0145-6008/02/2604-0535$03.00/0 Vol. 26, No.

4
ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH April 2002

Chronic Daily Ethanol and Withdrawal: 3. Forebrain

Pro-Opiomelanocortin Gene Expression and
Implications for Dependence, Relapse, and
Deprivation Effect
Dennis D. Rasmussen, Brian M. Boldt, Charles W. Wilkinson, and Dennis R. Mitton

Background: Although forebrain pro-opiomelanocortin (POMC)–producing neurons seem to mediate or

modulate many responses to ethanol consumption, changes in activity of this opiomelanocortinergic system in
response to chronic ethanol consumption, withdrawal, and subsequent abstinence remain unresolved.
Methods: We investigated the effects of chronic daily ethanol consumption, withdrawal, and subsequent
abstinence on adult male Sprague-Dawley rat forebrain opiomelanocortinergic activity as reflected by
changes in hypothalamic POMC messenger RNA (mRNA) content by using a well characterized liquid diet
model that we have previously demonstrated to accurately simulate not only daily oral ethanol consumption
quantity and pattern, but also both neuroendocrine and behavioral changes characteristic of actively
drinking and subsequently abstinent alcoholics.
Results: After 7 weeks of daily ethanol consumption at night and withdrawal during the day, evening
mediobasal hypothalamus POMC mRNA concentrations were suppressed versus both ad libitum–fed and
pair-fed controls. Morning POMC mRNA concentrations were also suppressed versus ad libitum–fed
controls and tended to be decreased versus pair-fed controls. Three weeks after gradual removal of ethanol
from the diet, mediobasal hypothalamus POMC mRNA concentrations were increased relative to ad
libitum–fed and pair-fed controls. Plasma concentrations of corticosterone, testosterone, and leptin were
also altered by the daily ethanol/withdrawal treatment and by subsequent abstinence.
Conclusions: Because each of these hormones has been demonstrated to modify forebrain POMC gene
expression under some conditions, the overall changes in forebrain opiomelanocortinergic regulation in
response to chronic daily ethanol/withdrawal and subsequent abstinence probably reflect, at least in part,
regulation by multiple endocrine mechanisms, together with responses to stress, development of tolerance
during chronic daily ethanol consumption, and rebound of function after termination of this consumption.
Overall, the demonstrated changes in forebrain POMC gene expression are consistent with significant roles
for forebrain opiomelanocortinergic regulation in mediating alcohol dependence, propensity to relapse,
and the alcohol deprivation effect.
Key Words: Pro-Opiomelanocortin, mRNA, Alcohol, Abstinence, Leptin.

T HE FOREBRAIN OPIOMELANOCORTINERGIC
system originates from neuronal perikarya in the arcu-
ate region of the mediobasal hypothalamus (MBH). These
From the Veterans Affairs Puget Sound Health Care System Mental Illness
Research, Education and Clinical Center (DDR) and the Geriatrics Re-
search, Education and Clinical Center (CWW), Seattle, Washington; and the
Departments of Psychiatry and Behavioral Sciences (DDR, CWW) and Med-
icine (DDR, BMB, DRM), University of Washington, Seattle, Washington.
Received for publication October 24, 2001; accepted January 14, 2002.
Supported by the Department of Veterans Affairs and by NIAAA/NIH
Grant AA10567 (DDR).
Presented in part at the 1999 Annual Meeting of the Society for Neuro-
science, Miami Beach, FL, October 23–28, 1999; and at the 2000 Annual
Meeting of the Research Society on Alcoholism, Denver, CO; June 24 –29,
2000.
Reprint requests: Dennis D. Rasmussen, PhD, Research Service 151,
American Lake VA Medical Center, Tacoma, WA 98493; Fax: 253-589-4004;
E-mail: drasmuss@u.washington.edu
Copyright © 2002 by the Research Society on Alcoholism.
Alcohol Clin Exp Res, Vol 26, No 4, 2002: pp 535–546
neurons produce the potent opioid ␤-endorphin and other
peptides derived from pro-opiomelanocortin (POMC) and
provide essentially all ␤-endorphinergic opioid innervation
to the forebrain, including the ventral tegmental area, nu-
cleus accumbens, septal area, and amygdala [for reviews,
see Gianoulakis and de Waele (1994) and O’Donohue and
Dorsa (1982)]. Activity of these opiomelanocortinergic
neurons has been reported to modulate many of the same
varied brain functions as are modulated by ethanol, includ-
ing psychomotor stimulation, reinforcement, pain percep-
tion, and the regulation of eating, reproduction, pituitary
hormone secretion, and immune function (Gianoulakis and
de Waele, 1994; O’Donohue and Dorsa, 1982).
We recently reported that acute induction and mainte-
nance of moderately elevated plasma ethanol levels (⬎100
mg/dl) in response to ethanol infusion via an indwelling
gastric cannula induced acute activation of the forebrain
535
536
opiomelanocortinergic system, as revealed by increases in ven-
tral tegmental area and nucleus accumbens ␤-endorphin con-
centrations within 30 min, coupled with more delayed (within
180 min) increases in MBH POMC messenger RNA
(mRNA) concentrations (Rasmussen et al., 1998). This ap-
parent induction of forebrain opiomelanocortinergic activity is
consistent with demonstrations that in vitro exposure to sim-
ilar ethanol concentrations acutely increased ␤-endorphin se-
cretion in rat MBH (Gianoulakis, 1990) and also increased
both ␤-endorphin secretion and POMC mRNA content in
cultured rat hypothalamic neurons (Pastorcic et al., 1994).
