The Journal of Neuroscience, August 1995, 15(B): 5439-5447
Increase of Extracellular
Restraint Stress and Ethanol Withdrawal
Levels in the Amygdala
of Awake Rats during
as Measured by
Lorang, Mark Yeganeh, Fernando Rodriguez de Fonseca,b Jacob Raber, George F.
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037
basal values within
added to the perfusion
weeks on a liquid
posed rats and 1.30 + 0.19 fmoV50
ing withdrawal, a progressive
over time was observed,
after the onset of withdrawal
1.15 + 0.30 fmoV50
peak of CRF-IR release
pearance of anxiogenic
ent data lend further
CRF-IR system in the amygdala
factor (CRF) in the anxiogenic
and ethanol withdrawal.
in a series of experiments
to monitor CRF-like
origin of CRF-IR release
in vitro by assessing
of rat amygdala.
in awake rats after the placement
in the amygdala.
stress (20 min)
4.54 + 1.33 fmoll50
1 hr. When 4-aminopyridine
(4.83 f 0.92 fmoV50
in rats previously
were 2.10 f 0.43 fmoV50
has suggested a role for corticotropin-
effects of stressful
of the rat amygdala.
in the amygdala
the Ca*+ dependency
In viva experiments
In the first experiment,
1.19 + 0.15 fmoV50
PI; p < 0.05) that returned
an increase of
(5 mM) was
PI in ethanol
PI in control
increase of CRF-IR
(10.85 + 0.49 fmoll50
PI of control rats, p < 0.01). Since
behavioral effects in rats, the pres-
support to the hypothesis
f.~l, p < 0.05).
at lo-12 reaching hr
of ap- to the time
in the media-
ment of Neurobiology,
Jan. 4, 1995; revised
March 20, 1995;
accepted april 3, 1995.
E E. Bloom,
We Talabot-Ayer for
Merlo Pith, M.D.,
Institute Molecular Biology, de
from of Internal Medicine, University of
of the Foundation Jaime de1 Amo, Universidad Compu-
1995 0 Society for Neuroscience 0270-6474/95/155439-09$05.00/O
tion of the emotional
enous or endogenous
of the response
Converging lines of evidence suggest that corticotropin-releas-
ing factor (CRF), a hypophysiotropic peptide of 41 amino acids
isolated from bovine hypothalamus (Vale et al., 1981), partici-
pates in the mediation of behavioral responses to stress in mam-
mals (Koob and Bloom, 1985; Dunn and Berridge, 1990; Nem-
eroff, 1991; Koob et al., 1993). High densities of CRF immu-
noreactive perikarya and terminals are present in limbic brain
structures (Swanson et al., 1983; Sakanaka et al., 1986; Gray,
1990). CRF binding sites (De Souza et al., 1987; Hauger et al.,
1987) and receptors (Perrin et al., 1993) are also widely distrib-
uted throughout the CNS. Although hypothalamic CRF is known
to play a major role in neuroendocrine adaptation to stress (Ri-
vier et al. 1982; Antoni, 1986; Plotsky, 1991), the role of CRF
in specific extrahypothalamic regions of the brain is not clear
yet. Changes of CRF-like immunoreactivity (CRF-IR) tissue lev-
els (Chappel et al., 1986) and CRF gene expression (Imaki et
al., 1991) are observed following transient or chronic stress in
several hypothalamic and limbic structures, indirectly suggesting
a response of CRF neurons to stress.
Exogenous administration of CRF induces physiological and
behavioral effects that resemble those produced by stress. Intra-
cerebroventricular (i.c.v.) injections of CRF increase the firing
of locus coeruleus neurons (Valentino and Foote, 1988), increase
peripheral sympathetic outflow (Brown et al., 1982) and respi-
ratory rate (Bohemer et al., 1990), and produce proconflict or
“anxiogenic-like” effects in a variety of paradigms of emotion-
ality in rodents (Britton et al., 1985; Dunn and File, 1987; Tak-
ahashi et al., 1989). Consistent with these observations, central
injections of CRF antagonists block the behavioral effects of
either exogenously administered CRE stress, or conditioned fear
(B&ton et al., 1986; Kalin et al., 1988; Swerdlow et al., 1989;
Liang et al., 1992a; Menzaghi et al., 1994).
Several of the stress-like responses associated with CRF ad-
ministration can be mimicked by stimulating the central amyg-
daloid nucleus (Ce), a structure that plays a role in orchestrating
various aspects of the emotional output (Swanson and Mogen-
son, 1981; Le Doux et al., 1988; Davis, 1992). Electrical stim-
ulation of the Ce increases heart rate, blood pressure, peripheral
sympathetic outflow, respiratory rate, and hypothalamo-pitui-
5440 Merlo Pith et al. * CRF Release from Amygdala in Stress and Ethanol Withdrawal
tary-adrenal (HPA) axis activity, and produce behavioral effects
similar of those seen in conditioned fear (Davis, 1986; Dunn
and Whitener, 1986; Iwata et al., 1987; Le Doux et al., 1988).
