Genetic basis for the psychostimulant effects of nicotine: a quantitative trait locus analysis in AcB/ BcA recombinant congenic mice

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1 Genes, Brain and Behavior (2005) 4: Copyright # Blackwell Munksgaard 2005 Genetic basis for the psychostimulant effects of nicotine: a quantitative trait locus analysis in AcB/ BcA recombinant congenic mice K. J. Gill and A. E. Boyle Research Institute of the McGill University Health Centre and Psychiatry Department, McGill University, Montreal, Quebec, Canada Corresponding author: K. J. Gill, PhD, Addictions Unit, 1604 Pine Avenue West, Montreal, Quebec, Canada H3G 1B4. kathryn.gill@mcgill.ca Genetic differences in sensitivity to nicotine have been reported in both animals and humans. The present study utilized a novel methodology to map genes involved in regulating both the psychostimulant and depressant effects of nicotine in the AcB/BcA recombinant congenic strains (RCS) of mice. Locomotor activity was measured in a computerized open-field apparatus following subcutaneous administration of saline (days 1 and 2) or nicotine on day 3. The phenotypic measures obtained from this experimental design included total basal locomotor activity, as well as total nicotine activity, nicotine difference, nicotine percent change and nicotine regression residual. The results indicated that the C57BL/6J (B6) were insensitive to nicotine over the entire dose response curve (0.1, 0.2, 0.4 and 0.8 mg/kg). However, the 0.8-mg/kg dose of nicotine produced a significant decrease in the locomotor activity in the A/ J strain and a wide and continuous range of both locomotor excitation and depression among the AcB/BcA RCS. Single-locus association analysis in the AcB RCS identified quantitative trait loci (QTL) for the psychostimulant effects of nicotine on chromosomes 11, 12, 13, 14 and 17 and one QTL for nicotine-induced depression on chromosome 11. In the BcA RCS, nicotine-induced locomotor activation was associated with seven putative regions on chromosomes 2, 7, 8, 13, 14, 16 and 17. There were no overlapping QTL and no genetic correlations between saline- and nicotine-related phenotypes in the AcB/BcA RCS. A number of putative candidate genes were in proximity to regions identified with nicotine sensitivity, including the a2 subunit of the nicotinic acetylcholine receptor and the dopamine D3 receptor. Keywords: A/J, AcB/BcA recombinant congenic strains, C57BL/6J, inbred strains, locomotor activity, nicotine, quantitative trait loci, RCS Received 3 August 2004, revised 19 November 2004, accepted for publication 26 November 2004 Recent studies estimate that there are approximately 46.2 million current smokers in the United States (CDC 2004). Cigarette smoking has been estimated to account for approximately 25% of the deaths in the United States (Bergen & Caporaso 1999; Peto & Doll 1992), resulting in a significant economic burden for society (Rice 1999). Overall, the use of tobacco products has been described as the largest single factor impacting on chronic disease and premature death (Bergen & Caporaso 1999). Estimates of the genetic and environmental effects on smoking initiation and maintenance have consistently shown that there are strong genetic factors influencing the onset of regular smoking (Stallings et al. 1999) as well as the persistence of smoking behavior (True et al. 1997). While smoking is pervasive, there are significant individual differences in responses to nicotine (Pomerleau 1995) and the development of dependence. One step toward the effective prevention and treatment for nicotine addiction would be to delineate factors that mediate individual differences in sensitivity to the drug. Strain differences in both nicotine consumption and sensitivity to the acute pharmacological effects of nicotine have been reported (Marks et al. 1986; Marks et al. 1989; Meliska et al. 1995; Robinson et al. 1996). For example, Meliska et al. (1995) examined the consumption of a number of drugs of abuse in the C57BL/6J (B6) and DBA/2 (D2) inbred strains of mice. The B6 showed a greater preference for and absolute consumption of ethanol, nicotine and amphetamine compared with D2 mice. Robinson et al. (1996) examined the extent to which A, BUB, C3H, B6, D2 and St/B strains of inbred mice were differentially sensitive to the effects of nicotine. The B6 consumed the highest levels of nicotine when presented with nicotine solutions ranging in concentration from 20 to 200 mg/ml, compared with the other strains. In the same study, the B6 showed less sensitivity to the seizure-inducing properties of nicotine, demonstrating that there was an inverse relationship between consumption and sensitivity to nicotine (Robinson et al. 1996). Finally, several studies (Marks et al. 1989; Miner & Collins 1989) have examined the effects of nicotine in 19 different strains of inbred mice across a comprehensive battery of doi: /j X x 401

