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The Journal of Neuroscience, May 1, 2000, 20(9):3157-3164
Ethanol-Associated Behaviors of Mice Lacking Norepinephrine
David
Weinshenker,
Nicole C.
Rust,
Nicole S.
Miller, and
Richard D.
Palmiter
Howard Hughes Medical Institute and Department of Biochemistry,
University of Washington, Seattle, Washington 98195
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ABSTRACT |
Although norepinephrine (NE) has been implicated in animal models
of ethanol consumption for many years, the exact nature of its
influence is not clear. Lesioning and pharmacological studies examining
the role of NE in ethanol consumption have yielded conflicting results.
We took a genetic approach to determine the effect of NE depletion on
ethanol-mediated behaviors by using dopamine  -hydroxylase knockout
(Dbh  /) mice that specifically lack the ability to synthesize NE. Dbh / males have reduced ethanol
preference in a two-bottle choice paradigm and show a delay in
extinguishing an ethanol-conditioned taste aversion, suggesting that
they drink less ethanol in part because they find its effects more
aversive. Both male and female Dbh / mice are
hypersensitive to the sedative and hypothermic effects of systemic
ethanol administration, and the sedation phenotype can be rescued
pharmacologically by acute replacement of central NE. Neither the
decreased body temperature nor changes in ethanol metabolism can
explain the differences in consumption and sedation. These results
demonstrate a significant role for NE in modulating ethanol-related
behaviors and physiological responses.
Key words:
norepinephrine; dopamine -hydroxylase; mice; ethanol; conditioned taste aversion; sedation; hypothermia
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INTRODUCTION |
Although previous lesioning and
pharmacological studies have suggested a role for norepinephrine (NE)
in ethanol-mediated behaviors, they have, taken together, failed to
reach solid conclusions. Acute administration of ethanol can modulate
the synthesis, turnover, and release of central NE (Corrodi et al.,
1966 ; Carlsson and Lindqvist, 1973; Hunt and Majchrowicz, 1974;
Pohorecky and Jaffe, 1975; Karoum et al., 1976) and the activity of
noradrenergic neurons (Aston-Jones et al., 1982; Pohorecky and Brick,
1987; Verbanck et al., 1990). However, the function
of these changes in noradrenergic transmission in response to ethanol
is controversial because of conflicting behavioral results. Depending
on site of administration and strain of rat used, chemical lesions of
noradrenergic neurons with 6-hydroxydopamine (6-OHDA) increase
(Kiianmaa et al., 1975; Melchior and Myers, 1976; Kiianmaa, 1980),
decrease (Myers and Melchior, 1975; Melchior and Myers, 1976), or have
no effect (Melchior and Myers, 1976; Richardson and Novakovski, 1978)
on voluntary ethanol consumption. There are a number of
caveats associated with this technique. For example, 6-OHDA ablates
entire neurons, resulting in the loss of neuropeptides such as galanin
and neuropeptide Y (NPY) that are colocalized with NE in noradrenergic
neurons (Everitt et al., 1984; Melander et al., 1986; Xu et al., 1998). 6-OHDA can also affect dopaminergic function. Last, 6-OHDA does not
completely eliminate NE, and both presynaptic and postsynaptic compensation can occur (Segal and Geyer, 1976; De Montigny et al.,
1980). There are also conflicting findings based on dopamine -hydroxylase (DBH) inhibitors (Amit et al., 1977; Daoust et al., 1990) and adrenergic agonists (Andreas et al., 1983; Grupp et al.,
1989).
As a consequence of this confusion, NE has been largely discounted as
being an important mediator of ethanol-associated behaviors. We took a
genetic approach to this question by measuring ethanol consumption,
intoxication, and hypothermia in mice with a targeted disruption of the
dopamine -hydroxylase gene, which is required for NE synthesis.
