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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 280:911–918, 1997
Vol. 280, No. 2
Printed in U.S.A.
Common Quantitative Trait Loci for Alcohol-Related
Behaviors and Central Nervous System Neurotensin Measures:
Hypnotic and Hypothermic Effects1
V. GENE ERWIN, PAUL D. MARKEL,2 THOMAS E. JOHNSON,2 VAUGHN M. GEHLE and BYRON C. JONES
Alcohol Research Center and School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado
Accepted for publication October 21, 1996
Evidence indicates a heritable component to alcoholism
(Goodwin et al., 1973; Bohman et al., 1981) and, in human
(Schuckit, 1980, 1984; Wilson et al., 1984) and animal studies
(McClearn and Kakihana, 1981), individual differences in
sensitivity to ethanol are well established. Careful studies
have shown that individuals at high risk for alcoholism differ
from individuals at low risk in their subjective responses to
ethanol and sensitivity to body sway (Schuckit, 1984). Recent
findings (Schuckit, 1994) provide strong evidence that initial
sensitivity to ethanol is a good predictor of the development
of alcoholism. Thus, identification of genes regulating ethanol sensitivity has been the subject of much recent research.
Quantitative genetic studies of ethanol-related behaviors
have shown that measures of hypnotic sensitivity, ST or
BECRR, and hypothermia are continuously and normally
distributed as a result of polygenic determination of the
phenotypes (DeFries et al., 1989; Erwin et al., 1990a). Indeed,
Received for publication February 19, 1996.
1
This work was supported, in part, by USPHS Grants AA 03527, AA 08940
and AA 07330.
2
Current address: Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309.
for NTRH density were found in common with confirmed QTL
for hypnotic sensitivity on chromosomes 1 (43 cM), 11 (57 cM)
and 15 (56 cM) and with an unconfirmed QTL on chromosome
3 (19 cM). Two common QTL for NT-ir levels, but not NTRH or
low-affinity neurotensin receptor receptors, and ethanol-induced hypothermia were observed on chromosomes 4 (43 cM)
and 6 (41 cM). Two common QTL for NT-ir levels and sleep time
were identified on chromosomes 3 (19 cM) and 9 (55 cM).
Common QTL indicate that genes regulating NT receptor
and/or NT-ir expression may be the same as those regulating
sensitivity to ethanol.
results obtained with the LSXSS RI strains indicate that
hypnotic sensitivity and hypothermia are regulated by a
minimum of seven and four genes, respectively (DeFries et
al., 1989; Erwin et al., 1990a). These RI strains were derived
from Long Sleep (LS/lbg) and Short Sleep (SS/lbg) lines of
mice, selectively bred for differences in hypnotic sensitivity to
ethanol and found to differ also in ethanol-induced hypothermia (DeFries et al., 1989).
Several studies have used QTL analyses to identify chromosomal regions linked to genes that regulate differences in
ethanol-related behaviors. Using the BXD RI derived from
the C57BL/6J and DBA/2J inbred strains of mice, investigators (Plomin et al., 1991; Rodriguez et al., 1995) found QTL at
the P , .05 level for ethanol consumption and hypnotic
sensitivity. Subsequently, other investigators (Buck et al.,
1994; Crabbe et al., 1994a; Phillips et al., 1994, 1995) have
reported finding provisional, P , .05, QTLs for ethanol-related behaviors including preference, locomotor activation,
tolerance and acute withdrawal seizures. Markel et al. (1996)
recently reported the use of SSLP markers to identify QTL
for ethanol-induced sleep time in LSXSS RI strains of mice.
These investigators found several provisional, P , .05, QTL
ABBREVIATIONS: NT, neurotensin; NTRH and NTRL, high- and low-affinity neurotensin receptor, respectively; NT-ir, neurotensin-immunoreactivity; QTL, quantitative trait loci; RI, recombinant inbred; cM, centiMorgan; VMB, ventral midbrain; STR, striatum (includes nucleus accumbens,
NA, and caudate putamen); HYP, hypothalamus; ST, sleep time (duration of loss of righting response after 4.2 g/kg ethanol) and BECRR, blood
ethanol concentration at regaining righting response; FC, frontal cortex; SSLP, simple sequence length polymorphism.
911
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ABSTRACT
Genetic correlations were found between high-affinity neurotensin receptor (NTRH) densities and NT-immunoreactivity (NTir) levels in specific brain regions and sensitivity to hypnotic and
hypothermic effects of ethanol in LSXSS recombinant inbred
strains of mice. Simple sequence length polymorphisms were
used to identify quantitative trait loci (QTL) influencing hypnotic
and hypothermic sensitivity to ethanol, NTRH and low-affinity
neurotensin receptor densities and NT-ir levels in LSXSS recombinant inbred strains. Common QTL for NTRH receptor
densities, NT-ir levels and these ethanol actions were identified.