2 Gill and Boyle physiological and behavioral measures. Substantial strain differences were observed; the A/J/Ibg strain was more sensitive to the effects of nicotine as measured by seizure activity, change in body temperature and heart rate compared with the C57BL/Ibg strain. Recent studies in molecular genetics have examined the role of nicotinic acetylcholine receptor (nachr) subunits in mediating the effects of nicotine. Knockout (KO) mice have been produced which lack the expression of various nachr receptor genes (e.g. Marubio et al. 1999; Picciotto et al. 1995). KO mice lacking the (4 nachr subunit exhibit less sensitivity to the locomotor-depressant effects of nicotine (Ross et al. 2000). Additionally, KO mice lacking the (2 nachr subunit are insensitive to the reinforcing effects of nicotine relative to wild-type mice (Epping-Jordan et al. 1999). At the present time, however, it is not clear whether endogenous variation in the expression of these receptor subunits is responsible for the individual differences in consumption and sensitivity to nicotine exhibited by various inbred strains. In addition, issues related to the development and compensation complicates the interpretation of the studies of KO mice (Gingrich & Hen 2000). Using a different methodology, the present study examined natural variation in sensitivity to the effects of nicotine on activity and the potential genetic factors mediating this variation. Activity-related phenotypes were examined as a result of the robust relationship demonstrated between the propensity of drugs to elicit psychomotor activation and their inherent capacity to act as a reinforcer in man (Wise 1988, 2004). The literature indicates that psychomotor activation is related to central dopaminergic processes to a significant extent (Tzschentke 2001; Vezina 2004). For example, administration of nicotine in rodents has been shown to result in a significant release of dopamine in the midbrain (Koob 1992; Leshner & Koob 1999). Thus, our understanding of the genetic factors mediating the expression of nicotine-induced activation may provide insight into those factors mediating nicotine reinforcement and/or tobacco abuse. In the present study, the A/J and B6 progenitors, as well as AcB/BcA recombinant congenic strains (RCS) of mice derived from them, were utilized to identify genetic markers and potential candidate genes associated with differential sensitivity to the locomotor effects of nicotine. The AcB/ BcA RCS of mice have been used previously to identify and confirm quantitative trait loci (QTL) associated with the stimulant effects of cocaine (Gill & Boyle 2003). The AcB/BcA RCS were produced by inbreeding mice from the second backcross generation of reciprocal backcrosses between the B6 and A/J progenitor strains (Fortin et al. 2001). Each AcB/BcA RC strain carries a different set of approximately 13.25% of the genes from the donor strain with the remaining 86.75% from the background strain. For example, each of the 22 BcA RCS carries different donor alleles from the A/J, with the B6 serving as the background strain. Recombinant congenic strains are considered to have an advantage in mapping and isolating the individual genes contributing to complex quantitative traits (Demant & Hart 1986) because of the fact that non-linked genes controlling a complex multigenic trait are likely to be separated into different RCSs. The chromosomal maps for the AcB/BcA RCS were used to identify chromosomal regions linked to the quantitative phenotypes displayed by the RCSs. Materials and Methods Subjects Male and female C57BL/6J and A/J mice were purchased from Jackson Laboratory (Bar Harbour, ME). Thirteen strains of the AcB and 22 strains of the BcA RC mice were provided by the MUHC (Montreal, Quebec, Canada) Research Institute Complex Traits Analysis Core Facility (an SPF facility). Pups were housed with same-sex littermates until 8 weeks of age when behavioral testing began. All mice were habituated to the same housing and environmental conditions for a minimum of 2 weeks prior to testing. Animals were housed in a colony controlled for temperature and humidity on a 12-h/12-h light/dark cycle (lights on at 0600 h). The mice were housed with same-sex littermates in standard polycarbonate solid-bottom cages with b-chip bedding and access to mouse chow and water ad libitum. Animals were tested under identical conditions in mixed squads (sex and strain) of approximately mice over the course of a 1.5-year period. Drug injections Nicotine solutions were prepared from nicotine salt (Sigma Chemical Co., St Louis, MO) dissolved in a sterile saline solution to concentrations including 0.1, 0.2, 0.4 and 0.8 mg/kg (0.035, 0.07, 0.14 and 0.28 mg/mg base). All nicotine doses were injected subcutaneously in a fixed volume of 0.01 ml/g body weight. The nicotine dose range selected was chosen based on pilot data indicating a significant reduction in activity observed following administration to the A/J strain of mice. Locomotor activity testing Locomotor activity was measured in custom-designed open-field boxes constructed of Plexiglas, measuring cm high. Six intersecting light-photocell assemblies placed inside the walls of the chambers, 3 cm above the floor, monitored the locomotor activity by automatic registration on a computer connected to the photocells. Counts were automatically registered at 1-min intervals throughout the sessions. A custom-designed computer software program monitored activity on the photocells, providing measures of horizontal activity and stereotypy. Horizontal activity was operationally defined as the number of beam breaks made over the course of the test period. Horizontal locomotor counts were automatically filtered to suppress the activity resulting from the repetitive breaks of a single beam that is 402 Genes, Brain and Behavior (2005) 4:

