Genes, Brain and Behavior (2005) 4: 401–411
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.
E-mail: [email protected]
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 scores, nicotine percent change and nicotine regression residual scores. 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
doi: 10.1111/j.1601-183X.2005.00116.x
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
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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
402
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 24–32 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
30 30 40 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
Genes, Brain and Behavior (2005) 4: 401–411
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 scores (day 2 day 1) and
habituation percent change scores (day 2 saline expressed
Genes, Brain and Behavior (2005) 4: 401–411
as the percent change from day 1 saline). Additionally, the
following measures of nicotine-induced activity were utilized
in all analyses: total nicotine activity scores (day 3), nicotine
difference scores (day 3 nicotine day 2 saline), nicotine
percent change scores (expressed as the percent change
from day 2 saline), nicotine percent change scores 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 scores on baseline saline scores. SPSS was used
to calculate the deviations of the observed nicotine scores
from the values predicted by the regression of total nicotine
scores on baseline saline scores. The latter nicotine activity
phenotypes (difference scores, nicotine percent change
scores 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 scores 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)
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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 MANAGER QTX was set to detect simple locus associations at a
threshold value of P 0.01. 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 (http://www.informatics.jax.org).
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 scores obtained on days 3–6. There was a significant
main effect for strain (F1,33 ¼ 25.09, P < 0.001), but no
significant main effect for sex (F1,33 ¼ 0.29, P > 0.05). In addition, there were no significant interactions (strain dose:
F1,140 ¼ 0.48, P > 0.05; strain sex: F1,33 ¼ 1.02, P > 0.05;
strain sex dose: F4,132 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
scores. 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.
404
The analysis failed to indicate a significant three-way interaction (F16,205 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 scores using two-way
ANOVA with repeated measures (strain nicotine dose)
revealed a significant effect of strain (F1,36 ¼ 4.34, P < 0.05)
and no significant strain dose interaction (F4,144 ¼ 0.127,
P > 0.05) Similarly, analysis of nicotine percent change
scores yielded a significant main effect for strain
(F1,35 ¼ 4.64, P < 0.04), with no strain dose interaction
(F4,140 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 (F1,36 ¼ 4.339, P < 0.05), as well as a
strain dose time interaction (F20,720 ¼ 1.652, P < 0.05)
for the nicotine difference scores. Similarly, analysis of nicotine percent change scores found a significant main effect
for strain (F1,35 ¼ 4.64, P < 0.04) and a significant
strain dose time interaction (F20,700 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
scores.
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 0.890 (P < 0.001) for
total day 1 saline, 0.926 (P < 0.001) for total day 2 saline,
0.830 (P < 0.001) for habituation difference and 0.825
(P < 0.001) for habituation percent change scores.
Nicotine-induced changes in locomotor activity
Analysis of nicotine difference scores among the RCS
using two-way ANOVA (strain sex) yielded a significant main
effect for strain (F37,371 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 scores using a two-way
ANOVA (strain sex) yielded a significant main effect for strain
(F37,384 ¼ 2.968, P < 0.001), but no sex or strain sex interaction effects (all P values > 0.05). Mean nicotine percent
change scores for (males and females combined) the AcB
Genes, Brain and Behavior (2005) 4: 401–411
Nicotine-induced locomotor activation
Activity scores (nicotine %
change)
A/J
Nic 0.0
260
240
220
200
180
160
140
120
100
80
60
40
20
0
–20
–40
–60
–80
–100
Nic 0.1
Nic 0.2
Nic 0.4
Nic 0.8
5
10
15
20
25
30
Activity scores (nicotine %
change)
Time course
C57BL/6J
260
240
220
200
180
160
140
120
100
80
60
40
20
0
–20
–40
–60
–80
–100
Nic 0.0
Nic 0.1
Nic 0.2
Nic 0.4
Nic 0.8
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 scores: F14,152 ¼ 3.76, P < 0.001;
nicotine percent change scores: F13,152 ¼ 3.89, P < 0.001)
and BcA RCS (nicotine difference scores: F22,299 ¼ 2.130,
P < 0.01; nicotine percent change: F22,319 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 0.824
(P < 0.001) for nicotine differences scores and 0.830
(P < 0.001) for nicotine percent change scores.
