Genetic variation within the Chrna7 gene modulates nicotine reward-like phenotypes in mice

Abstract

Mortality from tobacco smoking remains the leading cause of preventable death in the world, yet current cessation therapies are only modestly successful, suggesting new molecular targets are needed. Genetic analysis of gene expression and behavior identified Chrna7 as potentially modulating nicotine place conditioning in the BXD panel of inbred mice. We used gene targeting and pharmacological tools to confirm the role of Chrna7 in nicotine conditioned place preference (CPP). To identify molecular events downstream of Chrna7 that may modulate nicotine preference, we performed microarray analysis of α7 knock-out (KO) and wild-type (WT) nucleus accumbens (NAc) tissue, followed by confirmation with quantitative polymerase chain reaction (PCR) and immunoblotting. In the BXD panel, we found a putative cis expression quantitative trait loci (eQTL) for Chrna7 in NAc that correlated inversely to nicotine CPP. We observed that gain-of-function α7 mice did not display nicotine preference at any dose tested, whereas conversely, α7 KO mice demonstrated nicotine place preference at a dose below that routinely required to produce preference. In B6 mice, the α7 nicotinic acetylcholine receptor (nAChR)-selective agonist, PHA-543613, dose-dependently blocked nicotine CPP, which was restored using the α7 nAChR-selective antagonist, methyllycaconitine citrate (MLA). Our genomic studies implicated a messenger RNA (mRNA) co-expression network regulated by Chrna7 in NAc. Mice lacking Chrna7 demonstrate increased insulin signaling in the NAc, which may modulate nicotine place preference. Our studies provide novel targets for future work on development of more effective therapeutic approaches to counteract the rewarding properties of nicotine for smoking cessation.

Keywords: BXD panel; Chrna7; behavioral genetics; conditioned place preference; genomics; insulin; nicotine; quantitative trait loci; reward; α7 Nicotinic acetylcholine receptor.

Figure 1. BXD strain distribution for place conditioning for 0.5mg/kg nicotine

a) Following conditioning with either saline-saline (0.9%) or saline-nicotine (0.5mg/kg), nicotine place conditioning scores (black) on test day for BXD strains follow a continuous distribution, indicative of a quantitative trait. Progenitor strains, C57BL/6J and DBA/2J, show divergent phenotypes for this trait. Each point represents the mean ± SEM of n=6-12 mice per group. b) Transformed distribution, nicotine minus saline BXD strain means.

Figure 2. The nicotine place conditioning phenotype is genetically correlated to Chrna7 basal mRNA expression in the nucleus accumbens, but not the prefrontal cortex, or ventral midbrain

Conditioning scores (nicotine-saline) significantly correlate with basal Chrna7 expression (Chrna7 Probeset ID = 1440681_at, panel a) in the nucleus accumbens and prefrontal cortex (Chrna7 Probeset ID = 1450229_at, panel b), but not ventral midbrain (denoted as VTA). Correlation scattergrams (left of diagonal), univariate density plots (in white, along the diagonal), and Pearson's r values (right of diagonal) are displayed. For the correlation scattergrams, the linear fits are plotted in red with 50% confidence intervals in blue. Each point represents the mean for a BXD strain. A blue r value denotes a significant correlation, while grey r values are non-significant at an alpha = 0.05. All expression data are saline RMA values from the VCU BXD NA, PFC, and VMB Datasets, with probes containing SNPs between B6 and D2 genotypes removed.

Figure 3. Genome-wide interval map for Chrna7 mRNA levels across the BXD RI panel

a) A suggestive cis expression QTL (blue solid line) exists on chromosome 7 for Chrna7 mRNA levels in the nucleus accumbens (VCU BXD NA Dataset Saline RMA Values, Probeset ID 1440681_at). The cis eQTL remained after removal of the probes containing SNPs between B6 and D2 mice (red dotted line). The DBA/2J genotype for Chrna7 increases its expression. b) An enlargement of Chromosome 7 reveals two possible QTL peaks driving the mRNA expression of Chrna7, of which the proximal peak harbors Chrna7 (at 70.24Mb). C, qRT-PCR validation of microarray results. Basal mRNA expression of Chrna7 in the nucleus accumbens is significantly greater in D2 mice compared to B6 mice. Each point represents the mean ± SEM (* = p<0.01).

Figure 4. Deletion of the alpha7 nAChR results in increased sensitivity to nicotine place preference and knock-in or agonism of the alpha7 nAChR prevents nicotine place preference

Panel a), a significant increase in place preference scores for 0.5mg/kg nicotine was observed in both alpha7 KO and WT mice. Alpha7 KO mice display place preference for 0.1mg/kg nicotine; preference for 0.1mg/kg nicotine was of significantly lower magnitude than preference for 0.5mg/kg nicotine. Panel b), Only wildtype (WT) mice, but not alpha7 knock-in (KI) mice, show nicotine place preference for 0.5mg/kg of nicotine. c), Place preference for 0.5 mg/kg nicotine was significantly higher than preference for saline in B6 mice. Pretreatment with PHA-543613 (PHA), dose-dependently blocked place preference for 0.5mg/kg nicotine at 8.0 and 12.0 mg/kg PHA. d), For CPP for 0.5mg/kg of nicotine, 12.0mg/kg PHA blocked preference; this was reversed by pretreatment with 10.0mg/kg of methyllycacontinine (MLA). MLA alone did not alter nicotine preference. Each point represents the mean ± SEM (*/‡ = p<0.01, # = p<0.05). Unless otherwise denoted, all symbols of significance denote comparisons for within-group vehicle treatment.

Figure 5. Alpha7 knock-in and knock-out mice develop normal place preference to a 10mg/kg dose of cocaine; pre-treatment with PHA in C57BL/6J mice does not alter cocaine place preference

Panels a and b, a significant increase in place preference scores for 10mg/kg cocaine compared to within-genotype saline treatment was observed for all genotypes tested. Panel c, C57BL/6J mice develop place preference to 10mg/kg cocaine. Pre-treatment with PHA did not block cocaine place preference in C57BL/6J mice. Each point represents the mean ± SEM (* = p<0.01, # = p<0.05).

Figure 6. Knock-out of the α7 nAChR results in alterations to an insulin-related gene network

a) Top-ranked biological network of genes differentially regulated in the NAc between α7 KO and WT mice (red = upregulated, green = downregulated, colorless = imputed gene, number below each gene = KO/WT s-score, red arrows denote insulin-related genes). b) Genetic correlation network performed using BXD mRNA expression data, displaying co-regulation of Chrna7 and multiple insulin-related genes in the NAc. (All correlations drawn are significant and used Pearson's r, red = positive, blue = negative. bold = r ≥ |0.5|, solid = |.41| ≥ r < |0.49|).

Figure 7. Alpha7 knockout mice display differential basal expression of insulin-related proteins in the nucleus acumbens

a) Representative immunoblots of nucleus accumbens samples from individual mice. IDE=insulin-degrading enzyme, pIR=phosphorylated insulin receptor (Y= tyrosine residue phosphorylated), INSR/IR=β subunit of the insulin receptor, ACTB=β-actin). b) Quantitation and statistical analyses of immunoblot samples (n_KO=6, n_WT=5) revealed that IDE protein levels as well the degree of phosphorylation of the total insulin receptor were significantly increased in alpha7 KO mice compared to WT mice, while total insulin receptor levels showed a trend for being decreased compared to WT mice. All proteins were normalized to ACTB (* = p<0.01).