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Elevated extracellular CRF levels in the bed nucleus of the stria terminalis
during ethanol withdrawal and reduction by subsequent ethanol intake
M. Foster Olive*, Heather N. Koenig, Michelle A. Nannini, Clyde W. Hodge
1
Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco,
5858 Horton Street, Suite 200, Emeryville, CA 94608, USA
Received 31 July 2001; received in revised form 24 October 2001; accepted 6 November 2001
Abstract
Corticotropin-releasing factor (CRF) is widely distributed throughout the brain and has been shown to mediate numerous endocrine
and behavioral responses to stressors. During acute ethanol withdrawal, CRF release is increased in the central nucleus of the amygdala
(CeA), and there is evidence to suggest that this activation of amygdala CRF systems may mediate the anxiogenic properties of the
ethanol withdrawal syndrome. The present study was conducted to determine if another CRF-containing limbic structure, the bed
nucleus of the stria terminalis (BNST), we would exhibit similar increases in CRF neurotransmission during ethanol withdrawal. Rats
were administered an ethanol-containing (6.7% v/v) or control liquid diet for 2 weeks and subsequently implanted with microdialysis
probes into the lateral BNST. A 50– 75% increase in dialysate CRF levels was observed following removal of the ethanol-containing
diet, while no changes were observed in control animals. When ethanol-withdrawn animals were given subsequent access to the
ethanol-containing diet, dialysate CRF levels returned to basal levels. However, when ethanol-withdrawn animals were given subsequent
access to the control diet, dialysate CRF levels increased further to 101% above basal levels. These data demonstrate that extracellular
CRF levels are increased in the BNST during ethanol withdrawal, and that these increases are reduced by subsequent ethanol intake.
D2002 Elsevier Science Inc. All rights reserved.
Keywords: Microdialysis; Extended amygdala; Ethanol; Withdrawal; Bed nucleus of stria terminalis; Liquid diet
1. Introduction
The neuropeptide corticotropin-releasing factor (CRF)
plays an integral role in the behavioral and neuroendocrine
responses to physiological or psychological stressors
(Koob and Heinrichs, 1999; Smagin et al., 2001). CRF
is widely distributed throughout the brain, with highest
concentrations found in the hypothalamus and subcortical
limbic structures (Cummings et al., 1983; Morin et al.,
1999; Olschowka et al., 1982; Swanson et al., 1983).
While hypothalamic CRF is the primary initiator of the
hypothalamic–pituitary– adrenal (HPA) axis response
(Rivier and Plotsky, 1986; Vale et al., 1981), extrahypo-
thalamic CRF systems appear to mediate the behavioral
and autonomic responses to stress (Dunn and Berridge,
1990; Koob and Heinrichs, 1999; Koob et al., 1994;
Sutton et al., 1982).
Withdrawal from chronic intake of alcohol and other
drugs of abuse is often associated with severe physiological
and psychological manifestations of stress and anxiety.
Studies have demonstrated that these withdrawal symptoms
are largely mediated by limbic CRF-containing structures.
For example, antagonism of central CRF neurotransmission
can attenuate behavioral signs of drug and alcohol with-
drawal (Baldwin et al., 1991; Brugger et al., 1998; Sarnyai
et al., 1995). Other studies have shown that neuronal CRF
release is increased in the central nucleus of the amygdala
(CeA) during acute withdrawal from ethanol (Merlo-Pich
et al., 1995), cocaine (Richter and Weiss, 1999) and canna-
binoids (Rodrı
´guez de Fonseca et al., 1997), and that
antagonism of CRF neurotransmission in the CeA attenuates
the behavioral signs of drug and alcohol withdrawal (Hein-
richs et al., 1995; Rassnick et al., 1993). Thus, amygdalar
0091-3057/02/$ – see front matter D2002 Elsevier Science Inc. All rights reserved.
PII: S 0091-3057(01)00748-1
* Corresponding author. Tel.: +1-510-985-3922; fax: +1-510-985-
3101.
