid receptors in the hippocampal formation of male animals (75–77), whereas in females, some studies indicate a reduction (77), whereas others report no change (75, 76) in basal expression of glucocorticoid and mineralocorticoid receptor mRNA. However, removal of the inhibitory feedback effects of corticosterone on the HPA axis by adrenalectomy unmasked alterations in basal HPA regulation not apparent in intact animals. Glucocorticoid receptor expression was differentially increased in the hippocampus of PAE males (75) whereas mineralocorticoid receptor expression was differentially increased in the hippocampus of PAE females compared to their control counterparts (75). Moreover, corticosterone replacement was shown to be ineffective at normalizing the adrenalectomyinduced increase in hippocampal mineralocorticoid mRNA levels in PAE males. Together, these results indicate that altered basal regulation of the HPA axis may play a critical role in the hyperresponsivity to stress observed in individuals exposed to alcohol during the prenatal period.

Our results also indicate that CMS reduced the neural activity in the hippocampal formation of control males. This is not surprising given the previous finding that CMS can reduce the number of hippocampal granule cells (78), which could be related to reduced survival of newborn cells (79).

#### **MEDIAL PREFRONTAL CORTEX**

The mPFC also plays a dual role in modulating stress and emotional responses. Abnormal volume and activation of the PFC are commonly observed in individuals with anxiety and mood disorders (16, 52). Importantly, functional neuroimaging studies indicate that individuals exposed to alcohol during gestation show greater blood oxygen level-dependent (BOLD) responses in the PFC when performing a behavioral inhibition task (18), suggesting that the PFC could be responding differentially when challenged. In rodents, reduced function of the mPFC, induced by electrolytic lesions or temporary deactivation, results in variable effects: some authors report decreased anxiety-like behaviors (80– 82) while others report increased anxiety-like behaviors (83, 84). These discrepancies on the effects of the mPFC on anxiety-like behavior may be due to the extent of the lesions/deactivation and on the heterogeneous functions of the mPFC subregions. However, overall dysregulation of the mPFC can lead to an increased predisposition to psychopathologies. Our results show that PAE and control females exhibit different mPFC responses to the acute stress. Despite the fact that mPFC neural activity is increased in PAE non-CMS females, their behavioral performance was not different from controls in the elevated plus maze (31). Conversely, the neural activity within the mPFC of PAE males was not different from controls; however, PAE increased the expression of anxiety-like behaviors in male rats (31). This dissociation between behavioral and mPFC neural outcomes likely reflects the complex nature of anxiety- and depressive-like disorders, and suggests that other brain areas besides the mPFC may play a role in supporting increased anxiety-like behaviors observed in PAE animals.

The mPFC also plays a major role in modulating the HPA response to stressful events. However, its role is complex due to the fact that the PrL and ACC subregions are involved in inhibition while the IL is involved in excitation of the HPA axis (15, 85–87). Regardless of these opposing roles, our results show that the neural activity within the PrL and ACC in PAE and control animals follows a pattern similar to that within the IL. Specifically, PAE non-CMS females showed a higher response to acute stress in all mPFC areas than their female counterparts, with CMS reducing this neural activity. For the PrL, it is possible that the

reduced neural activity in PAE females exposed to CMS may be associated with a reduction in CRH mRNA expression within this area (77), suggesting that the inhibitory action of the PrL may be reduced following CMS, contributing to the hyperresponsivity to stress in PAE females. Similar to the PrL, the ACC is also associated with inhibition of the HPA axis (15, 85–87) and the reduced neural activity of the ACC in PAE females exposed to CMS could also contribute to the hyperresponsivity to stress in those animals. Despite an opposite role, the IL – which is involved in excitation of the HPA axis (15, 85–87) – also showed reduced neural activity in PAE females exposed to CMS, which suggests a possible alteration in the excitatory/inhibitory balance within the mPFC of PAE females.

#### **MEDIAL PARVOCELLULAR DORSAL DIVISION OF THE PARAVENTRICULAR NUCLEUS OF HYPOTHALAMUS**

The mpdPVN is composed of a discrete set of neurons that integrate all direct and indirect stress-related inputs from the amygdala, mPFC, and hippocampal formation in order to launch an appropriate response to stress (15, 62). These neurons synthesize and secrete CRH, the primary regulator of the pituitary–adrenal axis (88). Our results indicate that, independent of sex, PAE, and control rats showed similar neural activity in the mpdPVN in response to the acute stress of exposure to the elevated plus maze. However, when facing a noxious stressor, such as shock, or a major challenge to HPA regulation, such as adrenalectomy, PAE has been shown to induce enhanced neural activity within the PVN (75, 89). Studies have demonstrated that exposure to the elevated plus maze induces small increases in neural activity within the PVN of control animals when compared to the neural activity of animals exposed to shock (34). These results suggest that when PAE animals are tested under basal conditions, or face a mild stressor such as the elevated plus maze, the PVN is capable of responding appropriately; however, when faced with a more severe stressor such as shock, the PVN launches an inappropriately large response.

#### **CONSTRAINED PRINCIPAL COMPONENT ANALYSIS**

The univariate results above indicate that PAE is associated with changes in neural activity across a wide range of brain regions; however, it is important to examine not only individual brain regions, but also how these regions interact to form networks and whether these networks might be differentially altered by PAE and/or CMS. In order to examine this possibility, we employed CPCA, which allowed for the identification of combinations of brain regions that were affected by PAE. CPCA is an ideal statistical technique for examining networks of brain regions that are related to one or more variables of interest (in the current study, prenatal treatment and CMS exposure) because networks are defined only from the portion of the overall variance that is predictable from these independent variables. As such, it was possible to identify networks of brain regions that were associated with PAE (and CMS) in the current study. Comparison between the univariate and multivariate results shows several similarities in terms of the brain regions affected by PAE and CMS; however, the aggregation of these brain regions into networks through CPCA facilitates interpretation.