One of the QTL (chromosome 2, 80 cM) for NTRH density and
hypnotic sensitivity is linked to the NTRH gene, Ntsr. Also, QTL
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Erwin et al.
Vol. 280
Methods
Animals. Male LSXSS RI strains of mice were obtained from the
Institute for Behavioral Genetics, University of Colorado, Boulder,
CO. All experiments were conducted with mice (60–80 days of age)
which were maintained in a constant temperature (22°C), humidity
(20%) and light (12L/12D) environment. Separate groups of mice
were used for each phenotype determination.
Hypnotic and hypothermic sensitivity to ethanol. Hypnotic
sensitivity to ethanol was measured by determining the duration of
loss of righting response (sleep time) and the blood ethanol concentration, in mg ethanol per dl blood (mg/100 ml), at regaining righting
response following 4.2 g/kg ethanol. Hypothermia was measured as
the difference in rectal temperature immediately before, and at 60
min after administration of ethanol (4.2 g/kg i.p.). The procedures
and the mean values for these behavioral phenotypes in each LSXSS
RI strain have been published previously (DeFries et al., 1989; Erwin
et al., 1990a) and the data from those studies were used to determine
genetic correlations and perform QTL analyses described in the
present paper.
Neurotensin extraction and radioimmunoassay. Naive mice,
housed in groups of two to six per cage, were killed by cervical
dislocation and decapitation. Brains were dissected quickly (,1 min)
and specific regions punched according to the anatomical guidelines
of Slotnick and Leonard (1975). Neurotensin was extracted and
assayed as previously described (Erwin and Jones, 1989; Erwin et
al., 1990b). Regions were weighed and homogenized in 10 to 20
volumes of 0.01 N HCl, and placed in a boiling water bath for 5 min.
Homogenates were centrifuged at 20,000 3 g for 20 min, and the
resulting supernatant extracts were lyophilized and stored at 270°C
for less than 2 wk before radioimmunoassay. Standard double antibody radioimmunoassays (were used to measure NT concentrations
(Erwin and Jones, 1989; Erwin et al., 1990b). Details of the procedures and the mean values for NT levels in specific brain regions
including hypothalamus, nucleus accumbens and ventral midbrain
from LSXSS RI strains of mice, have been published previously
(Erwin et al., 1993).
Neurotensin binding assays. Animals were killed and brains
dissected as described above; dissected and/or punched regions were
rapidly chilled in cold (4°C) 50 mM Tris buffer, pH 7.4, containing 40
mg/ml bacitracin and 1 mM EDTA. Pooled regions from two to four
brains were homogenized in ten volumes of buffer; the homogenates
were centrifuged at 100,000 3 g for 30 min. The resulting membrane
pellet was rehomogenized and centrifuged; this wash procedure was
repeated twice.
Binding assays were performed essentially as described by Kitabgi
et al. (1977) and as previously published (Campbell and Erwin, 1993;
Erwin et al., 1993). Specific binding of saturating concentrations of
NT (20 nM 3H-NT) was determined in the presence of 10 mM levocabastine, a selective blocker of NTRL, to determine NTRH densities.
Total specific binding minus NTRH was defined as NTRL. Mean
values for densities of both NTRH and NTRL in specific brain regions
including frontal cortex, striatum (combined nucleus accumbens and
caudate putamen), and ventral midbrain from LSXSS RI strains of
mice, have been published previously (Erwin et al., 1993).
QTL analysis. LSXSS RI strains were genotyped as previously
published (Markel et al., 1996) using 120 simple sequence length
polymorphism (SSLP) markers (Research Genetics, Huntsville, AL)
found to be polymorphic in LS and SS parental strains. These markers covered the mouse genome at an average marker interval of 15
cM. Strain distribution patterns were established for all 20 linkage
groups in 24 RI strains (Markel et al., 1996). Because the LS and SS
parental lines were not completely inbred, more than two alleles
exist for some markers among the RI strains. Ten percent (12 of 120)
markers gave three or more alleles and as a result of being unable to
know the exact genotype (frequency of alleles) in the outbred LS and
SS progenitors of the Ris, we were unable to use the interval mapping method described by Markel et al. (1996) in identifying QTL.
Therefore, one-way analysis of variances were carried out with the
phenotypic measure as the dependent variable and the RI strains
were grouped by allele type. In approximately 6% (8 of 120) of the
markers, a given allele was represented only by one RI strain.
Because the unique strain may be the result of a new microsatellite
mutation, the resulting QTL are suspect; those instances are indicated in the tables. Analyses were performed with SPSS version 6.0
for Windows. Because this is not only an exploratory study to identify
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for hypnotic sensitivity and have identified and confirmed
significant QTL in a large panel of F2 mice derived from the
ISS and ILS inbred strains (Markel et al., 1996). One of the
highly significant QTL was for the marker, D2MIT21, located
at 80 cM on chromosome 2 (Markel et al., 1996), a locus
linked to the region containing the high-affinity NT receptor
gene, Ntsr (Laurent et al., 1994).