The results of our study with outbred Sprague-Dawley rats
were also consistent with a previous demonstration that daily
voluntary ethanol consumption increased hypothalamic
␤-endorphin and POMC mRNA contents in the alcohol-
preferring C57BL/6 strain of mice (de Waele and Gianou-
lakis, 1994). All of these results are consistent with an hypoth-
esis that ␤-endorphinergic opioid mechanisms mediate or
modulate some responses to alcohol ingestion. This hypothe-
sis is further supported by extensive evidence, with thorough
discussion and citations provided in recent reviews (Froehlich
and Li, 1994; Gianoulakis, 1996; Gianoulakis and de Waele,
1994; Ulm et al., 1995; Van Ree, 1996), that (1) many of the
behavioral and pharmacological effects of ethanol are similar
to those produced by opiates, and cross-tolerance has been
demonstrated to develop between ethanol and opiates for
many of these effects; (2) antagonists to either ␮- or ␦-opioid
receptors (which are the two high-affinity binding sites for
endogenous ␤-endorphin) can reduce intake of, or reinforce-
ment by, ethanol, independent of effects on consumption of
nutrients such as water; (3) responses to ␤-endorphin are
often mediated by ␮-opioid receptors, ␤-endorphin is the
endogenous ligand with the greatest affinity for ␮-opioid re-
ceptors, and ethanol self-administration and reward are de-
creased or eliminated in ␮-opiate receptor knock-out mice
(Hall et al., 2001; Roberts et al., 2000); (4) alcohol treatment
alters both forebrain ␤-endorphin concentrations and ␮- and
␦-opioid receptor binding; (5) the opioid antagonist naltrex-
one is an effective adjunct to detoxification of alcohol-
dependent patients, reducing alcohol craving, decreasing the
alcohol high, and decreasing relapse when the patients again
sample alcohol; and (6) rat and mouse strains that display
increased preference for alcohol have increased hypothalamic
POMC mRNA levels and release more ␤-endorphin from the
in vitro hypothalamus, spontaneously and in response to eth-
anol. Nonetheless, specific roles for ␤-endorphinergic mech-
anisms remain to be resolved; as suggested by Froehlich and
Li (1994) and Gianoulakis and de Waele (1994) and as sup-
ported by recent evidence from Cunningham et al. (1998) and
Froehlich et al. (2001), alcohol-induced activation of endog-
enous opioid systems may mediate or modulate the rewarding
properties of alcohol, attenuate the aversive properties of
alcohol, or both.
The acute alcohol–induced changes in ␤-endorphin con-
tents in the ventral tegmental area and nucleus accumbens
were especially interesting because these areas represent
RASMUSSEN ET AL.
the origin and principal projection site, respectively, of the
mesolimbic dopaminergic system, which has been demon-
strated to play a key role in mediating behavioral responses
to alcohol and other drugs of abuse [see Koob (1992) and
Samson et al. (1992) for reviews]. Furthermore, ␤-endor-
phin– containing fibers project from the arcuate nucleus of
the MBH to both the ventral tegmental area and the nu-
cleus accumbens (Khachaturian et al., 1985; Mansour et al.,
1988), and there is compelling evidence that opioid mech-
anisms in these two key brain sites have roles in mediating
the mesolimbic dopaminergic responses to ethanol [recent-
ly reviewed by Herz (1997) and Gianoulakis (1996)]. In
addition, increased extracellular dopamine in the nucleus
accumbens in response to intracerebroventricular (ICV)
␤-endorphin administration was antagonized by ICV pre-
treatment with either a selective ␮-opioid antagonist or a
selective ␦-opioid antagonist (Spanagel et al., 1990); this is
consistent with the roles of ␮- and ␦-opioid receptors both
in mediating responses to endogenous ␤-endorphin and in
modulating intake of, or reinforcement by, ethanol.
Low-dosage ICV ␤-endorphin is reinforcing for rats
(Amalric et al., 1987; Spanagel et al., 1991). In addition, the
behavioral activation associated with the administration of
low, nonsedative doses of ethanol (Di Chiara and Im-
perato, 1986) is consistent with evidence that (1) low-dose
ICV ␤-endorphin stimulates locomotion (Amalric et al.,
1987; Spanagel et al., 1991), (2) ventral tegmental injection
of opiates increases arousal (Joyce and Iversen, 1979), and
(3) naloxone reduces social locomotion (Dokla, 1992) in
rats. Furthermore, ␤-endorphin released by projections
from the MBH to the periaqueductal gray suppresses no-
ciception (Sim and Joseph, 1991), consistent with analgesic
responses to ethanol. Thus, forebrain opiomelanocortiner-
gic mediation is also consistent with diverse behavioral
responses to alcohol.
␤-Endorphin–mediated opiomelanocortinergic mecha-
nisms likewise seem to mediate some responses to other
drugs of abuse, suggesting common ␤-endorphinergic
mechanisms. For example, reinforcing responses to opiates
are mediated by forebrain ␮- and ␦-opioid receptors (Self
and Stein, 1992), and opioid receptor antagonists such as
naloxone or naltrexone suppress self-administration of opi-
ates such as heroin or morphine (Goldberg et al., 1971;
Killian et al., 1987; Koob and Bloom, 1988; Vaccarino et
al., 1985). Similarly, studies with rats, monkeys, and hu-
mans have demonstrated that naloxone or naltrexone can
reduce self-administration of (Corrigall and Coen, 1991;
De Vry et al., 1989; Kosten et al., 1989, 1992; Mello et al.,
1990; Ramsey and Van Ree, 1991) or reinforcement by
(Bain and Kornetsky, 1987; Bilsky et al., 1992; Houdi et al.,
1989) cocaine, consistent with evidence that cocaine alters
␮-opioid receptor binding in brain areas associated with
reinforcement (Cox et al., 1996; Hammer, 1989; Itzhak,
1993; Itzhak et al., 1992; Unterwald et al., 1992). Because
this effect of cocaine is selective for ␮-opioid (Cox et al.,
1996; Hammer, 1989; Itzhak, 1993; Itzhak et al., 1992;
ALCOHOL AND ABSTINENCE EFFECTS ON BRAIN POMC MRNA
Unterwald et al., 1992) receptors, it is likely that at least
some opioid effects of cocaine use are mediated by the
␤-endorphinergic system. Consistent with this hypothesis,
cocaine self-administration altered the concentration of
␤-endorphin in some of these same areas (Sweep et al.,
1988, 1989). Likewise, naloxone can reduce the number of
cigarettes smoked (Karras and Kane, 1980) and elicit a
withdrawal syndrome in nicotine-dependent rats (Malin et
al., 1992). Administration of nicotine to rats protected
against subsequent brain ␮-opioid receptor inactivation by
␤-funaltrexamine (Davenport et al., 1990) [which selec-
tively alkylates and inactivates unoccupied ␮-opioid recep-
tors (Ward et al., 1982)]; this suggests that nicotine induced
the release of ␤-endorphin, which then occupied the
␮-opioid receptors. Nicotine acutely (within 60 min) al-
tered ␤-endorphin content in the hypothalami of mice
(Gudehithlu et al., 1989), and nicotine acutely stimulated
␤-endorphin release into the medium bathing rat hypotha-
lamic neurons maintained in culture (Boyadjieva and
Sarkar, 1997). In morphine-dependent mice, withdrawal
was attenuated by nicotine (putatively increasing endoge-
nous opioid secretion; Brase et al., 1974). Likewise, nico-
tine was heroin addicts’ most desired substitute for heroin
(Blumberg et al., 1974). Acupuncture [which increases ce-
rebrospinal fluid (CSF) ␤-endorphin levels (Jones et al.,
1980)] helped smokers quit, and they experienced an in-
tense desire to resume smoking after naloxone administra-
tion (Malizia et al., 1978). These results suggest that fore-
brain ␤-endorphinergic opioid activity also mediates or
modulates some responses to nicotine, as we have reviewed
more extensively elsewhere (Rasmussen, 1995). Similarly,
tetrahydrocannabinol’s enhancement of brain stimulation
reinforcement is blocked by naloxone dosages that, them-
selves, have no effect (Gardner et al., 1988), suggesting that
the response to tetrahydrocannabinol is in part opioid;
other effects of tetrahydrocannabinol are also blocked by
opioid-receptor antagonists (Tulunay et al., 1981; Wilson
and May, 1975). Tetrahydrocannabinol blocked naloxone-
precipitated morphine withdrawal syndromes (further sug-
gesting that tetrahydrocannabinol stimulates endogenous
opioid release; Bhargava, 1976; Hine et al., 1975), and
naloxone precipitated opiate-like withdrawal in rats that
had been repeatedly treated with tetrahydrocannabinol
(Hirschhorn and Rosecrans, 1974). These interactions are
consistent with reports that tetrahydrocannabinol can alter
␤-endorphin concentrations in the hypothalamus (Kumar
et al., 1984; Wiegant et al., 1987). Thus, there is compelling
evidence that some responses—not only to ethanol, but
also to varied other drugs of abuse, including opiates, co-
caine, nicotine, and tetrahydrocannabinol—are probably
mediated in part by interactions with the opiomelanocort-
inergic opioid system.