3 Nicotine-induced locomotor activation due to grooming. Testing was performed under dim red light (40 W) in a sound-insulated room, immediately prior to lights off. Naive male and female mice were habituated to the handling and injection procedures as well as the test chambers prior to nicotine testing. Habituation was carried out on 2 successive days (days 1 and 2) where naive animals were transported to the testing room, weighed and injected with sterile 0.9% NaCl saline solutions (0.01 ml/g body weight). Activity was monitored in the test chambers for 30 min following each subcutaneous saline injection. A/J and B6 progenitors While the QTL association approach does not require that the A/J and C57BL/6J progenitors differ with regard to the phenotypic measure (i.e. nicotine-induced locomotor activation) (Gora-Maslak et al. 1991), they were assessed, however, to establish the optimal dose of nicotine to be administered in the subsequent QTL analysis. A repeated measures design was used to assess the effects of nicotine in a group of naive A/J (final n ¼ 12 females and 14 males) and B6 (final n ¼ 12 females and 13 males). Mice were habituated to the handling and injection procedures as described above on days 1 and 2. On day 3, nicotine testing began where each mouse received all doses of nicotine (0, 0.1, 0.2, 0.4 and 0.8 mg/kg) in a randomized order on an alternate day schedule. Activity was monitored for 30 min immediately following each subcutaneous injection. A randomized order of nicotine doses was utilized to minimize the influence of any order or carry-over effects. In addition to this methodological precaution, a post hoc statistical analysis was performed to assess the extent to which repeated nicotine injections (across four sessions on alternate days) altered the expression of nicotine-induced locomotor activation, as discussed below. AcB/BcA recombinant congenic strains Response to nicotine in naive male and female RC mice was examined using a repeated measures design in which animals were habituated to the handling and injection procedures as described above on days 1 and 2, followed by nicotine (0.8 mg/kg) on day 3. The dose of nicotine was chosen based on significant reductions in activity observed following its administration to the A/J strain of mice. A total of 449 RC mice were tested, comprised of 148 AcB (71 males and 77 females) and 301 BcA (155 males and 146 females). There was a mean of 14 mice per RCS. Approximately equal numbers of male and female mice of each strain were tested. Statistical analysis The phenotypic measures obtained from this experimental design included total basal activity on saline days 1 and 2, as well as habituation difference (day 2 day 1) and habituation percent change (day 2 saline expressed as the percent change from day 1 saline). Additionally, the following measures of nicotine-induced activity were utilized in all analyses: total nicotine activity (day 3), nicotine difference (day 3 nicotine day 2 saline), nicotine percent change (expressed as the percent change from day 2 saline), nicotine percent change for each 5-min time block over the 30-min session (total of six time bins) and regression residuals derived from the regression of total nicotine on baseline saline. SPSS was used to calculate the deviations of the observed nicotine from the values predicted by the regression of total nicotine on baseline saline. The latter nicotine activity phenotypes (difference, nicotine percent change and regression residuals) were assessed to take into account strain differences in basal locomotor activity and habituation. Genetic correlations between the strain means for the saline- and nicotine-related phenotypes were computed using Pearson s r value. Data for each mouse tested were coded and entered into a database using MICROSOFT EXCEL. Statistical analyses were performed with SPSS version 11.5 for Windows (SPSS Inc., Chicago, IL). All activity data were tested for normality and outliers using the SPSS explore function. On the basis of outlier analysis, animals with outside the 95% confidence interval for the strain mean were eliminated. Those removed from the analysis were randomly distributed by sex and strain. Comparisons between strains of mice were carried out using ANOVA with strain and sex as fixed factors, followed by multiple-comparison analysis. Note that, where appropriate, ANOVA analyses included data from both progenitors (A/J and B6) and RCS to identify informative RCSs (i.e. those RCS carrying donor alleles that produced a significantly different phenotype compared with the background strain). These analyses were conducted using ANOVA followed by pairwise Dunnett s contrasts between the mean phenotypic values for each RCS and the appropriate progenitor strain (A in the case of the AcB strains and B6 in the case of the BcA strains). Genetic analysis of the AcB/BcA RCS The genomic mapping of the progenitors and RCS was conducted using simple sequence length polymorphism (SSLP) microsatellite markers. Oligonucleotide primer pairs for each marker were purchased from Research Genetics (Huntsville, AL). The markers were typed in all strains using standard polymerase chain reaction-based methods, as described in detail by Fortin et al. (2001). To date, a total of 625 markers have been typed in each strain, providing coverage of the entire genome with an average spacing of 2.6 cm. The database of genetic linkage markers was produced at the Research Institute of the McGill University Health Centre using map positions based on the Mouse Genome Database (Jackson Laboratory) (Fortin et al. 2001). Before genetic analysis, a randomly selected split-half correlational analysis was performed (corrected with the Spearman Brown formula) Genes, Brain and Behavior (2005) 4:

4 Gill and Boyle on the phenotypic measures to verify the reliability of the RCS means. The strain distribution pattern (SDP) of mean phenotypic values and the chromosomal maps of SSLP markers for the AcB/BcA RCS were used to compute single-locus associations with MAP MANAGER QTX (Manly et al. 2001). The significance of the association for each marker and the identification of QTL was tested using the likelihood ratio statistic, as described by Manly and Olson 1999). It is acknowledged that this analytic strategy is likely to produce a high type I error rate because of large number of computations, and as such the QTL identified in the present analysis must be considered provisional until confirmed through additional research (Boyle & Gill 2001; Gill et al. 1998). MAP MAN- AGER QTX was set to detect simple locus associations at a threshold value of P In addition, a more stringent statistical criterion based on the permutation test (Churchill & Doerge 1994) was used to establish the genome-wide significance threshold for each phenotype. Loci exceeding the suggestive or significant threshold calculated by the permutations tests were indicated in the tabled data. This was followed up by the computation of bivariate correlations between the SDP of mean phenotypic values and the significant markers. AcB and BcA RCS were analysed separately. All genetic analyses included only data from RCSs, excluding the A/J or B6 progenitors. Potential candidate genes in the entire region of the donor QTL were identified using the Mouse Genome Database ( Plausible candidates were chosen based on the known psychopharmacological actions of nicotine. Results A/J and B6 progenitors Total nicotine activity A three-way ANOVA with repeated measures (strain sex nicotine dose) was performed on the total nicotine activity obtained on days 3 6. There was a significant main effect for strain (F 1,33 ¼ 25.09, P < 0.001), but no significant main effect for sex (F 1,33 ¼ 0.29, P > 0.05). In addition, there were no significant interactions (strain dose: F 1,140 ¼ 0.48, P > 0.05; strain sex: F 1,33 ¼ 1.02, P > 0.05; strain sex dose: F 4,132 ¼ 0.63, P > 0.05). Because there were no sex differences on any measure in the A/J and B6, sex was not considered as a factor in any subsequent analyses. Because of the repeated nicotine administrations in the progenitor A/J and B6 strains (days 3 6), a post hoc analysis was performed to assess the potential for sensitisation or tolerance effects. A three-way ANOVA with repeated measures (strain nicotine dose day of nicotine presentation: day 3 day 6) was performed using the total nicotine activity. In this manner, it was possible to determine whether the effects of the nicotine administration changed as a function of the order in which the doses were presented. The analysis failed to indicate a significant three-way interaction (F 16,205 ¼ 0.81, P > 0.05). Thus, the results of this analysis did not provide any evidence of sensitization or tolerance to the effects of nicotine in the A/J or B6 mice within the context of the present paradigm. Nicotine-induced changes in locomotor activity Analysis of the nicotine difference using two-way ANOVA with repeated measures (strain nicotine dose) revealed a significant effect of strain (F 1,36 ¼ 4.34, P < 0.05) and no significant strain dose interaction (F 4,144 ¼ 0.127, P > 0.05) Similarly, analysis of nicotine percent change yielded a significant main effect for strain (F 1,35 ¼ 4.64, P < 0.04), with no strain dose interaction (F 4,140 ¼ 0.347, P > 0.05). To examine the temporal responses to nicotine over the entire 30-min testing period, the data were divided into 5-min time blocks. A three-way ANOVA with repeated measures (strain nicotine dose time block) yielded significant effects of strain (F 1,36 ¼ 4.339, P < 0.05), as well as a strain dose time interaction (F 20,720 ¼ 1.652, P < 0.05) for the nicotine difference. Similarly, analysis of nicotine percent change found a significant main effect for strain (F 1,35 ¼ 4.64, P < 0.04) and a significant strain dose time interaction (F 20,700 ¼ 3.02, P < 0.001), as shown in Fig. 1. These results demonstrate that the strain and dose effects varied as a function of the time after injection. A posteriori comparisons (Tukey) of the time course data in Fig. 1 indicated that the 0.8 mg/kg dose of nicotine produced a significant (P < 0.05) reduction in locomotor activity from 10 to 25 min after injection in the A/J, compared with saline-treated controls. In contrast, the B6 mice failed to exhibit any significant dose- or time-related changes in