Consideration of Ntsr as a candidate gene for regulation of
ethanol sensitivity is consistent with a number of pharmacological and behavioral effects of NT. Central administration
of NT produces effects similar to those observed after acute
ethanol administration (Erwin et al., 1990a) including hypothermia (Nemeroff et al., 1977; Jolicoeur et al., 1981; Hernandez et al., 1984) and locomotor activation (Kalivas et al.,
1981; Kalivas et al., 1982) or inhibition (Nemeroff et al., 1977;
Clineschmidt et al., 1979; Jolicoeur et al., 1981), depending
on the dose and site of administration. Moreover, centrally
administered NT markedly enhances hypnotic, locomotor inhibitory and hypothermic effects of ethanol (Luttinger et al.,
1981; Erwin et al., 1987; Widdowson, 1987; Erwin and Su,
1989) in both rats and mice. Recent studies have shown a
cross-tolerance between ethanol- and neurotensin-induced
locomotor inhibition or hypothermia (Erwin et al., 1995).
Neurotensin receptors have been well-characterized from
rat and mouse brain (Mazella et al., 1983; Kitabgi et al., 1985;
Campbell et al., 1991). These studies revealed biphasic binding isotherms best described by two independent, NTRH and
NTRL, binding sites. The H1 histamine antagonist, levocabastine, selectively inhibits binding of NT to NTRL (Kitabgi
et al., 1985; Campbell et al., 1991), providing a valuable tool
to distinguish between NTRH and NTRL. Levocabastine has
been used to demonstrate differences in densities of NTRH
and NTRL in brain regions from LS and SS mice (Campbell et
al., 1991) and from the LSXSS RI strains of mice (Erwin et
al., 1993). The results indicated that differences in NT receptor densities and in NT-ir levels were mediated by multiple
genes with additive effect (Erwin and Jones, 1993; Erwin et
al., 1993) and significant genetic correlations were observed
between hypnotic sensitivity to ethanol and FC NTRH density (Erwin and Jones, 1993). Genetic correlations between
NT-ir levels, or NT receptor densities, and ethanol-related
behaviors provides a rational basis for searching for common
QTL between NT receptor densities or NT-ir levels and ethanol sensitivity.
Identification of common QTL regulating NTR binding capacities, NT-ir levels and ethanol actions would support the
hypothesis that genetic differences in ethanol sensitivity are
mediated, in part, by differences in these NTergic processes.
Thus, the research presented herein had three aims: 1) to
identify, in LSXSS RI strains, QTL for BECRR and hypothermia; 2) to determine QTL for NT receptor densities and NT-ir
levels and 3) to determine if any QTL for hypnotic or hypothermic effects of ethanol are in common with QTL for NT
receptor densities and NT-ir levels in specific brain regions.
1997
913
QTL for Alcohol Sensitivity and NT
provisional QTL for NT measures, but is intended to identify common QTL between independent phenotypes, levels of significance,
P , .05, is used. It is recognized that this level of significance,
particularly with limited numbers of RI strains (22–26, depending on
the phenotype), will produce type I errors (Lander and Schork, 1994).
However, the goal of this study is similar to those of Crabbe et al.
(1994a) and Phillips et al. (1995), i.e., to identify any potentially
important QTL by using an a level of 0.05 because it reduces the type
II error rate. Protection against type I error depends on future LS
and SS inbred strains (Belknap et al., 1996) and on finding QTL in
common with those reported by other investigators using independent panels of RI strains. Candidate genes that are associated with
a linkage group 615 cM (the average map interval for markers) were
taken from 1994 Chromosome Committee Report (Committee on the
Mouse Genome, 1994).
Results
TABLE 1
Genetic correlations between measures of ethanol sensitivity
and neurotensin-ir levels and receptor densities in the LSXSS
recombinant inbred strains of mice
Sleep Time
NT-ir
HYP
NA
VMB
20.38 (0.07)
20.24 (0.28)
20.62
(0.002)
NTRL
FC
STR
VMB
NTRH
FC
STR
VMB
BECRR
Hypothermia
0.21 (0.34)
0.06 (0.78)
0.41 (0.05)
20.20 (0.35)
20.30 (0.17)
0.07 (0.76)
0.22 (0.29)
20.02 (0.93)
0.13 (0.54)
20.18 (0.33)
0.11 (0.56)
0.06 (0.77)
20.05 (0.80)
0.07 (0.75)
20.06 (0.78)
0.39 (0.05)
0.40 (0.04)
0.13 (0.51)
20.12 (0.56)
20.38 (0.06)
20.12 (0.55)
20.05 (0.80)
20.28 (0.16)
0.05 (0.79)
Values represent Pearson product moment correlations among LSXSS RI
strain means (n 5 22–26) for each phenotype. The level of significance (P value)
is shown in parentheses.