#### **Networks regulating stress and emotion in males**

In males, CPCA revealed two functional networks that were collectively responsible for 34.17% of the total variation. The first network included the majority of the amygdala nuclei (medial, cortical, basal, and lateral) and all of the hippocampal formation areas analyzed (CA1, CA3, DG, and ventral subiculum). This *Amygdala* + *Hippocampal Formation network* was strongly active in control animals that were not exposed to CMS, but showed decreased activation in controls subjected to CMS. These findings suggest that the *Amygdala–Hippocampal Formation network* is critically involved in stress and emotional regulation in control males, and that exposure to chronic unpredictable stress induces functional changes in this network by preventing typical neural activation of the amygdala and hippocampal formation in response to acute stress. Dysregulation in this network may partially explain the increased susceptibility to the development of psychopathologies and abnormal stress responses in animals subjected to CMS (90–93). Importantly, in contrast to controls, the *Amygdala* + *Hippocampal Formation network* showed decreased activation for PAE non-CMS animals, indicating that PAE may alter the typical neural activity in these areas such that PAE animals in the non-CMS conditions look similar to controls in the CMS condition.

The second network extracted from the CPCA included all of the mPFC areas analyzed (PrL, ACC, and IL) and the mpdPVN. This *Prefrontal Cortex* + *Paraventricular Nucleus network* was activated to some degree in all prenatal groups in the non-CMS condition, although activation of this network was statistically significant only in the PAE non-CMS group. These data suggest that, independent of prenatal treatment, the mPFC and the mpdPVN form a network of structures that work together in the regulation of stress and emotion in males, but that PAE animals engage this network to a greater degree than control animals. This phenomenon could be a compensatory mechanism for the reduced involvement of the *Amygdala* + *Hippocampal Formation network*, as indicated by the negative correlation for PAE non-CMS males with this network. Additionally, all prenatal groups subjected to CMS in adulthood showed reduced activity in the *Prefrontal Cortex* + *Paraventricular Nucleus network* following acute stress. This switch between positive and negative associations with this network following exposure to CMS highlights the vulnerability of the mPFC and mpdPVN to chronic and unpredictable stress in adulthood. Indeed, the preclinical literature has demonstrated that the PFC is extremely susceptible to the effects of CMS, as chronic stress results in morphological alterations to mPFC neurons, including retraction of the apical dendritic branches and spine loss in layer II/III neurons (94–96). Additionally, stress-related morphological changes in the mPFC have been associated with increased anxietyand depressive-like behaviors (90, 97) as well as with dysfunction in attentional set shifting and working memory (98, 99).

#### **Networks regulating stress and emotion in females**

Similar to males, CPCA indicated two functional neural networks in females. However, these networks were more restricted and only included major subdivisions of the same brain regions. The first network (*Prefrontal Cortex*) included all areas of the mPFC (ACC, IL, and PrL); the second network (*Amygdala*) included four nuclei of the amygdala (central, lateral, medial, and basal) and the IL. Together, these two functional neural networks in females accounted for 25.72% of the total variation, in contrast to 34.17% accounted for by the two functional networks in males.

The *Prefrontal Cortex network* was strongly activated in PAE non-CMS females, which contrasts with the decreased activity observed among control non-CMS females. Moreover, exposure to CMS resulted in a negative association for PAE females with the *Prefrontal Cortex network*, whereas there was no significant change in activation of this network in control females. These results indicate that PAE females rely on the *Prefrontal Cortex network* more than control females for stress and emotional regulation. This abnormal engagement of the *Prefrontal Cortex network* in response to acute stress is also observed in PAE males and indicates that alcohol exposure during the gestational period results in dysregulation of mPFC function in both sexes. Additionally, our results suggest that the mPFC of PAE females is more susceptible to the effects of CMS when compared to controls. The switch between positive and negative associationsfor the *Prefrontal Cortex network* following exposure to CMS highlights the vulnerability of the mPFC to chronic and unpredictable stress.

Similarly, the *Amygdala network*, which also contains the IL region, showed decreased activity in controls,independent of CMS condition, but was significantly positively activated in PAE females independent of CMS. As the amygdala and the IL subregion of the mPFC activate the HPA axis (14, 15), this abnormal increased activity of the *Amygdala network* following PAE may underlie, at least in part, the hyperresponsivity to stress observed in those animals. Additionally, the activity of the *Amygdala network* in response to acute stress in control and PAE females was not altered by CMS, indicating that, regardless of the level of activity, the amygdala of controls and PAE females is more resistant to chronic and unpredictable stress.

#### **PAIR-FEEDING**

The PF group is used to control the reduced food intake typically observed in alcohol-consuming dams. However, pair-feeding is an imperfect control procedure as it cannot control for the effects of alcohol on absorption and utilization of nutrients. Additionally, despite receiving optimal nutrition during pregnancy (43), the PF dams are underfed compared to the *ad libitum*-controls, as they receive a reduced ration matched to that of an alcoholconsuming partner. As a result, they are hungry, and typically consume their entire daily food ration within a few hours of presentation, remaining food deprived for the rest of the day. This pair-feeding regimen is thus a mild prenatal stressor, and its effects on offspring behavioral and physiological responsiveness represent, at least partially, an effect of stress above and beyond the nutritional aspect of receiving a reduced food ration. Our results suggest that PF males showed a pattern of neural activity within the amygdala and hippocampal formation similar to PAE males, while the pattern of neural activity within the mPFC and mdpPVN was similar to both PAE and controls. For females, on the other hand, the pattern of neural activity for PF animals was similar to the pattern displayed by the control group. Taken together, these results suggest that the males may be more susceptible to the effects of pair-feeding than females. However, the only effect exclusively observed in PF animals was a significant negative correlation of the *Amygdala network* with the non-CMS PF female condition, which suggests that stress and emotional regulation rely less on the amygdala in PF than PAE or control females.

#### **SUMMARY AND CONCLUSION**

The results presented here build on and expand our knowledge of the differential neural regulation of stress and emotion in individuals exposed to alcohol during gestation. Importantly, despite a relatively low number of animals in one of the experimental groups, the use of CPCA provided a powerful approach for assessing global changes in functional neural networks in PAE compared to control animals, allowing us to go beyond simple assessments of changes in individual brain areas. Indeed, our results highlight how PAE and control males and females recruit different brain networks in the regulation of stress responses and emotion. Specifically, CPCA indicates that while control males rely more on the amygdala and hippocampal formation, PAE males rely more on the mPFC and mpdPVN when facing a stressful situation. In contrast, the functional neural networks underlying stress and emotional regulation in females are more restricted, as PAE females rely primarily on the mPFC and amygdala. Additionally, our results indicate that CMS differentially affected the neural networks regulating stress and emotion in PAE and control animals. Indeed, exposure to CMS reduced the activity of the*Amygdala* + *Hippocampal Formation network* in control males, but reduced the activity of the *Prefrontal Cortex* + *Paraventricular Nucleus network* in PAE males. For females, CMS only reduced the activity of the *Prefrontal Cortex network* in PAE animals. Together, our results suggest that PAE, regardless of its association with CMS, results in a sexually dimorphic dysregulation of the neurocircuitry that underlies stress and emotional regulation, which may be implicated in the stress hyperresponsivity and increased vulnerability to anxiety and depressive disorders observed among individuals exposed to alcohol during gestation (5, 9, 22, 23, 31). Finally, understanding the underlying neural mechanisms of deficits induced by PAE is a crucial step toward the establishment of specific strategies for treating resultant psychopathologies in these individuals.