Consistent with these observations, lesions of the Ce attenuate
the conditioned fear response (Sananes and Davis, 1992) and
produce anti-conflict effects (Shibata et al., 1989). Since a rich
plexus of CRF-containing terminals and neurons projecting to
the brainstem is present in the Ce (Gray and Magnuson, 1987),
it has been suggested that CRF in the amygdala participates in
the mediation of the increased emotionality produced by expo-
sure to stressors.
Prolonged increase in emotionality and HPA axis activation
that resemble the effects of stress have been also described dur-
ing ethanol withdrawal (Freand, 1969; Tabakoff et al., 1978; De
Sota et al., 1985). In spite of the evidence suggesting a role of
CRF in the ethanol withdrawal
1987; Rivier et al., 1990; Baldwin et al., 1991), direct evidence
on CRF release in the amygdala during exposure to stress or
ethanol withdrawal is still lacking.
Recently, a microdialysis procedure to measure in vivo extra-
cellular levels of CRF-IR in freely moving rats was developed
(Merlo Pith et al., 1993). In the present work, this method was
used to investigate the effects of restraint stress and ethanol
withdrawal on CRF-IR release from the amygdala of awake rats.
syndrome (Ehlers and Chaplin,
Materials and Methods
Animals. Fifty-five male Wistar rats (Charles River), weighing 200-225
gm at the beginning of the experiments, were housed in groups of three
in standard rat cages in a humidity and temperature (22°C) controlled
vivarium with l2/12 hr L/D cycle (lights on at 1900 hr). Immediately
after surgery or at the beginning of the experiments involving liquid
diet consumption, the rats were caged individually. Food and water were
provided ad libitum for the animals included in experiments I and 2,
whereas nutritionally balanced liquid diets were used in experiment 3,
as described below.
Dissection of amygdala for
in vitro experiments. Rats were sacrificed
by decapitation between 1000 hr and 1100 hr, their brain rapidly re-
moved, and placed in a brain slicer (San Diego Instruments, San Diego,
CA). A 3 mm thick coronal slice was cut, starting at 1.8 mm posterior
to bregma (Paxinos and Watson, 1986). The amygdaloid regions were
dissected out bilaterally from the slice under a low-magnification
croscope by a vertical cut tangential to the external capsule and a di-
cut along the medial border of the ipsilateral optic tract, as pre-
viously described (Raber et al., 1994). Amygdaloid
placed on a Brinkmann tissue chopper to obtain 300 pm slices. Minced
amygdaloid regions were placed in tubes containing balanced Earle’s
salt solution (GIBCO, Gaithersburg, MD) supplemented with 0.1% bo-
vine serum albumin (BSA, Pentex, Miles Inc., Kankakee, IL), 60 kg/
ml ascorbic acid (Aldrich, Milwaukee,
bacitracin, I pM phenylmethylsulfonyl
and 200 kIU/ml aprotinin (Boehringer-Mannheim,
Slices of the whole amygdaloid regions were incubated at 37°C under
O&O, (95:5%) or 60-80 min before starting the experiment.
in vivo experiments with microdialysis.
fore the microdialysis experiments, rats were anesthetized (1% halo-
thane in OZ/COz, 95:5%) and placed in a stereotaxic apparatus (David
Kopf Instruments, Tujunga, CA). A guide cannula (CMA/IO,
ytical Systems, West Lafayette, IN) was unilaterally
above the final location of the microdialysis probe tip, and secured with
stainless steel skull screws and dental cement. Final stereotaxic coor-
dinates with respect to the microdialysis probe tip were 2.6 mm pos-
terior to bregma, 4.2 mm lateral to the midline, -8.7 mm below dura
(Paxinos and Watson, 1986).
Microdialysis apparatus and perfusion media. Experiments were per-
formed using commercially available microdialysis probes (CMA/lO,
external diameter of 0.5 mm, 2 mm dialysis membrane tip, molecular
cutoff of 20,000 Da). Perfusion medium was loaded into Hamilton sy-
ringes operated by a pulseless microinfusion pump (CMA/lOO).
uid switch (CMA/l 10) was used to permit rapid changes of the perfu-
sion medium. Perfusate was collected in polyethylene tubes kept in ice
regions were then
WI) 0.54 mg/kg glucose, 20 mM
fluoride (Sigma, St. Louis, MO),
Seven days be-
implanted 7 mm
during the perfusion interval (20 min). Perfusion fractions were frozen
on dry ice immediately after collection and stored at -70°C until ra-
dioimmunoassay. Before each experiment, microdialysis probes were
perfused at room temperature with artificial cerebrospinal fluid contain-
ing 1% BSA to saturate nonspecific binding sites for peptides within
the perfusion system.