Finally, as we have discussed in a previous paper in this
series (Rasmussen et al., 2000), ethanol and other drugs of
abuse can acutely stimulate secretion of pituitary adreno-
corticotropin (ACTH) and adrenal glucocorticoids (pri-
537
marily corticosterone in the rat and cortisol in humans),
apparently largely mediated by increased hypothalamic
corticotropin-releasing factor (CRF) secretion. Hypothalamic-
pituitary-adrenal (HPA) secretory activity (i.e., the secretory
cascade of hypothalamic CRF and arginine vasopressin, pitu-
itary ACTH, and adrenal glucocorticoids) is also increased by
acute stress, and there is considerable evidence that this HPA
secretory response to stresses, ethanol, and other drugs of
abuse can play a role in mediating or modulating psychostimu-
lation and reinforcing responses (Piazza and Le Moal, 1997).
Because acute stress increases hypothalamic CRF and
␤-endorphinergic activity (Harbuz and Lightman, 1992; Mil-
lan et al., 1981; Morimoto et al., 1993; Patel and Pohorecky,
1988) [which may be related, because there is evidence that
hypothalamic CRF secretion induces not only release of
␤-endorphin and ACTH from the pituitary, but also
␤-endorphin release within the MBH (Burns et al., 1989;
Nikolarakis et al., 1988)], but increased glucocorticoids (char-
acteristic of the stress response) can decrease forebrain ␤-en-
dorphinergic/opiomelanocortinergic activity (Almeida et al.,
1992; Beaulieu et al., 1988), stress and associated HPA acti-
vation could clearly exert complex effects on alcohol- and
other drug-induced changes in a forebrain opiomelanocortin-
ergic mechanism. Furthermore, because hypothalamic dopa-
minergic activation seems to stimulate hypothalamic CRF
(Borowsky and Kuhn, 1991, 1993; Rasmussen, 1991) and
␤-endorphin (Matera and Wardlaw, 1993; Rasmussen, 1991,
1992; Rasmussen et al., 1987, 1992) neurosecretory activity,
the modulation of dopaminergic, HPA, and ␤-endorphinergic
mechanisms may be integrated, as we have proposed for
responses to nicotine (Rasmussen, 1995).
Thus, the hypothesis that a forebrain opiomelanocortin-
ergic opioid mechanism has a key role in mediating/mod-
ulating some responses to alcohol is supported by multiple
complementary and reinforcing lines of evidence: (1) sim-
ilarity of behavioral and pharmacological effects of ethanol
and opiates, with demonstrations of cross-tolerance; (2) ␮-
and ␦- opioid receptor antagonist–induced suppression of
alcohol self-administration and reinforcement; (3) alcohol-
induced alteration of ␮- and ␦-opioid receptor binding and
␤-endorphin concentrations in brain areas associated with
reinforcement; (4) effects of alcohol on release of
␤-endorphin, and corresponding changes in POMC gene
expression, in forebrain opiomelanocortinergic neurons in
vitro and in vivo; (5) correlations between ethanol prefer-
ence and indices of forebrain opiomelanocortinergic activ-
ity in different rat and mouse strains; (6) alcohol-induced
stimulation of CRF neurosecretion, because CRF is an intra-
hypothalamic activator of the forebrain ␤-endorphinergic sys-
tem; (7) compatibility of the opiomelanocortinergic mecha-
nism with behavioral responses to alcohol; (8) evidence that
this opiomelanocortinergic system probably also mediates or
modulates some responses to opiates, cocaine, nicotine, and
tetrahydrocannabinol; and (9) evidence that the forebrain
opiomelanocortinergic system is functionally integrated with
both the HPA and mesolimbic dopaminergic systems, which
538
have central roles in mediating or modulating the behavioral,
neuroendocrine, and pathologic responses to alcohol and
other drugs of abuse. Our long-term goal is to resolve the
roles that interactions among these three key systems play in
mediating responses to alcohol and other drugs of abuse,
revealing potential treatments, interventions, and predictive
indices. Our current study advances this process by resolving
chronic changes in the forebrain opiomelanocortinergic sys-
tem in response to chronic daily ethanol consumption and
withdrawal by outbred male Sprague-Dawley rats.