#### **ACKNOWLEDGMENTS**

This research was funded by NIH/NIAAA grants R37 AA007789 and R01 AA022460, and NeuroDevNet (Canadian Networks of Centers of Excellence) grant 20R64153 to Joanne Weinberg, and Canadian Foundation of Fetal Alcohol Research (CFFAR) grant to Charlis Raineki and Joanne Weinberg. Kim G. C. Hellemans was funded by awards from the Michael Smith Foundation for Health Research and IMPART (Canadian Institute if Health Research STIHR). Tamara Bodnar wasfunded by Natural Sciences and Engineering Research Council of Canada (NSERC) graduate scholarship. Todd S. Woodward was funded by career investigator awards from CIHR and the Michael Smith Foundation. Katie M. Lavigne was funded by a CIHR award in partnership with the British Columbia Schizophrenia Society.

#### **REFERENCES**

1. Davidson RJ,McEwen BS. Social influences on neuroplasticity: stress and intervention to promote well-being. *Nat Neurosci* (2012) **15**:689–95. doi:10.1038/ nn.3093

hippocampus activity. *Neuron* (2011) **71**:898–910. doi:10.1016/j.neuron.2011. 07.027

**Conflict of Interest Statement:** 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.

*Received: 31 October 2013; accepted: 10 January 2014; published online: 13 February 2014.*

*Citation: Raineki C, Hellemans KGC, Bodnar T, Lavigne KM, Ellis L, Woodward TS and Weinberg J (2014) Neurocircuitry underlying stress and emotional regulation in animals prenatally exposed to alcohol and subjected to chronic mild stress in adulthood. Front. Endocrinol. 5:5. doi: 10.3389/fendo.2014.00005*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2014 Raineki, Hellemans, Bodnar, Lavigne, Ellis, Woodward and Weinberg . This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### Glucocorticoid programing of the mesopontine cholinergic system

#### **Sónia Borges 1,2, Bárbara Coimbra1,2, Carina Soares-Cunha1,2, Ana P. Ventura-Silva1,2, Luisa Pinto1,2 , Miguel M. Carvalho1,2, José-Miguel Pêgo1,2, Ana João Rodrigues 1,2\* and Nuno Sousa1,2\***

<sup>1</sup> Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal

2 ICVS/3B's – PT Government Associate Laboratory, Braga/Guimarães, Portugal

#### **Edited by:**

Nikolaos P. Daskalakis, Icahn School of Medicine at Mount Sinai, USA

#### **Reviewed by:**

Therese Jay, INSERM U894, France Li-Tung Haung, Chang Gung University College of Medicine, Taiwan

#### **\*Correspondence:**

Ana João Rodrigues and Nuno Sousa, Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal e-mail: ajrodrigues@ ecsaude.uminho.pt<EMAIL_ADDRESS>
Stress perception, response, adaptation, and coping strategies are individually distinct, and the sequel of stress and/or glucocorticoids (GCs) is also distinct between subjects. In the last years, it has become clear that early life stress is a powerful modulator of neuroendocrine stress-responsive circuits, programing intrinsic susceptibility to stress, and potentiating the appearance of stress-related disorders such as depression, anxiety, and addiction. Herein we were interested in understanding how early life experiences reset the normal processing of negative stimuli, leading to emotional dysfunction. Animals prenatally exposed to GCs (in utero glucocorticoid exposure, iuGC) present hyperanxiety, increased fear behavior, and hyper-reactivity to negative stimuli. In parallel, we found a remarkable increase in the number of aversive 22 kHz ultrasonic vocalizations in response to an aversive cue. Considering the suggested role of the mesopontine tegmentum cholinergic pathway, arising from the laterodorsal tegmental nucleus (LDT) and pedunculopontine tegmental nucleus (PPT), in the initiation of 22 kHz vocalizations and hypothetically in the control of emotional arousal and tone, we decided to evaluate the condition of this circuit in iuGC animals. Notably, in a basal situation, iuGC animals present increased choline acetyltransferase (ChAT) expression in the LDT and PPT, but not in other cholinergic nuclei, namely in the nucleus basalis of Meynert. In addition, and in accordance with the amplified response to an adverse stimulus of iuGC animals, we found marked changes in the cholinergic activation pattern of LDT and PPT regions. Altogether, our results suggest a specific cholinergic pathway programing by prenatal GC, and hint that this may be of relevance in setting individual stress vulnerability threshold.

**Keywords: glucocorticoids, stress, acetylcholine, anxiety, fear, pedunculopontine tegmental nucleus, laterodorsal tegmental nucleus, ultrasonic vocalizations**

#### **INTRODUCTION**

Exposure to stressful events or synthetic glucocorticoids (GCs), such as dexamethasone, early in life, are a risk factors for the development of different neuropsychiatric disorders in adulthood, namely depression and anxiety (1). Such effects are partially mediated by de-regulation of the hypothalamic-pituitaryadrenal (HPA) axis, leading to altered GC secretion (2, 3), which can induce long-term molecular and functional changes in GCsensitive nuclei. Importantly, several recent studies showed that high levels of GCs have a deleterious effect in the developing brain, inducing prominent neurochemical, structural, and molecular changes in several brain regions (4–8) culminating in the development of anxious and depressive-like traits (9–13).

