Prenatal alcohol exposure modifies glucocorticoid receptor subcellular distribution in the medial prefrontal cortex and impairs frontal cortex-dependent learning

Abstract

Prenatal alcohol exposure (PAE) has been shown to impair learning, memory and executive functioning in children. Perseveration, or the failure to respond adaptively to changing contingencies, is a hallmark on neurobehavioral assessment tasks for human fetal alcohol spectrum disorder (FASD). Adaptive responding is predominantly a product of the medial prefrontal cortex (mPFC) and is regulated by corticosteroids. In our mouse model of PAE we recently reported deficits in hippocampal formation-dependent learning and memory and a dysregulation of hippocampal formation glucocorticoid receptor (GR) subcellular distribution. Here, we examined the effect of PAE on frontal cortical-dependent behavior, as well as mPFC GR subcellular distribution and the levels of regulators of intracellular GR transport. PAE mice displayed significantly reduced response flexibility in a Y-maze reversal learning task. While the levels of total nuclear GR were reduced in PAE mPFC, levels of GR phosphorylated at serines 203, 211 and 226 were not significantly changed. Cytosolic, but not nuclear, MR levels were elevated in the PAE mPFC. The levels of critical GR trafficking proteins, FKBP51, Hsp90, cyclophilin 40, dynamitin and dynein intermediate chain, were altered in PAE mice, in favor of the exclusion of GR from the nucleus, indicating dysregulation of GR trafficking. Our findings suggest that there may be a link between a deficit in GR nuclear localization and frontal cortical learning deficits in prenatal alcohol-exposed mice.

Figure 1. Y- Maze Reversal learning performance in naïve male 45–50 day old prenatal alcohol exposure (PAE, n = 6) and saccharin control (SAC, n = 6) mice.

Data presented are mean percent correct arm choice (±SEM). Details of the methods for these procedures are presented in Materials and Methods. Analyses are presented in the Results section.

Figure 2. Cytosolic levels of the glucocorticoid receptor (GR), phospho-serine 226 (pS226) GR (left column), phospho-serine 211 (pS211) GR (center column) and phospho-serine 203 (pS203) GR (right column) in the medial frontal cortex of saccharin (SAC) control and prenatal alcohol exposure (PAE) offspring.

Cytosolic fractions were prepared and anti-GR (top row graphs) and anti-phospho-specific GR (middle row graphs) immunoreactivities were determined as described in the Materials and Methods. Anti-total GR and anti-phospho-GR data are presented as the mean (± SEM) immunoreactivities corrected to Coomassie stain. The ratio of the anti-phospho-GR to anti-total GR immunoreactivities (bottom row graphs) was calculated as described in the Materials and Methods. The levels of total GR and all three phospho-GRs, as well as the ratios of all three phospho-GR/total GR, were not different in SAC (n = 7) and PAE (n = 7) mice. Representative Western blots are presented below.

Figure 3. Nuclear levels of the glucocorticoid receptor (GR), phospho-serine 226 (pS226) GR (left column), phospho-serine 211 (pS211) GR (center column) and phospho-serine 203 (pS203) GR (right column) in the medial frontal cortex of saccharin (SAC) control and prenatal alcohol exposure (PAE) offspring.

Nuclear fractions were prepared and anti-GR (top row graphs) and anti-phospho-specific GR (middle row graphs) immunoreactivities were determined, as described in the Materials and Methods. Anti-GR and anti-phospho-GR data are presented as the mean (± SEM) immunoreactivities from PAE (n = 9–10) and SAC (n = 9–10) mice corrected to Coomassie stain. The ratio of the anti-phospho-GR to anti-total GR immunoreactivities (bottom row graphs) was calculated as described in the Materials and Methods. Total nuclear GR levels were significantly lower in the PAE mice (p = .05 for pS226 GR, left top panel; p = .008 for pS211 GR, center top panel; p = .005 for pS203 GR, right top panel). The ratio of each phospho-GR to total GR was significantly higher in the PAE (pS226 GR/total GR, p = .04; pS211 GR/total GR, p = .04; pS203 GR/total GR, p = .02). Representative Western blots are presented below.

Figure 4. Cytosolic (A) and nuclear (B) levels of the mineralocorticoid receptor (MR) in the medial frontal cortex of saccharin (SAC) control and prenatal alcohol exposure (PAE) offspring.

Representative immunoblots are shown below each figure. Subcellular fractions and anti-MR immunoreactivities were prepared as described in the Materials and Methods section. Data are presented as mean immunoreactivity corrected to Coomassie stain ± SEM in the PAE (n = 8–10) and SAC (n = 9–10) mice. Cytosolic MR levels were increased (p<0.05) in the PAE mice, whereas nuclear MR levels were not different in the two groups.

Figure 5. Cytosolic levels of the FKBP51 (A), FKPB52 (B), Cyclophilin 40 (C) and Hsp90 (D) in the medial frontal cortex of saccharin (SAC) control and prenatal alcohol exposure (PAE) offspring.

Corresponding representative immunoblots are shown below each figure. Cytosolic fractions were prepared, and specific immunoreactivities were determined as described in the Materials and Methods. Data are presented as mean immunoreactivity corrected to Coomassie stain ± SEM in the PAE (n = 7) and SAC (n = 7) mice. Cytosolic FKBP51 (A) levels were increased (p = .02) and cytosolic FKBP52 (B) levels were not different between the PAE and SAC mice. Cytosolic cyclophilin 40 (C) levels were lower (p = .03) in the PAE offspring compared to SAC. Cytosolic Hsp90 (D) levels (p = .02) were lower in SAC mice.

Figure 6. Membrane levels of dynamitin (A), Dynein IC1 (B) and Dynein IC2 (C) in the medial frontal cortex of saccharin (SAC) control and prenatal alcohol exposure (PAE) offspring.

Corresponding representative immunoblots are shown below the respective figures. The cytosolic fraction was prepared and immunoreactivities were determined as described in the Materials and Methods. Data are presented as mean (± SEM) immunoreactivity corrected to Coomassie stain in PAE (n = 7) and SAC (n = 7) mice. Membrane dynamitin levels were lower (p = .01) in the PAE compared to SAC offspring. Membrane dynein C-1 levels were elevated (p = .002), while dynein IC-2 levels were lower (p = .012), in the PAE compared to SAC.