E-mail address: folive@itsa.ucsf.edu (M.F. Olive).
1
Present address: Department of Psychiatry, Bowles Center for
Alcohol Studies, School of Medicine CB No. 7178, University of North
Carolina at Chapel Hill, Chapel Hill, NC 27599-7178, USA.
www.elsevier.com/locate/pharmbiochembeh
Pharmacology, Biochemistry and Behavior 72 (2002) 213 – 220
CRF systems appear to contribute largely to the behavioral
signs of drug withdrawal.
The bed nucleus of the stria terminalis (BNST) is
considered to be an integral part of the extended amygdala
complex and shares various neuroanatomical and neuro-
chemical homologies with the CeA (Alheid et al., 1995,
1998; de Olmos and Heimer, 1999). The BNST contains
numerous CRF-immunopositive neuronal cell bodies (Cum-
mings et al., 1983; Morin et al., 1999; Olschowka et al.,
1982; Phelix and Paull, 1990; Swanson et al., 1983) and
also receives CRF-containing projections from the CeA
(Sakanaka et al., 1986). The BNST has been implicated
in neuronal (Bonaz and Tache, 1994) and behavioral
(Gewirtz et al., 1998; Walker and Davis, 1997) responses
to stress. In particular, CRF systems in this region appear to
mediate behavioral responses to stressors (Lee and Davis,
1997), as well as stress-induced relapse to drug-seeking
behaviors (Erb and Stewart, 1999). The goal of the present
study was to use microdialysis to assess changes in extrac-
ellular CRF levels in the BNST during acute ethanol
withdrawal. In addition, we sought to determine if extrac-
ellular CRF levels in this region could be modulated by
voluntary ethanol consumption following the acute with-
drawal phase.
2. Materials and methods
2.1. Animals
Male Long–Evans rats (250– 400 g, Harlan, Madison,
WI) were housed individually in cylindrical Plexiglas
microdialysis cages (30 cm diameter, Instech Laboratories,
Plymouth Meeting, PA) under a 12:12 light– dark cycle
with lights on at 06:00 h. All experiments were performed
during the light portion of the light–dark cycle and were
performed in accordance with approved institutional proto-
cols and the National Institutes of Health Guide for Care
and Use and Laboratory Animals (NIH Publication No. 85-
23, revised 1985).
2.2. Surgical procedures
Animals were anesthetized with 2% halothane vaporized
in a 1:1 mixture of O
2
and N
2
O, implanted with guide
cannulae (SciPro, North Tonawanda, NY) aimed at the
lateral region of the BNST (stereotaxic coordinates AP
0.3 mm, ML ± 1.6 mm from bregma, DV 6.0 mm from
the skull surface, according to the atlas of Paxinos and
Watson, 1997) and secured with skull screws and dental
cement. The wound was treated with 2% bacitracin and 2%
xylocaine topical ointments, sutured closed with 3-0 vicryl
sutures, and animals were allowed to recover in home
microdialysis cages for at least 5 days prior to the adminis-
tration of the liquid diet. Food and water were available ad
libitum during recovery from surgical procedures.
2.3. Administration of liquid diet
Following recovery from surgery, rats were placed on a
Lieber–DeCarli liquid ethanol diet (No. 710260, Dyets,
Bethlehem, PA) or control diet (No. 710027, Dyets) (Lieber
and DeCarli, 1982) in the home microdialysis cage as the
sole source of nutrients for 2 weeks. The ethanol diet
contained 6.7% (v/v) ethanol, while the control diet con-
tained an equicaloric amount of maltose dextrin (both
diets = 1 kcal/ml). Body weight and amount of diet con-
sumed were recorded daily during diet administration.
During microdialysis procedures, diets were removed from
home cages following 90 min of baseline sample collection
and were replaced 7.5 h later followed by an additional
90 min of postwithdrawal sample collection. Following the
withdrawal period, control diet-fed animals were fed the
control diet (CTRL–CTRL), while ethanol-fed animals
were fed either the ethanol-containing diet (ETOH – ETOH)
or control diet (ETOH–CTRL).