The basic perfusion medium consisted of artificial cerebrospinal fluid
(ACSF) containing NaCl (149.0 mM),
CaCl, (1.2 mM), NaH,PO, (1.2 mM), and BSA (1%). The ACSF (pH
7.4) was filtered sterile with nylon filters (Gelman Acrodisc 4192, Ann
Arbor, MI). This medium was used for rinsing the probe, and perfusion
of probes implanted into the animals during periods when CRF levels
were not monitored. The perfusion medium used for measuring CRF
was prepared by adding specific anti-CRF serum (rC70 or rC69,kindly
provided by W. Vale) to ACSF at a dilution of 1:80.000. Anti-CRF
previous observations have shown that the
addition of a specific, well-characterized
perfusion medium can improve the recovery of CRF-IR (Merlo Pith et
al., 1993). At the end of each experiment, an aliquot of remaining per-
fusion medium containing anti-CRF serum was frozen together with the
collected fractions, and stored at -70°C. This medium was then used
in the construction of the standard curve of the radioimmunoassay.
CRF radioimmunoassays. Detection and quantification of CRF was
adapted from Vale et al. (1983). This procedure of CRF measurement
is based on competitive binding radioimmunoassay, and was validated
with independent HPLC studies. However, in our study, no data veri-
fying of the identity of the immunoreactivity
low amounts recovered by the experimental techniques we used. There-
fore, the measurements are reported as CRF-like
The CRF radioimmunoassay standard method was employed to mea-
sure the concentrations of CRF-IR released from amygdaloid region
tissue in vitro,
as previously described (Smith et al., 1986; Calogero et
This procedure has been extended to microdialysis applications in a
series of in vitro
and in vivo validations (Merlo Pith et al., 1993).
Briefly, the concentration of specific anti-CRF serum within the perfu-
sion medium was calculated in order to optimize the radioimmunoassay
for CRE For each experiment standard curves were prepared in dupli-
cate using 50 $/tube of the same perfusion medium containing anti-
CRF serum used for microdialysis probe perfusion and fraction collec-
tion. Final anti-CRF serum dilution was 1:800,000, and the final incu-
bation volume was 500 @l/tube. Standards and samples were incubated
in borosilicate glass tubes for 24 hr at +4”C before addition of 16,000
cpm/tube of the tracer, [‘251-Tyr’]rCRF (DuPont/NEN, Boston, MA) di-
luted in normal rabbit serum (Peninsula, Belmont, CA). A second in-
cubation of 24 hr was followed by precipitation of the antigen-antiserum
complex with pretitrated goat anti-rabbit antiserum (Peninsula), poly-
ethylene glycol 8000, and 30 min centrifugation
decanting the supernatant, residual radioactivity was counted in each
tube using a gamma-counter (APEXIO,
CA). A logistic four-parameter model was used for interpolation of stan-
Sensitivity of the assay was 0.35 fmol/tube or less.
Verijcation of microdialysis probe placement. The day after the ex-
periment, rats were deeply anesthetized with halothane, and microdi-
alysis probes were gently removed and inspected for viability. The an-
imals were then decapitated, the brain dissected out, and immediately
frozen on dry ice. Unfixed brains were cut on a cryostat.
The brain of one group of rats was cut in thin sections (20 pm),
postfixed in 4% paraformaldehyde for 1 min, and stained with cresyl
violet for rostrocaudal analysis of probe trace location within the amyg-
dala. Since the anatomical plane of cutting was different from the plane
used for stereotaxic guide cannula implantation, the localization of the
active zone of microdialysis probe within the amygdala was obtained
by inspecting various adjacent sections. The analysis started from the
more caudal level that displayed the deepest lesion trace produced by
the microdialysis probe, corresponding to the location of the probe tip.
Rostrocaudal and dorsoventral location of probes active zone in each
rat brain was identified by selecting the section with a trace positioned
at approximately 1 mm distance from the tip. The rostrocaudal levels
of this section was attributed using a stereotaxic atlas (Paxinos and
Watson, 1986), and represented as a 2 mm segment on a semianatomical
scheme of the same stereotaxic level.
In a second group of rats, an alternative approach was used to eval-
uate the anatomical probe location in the rat brains. Thick sections (200
KC1 (3.7 mM), MgCI, (0.9 mM),
anti-CRF rabbit serum to the
are presented, due to the
at 3000 X g. After
ICN Biomedicals, Costamesa,
The Journal of Neuroscience, August 1995, E(8) 5441
pm) were collected and postfixed in 4% paraformaldehyde
1 hr. After rinsing with PBS, wet sections were mounted on slides, put
on a negative carrier of a photographic enlarger, and projected onto the
easel to obtain “negative” photographic prints. Adjacent sections were
also cut and stained with cresyl violet to provide additional information
about the anatomical localization of the microdialysis probe tip. The
location of the active zone of the microdialysis probes was then iden-
tified as described above.
Diet, ethanol administration, and blood alcohol level determination.
Seventeen rats selected for experiment 3 were maintained on a nutri-
tionally balanced liquid diet consisting of a Sustacal (Mead Johnson,
Evansville, IN) base supplemented with vitamins (ICN Nutritional Bio-
chemicals, Aurora; 0.3 g/l00 ml) and minerals (ICN Nutritional
chemicals; 0.5 g/100 ml) as previously described (Baldwin et al., 1991).