Genetic analysis in the RC strains
5
10
15
20
25
30
Time course
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 scores).
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 scores
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
300
Genes, Brain and Behavior (2005) 4: 401–411
*
200
*
150
*
* *
100
*
50
0
–50
–100
AcB (A/J background)
BcA (B6 background)
–150
Ac
AcB60
AcB51
AcB62
AcB54
AcB64
AcB65
B5
2
Ac A/J
Ac B53
AcB55
AcB58
B
Ac 57
B
Ac 56
BcB61
BcA84
A
C Bc 77
57 A
BL 67
Bc /6J
BcA87
BcA69
BcA72
BcA83
BcA81
BcA70
BcA80
BcA66
BcA82
BcA86
BcA75
BcA74
BcA85
BcA76
BcA79
BcA68
BcA71
BcA73
A7
8
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 scores are
expressed as a percent change from
day 2 saline controls (nicotine percent
change scores). Strain means are
plotted in the order of ascending
activity scores. Informative strains are
denoted by asterisks.
Activity scores (nicotine % change)
250
Progenitor or recombinant strain
405
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 scores and nicotine percent change
scores 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 scores. The putative chromosomal regions
containing A/J donor alleles that were associated with both
nicotine difference scores and nicotine percent change
scores 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 scores 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 scores, 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 scores
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 scores) in the AcB recombinant congenic strains (RCS)
Chromosome
Peak marker*
cM†
Correlation‡ (P value)
Region containing
donor QTL (cM)
Candidate
gene (cM)
Description
11
D11Mit82
14.0
þ0.584 (0.0002)¶
1.5–18.0
Gabra1 (19.0)
g-Aminobutyric acid (GABA-A)
receptor, subunit a1
g-Aminobutyric acid (GABA-A)
receptor, subunit a6
Loss of righting induced by
ethanol 4 (QTL)
Dopamine receptor binding 6
(QTL)
Gabra6 (23.0)
11
D11Mit39
49.0
0.695 (0.003)
47.67–54.0
Lore4 (49.0)
12
D12Mit233
52.0
þ0.728 (0.002)¶
52.0–59.0
Drb6 (59.0)
13
14
17
D13Mit218
D14Mit165
D17Mit221
9.0
52.0
56.7
þ0.609 (0.01)
þ0.643 (0.008)¶
þ0.656 (0.007)¶
8.0–11.0
52.0–60
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: 401–411
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
D11Mit74
D11Mit62†
D11Mit82†
D11Mit135†
D11Mit51†
D11Mit268
D11Mit119
D11Mit39†
D11Mit285†
D11Mit70
D11Mit263
0.00
1.50†
16.00†
17.00†
18.00†
19.00
47.67
49.00†
52.00†
54.00
55.60
AcB51
AcB62
AcB54
AcB64
AcB65
AcB52 AcB53 AcB55 AcB58* AcB57* AcB56* AcB61*
75.262 61.39 51.228 46.757 28.89 27.012 2.75 38.44
40.116 95.96
100.89
124.568 192.668
A
A
A
A
A
A
B
B
B
B
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
A
A
A
A
A
B
B
B
B
A
A
A
A
A
B
A
A
A
A
A
A
B
B
B
B
B
A
A
A
A
A
A
B
B
B
A
A
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
A
A
A
A
A
l*Informative 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 scores.