GC programing effects in emotional behavior are far from being completely understood at a mechanistic level, but it has been shown that GCs modulate on the long-term the activity of particular neural pathways. In this perspective it is interesting to refer that early life stress/GC exert a powerful effect in the developing dopaminergic neurons, especially at the mesolimbic level (7). For example, we have shown that animals prenatally exposed to GC present marked hypodopaminergia and D2 epigenetic/expression changes, responsible for their anhedonia and motivational deficits, since administration of a dopamine precursor, l-DOPA, fully reverted the molecular and behavioral impairments (14, 15). Obviously, this dopaminergic de-regulation may also occur indirectly through modulation of upstream neurotransmitter systems. One promising candidate is the ascending cholinergic pathway, comprising projections from acetylcholine (ACh)-rich nuclei within the pons, particularly the laterodorsal (LDT) and pedunculopontine tegmental nuclei (PPT) to the ventral tegmental area (VTA) (16–18). In support of this, it was already shown that stress/GC strongly elicit cholinergic activity of these regions (19), and that they, in turn, critically affect basal and phasic activity of VTA neurons (20–23). Additionally, GC can putatively bind to GC-responsive elements of cholinergic players and control their expression, namely choline acetyltransferase (ChAT) (24) and acetylcholine esterase (AChE) (25), two enzymes of the cholinergic pathway. Also relevant is to note that the ascending cholinergic system is implicated in the control of the stress response by modulating hypothalamic pituitary adrenal (HPA) axis function (26–28) and in mediating the anxiogenic effects of stress (29–31).

Surprisingly, very few studies have focused on the impact of stress/GC exposure in the cholinergic system. Considering the lack of evidence, herein, we focused on the impact of prenatal exposure to GCs in the ascending cholinergic system, and the relevance of such changes in stress-related anxious and fear behaviors.

#### **MATERIALS AND METHODS**

#### **ANIMALS AND TREATMENTS**

All manipulations were conducted in accordance with local regulations on animal care and experimentation (European Union Directive 2010/63/EU). Pregnant Wistar Han rats were subcutaneously injected with the synthetic GC dexamethasone at 1 mg kg−<sup>1</sup> (*in utero* glucocorticoid exposure, iuGC animals), or with vehicle (CONT; control animals), on days 18 and 19 of gestation.

Dexamethasone dosage was selected based on our previous studies showing that this regimen effectively impairs HPA axis activity in a long-term basis (12). From a clinical perspective, guidelines on prenatal corticotherapy (32, 33) recommend single course administration (0.3–0.5 mg/kg), however, multiple courses of GCs are often administered (34), despite the lack of evidence of increased therapeutic efficacy. Nevertheless, we must consider the difficulty in the transposition of human doses to rodents due to ADME species specificity.

At weaning day, male offspring were house-paired randomly, according with prenatal treatment, under standard laboratory conditions: artificial 12 h light/dark cycle (lights on from 08:00 a.m. to 08:00 p.m.); room temperature 22°C; food and water were provided *ad libitum*. Animals derived from at least three different litters were used for all the experimental procedures.

#### **BEHAVIORAL TESTS**

All tests were performed during the day period, except the confined cage and fear-conditioning protocols that were conducted during the night period (08:30 p.m. to 03:00 a.m.). All behavioral equipment was cleaned between animals (ethanol 10%) in order to remove any olfactory cues.

#### **OPEN FIELD**

The open field (OF) test was conducted in an arena (43.2 cm × 43.2 cm) with transparent acrylic walls and white floor (Med Associates Inc., St. Albans, VT, USA). Rats were placed in the center of the arena and movement was monitored over a period of 10 min with the aid of two 16-beam infrared arrays. Total distance traveled was used as an indicator of locomotor activity.

#### **ELEVATED PLUS MAZE**

The elevated plus maze (EPM) test was carried out under bright white light. Animals were placed individually for 5 min in the center of a black polypropylene plus-shaped platform elevated 72.4 cm above the floor. The apparatus consisted of two open arms (50.8 cm × 10.2 cm) and two closed arms (50.8 cm × 10.2 cm × 40.6 cm) (MedAssociates Inc., St. Albans, VT, USA). The number of entries into each arm and the time spent therein were recorded.

#### **LIGHT/DARK BOX TEST**

The light/dark box (L/D) test was performed inside the OF arena (43.2 cm × 43.2 cm) (MedAssociates Inc., St. Albans, VT, USA). A dark compartment was attached to one side with an opening facing the center of the arena. Animals were individually placed in the center of the illuminated part. The distance traveled and time spent in each compartment was recorded in a single trial of 10 min.

#### **CONFINED CAGE TEST**

The confined cage test was performed in a non-restrictive Plexiglas cylinder (inner diameter 8.8 cm, length 22.2 cm), mounted on a Plexiglas platform and placed in a ventilated, sound-attenuated chamber (SR-LAB, San Diego Instruments, San Diego, CA, USA). A stainless steel grid was placed inside the cylinder, through which an electric current could be passed (shock chamber). A microphone and a video camera were placed inside the sound-attenuated chamber. The protocol was performed in two consecutive days, in which the animals were placed inside the shock chamber for 3 min. The ultrasonic vocalizations (USVs) and the percentage of total freezing time were measured.

#### **FEAR-CONDITIONING PARADIGM**

The fear-conditioning test was performed in a non-restrictive Plexiglas cylinder (inner diameter 8.8 cm, length 22.2 cm), mounted on a Plexiglas platform and placed in a ventilated, sound-attenuated chamber (SR-LAB, San Diego Instruments, San Diego, CA, USA). The protocol was performed in three consecutive days (35). On the first day (habituation), each animal was placed in the shock chamber for 11 min. On the second day (conditioning), each subject was positioned inside the shock chamber for 3 min (no light, no shock). Afterwards, animals were exposed to six lights/shock pairings (0.4 ± 0.1 mA), with an inter-stimulus-interval (ISI) of 60 s. The shock was given for 500 ms, immediately after the cue light was turned off. On the following day (test day), after an initial phase of 3 min without light, animals were presented with a 20 s cue light for six times, but no shock was given (ISI of 60 s). During all procedures, USVs and freezing behavior were recorded.

#### **USVs ANALYSIS**

An ultrasound microphone (CM16/CMPA, Avisoft Bioacoustics, Berlin, Germany) sensitive to frequencies of 10–200 kHz, placed 15 cm above the floor, was used in all experiments. The microphone was connected via an Avisoft UltrasoundGate 416H (Avisoft Bioacoustics) to a personal computer; USVs were recorded using the Avisoft-Recorder (version 5.1.04) with the following settings: sampling rate: 250,000; format: 16 bit. For acoustical analysis, recordings were transferred to Avisoft SASLab Pro (version 5.1.22, Avisoft Bioacoustics). This program was used in order to produce spectrograms of USVs by conducting a fast Fourier transformation (256 FFT-length, 100% frame, Hamming window filter, 50% time window overlap). These spectrograms had a frequency resolution ∼1.2 kHz and a temporal resolution ∼0.4 ms.