Ethanol (8.5% v/v) was added to the liquid diet of experimental rats.
Control diet was made equicaloric
control and ethanol liquid diets were given for 2-3 weeks, and the diets
were available ad lib&urn. The volume of diet consumed was measured
each morning. A “pair-feeding” procedure was instituted for control
rats by keeping the volume of available control diet equal to the volume
of ethanol-containing diet consumed by the rats of the ethanol group
on the previous day. The effect of this diet regimen on body weight
was monitored regularly.
Blood alcohol levels (BAL) were measured in each rat 10 d after the
beginning of the ethanol diet exposure, and 3 d before the experiment.
Blood (0.5 ml) was taken from the tail, collected into 4 p,I heparin (1000
USP units/ml), and centrifuged for 5 min at 10,000 rpm. The plasma
was acid extracted and alcohol content was determined using the NAD/
ADH method (Sigma). Rats were included into the experimental group
when the BAL measured on the last sampling day was higher than 100
Experiment I: in vitro measurements of CRF-IR release from amyg-
dala. In vitro
experiments were performed according to the method of
Calogero et al. (1989), with modification.
ducted at 37°C under O,/CO, (95:5%), and consisted of serial passage
of the minced tissue through different wells of a 48-multiwell
(Costar, Cambridge, MA) previously precoated with incubation buffer
containing 1% BSA. Each well contained 700 p,l medium, and tissue
minces were passed through wells at 20 min interval. At the end of
each incubation interval the tissue was removed and placed on the next
well for the following 20 min interval. Immediately after removing the
tissue, the incubation medium was collected in tubes, frozen on dry ice,
and stored at -70°C until CRF radioimmunoassay. A set of preliminary
experiments indicated that stabilization of spontaneous CRF-IR release
occurs between 80 and 100 min after the beginning of the experiment.
The levels of CRF-IR measured in the following
(100-120 min) was considered the reference basal value for comparison
with the stimulated levels. Stimulation was performed in the subsequent
interval (120-140 min) incubating the sections in medium containing
the K+-channel blocker 4-aminopyridine.
obtained by incubating minced amygdala with 4-aminopyridine
centrations of 0.1, 1.0, or 10.0 mu. The Ca*+-dependency of CRF-IR
release produced by 4-aminopyridine
bating the tissue with the Ca?+ channel blocker CoCI, (IO mM). Incu-
bation with CoCl, begun during the interval before exposure to 4-ami-
nopyridine. Each experimental group consisted of amygdala minces col-
lected from five to six animals, each tube containing slices from a single
Experiment 2: effects of restraint stress on CRF-IR release from the
amygdala. This experiment was performed in the home cage of seven
rats previously implanted with a guide cannula. Microdialysis
were slowly inserted 12 hr prior to the start of sampling and secured in
the guide cannula
under brief, shallow halothane anesthesia. During this
period, the probe was perfused with ACSF at a flow rate of 0.7 kl/min.
One hour before sampling, the perfusion medium was changed to a
freshly prepared medium containing anti-CRF serum, and the flow rate
was increased to 3.0 pl/min. This flow rate was previously shown to
yield optimum recovery (Merlo Pith et al., 1993). Fractions were col-
lected every 20 min, at a volume of approximately 60 ~1. After a period
of basal collection (five to six samples), animals were restrained for 20
min by immobilizing their paws. At the end of the stress period rats
were returned to their home cage and sampling was continued for ap-
proximately 2-3 hr. Perfusion medium was then substituted with a so-
lution containing 10 mu 4-aminopyridine,
for at least
by adding sucrose (Sigma). Both
The experiment was con-
A dose-response curve was
(1.0 mM) was studied by incu-
and perfusion was continued
for 1 hr. At the end of the test the animals were anesthetized and sac-
rificed as described.
Experiment 3: effects of ethanol withdrawal
the amygdala. In this experiment, rats exposed to ethanol diet (n = 9)
or control diet (n = 8) were implanted with guide cannulae 1 week
before the experiment. On the testing day, 12 hr prior to the start of
sampling, microdialysis probes were secured in the guide cannula under
halothane anesthesia. The probe was perfused with ACSF at a rate of
0.7 yl/min overnight. On the next morning, 1 hr before sampling, the
perfusion medium was changed to a freshly prepared medium contain-
ing anti-CRF serum, and the flow rate was increased to 3.0 pl/min.
