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The Journal of Neuroscience, April 1, 2002, 22(7):2926-2935
Reduction of Stress-Induced Behavior by Antagonism of
Corticotropin-Releasing Hormone 2 (CRH2) Receptors in
Lateral Septum or CRH1 Receptors in Amygdala
Vaishali P.
Bakshi1,
Stephanie
Smith-Roe2,
Sarah
M.
Newman1,
Dimitri E.
Grigoriadis3, and
Ned H.
Kalin1
1 Department of Psychiatry, University of Wisconsin,
Madison, Wisconsin 53719, 2 Department of Environmental
Toxicology, Oregon State University, Corvallis, Oregon 97330, and
3 Neurocrine Biosciences Inc., San Diego, California
92121
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ABSTRACT |
Although corticotropin-releasing hormone (CRH), a regulator of
stress responses, acts through two receptors (CRH1 and
CRH2), the role of CRH2 in stress
responses remains unclear. Knock-out mice without the CRH2
gene exhibit increased stress-like behaviors. This profile could result
either directly from the absence of CRH2 receptors or
indirectly from developmental adaptations. In the present study,
CRH2 receptors were acutely blocked by  -helical CRH
(hCRH, CRH1/CRH2 antagonist; 0, 30, 100, and 300 ng) infusion into the lateral septum (LS), which
abundantly expresses CRH2 but not CRH1
receptors. Freezing, locomotor activity, and analgesia were tested
after infusion. Intra-LS hCRH blocked shock-induced freezing without
affecting activity or pain responses; infusions into lateral ventricle
or nucleus of the diagonal band had no effects. The same behavioral
profile was obtained with D-Phe-CRH(12-41) (100 ng), another CRH1/CRH2 antagonist.
A selective CRH1 antagonist (NBI27914), in doses
that reduced freezing on intra-amygdala (central nucleus) infusion (0, 0.2, and 1.0 µg), did not affect freezing when infused into the LS.
Ex vivo autoradiography revealed that binding of
[125I]sauvagine, a mixed
CRH1/CRH2 agonist, was prevented in the
LS by previous intra-LS infusion of hCRH but not NBI27914. In
vitro studies demonstrated that
[125I]sauvagine binding in the LS could be
inhibited by a CRH1/CRH2 antagonist but
not by the selective CRH1 receptor antagonist, confirming
that in the LS, hCRH antagonized exclusively CRH2 receptors. Acute antagonism of CRH2 receptors in the LS
thus produces a behaviorally, anatomically, and pharmacologically
specific reduction in stress-induced behavior, in contrast to results
of recent knock-out studies, which induced congenital and permanent
CRH2 removal. CRH2 receptors may thus represent
a potential target for the development of novel CRH system anxiolytics.
Key words:
CRF; anxiety; corticotropin-releasing hormone; corticotropin-releasing factor; defensive behavior; freezing; behavioral inhibition
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INTRODUCTION |
Corticotropin-releasing hormone
(CRH) coordinates various aspects of the stress response (Vale et al.,
1981 ). CRH elicits behaviors normally exhibited in response to stress,
whereas CRH receptor antagonists prevent stress-induced behaviors (Koob
and Heinrichs, 1999). Patients with stress-related problems such as depression often have alterations in their CRH system, suggesting that
it may play an important role in stress-related psychopathology (Nemeroff et al., 1984; Mitchell, 1998).
Recently, CRH receptor antagonists have been developed as a novel class
of anxiolytics and antidepressants (McCarthy et al., 1999). A
preliminary open-label clinical trial indicated that these compounds
alleviate symptoms in depressed patients (Zobel et al., 2000). Although
there are two cloned CRH receptors (CRH1 and
CRH2 , , ), most studies suggesting that
CRH receptor antagonists may be psychotherapeutic agents have focused
on the CRH1 subtype, perhaps because highly
selective nonpeptide antagonists for CRH2
receptors (splice variant expressed in brain) have not been identified
(Perrin and Vale, 1999). Nonetheless, the very recent discovery of
endogenous and highly selective CRH2-receptor ligands in rodent and human brain suggests that this receptor may play
some intrinsic functional role (Hsu and Hsueh, 2001; Lewis et al.,
2001; Reyes et al., 2001). Nonetheless, the role of
CRH2 receptors in stress and anxiety remains unclear.
Antisense oligonucleotides have been used to study
CRH2 receptor functioning in stress; however,
interpretation of these studies is difficult, because they failed to
demonstrate an appreciable reduction in CRH2
receptors after oligonucleotide infusion (Heinrichs et al., 1997;
Liebsch et al., 1999). Paradoxically, knock-out studies indicate that
deletion of the CRH2 gene produces a phenotype
characterized by increased anxiety-like behaviors (Bale et al., 2000;
Coste et al., 2000; Kishimoto et al., 2000). One problem with
interpreting these studies, however, is that the observed phenotype
could be attributable to indirect developmental alterations resulting
from the mutation rather than being a direct result of the gene
deletion (Gingrich and Hen, 2000). Measurement of stress-related
behavior after acute antagonism of CRH2 receptors circumvents these confounds.
The distributions of CRH1 and
CRH2 receptors in rodent brain are mostly
nonoverlapping (Chalmers et al., 1995; Primus et al., 1997), suggesting
that the role of CRH2 receptors in stress
might be determined by antagonizing CRH receptors in a brain region
selectively expressing the CRH2 receptor
subtype. The lateral septum (LS) contains a high density of
CRH2 receptors but is devoid of
CRH1 receptors (Chalmers et al., 1995; Primus et
al., 1997). Although learning a conditioned fear response involves CRH
receptors in the LS (Lee, 1995; Radulovic et al., 1999), a selective
role for the CRH2 receptor subtype in
stress-induced behavior remains unclear.