Twenty-two kilohertz call detection was provided by an automated threshold-based algorithm (threshold: −40 dB) and a hold time mechanism (hold time: 20 ms). A lower-cut-off-frequency of 18 kHz was used to reduce background noise.

Calls were also inspected manually to ensure that, when necessary, USVs not detected automatically could be subsequently included in the automatic parameter analysis.

#### **IMMUNOHISTOCHEMISTRY**

For immunohistochemistry (IHC), 11 rats were sacrificed by pentobarbital (Eutasil, Sanofi) anesthesia and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (pH 7.4 in 0.1 M phosphate buffer). Brains were removed and post-fixed for 48 h in 4% paraformaldehyde and then rinsed and stored in 30% of sucrose until sectioning. Brains were sectioned coronally, at a thickness of 50µm, on a vibratome (VT1000S, Leica, Germany) and stored in cryoprotectant solution at −20°C until use.

Briefly, free-floating sections were pre-treated with 3% H2O<sup>2</sup> in PBS, thoroughly rinsed in PBS, blocked with 2.5% fetal bovine serum (FBS) in PBS-Triton 0.3% for 2 h at room temperature, and then incubated overnight at 4°C with primary antibody goat anti-ChAT (Millipore, MA, USA; 1:1000). Afterwards, sections were washed, incubated with the secondary biotinylated anti-goat (Vector Lab., USA; 1:200) for 1 h, and processed with an avidin-biotin complex solution (ABC-Elite Vectastain reagent; Vector Lab, USA). Detection was done using 0.5 mg/ml 3,3<sup>0</sup> -diaminobenzidine. Sections were washed and mounted on glass slides, air-dried, counterstained with hematoxylin, and cover slipped with Entellan-New (Merck, Darmstadt, Germany).

#### **IMMUNOFLUORESCENCE**

Ninety minutes after completion of the fear-conditioning paradigm, 12 animals were deeply anesthetized with pentobarbital (Eutasil, Sanofi) and were transcardially perfused with 0.9% saline followed by 4% paraformaldehyde. Brains were removed and post-fixed in 4% paraformaldehyde. Coronal vibratome sections (50µm) were first incubated with rabbit anti-c-fos primary antibody (1:1000; Ab-5, Calbiochem, USA), followed by incubation with goat anti-ChAT primary antibody (1:1000; anti-ChAT, Millipore, MA, USA).  Finally, all sections were stained with 4<sup>0</sup> ,6-diamidino-2-phenylindole (DAPI; 1 mg/ml). For each animal, c-fos-positive cells within the PPT and LDT were analyzed after double staining with cholinergic (ChAT) marker and cell counts were performed by confocal microscopy (Olympus FluoViewTMFV1000, Hamburg, Germany). Estimation of cell density was obtained by crossing cell number values with the corresponding areas, determined using an Olympus BX51 optical microscope and the StereoInvestigator software (Microbrightfield, VT, USA).

#### **STEREOLOGICAL PROCEDURES**

Eleven animals were perfused transcardially with 4% paraformaldehyde, under deep pentobarbital (Eutasil, Sanofi) anesthesia. Brains were included in glycolmethacrylate (Tecnovit 7100, Heraeus Kulzer, Wehrheim, Germany) and sectioned on a microtome as described in detail elsewhere (36). Every other 30µm thick coronal section was collected on a gelatinized slide, stained with Giemsa, mounted with Entellan-New (Merck, Darmstadt, Germany), and

cover slipped. Stereological procedures were performed by a blind observer.

Laterodorsal tegmental and PPT regions were outlined according to the atlas of Paxinos and Watson (37) and based on noticeable cytoarchitectural differences, namely density of cells and size of the perikarya.

Volume and neuronal number estimations were performed using StereoInvestigator software (Microbrightfield,VT, USA) and a camera attached to a motorized microscope (Axioplan 2, Carl Zeiss, Germany). The Cavalieri's principle was used to assess volume. Briefly, every second section was used and the cross-sectional area was estimated by point counting (final magnification 112×). We used a test point system in which the interpoint distance, at the tissue level, was 150µm for LDT and 200µm for PPT. The volume of the region of interest was calculated from the number of points within its boundaries and the distance between sampled sections.

Average cell numbers were estimated using the optical fractionator method (38). Briefly, a grid of virtual 3D-boxes (LDT: 30µm × 30µm × 20µm; PPT: 40µm × 40µm × 20µm) equally spaced (using the same grid spacing as for volume estimations) was superimposed on every second section of the lamina of interest and cells within boxes were counted. Coefficients of error (CE) were automatically computed, according to the formulas of Gundersen et al. (39) for cell numbers and Gundersen et al. (40) for volume estimations. Glial cells were not included in the estimations, and the discrimination between neuronal and glial cell body profiles was based on the criteria described by Ling et al. (41) and Peinado et al. (42).

#### **STATISTICAL ANALYSIS**

Statistical analysis was performed in GraphPad Prism 5.0 (Graph-Pad Software, Inc., La Jolla, CA, USA). Statistical analysis between two groups was made using Student's *t*-test or Mann–Whitney tests. Two-way analysis of variance (ANOVA) was used when appropriate. Bonferroni's *post hoc* multiple comparison test was used for group differences determination. Non-parametric analysis (Mann–Whitney test) was used when normality of data was not assumed. Results are presented as mean ± SEM. Statistical significance was accepted for *p* ≤ 0.05.

#### **RESULTS**

#### **IN UTERO GLUCOCORTICOID EXPOSURE IMPAIRS EMOTIONAL BEHAVIOR**

Animals were exposed to a battery of behavioral tests that consisted of paradigms studying spontaneous exploratory behavior (OF), tasks of innate anxiety (EPM, L/D test, confined cage), and reactivity to adverse stimulus (version of fear-conditioning paradigm). Since USVs can give information on the emotional status of the animal, we decided to further complement the behavioral characterization by measuring USVs in these different paradigms.

In the OF, iuGC animals presented a decrease in the number of ambulatory counts (**Figure 1A**, *t* = 2.197, *p* = 0.037) and total distance traveled (**Figure 1B**, *t* = 3.002, *p* = 0.006) when compared with control animals. In addition, iuGC animals exhibited a decrease in the percentage of time spent in the center of the arena (**Figure 1C**, *t* = 2.416, *p* = 0.023).