Fractions of 60 ~1 were then collected every 20 min. All experiments
started with collection of five basal fractions, during a period of 2 hr
in which ethanol diet was available. At the end of this period the bottle
containing ethanol diet was removed and replaced by control diet. Per-
fusion medium was then changed, and perfusion continued at the same
flow rate using regular ACSE Standard ACSF and solution containing
anti-CRF serum were substituted every 2 hr, and this procedure was
repeated four times during the experiment. Thus, CRF-IR measurements
were performed over four periods of 2 hr interspersed with nonsampling
2 hr periods over a total of 12 hr. The first fraction collected after each
change of the perfusion medium was discarded to avoid carryover ef-
fects from dialysate remaining in the tubing dead volume. The four-
fraction collection periods corresponded to the 24 hr, 68 hr, and 10-
12 hr intervals afte; withdrawal. I\t the end of the experiment, rats were
Derfused with ACSF over nirrht at 0.7 u,l/min before sacrifice. Durine
hithdrawal each rat was obierved fo; physical signs of withdraw;
(Baldwin et al., 1991).
Statistical analysis. The effects of various doses of 4-aminopyridine
on CRF release in vitro were analyzed using one-way ANOVA,
a mixed factorial ANOVA was used to study the interaction of CoC12
on 4-aminopyridine-stimulated release. The effect of restraint stress on
CRF-IR release in vivo was assessed using one-way ANOVA
peated measurements. The data collected in the experiment performed
on ethanol-dependent rats were analyzed using a mixed factorial ANO-
VA with “diet” as between-subject factor and “time of withdrawal”
within-subject factor. In all experiments significant differences among
individual means were determined by Tukey’s post hoc test.
on CRF-IR release from
Figure 1 shows the effects of 4-aminopyridine on CRF release
from slices of amygdala tissues in vitro. Inclusion of 4-amino-
pyridine in the incubation medium significantly increased CRF-
IR release in a dose-response fashion (p < 0.01). Significant
effects were observed at 1.0 ttIM @ < 0.05) and 10.0 mM
0.01) 4-aminopyridine concentrations, while the lowest dose (0.1
mM) was without significant effect. In the presence of CoCl,,
the stimulating effects of 1.0 mM 4-aminopyridine were com-
pletely antagonized (p < 0.05). These results confirm the Ca2+
dependency of CRF-IR release from slices of rat amygdala in
I: in vitro CRF-IR release from amygdala
from the amygdala
Postmortem anatomical evaluation of the placement of the “ac-
tive” zone of the microdialysis probes in rats exposed to re-
straint stress revealed that the target region was an area defined
by the lateral component of the Ce and the medial border of the
BL (Fig. 2). A graphical representation of the locations of the
microdialysis membrane region in the brain of all rats of this
experiment is shown in Figure 3B. Two rats were not included
in the data analysis because of anatomical misplacement of the
Basal and stress-induced CRF-IR levels in dialysate from the
amygdala of awake rats over time are shown in Figure 3A. Basal
CRF-IR dialysate concentrations in undisturbed rats were 1.19
+ 0.15 fmol/50 ~1. These values represent the mean and the
standard error of all the fraction values measured before stress
2: effects of restraint stress on CRF-IR release
5442 Merlo Pith et al. - CRF Release from Amygdala in Stress and Ethanol Withdrawal
0 0.1 1.0 10.0
n regular medium
0 CoCI, (1 OmM)
0 1 .o
Figure 1. A, Effects of incubation with 4-aminopyridine
in vitro. Amygdala slices were incubated in regular medium, and then exposed to 4-aminopyridine
basal CRF-IR levels did not differ from those measured in the stimulation interval when 4-aminopyridine
obtained during the stimulation intervals are shown. Each bar represent the mean t- SEM of four to six rats (*, ,V < 0.05; **, p < 0.01; vs basal
CRF-IR levels, Tukey’s test). B, Effects of cobalt chloride (CoCl,) on CRF-IR release induced by 4-aminopyridine
in vitro. Basal CRF-IR levels are represented on the left, when 4-aminopyridine
in the presence of 1 mM 4-aminopyridine. Incubation with 10 mM CoCl, (open bars) abolished the effects of 4-aminopyridine.
the mean 2 SEM of five rats (*, p < 0.05, stimulated vs basal CRF-IR levels; #, p < 0.05; regular medium vs CoCl, during stimulation).
at different concentrations (mM) on CRF-IR release from slices of the rat amygdala tissue
during the following 20 min interval. Since
was absent (0 mM), only the results
from slices of amygdala tissue
was absent, while stimulated CRF-IR levels are shown on the right,
Each bar represents
in all rats. Restraint stress (20 min) produced a significant in-
crease of CRF-IR dialysate levels to 4.54 +- 1.33 fmolR0 ~1 (p
< 0.01). After stress, dialysate CRF-IR levels returned to levels
close to the basal values in about 1 hr. During the following
intervals, perfusion with 4-aminopyridine markedly increased
CRF-IR concentrations in the dialysate collected over a 1 hr
sampling period (4.83 ? 0.92 fmoV50 ~1, p < 0.01). All rats in
which CRF-IR release was increased by restraint stress also
showed significant increases of CRF-IR levels during 4-amino-
pyridine perfusion. Observation of the rats during the experi-
ment indicated that perfusion with 4-aminopyridine produced
behavioral signs of increased arousal, inducing episodes of lo-
comotor activity alternated with periods of immobility, and oc-
casional limbic seizures.