†
(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 30min 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 scores) in the BcA recombinant congenic strains
Chromosome
Peak marker*
cM†
Correlation‡ (P value)
Region containing
donor QTL (cM)
Candidate
gene (cM)
Description
2
D2Mit311
D7Mit66
D8Mit124
D13Mit59
D14Mit155
83.1
57.5
6.0
16.0
25.0
þ0.513
þ0.642
þ0.584
þ0.514
þ0.783
71.0–96.0
57.5–65.6
6.0
16.0–21.0
14.5–43.0
Smstr4 (84)
Somatostatin receptor 4
Chrna2 (20)
Cholinergic receptor, nicotinic,
alpha polypeptide 2 (neuronal)
5-Hydroxytryptamine (serotonin)
receptor 2A
Dopamine receptor 3
7
8
13
14
(0.009)
(0.0006)
(0.01)
(0.009)
(<0.00001)¶
Htr2a (41.5)
16
17
D16Mit131
D17Mit76
4.3
54.6
þ0.814 (<0.00001)¶
þ0.695 (0.0001)¶
4.3–66.0
44.5–56.0
Drd3 (23.3)
*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: 401–411
407
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 scores were significantly correlated with day 2 saline as well as the habituation measures (P < 0.01). Only the
total untransformed nicotine activity scores were correlated
with the basal activity levels as measured on saline (days 1
and 2). In contrast, the SDP for the nicotine difference
scores, nicotine percent change scores 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 scores (chromosomes 1, 6 and 19)
and habituation percent change scores (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 scores (chromosomes 3, 6 and 15) and habituation percent change scores
(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 scores and regression residuals)
were not genetically correlated with total day 1 or 2 saline
activity or habituation scores. 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
scores
Day 2
saline
scores
Habituation
difference
scores
Habituation
percent change
scores
Total
nicotine
scores
Nicotine
difference
scores
Nicotine
percent change
scores
Nicotine
regression
residuals
Day 1 saline
Day 2 saline
Habituation difference
Habituation percent change
Total nicotine
Nicotine difference
Nicotine percent change
Nicotine regression residuals
—
0.767*
0.838*
0.523*
0.585*
0.067
0.183
0.138
—
—
0.318
0.003
0.585*
0.281
0.304
0.030
—
—
—
0.805*
0.323
0.113
0.031
0.208
—
—
—
—
0.068
0.213
0.072
0.094
—
—
—
—
—
0.316
0.266
0.465*
—
—
—
—
—
—
0.635*
0.596*
—
—
—
—
—
—
—
0.580*
—
—
—
—
—
—
—
—
*Correlations are significant at P < 0.01 (two tailed).
408
Genes, Brain and Behavior (2005) 4: 401–411
Nicotine-induced locomotor activation
Table 5: Chromosomal regions associated with saline- and
habituation-related phenotypes in the AcB/BcA recombinant
congenic strains
Chromosome
Day 1 and day 2 saline
activity scores
1
3
6
15
19
Habituation difference and
percent change scores
1
3
6
9
10
15
Peak marker* cM† Region containing
donor QTL (cM)
D1Mit435
D3Mit335
D6Mit184
D15Mit68
D19Mit106
36.9 32.8–48.8
29.5 29.5
26.3 0.5–29.0
44.1 39.1–48.2
22.0 0.5–22
D1Mit435
D3Mit224
D6Mit340
D9Mit279
D10Mit42
D15Mit68
36.9
22.0
72.0
67.0
44.0
44.1
32.8–36.9
22.0–29.5
65.5–74.1
55.0–67
36.0–52.0
39.1–47.9
QTL, quantitative trait loci.
*Peak marker in significant region identified by MAP MANAGER
(P 0.01).
†
Recombinant distance in centimorgans from centromere.
QTX
(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
Genes, Brain and Behavior (2005) 4: 401–411
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. While
a number of interesting candidate genes including the a2
subunit of the nicotinic receptor, the dopamine D3 receptor
and the GABA-A a1 and a6 subunits were located in proximity to regions associated with nicotine-induced locomotor
activation, further confirmation and fine mapping are
required.
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Acknowledgments
This research was supported by grants awarded to K. J. Gill from
the Canadian Institutes of Health Research and the Alcohol
Beverage Medical Research Foundation. The authors thank
Emerillon Therapeutics Inc., Canada, as well as Dr E. Skamene,
Director of the Research Institute of the McGill University Health
Centre, for providing access to the AcB/BcA mice and genetic
maps. The authors thank G. Gauthier for her technical assistance
in testing animals.
411