2.4. Microdialysis procedures
Following 2 weeks of diet consumption, animals were
lightly reanesthetized as described above and implanted
with microdialysis probes with 2 mm polyethylsulfone
membranes (15 kDa cut-off, 0.6 mm o.d., SciPro) to a
final depth of 8.0 mm from the skull surface. These
probes have an in vitro recovery rate of 13.5% for CRF
(Olive and Hodge, 2001). Probes were continuously
perfused with artificial cerebrospinal fluid (aCSF), contain-
ing 125 mM NaCl, 2.5 mM KCl, 0.5 mM NaH
2
PO
4
H
2
O,
5mMNa
2
HPO
4
, 1 mM MgCl
2
6H
2
O, 1.2 mM CaCl
2
2H
2
O, 5 mM D-glucose, 0.2 mM L-ascorbic acid and
0.025% (w/v) bovine serum albumin, pH = 7.3–7.5.
Probes were attached to dual channel liquid swivels
(Instech Laboratories) with FEP tubing (0.005 in. i.d.,
CMA/Microdialysis, North Chelmsford, MA) for freely
moving microdialysis procedures. Animals were allowed
to recover from probe implantation overnight prior to
withdrawal experiments. On the following day, the aCSF
flow rate was set at 2.0 ml/min, and microdialysis samples
were collected into polypropylene microcentrifuge tubes in
a refrigerated microsampler (SciPro) at 30-min intervals.
Samples were immediately stored on dry ice following
collection and later frozen at 70 C until analysis by
radioimmunoassay (RIA).
2.5. Brain histology
Following microdialysis procedures, animals were deeply
anesthetized with Nembutal (150 mg/kg ip) and perfused
transcardially with 100 ml of 0.9% NaCl followed by 250 ml
of Streck Tissue Fixative (Streck Laboratories, La Vista,
NE). Brains were then removed and placed in the same
fixative for at least 48 h at 4 C. Coronal brain sections
(30 mm thickness) were cut on a cryostat (Leica, Deerfield,
M.F. Olive et al. / Pharmacology, Biochemistry and Behavior 72 (2002) 213–220214
IL), placed onto gelatin-coated slides and coverslipped.
Probe placement was verified under light microscopy, and
data from animals with probe placements outside of the
target region were discarded.
2.6. CRF radioimmunoassay
CRF content in microdialysates was measured using a
commercially available RIA kit (RK-019-06, Phoenix
Pharmaceuticals, Mountain View, CA) adapted to solid-
phase procedures (Olive and Hodge, 2001). Briefly, 96-well
microtiter plates (Dynex Microlite 2+, Dynex Technolo-
gies, Chantilly, VA) were incubated with a protein A
solution (0.4 mg/50 ml, in 0.1 M NaHCO
3
, pH = 9.0) for at
least 24 h at 4 C to facilitate binding of the antisera to
the plate wells. Plates were then washed with assay buffer
(0.15 M K
2
HPO
4
, 0.2 mM ascorbic acid, 0.1% Tween-20,
0.1% gelatin, pH = 7.4, with phenol red added for
enhanced visualization), blotted dry on a paper towel
and incubated with 50 ml/well of rabbit antisera to rat/
human CRF (diluted 1:25 from stock in assay buffer) for
24 h at 4 C. According to the manufacturer, this antisera
crossreact 100% with rat/human CRF and 0% with
urocortin, adrenocorticotropic hormone, Arg
8
-vasopressin,
pituitary adenylate cyclase activating polypeptide and
luteinizing hormone-releasing hormone. Following incuba-
tion with the antisera, plates were washed and incubated
with 0–50 fmol/50 ml (in quadruplicate) of synthetic rat
CRF standards diluted in aCSF. Microdialysis samples
(50 ml) were also added at this time. Standards and
samples were incubated at 4 C for 24 h. Next, approx-
imately 5000 cpm/50 mlof
125
I-labelled rat/human Tyr
0
-
CRF (diluted in assay buffer) was added to each well, and
the plates were incubated at 4 C for 48 h. Finally, plates
were washed with assay buffer and blotted dry on a paper
towel, and 100 ml of Microscint 40 scintillation fluid
(Packard Instrument, Meriden, CT) was added to all
wells. The plates were covered with TopSeal film, agi-
tated for 1 min on an orbital shaker and counted on a
TopCount Microplate Scintillation Counter (Packard
Instrument). Data from microdialysis samples falling out-
side of the linear range for this assay (1.5–50 fmol/50 ml)
were discarded.