In the EPM, iuGC animals spent significantly less time in the open arms (**Figure 1D**, *t* = 2.947, *p* = 0.009) and presented a reduction in the number of open arms entries (**Figure 1E**, *t* = 2.375, *p* = 0.031), when compared with control animals. No differences were found in the time and entries in the closed arms (data not shown; time: *t* = 0.479, *p* = 0.637; entries: *t* = 0.872, *p* = 0.392).

In the L/D test, iuGC animals presented an increase in the ratio dark/light (**Figure 1F**, *t* = 2.765, *p* = 0.014) and no differences in distance traveled in both compartments (**Figure 1G**, dark: *t* = 0.117, *p* = 0.909; light: *t* = 0.137, *p* = 0.893).

In the confined cage, while control animals rarely emitted aversive 22 kHz USVs, iuGC animals vocalized throughout the exposure (**Figure 1H**; *U* = 81, *p* = 0.016). Similarly, iuGC group presented increased freezing behavior (**Figure 1I**; *t* = 2.846, *p* = 0.013). Upon habituation to the cage (2 days of exposure), iuGC animals no longer presented this anxious-like response.

To investigate reactivity to an adverse stimulus, we performed a variation of the classical fear-conditioning paradigm. Animals were conditioned to a cue (light) predicting painful electric shocks. After cage habituation, animals were given six pairs of cue-shock. No emission of 22 kHz USVs was observed in the baseline phase (0–3 min), but upon light/shock pairings, as expected, both groups significantly emitted more negative USVs. iuGC animals emitted negative USVs to a greater extent than control animals (**Figure 2A**, *t* = 1.562, *p* = 0.130) and also presented an increase in freezing behavior time (**Figure 2B**, *t* = 2.355, *p* = 0.034). After this conditioning session, in the next day, animals were exposed to the cue, but no shock was given. In the initial period, both groups emitted more 22 kHz USVs than the day before (*F*1,58 = 4.10, *p* = 0.047); cue exposure elicited more negative USVs in both groups, but again, iuGC animals were over-reactive (**Figure 2C**, *t* = 2.804, *p* = 0.011). Analysis of freezing behavior further confirmed the phenotype of iuGC group (**Figure 2D**, *t* = 2.087, *p* = 0.049). Plotting the number of negative USVs along time further confirms that iuGC animals were over-reactive to the cue predicting the adverse stimulus, since iuGC group emitted more context-induced 22 kHz vocalizations than control group, especially in the first 60 s (**Figure 2E**, *F*1,131 = 104.42, *p* < 0.0001). Upon the first cue exposure (ON period), both groups increased the number of USVs with no major differences between them. However, upon second cue exposure, iuGC animals emitted more negative USVs than control group and remained over-reactive throughout. The pattern of 22 kHz USVs emission in both groups is interesting since immediately after the light is turned off, both groups emit more negative vocalizations, indicative of the consolidated association of the cue with the electric shock.

#### **iuGC EXPOSURE INDUCES PROMINENT CHOLINERGIC ALTERATIONS**

Pharmacological studies suggest that the ascending cholinergic tegmental system is responsible for the initiation and production of negative vocalizations in rodents (43–45). In addition, cholinergic signaling is highly responsive to stress/GC (46) and is important for the manifestation of aversive behaviors (47). Considering these findings, we decided to further explore the impact of iuGC exposure in the cholinergic circuitry. To do so,

we have performed IHC against ChAT, the key enzyme in ACh synthesis. The number of ChAT-positive cells in the LDT was significantly higher in iuGC animals when compared to control group (**Figures 3A,B**; 49% increase; *U* = 3, *p* = 0.026). The PPT, a LDT adjacent region, also presented substantial increase in the number of ChAT-positive cells (**Figures 3C,D**; *U* = 2, *p* = 0.024).

On the contrary, the nucleus basalis of Meynert did not show any significant alteration in the number of cholinergic positive cells (**Figures 3E,F**; *U* = 8, *p* = 0.315). Other regions such as the NAc (core and shell), rich in cholinergic interneurons, also did not show any differences in the number of ChAT-positive cells (**Figures 3G,H**; core: *U* = 13, *p* = 0.927; shell: *U* = 6, *p* = 0.164).

Because iuGC can induce relevant structural changes, we measured the volume and number of cells in the LDT and PPT nuclei. No statistical differences regarding the volume of LDT were found between control and iuGC animals (**Figures 4A–C**; volume: *U* = 5, *p* = 0.486; cell numbers: *U* = 3, *p* = 0.200). In the PPT, no significant differences in volume and cell numbers were found

(**Figures 4D–F**; volume: *U* = 2, *p* = 0.114; cell numbers: *U* = 2, *p* = 0.229).

To assess if the observed increase in ChAT-positive cells in iuGC group was translated into augmented ACh release, we measured the levels of ACh in cholinoceptive regions. A trend for increased ACh levels in the hypothalamus and the amygdala was found (data not shown).

#### **CHOLINERGIC NEURONS IN THE LDT AND PPT ARE DIFFERENTIALLY ACTIVATED UPON ADVERSE STIMULUS IN iuGC ANIMALS**

To better determine the impact of iuGC in cholinergic neurons, and the relevance of such changes in reaction to adverse stimuli, we evaluated neuronal activation patterns using c-fos labeling in combination with ChAT after the fear-conditioning protocol.

Briefly, animals were subjected to the modified fearconditioning protocol, and sacrificed 90 min after stimuli on the test day. As depicted in **Figure 5**, after an adverse stimulus, iuGC animals presented a significant increase (85%) in the number of c-fos-positive cells in the LDT (**Figures 5A,B**; *U* = 0, *p* = 0.008). The number of ChAT-positive cells was also substantially augmented (64%) in iuGC animals when compared to control animals (**Figure 5C**; *U* = 0, *p* = 0.014).

Regarding the PPT, after the adverse stimuli, iuGC animals present 2.5 times more c-fos-positive cells than control animals (**Figures 5E,F**; *U* = 0, *p* = 0.002). Similarly, the number of ChATpositive cells was also increased in iuGC animals (**Figure 5G**; *U* = 0, *p* = 0.002).