The present studies tested the hypothesis that
CRH2 receptor blockade in the LS would
decrease stress-induced behavior by determining whether intra-LS
infusion of hCRH or
D-Phe-CRH(12-41), CRH1/CRH2 receptor
antagonists, reduced shock-induced freezing, a CRH-receptor-mediated
defensive behavior displayed in response to fear (Kalin et al., 1988;
Swiergiel et al., 1992, 1993). Effects of hCRH on other behaviors or
in neighboring regions were measured to assess the behavioral and
anatomical specificity of LS-mediated effects. Receptor subtype
specificity of LS-mediated effects was determined by comparing the
effects of CRH1/CRH2
antagonists with those of a selective CRH1
antagonist, NBI27914 (Chen et al., 1996). Of particular interest was
whether acute antagonism of CRH2 receptors
would produce a different behavioral profile than that seen after
CRH2 gene knock-out.
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MATERIALS AND METHODS |
Animals
One hundred seventy-four male Sprague Dawley rats (Charles River
Laboratories) were used in the present studies. Rats were housed in
pairs in clear plastic cages in a temperature- and humidity-controlled vivarium and were maintained on an ad libitum diet of lab
chow (Harlan Teklad, Madison, WI) and water. Lights in the animal
colony came on at 7 A.M. and turned off at 7 P.M.; all testing occurred between 11 A.M. and 4 P.M. On arrival, rats were handled gently by the
experimenter to minimize stress during the experiments. Animal
facilities were approved by the Association for the Assessment and
Accreditation of Laboratory Animal Care; protocols were in accordance with the Guiding Principles in the Care and Use of Animals
provided by the American Physiological Society and the guidelines of
the National Institutes of Health. All efforts were made to prevent
animal suffering and minimize the number of animals used for the studies.
Surgery
Within 1 week of arrival, animals were anesthetized with sodium
pentobarbital (65 mg/kg; Butler Co., Columbus, OH), treated with 0.1 ml
of atropine sulfate (Phoenix Pharmaceuticals, St. Joseph, MO) to
minimize respiratory distress, and placed into a stereotaxic apparatus
(Kopf Instruments, Tujunga, CA). Stainless steel cannulas (23 gauge)
were implanted bilaterally and affixed to the skull with dental cement
(Lang Dental, Wheeling, IL) and skull screws (Small Parts, Miami Lakes,
FL). Cannulas were aimed at the LS [coordinates were anteroposterior
(AP), +0.4 mm from bregma; lateromedial (LM), ±0.8 mm from midline,
and dorsoventral (DV),  3.5 mm from skull surface], the lateral
ventricle (LV; coordinates were AP, 0.4 mm from bregma; LM, ±1.5 mm
from midline; and DV, 2.2 mm from skull surface), the nucleus of the
diagonal band (NDB; coordinates were AP, +0.4 mm from bregma; LM, ±0.8 mm from midline; and DV, 3.5 mm from skull surface), or the central nucleus of the amygdala (CeA; coordinates were AP, 2.5 mm from bregma; LM, ±4.2 mm from midline; and DV, 5.2 mm from skull surface) (all coordinates based on the atlas of Paxinos and Watson, 1998). After
surgery, rats were allowed 5-7 d to recover, during which time daily
health checks were performed by the experimenter.
Drugs
-Helical CRH (hCRH) and
D-Phe-CRH(12-41) were obtained from
Bachem-Peninsula Laboratories (Torrance, CA) and were dissolved in
sterile distilled water, pH 6.5. Thus, the vehicle treatment for all
experiments in which hCRH and
D-Phe-CRH(12-41) were administered
was distilled water, pH 6.5. NBI27914 was synthesized at Neurocrine
Biosciences (Chen et al., 1996) and dissolved with sonication in a
vehicle solution of 90% distilled water, 5% ethanol, and 5%
cremophor EL (Sigma, St. Louis, MO). This vehicle solution was
used as the control treatment for all experiments in which NBI27914 was
administered into the brain. All doses (see below) were calculated
using the HCl salt weight.
Microinfusion procedure
On all test days, animals were gently held, and their stylets
were removed and placed into 70% ethanol. Cannulas were cleaned with a
dental broach, and stainless steel injectors (30 gauge) were lowered so
that they extended 1.5-5 mm below the tips of the cannulas. Thus, the
final DV coordinates from skull surface were 6.0 mm for the LS, 3.7
mm for the LV, 8.5 mm for the NDB, and 8.2 mm for the CeA. The
injectors were attached to polyethylene tubing, which was connected to
10 µl Hamilton microsyringes (Fisher Scientific, Pittsburgh, PA) that
were mounted on a motorized pump (Harvard Apparatus, Holliston, MA). A
total of 0.5 µl of vehicle or drug per side was delivered over 93 sec
in each infusion. The pump was then shut off, and injectors were kept
in place for an additional 60 sec to allow for absorption of the
injection bolus into the tissue. Injectors were then removed; stylets
were replaced; and animals were placed immediately into chambers for
behavioral testing. Two to 3 d before drug testing, all rats
received a mock infusion in which injectors were lowered but no
solution was delivered to acclimate rats to the infusion procedure and
to minimize stress attributable to injections on the test days.
Behavioral Testing
After drug infusions, rats were tested in one of the three
following behavioral paradigms. For all studies, rats were placed into
the behavioral testing apparatus immediately after drug infusion. The
experimenter was blind to the treatment condition of the rats for all testing.