locomotor activity at any time during testing. Comparable results were obtained with the analysis of nicotine difference. AcB/BcA RCS The reliability of the strain means for the entire AcB and BcA RC series of strains (calculations described above) yielded a value (males and females combined) of (P < 0.001) for total day 1 saline, (P < 0.001) for total day 2 saline, (P < 0.001) for habituation difference and (P < 0.001) for habituation percent change. Nicotine-induced changes in locomotor activity Analysis of nicotine difference among the RCS using two-way ANOVA (strain sex) yielded a significant main effect for strain (F 37,371 ¼ 2.377, P < 0.001), but no sex or strain sex interaction effects (all P values > 0.05) Similarly, analysis of nicotine percent change using a two-way ANOVA (strain sex) yielded a significant main effect for strain (F 37,384 ¼ 2.968, P < 0.001), but no sex or strain sex interaction effects (all P values > 0.05). Mean nicotine percent change for (males and females combined) the AcB 404 Genes, Brain and Behavior (2005) 4:

5 Nicotine-induced locomotor activation Activity (nicotine % change) Activity (nicotine % change) A/J Time course C57BL/6J Time course Nic 0.0 Nic 0.1 Nic 0.2 Nic 0.4 Nic 0.8 Nic 0.0 Nic 0.1 Nic 0.2 Nic 0.4 Nic 0.8 Figure 1: Time course for the effects of nicotine (Nic; 0.0, 0.1, 0.2, 0.4 and 0.8 mg/kg) on locomotor activity in A/J and C57BL/6J mice. Values represent means expressed as the percent change from day 2 saline controls (nicotine percent change ). and BcA strains are presented in Fig. 2. AcB and BcA strains are independently presented in the ascending order of locomotor activity, and the progenitors A/J and B6 are included for comparison. Because of the lack of sex differences on any measure among the RCS, sex was not considered in subsequent analyses. Analysing the AcB and BcA separately using one-way ANOVA yielded significant strain differences among the AcB RCS (nicotine difference : F 14,152 ¼ 3.76, P < 0.001; nicotine percent change : F 13,152 ¼ 3.89, P < 0.001) and BcA RCS (nicotine difference : F 22,299 ¼ 2.130, P < 0.01; nicotine percent change: F 22,319 ¼ 2.28, P < 0.01). Four informative strains (described above) were observed among the AcB set (compared to the A/J) and two among the BcA set (compared to the B6). The informative strains are denoted in Fig. 2 with asterisks. The reliability of the strain means for the entire AcB and BcA RC series of strain yielded a value (males and females combined) of (P < 0.001) for nicotine differences and (P < 0.001) for nicotine percent change. Genetic analysis in the RC strains Chromosomal regions carrying donor alleles modifying the nicotine activity phenotypes were identified through the calculation of single-locus associations with MAP MANAGER QTX (Manly et al. 2001) and confirmed through the calculation of bivariate correlations. The analysis of nicotine difference, nicotine percent change and regression residual yielded comparable results. In many cases, multiple markers were identified; however, only the peak markers (with the largest correlations) were included in the tables. The genetic maps were used to determine the minimum interval (in cm) containing the donor QTL by identifying the common region Figure 2: Strain distribution of nicotine-induced activity among progenitor A/J and B6, as well as the AcB and BcA recombinant congenic strains. Activity are expressed as a percent change from day 2 saline controls (nicotine percent change ). Strain means are plotted in the order of ascending activity. Informative strains are denoted by asterisks. Activity (nicotine % change) AcB (A/J background) AcB60 AcB51 AcB62 AcB54 AcB64 AcB65 AcB52 A/J AcB53 AcB55 AcB58 AcB57 AcB56 AcB61 Progenitor or recombinant strain BcA (B6 background) BcA84 BcA77 BcA67 C57BL/6J BcA87 BcA69 BcA72 BcA83 BcA81 BcA70 BcA80 BcA66 BcA82 BcA86 BcA75 BcA74 BcA85 BcA76 BcA79 BcA68 BcA71 BcA73 BcA78 Genes, Brain and Behavior (2005) 4:

6 Gill and Boyle across all strains that possessed donor alleles at the significant markers identified by MAP MANAGER. Table 1 summarizes the putative chromosomal regions containing B6 donor alleles that were associated with the nicotine difference and nicotine percent change among the AcB RCS. Analysis resulted in the identification of six common putative regions on chromosomes 11, 12, 13, 14 and 17. In general, B6 donor alleles on the A/J background of the AcB RCS increased mean phenotypic values as shown by the positive correlations in Table 1. However, the correlation at D11Mit39 was negative, indicating that the B6 donor allele at this locus produced locomotor depression among the AcB strains. Haplotypes at significant markers on chromosomes 11 that illustrate the varying effects of B6 loci on the A/J background are summarized in Table 2. The allelic status at each locus is represented by an A for the A/J and a B for the B6 allele. The strains were ordered based on increasing phenotypic values for nicotine percent change. Those strains possessing the B6 alleles on chromosome 11 near D11Mit82 were observed to exhibit significantly greater nicotine-induced activation than those strains that possessed the A/J allele at this locus. However, B6 alleles on chromosome 11 near D11Mit39 were observed to exhibit the reverse greater