2.7. Data analysis
Femtomole values of CRF content for each 30-min
sample were transformed to percentage of basal CRF
release, assigning a value of 100% to the average CRF
level in the three 30-min baseline samples collected prior
to diet removal. Percent baseline data were then collapsed
into 90-min time blocks. All data are presented as mean ±
S.E.M. and were analyzed using a two-way repeated-
measures analysis of variance (ANOVA) followed by a
Neuman–Keuls post hoc test (SigmaStat, SPSS Science,
Chicago, IL).
3. Results
3.1. Placement of microdialysis probes
As shown in Fig. 1, the majority of dialysis probes were
placed in the rostrolateral portion of the BNST. Probes often
extended ventrally beyond the anterior commissure into the
ventral portions of the BNST as well.
3.2. Diet consumption
Rats placed on the ethanol-containing diet consumed an
average of 10.3 ± 0.8 g/kg/day of ethanol (65.8 ± 4.9 ml of
Fig. 1. Diagram of coronal sections of the rat brain indicating location of
dialysis probe placements in the lateral BNST. Vertical lines indicate
approximate location of probe membrane derived from histological
sections. Numbers indicate distance (in mm) from bregma. Figure adapted
from Paxinos and Watson (1997).
M.F. Olive et al. / Pharmacology, Biochemistry and Behavior 72 (2002) 213–220 215
diet/day). Rats administered the control diet consumed
102.5 ± 4.8 ml of diet/day. The body weights of animals in
the three different treatment groups before and after diet
administration are shown in Table 1. Control diet-fed
animals gained approximately 40 g during the 2 weeks of
diet administration, while the body weights of ethanol-fed
animals did not change.
When ethanol-fed rats were given access to the ethanol-
containing diet during the 90-min postwithdrawal period
(ETOH –ETOH group), 1.3 ± 0.3 g/kg ethanol was con-
sumed. When ethanol-fed rats were given access to the
control diet during the 90-min postwithdrawal period
(ETOH –CTRL group), 15.9 ± 4.6 ml of diet was consumed.
When control-fed rats were given access to the control diet
during the 90-min postwithdrawal period (CTRL – CTRL
group), 12.7 ± 1.6 ml of diet was consumed.
3.3. Radioimmunoassay of CRF
The IC
50
of the CRF RIA ranged from 8 to 12 fmol/50 ml,
and the limit of detection was approximately 1.5 fmol/50 ml
(Olive and Hodge, 2001). Absolute basal levels of dialysate
CRF content in each of the three treatment groups are shown
in Table 1 and did not differ across treatment groups.
Microdialysis data from one animal in each treatment group
had to be discarded as the dialysate CRF concentrations
were outside of the linear range for this assay (1.5 – 50 fmol/
50 ml).
3.4. Effects of removal and replacement of liquid diet on
extracellular CRF levels in the BNST
Two-way ANOVA tests revealed significant main effects
of time [ F(6,403) = 5.62, P< .001] and treatment group
[F(2,403) = 18.83, P< .001]. A significant interaction be-
tween time and treatment was also found [ F(12,403) = 2.17,
P< .05]. Pairwise multiple comparison procedures showed
that dialysate CRF levels were increased during withdrawal
only in ethanol-fed anim als ( P< .001). As seen in Fig. 2,
dialysate CRF levels in ethanol-withdrawn rats were
increased approximately 50 –75% above baseline and con-
trol-fed animals starting at 4.5 h following diet removal.