Shock-induced freezing. Rats were placed individually in a
black Plexiglas chamber (21 × 11 × 6 inches) with a metal
floor grid and overhead houselights (San Diego Instruments, La Jolla, CA). After a 2 min acclimation period to the chamber, three mild foot
shocks were delivered (1 sec, 1.5 mA, separated by 20 sec). The onset
and duration (in seconds) of freezing behavior (cessation of all body
movements except that required for respiration) were rated for 15 min
immediately after the final shock. To be counted as a bout of freezing,
freezing behavior had to occur continuously for a minimum of 5 sec.
This criterion was applied to minimize the possibility of obtaining
spurious counts of freezing.
Locomotor activity. Rats were placed individually in clear
polycarbonate cages (19 × 10.5 × 8 inches) equipped with
computer-interfaced photocells along their long axis and cage top to
measure unconditioned locomotor activity (San Diego Instruments).
Rearing (vertical activity, total cage top photobeam breaks),
ambulation (number of cage crossings), and total activity (total
photobeam breaks) were recorded over 20 min to match the time course of
the freezing test.
Analgesia. Rats were placed individually on a metal plate
(prewarmed to 50°C) within a clear Plexiglas chamber (11 × 11 × 8 inches). The latency (in seconds) for the rat to lick its
hindpaws after placement onto the plate was recorded. If licking did
not occur, rats were removed by the experimenter after 60 sec had elapsed; these animals were given a score of 60.
Experimental design
Eleven experiments were conducted in separate groups of rats.
Effects of intra-LS CRH1/CRH2
antagonists on shock-induced freezing. In experiment 1, rats were
given infusions of either vehicle (distilled water; n = 10) or hCRH (30 ng; n = 10) into the LS and placed
into the freezing apparatus. In experiment 2, rats received either
vehicle (n = 12) or a higher dose of hCRH (100 ng;
n = 12) into the LS and were tested for shock-induced freezing. In experiment 3, either vehicle (n = 6) or
300 ng of hCRH (n = 6) was infused into the LS
before testing in the freezing apparatus. The doses of hCRH used in
the present experiments were chosen on the basis of previous reports
that infusion of 100-200 ng of this antagonist per side into either
the locus ceruleus or amygdala reduces shock-induced freezing
(Swiergiel et al., 1992, 1993). Finally, an additional experiment was
performed to confirm that a different more potent
CRH1/CRH2 antagonist would have the same effects on freezing as hCRH. Thus, in experiment 4, either vehicle (n = 8) or 100 ng of
D-Phe-CRH(12-41) (Menzaghi
et al., 1994) (n = 7) was infused into the LS, and rats were then tested for shock-induced freezing. In this experiment, a 1.0 mA shock intensity was used instead of 1.5 mA to ascertain that
baseline latency and duration of freezing in vehicle-treated rats were
not influenced by this difference in shock intensity.
Effects of intra-LS CRH1/CRH2
antagonists on locomotor activity and analgesia. Because potential
changes in freezing may not necessarily reflect changes in
stress-induced behavior but rather may simply be an artifact of altered
motor activity levels or pain responses caused by the drug, the effects
of CRH1/CRH2 antagonists on
baseline locomotor activity and analgesia were tested. Thus, in
experiment 5, rats were given a wide dose range of hCRH (0, 30, 100, or 300 ng; n = 11) into the LS and placed into
photocell cages. All animals received all hCRH doses in a
counterbalanced order over 4 test days. All rats had been habituated to
the test cages and infusion procedure a few days before testing;
successive tests were separated by 3 d. In a separate set of rats
that were naive to the testing chambers, the effects of
D-Phe-CRH(12-41) (0 or 100 ng; n = 6 per group) were evaluated to corroborate
hCRH findings and also to be certain that the rat's level of
familiarity with the given testing chamber did not influence the CRH
antagonist-induced effects (experiment 6). In this experiment, rats
were tested only once, and separate animals were used for the different
treatment groups so that all testing would occur on the first day that
the rats were introduced to the testing chamber; this protocol was chosen to match that of the freezing experiments. In experiment 7, rats
were tested for potential changes in pain sensitivity after receiving
intra-LS infusion of hCRH (0 or 100 ng). Testing was conducted over
2 test days that were separated by 1 week. On the first test day, half
of the rats received vehicle, and the other half got hCRH before
being placed onto the hotplate. One week later, this protocol was
repeated, balancing the treatments such that animals that had
previously received hCRH got vehicle and rats that previously
received vehicle got hCRH. Three separate sets of rats were used for
this experiment: one was tested 3 min after the infusions
(n = 7; to correspond to the time point at which shock
was delivered in the freezing paradigm); another was tested 10 min
after the infusions (n = 6); and the final group was
tested 15 min after the infusions (n = 5). These
different time points were selected to map out the duration of the
freezing test and to determine whether there were any alterations in
pain sensitivity produced by hCRH at any time during this 15 min period.
Effects on shock-induced freezing of hCRH infusion into
regions neighboring the LS. To confirm that the behavioral effects observed after intra-LS infusion of hCRH were localized specifically to the LS and not attributable to diffusion of the drug to other areas,
the effects on freezing of hCRH infusion into regions neighboring
the LS were determined. Because the LS is bordered by the lateral
ventricle, experiment 8 was performed to determine whether direct
intra-LV infusion of hCRH in the present dose range would affect
freezing. The same protocol as in experiment 2 was used, except that
intracranial infusions were made into the LV (vehicle,
n = 9; 100 ng of hCRH, n = 9).
Experiment 9 was also identical to experiment 2, except that infusions
were delivered into the NDB (vehicle, n = 8; 100 ng of
hCRH, n = 10). The NDB is ventrally adjacent to the LS.