To determine if the observed increase in c-fos-positive cells was due to enhanced cholinergic activation, we also quantified the number of c-fos/ChAT-positive cells after fear conditioning. Remarkably, iuGC animals presented a substantial increase (186%) in the number of c-fos/ChAT-positive cells in the LDT in comparison to control animals (**Figure 5D**; *U* = 4, *p* = 0.029). Similarly, in the PPT, we observed that iuGC animals display a four-time increase in the number of double c-fos/ChAT-positive cells (**Figure 5H**; 3.358 vs. 0.801%, *U* = 4.5, *p* = 0.030).

#### **DISCUSSION**

Confirming previous findings (12, 14, 48), we observed that iuGC animals present an anxious phenotype. In the EPM, iuGC animals presented a decrease in the time and entries in the open arms when compared with control animals, in accordance with the L/D test, where they spent less time in the anxiogenic compartment. Moreover, iuGC animals emitted more 22 kHz negative calls and presented enhanced freezing behavior in the confined cage paradigm. These differences were eliminated by habituation. This is in accordance with previous work showing that rats that are highly anxious tend to vocalize more often and present augmented freezing time during aversive stimuli than rats that display low anxiety-like trait (35). In the fear-conditioning paradigm, iuGC animals emitted more negative calls and enhanced freezing behavior on the conditioning day. This suggests an emotional overreactivity of iuGC animals to adverse stimulus. In further support of this idea, iuGC animals emitted more negative calls than control subjects in response to the cue predicting the harmful stimulus and during ISI, suggesting an over-reactive response. Altogether, our data confirms that iuGC exposure leads to anxious behavior and exacerbated response to stressful events in adulthood, a finding also observed in other stress models (49–51). Similarly, a rat line that displays signs of extreme trait anxiety also presents increased stress vulnerability and reactivity (52).

One remarkable finding was the increased emission of negative calls in iuGC animals, hinting differential activity of the mesopontine cholinergic circuitry, that mainly comprises projections from the LDT region (43, 44, 53, 54). In accordance, we found increased ChAT expression in the LDT of iuGC animals in a basal situation, suggesting overproduction of ACh. Upon exposure to a cue predicting an adverse stimulus, there was a marked increase in the number of c-fos/ChAT-positive cells, potentially explaining iuGC behavioral response. Indeed it was shown that LDT activation induces a complex state of defensiveness, critical for alarm responses to dangerous stimuli (44, 45). Our results are in accordance with previous work, in which they have shown that pharmacological stimulation of the LDT lead to an increase in the emission of 22 kHz USVs (44). In addition, the increased LDT cholinergic signaling could also be implicated in the impaired negative HPA axis feedback of iuGC animals (12) since LDTarising ACh enhances ACTH and CRF release (28, 55, 56). On the other hand, GC also modulate cholinergic signaling (19), suggesting a reciprocal ACh-GC control that may culminate in a vicious loop.

Alike the LDT, iuGC animals presented increased PPT cholinergic signaling both in a basal situation and after exposure to the cue predicting an adverse stimulus. Apart from the classical role of PPT in sleep and arousal, lesion studies suggest a role in anxiety modulation, although both anxiogenic and anxiolytic effects were found depending on the degree and type of lesion. Excitotoxic and electrolytic PPT lesions induce an anxiogenic-like status (57, 58), contrary to one study that suggests a slight anxiolytic effect (59). On the other hand, ibotenic lesions seem to reduce anxious-like phenotype (60). Apart from distinct technical procedures, it has been suggested that opposing results may arise from damage in different sub-regions within the PPT (pars compacta vs. dissipata) or in surrounding regions, namely the cuneiform nucleus (59, 61).

This programing effect of iuGC exposure is not so surprising considering the importance of GC receptors for the maturation of medial septal and hippocampal cholinergic neurons (62, 63), yet, more studies need to be performed to understand how GC exert long-lasting functional and molecular changes in these neurons.

*In utero* glucocorticoid exposure-induced alterations in the mesopontine cholinergic pathway may go beyond a direct effect of cholinergic inputs on behavioral output. For instance, LDTdependent cholinergic activation of VTA evokes dopamine release in the NAc (20, 21, 64) driving motivational and reward behaviors (65, 66). However, these findings are somewhat contradictory to the VTA-accumbal hypodopaminergia and anhedonia observed in iuGC animals (6, 14, 15). One possible explanation is that the sustained augmented LDT-VTA ACh signaling could desensitize or alter the expression/epigenetic status of the nicotinic/muscarinic receptors as a compensatory mechanism. Indeed, GC or acute stress can induce prominent ACh release in specific brain regions (46, 67, 68), and transiently change the expression levels of different cholinergic players through c-fos binding to promoter regions of target genes in order to maintain the homeostasis (19). Additional studies focusing on the regulation of the expression of different cholinergic players by GC will be critical to better comprehend our findings.

In summary, our results show for the first time that prenatal GC exposure programs the mesopontine cholinergic pathway, leading to cholinergic hyperactivation of both the LDT and PPT, which in turn can underlie the anxious behavior and enhanced stress reactivity observed in these animals.

#### **ACKNOWLEDGMENTS**

Authors would like to thanks Tircia Santos and Mariana Cerqueira (3B's group) for the technical help with the fluorimeter. This project was supported by a grant of Institute for the Study of Affective Neuroscience (ISAN) and by Janssen Neuroscience Prize. Sónia Borges, Carina Soares-Cunha, Miguel M. Carvalho, Ana P. Ventura-Silva, and Ana João Rodrigues have Fundação para a Ciência e Tecnologia (FCT) fellowships (Sónia Borges: SFRH/BD/89936/2012; Carina Soares-Cunha: SFRH/BD/51992/2012; Miguel M. Carvalho: SRFH/BD/51061/2010, Ana P. Ventura-Silva: SFRH/BD/52078/ 2013; Ana João Rodrigues: SFRH/BPD/33611/2009).

