Hypothalamic-pituitary-adrenal axis and behavioral dysfunction following early binge-like prenatal alcohol exposure in mice

Abstract

The range of defects that fall within fetal alcohol spectrum disorder (FASD) includes persistent behavioral problems, with anxiety and depression being two of the more commonly reported issues. Previous studies of rodent FASD models suggest that interference with hypothalamic-pituitary-adrenal (HPA) axis structure and/or function may be the basis for some of the prenatal alcohol (ethanol) exposure (PAE)-induced behavioral abnormalities. Included among the previous investigations are those illustrating that maternal alcohol treatment limited to very early stages of pregnancy (i.e., gestational day [GD]7 in mice; equivalent to the third week post-fertilization in humans) can cause structural abnormalities in areas such as the hypothalamus, pituitary gland, and other forebrain regions integral to controlling stress and behavioral responses. The current investigation was designed to further examine the sequelae of prenatal alcohol insult at this early time period, with particular attention to HPA axis-associated functional changes in adult mice. The results of this study reveal that GD7 PAE in mice causes HPA axis dysfunction, with males and females showing elevated corticosterone (CORT) and adrenocorticotropic hormone (ACTH) levels, respectively, following a 15-min restraint stress exposure. Males also showed elevated CORT levels following an acute alcohol injection of 2.0 g/kg, while females displayed blunted ACTH levels. Furthermore, analysis showed that anxiety-like behavior was decreased after GD7 PAE in female mice, but was increased in male mice. Collectively, the results of this study show that early gestational alcohol exposure in mice alters long-term HPA axis activity and behavior in a sexually dimorphic manner.

Keywords: Anxiety; Depression; FASD; Hypothalamic-pituitary-adrenal axis; Prenatal alcohol exposure; Stress.

Figure 1. PAE increases HPA axis activity after acute restraint stress

CORT (A,B) and ACTH (C,D) were measured under basal conditions and after 5 min and 15 min of restraint stress in males (left column) and females (right column). Letters above each bar (a, b, c, or d) denote the groups that are statistically similar, e.g., a bar with the letter ‘a’ above it is statistically similar to all other bars with an ‘a’ above them. All groups that are statistically different have p’s < 0.05. Male (n’s) = baseline (13–17), 5 min (7–11), 15 min (47); female (n’s) = baseline (12–17), 5 min (7–9), 15 min (5–7).

Figure 2. PAE increases HPA axis activity over time following acute restraint stress

CORT (A,B) and ACTH (C,D) were measured 60 and 90 min following a 15-min restraint period in males (left column) and females (right column). The baseline and 15-min restraint time points shown in Fig. 1 were graphed in Fig. 2 as B and 0 min, respectively, in order to better depict the profile of CORT and ACTH levels over time. *with bracket p < 0.05, significant main effect of prenatal treatment; *p < 0.05, GD7 Alc vs. GD7 Con; Male (n’s) = baseline (13–17), 5 min (7–11), 15 min (4–7), 60 min (4–7), 90 min (3––6); female (n’s) = baseline (12–17), 5 min (7–9), 15 min (5–7), 60 min (5–7), 90 min (4–7).

Figure 3. PAE causes HPA axis dysfunction after an acute alcohol injection

CORT (A,B) and ACTH (C,D) were measured under basal conditions and 20, 60, and 120 min after a 2.0 g/kg alcohol injection in males (left column) and females (right column). BACs (E,F) were measured at 20, 60, and 120 min. *with bracket p < 0.05, significant main effect of prenatal treatment; Male (n’s) = 20 min (7–8), 60 min (4–6), 90 min (6–8); female (n’s) = 20 min (6–7), 60 min (7–8), 90 min (8).

Figure 4. PAE and sex interact to influence the relationship between BACs and ACTH levels

Regression analysis of BACs and ACTH levels in males (left column) and females (right column) exposed prenatally to vehicle (Ringer’s solution) (A,B) or alcohol (C,D). BACs and ACTH levels positively correlate in male GD7 Con and female GD7 Alc mice but not male GD7 Alc and female GD7 Con mice. Male (n’s) = GD7 Con (19), GD7 Alc (17); female (n’s) = GD7 Con (21), GD7 Alc (21).

Figure 5. PAE affects anxiety-like behavior in a sexually dimorphic manner

Mice were tested on two procedures that measure anxiety-like behavior, light-dark testing (A–F) and elevated plus maze (G–H). Light entries (A,B), light duration (C,D) and percent light distance ([light distance/(light + dark distance)] × 100) (E,F) were measured during light-dark testing, while percent time spent in the open arms ([open arms time/(closed + open arms time)] × 100) (G,H) was measured on the elevated plus maze in males (left column) and females (right column). *with bracket p < 0.05, significant main effect of prenatal treatment; *p < 0.05, GD7 Alc vs. GD7 Con; male n’s = 8–9; female n’s = 9–12.

Figure 6. PAE affects depression-like behavior in a sexually dimorphic manner

Immobility duration on the forced swim test was measured in males (A) and females (B) to test depression-like behavior. *p < 0.05, GD7 Alc vs. GD7 Con; Male n’s = 10–11; Female n’s = 13–15.