An especially effective way to examine chronic changes
in overall forebrain opiomelanocortinergic activity is to
evaluate associated changes in POMC gene expression in
the arcuate nucleus. However, previous studies that used
this approach to examine responses to chronic ethanol
treatment have yielded results that seem to conflict. For
example, some demonstrated increases (Angelogianni and
Gianoulakis, 1993; de Waele and Gianoulakis, 1994), some
demonstrated decreases (Scanlon et al., 1992a), and some
revealed no change (Zhou et al., 2000) in hypothalamic
POMC mRNA content after long-term (e.g., ⬎2 weeks)
chronic daily ethanol administration to rats. Variability in
responses to chronic ethanol treatment of rats could be due
to differences in dosages, differences in routes and vehicles
of ethanol administration, varying durations of treatment,
whether analyses were made when plasma ethanol levels
were increased versus during withdrawal, circadian/
photoperiod-dependent variability in regulation, whether
the ethanol was self-administered versus investigator-
administered, and other unrecognized confounding vari-
ables. Consequently, we have investigated the effects of
chronic ethanol and withdrawal on rat hypothalamic
POMC mRNA content by using a well characterized liquid
diet model that we have previously demonstrated (Rasmus-
sen et al., 2000, 2001) to (1) provide reproducible daily oral
ethanol consumption that produces behaviorally relevant
plasma ethanol levels (approximately 135 mg/dl) during the
active (awake) stage of the photoperiod, alternating with
daily withdrawal to undetectable plasma ethanol levels dur-
ing the inactive (sleep) stage; (2) establish physical depen-
dence on ethanol; and (3) produce not only HPA axis, but
also behavioral (anxiety-like behavior, response to novelty,
sucrose preference), changes consistent with those of ac-
tively drinking and subsequently abstinent alcoholics, with-
out altering any of these parameters in pair-fed control rats.
We reasoned that using this model which has been con-
firmed to accurately simulate not only daily oral ethanol
consumption quantity and pattern, but also both neuroen-
docrine and behavioral changes characteristic of actively
drinking and subsequently abstinent alcoholics, would al-
low determination of chronic ethanol– and abstinence-
induced changes in forebrain opiomelanocortinergic activ-
ity that are especially relevant to alcoholics and alcohol
abusers. We further reasoned that evaluating changes in
forebrain opiomelanocortinergic gene expression not only
when plasma ethanol levels were high [at the end of the
RASMUSSEN ET AL.
active (awake) stage of the photoperiod], but also during
daily withdrawal when plasma ethanol levels were unde-
tectable [at the end of the inactive (sleep) stage], would
probably reveal additional complexities of the effect of this
daily ethanol consumption/withdrawal on regulation of
forebrain opiomelanocortinergic activity. Finally, this daily
ethanol treatment produces a slight decrease in body
weight relative to ad libitum–fed controls, so we also de-
termined changes in plasma levels of leptin, a hormone that
(1) is produced by fat cells (Ugrumov et al., 1989), (2) is
maintained at plasma levels that generally correlate with
adiposity (Ugrumov et al., 1989), and (3) seems to have a
stimulatory role in modulating hypothalamic POMC gene
expression (Cheung et al., 1997; Schwartz et al., 1997) and
thus could contribute to the overall effects of chronic
ethanol consumption on hypothalamic POMC mRNA
content.
MATERIALS AND METHODS
Animals
Adult male Sprague-Dawley rats obtained from Simonsen Laboratories
(Gilroy, CA) were individually housed in 12-hr light/12-hr dark cycles
(lights off at 1700 hr) for 2 weeks before and throughout the study. The
rats weighed 260 to 280 g at the start of the study. All procedures were
performed under a University of Washington IACUC-approved protocol
in accord with the NIH Guide for the Care and Use of Laboratory Animals
(National Research Council, 1985).
Liquid Diet
Bio-Serv Liquid Rat Diet L/D’82 (Bio-Serv, Frenchtown, NJ) was
provided daily during the final 15 min of the light period in graduated
Liquidiet Feeding Tubes (Bio-Serv) and was removed the next day at
approximately 3 hr after lights on. On weekends, the liquid diet was
provided at the start of the dark period but was not removed until fresh
diet was provided at the same time on the next day. Consequently, on 5
days/week, the liquid diet ⫾ ethanol available during the dark period was
removed early in the subsequent light period, whereas on the weekends it
was available continuously. Supplemental water was available at all times
for all rats. Each rat was also provided with a nonnutritive nylon Nylabone
(Bio-Serv) to maintain gnawing and chewing behaviors.
Treatments
There were three treatment groups: ethanol-treated, pair-fed control,
and ad libitum–fed control. Ethanol-treated rats were introduced gradu-
ally to 5% ethanol (w/v, prepared with 95% ethanol) in the liquid diet over
a 3-week period (i.e., 4 days 0% ethanol, 1 day 0.8%, 1 day 1.7%, and then
3 days each at 2.5, 2.9, 3.3, 3.8, 4.2, and 4.6% before finally achieving 5%).
After an additional 4 weeks at 5% ethanol, the ethanol-treated rats were
withdrawn gradually from ethanol over a 1-week period (i.e., 1 day each at
4.3, 3.3, 2.5, 1.7, 0.8, and 0% ethanol). Control rats were individually
pair-fed or were fed ad libitum with an isocaloric control liquid diet in
which the calories from ethanol were replaced by maltose dextrin. We
accomplished pair-feeding by daily measuring the amount of diet con-
sumed by each ethanol-treated rat and providing that amount of control
diet to the individually matched pair-fed control animal for the subsequent
day. After gradual withdrawal of ethanol from the liquid diet of the
ethanol-treated rats, the rats in all three treatment groups were switched
to ad libitum chow for the remaining 3 weeks of the study.
ALCOHOL AND ABSTINENCE EFFECTS ON BRAIN POMC MRNA
Sample Collection
Nine rats from each treatment group (i.e., ethanol-treated, pair-fed
control, and ad libitum–fed control) were decapitated in the morning (2–3
hr after lights on), and another nine from each treatment group were
decapitated in the evening (in the last hour before lights off) at the end of
the 4 weeks of chronic 5% ethanol treatment. Nine more rats from each
treatment group were decapitated in the morning (2–3 hr after lights on)
3 weeks after complete cessation of ethanol consumption (i.e., 3 weeks
after the return to chow). At decapitation, trunk blood was collected into
chilled polypropylene tubes containing 100 ␮l of 100 mg ethylenediami-
netetraacetic acid per milliliter of water; and plasma was stored at ⫺70°C
in polypropylene tubes. The brain was rapidly removed, snap-frozen in
isopentane on dry ice, and stored at ⫺70°C.