Effects of a selective CRH1 antagonist on
shock-induced freezing. To determine the receptor subtype
specificity of LS-mediated effects, the effects of intra-LS infusion of
a selective CRH1 antagonist (NBI27914;
CRH1:CRH2 affinity,
>10,000; Chen et al., 1996) were measured. First, to identify a dose
of NBI27914 that is sufficient to block shock-induced freezing after
intracranial administration, several doses of the
CRH1 antagonist were infused into the CeA, a
structure that contains high levels of the CRH1 receptor subtype and through which hCRH reduces shock-induced freezing (Swiergiel et al., 1993). Thus, in experiment 10, rats received either vehicle (n = 6) or 0.2 µg
(n = 6) or 1.0 µg (n = 7) of NBI27914
into CeA before testing in the freezing apparatus. Because the 1 µg
dose was found to potently reduce shock-induced freezing, this dose was
used for the LS experiment. Thus, in experiment 11, rats were given
either vehicle (n = 7) or 1.0 µg of NBI27914 (n = 7) into the LS and were then placed in freezing
chambers. At the end of the shock-induced freezing test in experiment
11, rats were immediately killed, and their brains were prepared
for autoradiography of CRH receptors in the LS. In addition, a few rats
that received 100 ng of hCRH into the LS before testing in the
freezing chamber (n = 5) were included in experiment 11 to compare brain sections from hCRH-treated rats with those from NBI27914-treated rats in autoradiographic analyses. Because the behavioral data from these hCRH-treated rats were identical to those
from experiment 2, their freezing data are not displayed for the sake
of brevity. Autoradiographic results from these animals are depicted in
Figure 6.
Histology
At the end of the experiments (except experiments 8 and 11),
rats were given an overdose of pentobarbital (130 mg/kg) and perfused
transcardially with isotonic saline followed by 10% formalin. Brains
were removed, stored in formalin, and subsequently sectioned into 60 µm coronal sections using a cryostat (Leica Instruments, Deerfield,
IL). After staining with cresyl violet, sections were examined under a
microscope for the location of injector tip placements. Animals whose
injector placements fell outside of the targeted brain regions were
excluded from analyses of behavioral data. At the time of histological
verification of injector tip placements, the experimenter was blind to
the pharmacological treatment as well as the behavioral data for each
animal. For experiment 8, rats were deeply anesthetized with sodium
pentobarbital and were then given a 5 µl infusion of Chicago blue dye
(Sigma) through their ventricular cannulas. After a 3 min diffusion
period, rats were decapitated, and brains were removed and sectioned
into 2 mm slices. The appearance of dye within the ventricular system distal to the injection site was verified for each rat; rats without dye in their ventricles were excluded from analysis of behavioral data.
Autoradiography
For experiment 11, instead of perfusion, rats were killed by
rapid decapitation immediately after they were tested in the freezing
apparatus, and brains were quickly removed and frozen in chilled
2-methylbutane (20 to 30°C). Brains were then mounted onto a
cryostat block with Tissue-Tek (Hacker Instruments) and sectioned using
a Leica cryostat. Twenty micrometer sections were thaw-mounted onto
Fisher Scientific "plus-charged" slides, allowed to air dry, and
stored at 80°C until use. On the day of assay, slides were thawed
to room temperature and allowed to completely dry for a further 20 min.
The area around each section was outlined using a grease marker, and
300 µl of [125I]sauvagine (50-100
pM final concentration in PBS containing 10 mM
MgCl2 and 2 mM EGTA, pH 7.0) was
gently applied directly onto each section. Nonspecific binding was
determined in adjacent sections by the addition of 1 µM
NBI27914, the selective CRH1 receptor antagonist
with a Ki of 2 nM (Chen et al., 1996), for the determination of
the CRH1-specific binding, or 1 µM
D-Phe-CRH(12-41), which is
an antagonist with equal affinity for the CRH1
and CRH2 receptors
(Ki, ~30 nM),
in the buffer for determination of both CRH1 and
CRH2 receptor-specific binding. The slides were
placed in a covered humidified chamber to reduce evaporation and
incubated at 22°C for 40-45 min. After the incubation, the solution
was gently aspirated from the section under vacuum, and the slides were
washed using two 5 min dips in ice-cold PBS and Triton X-100 (0.01%),
pH 7.0. Slides were then air-dried and apposed to Biomax MR x-ray film
(Eastman Kodak Co., Rochester NY) for 4-5 d. Images were captured
using a light box and digital camera (Northern Lights, St. Catharines,
Ontario, Canada) and visualized using NIH Image (National Institutes of
Health, Bethesda, MD). One set of adjacent sections from each animal
was stained with cresyl violet and used to verify the location of
injector tips in the LS.
Data analysis
For all freezing experiments, the interval (in seconds) between
the final foot shock and the commencement of freezing (latency to
freeze) and the total number of seconds the rat spent freezing during
the 15 min test session were calculated for each animal. In the
analgesia experiment, the number of seconds (of a maximum of 60) that
it took for the rat to lick its hindpaws after being placed on the hot
plate was measured for each rat. Because freezing and analgesia data
were not normally distributed (there was an upper limit to the scores
imposed by the length of the testing session), nonparametric statistics
were used to analyze these measures instead of parametric tests. For
all freezing data, separate Mann-Whitney U tests (vehicle
group vs drug group) were performed for each experiment. In experiment
10, where multiple comparisons were made, the level for statistical
significance was adjusted to p < 0.02. Hot plate data
were analyzed with a Wilcoxon matched pairs signed ranks test (vehicle
vs drug treatment) because of the within-subjects design of this
experiment. For locomotor activity data, the numbers of rears,
ambulations (cage crossings), and total activity (total photo beam
breaks) were calculated for each 10 min period of a 90 min test
session. These data were analyzed with separate two-factor ANOVAs for
each activity index, with drug treatment and time point as
within-subjects factors.
