Region-specific transcriptional response to chronic nicotine in rat brain

Abstract

Even though nicotine has been shown to modulate mRNA expression of a variety of genes, a comprehensive high-throughput study of the effects of nicotine on the tissue-specific gene expression profiles has been lacking in the literature. In this study, cDNA microarrays containing 1117 genes and ESTs were used to assess the transcriptional response to chronic nicotine treatment in rat, based on four brain regions, i.e. prefrontal cortex (PFC), nucleus accumbens (NAs), ventral tegmental area (VTA), and amygdala (AMYG). On the basis of a non-parametric resampling method, an index (called jackknifed reliability index, JRI) was proposed, and employed to determine the inherent measurement error across multiple arrays used in this study. Upon removal of the outliers, the mean correlation coefficient between duplicate measurements increased to 0.978±0.0035 from 0.941 ±0.045. Results from principal component analysis and pairwise correlations suggested that brain regions studied were highly similar in terms of their absolute expression levels, but exhibited divergent transcriptional responses to chronic nicotine administration. For example, PFC and NAs were significantly more similar to each other (r=0.7; P<10 −14 ) than to either VTA or AMYG. Furthermore, we confirmed our microarray results for two representative genes, i.e. the weak inward rectifier K + channel (TWIK-1), and phosphate and tensin homolog (PTEN) by using real-time quantitative RT-PCR technique. Finally, a number of genes, involved in MAPK, phosphatidylinositol, and EGFR signaling pathways, were identified and proposed as possible targets in response to nicotine administration.

Nicotine coregulates multiple pathways involved in protein modification/degradation in rat brain

“…Microarray production, cDNA probe labeling, and array hybridization A pathway-focused microarray consisting of 638 sequence-verified genes that was designed by our laboratory (called the homeostatic pathway-focused array) was used [52]. This array contains 638 sequence-verified genes selected from rat cDNA clones (Research Genetics, AL) and the 15K NIA mouse cDNA gene set [52] on the basis of our previous studies [28,37]. It was shown that cross-species hybridization (i.e., mouse clones against rat target samples) using a cDNA platform is accurate and specific [59].…”

“…The slides were printed by the GMS 417 arrayer (Affymetrix, Santa Clara, CA) according to the protocols suggested by the manufacturer. cDNA probe labeling and array hybridization were performed as described previously with slight modifications [28,29].…”

“…Our previous investigations have demonstrated the value of microarrays in tracking nicotine-induced changes in expression of genes that belong to the phosphatidylinositol-driven pathways, both in the brain and in PC-12 cells [28,37]. These initial findings implicated nicotine as an important modulator of cellular homeostasis, thus warranting its further study in relation to other homeostatic pathways such as those of protein modification and degradation.…”

“…The recent development of microarray technologies, allowing comparison of expression profiles of thousands of genes simultaneously, has revolutionized almost all biomedical research fields, including nicotine abuse research [16,28,37]. Previously, by using cDNA microarrays, we have shown that several genes such as rap 1, Homer, and NF-kB and biochemical signaling pathways such as phosphatidylinositol and JNK cascades may play significant roles in nicotine dependence [28,37].…”

“…Previously, by using cDNA microarrays, we have shown that several genes such as rap 1, Homer, and NF-kB and biochemical signaling pathways such as phosphatidylinositol and JNK cascades may play significant roles in nicotine dependence [28,37]. Using the same approach, in an accompanying paper [34], we followed the expression levels of 638 sequence-verified genes in five regions of the rat brain over time to gain a better understanding of how nicotine influences the molecular environment in physiologically pertinent brain regions.…”