Dbh / mice completely lack NE and have been useful in
determining many critical functions of NE in vivo. These include roles predicted by pharmacology such as cardiovascular function
(Cho et al., 1999), smooth muscle contraction, and brown fat
thermogenesis (Thomas and Palmiter, 1997a), and novel roles such as
embryonic development (Thomas et al., 1995), maternal behavior (Thomas
et al., 1997b), and immune function (Alaniz et al., 1999).
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MATERIALS AND METHODS |
Animals. Dbh knockout mice, maintained on
a 129/SvEv and C57BL/6J hybrid background, were developed and generated
as described (Thomas et al., 1995, 1998). Dbh / males
that were rescued for their fertility defect by
L-threo-3,4-dihydroxyphenylserine (DOPS) were
bred to Dbh +/ females, and 9th-12th generation
littermates between 3 and 6 months of age were used in all experiments.
Results were similar across generations.
Dbh / mice were identified by the delayed growth and
ptosis phenotype, which perfectly correlates with the Dbh
/ genotype (data not shown). A subset of genotypes was confirmed by
PCR. Dbh +/ mice have normal levels of epinephrine and NE,
and they are indistinguishable from wild-type littermates for all
previously tested behaviors (Tafari et al., 1997; Thomas and Palmiter,
1997a,b; Thomas et al., 1998; Alaniz et al., 1999; our unpublished
data). Therefore, heterozygous (Dbh +/) littermates were
used as controls for all experiments in this study.
Experimental protocols were approved by the Animal Care Committee at
the University of Washington and meet the guidelines of the American
Association for Accreditation of Laboratory Animal Care.
Ethanol-induced hypothermia. Dbh +/ and Dbh
/ mice of both sexes were anesthetized with 425 mg/kg of a 2.5%
tribromoethanol solution (Sigma, St. Louis, MO), and transmitters
(Mini-Mitter Company, Sunriver, OR) were surgically implanted into the
peritoneal cavity. Mice were allowed to recover for 7 d before
body temperature measurement. Body temperature was recorded every 5 min
by an ER-4000 Energizer/Receiver via radio telemetry. Experiments were
separated by at least 7 d. To measure hypothermia to acute ethanol
administration, body temperature was measured before and after a 3 gm/kg ethanol injection, at either 22 or 30°C. To measure changes in
body temperature during oral consumption of ethanol, it was recorded
over a 12 hr light/dark cycle at room temperature (22°C) in response
to ingestion of a 3% (w/v) ethanol solution available ad
libitum as the only fluid source.
Ethanol-induced sedation. Naïve male and female mice
were either untreated or injected with 2 mg/ml ascorbic acid. Five
hours later, mice were injected with 3 gm/kg (i.p.) of a
filter-sterilized solution containing 20% (w/v) ethanol and 0.9%
NaCl. Mice were placed on their backs in a plastic U-shaped trough
until they lost their righting reflex. The number of minutes it took
for a mouse to right itself on all four paws, three times in 30 sec, was recorded as latency to regain righting reflex. There were no sex
differences or differences between untreated and ascorbic acid-injected
animals, and these groups were combined. This experiment was performed
at both 22 and 30°C. For the DOPS rescue, mice received 1 mg/gm DOPS,
0.125 mg/gm S-()-carbidopa (ICN Biomedicals, Aurora, OH),
and 2 mg/ml ascorbic acid subcutaneously 5 hr before ethanol injection.
Solutions were made in 0.2 M HCl and neutralized
with NaOH just before injection. Data were analyzed with the
Wilcoxon-Mann-Whitney U test using a Bonferroni correction
for multiple pairwise comparisons.