Figure 2. Anatomical estimation of the “active” zone of microdialysis probes implanted in rats and aimed at the amygdala. A series of consecutive
brain sections bearing the trace of the microdialysis probe were stained with cresyl violet. Sections were selected on the basis of the most ventral
location of the probe tip. The sections with the deepest trace was selected (A), and a segment of 2 mm then drawn vertically, tracing the estimated
location of the active zone (broken line). The same procedure was performed on more rostra1 sections, maintaining the stereotactic and anatomical
alignment (B and C). From this analysis the estimated region of location of the microdialysis membrane was an area defined by the lateral component
of the central nucleus of the amygdala (Ce) and the medial border of the basolateral nucleus of the amygdala (BL).
The Journal of Neuroscience, August 1995, 1~78) 5443
fractions (20 min)
Figure 3. A, Effects of restraint stress (20 min) and 4-aminopyridine
microdialysis in the rat amygdala. Fractions were collected every 20 min. CRF-IR levels measured during stress and 4-AP were significantly
different from basal levels 0, < 0.05). B, Distribution of the anatomical localizations
anatomical trace analysis carried out as described in Figure 2. Only microdialysis probe traces of rats included in the restraint stress experiment
are reported (n = 5).
(4-AP, 10 mM) perfusion on CRF-IR levels as measured by intracranial
of the “active” zone of microdialysis probes according to
Experiment 3: effects of ethanol withdrawal on CRF-IR
release from the amygdala
Exposure to the ethanol diet produced
levels in exposed rats (126 +- 13 mg%). No differences
weight between the ethanol and control
the day before the experiment
perimental rats during microdialysis
during the 6-12 hr period of withdrawal,
were awake most of the time, displayed
neous locomotor activity, exhibited
stiffness of the tail. Conversely,
sleeping, and no body tremor
moderately high BAL
diet groups as assessed
was noted. Observation
low levels of sponta-
occasional body tremor and
control rats were frequently
or stiffness of the tail were ob-
served. Seven rats (three control
rats) were excluded
failure (n = 2), probe disconnection,
2), resulting a sample of five ethanol-dependent
control rats. The initial
period of three rats included
for assay, and therefore
from the analysis. Postmortem
dialysis probe placements
between the Ce and the BL (Fig. 4), as observed in rats exposed
to restraint stress.
and four ethanol
analysis because of surgical
errors (n = l), or inadequate
rupture (n =
rats and five
the 2-4 hr
were not available
interval was excluded
that the tips were located
in the experiment
Figure 4. Anatomical localizations of
zone of microdialysis
probes according to anatomical trace
analysis carried out as described in Fig-
ure 2. Only the microdialysis
traces of rats included in the ethanol
experiment are reported (n = 10).
5444 Merlo Pith et al.
l CRF Release from Amygdala in Stress and Ethanol Withdrawal
Figure 5. Effects of ethanol with-
drawal on CRF-IR levels in the rat
amygdala as determined by microdi-
was collected over
four 2 hr periods regularly alternated
with nonsampling 2 hr periods. The
four sampling periods correspond to
the basal collection (before removal of
ethanol), and 2Z4 hr, 6-8 hr, and 1%
12 hr after withdrawal. Fractions were
collected every 20 min. Data are rep-
resented as mean t
S/group). ANOVA confirmed signifi-
differences between the two
groups over time (JJ < 0.05).
SEM (n =
2-4 h 6-8 h /
20 0 2 4 6 8 10 12 14 16 18
fractions (20 min)
The results of the microdialysis
Figure 5. The average basal CRF-IR levels measured in dialysate
from the amygdala were 1.30 + 0.19 fmol/SO p.1 and 2.11 -C
0.43 fmolR0 p,l in control diet and ethanol diet-exposed rats,
respectively. Although this difference was not statistically sig-
nificant, ANOVA indicated that the overall difference between
the two treatment groups during withdrawal
[F(1,8) = 7.65, p < 0.051. Accordingly,
time-dependent increase in CRF-IR levels measured in dialysate
(p < 0.01). This effect over time was mainly due to the increase
of CRF-IR levels measured in ethanol-exposed rats during with-
drawal, as indicated by significant “treatment X time” interac-
tion @ < 0.01). Post hoc analysis of simple effects showed that
the two groups were significantly different during the lo-12 hr
period of withdrawal (p < O.Ol), while only marginally signif-
icant increases in CRF-IR levels were obtained during the 6-8
hr period of withdrawal (0.10 > p > 0.05).
experiments are shown in
there was a significant
The results presented in this article confirm and extend previous
observations that depolarizing agents can induce CRF-IR release
from slices of rat amygdala in vitro in a Ca*+-dependent manner.