TABLE 2
QTL for high-dose ethanol actions in the LSXSS RI strains
Chromosome
No.
1
2
3
4
6
9
11
14
15
18
Marker
cM
D1Mit45
D1Mit17
D2Mit21
D3Mit21
D4Mit205
D6Mit67
D6Mit36
D9Mit42
D9Mit12
GABRA1
D14Mit1
D15Mit12
D18Mit7
58
103
80
19
43
41
46
8
55
19
3
6
50
Sleep
Time
0.09a
0.03
0.005a
BECRR
Hypothermia
0.04
0.03
0.05
0.006
0.01
0.02
0.03
0.04
a
0.03
0.02
0.05
a
0.03
0.04
The level of significance is shown for RI mean values grouped by allele.
Significant differences were detected with one-way ANOVA. a Denotes QTL confirmed in ILS 3 ISS F2 generation. This F2 study also found significant QTL for
sleep time on chromosome 11 at 56 cM and chromosome 15 at 61 cM (Markel et
al., submitted).
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Genetic correlations between ethanol sensitivity
and NT measures. The genetic correlations between NTRH
and NTRL and hypnotic sensitivity to ethanol (ST) have been
reported previously (Erwin and Jones, 1993). In our study,
additional NT receptor binding data were included for some
RI strains so that mean values were based on at least four
degrees of freedom for those strains. Therefore, in table 1, we
have included the correlations between NTR densities and
ST as well as between all NT measures and BECRR and
hypothermia. Among the LSXSS RI strains, ST and BECRR
are negatively correlated measures of hypnotic sensitivity
(DeFries et al., 1989) with a high BECRR being related to a
short ST, i.e., insensitivity to ethanol. Thus, as observed in
table 1, NT measures positively correlated with ST are negatively correlated with BECRR and vice versa. Correlations
between NT-ir in HYP or NA and ST were not significant, but
showed a consistent trend with r values of 20.38 and 20.24,
respectively. Significant positive correlations were observed
between ST and NTRH densities in frontal cortex (FC) and
STR, but not in VMB. The results showed 4 correlations of 27
with values of P , .05 and the cumulative Poisson distribution was used to determine the probability of observing 4
correlations at P , 0.05 when only 1.35 (0.05 3 27) are
expected by chance. The probability was calculated to be p 5
0.048 [P(4 or more) 5 1–0.9517]. Thus, it is highly likely that
one or more of these genetic correlations indicate common
underlying mechanisms regulating hypnotic sensitivity to
ethanol and NT measures.
Provisional QTL for ethanol actions. It should be recognized that QTL analyses allow only a rough identification
of genetic map locations of genes that exert modest effects on
continuously distributed phenotypes. Results presented in
table 2 show provisional QTL identified by specific SSLP
markers and map distance (cM), for ST, BECRR and hypothermia. QTL for ST in the LSXSS RI strains have been
published elsewhere (Markel et al., 1996) and are presented
for comparison with other phenotypes and for identification
of common QTL. As indicated in table 2, two of the six QTL
for ST have been confirmed (Markel et al., submitted) in a
large panel of F2 mice derived from inbred strains of LS and
SS. The two confirmed QTL for ST were on chromosomes 1 at
58 cM (D1MIT45 marker) and 2 at 80 cM (D2MIT21 marker).
There were three common QTL for ST and BECRR, an expected result, because these phenotypes both measure hypnotic sensitivity at the same endpoint. The QTL for ST on
chromosome 2 was observed also for BECRR and there were
two other, as yet unconfirmed, QTL in common for ST and
BECRR on chromosomes 1 and 18. The five QTL for hypothermia were on chromosomes 4, 6, 11, 14 and 15.
Provisional QTL for neurotensin receptors. The densities of NTRH and NTRL in the FC or STR have been shown
to differ up to 1.6- and 1.9-fold, respectively, among the
LSXSS RI strains (Erwin et al., 1993). These differences are
continuously and normally distributed and show a significant effect of RI strain (genetic effect) on NT receptor density
in all brain regions. Estimates indicate a minimum of six and
four genes controlling the differences in densities of NTRH
and NTRL, respectively (Erwin et al., 1993) for STR and FC.
In VMB, four and three genes were estimated to account for
differences in NTRH and NTRL, respectively. Provisional
QTL for genes regulating differences in NTRL and NTRH
densities are shown in tables 3 and 4. For NTRH, there were
4, 9 and 8 QTL for VMB, STR and FC, respectively. This
number of QTL for each brain region is reasonable considering the number of genes estimated to regulate densities of
these receptors. There are common QTL for NTRH in STR
and FC on chromosomes 2 at 80 cM and 8 at 8 cM and one
QTL in the same linkage group on chromosome 8 for VMB,
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Erwin et al.
Vol. 280
TABLE 3
QTL for densities of high-affinity neurotensin receptors in brain
regions from the LSXSS RI strains
Chromosome
No.