Ethanol preference test. Throughout the experiment, fluid
intake, food intake, and body weight were measured daily. Because of
the increased metabolic rate of Dbh / mice (Thomas and
Palmiter, 1997a), breeder chow (Purina 5015 Irradiated Mouse Diet,
Animal Specialties, Hubbard, OR) was provided ad libitum to
discourage calorically driven ethanol intake during this study. Twelve
Dbh +/ and 12 Dbh / naïve male mice
were housed individually and given continuous access to two water
bottles. The first bottle always contained water, and the second bottle
contained the following solutions in the order listed: water (8 d),
1.7% sucrose (2 d), 4.25% sucrose (2 d), 0.03 mM quinine (2 d), 0.1 mM
quinine (2 d), water (6 d), 3% ethanol (8 d), 6% ethanol (8 d), and
10% ethanol (8 d). The positions of the bottles were changed every day
to control for side preferences. In a separate study, six male
Dbh +/, six male Dbh /, six female
Dbh +/, and six female Dbh / naïve
mice were subjected to the same paradigm without the sucrose and
quinine. No differences were found between the male mice in the two
experiments, and results were combined. Data were analyzed by comparing
the average daily intake of Dbh +/ with Dbh
/ animals at each ethanol concentration with the
Wilcoxon-Mann-Whitney U test.
Ethanol metabolism. Male and female mice were injected
intraperitoneally with ethanol [3 gm/kg; 20% (w/v) in 0.9% NaCl
solution and filtered]. Trunk blood was collected 1 or 3 hr later in
heparinized tubes and centrifuged, and plasma was frozen on dry ice and
stored at 80° until assayed. Blood ethanol levels were measured
spectrophotometrically with an alcohol (ethanol) kit (Sigma). Two
measurements were taken for each sample, and the results were averaged.
No sex differences were found, and results were combined. Data were
analyzed by Student's t tests.
Conditioned taste aversion. Dbh / and Dbh +/
males were put on a limited access drinking schedule, where water
was available only from 8:00-8:30 A.M. and 4:00-5:00 P.M. Food was
available ad libitum. On the first conditioning day, a
0.15% saccharin solution was given instead of water during the morning
drinking session. Saccharin consumption was measured after 30 min, and
mice in the "paired" groups were injected with either ethanol (2 gm/kg, i.p.) or 0.15 M LiCl (20 ml/kg, i.p.).
Mice in the "unpaired" groups were injected with saline. The next
day, water was given during the morning drinking session, and unpaired
mice were injected with ethanol or LiCl, and paired groups were
injected with saline ("reverse injections"). Thus, all mice
received identical treatments, except that only the paired groups
experienced the pairing of the novel taste of saccharin with the
ethanol or LiCl. Water was given during the morning drinking session on
days 3 and 4. On day 5, the first test day, saccharin was given during
the morning session and consumption was measured and compared with
preconditioning saccharin consumption. Because no differences were
found for either the paired or unpaired groups, the paired groups were
injected with ethanol or LiCl again, and the first test day also served as the second conditioning day. Water and reverse injections were given
on day 6. Saccharin consumption was measured on day 7, the second test
day, and all paired groups showed similar and significant aversions.
However, because the unpaired ethanol groups also showed aversions,
ethanol and LiCl were administered to the paired groups for a third
time. On day 8, water and reverse injections were given. All groups
were given 3 d of rest before the final test on day 12. At this
time, all paired groups showed aversions, whereas none of the unpaired
groups did.
To measure aversion extinction, all mice were given access to both
water and saccharin ad libitum. Intake of each fluid was measured daily, and saccharin preference ratios were calculated by
dividing the amount of saccharin solution consumed by the total amount
of fluid consumed. After 10 d, the water was removed, and saccharin was available ad libitum as the only fluid source
to force aversion extinction. Data were analyzed by ANOVA followed by
Student Newman-Keuls post hoc tests.