#### **REFERENCES**

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accumbens or amygdala. *Neuroscience* (1996) **74**:767–74. doi:10.1016/0306- 4522(96)00211-4

**Conflict of Interest Statement:** 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.

*Received: 28 October 2013; paper pending published: 16 November 2013; accepted: 25 November 2013; published online: 13 December 2013.*

*Citation: Borges S, Coimbra B, Soares-Cunha C, Ventura-Silva AP , Pinto L, Carvalho MM, Pêgo J-M, Rodrigues AJ and Sousa N (2013) Glucocorticoid programing of the mesopontine cholinergic system. Front. Endocrinol. 4:190. doi: 10.3389/fendo.2013.00190*

*This article was submitted to Neuroendocrine Science, a section of the journal Frontiers in Endocrinology.*

*Copyright © 2013 Borges, Coimbra, Soares-Cunha, Ventura-Silva, Pinto, Carvalho, Pêgo, Rodrigues and Sousa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Context modulates outcome of perinatal glucocorticoid action in the brain

#### **E. Ronald de Kloet 1,2\*, Sanne E. F. Claessens <sup>1</sup> and Jiska Kentrop<sup>1</sup>**

<sup>1</sup> Department of Medical Pharmacology, Leiden University Medical Center, Leiden University, Leiden, Netherlands <sup>2</sup> Department of Endocrinology and Metabolism, Leiden University Medical Center, Leiden, Netherlands

#### **Edited by:**

Nikolaos P. Daskalakis, Icahn School of Medicine at Mount Sinai, USA

#### **Reviewed by:**

Gábor B. Makara, Hungarian Academy of Sciences, Hungary Nuno Sousa, University of Minho, Portugal

#### **\*Correspondence:**

E. Ronald de Kloet, Department of Endocrinology and Metabolism, Division of Medical Pharmacology, LACDR, Leiden University Medical Center, Leiden University, PO Box 9503, Leiden 2300 RA, Netherlands e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
Prematurely born infants may be at risk, because of inadequate maturation of tissues. If there are signs of preterm birth, it has become common practice therefore to treat either antenatally the mother or postnatally the infant with glucocorticoids to accelerate tissue development, particularly of the lung. However, this life-saving early glucocorticoid treatment was found to increase the risk of adverse outcome in later life. In one animal study, the authors reported a 25% shorter lifespan of rats treated as newborns with the synthetic glucocorticoid dexamethasone, but so far this finding has not been replicated. After a brief clinical introduction, we discuss studies in rodents designed to examine how perinatal glucocorticoid action affects the developing brain. It appears that the perinatal action of the glucocorticoid depends on the context and the timing as well as the type of administered steroid. The type of steroid is important because the endogenous glucocorticoids cortisol and corticosterone bind to two distinct receptor populations, i.e., mineralocorticoid and glucocorticoid receptors (GR), while synthetic glucocorticoids predominantly bind to the GR. In addition, if given antenatally hydrocortisone is inactivated in the placenta by 11β-HSD type 2, and dexamethasone is not. With respect to timing, the outcome of glucocorticoid effects is different in early vs. late phases of brain development. The context refers to the environmental input that can affect the susceptibility to glucocorticoid action in the newborn rodent brain; early handling of pups and maternal care obliterate effects of post-natal dexamethasone treatment. Context also refers to coping with environmental conditions in later life, for which the individual may have been programed epigenetically by early-life experience. This knowledge of determinants affecting the outcome of perinatal glucocorticoid exposure may have clinical implications for the treatment of prematurely born infants.

**Keywords: preterm birth, stress, glucocorticoids, antenatal, post-natal, human, rodent, brain**

#### **INTRODUCTION**

The endogenous glucocorticoids cortisol and corticosterone promote the development of various tissues and organ systems during development. However, in prematurely born infants this effect of the steroids on maturation is abrogated resulting in a lifethreatening situation. Therefore, it has become common practice to administer synthetic glucocorticoids such as dexamethasone to accelerate maturation, particularly of lung tissue (1, 2). If given antenatally to the mother the mortality of the prematurely born infant is decreased. While the infants obviously benefit from perinatal glucocorticoids, the treatment reportedly can have negative implications for later life outcome, particularly with respect to dexamethasone or its analogs (3–5). This health risk at adulthood includes impaired cardiovascular and immune functions as well as deficits in motor and cognitive performance, altered emotional reactivity, and neuroendocrine regulations.

In this contribution, data from animal and human studies with respect to the perinatal (antenatal and post-natal) action of glucocorticoids are presented. The action of the naturally occurring glucocorticoids is mediated by mineralocorticoid receptors (NR3C2, MR) and glucocorticoid receptors (NR3C1, GR) (6, 7). These glucocorticoids, cortisol, and corticosterone (the latter steroid only in rodents), bind to MR and GR with an order of magnitude difference in binding affinity (6). This implies that corticosterone and cortisol bind to the brain MR with high affinity resulting in substantial occupation even during the very low circulating hormone levels that are typically observed during the circadian trough and in rodents during the early post-natal stress-hyporesponsiveperiod (SHRP). These same steroids bind to GR only in adequate amounts when in adult animals their concentration has increased after stress and at the circadian peak. The membrane variants of MR and GR also display a lower affinity suggesting that their rapid effects preferably occur only at rising and high steroid concentrations during stressful situations and circadian peaks (8). No data exist on the role of these membrane variants during development. The synthetic glucocorticoids preferentially bind to GR, usually with high affinity (9).

We first review the human studies on antenatal activity of the hypothalamic–pituitary–adrenal (HPA)-axis, development of corticosteroid receptors and the effect of antenatal glucocorticoid treatment. Then, after an overview of antenatal rodent data, we will discuss the action of dexamethasone during the first post-natal days (pnds) of the newborn rodent, since this resembles most the antenatal treatment protocol in the human. Most animal studies have been performed with male animals; if females are used this is indicated. This contribution is concluded with a translational perspective that may be helpful in the clinical management of perinatal glucocorticoid treatment.

#### **HUMAN STUDIES**

#### **ANTENATAL HPA-AXIS DEVELOPMENT**

During pregnancy, the activity of the maternal HPA-axis gradually increases over time toward a state of hypercortisolism that is caused by the steadily increasing production of corticotropinreleasing hormone (CRH) in the placenta and fetal membranes (10–12)). Fetal levels of CRH, adrenocorticotropic hormone (ACTH), and cortisol also rise, but not as much as the maternal values (13). Most of the cortisol present in amniotic fluid has a maternal origin and has successfully traversed the placenta. However, this accounts for only 10–20% of maternal cortisol because the remaining 80–90% is converted to inactive cortisone by the enzyme 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) to protect the fetus from excess glucocorticoid exposure (14, 15). During the third trimester, fetal 11β-HSD2 levels fall and the fetus is exposed to high levels of CRH and endogenous cortisol (16). In this period, cortisol is able to exert its positive influence on the maturation of several organ systems, in preparation of survival after birth (17, 18).

#### **ANTENATAL MR AND GR EXPRESSION**