1
2
3
6
7
8
9
11
16
Map
Distance
(cM)
D1Mit45
D2Mit21
D3Mit21
D3Mit22
D3Mit15
D6Mit48
D7Mit21
D7Mit71
D8Mit18
D8Mit4
D9Mit4
HOXB
D11Mit52
D11Mit105
D13Mit55
D14Mit35
D15Mit3
D15Mit41
D16Mit34
58
80
19
39
66
10
1
65
8
15
29
56
62
80
10
44
38
61
12
Ventral
Midbrain
Striatum
Frontal
Cortex
0.05
0.004
0.03
0.01
0.005
0.02
0.03*
0.05
0.04
0.04
0.03
0.05
0.03
0.04
0.006
0.01
0.002
0.02
0.02
0.02
0.009
0.008
The level of significance, where P , .05, is shown for RI mean values grouped
by allele. Significant differences were detected with one-way ANOVA. a denotes
a provisional QTL driven by an allele found in only one of the 26 RI.
TABLE 4
QTL for densities of low-affinity neurotensin receptors in brain
regions from the LSXSS RI strains
The level of significance, where P , .05, is shown for RI mean values grouped by
allele. Significant differences were detected with one-way ANOVA. a denotes
provisional QTL driven by an allele found in only one of the 26 RI.
Chromosome
No.
1
2
3
4
5
6
8
9
11
12
13
15
16
19
Marker
Map
Distance
(cM)
D1Mit1
D1Mit42
D1Mit17
D2Mit56
D3Mit18
D4Mit1
D5Mit24
D6Mit15
D8Mit42
D9Mit2
D9Mit4
D11Mit2
D12Mit46
D12Mit33
D13Mit19
D13Mit35
D15Mit41
D15Mit14
D6Mit6
D19Mit16
14
78
103
39
76
9
66
78
71
17
29
2
17
29
25
72
61
64
58
15
Ventral
Midbrain
Striatum
Frontal
Cortex
0.02
TABLE 5
QTL for neurotensin-immunoreactivity in brain regions from the
LSXSS RI strains
0.01
The level of significance, where P , .05, is shown for RI mean values grouped by
allele. Significant differences were detected with one-way ANOVA. a denotes
provisional QTL driven by an allele found in only one of the 26 RI.
0.04
0.04
0.04
0.01
0.0002
0.00001
0.01
0.01
0.04
0.03a
0.04
0.008
0.04
0.01
0.0001
0.02
0.004
0.03
0.02
0.04
0.01
0.04
Chromosome
No.
2
3
0.0004
4
0.006
0.04
0.03
5
6
7
8
9
STR and FC. Several candidate genes are located in the
region of the QTL on chromosome 2 including Ntsr, NTRH;
Avp, arginine vasopressin; and Pdyn, prodynorphin genes.
Previous studies showed genetic correlations for NTRH density across brain regions were relatively low, r 5 0.24 to 0.37
(Erwin et al., 1993). Similarly, our study shows a limited
number of common QTL for NTRH across brain regions indicating differences in regulation of densities of the receptors
in these brain regions.
The results in table 4 show a number of significant, P ,
.01, QTL for NTRL. Common QTLs were observed for NTRL
12
13
14
18
19
X
Marker
D2Mit7
D3Mit21
D3Mit22
D3Mit18
D4Mit27
D4Mit205
D5Mit24
D6Mit67
D6Mit15
D7Mit21
D8Mit4
D8Mit42
D9Mit35
D9Mit12
D12Mit33
D12Mit27
D13Mit19
D13Mit23
3
D13Mit27
D14Mit35
D18Mit7
D19Mit33
D19Mit6
DXMit27
DXMit48
Map
Distance
(cM)
Ventral
Midbrain
27
19
39
76
31
43
66
41
78
1
15
71
52
55
29
52
25
29
0.05
0.008
42
44
50
42
54
3
14
0.03
0.04
0.003
Nucleus
Accumbens
Hypothalamus
0.04
0.04
0.002
0.02a
0.01a
0.03
0.0005
0.02
0.05
0.002
0.03
0.03
0.006
0.0006a
0.0006a
0.0002
0.03a
0.03
0.01
0.04
0.01
0.04
0.02
0.02
0.01
0.05
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13
14
15
Marker
in VMB, STR and FC on chromosomes 6, 8 and 13. There
were common QTL for NTRH and NTRL on two chromosomes,
chromosome 9 at 29 cM and chromosome 15 at 61 cM. Candidate genes for these QTL include Drd2 dopamine D2 receptor and Tcfcp2, transcription factor CP2. Evidence that the
Drd2 locus may be a candidate gene is consistent with studies showing that administration of dopamine D2 antagonists
alters NTRH binding in rats (Bolden-Watson et al., 1993).