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RESULTS |
Ethanol-induced hypothermia is more severe in Dbh
/ mice
To determine the effect of NE deficiency on ethanol-induced
hypothermia, Dbh / mice and littermate controls were
implanted with temperature monitors, and core body temperature was
measured in response to an injection of ethanol (3 gm/kg, i.p.). Body
temperature averaged over 60 min before ethanol injection in
Dbh / mice was slightly higher than controls at 22°C
(Dbh /, 37.11 ± 0.07°; Dbh +/,
36.21° ± 0.05°) and 30°C (Dbh /, 37.43° ± 0.82°; Dbh +/, 36.47° ± 0.27°). When ethanol was
administered at 22°C, Dbh / mice experienced prolonged
hypothermia (~6 hr) and an approximately sixfold greater decrease in
body temperature than controls (Fig. 1A,C).
Presumably as a direct result of the decreased core body temperature,
ethanol was metabolized more slowly in Dbh / mice (Fig.
2A). When this
experiment was repeated at thermoneutrality (30°C), there were no
gross differences between genotypes in the average hypothermic effect
of ethanol (Fig. 1B,C) or ethanol
metabolism (Fig. 2B). These results demonstrate that
the increased ethanol-induced hypothermia and lower rate of ethanol
metabolism in Dbh / mice are dependent on ambient
temperature.
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Figure 1.
Ethanol-induced changes in body temperature.
Representative hypothermic response to acute administration of 3 gm/kg
ethanol at (A) 22° ambient temperature and
(B) 30° ambient temperature. Each data point is
an average of three 5 min measurements. C, Mean
hypothermic response at 22° (Dbh +/,
n = 4; Dbh /,
n = 3) and 30° (Dbh +/,
n = 3; Dbh /,
n = 3). No sex differences were found, and results
were combined.
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Figure 2.
Ethanol metabolism. Serum ethanol concentration 1 and 3 hr after an acute injection of 3 gm/kg ethanol at
(A) 22° ambient temperature and
(B) 30° ambient temperature
(n = 6 for each genotype at each time point).
*p < 0.05.
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Dbh / mice are hypersensitive to
ethanol-induced sedation
The role of NE in modulating ethanol sedation was tested by
measuring the latency to regain righting reflex after an acute intraperitoneal injection of 3 gm/kg ethanol. At 22°C, Dbh
/ mice took more than three times as long to regain righting reflex than controls (Fig. 3A). To
rule out the possibility that this phenotype was a secondary effect of
increased hypothermia and decreased metabolism, the experiment was
repeated at 30°C. Despite no difference in heat loss or ethanol
metabolism at this temperature (Figs. 1B,
2B), Dbh / mice were still unconscious
almost three times as long as controls (Fig. 3B),
demonstrating that NE is critical for antagonizing ethanol-induced
sedation independent of its role in temperature homeostasis and
metabolic rate. No sex differences were observed (data not shown).
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Figure 3.
Ethanol-induced sedation. Latency to regain
righting reflex after an acute injection of 3 gm/kg ethanol at
(A) 22° ambient temperature (Dbh
+/, n = 6; Dbh /,
n = 6) and (B) 30° ambient
temperature (Dbh +/, n = 18;
Dbh /, n = 20). DOPS (1 mg/gm) + carbidopa (0.125 mg/gm) (Dbh +/, n = 13; Dbh /, n = 12) was
administered 5 hr before ethanol injection. *p < 0.05; **p < 0.01;
 p < 0.0001.
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To determine whether restoring NE centrally to Dbh /
mice could reduce the sedation hypersensitivity, DOPS and carbidopa were administered 5 hr before ethanol injection. DOPS can be converted to NE by L-aromatic amino acid decarboxylase
(AADC), thus bypassing the requirement for DBH, and carbidopa is an
inhibitor of AADC that cannot cross the blood-brain barrier. Similar
paradigms restore central but not peripheral NE levels to between 11 and 26% of wild-type levels (Thomas and Palmiter, 1997b; Thomas et
al., 1998). The increased latency to regain righting reflex in
knockouts was rescued by DOPS + carbidopa treatment (Fig.
2B). This result demonstrates that a central NE
deficiency in Dbh / mice is responsible for the
hypersensitivity to ethanol-induced sedation.
