Prenatal alcohol exposure alters steady-state and activated gene expression in the adult rat brain

Abstract

Background: Prenatal alcohol exposure (PAE) is associated with alterations in numerous physiological systems, including the stress and immune systems. We have previously shown that PAE increases the course and severity of arthritis in an adjuvant-induced arthritis (AA) model. While the molecular mechanisms underlying these effects are not fully known, changes in neural gene expression are emerging as important factors in the etiology of PAE effects. As the prefrontal cortex (PFC) and hippocampus (HPC) play key roles in neuroimmune function, PAE-induced alterations to their transcriptome may underlie abnormal steady-state functions and responses to immune challenge. This study examined brains from adult PAE and control females from our recent AA study to determine whether PAE causes long-term alterations in gene expression and whether these mediate the altered severity and course of arthritis in PAE females.

Methods: Adult females from PAE, pair-fed (PF), and ad libitum-fed control (C) groups were injected with either saline or complete Freund's adjuvant. Animals were terminated at the peak of inflammation or during resolution (Days 16 and 39 postinjection, respectively); cohorts of saline-injected PAE, PF, and C females were terminated in parallel. Gene expression was analyzed in the PFC and HPC using whole-genome mRNA expression microarrays.

Results: Significant changes in gene expression in both the PFC and HPC were found in PAE compared to controls in response to ethanol exposure alone (saline-injected females), including genes involved in neurodevelopment, apoptosis, and energy metabolism. Moreover, in response to inflammation (adjuvant-injected females), PAE animals showed unique expression patterns, while failing to exhibit the activation of genes and regulators involved in the immune response observed in control and pair-fed animals.

Conclusions: These results support the hypothesis that PAE affects neuroimmune function at the level of gene expression, demonstrating long-term effects of PAE on the central nervous system response under steady-state conditions and following an inflammatory insult.

Keywords: Arthritis; Ethanol; Gene Expression; Inflammation; Prenatal Alcohol Exposure; Rat.

Figure 1. Prenatal treatment alters gene expression patterns under steady-state conditions

Venn diagram of the number of the number of probes significantly altered in each contrast at Day 16 post-saline injection, with moderated F-statistic q <0.25 and moderated t-statistic p <0.05 (80 in the PFC, 30 in the HPC). The number of probes with unique effects in PAE versus both PF and C animals are highlighted in grey, and listed in Table 1. The center of each Venn diagram shows the number of probes differentially expressed among all three prenatal treatment groups. The intersection on the left of each diagram shows the number of probes with a common effect of prenatal ethanol exposure and pair-feeding. The intersection on the right of each diagram shows the number of probes with a unique effect of pair-feeding.

Figure 2. Prenatal alcohol exposure alters steady-state gene expression at Day 16 post-saline injection

In the prefrontal cortex (a), 15 genes were differentially expressed in response to ethanol. In the hippocampus (b), 4 genes were differentially expressed in response to ethanol. F-statistic q-value <0.25 for all genes identified.

Figure 3. RT-qPCR verification of genes altered by prenatal alcohol exposure

(a) Three genes were significantly upregulated in PAE animals (Dusp6 and Ap1s2 in PFC; Rgs3 in HPC). Graphs were plotted as fold change to control animals (where C animals expression = 1) ± SEM. ** = p<0.01, * = p<0.05, # = p<0.1. (b) Fold-changes in expression were positively correlated between microarray and RT-qPCR results for E vs C animals (r²=0.3552, p<0.02). Annotated data points represent genes identified as significant in both methods. (c) Bland-Altman plot of genes identified by microarray analysis. Dotted lines represent the 95% limits of agreement (Bias = 0.06467) and annotated data points represent genes identified as significant in both methods.

Figure 3. RT-qPCR verification of genes altered by prenatal alcohol exposure

(a) Three genes were significantly upregulated in PAE animals (Dusp6 and Ap1s2 in PFC; Rgs3 in HPC). Graphs were plotted as fold change to control animals (where C animals expression = 1) ± SEM. ** = p<0.01, * = p<0.05, # = p<0.1. (b) Fold-changes in expression were positively correlated between microarray and RT-qPCR results for E vs C animals (r²=0.3552, p<0.02). Annotated data points represent genes identified as significant in both methods. (c) Bland-Altman plot of genes identified by microarray analysis. Dotted lines represent the 95% limits of agreement (Bias = 0.06467) and annotated data points represent genes identified as significant in both methods.

Figure 3. RT-qPCR verification of genes altered by prenatal alcohol exposure

(a) Three genes were significantly upregulated in PAE animals (Dusp6 and Ap1s2 in PFC; Rgs3 in HPC). Graphs were plotted as fold change to control animals (where C animals expression = 1) ± SEM. ** = p<0.01, * = p<0.05, # = p<0.1. (b) Fold-changes in expression were positively correlated between microarray and RT-qPCR results for E vs C animals (r²=0.3552, p<0.02). Annotated data points represent genes identified as significant in both methods. (c) Bland-Altman plot of genes identified by microarray analysis. Dotted lines represent the 95% limits of agreement (Bias = 0.06467) and annotated data points represent genes identified as significant in both methods.

Figure 4. Adjuvant exposure alters gene expression at Day 16 post-injection

8 genes showed significant changes in expression among treatment groups in prefrontal cortex (a). 4 genes demonstrated significant changes among treatment groups in the hippocampus (b). F-statistic q-value <0.25 for all genes identified.

Figure 5. Ethanol-exposed animals show altered response to adjuvant

In a subset of genes, Ethanol-exposed animals showed no response to Adjuvant, although pair-fed and control animals responded with an upregulation of the gene (Lcn2, Sgk). In others, gene expression levels in ethanol animals were already elevated compared to pair-feds and controls, but did not change in response to the extent of their control counterparts (Ctgf, Vwf).