Densities of NTRL and NTRH appear, for the most part, to be
regulated independently, because there was only one significant genetic correlation between densities of these receptors
(Erwin et al., 1993), and there is little overlap in QTL for
these receptors.
Provisional QTL for NT-ir levels. Levels of NT-ir in
HYP, NA or VMB have been shown to differ by 4.7-, 5.4- and
3.0-fold, respectively, among the LSXSS RI strains (Erwin et
al., 1993). The levels of NT-ir were normally and continuously distributed with estimates of five, four and three genes
regulating the levels in HYP, NA and VMB, respectively. The
QTL for NT-ir levels in HYP, NA and VMB are shown in table
5; there are 7, 7 and 10 QTL for those brain regions, respectively. Previous studies showed that NT-ir levels in various
brain regions were only moderately correlated (Erwin et al.,
1993) indicating a mostly region-specific regulation of NT-ir
levels. Those observations are consistent with results of the
present study showing only three of the 24 NT-ir QTL in
common among brain regions; two of those three are in common for NT-ir in HYP and VMB. Some of the QTL in table 5
are linked to candidate genes of interest including: mme,
neutral metallo endopeptidase on chromosome 3 at 34 cM;
Pkcg, protein kinase C, gamma on chromosome 7 at 2 cM and
1997
QTL for Alcohol Sensitivity and NT
positives is .72. Thus, .72 markers would be expected to
attain P , .05 for both phenotypes by chance. The cumulative
Poisson distribution was used to determine the probability of
observing 3 or 4 common markers at P , .05 (QTL) between
sleeptime and striatal NTRH binding when only .7 are expected by chance. The P(3 or more) 5 .03 and P(4 or more) 5
.005. These results show, by conservative estimates, that the
number of common QTL is significantly greater than that
due to chance. Common QTL between other NT measures
and hypnotic sensitivity to ethanol are not significantly
greater than those expected by chance at P , .05.
Discussion
Our study involved new determinations of genetic correlations between ethanol-induced hypnotic effects or hypothermia and NT measures. These observations were followed by
the use of QTL analyses to identify tentative chromosomal
loci that regulate ethanol effects and levels of NTR and NT-ir
expression. The second step in these ongoing studies will be
to confirm the QTL for NT measures and the common QTL
for ethanol effects and NT measures in an independent investigation using a panel of F2 mice derived from the LS and
SS inbred strains.
Hypnotic sensitivity to ethanol was correlated with NTRH
densities in the STR or FC (r 5 0.40 or 0.39, P , .05,
respectively), but ethanol-induced hypothermia did not correlate with NTRH density. Significant positive correlations
between hypnotic sensitivity to ethanol and NTRH density in
these brain regions are consistent with the findings of motor
inhibitory effects of NT when the peptide is administered into
the NA area (Erwin and Su, 1989). Similarly, NT, injected
into the NA, inhibits the locomotor activating effects of dopaminergic drugs (Kalivas et al., 1984) and, when administered intracerebro ventricularly, NT markedly potentiates
hypnotic effects of ethanol (Erwin et al., 1987). Another study
has shown a positive association between NT receptor density and ethanol sensitivity (Erwin and Jones, 1993). Chronic
TABLE 6
Common QTL for neurotensin measures and ethanol sensitivity
QTL were determined as described in the tables and “Methods.” Candidate genes and QTL map locations were obtained from the mouse genome database,
chromosome committee.
Chromosome
No.
1
2
3
4
6
9
11
15
18
Marker
Map Distance
(cM)
D1Mit45
D1Mit7
D2Mit21
D3Mit21
D4Mit205
D6Mit67
D9Mit12
HoxB
D15Mit41
D18Mit7
58
103
80
19
43
41
55
56*
61*
50
Phenotypes
NTRH, ST
NTRL, ST, BECRR
NTRH, ST, BECRR
NTRH, NT-ir, BECRR
NT-ir, Hypothermia
NT-ir, Hypothermia
NT-ir, ST
NTRH, ST
NTRH, ST
NT-ir, ST, BECRR
Candidate Genes
Cchl1a3a
Atpla2,Akp1b
Ntsr, Pdyn, Avpc
Mmed
June
Itpr1f
Acra3, Gnai2g
Htt, Nos2, Cnp1, Pkcah
Spl1, Tcfcp2i
Adrb2, Gpcr7j
* These QTL for ST were not observed in LSXSS RI strains, but were observed in F2 mice derived for LS and SS inbred strains (Markel et al., submitted).
a
Cchl1a3, calcium channel L type subunit A3.
b
Atpla2, ATPase (NA,K) alpha 2; Akp1, alkaline phosphatase 1.
c
Ntsr, neurotensin receptor high-affinity; Pdyn, prodynorphin; AVP, arginine vasopressin.
d
Mme, neutral metallo endopeptidase.
e
Jun, cjun oncogene.
f
Itpr1, inositol 1,4,5-trisphosphate receptor.