nicotine-induced depression. Stepwise multiple regression analysis demonstrated that a subset of markers including D11Mit82 and D12Mit233 accounted for 81.7% of the genetic variance in nicotine percent change. The putative chromosomal regions containing A/J donor alleles that were associated with both nicotine difference and nicotine percent change among the BcA RCS are summarized in Table 3. In general, A/J alleles inherited by the BcA were associated with nicotine-induced activation, rather than depression. Analysis resulted in the identification of seven putative regions on chromosomes 2, 7, 8, 13, 14, 16 and 17. Stepwise multiple regression analysis demonstrated that the markers D14Mit155 and D16Mit131 accounted for 73% of the genetic variance in nicotine percent change in the BcA strains. It should be noted that because of the small interstrain variation observed in the BcA panel (as opposed to the robust differences between the AcB RCS), the outcome of the QTL analysis will be significantly influenced by the small number of informative strains. A potential confound in the use of drug-change phenotypes, including difference and percent change, is the influence of strain differences in baseline activity measures. To address this issue, we performed an analysis of derived regression residuals to evaluate a measure of nicotine-induced changes in locomotor activity, which by definition is independent of baseline values. The resulting QTL analyses indicated that in the AcB RCS three putative regions on chromosome 11 and 14 were identified (P < 0.01). In the BcA RCS, four putative regions on chromosomes 2, 8, 14 and 16 were identified. The loci were identical to those found using nicotine difference and nicotine percent change summarized in Tables 1 and 3. Additionally, analyses of the time course of nicotine effects, expressed as the percent change from day 2 saline, were examined. In the AcB RCS, the strain means for time blocks (min) 5, 10, 15 and 20 min were significantly Table 1: Chromosomal regions containing unique B6 donor alleles associated with nicotine-related phenotypes (nicotine difference and nicotine percent change ) in the AcB recombinant congenic strains (RCS) Chromosome Peak marker cm Correlation (P value) Region containing donor QTL (cm) Candidate gene (cm) Description 11 D11Mit þ0.584 (0.0002) Gabra1 (19.0) g-aminobutyric acid (GABA-A) receptor, subunit a1 Gabra6 (23.0) g-aminobutyric acid (GABA-A) receptor, subunit a6 11 D11Mit (0.003) Lore4 (49.0) Loss of righting induced by ethanol 4 (QTL) 12 D12Mit þ0.728 (0.002) Drb6 (59.0) Dopamine receptor binding 6 (QTL) 13 D13Mit þ0.609 (0.01) D14Mit þ0.643 (0.008) D17Mit þ0.656 (0.007) 56.7 Peak marker in significant region identified by MAP MANAGER QTX (P < 0.01). Recombinant distance in centimorgans from centromere. Correlations between strain distribution pattern for marker and strain means. It should be noted that in many cases multiple markers in a given region were identified by MAP MANAGER QTX; however, only the peak marker (with the largest correlation) was included in the table. The genetic maps were used to determine the interval (in centimorgans) containing the quantitative trait loci (QTL) by identifying all strains that possessed donor alleles at the significant markers. Positive correlations indicate that the B6 alleles increased the mean phenoytypic values when placed on an A/J background. Indicate loci which exceed the statistical threshold of suggestive or significant established through the use of the permutation test (MAP MANAGER QTX). 406 Genes, Brain and Behavior (2005) 4:

7 Nicotine-induced locomotor activation Table 2: Haplotypes at significant marker loci for nicotine activation and depression in the AcB recombinant congenic strains (RCS) Marker cm AcB RCS (nicotine percent change) AcB60 AcB51 AcB62 AcB54 AcB64 AcB65 AcB52 AcB53 AcB55 AcB58 AcB57 AcB56 AcB D11Mit A A A B A A A A A A A A B D11Mit A A A A A A A A A A A B B D11Mit A A A A A A A A A A A B B D11Mit A A A A A A A B A A A B B D11Mit A A A A A A A B A A A B B D11Mit A A A A A A A B A A A A B D11Mit B B B A A A A A A A B A A D11Mit B B B A A A A A A A A A A D11Mit B B B A A A A A A A A A A D11Mit B B A A A A A A A A A A A D11Mit B B A A A A A A A A A B A linformative strains with a significantly higher mean phenotypic values than the background A/J. Significant marker loci. Donor alleles inherited from the B6 strain are designated with a B. The table is arranged in ascending order of mean phenotypic values for nicotine percent change. (P < 0.05) correlated with the 30-min total session values (r ¼ 0.77, 0.74, 0.60 and 0.77, respectively). In the BcA RCSs, the strain means for time blocks (min) 5, 10, 15 and 20 were significantly (P < 0.05) correlated with the 30- min total session values (r ¼ 078, 0.88, 0.65 and 0.81, respectively). The results of the analysis of individual time blocks indicated that the identified QTL from 5 through 20 min in the AcB and BcA RCSs were consistent with those identified with the total nicotine percent change phenotype. Six putative chromosomal regions containing the B6 donor alleles on chromosomes 11, 12, 13, 14 and 17 were identified among the AcB RCS. In the BcA RCS, four putative regions on chromosomes 2, 7, 8, 14, 16 and 17 were identified. Table 3: Chromosomal regions containing unique A/J donor alleles associated with nicotine-related phenotypes (nicotine difference and nicotine percent change ) in the BcA recombinant congenic strains Chromosome Peak marker cm Correlation (P value) Region containing donor QTL (cm) Candidate gene (cm) Description 2 D2Mit þ0.513 (0.009) Smstr4 (84) Somatostatin receptor 4 7 D7Mit þ0.642 (0.0006) D8Mit þ0.584 (0.01) D13Mit þ0.514 (0.009) D14Mit þ0.783 (< ) Chrna2 (20) Cholinergic receptor, nicotinic, alpha polypeptide 2 (neuronal) Htr2a (41.5) 5-Hydroxytryptamine (serotonin) receptor 2A 16 D16Mit þ0.814 (< ) Drd3 (23.3) Dopamine receptor 3 17 D17Mit þ0.695 (0.0001) Peak marker in significant region identified by MAP MANAGER QTX (P < 0.01). Recombinant distance in centimorgans from centromere. Correlations between strain distribution pattern for marker and strain means. It should be noted that in many cases multiple markers in a given region were identified by MAP MANAGER QTX; however, only the peak marker (with the largest correlation) was included in the table. The genetic maps were used to determine the interval (in centimorgans) containing the quantitative trait loci (QTL) by identifying all strains that possessed donor alleles at the significant markers. Positive correlations indicate that the A/J alleles increased mean phenoytypic values when placed on a B6 background. Loci which exceed the statistical threshold of suggestive or significant established through the use of the permutation test (MAP MANGER QTX). Genes, Brain and Behavior (2005) 4:

8 Gill and Boyle Comparison of saline-, habituation- and nicotine-related phenotypes among the RCS Potential genetic overlaps among the saline-, habituationand nicotine-related phenotypes were examined by computing genetic correlations and by comparing the location of mapped QTL for each phenotype. Genetic correlations were computed using the strain means for the entire AcB/ BcA RC series (excluding progenitors) for all phenotypes as summarized in Table 4. The results demonstrated that the total day 1 saline activity were significantly correlated with day 2 saline as well as the habituation measures (P < 0.01). Only the total untransformed nicotine activity were correlated with the basal activity levels as measured on saline (days 1 and 2). In contrast, the SDP for the nicotine difference, nicotine percent change and regression residuals were not significantly correlated with any of the other phenotypes. Chromosomal regions carrying donor alleles modifying the saline baseline and habituation phenotypes among the RCS were identified through the calculation of single-locus associations, as described above. In the AcB RCS, significant (P < 0.01) QTL were identified for day 1 saline (chromosomes 1, 6 and 19), day 2 saline (chromosome 6), habituation difference (chromosomes 1, 6 and 19) and habituation percent change (chromosomes 10 and 15). In the BcA RCS, QTL were identified for day 1 saline (chromosomes 3, 6 and 15), day 2 saline (chromosomes 3 and 15), habituation difference (chromosomes 3, 6 and 15) and habituation percent change (chromosomes 6, 9, 10 and 15). A combined summary of all the chromosomal regions associated with saline and habituation phenotypes in the AcB/BcA RCS is summarized in Table 5. The loci identified in Table 5 were compared with those associated with nicotine-related phenotypes summarized in Tables 1 and 3, to identify the presence or absence of commonalities. The results of the analysis failed to identify any overlapping QTL. Discussion The present study examined genetic differences in the sensitivity to nicotine among the A/J, B6 and AcB/BcA RCS. Temporal analysis of progenitor data revealed that the A/J exhibited a significant decrease in locomotor activity following nicotine administration. In contrast, the B6 failed to exhibit locomotor excitation or depression and was generally insensitive to the effects of nicotine. This strain difference is consistent with the literature across multiple behavioral and physiological measures (Crawley et al. 1997; Marks et al. 1989; Miner & Collins 1989). On the other hand, the AcB/BcA RCS displayed a wide and continuous range of nicotine-induced activity, suggesting the presence of a quantitative trait involving the additive effects of several genes. In addition, a degree of nicotine-induced locomotor excitation was observed among the RCS, in contrast to the progenitors. As shown in Fig. 2, recombination of the parental genomes produced new strains of inbred mice that showed locomotor excitation in response to nicotine. Significantly, alleles originating from both the A/J and B6 progenitors were associated with nicotine-induced activation. Specifically, B6 alleles on an A/J background were associated with nicotine-induced activation in the RCS AcB61, AcB56, AcB57 and AcB58. The results of the full-genome screen for the effects of nicotine on locomotor activation revealed 12 loci that were associated with increased nicotine-induced activity in the AcB or BcA RCS and one B6 allele associated with locomotor depression in the AcB strains. It is important to note that QTL for nicotine-induced activation did not overlap with those identified for basal activity or the habituation measures. This finding is consistent with the observation that