Dbh / males have reduced ethanol preference
We measured ethanol consumption in males using a two-bottle
preference paradigm. At the beginning of the experiment, control mice
were larger than Dbh / mice. However, during the course of the experiment, Dbh / mice ate more food, drank more
water and total fluid, and gained more weight than controls and were of
comparable weight by the end of the study (Fig.
4). Male Dbh / mice had a
reduced ethanol preference at all ethanol concentrations tested (Fig.
5A). Despite having an
increase in other ingestive behaviors (food and fluid consumption),
Dbh / males consumed less ethanol at the 6 and 10%
concentrations (Fig. 5B). Preliminary results with a small
group of Dbh / females suggest that they have no
differences in ethanol preference compared with controls (data not
shown).
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Figure 4.
Body weight and consumption measurements during
preference study. A, Body weight before start of
preference study. B, Body weight after conclusion of
preference study. C, Weight gain during preference study
calculated as a percentage of starting weight. D,
Average food consumption over course of preference study.
E, Average water consumption over course of preference
study. F, Average total fluid (water + ethanol) over
course of preference study. G, Sucrose preference ratio
(weight of sucrose solution consumed/weight of total fluid consumed).
H, Quinine preference ratio (weight of quinine solution
consumed/weight of total fluid consumed). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
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Figure 5.
Ethanol preference and consumption.
A, Ethanol preference ratio (weight of ethanol
consumed/weight of total fluid consumed) of males (Dbh
+/, n = 18; Dbh /,
n = 18). B, Male ethanol consumption
calculated as grams of ethanol per kilogram of body weight.
*p < 0.05; **p < 0.01.
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To rule out differences in taste preference, mice were tested for their
preference for sweet (sucrose) and bitter (quinine) solutions (Fig. 4).
There were no differences between genotypes in preference for either
compound, demonstrating that the reduced ethanol preference and
consumption in Dbh / males is not generalized to all
tastants or related to caloric value.
Because ethanol-induced hypothermia was more severe in Dbh
/ mice, it was possible that the decreased consumption was
secondary to a reduction in body temperature. We tested this hypothesis by measuring body temperature over 12 hr when the mice were given only
ethanol to drink. Dbh / mice experienced no hypothermia despite consuming more ethanol than animals drank during the preference study (Fig. 6) (data not shown). We
conclude that the decreased voluntary ethanol consumption in
Dbh / males is independent of the hypothermic
effect.
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Figure 6.
Body temperature during voluntary ethanol
consumption. Shown is measurement of body temperature over 12 hr with
3% ethanol the only fluid available (Dbh +/,
n = 2; Dbh /,
n = 2).
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Extinction of a conditioned taste aversion to ethanol is delayed in
Dbh / males
Because ethanol has both rewarding and aversive effects, we
reasoned that Dbh / males drink less ethanol either
because they find it less rewarding or because they find it more
aversive. We used a conditioned taste aversion paradigm to test the
latter hypothesis. During the conditioning sessions, a novel taste
(saccharin) was paired with either a 2 gm/kg ethanol injection (paired
groups) or a saline injection (unpaired groups). LiCl was also paired with saccharin. If the aversive effects of the ethanol or LiCl injection are associated with the taste of saccharin, animals should
drink less saccharin during subsequent sessions. Saccharin consumption
was measured after the conditioning sessions and was compared with
preconditioning saccharin consumption levels. Dbh +/ and
Dbh / males in the ethanol- and LiCl-paired groups
required two conditioning sessions to establish an aversion and
displayed a comparable reduction in saccharin consumption after three
conditioning sessions (Fig.
7A) (data not shown). No
differences in preconditioning or postconditioning saccharin
consumption were manifested in the unpaired groups. To assess
extinction of taste aversion, saccharin and water consumption were
measured in a two-bottle preference paradigm. Dbh +/ males
in the paired ethanol group reached a steady-state extinction after
3 d (i.e., showed no preference for water over saccharin).