MBH Dissection
The MBH was dissected from the frozen brain; the brain was first
equilibrated to ⫺10°C, and a chilled Rodent Brain Matrix (Activational
Systems, Warren, MI) was used to remove a coronal section extending 3
mm from the caudal optic chiasm to the rostral mamillary body. This
coronal slice was placed on a stainless-steel plate on dry ice, and a
triangular piece of tissue (base extending bilaterally approximately 1.5 mm
from the midline of the median eminence, apex at the midpoint of the
third ventricle) was dissected.
POMC mRNA Solution Hybridization/RNase Protection Assay
Total cytoplasmic RNA was extracted from the MBH and then assayed
for POMC and cyclophilin mRNA contents by solution hybridization/
RNase protection assay, as we have described elsewhere (Rasmussen et
al., 1992, 1998); template DNAs for the in vitro synthesis of 32P-labeled
probe (antisense) and unlabeled reference (sense) RNAs were comple-
mentary DNA fragments spanning 220 base pairs (bp) (POMC) or 111 bp
(cyclophilin). In short, 32P-labeled probes were allowed to hybridize with
extracted cytoplasmic RNA (from samples) and with known amounts of
reference RNAs (for standard curves); the hybridizations were digested
with RNases and purified by proteinase K digestion, phenol/chloroform
extraction, and ethanol precipitation; and the RNA:RNA hybrids were
electrophoresed through nondenaturing polyacrylamide gels. The gels
were dried and exposed to x-ray film, and the protected bands were cut
from the gel (with use of the autoradiogram as a guide) and counted by
liquid scintillation. The counts per minute scores were plotted against the
known amounts of reference RNAs, yielding regression lines from which
the contents of protected cyclophilin and POMC mRNA in each sample
were computed. The cyclophilin mRNA contents of the MBHs were not
significantly altered by the experimental treatments and served as internal
references for expressing changes in POMC mRNA concentrations. Two
assays were performed; the first included all samples from rats killed at the
end of the chronic 5% ethanol treatment (morning and afternoon), and
the second included all samples from rats killed 4 weeks after cessation of
ethanol consumption.
Leptin Radioimmunoassay
Plasma leptin concentrations were determined with a Rat Leptin RIA
Kit purchased from Linco Research, Inc. (St. Charles, MO). All samples
were quantitated in a single assay.
Statistical Analyses
Data are presented as mean ⫾ SEM. Statistical tests are noted in the
figure legends.
RESULTS
We have reported in a previous article in this series that
the gradual addition of 5% (w/v) ethanol to the liquid diet
539
over a 3-week period maintained continuous weight gain by
the rats evaluated in this study, although at a moderately
decreased rate compared with ad libitum–fed controls [data
previously presented in Rasmussen et al. (2000)]. This de-
crease in rate of weight gain was associated with an initial
15–25% decrease in liquid diet consumption, which stabi-
lized at a 10 –15% decrease after approximately 2 weeks’
consumption of 5% ethanol; removal of ethanol from the
diet led to a transient rebound increase in consumption
[data previously presented in Rasmussen et al. (2000)].
There were no significant differences in body weights of
pair-fed control rats versus ethanol-treated rats at any time
point in the study [data previously presented in Rasmussen
et al. (2000)]. Daily provision of 5% ethanol in the liquid
diet produced nocturnal plasma ethanol levels of approxi-
mately 135 mg/dl, and daily removal of the liquid diet early
in the light period produced complete plasma ethanol with-
drawal by the end of the light period (i.e., plasma ethanol
concentrations were undetectable) [data previously pre-
sented in Rasmussen et al. (2000)]. We also reported that
chronic daily ethanol consumption produced moderate hy-
percorticosteronemia (relative to ad libitum–fed and pair-
fed controls), followed by hypocorticosteronemia after 3
weeks of abstinence after complete removal of ethanol
from the diet [data previously presented in Rasmussen et
al. (2000)].
After 7 weeks of daily nocturnal ethanol consumption
and diurnal withdrawal (i.e., 3 weeks of gradual introduc-
tion plus 4 weeks of daily 5% ethanol), evening MBH
cytoplasmic POMC mRNA concentrations were sup-
pressed 24% (p ⬍ 0.01) versus both ad libitum–fed and
pair-fed controls, which were not significantly different
(Fig. 1). Morning cytoplasmic POMC mRNA concentra-
tions were also suppressed (36%; p ⬍ 0.01) versus ad
libitum–fed controls and tended to be decreased (18%; p ⫽
0.06) versus pair-fed controls (Fig. 1). POMC mRNA con-
centrations were greater (p ⬍ 0.05) in the morning than in
the evening in the ad libitum–fed control rats, but not in the
pair-fed control rats or the rats consuming ethanol (Fig. 1).
Seven weeks of daily ethanol/withdrawal also suppressed
plasma leptin levels in both ethanol-treated and pair-fed
control rats, both in the morning (p ⬍ 0.01) and in the
afternoon (p ⬍ 0.05; Fig. 1). Plasma leptin levels were
greater in the morning than in the evening (p ⬍ 0.05) in ad
libitum–fed controls, but not in pair-fed control rats or in
rats consuming ethanol (Fig. 1).
Three weeks after cessation of chronic daily ethanol
consumption and withdrawal, morning MBH cytoplasmic
POMC mRNA concentrations were increased (p ⬍ 0.05)
versus both ad libitum–fed and pair-fed controls, which
were not significantly different (Fig. 2). Three weeks after
cessation of chronic daily ethanol consumption and with-
drawal, morning plasma leptin levels in ad libitum–fed
control, ethanol-treated, and pair-fed control rats were not
significantly different, although there was a modest trend (p
540
Fig. 1. MBH POMC mRNA concentrations (upper panel) and plasma leptin
concentrations (lower panel) after 7 weeks of daily ethanol/withdrawal. Each bar
represents the mean ⫾ SE of nine rats; analyses by two-way ANOVA were
followed, when appropriate, by one-way ANOVA and Newman-Keuls multiple
comparisons. Upper panel: a ⬎ b, p ⬍ 0.05; c, p ⬍ 0.01 versus both control and
pair-fed; d, p ⬍ 0.01 versus control; e, p ⬍ 0.05 versus control; ⫹p ⫽ 0.06 versus
pair-fed. Lower panel: a ⬎ b, p ⬍ 0.05; c, p ⬍ 0.01 versus control; d, p ⬍ 0.05
versus control. EtOH, ethanol.
⫽ 0.29) for leptin levels to be increased in the rats that had
previously consumed ethanol (Fig. 2).