When ethanol-withdrawn animals were given subsequent
access to the ethanol-containing diet, dialysate CRF levels
declined to basal values and were not significantly different
from that of control-fed animals. However, when ethanol-
withdrawn animals were given access to the control diet,
dialysate CRF levels increased to 101 ± 21% above base-
line. These values were significantly higher than those
at the same time point of control-fed animals and those
of ethanol-withdrawn animals given access to the ethanol
diet ( P< .001).
4. Discussion
In the present study, we demonstrated an increase in
extracellular CRF levels in the BNST during the acute
withdrawal phase following chronic ethanol ingestion.
These data parallel the results of an earlier study dem-
onstrating increases in extracellular CRF levels in the
CeA during acute ethanol withdrawal (Merlo-Pich et al.,
1995). These investigators demonstrated that extracellular
CRF levels in the CeA begin to increase approximately at
6–8 h following diet removal and peak at 10– 12 h
postwithdrawal. Yet, in the present study, we observed
significant increases in extracellular CRF levels starting
at 4.5 h following diet removal and apparently peaking at
6 h postwithdrawal (although we did not measure CRF
Table 1
Body weight and basal dialysate levels of CRF in each treatment group
a
Treatment group Prediet body weight (g) Postdiet body weight (g) Basal dialysate CRF levels (fmol/50 ml)
CTRL –CTRL 360.12 ± 4.53 400.25 ± 4.09 9.92 ± 1.17
ETOH –ETOH 344.12 ± 3.68 339.38 ± 4.39 7.14 ± 1.06
ETOH –CTRL 334.71 ± 5.58 341.43 ± 5.44 7.26 ± 0.50
Data are presented as means ± S.E.M.
a
See Section 2.3 for description of treatment groups.
Fig. 2. Effect of acute ethanol withdrawal and subsequent access to ethanol-
containing or control liquid diet on extracellular CRF levels in the BNST.
Each data point represents the mean ± S.E.M. dialysate level of CRF
(expressed as a percent of basal levels) in three 30-min microdialysis
samples for each animal. Treatment groups are designated as control-fed
rats with subsequent access to the control diet (.,n= 7), ethanol-fed rats
with subsequent access to the ethanol-containing diet (~,n= 7), and
ethanol-fed rats with subsequent access to the control diet (5,n= 7).
*P< .05 vs. baseline.
#
P< .05 vs. control-fed animals at the same time
point.
+
P< .05 vs. ethanol-fed animals at the same time point.
M.F. Olive et al. / Pharmacology, Biochemistry and Behavior 72 (2002) 213–220216
release at 8 –12 h after diet removal). Thus, possible differ-
ences in the temporal dynamics of CRF release during
ethanol withdrawal may exist between different regions of
the extended amygdala, with CRF systems in the BNST
being activated earlier in the withdrawal phase than in
the CeA.
In order to adequately compare the present results
with those of Merlo-Pich et al. (1995), a few minor proce-
dural differences should be noted and addressed. First,
although both studies administered the liquid diet for at least
2 weeks, a Lieber –DeCarli liquid diet containing 6.7%
(v/v) ethanol was used in the present study, whereas a
Sustacal diet containing 8.5% (v/v) ethanol was used by
Merlo-Pich et al. (1995). Second, the present study used
Long–Evans rats as subjects while Merlo-Pich et al. used
Wistar rats. Third, control diet-fed animals were not pair-
fed in the present study; that is, the volume of control
diet consumed was not yoked to the volume consumed by
ethanol-fed animals. Other procedural variations include
minor differences in aCSF composition and flow rate,
probe membrane type and relative CRF recovery, and
RIA procedures. Thus, any of these procedural disparities
may have contributed to the slight temporal differences in
CRF release in the BNST during ethanol withdrawal
observed here versus those observed in the CeA by
Merlo-Pich et al. (1995). Future studies examining CRF
release in both regions using the exact same experimental
paradigms will shed light on whether the BNST CRF
systems are indeed activated prior to those in the CeA
during acute ethanol withdrawal.