Dbh / males took 6 d to achieve similar amounts of
extinction (Fig. 7B). No differences were found between genotypes in the paired LiCl groups (Fig. 7C). After 10 d, some paired mice of each genotype fully or partially extinguished
the aversion, whereas others failed to do so at all. The unpaired groups of either genotype manifested a preference for saccharin, in
that ~80% of fluid consumed was from the saccharin bottle throughout the experiment. To force extinction in the paired animals so that saccharin consumption was comparable to that of the unpaired groups, the water bottle was removed and saccharin was the only fluid available. Within 1 d, all paired groups were consuming amounts of
saccharin comparable to the unpaired groups, demonstrating that all
mice were capable of extinguishing the taste aversion (data not
shown).
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Figure 7.
Conditioned taste aversion. A,
Percentage of preconditioning saccharin solution consumed after the
third conditioning trial (n = 6 for each group
except for EtOH unpaired Dbh /,
n = 4). Saccharin preference ratio (weight of
saccharin solution consumed/weight of total fluid consumed) after the
third conditioning trial for (B) ethanol-paired
mice and (C) LiCl-paired mice.
*p < 0.05.·
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DISCUSSION |
Ethanol-induced hypothermia
A decrease in body temperature in response to acute administration
of ethanol has been well documented in rodents (Nikki et al., 1971;
Lomax et al., 1980) and is evident in humans under conditions of low
ambient temperature and strenuous activity (for review, see Pohorecky
and Brick, 1987). There is also support for the notion that ethanol
plays a role in deaths resulting from accidental hypothermia (Weyman et
al., 1974; Hirvonen, 1979). We show that the magnitude and duration of
ethanol-induced hypothermia are augmented in mice lacking NE. Previous
studies have described the inability of Dbh / mice to
regulate their body temperature in cold environments because of
impaired vasoconstriction and inability to activate brown fat
thermogenesis (Thomas and Palmiter, 1997b). Because brown fat
thermogenesis is not involved in ethanol-induced hypothermia (Huttunen
et al., 1998) and ethanol facilitates vasodilation (Gillespie, 1967;
Ritchie, 1980), we hypothesize that the hypersensitivity of
Dbh / mice to ethanol-induced hypothermia is primarily a result of defective heat conservation rather than an absence of heat
production. It is also possible that depletion of hypothalamic NE in
the Dbh / mice has compromised central temperature
regulation and contributes to the increased hypothermia.
Previous studies have shown that Dbh / mice have a
slightly lower body temperature than controls (Thomas and Palmiter,
1997a), whereas we observed a slightly elevated body temperature. A
difference in temperature measurement (single rectal measurements vs
continuous measurements from the intraperitoneal cavity) could account
for this difference.
Ethanol-induced sedation
To our knowledge, the nearly threefold difference between
Dbh / and Dbh +/ animals in sedation
sensitivity is the largest described to date. For example, rats bred
for ethanol preference regain their righting reflex about twice as
quickly as rats bred for nonpreference (Kurtz et al., 1996), and NPY
/ mice differ from controls by only ~25% (Thiele et al., 1998).
There exists a close parallel between the effects of ethanol on neurons
of the locus coeruleus (LC), the primary noradrenergic brain nucleus, and on behavior. NE turnover and locomotor activity are increased after
low doses of ethanol, whereas high doses of ethanol decrease NE
turnover and are sedative (for review, see Pohorecky, 1977; Pohorecky
and Brick, 1987). Because activation of the LC is associated with
consciousness and arousal (Jouvet, 1969; Hobson et al., 1975; Aston-Jones and Bloom, 1981; Robbins, 1984), it is possible that the
behavioral effects of ethanol are mediated in part by its effects on LC
activity and NE release. Our result that Dbh / mice that
lack NE are hypersensitive to ethanol-induced sedation is consistent
with this idea.