g
Acra3, acetylcholine receptor a 3; Gnai2, guanine nucleotide binding protein a inhibitory subunit.
h
Htt, 5-hydroxytryptophane transporter; Nos2, nitric oxide synthase; Cnp1, cyclic nucleotide phosphodiesterase; Pkca, protein kinase C a.
i
Spl1, Sp1 transacting transcription factor; Tcfcp2, transcription factor CP2.
j
Adrb2, adrenergic receptor b2; Gpcr7, G-protein coupled receptor 7.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Gria3, glutamate receptor AMPA3, alpha 3 on chromosome X
at 5 to 12.5 cM. Neurotensin has been shown to be degraded
by metallo endopeptidases (Checler et al., 1988; Kitabgi et al.,
1992) and potential catalytic differences would be expected to
regulate, in part, differences in levels of the peptide. The
chromosomal location of the NT precursor gene for mouse has
not been determined; therefore, it remains to be seen
whether this gene might be a candidate for an NT-ir QTL.
Whether there are polymorphisms in mme, Ntsr or the NT
precursor genes in the LSXSS RI strains is unknown, but is
the subject of current investigation.
Common QTL for NT measures and ethanol sensitivity. Common QTL for these phenotypes are summarized in
table 6. Four QTL for NTRH density were found in common
with confirmed QTL for hypnotic sensitivity on chromosomes
1 (58 cM), 2 (80 cM), 11 (56 cM) and 15 (61 cM) and one was
in common with an unconfirmed QTL on chromosome 3 (19
cM). As noted in tables 2 and 6, the QTL for ST on chromosomes 11 and 15 were not observed in the LSXSS RI strains,
but, were identified in a study with F2 mice derived from LS
and SS inbred strains (Markel et al., submitted). These QTL
for ST are shown here because of they are in common with
QTL for NTRH. Only one common QTL was found for NTRL
and hypnotic sensitivity to ethanol on chromosome 1 at 103
cM. There were no common QTL for NTRL or NTRH and
ethanol-induced hypothermia. Candidate genes linked to
these common QTL are listed in table 6.
An important question is whether the number of common
QTL between pairs of phenotypes (NT measure and ethanol
sensitivity) exceeds that expected by chance. This question
was assessed for ethanol-induced sleep time and striatal
NTRH. The probability of one common QTL due to chance
would be the product of the relative frequency of QTL for the
two phenotypes. The frequency for sleep time is 8% (8 QTL
out of an estimated 100 unlinked markers) and the frequency
for NTRH is 9%. The probability of markers at P , .05 for
these phenotypes being in common is .0072 (0.08 3 0.09) and
for 100 independent markers the expected number of false
915
916
Erwin et al.
in ILSXISS F2 generations, and on chromosome 3 (D3MIT21,
19 cM).
Common QTL for hypnotic sensitivity and NT-ir levels
were observed on chromosomes 3 (D3MIT21, 19 cM), 9
(D9MIT12, 55 cM) and 18 (D18MIT7, 50 cM). Candidate
genes that might regulate ethanol actions and NT receptor or
NT-ir precursor gene expression and that are linked to these
common QTL include: Mme, membrane metalloendopeptidase on chromosome 3; Gnai2, guanine nucleotide binding
protein, alpha inhibiting-2 and Acra3, acetylcholine receptor
alpha-3 neural on chromosome 9 and Adrb2, adrenergic receptor, beta-2 and G-protein coupled receptor 7 on chromosome 18 (Committee on the Mouse Genome, 1994). These
candidate genes are consistent with reports that the promoter region of the NT precursor gene contains a cyclic AMP
responsive element, a glucocorticoid responsive element, and
an AP-1 site (Kislauskis and Dobner, 1990; Dobner et al.,
1992). The gene for preproNT/neuromedin N (preproNT) has
been cloned from rat, dog, cow and human and in rats, levels
of mRNA in specific brain regions, except for frontal cortex,
correspond reasonably well, with levels of the peptide (Alexander et al., 1989). This gene has been mapped to human
chromosome 12 (Gerhard et al., 1989), but to our knowledge
has not been mapped in the mouse genome. Kislauskis and
Dobner (1990) have provided evidence for cooperative regulation of preproNT gene expression by transcription factors
including c-fos, CREB (cAMP responsive element binding
protein), and glucocorticoids. In brain tissues, drugs that
elevate levels of NT-ir, e.g., haloperidol, produce increases in
c-fos mRNA before increases in preproNT mRNA (Merchant
and Miller, 1994). Ethanol administration alters most, if not
all of these factors that regulate preproNT expression. For
example, it is well known that ethanol causes a rapid increase in plasma glucocorticoids in mice (Zgombick and Erwin, 1987) and in cells and brain tissues ethanol stimulates
cAMP production via dopamine-coupled adenylyl cyclase
(Rabin and Molinoff, 1981; Rabe et al., 1990). Ethanol increases dopamine overflow in nucleus accumbens and striatum (Lai et al., 1979; Imperato and Dichiara, 1986) and
evidence has been reported indicating that chronic ethanol
exposure alters c-fos mRNA levels (Dave et al., 1989). To
date, little is known regarding regulation of expression of the
high-affinity NT receptor gene.