DISCUSSION
This paradigm of repetitive daily ethanol consumption
and withdrawal was designed to simulate the ethanol expo-
sure experienced by many chronic ethanol abusers. With
this model, MBH POMC gene expression was decreased in
response to long-term daily ethanol consumption but was
increased 3 weeks after complete cessation of ethanol con-
sumption. Although the exact timing and maximal magni-
tude of this opioid gene modulation remain to be resolved,
and it cannot currently be determined whether transcrip-
tion, translational efficiency, or mRNA stability were al-
tered, these results suggest that chronic daily episodes of
ethanol consumption decreased overall opiomelanocortin-
ergic activity in the forebrain, but that this opiomelanocort-
inergic activity was conversely increased 3 weeks after ces-
sation of ethanol consumption.
There are two prominent hypotheses regarding roles for
RASMUSSEN ET AL.
Fig. 2. Morning MBH POMC mRNA concentrations (upper panel) and plasma
leptin concentrations (lower panel) 4 weeks after cessation of chronic daily
ethanol consumption and withdrawal. Each bar represents the mean ⫾ SE of nine
rats; analyses were performed by one-way ANOVA with Newman-Keuls multiple
comparisons. *p ⬍ 0.05 versus both control and pair-fed.
the POMC-derived opioid ␤-endorphin in mediating exces-
sive alcohol consumption, as have been reviewed previously
(Gianoulakis, 1993; Herz, 1997; Reid et al., 1991). The
opioid-deficiency hypothesis suggests that a relative deficit
in basal activity of the endogenous opioid systems favors
increased consumption of ethanol as self-medication to
restore this opioid activity to normal, i.e., negative rein-
forcement. This hypothesis is supported by evidence that
chronic alcoholics have decreased hypothalamic (Gambert
et al., 1981) and CSF (Savoldi et al., 1983) ␤-endorphin
concentrations, that ethanol administration acutely in-
creases ␤-endorphin secretion by rat forebrain neurons
(Boyadjieva and Sarkar, 1994; Gianoulakis and de Waele,
1994; Rasmussen et al., 1998), and that mice expressing low
basal levels of ␤-endorphin (50% of normal) drink more
ethanol than wild-type mice (Grisel and Low, 2000). In
contrast, the opioid surfeit hypothesis suggests that in-
creased activity of the endogenous opioid systems increases
self-administration of ethanol. According to this hypothe-
sis, when ethanol drinking occurs, the enhanced ethanol-
induced stimulation of opioid activity provides reinforce-
ment to continue drinking. This opioid-surfeit hypothesis is
ALCOHOL AND ABSTINENCE EFFECTS ON BRAIN POMC MRNA
supported by evidence that administration of small
amounts of morphine can increase alcohol consumption,
probably by stimulating the opioid system and initiating the
desire for more opiate stimulation (Gianoulakis and de
Waele, 1994; Reid et al., 1991). Our results suggest that
chronic alcohol abuse may produce conditions consistent
with both hypotheses at different times in the alcohol
abuse/abstinence cycle. A relative deficit in basal opiomela-
nocortinergic opioid activity in response to repetitive alco-
hol/withdrawal (as reflected by decreased POMC mRNA
during the chronic alcohol consumption phase of this
study) could lead either to increased motivation to initiate
additional drinking episodes to acutely, although tran-
siently, restore more increased endogenous opioid activity
(i.e., self-medication), or to increased relative amplitude of
the acute opioid response (due to the lower basal activity,
and perhaps facilitated by ␮- and ␦-opioid receptor com-
pensatory upregulation in response to the chronic
␤-endorphin deficit). This acute reinforcement in the con-
text of putatively up-regulated opioid receptors could even
be mediated or facilitated by acute ethanol–induced release
of non-POMC opioids, e.g., the enkephalins, consistent
with reports that genetically ␤-endorphin– deficient mice
acquired operant responding for ethanol more effectively
than controls (Grahame et al., 1998) and also consumed
more ethanol under a limited-access paradigm (Grahame
et al., 2000). Conversely, a relative excess in basal opi-
omelanocortinergic opioid activity during subsequent pro-
longed abstinence, as reflected by increased forebrain
POMC mRNA at 3 weeks after cessation of repetitive
alcohol/withdrawal cycles in this study, could provide a
basis for increased opioid priming and potentially en-
hanced total magnitude of reinforcing opioid response to
ethanol, consistent with evidence that genetically alcohol-
preferring rat strains also exhibit increased hypothalamic
POMC gene expression (Gianoulakis et al., 1992) and that
genetically alcohol-preferring mouse strains exhibited in-
creased basal and ethanol-stimulated ␤-endorphin release
from hypothalamic tissue in vitro (Gianoulakis, 1996).
The alcohol deprivation effect, a phenomenon demon-
strated in rats that have had previous experience consum-
ing ethanol and then exhibit a transitory increase in con-
sumption after being deprived of ethanol consumption for
varying time periods (Sinclair, 1972; Sinclair and Senter,
1968), has been suggested to contribute to relapse and an
increased quantity of alcohol drinking after periods of ab-
stinence. Naltrexone has been demonstrated to dose-
dependently prevent expression of the alcohol deprivation
effect in monkeys (Kornet et al., 1991), suggesting that
persistently increased forebrain opiomelanocortinergic ac-
tivity after cessation of ethanol consumption, as demon-
strated in this study, may contribute to the neuroadapta-
tions in the reinforcing properties of ethanol that have been
suggested to underlie the alcohol deprivation effect (Hey-
ser et al., 1997).
Exposure to a novel environment has been reported to
541
activate the forebrain ␤-endorphinergic system (Dalmaz et
al., 1991), and forebrain administration of low doses of
␤-endorphin has been reported to stimulate locomotion
(Radcliffe and Erwin, 1998). Thus, our demonstration that
forebrain POMC gene expression was increased 3 weeks
after cessation of chronic ethanol/withdrawal is also con-
sistent with our previous demonstration that locomotor
activities in novel environments were increased at this same
time in another study that used an identical chronic etha-
nol/withdrawal model (Rasmussen et al., 2001).
Although mechanisms responsible for the demonstrated
changes in forebrain POMC gene expression cannot be
resolved from these results, several plausible candidates
can nonetheless be identified. For example, because repet-
itive immobilization stress has been demonstrated to de-
crease MBH POMC mRNA content in rats (Makino et al.,
1999), it would be reasonable to suggest that the suppres-
sion of POMC mRNA content by chronic daily ethanol/
withdrawal in this study may have represented, entirely or
in part, a response to chronic repetitive stress associated
with repetitive daily ethanol consumption and withdrawal.
