Long-term effects of gestational nicotine exposure and food-restriction on gene expression in the striatum of adolescent rats

Abstract

Gestational exposure to environmental toxins such as nicotine may result in detectable gene expression changes in later life. To investigate the direct toxic effects of prenatal nicotine exposure on later brain development, we have used transcriptomic analysis of striatal samples to identify gene expression differences between adolescent Lister Hooded rats exposed to nicotine in utero and controls. Using an additional group of animals matched for the reduced food intake experienced in the nicotine group, we were also able to assess the impact of imposed food-restriction on gene expression profiles. We found little evidence for a role of gestational nicotine exposure on altered gene expression in the striatum of adolescent offspring at a significance level of p<0.01 and |log2 fold change >0.5|, although we cannot exclude the possibility of nicotine-induced changes in other brain regions, or at other time points. We did, however, find marked gene expression differences in response to imposed food-restriction. Food-restriction resulted in significant group differences for a number of immediate early genes (IEGs) including Fos, Fosb, Fosl2, Arc, Junb, Nr4a1 and Nr4a3. These genes are associated with stress response pathways and therefore may reflect long-term effects of nutritional deprivation on the development of the stress system.

Figure 1. Volcano plots representing group comparisons for all genes included in the analysis.

x-axes represent log2 fold-changes and y-axes represent the –log10(p-values) associated with the t-statistic. Vertical dotted lines are positioned at a log2 fold-change of 0.5 or −0.5 and horizontal dotted lines are positioned at the equivalent of p = 0.01. In red are those genes that are differentially expressed at p<0.01 and log2 fold-change>0.5 or <−0.5. A) Nic vs. Con B) Nic vs. Con-Pf and C) Con vs. Con-Pf.

Figure 2. qRT-PCR validation of 9 food-restriction-sensitive genes.

The y-axis represents the log2(fold changes) observed in both comparisons involving the food restricted group. Differences in gene expression using qRT-PCR were consistent with microarray data. The dashed line represents the fold change cut-off (log2(fold change) >0.5) used in the microarray analysis.

Figure 3. Ingenuity Pathways Analysis of genes identified as differentially expressed in the Nic vs. Con-Pf and the Con vs. Con-Pf comparisons.

On the left is the top network identified consisting of 7/12 genes in our list, and on the right is the top associated canonical pathway. Highlighted red are the genes in our list that were over-expressed due to food-restriction.

Figure 4. Gene set enrichment analysis (GSEA) of the GO pathway “RESPONSE_TO_STRESS” in the Con vs. Con-Pf comparison.

The input gene list was all genes in the microarray analysis ranked by –log10(p-value)×log2(fold change). The enrichment score profile displays an enrichment of pathway hits at the top of the list, suggesting multiple top-ranked genes involved in the “Response to stress” pathway.