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RESULTS |
Effects on freezing of CRH1/CRH2
receptor antagonist infusion into the LS
The results of experiment 1 are displayed in Figure
1C. Infusion of 30 ng of
hCRH into the LS had no effect on either index of freezing behavior
(latency to freeze, p = 0.427; total duration of
freezing, p = 0.705, Mann-Whitney U test).
In contrast, a higher dose of hCRH (100 ng/side) significantly
increased the latency to begin freezing (p < 0.004) and decreased the total duration of freezing
(p < 0.018) after infusion into the LS
(experiment 2, seen in Fig. 1A). Rats that received
100 ng of hCRH took approximately three times as long to begin
freezing and spent approximately half as much time freezing compared
with vehicle-treated controls. As depicted in Figure
1B, hCRH decreased freezing in the first as well
as the second and third portions of the test session, indicating that
this reduction was not simply attributable to the antagonist-induced
increase in the latency to begin freezing. Thus, infusion of 100 ng of
hCRH into the LS significantly reduced this measure of
stress-induced behavior. Interestingly, infusion of a higher dose of
hCRH into the LS (experiment 3; 300 ng) failed to affect either the
latency to freeze (p = 0.748) or the total duration of freezing (p = 0.521), indicating
that hCRH displays an inverted U-shaped dose-response profile for
reducing shock-induced freezing in the LS (Fig. 1D).
Finally, Figure 1E illustrates the results of
experiment 4, which demonstrated that another more potent
CRH1/CRH2 receptor
antagonist,
D-Phe-CRH(12-41), also
increased the latency to freeze (p < 0.011) and
decreased the total duration of freezing after infusion into the LS
(p < 0.05). This reduction in freezing also
occurred throughout the test session (Fig. 1F). It
should be noted that this decrease in freezing was identical to that
seen with hCRH and that the latency and total freezing values in
vehicle-treated rats in both experiments were similar. This similarity
in profile between experiments 2 and 4 indicates that no effect on
freezing latency or duration was produced by using a 1.0 versus a 1.5 mA shock intensity. These findings with
D-Phe-CRH(12-41) confirm
the results with hCRH and furthermore indicate that behavioral
effects of these antagonists in the LS cannot be attributed to indirect
actions mediated through the CRH-binding protein, which binds with
moderate affinity to hCRH but has no affinity for
D-Phe-CRH(12-41) (Chan et al., 2000).
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Figure 1.
Effects on freezing of -helical CRH or
D-Phe-CRH(12-41) infusion into the lateral
septum. Values represent means ± SEM for each group.
VEH, Vehicle (distilled water). All doses are in 0.5 µl/side. Bin, Each 5 min portion of the freezing test
session. A, Effects of 100 ng of hCRH on latency and
total duration of freezing. B, Time course of freezing
with 100 ng of hCRH. C, Effects of 30 ng of hCRH.
D, Effects of 300 ng of hCRH. E,
Effects of 100 ng of D-Phe-CRH(12-41).
F, Time course of freezing with 100 ng of
D-Phe-CRH(12-41). *p < 0.05, **p < 0.01 compared with VEH
group.
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Effects on locomotor activity and analgesia of intra-LS
hCRH infusion
In contrast to the potent effects on shock-induced freezing
behavior, hCRH infusion into the LS produced no changes in locomotor activity (experiment 5). ANOVAs failed to indicate main effects of
hCRH on rearing [F(3,30) = 1.66;
NS], ambulation [F(3,30) = 1.01;
NS], or total activity [F(3,30) = 0.55; NS]. Moreover, no significant treatment × time
interactions were seen. Because the effects were identical for all
three indices of locomotor activity, only the data for total activity
counts (total photo- beam breaks) are displayed (Fig.
2A). Similarly, in
experiment 6, D-Phe-CRH(12-41) failed to
affect any index of locomotor activity; total activity counts are shown
in Figure 2B
[F(1,10) = 0.10; NS]. Analysis of
data from experiment 7 indicated that pain thresholds were similarly
unaffected by infusion of 100 ng of hCRH into the LS (Fig.
2C). A Wilcoxon signed ranks test revealed that the latency
to lick hindpaws after being placed on the hot plate did not differ
between the vehicle condition and the drug condition 3 min
(p = 0.917), 10 min (p = 0.631), or 15 min (p = 0.464) after infusion,
thereby mapping out the time course of the freezing test. Thus,
intra-LS infusion of hCRH had no effect on pain thresholds or
locomotor activity at any time during the length of the freezing test
session, suggesting that changes in shock-induced freezing that were
produced by this treatment were not simply an artifact of altered
sensitivity to the foot shock or altered baseline activity levels.
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Figure 2.
Effects on other behaviors of -helical CRH or
D-Phe-CRH(12-41) infusion into the lateral
septum. Values represent means ± SEM for each group.
VEH, Vehicle (distilled water). All doses are in
nanograms per 0.5 µl/side. A, Effects of hCRH on
locomotor activity (total photobeam breaks in activity cages).
B, Effects of D-Phe-CRH on locomotor
activity. C, Effects of hCRH on pain sensitivity
(latency to lick hindpaws in the hotplate test).