The present study did not quantify physical withdrawal
symptoms during the 7.5-h period following diet removal
so as to minimize disturbance of the animals, which might
lead to confounding alterations in CRF release. However,
numerous studies have shown that administration of an
ethanol-containing liquid diet for at least 2 weeks produces
overt physical signs of withdrawal such as anxiety (Bald-
win et al., 1991; Rassnick et al., 1993), decreased loco-
motor activity (Merlo-Pich et al., 1995), body tremor
(Merlo-Pich et al., 1995), acoustic startle (Rassnick et al.,
1992), ultrasonic vocalizations (Knapp et al., 1998) and
audiogenic or handling-induced seizures (Frye et al., 1983;
Olive et al., 2001) at 6 –8 h following diet removal. Thus, it
is highly likely that the animals in the present study were
experiencing one or more symptoms of ethanol withdrawal
during this period, when peak increases in CRF release
were observed.
Likely sources of extracellular CRF in the BNST are the
CRF-containing projections from the CeA (Sakanaka et al.,
1986). Thus, the BNST –CeA pathway may be activated
during ethanol withdrawal. However, given that numerous
CRF-immunopositive neuronal cell bodies have been
observed in the BNST (Cummings et al., 1983; Morin et
al., 1999; Olschowka et al., 1982; Phelix and Paull, 1990;
Swanson et al., 1983), it is possible that extracellular CRF
could arise from local somatodendritic release of this
peptide. Thus, the precise source of basal and withdrawal-
induced increases in extracellular CRF levels in the BNST
remains to be determined.
The neurochemical mechanism(s) governing limbic CRF
release also need to be assessed. It has been demonstrated
that stress increases norepineprhine (NE) release in the
BNST (Pacak et al., 1995), and numerous studies have
demonstrated reciprocal interactions between NE, CRF and
stress (for review, see Koob, 1999). In addition, acute
ethanol administration was recently demonstrated to increase
dopamine release in the BNST (Carboni et al., 2000). Thus,
catecholaminergic mechanisms may contribute to with-
drawal-induced release of CRF in the BNST.
The present study also demonstrated that acute ethanol
intake, but not control diet intake, following the withdrawal
period reduced withdrawal-induced increases in extracellu-
lar CRF levels in the BNST. Thus, endogenous CRF
release can be modulated by acute ethanol intake. The
mechanisms by which ethanol suppresses withdrawal-
induced increases in extracellular CRF in the BNST are
currently unknown. To our knowledge, the current study is
the first in vivo determination of CRF release in the BNST.
However, other studies have implicated numerous neuro-
transmitter systems in the secretion of hypothalamic CRF
in vitro (Grossman and Costa, 1993; Grossman et al.,
1993), including inhibition of CRF secretion by GABAer-
gic mechanisms (Calogero, 1995; Calogero et al., 1988;
Grossman et al., 1993). Thus, if similar regulatory mech-
anisms govern CRF release in the BNST, it could be
postulated that acute ethanol could inhibit CRF release
via facilitation of GABA
A
receptor function. Exploration
of this possibility is clearly warranted.
The precise physiological and behavioral ramifications of
the observed increased extracellular levels of CRF in the
BNST during ethanol withdrawal are unknown. Given the
intricate connections of the BNST with other limbic brain
regions, it is tempting to speculate that the increased CRF
neurotransmission in the BNST contributes to the anxio-
genic and negative emotional aspects of the acute ethanol
withdrawal phase. While other studies have suggested that
the anxiogenic properties of ethanol withdrawal are medi-
ated by CRF systems in the CeA (Rassnick et al., 1993),
contributions of CRF systems in the BNST cannot be ruled
out at this point. Indeed, it has been demonstrated that intra-
BNST infusions of CRF enhance fear-potentiated acoustic
startle reflexes, and that these effects are specifically medi-
ated by CRF receptors in this region (Lee and Davis, 1997).