The stimulant action of 4-aminopyridine on CRF-IR release was
also present in awake rats studied with intracranial microdialy-
sis. More interestingly, a transient increase of CRF-IR release
was observed when rats implanted with a microdialysis probe
in the amygdala were exposed to restraint stress for 20 min. In
contrast, a progressive and prolonged increase of CRF-IR release
was measured in ethanol-dependent rats during ethanol with-
drawal, with a time course corresponding to the emergence and
progression of behavioral signs of withdrawal. These findings
indicate that CRF-IR can be released from the rat amygdala in
response to stress produced by exogenous or endogenous stimuli
that result in increased arousal and emotionality.
The origin of CRF-IR measured in the present experiments is
likely to be in the nerve terminals of the Ce. This interpretation
is based on immunocytochemical evidence that shows a dense
plexus of positive neurons, fibers, and terminals in the Ce (Sak-
anaka et al., 1988; Cassel and Gray, 1989; Gray, 1990). The
assessment of microdialysis probe locations within the amygdala
indicated that CRF-IR levels in perfusate were detectable when
the probe was placed in close proximity to the Ce. In two cases
of anatomical misplacement, in which the active part of the
probe was located in the cortex, the levels of CRF-IR were al-
most undetectable (data not ‘shown). The anatomical specificity
of CRF-IR measurement with microdialysis in the rat brain was
previously studied in anesthetized rats. CRF-IR levels were un-
detectable in perfusate from probes placed in the upper third
ventricle and dorsal striatum, while CRF-IR levels of about 1
fmol/50 ~1 were measured in the mediobasal hypothalamus and
Ce (Merlo Pith et al., 1993). CRF-IR release from the medioba-
sal hypothalamus was also measured in anesthetized rats (Gabr
et al., 1994), and in awake rats using the push-pull cannula (Ixart
et al., 1987). In both present experiments involving awake rats,
basal dialysate CRF-IR levels were within the range measured
in the perfusate from the amygdala of anesthetized rats.
The synaptic origin of CRF-IR is further supported by the
observation of enhanced CRF-IR release by depolarizing stimuli,
and its dependency upon Ca*+ availability. Ca*+-dependent K+-
induced release of CRF has been demonstrated in amygdala pri-
mary culture from embryonic explants (Cratty and Brikle, 1994),
in slices of amygdala tissue from adult rat brain (Smith et al.,
1986), and in perfusate from a microdialysis probe located in
the Ce of anesthetized rats (Merlo Pith et al., 1993; Richter et
The Journal of Neuroscience, August 1995, 75(E) 5445
al., submitted). In the present work,
extended by showing that also the K+-channel blocker 4-ami-
nopyridine stimulates CRF-IR release in the amygdala. CRF-IR
increases produced by 4-aminopyridine
blocked by the Ca2+ antagonist CoCl, in vitro. Preliminary ob-
servations indicate that the same blocking effect of CoCl, was
present in anesthetized rats implanted with microdialysis probes
in the amygdala (R. Richter, unpublished data). It is worth to
note that the 4-aminopyridine-dependent
not be a direct effect. In fact, 4-aminopyridine
lease norepinephrine and acetylcholine in vitro (Drukarch
1989; Heemskerk et al., 1990). Since norepinephrine or acetyl-
choline can produce CRF-IR release from the CNS neurons
(Tsagarakis et al., 1988; Hu et al., 1992), it cannot be excluded
that these or other neurotransmitters
4-aminopyridine on CRF release within the amygdala.
Previous findings of high concentrations of CRF binding sites
within the amygdaloid nuclei, particularly in the basolateral nu-
cleus (BL) (De Souza, 1987; Perrin et al., 1993) strongly sug-
gest a role for locally released CRF-IR on amygdaloid neuron
activity. In brain slice preparations CRF produces postsynaptic
depolarization in most of amygdala cells (Eberly et al., 1983;
Rainnie et al., 1992) but presynaptic inhibitory effects on CRF-
IR neurons have also been proposed (Wiersma et al., 1993). CRF
binding sites are located on the perikarya,
minals of peptide-containing medium spiny neurons of the Ce,
including CRF-IR neurons. Incidentally, receptors on CRF-IR
neurons may represent autoreceptors that regulate CRF-IR
lease, locally (Wiersma et al., 1993) or in their projection fields
in hypothalamic and brainstem nuclei. Thus, a rise in extracel-
lular CRF-IR levels in the Ce may contribute to activate peptide-
containing medium spiny neurons of the amygdala, activation
recently demonstrated using Fos immunostaining technique in
rats exposed to restraint stress (Honkaniemi,
by acting on autoreceptors, CRF may also participate in the shut-
down of CRF-IR release from CRF-IR neurons. To further com-
plicate the picture of the role of CRF in the amygdala, BL neu-
rons are also likely to be affected by CRF-IR.
alysis experiments have shown that CRF-IR levels increased in
a sampling region between Ce and BL during stress, it is rea-
sonable to suggest that locally released CRF-IR can diffuse and
modulate also BL neurons, possibly in a nonsynaptic way (Fuxe
and Agnati, 1991). Since BL neurons massively project to Ce,
but Ce neurons do not project to BL (McDonald,
possible that CRF-IR diffusing from Ce can retrogradely affect
BL neurons, providing a local nonsynaptic feedback mechanism.
