Whole genome expression was measured in the VTA of adolescent and adult male SD rats exposed to saline, chronic nicotine for 14 days, or 30 days withdrawal following chronic nicotine. A two-way ANOVA with 10% FDR revealed 4878 genes with a significant age X treatment interaction. Next, we applied template matching (also known as feature selection) (Pavlidis and Noble, 2001
) to search for patterns of expression across age and treatment, identifying three distinct temporal expression patterns referred to as “transient response”, “persistent response” and “late response” genes. Transient response genes displayed an initial response (up- or down-regulated) to chronic nicotine compared to saline/baseline as measured at the end of the two week treatment period, but had returned to baseline expression levels at the end of the 30 day withdrawal period (). There were 80 adolescent-specific genes () and 171 adult-specific genes () that exhibited a transient response to chronic nicotine. Persistent response genes displayed an initial response to chronic nicotine as measured at the end of the two week treatment period, and remained up- or down-regulated at the end of the 30 day withdrawal period (). Sixty two adolescent-specific genes () and 33 adult-specific genes () exhibited a persistent pattern of response. Late response genes showed no initial response to chronic nicotine at the end of the two week treatment period, but showed significant up- or down-regulation at the end of the 30 day withdrawal period (). A total of 532 adolescent-specific genes () and 101 adult-specific genes () exhibited a late response to chronic nicotine.
Figure 1 Gene expression correlation with chronic nicotine exposure. Heat maps of template-matched genes show positive and negative correlations with chronic nicotine treatment. The black graph above each heat map represents the specific template employed for (more ...)
A one-way ANOVA with 10% FDR revealed 973 adult genes and 3072 adolescent genes that were significant for chronic nicotine regulation. Of these, 425 genes were shared between the two age groups. Eleven shared genes exhibited a transient response (), 9 shared genes exhibited a persistent response (), and 352 shared genes exhibited a late response to chronic nicotine (). It is noteworthy that the majority of shared gene regulation (≈83%) occurred after the end of chronic nicotine treatment (late response shared genes). By comparison, the number of transient and persistent response shared genes accounted for less than 6% of all shared genes ().
Venn diagram of template-matched genes significantly regulated by chronic nicotine treatment in adolescent and adult rats.
Adolescent- and adult-specific genes (transient, persistent and late response) were further analyzed for biological significance. Ingenuity Pathway Analysis (IPA) was employed for the identification of top ranked biological functions, significant networks, and canonical pathways associated with these age-specific genes. shows high level biological functions identified by IPA that were significantly overrepresented in our adolescent-specific, adult-specific, and shared data sets, including genetic disorders, psychological disorders, neurological disease and behavior. Further analysis focused on genes that contribute to nervous system development and function. This category consisted of 42 adolescent-specific, 14 adult-specific, and 23 shared genes involved in a number of biological processes (). depicts an interaction network of adolescent- () and adult-specific genes () involved in nervous system development and function. The adolescent-specific genes form an extensive interaction network in contrast to adult-specific genes.
Over-represented biological functions with possible biological relevance to chronic nicotine treatment. These data were generated in Ingenuity Pathways Analysis and significance is expressed as the inverse log of the p-value.
Number and function of genes identified by Ingenuity Pathways Analysis involved in nervous system development and function in adolescent and adult animals including age-specific and shared genes.
Interaction network of genes significant for regulation by chronic nicotine falling within Nervous System Development and Function, showing Adolescent network (main figure) and corresponding Adult network (inset).
depicts the canonical pathways significantly over-represented by adolescent-specific, adult-specific or shared genes regulated by chronic nicotine treatment. Five of the seven canonical pathways shown are unique to either adolescents or adult animals. Of note is the synaptic long-term potentiation (LTP) canonical pathway, where 8 adolescent-specific genes were identified as part of the LTP canonical pathway (). By comparison, the LTP canonical pathway was not significantly overrepresented by adult-specific genes. In fact, no adult-specific genes were placed in this pathway, and two genes were shared between the age groups (not significant).
Canonical pathways regulated by chronic nicotine. These data were generated in IPA and are expressed as the inverse log of the p-value.
Synaptic long term potentiation canonical pathway in adolescent rats treated with chronic nicotine. Genes shown in red are positively correlated and genes shown in green are negatively correlated.
Shared genes (transient, persistent, and late response) analyzed for biological significance with IPA were similarly over-represented in the same biological functions first seen in the adolescent- and adult-specific data sets. High level biological functions identified as overrepresented by the shared genes () include nervous system development and function, cell morphology, cellular development, genetic disorder, neurological disease and psychological disorders. On the other hand, two overrepresented canonical pathways identified by IPA were unique to the shared data set. Top canonical pathways identified as overrepresented by the shared genes include semaphorin signaling in neurons, shown to function in synaptogenesis, axon pruning, and the density and maturation of dendritic spines (Pasterkamp and Giger, 2009
), and ephrin receptor signaling, shown to function in dendritic spine morphology (Lai and Ip, 2009
Quantitative real-time RT-PCR was performed to validate microarray results of 12 adolescent-specific genes differentially regulated by nicotine (1 transient, 1 persistent and 10 late response genes; see ). These include four genes in the LTP canonical pathway and eight genes involved in nervous system development and function. All twelve genes were validated by RT-PCR as significant by ANOVA and post-hoc Tukey test (P < 0.05).
Figure 7 Quantitative real time RT-PCR validation of microarray data. Comparison of individual template matched genes measured by microarray analysis (white bars) to real time RT-PCR (black bars). Expression values for adolescents treated with saline (Sal), nicotine (more ...)