In an earlier study in this series (Rasmussen et al., 2001),
we demonstrated that this chronic daily ethanol and with-
drawal paradigm produced clear signs of physical with-
drawal during the last hour before liquid diet and ethanol
were again provided at the start of the subsequent dark
period each day; this is consistent with the decrease of
plasma ethanol to undetectable levels at this time and
suggests that the chronic daily ethanol and withdrawal
model produced physical dependence and that the rats
experienced withdrawal daily. Consequently, the daily in-
creases in plasma ethanol or the daily withdrawals, or both,
may have been stressful. Repetitive ethanol intoxication,
dysphoria associated with repetitive withdrawal, commonly
associated nutritional deficits, and the psychosocial conse-
quences of ethanol abuse are likewise stressful for chronic
alcohol abusers, so a potential contribution by stress is
clinically relevant.
The increase in POMC gene expression 3 weeks after
cessation of chronic daily ethanol/withdrawal may likewise
have been due, at least in part, to a stress response. We
have previously demonstrated that rats treated with this
same paradigm exhibited increased anxiety-like behavior 3
to 4 weeks after cessation of the chronic daily ethanol/
withdrawal cycles (Rasmussen et al., 2001). We have also
reported that, 3 weeks after cessation of chronic daily
ethanol/withdrawal cycles, the rats investigated in this study
exhibited changes in HPA regulation that seem similar to
those of posttraumatic stress disorder (PTSD) patients and
abstinent alcoholics, who both characteristically exhibit in-
creased anxiety (Rasmussen et al., 2000). PTSD patients
also exhibit increased CSF ␤-endorphin concentrations
(Baker et al., 1997), which may be part of an adaptive
coping response to PTSD symptoms, because there is a
negative correlation between CSF ␤-endorphin concentra-
tions and intrusive and avoidant symptoms of PTSD (Baker
542
et al., 1997). Thus, it would be reasonable to hypothesize
that the increased POMC gene expression 3 weeks after
cessation of chronic daily ethanol/withdrawal cycles in this
study may similarly reflect a response to increased anxiety
at that time. However, activation of the ␮-opioid receptors
that seem to mediate many of the ␤-endorphinergic re-
sponses to ethanol has been demonstrated to increase
anxiety-like behavior in rats (Calenco-Choukroun et al.,
1991; Filiol et al., 2000), so it is also reasonable to suggest
that the increased forebrain opiomelanocortinergic activity
demonstrated in this study could, conversely, be responsi-
ble for the increased anxiety-like behavior. This putative
role of opiomelanocortinergic activity in producing anxiety
during abstinence is intriguing because increased anxiety
contributes to the tendency for abstinent alcoholics to re-
lapse [reviewed in Rasmussen et al. (2001)].
The diurnal changes in POMC mRNA content and
plasma leptin levels suggest another potential mechanism
by which ethanol consumption may have altered forebrain
POMC gene expression. Ad libitum–fed control rats exhib-
ited MBH POMC mRNA contents that were greater in the
morning than in the afternoon, consistent with previous
reports of MBH POMC mRNA diurnal rhythmicity
(Steiner et al., 1994). In contrast, both the rats that had
received 7 weeks of daily ethanol/withdrawal treatment and
the corresponding pair-fed rats did not. Likewise, plasma
leptin levels were greater in the morning than in the after-
noon in the ad libitum–fed controls, but 7 weeks of daily
ethanol/withdrawal suppressed plasma leptin levels in both
ethanol-treated and pair-fed rats, both in the morning and
in the afternoon, consistent with the moderately decreased
body weight of these animals. MBH POMC neurons have
been demonstrated to have high concentrations of leptin
receptors (Cheung et al., 1997), leptin has been demon-
strated to have a stimulatory role in regulating MBH
POMC mRNA content (Schwartz et al., 1997), the hypo-
thalamic opiomelanocortinergic system has been demon-
strated to mediate some responses to leptin (Fan et al.,
1997), and plasma leptin levels have been demonstrated to
be correlated with hypothalamic opioid tone (Wand and
Schumann, 1998). Consequently, it is reasonable to suggest
that the lack of a diurnal increase in morning plasma leptin
concentrations may have been in part responsible for the
lack of a morning increase in POMC mRNA concentra-
tions in the pair-fed control animals. Likewise, a trend for
the ethanol-treated rats to retain a slight morning versus
evening increase in plasma leptin levels (p ⫽ 0.07; Fig. 1,
lower panel) may explain why POMC mRNA levels in the
ethanol-treated rats were very significantly (p ⬍ 0.01) sup-
pressed relative to pair-fed rats in the evening but less so (p
⫽ 0.06) in the morning. It should be noted that the appar-
ent loss or significant attenuation of the diurnal pattern of
both leptin and POMC mRNA in response to alcohol
treatment and restricted nutrition (pair-feeding) could also
reflect a time shift in these diurnal patterns. The moderate
decreases in body weight and plasma leptin levels in the
RASMUSSEN ET AL.
daily ethanol/withdrawal-treated rats and the putatively re-
sultant alterations of diurnal POMC mRNA rhythmicity
may have clinical relevance because alcoholics commonly
exhibit malnutrition and lower fat mass (Addolorato et al.,
1998).
We did not detect significant differences in plasma leptin
levels determined at a single time point in the morning
after 3 weeks of abstinence, although there was a trend for
leptin levels to be increased in the rats that had previously
received chronic daily ethanol/withdrawal treatment. How-
ever, an inverse relationship between plasma ACTH and
plasma leptin concentrations has been reported in rats
(Spinedi and Gaillard, 1998), and we have previously dem-
onstrated that the rats investigated in this study had signif-
icantly suppressed anterior pituitary POMC (precursor to
ACTH) mRNA concentrations after 3 weeks of abstinence
(Rasmussen et al., 2000). This suggests that leptin levels at
other times of the day may indeed have been significantly
increased in the abstinent rats and, as seems to be the case
during chronic daily ethanol/withdrawal treatment, that
leptin may likewise have had a role in mediating the effects
of prior ethanol consumption on forebrain POMC mRNA
levels during subsequent abstinence. If so, it is intriguing
that leptin increases the alcohol deprivation effect (Kiefer
et al., 2001), consistent with our hypothesis that increased
forebrain POMC gene expression may likewise have a role
in mediating the alcohol deprivation effect. Leptin has been
demonstrated to decrease the HPA response to stress
(Heiman et al., 1997), so it is also plausible that putatively
increased leptin levels at some time points during absti-
nence could conversely be responsible for the decreased
HPA activity that we have previously demonstrated (Ras-
mussen et al., 2000).