Evidence from both Drosophila and vertebrates has implicated
cAMP signaling in modulating ethanol intoxication (for review, see
Bellen, 1998). The 1,
2, and  2
adrenoreceptors are G-protein-coupled receptors that regulate cAMP
production through adenylate cyclase. The sedation phenotype of
Dbh / mice could be explained by a deficiency in this
signaling pathway, suggesting that ethanol sedation is similarly
controlled by cAMP in this model.
DOPS rescue of sedation hypersensitivity
The ability of DOPS to restore normal sedation sensitivity to
Dbh / mice is critical because it addresses four caveats
of our experimental design. Dbh is required to convert
dopamine (DA) to NE. Consequently, the Dbh / mice
produce DA instead of NE in noradrenergic neurons, albeit in very small
quantities (Thomas et al., 1998); thus, any phenotype we observe could
be the result of ectopic DA production. DOPS is converted to NE by
AADC, thus bypassing DBH and leaving the ectopic DA unaffected (Thomas
et al., 1998). The efficacy of DOPS in this experiment demonstrates that the sedation hypersensitivity is a result of NE deficiency rather
than ectopic DA production. Because our mice are a hybrid of two
strains with different ethanol sensitivities, it is possible that
strain background effects or Dbh-linked genes cause the
altered ethanol response rather than NE. This hypothesis is ruled out by the observation that DOPS rescues sedation hypersensitivity in
Dbh / mice without affecting the performance of controls in this assay. The ability of a single injection of DOPS to rescue the
sedation phenotype of Dbh / adults also demonstrates
that this phenotype is caused by an acute requirement for NE and not a
defect with a developmental basis. Carbidopa, an inhibitor of AADC that
cannot cross the blood-brain barrier, was included to show that the
rescue, and therefore the sedation hypersensitivity, is a central
effect of ethanol. Dbh / mice lack epinephrine in
addition to norepinephrine (Thomas et al., 1995). Because DOPS + carbidopa treatment partially restores both catecholamines (Thomas et
al., 1998), our experiments cannot distinguish between a requirement for either transmitter in ethanol-related behaviors. However, the
amount of epinephrine produced in the CNS is very small relative to NE.
Because of technical constraints, we were unable to test whether DOPS + carbidopa treatment could rescue the reduced ethanol preference in
Dbh / mice. However, because nearly all previously described phenotypes of Dbh / mice can be rescued by
DOPS treatment (Thomas et al., 1995; Thomas and Palmiter, 1997a; Thomas
et al., 1998), we suspect that the ethanol consumption phenotype is
also caused by an acute lack of central NE.
Ethanol preference and consumption
Despite dozens of studies over the last 30 years suggesting a role
for NE in ethanol consumption, NE has been conspicuously absent from
recent reviews on ethanol neurobiology (Diamond and Gordon, 1997;
McBride and Li, 1998). Perhaps this is because of a lack of consensus
produced by studies with conflicting results (see introductory
remarks). Our results clearly demonstrate that NE influences normal
ethanol consumption in male mice. The reduced ethanol consumption in
male Dbh / mice is striking considering that other
ingestive behaviors (food and water intake) were increased by the lack
of NE. Previous work demonstrated that Dbh / mice have
an increased metabolic rate, which could account for the increased food
and water intake (Thomas and Palmiter, 1997b).
It has recently been shown that NPY levels are inversely related to
ethanol consumption (Thiele et al., 1998). Because a subset of
noradrenergic neurons uses NPY as a cotransmitter, it is possible that
the decreased ethanol intake in Dbh / mice is a direct result of a compensatory upregulation of NPY in noradrenergic neurons.
However, an examination of NPY levels in Dbh / mice using mRNA in situ hybridization and RT-PCR argues against
this hypothesis (data not shown).