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Effects on freezing of hCRH infusion into sites adjacent to
the LS
Figure 3 depicts the effects on
shock-induced freezing of 100 ng of hCRH infusion into either the LV
or the NDB. Mann-Whitney U tests indicated that for the LV,
hCRH-treated rats performed no differently from vehicle-treated rats
on either the latency to begin freezing (p = 0.965) or the total duration of freezing (p = 0.453) (experiment 8, seen in Fig. 3A). Likewise, it is
shown in Figure 3B that infusion of hCRH into the NDB
(experiment 9) had no effect on either measure of freezing
(p = 0.894 for latency, and p = 0.248 for total duration). Thus, although this dose of hCRH markedly
reduced freezing on intra-LS infusion, it had no effect on
shock-induced freezing when delivered into regions that are adjacent to
the LS, suggesting that the effects of hCRH after intra-LS infusion
were not mediated by actions at other adjacent brain regions.
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Figure 3.
Effects on freezing of -helical CRH infusion
into regions adjacent to the lateral septum. Values represent
means ± SEM for each group. VEH, Vehicle
(distilled water); hCRH is 100 ng/0.5 µl per side.
A, Infusion into the lateral ventricle.
B, Infusion into the nucleus of the horizontal limb of
the diagonal band.
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|
Effects of a selective CRH1 receptor antagonist
on freezing
The effects on freezing of NBI27914 (a selective
CRH1 antagonist) administration into the CeA were
investigated in experiment 10 (Fig.
4A). It was found that
the 1.0 µg dose (p = 0.015) but not the 0.2 µg dose (p = 0.037) significantly increased
the latency to begin freezing after NBI27914 infusion into the CeA. The
high (p = 0.010) but not the low
(p = 0.150) dose of NBI27914 also decreased the
total duration of freezing after intra-CeA infusion; freezing was
reduced throughout the test (Fig. 4B). Thus, infusion of a highly selective CRH1 antagonist into the
CeA profoundly reduced shock-induced freezing, suggesting that within
this structure, the CRH1 receptor subtype at
least in part mediates stress-induced behavioral responses. In
contrast, when infused into the LS, NBI27914 had no effect on the
latency to begin freezing (p = 0.338) or the
total duration of freezing (p = 0.655)
(experiment 11; Fig. 4C). Thus, a dose of a
CRH1 receptor antagonist that markedly reduced
freezing on intra-CeA infusion failed to alter this behavior when
infused into the LS.
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Figure 4.
Effects on freezing of a selective
CRH1 receptor antagonist. Values represent means ± SEM for each group. VEH, Vehicle solution;
NBI, NBI27914. Doses are in micrograms per 0.5 µl/side. A, Effects on latency and total duration of
freezing after infusions into the central nucleus of the amygdala.
B, Time course of freezing with NBI27914 infusion into
the CeA. C, Effects of infusions into the lateral
septum. *p < 0.02 compared with VEH
group.
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Histological analysis
Figure 5 displays the location of
injector tips in the various brain regions that were studied. Injector
tips for all animals except two in the LS experiments, one in the NDB
experiment, and one in the CeA experiment were found to be within the
targeted regions. Data from these anatomical outliers were excluded
from statistical analysis; the sample sizes reported in Materials and Methods reflect the omission of these rats. The photomicrographs in
Figure 5 depict a section from each brain region under investigation; the location of injector tips in these images is representative of
placements within that region. As can be seen in Figure 5, excessive
tissue damage was not observed with infusion into any region.
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Figure 5.
Histological verification of injector tip
placements. Photomicrographs show Nissl-stained coronal sections
through the lateral septum (A), the nucleus of
the horizontal limb of the diagonal band (B), and
the central nucleus of the amygdala (C).
Black arrows indicate the location of injector tips.
Sections illustrate representative injector tip placements for each
region. Note the absence of necrosis or lesioning at the injection
sites after infusions.
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Autoradiography for CRH receptors in the LS
Ex vivo receptor autoradiography was used as described
above for assessing [125I]sauvagine
binding in brain sections from animals that had received either vehicle
or CRH receptor antagonists into the LS. This analytical technique was
used to provide a qualitative assessment of relative levels of
[125I]sauvagine binding in sections from
different treatment groups. It should be noted that although there is
some unavoidable variability in the precise rostral-caudal location of
the selected sections, these differences are small (~100 µm), and
that CRH2 receptors are distributed
homogeneously throughout the entire extent of the LS (Chalmers et al.,
1995). Thus, the sections displayed in the following figures provide a
representative example of receptor labeling within this structure after
various CRH antagonist treatments.
Figure 6 displays representative sections
from rats that were given vehicle (Fig. 6A), 1 µg
of NBI27914 (Fig. 6B), or 100 ng of hCRH (Fig.
6C) directly into the LS before behavioral testing. The
binding of
[125I]Tyr0
sauvagine to CRH receptors in the LS was nearly undetectable in rats
that had received the nonselective antagonist hCRH into this region
(Fig. 6C). As can be seen clearly in Figure
6B, there was no inhibition of
[125I]sauvagine binding in the LS in
animals that had received intra-LS NBI27914; the level of binding in
NBI27914-treated rats was equivalent to that observed in
vehicle-treated rats (Fig. 6A). Thus, the observation
that a mixed CRH1/CRH2
(hCRH) antagonist prevented radioligand binding in the LS but a
selective CRH1 antagonist did not suggests that
the CRH receptor to which the infused hCRH is binding is of the
CRH2 subtype.
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Figure 6.