Other endocrine parameters have also been demon-
strated not only to modify forebrain POMC gene expres-
sion, but also to be altered by chronic daily ethanol/with-
drawal, as well as by subsequent abstinence, suggesting
additional potential interactions that may be at least in part
responsible for the changes in POMC gene expression
demonstrated in these studies. For example, we have pre-
sented preliminary results (Rasmussen et al., 1999) from
two separate experiments (including the animals investi-
gated in this study) that used this chronic daily alcohol/
withdrawal model to demonstrate that although plasma
testosterone levels were suppressed by chronic daily etha-
nol cycles, they were increased relative to both ad libitum–
fed and pair-fed controls at 3 weeks after cessation of the
daily ethanol consumption, compatible with a recent report
that high testosterone levels during abstinence are associ-
ated with type II alcoholism (Stålenheim et al., 1998).
Because testosterone has been reported to facilitate fore-
brain POMC gene expression (Chowen-Breed et al., 1989),
it is reasonable to suggest that the decreased testosterone
levels in response to chronic daily ethanol/withdrawal may
have contributed to the decreased forebrain POMC mRNA
concentrations during chronic ethanol treatment and, con-
ALCOHOL AND ABSTINENCE EFFECTS ON BRAIN POMC MRNA
versely, that increased testosterone levels 3 weeks after
cessation of ethanol consumption may have contributed to
the increased forebrain POMC mRNA during abstinence.
There are conflicting reports regarding the effects of
glucocorticoids on forebrain opiomelanocortinergic activ-
ity. Although removal of glucocorticoids by adrenalectomy
has been reported not to alter forebrain POMC mRNA
concentrations (Scanlon et al., 1992b), there is also evi-
dence that decreased circulating glucocorticoids can in-
crease, and increased glucocorticoids can decrease, fore-
brain opiomelanocortinergic activity (Almeida et al., 1992;
Beaulieu et al., 1988; Patchev et al., 1991). An inhibitory
role for increased glucocorticoids is also consistent with
evidence that increased glucocorticoids can suppress hypo-
thalamic CRF neurosecretion (Owens and Nemeroff,
1991), putatively diminishing CRF-mediated stimulation of
forebrain opiomelanocortinergic activity (Burns et al.,
1989; Nikolarakis et al., 1988). Thus, it is plausible that the
moderate chronic hypercorticosteronemia during chronic
daily ethanol/withdrawal treatment that we have previously
reported elsewhere (Rasmussen et al., 2000) for these same
rats may have contributed to the decreased forebrain
POMC mRNA concentrations during chronic ethanol
treatment; conversely, decreased basal glucocorticoid activ-
ity 3 weeks after cessation of ethanol consumption by these
rats [also previously reported elsewhere (Rasmussen et al.,
2000)] may have permitted increased forebrain POMC
mRNA during abstinence. Because ␤-endorphin released
from POMC neurons inhibits the activity of hypothalamic
CRF neurons (Pechnick, 1993), it is also plausible that the
decreased POMC gene expression and associated opi-
omelanocortinergic activity during chronic daily ethanol/
withdrawal could lead to the chronic hypercorticosteron-
emia that we previously demonstrated (Rasmussen et al.,
2000) and that the conversely increased POMC gene ex-
pression and associated opiomelanocortinergic activity 4
weeks after cessation of ethanol/withdrawal treatment
could lead to the hypocorticosteronemia that we have like-
wise demonstrated during this abstinence (Rasmussen et
al., 2000).
The effects of chronic daily ethanol/withdrawal and sub-
sequent abstinence on forebrain POMC gene expression
may also have been modulated by development of toler-
ance to the effects of ethanol and a subsequent rebound
effect after cessation of the chronic ethanol treatment.
Boyadjieva and Sarkar (1994) have demonstrated that
when primary cultures of rat hypothalamic neurons were
exposed to ethanol for 2 days, desensitization to the acute
stimulatory effect of ethanol on ␤-endorphin secretion oc-
curred. Furthermore, with removal of ethanol from the
culture medium, ␤-endorphin secretion rebounded, and a
hypersecretory response was observed that persisted for 2
days. These results suggest that habituation of the opi-
omelanocortinergic neurons to the acute stimulatory effect
of ethanol could contribute to the decline in forebrain
POMC gene expression in response to chronic daily etha-
543
nol consumption, perhaps allowing the inhibitory effects of
stress, decreased leptin, increased glucocorticoids, de-
creased testosterone, and changes in other modulators to
be expressed. Likewise, a prolonged hypersecretory re-
sponse after cessation of the prolonged daily ethanol/with-
drawal regimen may have contributed to the subsequent
increase in POMC gene expression, perhaps facilitated by
decreased glucocorticoid, increased leptin, and increased
testosterone levels.
Finally, it is notable that we previously demonstrated that
chronic daily administration of moderate-dosage nicotine
also suppressed male rat forebrain POMC mRNA concen-
trations and that this opiomelanocortin gene expression
subsequently rebounded 2 weeks after cessation of treat-
ment but eventually decreased to levels significantly below
those of control rats (Rasmussen, 1997). These results
suggest that the increase in forebrain POMC mRNA levels
at 3 weeks after cessation of chronic daily ethanol/with-
drawal treatment in this study may not be either maximal or
permanent and could potentially even revert to lower levels
than those of control animals. Further studies will address
this issue.
In summary, the demonstrated changes in forebrain
POMC gene expression in response to chronic daily etha-
nol/withdrawal and subsequent cessation of this ethanol
consumption are consistent with significant roles for fore-
brain opiomelanocortinergic regulation in mediating alco-
hol dependence, propensity to relapse, and the alcohol
deprivation effect. These opiomelanocortinergic responses
probably reflect multifactorial integration of several mech-
anisms, including stress responses, regulation by multiple
endocrine systems, development of tolerance during
chronic daily ethanol consumption, and rebound of func-
tion after termination of this consumption.
ACKNOWLEDGMENTS
The authors thank Dr. J. L. Roberts for providing the POMC
and cyclophilin complementary DNA, and Elizabeth Colasurdo
for performing the leptin radioimmunoassay.
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