Thus, increased CRF release in the BNST may mediate
anxiety-like behaviors during ethanol withdrawal. Other
neurotransmitters in this region such as NE may also
contribute to the aversive nature of the acute withdrawal
phase (Delfs et al., 2000).
The BNST gives rise to extensive projections to the
paraventricular nucleus of the hypothalamus (Alheid et al.,
1995; Herman et al., 1994). Thus, increases in extracellular
CRF levels in the BNST may contribute to the HPA axis
M.F. Olive et al. / Pharmacology, Biochemistry and Behavior 72 (2002) 213–220 217
activation commonly observed during ethanol withdrawal
(Gallant and Pena, 1992; Rasmussen et al., 2000; Tabakoff
et al., 1978). The BNST also sends projections to various
brainstem regions known to regulate autonomic function
(Alheid et al., 1995; Moga et al., 1989). Indeed, a recent
study demonstrated that CRF signaling in the BNST medi-
ates stress-induced activation of cardiovascular function
(Nijsen et al., 2001). Thus, the observed increases in
extracellular CRF in the BNST may contribute to the
cardiovascular activation and dysregulation commonly
observed during ethanol withdrawal (Mehta and Sereny,
1979; Smile, 1984; Weise et al., 1985).
In animal models of drug dependence, exposure to
stressors, drug-paired environmental stimuli and priming
doses of the drug induce reinstatement of drug and ethanol
self-administration following extinction (Katner et al., 1999;
Le et al., 1998; Koob, 2000; Shaham et al., 2000; Stewart,
2000). Stress-induced ‘‘relapse’’ behavior can be attenuated
by administration CRF antagonists (Le
ˆet al., 2000; Sarnyai
et al., 2001; Shaham et al., 2000; Stewart, 2000), even when
microinjected into the BNST (Erb and Stewart, 1999). Thus,
the increased CRF release in the BNST observed in the
present study may play a role in the ability of stress to
induce relapse to ethanol-seeking behavior following
detoxification. However, other neuropeptide systems may
also be involved in stress-induced relapse to drug-seeking
behaviors (Martin-Fardon et al., 2000).
A particularly interesting aspect of the present study was
the finding that when animals that were previously fed the
ethanol-containing diet were exposed to the control diet
following withdrawal, CRF release in the BNST increased
to levels above those seen during the withdrawal period. It is
possible that this effect could be a form of conditioned
withdrawal or cue reactivity (for review, see Drummond,
2001). For instance, conditioning theories suggest that
neutral stimuli, such as the sensory cues associated with
the liquid diet, can elicit unconditioned responses after
repeated with a drug (i.e., ethanol). Disruption of this
pairing in ethanol-fed animals by the presentation and intake
of the control diet might have produced a stress response, as
reflected in increased CRF release. Indeed, there is ample
evidence that reactivity to learned ethanol-associated stimuli
(i.e., ‘‘cue reactivity’’) indeed can influence craving and
relapse to ethanol consumption during acute withdrawal, as
well as protracted abstinence (for reviews, see Drummond,
2000, 2001). Additional studies measuring CRF release
following explicit pairing of ethanol exposure and envir-
onmental stimuli are required to further address this issue.
In conclusion, the present study demonstrates that extra-
cellular CRF levels are elevated in the BNST during acute
ethanol withdrawal, and that this activation can, in turn, be
reduced by subsequent ethanol consumption or further
increased by the presentation of a nonalcohol containing
diet. Further investigations into the motivational, affective
and autonomic consequences of these increases in CRF
release in the BNST are clearly needed.
Acknowledgments
This research was supported by funds from the State of
California for medical research on alcohol and substance
abuse through the University of California at San
Francisco. The authors wish to thank Nigel Maidment
and Hoa Lam for their technical advice on solid-phase CRF
RIA procedures.
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