Sensitivity to and preference for ethanol are inversely related (Kurtz
et al., 1996; Thiele et al., 1998), and this is also true for Dbh
/ males. Therefore, it is surprising that Dbh / females show no decrease in ethanol preference yet still display the
sedation hypersensitivity, although more animals will need to be tested
to confirm this result. Genetic analyses of mouse strains that differ
in ethanol preference (Melo et al., 1996; Peirce et al., 1998) and
ethanol sedation (Bennett and Johnson, 1998) have revealed a number of
sex-specific loci.
Ethanol aversion
One reason that Dbh / males consume less ethanol
may be that NE is important for attenuating the aversive effects of
ethanol. The locus coeruleus is activated in response to aversive
stimuli, and it was the only brain region that showed a consistent
difference in c-fos immunoreactivity between genetically high and low
ethanol-preferring rats after acute ethanol administration (Thiele et
al., 1997). Ethanol-induced sedation and hypothermia could be
considered aversive, and the Dbh / mice are exquisitely
sensitive to these effects. NE-deficient mice were delayed in the
extinction of an ethanol-paired conditioned taste aversion compared
with controls. No differences between genotypes were found with a
LiCl-paired conditioned taste aversion, demonstrating that Dbh
/ mice do not have a general extinction defect using this
paradigm. The number of trials required to establish the ethanol-paired
aversion and the extent of the aversion were similar between genotypes.
The delayed extinction of the conditioned taste aversion supports the
idea that Dbh / males drink less ethanol because they
find it more aversive. However, this interpretation must be made with
caution because the phenotype is relatively subtle and the doses of
ethanol administered for the conditioned taste aversion are much
greater than the amount consumed voluntarily.
NE and ethanol-associated reward
Another possible explanation for the decreased ethanol consumption
by Dbh / males is that NE is important for mediating the
rewarding effects of ethanol. DA has received the bulk of the attention
regarding the rewarding effects of drugs of abuse, including ethanol
(Wise, 1980; Koob et al., 1998). Electrophysiological, pharmacological,
and genetic experiments have established a clear role for DA in
voluntary ethanol consumption (Koob et al., 1994; El-Ghundi et al.,
1998; Phillips et al., 1998). However, it has been suggested that NE,
and not DA, is critical for ethanol reinforcement (Amit and Brown,
1982). Support for this hypothesis comes from studies demonstrating
that (1) ethanol has a greater effect on NE turnover and release than
DA (Corrodi et al., 1966; Hunt and Majchrowicz, 1974); (2) chemical
lesioning of the NE system but not the DA system modulates voluntary
ethanol intake (Myers and Melchior, 1975; Kiianmaa et al., 1979;
Rassnick et al., 1993); and (3) blocking the conversion of DA to NE via
DBH inhibitors attenuates the positive reinforcing effects of ethanol
(Brown et al., 1977). One way to reconcile these data is to emphasize that the NE and DA systems need not be disassociated (Wise and Bozarth,
1985). NE modulates the activity of dopaminergic cells in the
substantia nigra and ventral tegmental area (Grenhoff et al., 1993) and
DA release in the nucleus accumbens (Lategan et al., 1990). Thus, NE
could influence ethanol intake via modulation of DA release or in
parallel to DA.
| |
FOOTNOTES |
Received Nov. 19, 1999; revised Feb. 15, 2000; accepted Feb. 23, 2000.
D.W., N.C.R., and N.S.M. were supported by the Howard Hughes Medical
Institute. We thank Todd Thiele, Don Marsh, Linda Ste. Marie, William
Alynick, Suzanne Weinshenker, and Michelle Brot for technical and
intellectual input, and Doug Kim and Mark Szczypka for critical reading
of this manuscript. We thank Ted Young for the use of his 30°C room
and Sumitomo Pharmaceuticals for the generous donation of DOPS.
Correspondence should be addressed to Dr. Richard D. Palmiter, Howard
Hughes Medical Institute, UW Biochemistry, Box 357370, University of
Washington, Seattle, WA 98195. E-mail:
palmiter{at}u.washington.edu.
| |
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ABSTRACT
INTRODUCTION