All these observations point to a complex modulatory role of
CRF within the amygdala.
The present results complement a series of behavioral exper-
iments aimed to understand the involvement of CRF in emo-
tionality. When the CRF antagonist aCRF(941)
jetted within the amygdala of rats exposed to social stress, a
dose-dependent decrease of the stress-induced
effect was observed (Heinrichs
stress-induced immobility was also produced by intra-amygdala
administration of aCRF(9-41) in the pM range (Swiergiel et al.,
1993). Thus, it may be concluded that CRF in the amygdala
mediates the emotional response to stress exposure (but see also
Liang et al., 1992a,b).
The results of the present experiments performed in ethanol-
dependent rats during ethanol withdrawal
hypothesis. Chronic ethanol exposure has been associated with
these observations were
release of CRF-IR may
is known to re-
mediate the effects of
dendrites, and ter-
1992), it is
et al., 1992). Attenuation of
partially support this
1979; Glue and Nutt, 1990). Stress-like
withdrawal from chronic ethanol have been previously studied
in rats using the social interaction test (File et al., 1989) and the
elevated plus-maze (Baldwin et al., 1991). In these experiments,
significant withdrawal signs are observed approximately 8-10
hr after ethanol withdrawal. Recent work has shown that intra-
amygdala administration of cxCRF(9-41)
like effects of ethanol withdrawal
hr after withdrawal (Rassnick et al., 1993). Interestingly,
period corresponds to the rise in CRF-IR dialysate levels from
amygdala observed in the present experiment. The most intense
effects of withdrawal on CRF-IR release were observed at lo-
12 hr. The peak of CRF-IR levels were two to three times higher
that the peak effect in rats exposed to restraint stress, suggesting
an increased activity of CRF-IR neurons in the amygdala. In rats
chronically exposed to ethanol, increased sensitivity to the lo-
comotor stimulating effects of CRF is also reported (Ehlers and
The mechanisms involved in the enhanced CRF-IR
are not known. Since acute administration of ethanol exerts an-
xiolytic-like effects, prolonged exposure to ethanol may produce
adaptive changes in central neurons to counteract this inhibitory
effect (Pohorecky, 1981; Koob and Bloom, 1988). According to
this hypothesis, the absence of ethano! during withdrawal
produce a rebound activation of previously
leading to their hyperactivity (Glue and Nutt, 1990). The in-
creased CRF release measured in the amygdala of dependent
rats during withdrawal may represent such a mechanism, in that
inhibition of CRF neurons of the Ce by chronic ethanol may
result in a downregulation of inhibitory receptors, such as CRF
autoreceptors or GABAlbenzodiazepine
zodiazepine receptors activity is known to be reduced by chronic
ethanol exposure (Morrow et al., 1988). Since both Ce and BL
neurons contain a very high density of GABAfbenzodiazepine
binding sites (Niehoff and Kuhar, 1988) it can be surmised that
CRF-IR neurons are normally under the inhibitory effects of
GABAergic transmission. Therefore, the reduced tonic intluence
of GABAergic transmission in chronic ethanol exposed rats may
result in disinhibition of CRF-IR neurons.
A third possible substrate mediating the CRF-IR release in the
amygdala during ethano) withdrawal
amines, in particular, norepinephrine. In both situations of stress
and ethanol withdrawal the triggering signal for CRF-IR release
may be due to norepinephrine released from the terminals in-
nervating the Ce (Fallon et al., 1988; Gavin, 1990). A recent
microdialysis experiment (Tanaka et al., 1991) showed that the
temporal profile of norepinephrine release from the amygdala of
rats exposed to 20 min restraint stress closely matches that of
CRF-IR described in the present work.
evokes CRF-IR release from hypothalamic neurons (Tsagarakis
et al., 1988; Hu et al., 1992), while microdialysis
show that CRF produces norepinephrine release from nerve ter-
minals in prefrontal cortex and hypothalamus
Dunn, 1993), suggesting the existence of a reciprocal stimulating
interaction between CRF and norepinephrine. Although the rel-
evance of central norepinephrine in anxiety is still under debate,
some of the behavioral effects produced by stress and ethanol
withdrawal involve adrenoreceptor
Dunn, 1989; Glue et al., 1989; Soderpalm and Engel, 1990).
In conclusion, a recently validated method to measure CRF-
IR release with microdialysis in freely moving rats was used to
in rodents and humans (Cooper et al.,
effects produced by
blocks the anxiogenic-
in the elevated plus-maze 8
is represented by mono-
In vitro norepinephrine
mediation (Berridge and
5446 Merlo Pith et al.
l CRF Release from Amygdala in Stress and Ethanol Withdrawal
neurons of the amygdala
effects of restraint
the effects of restraint stress and ethanol
release from the amygdala.
effects on CRF-IR release induced
effects. Together, these results suggest that CRF-IR
stress and ethanol
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