There were no common QTL for ethanol-induced hypothermia and NT receptor densities, but there were two for NT-ir
levels and hypothermia: on chromosomes 4 (43 cM) and 6 (41
cM). Potential candidate genes for these common QTL include the c-jun oncogene (Jun) on chromosomes 4 and inositol
1,4,5-trisphosphate receptor (ltpr1) on chromosome 6. A
number of investigators have shown that centrally administered NT produces marked hypothermia (Martin et al., 1980;
Bissette et al., 1982; Prange and Nemeroff, 1982; Erwin and
Jones, 1990); thus, these common QTL suggest a possible
mechanistic link between endogenous levels of NT in the
HYP or NA and ethanol-induced hypothermia. This hypothesis is supported by data showing a genotype-and dose-dependent effect of ethanol on NT-ir levels as well as on thermoregulation in the LS and SS mice (Erwin et al., 1990b).
Previous studies showed no significant genetic correlation
between NT-ir levels and NTRH or NTRL in the VMB (Erwin
et al., 1993). This finding is consistent with results in tables
3 to 5 showing only one common QTL for NT-ir and NTRH
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
ethanol administration produced a decrease in NT receptor
density in the NA, which was associated with development of
tolerance to locomotor inhibitory effects of ethanol (Erwin et
al., 1992). Thus, there are substantial data linking the inhibitory effects of ethanol to NTRH densities.
Significant correlations were observed between NT-ir levels in the VMB and hypnotic sensitivity to ethanol (r 5
20.62, P , .002) (table 1). The negative correlation is consistent with the observations that NT administered into the
VMB produces locomotor activation in rats and mice (Kalivas
et al., 1982; Erwin and Jones, 1989).
Analyses of associations between molecular markers and
ethanol sensitivity, NT receptor densities and NT-ir levels
were conducted using 24 LSXSS RI strains. Clearly, there is
limited statistical power with only 24 RI strains. Thus, except for those confirmed QTL for ST, the QTL shown in tables
2 to 4 must be considered provisional. There were no common
QTL for the hypnotic and hypothermic effects of ethanol.
These results are consistent with the absence of significant
genetic correlations between hypothermia and ST in the
LSXSS RI strains (Erwin and Jones, 1993) and with other
studies showing different QTL for hypothermia and hypnotic
sensitivity (Crabbe et al., 1994, a and b). Comparison of QTL
for hypothermia induced by 4.2 g/kg ethanol (table 2) with
those obtained by Crabbe et al. (1994a) at 4.0 g/kg indicate
one possible common QTL, located on chromosome 11 in the
region of 19 cM, for the LSXSS and BXD RI panels. Our
results could be considered a confirmation of a QTL for hypothermia in this region. This linkage group contains several
candidate genes that might be mechanistically linked to ethanol effects, including the gamma-aminobutyric acid receptor subunits alpha-1 (Gabra1) and gamma 2 (Gabrg2), and
the adrenergic receptor alpha-1 (Adra1) genes. One might
expect some overlap in QTL for ethanol effects between the
LSXSS RI and the BXD RI panels, because the C57BL/6J and
DBA/2J mice were two of the eight inbred strains that comprised the genetically heterogeneous stock from which the LS
and SS mice were derived. However, because there are undoubtedly allelic differences in the parental stocks used in
generating the RI panels, it is to be expected that some QTL
would differ between the two sets of RI strains.
Results in tables 3 and 4 show provisional QTL for NT
receptor densities and NT-ir levels in specific brain regions.
Common QTL for either NT receptor densities or NT-ir levels
were not always observed across brain regions, an expected
result if these NT processes are regulated by multiple factors
that differ by brain region. Alternatively, it is probable that
some of the QTL for each brain region are fortuitous, the
result of type I and type II errors. However, the finding of
common QTL for NTRH densities in both FC and STR and for
ST and BECRR on chromosome 2 (80 cM), a region containing the Ntsr locus, makes this a strong candidate gene. Certainly, the observation suggests pleiotropic effects of a locus
linked to the region containing the Ntsr gene. The presence of
a QTL for ethanol-induced sleep time on chromosome 2 (80
cM) has been confirmed in a panel of 186 F2 mice derived
from the ILS/lbg and ISS/lbg strains. This QTL accounts for
25% of the genetic variance with a LOD score of 6.1 (Markel
et al., submitted). It is important to confirm the QTL for
NTRH on this chromosome. Other common QTL for hypnotic
sensitivity to ethanol and NT receptor densities were found
on chromosomes 1 (D1MIT45, 58 cM), a QTL also confirmed
Vol. 280
1997
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