Ex vivo autoradiography for CRH
receptors after intra-lateral septum infusion of CRH receptor
antagonists. Autoradiograms show representative coronal sections
through the lateral septum from rats that had received vehicle
(A), 1.0 µg/side NBI27914
(B), or 100 ng/side -helical CRH
(C) into lateral septum before killing. Note the
high level of radioligand binding to CRH receptors in the lateral
septum in sections from rats that were treated with either vehicle or
NBI27914 and, in contrast, the absence of binding in sections from rats
that had received -helical CRH. Arrows indicate the
location of [125I]sauvagine binding within the lateral
septum.
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Figure 7 demonstrates the pattern of
binding of [125I]sauvagine to CRH
receptors in representative sections from rats treated with vehicle in
the LS. These were consecutive sections from the same animal. Sections
were incubated with
[125I]Tyr0
sauvagine in the absence of antagonists (Fig. 7A) or the
presence of 1 µM NBI27914 (Fig. 7B)
or 1 µM
D-Phe-CRH(12-41) (Fig. 7C). In the presence of 1 µM
NBI27914, [125I]sauvagine binding was
inhibited from CRH1 receptors in the cortex, with
no observable inhibition of binding to CRH2
receptors in the LS (arrows). In contrast, in the presence
of D-Phe-CRH(12-41), the
nonselective CRH1/CRH2
antagonist, radioligand binding was virtually abolished in both the
cortical and septal areas (Fig. 7C). Note the faint
intensity of the radioactive signal in Figure 7C; areas that
appear light or white indicate that little or no [125I]Tyr0
sauvagine bound to this section, suggesting that incubation with the
unlabeled D-Phe-CRH(12-41)
caused nearly all CRH receptors to become occupied and therefore
unavailable for
[125I]Tyr0
sauvagine binding. These results confirm that the identity of CRH
receptors within the LS is of the CRH2 but not
the CRH1 receptor subtype. These data are
consistent with previous reports indicating that there is a high
density of CRH2 receptors in the LS, but that
this structure is devoid of CRH1 receptors
(Chalmers et al., 1995; Primus et al., 1997). Taken together, these
findings indicate that the behavioral actions of intra-LS hCRH
infusion are likely mediated through CRH2 and
not CRH1 receptors.
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Figure 7.
In vitro autoradiography for CRH
receptors in the lateral septum. Autoradiograms show representative
coronal sections through the lateral septum from rats that had received
intra-lateral septum infusion of vehicle before killing.
A, Total binding of the
CRH1/CRH2 radioligand
[125I]sauvagine. B,
[125I]Sauvagine binding in the presence of excess
unlabeled NBI27914. C,
[125I]Sauvagine binding in the presence of excess
unlabeled CRH1/CRH2 receptor antagonist
D-Phe-CRH(12-41). Note the high intensity of
signal in the lateral septum in sections that were incubated with
NBI27914 but the absence of signal in sections incubated with the
CRH1/CRH2 antagonist
D-Phe-CRH(12-41). The light
appearance of C indicates that
[125I]sauvagine binding was virtually absent in
this section. Arrows indicate the location of
[125I]sauvagine binding within the lateral
septum.
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DISCUSSION |
In the present studies, microinfusion of the
CRH1/CRH2 receptor
antagonist hCRH into the LS decreased shock-induced freezing, a
measure of stress-induced behavior, without affecting general activity
levels or pain sensitivity. The same dose that decreased freezing on
intra-LS infusion had no effect after infusion into neighboring regions
such as the LV or NDB. An identical pattern of results was obtained
with the more potent
CRH1/CRH2 receptor antagonist D-Phe-CRH(12-41). In
contrast, a highly selective CRH1 receptor
antagonist that reduced freezing after intra-CeA infusion failed to
affect freezing when infused into the LS. Moreover, intra-LS
administration of the CRH1-selective antagonist
did not affect ex vivo binding of
[125I]sauvagine to CRH receptors in the
LS. Infusion of hCRH into this region, however, completely prevented
[125I]sauvagine binding in the LS.
In vitro studies demonstrated that [125I]sauvagine binding in the LS was
not affected after incubating sections with excess
CRH1 antagonist but was abolished after
incubating sections with excess
CRH1/CRH2 receptor
antagonist. These results confirm that on intra-LS infusion, the
actions of hCRH on freezing can be attributed to the interaction of
this antagonist with the CRH2 receptor. Taken
together, these findings indicate that blockade of
CRH2 but not CRH1
receptors within the LS decreases stress-induced behavior. To the best
of our knowledge, this is the first report to show that the acute and
selective antagonism of CRH2 receptors reduces
stress-induced defensive behavior.
The behavioral specificity of the CRH antagonist-induced reduction in
freezing is supported by the finding that locomotor activity
(irrespective of whether rats were previously habituated to the testing
chambers) and pain sensitivity (regardless of the postinfusion time
point for testing) were not affected by hCRH or
D-Phe-CRH(12-41). The finding that a
separate CRH receptor antagonist,
D-Phe-CRH(12-41), produced the exact
same behavioral profile as hCRH provides independent corroboration
for the notion that blockade of CRH receptors within the LS causes a
reduction specifically in stress-like behavior. CRH receptor blockade
caused an increase in the latency to begin freezing and a decrease in the duration of freezing at each time point of the test, indicating that this reduction in stress-induced freezing was long-lasting and
occurred throughout the entire extent of the test session. The finding
that a selective CRH1 antagonist blocked freezing when infused into the CeA but failed to alter freezing after intra-LS infusion provides novel and clear evidence (in conjunction with the
autoradiographic findings) that within the LS, it is the antagonism of
specifically the CRH2 receptor subtype that
reduces stress-induced behavior.