the nicotine-related phenotypes (nicotine difference, nicotine percent change and regression residuals) were not genetically correlated with total day 1 or 2 saline activity or habituation. In addition, the QTL for nicotine activation identified in the present study did not overlap with QTL identified for novelty/stress-induced activation mapped in the AcB/BcA RCS in the same laboratory Table 4: Genetic correlations between saline-, habituation- and nicotine-related phenotypes in the AcB/BcA recombinant congenic strains Phenotype Day 1 saline Day 2 saline Habituation difference Habituation percent change Total nicotine Nicotine difference Nicotine percent change Nicotine regression residuals Day 1 saline Day 2 saline Habituation difference Habituation percent change Total nicotine Nicotine difference Nicotine percent change Nicotine regression residuals Correlations are significant at P < 0.01 (two tailed). 408 Genes, Brain and Behavior (2005) 4:

9 Nicotine-induced locomotor activation Table 5: Chromosomal regions associated with saline- and habituation-related phenotypes in the AcB/BcA recombinant congenic strains Chromosome Peak marker cm Region containing donor QTL (cm) Day 1 and day 2 saline activity 1 D1Mit D3Mit D6Mit D15Mit D19Mit Habituation difference and percent change 1 D1Mit D3Mit D6Mit D9Mit D10Mit D15Mit QTL, quantitative trait loci. Peak marker in significant region identified by MAP MANAGER QTX (P 0.01). Recombinant distance in centimorgans from centromere. (unpublished data). Together, these results suggest that the genetic mechanisms mediating the expression of the nicotine phenotypes are distinct from those mediating the expression of basal or stress-induced locomotor responses. It is of interest that a number of the QTL associated with basal or stress-induced locomotor responses in the present study confirm those previously identified in the literature (Gershenfeld et al. 1997). Consistent with the present study, a number of open-field activity QTL including those on chromosome15 (42 cm) and 19 (19 cm) were identified in an A/J by C57BL/6J F2 cross (Gershenfeld et al. 1997). Potential candidate genes located in the significant QTL regions summarized in Tables 1 and 3 include the a2 nachr located at 20 cm on chromosome 14, the dopamine D3 receptor located a 23.3 cm on chromosome 16 and the g-aminobutyric acid (GABA-A) receptors Gabra1 (19 cm) and Gabra6 (23 cm) on chromosome 11. The identification of candidate genes is highly speculative at the present time because of the lack of resolution and the inherent risk of false positives in the process of QTL analysis. The candidates were chosen based on the known psychopharmacological actions of nicotine. Of interest is research showing that the dopamine-rich ventral tegmental area (VTA) of the brain is a central target for the effects of nicotine (Picciotto & Corrigall 2002) and its associated effects on locomotor activity (Panagis et al. 1996) and self-administration. It has been demonstrated that nicotine can influence the functioning of the dopamine system both directly and indirectly through the modulation of the GABA and glutamate neurotransmitter systems in the VTA (Mansvelder et al. 2002). Research indicates that the dopamine D3 receptor (Diaz et al. 2000), GABA-A a1 (Charlton et al. 1997) and the a2 nachr (Charpantier et al. 1998) are all expressed within the VTA. In the present study, a QTL associated with altered sensitivity to the effects of nicotine in the AcB/BcA RCS was identified in a region within the region coding for the a2 nachr subunit (20 cm on chromosome 14). Previous research has shown that KO mice lacking the a4 nachr subunit exhibit less sensitivity to the locomotor-depressant effects of nicotine (Ross et al. 2000). Similarly, Tritto et al. (2002) demonstrated that genetic differences in sensitivity to nicotine could be partially explained by polymorphisms in the neuronal nicotinic a4 and a6 genes. Overall, recent reviews have noted that the genes encoding the various nicotine receptor subunits are strong candidates for genes regulating nicotine dependence (Lerman & Berrettini 2003). A potential limitation of the present study is the lack of measurement of blood and brain nicotine levels and their relationship to the observed behavioral effects. At the present time, it is assumed that there are no differences among the AcB/BcA RCS in terms of nicotine pharmacokinetics; however, additional research is warranted to address this important issue. To date, there is little data to suggest that there are significant strain differences in nicotine absorption or elimination. In fact, Hatchell and Collins (1980) demonstrated that there were no pharmacokinetic differences across three inbred mouse lines, including the B6. Specifically, they failed to observe any consistent genetic influence between the expression of nicotine-induced locomotor activity and the rate of liver nicotine elimination or brain nicotine levels. In conclusion, single-locus association analysis identified QTL for nicotine-induced activation on chromosomes 11, 12, 13, 14 and 17 in the AcB strains. In the BcA strains, nicotineinduced activation was associated with seven putative regions on chromosomes 2, 7, 8, 13, 14, 16 and 17. 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