The present studies thus indicate that the LS is an important site for
the regulation of stress-induced freezing. The finding that 100 ng of
hCRH reduced freezing on intra-LS infusion but failed to alter this
behavior after intracerebroventricular administration is particularly
striking, given that the lateral ventricle borders the LS. Our previous
work indicates that 25 µg of this antagonist is required to reduce
shock-induced freezing via intracerebroventricular administration
(Kalin et al., 1988). Thus, the failure of 100 ng of hCRH to reduce
freezing after intracerebroventricular administration likely indicates
that hCRH-induced effects observed in LS-treated rats were highly
anatomically specific to the LS and did not arise from diffusion of the
antagonist into the ventricle or other brain regions. One interesting
feature of the hCRH dose-response profile was its inverted U shape
with regard to freezing; a middle dose but neither a low nor high dose
reduced this stress-induced behavior after infusion into the LS.
Although the specific mechanisms underlying this profile remain to be
determined, it should be noted that an identical dose-response profile
for hCRH on shock-induced freezing has been observed previously for
the CeA (Swiergiel et al., 1993).
The present findings extend previous reports indicating that hCRH
infusion into locus ceruleus or CeA decreases shock-induced freezing
behavior (Swiergiel et al., 1992, 1993). CRH receptor antagonism within
the CeA also prevents behaviors induced by a variety of other different
stressors (Heinrichs et al., 1992; Rassnick et al., 1993). The present
results further these findings by indicating that within the CeA, the
reduction of stress-induced behavior is likely mediated through
blockade of the CRH1 receptor subtype. Moreover,
the present findings suggest that decreases in stress-induced behaviors
after CRH1 receptor knock-out or systemic CRH1 receptor antagonist administration may
involve the CeA (Smith et al., 1998; Timpl et al., 1998; Contarino et
al., 1999; Okuyama et al., 1999; Habib et al., 2000). It is thus
possible that the recently described clinical efficacy of
CRH1 receptor antagonists in depression (Zobel et
al., 2000) involves the blockade of CRH1 receptors within the CeA. To the best of our knowledge, the present report is the first to identify the specific neuroanatomical substrates through which the different CRH receptor subtypes regulate defensive freezing.
It could be argued that the decreased freezing seen after intra-LS
hCRH infusion may result from diffusion of the drug into the medial
septum (MS), a structure devoid of CRH2
receptors but enriched in CRH1 receptors
(Chalmers et al., 1995). This possibility is unlikely, however, given
that intra-LS NBI27914 infusion (which could diffuse to the MS and
block CRH1 receptors) and intra-NDB hCRH
infusion (which would be equally likely to diffuse to the MS as would
intra-LS hCRH) failed to alter shock-induced freezing. Thus, it
seems that although both CRH1 and
CRH2 receptors regulate stress-induced
behaviors, they act through different brain regions. The present
findings are in agreement with a previous report in which intra-LS
administration of a putative CRH2-preferring antagonist [anti-sauvagine-30 (AS30)] was found to reverse stress- or
CRH-induced decreases in open arm entries in an elevated plus- maze in
mice (Ruhmann et al., 1998; Radulovic et al., 1999). High doses of AS30
have also been reported to decrease freezing behavior after
intracerebroventricular infusion (Takahashi et al., 2001), further
corroborating the findings of the present study, yet there are
conflicting reports about the CRH2 selectivity of
AS30 (Ruhmann et al., 1998; Higelin et al., 2001). The present studies
therefore clarify these previous behavioral findings by systematically
demonstrating that reductions in stress-induced behavior caused by CRH
antagonist administration into the LS are attributable to the
CRH2 and not the CRH1
receptor. Furthermore, the present findings indicate that these two
receptor subtypes may regulate stress-induced behavior through
different anatomical sites.
In contrast to the findings with acute CRH2
receptor antagonism, CRH2 knock-out mice
exhibit higher levels of stress-induced behaviors than wild-type
controls, although this profile is not consistent across behavioral
paradigms or across different laboratories (Bale et al., 2000; Coste et
al., 2000; Kishimoto et al., 2000). A major difficulty in interpreting
the results of constitutive gene knock-out studies is that the animal
matures without the gene of interest and can develop compensatory
alterations that contribute to the final behavioral phenotype
(Picciotto and Wickman, 1998; Gingrich and Hen, 2000). Thus, the
stress-like behavioral phenotype observed in the
CRH2 knock-out mice might not derive directly
from the absence of the CRH2 receptor but
rather may be caused through indirect compensatory alterations in the CRH and other systems. Increased gene expression of CRH (in the CeA)
and the CRH-like ligand urocortin has been reported in
CRH2 knock-out mice (Bale et al., 2000; Coste
et al., 2000). Urocortin decreases feeding and increases stress-like
responding in certain approach-avoidance-based behavioral tests (Spina
et al., 1996; Moreau et al., 1997; Jones et al., 1998; Cullen et al.,
2001). Likewise, the present findings indicate that stimulation of
CRH1 receptors in the CeA would increase
stress-like responses. The very novel discovery of
CRH2-selective members of the CRH family of
endogenous ligands (urocortin II, urocortin III, and stresscopin) offers an even more complex picture of the potential compensatory effects within this system (Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al., 2001). Thus, increases in these ligands in response to constitutive CRH2 gene deletion
could underlie the putative stress-like phenotype of the
CRH2 knock-out mice. Future studies using
novel gene-targeting approaches such as virally mediated gene transfer
or inducible knock-out techniques (Stark et al., 1998; Simonato et al.,
2000) will aid in clarifying the contributions of developmental factors
to the phenotype produced by CRH2 receptor
knock-out.
One previous report indicates that subchronic exposure to
benzodiazepines decreased CRH1 receptor levels
but increased CRH2 receptor levels in rat
brain, indicating that CRH1 and
CRH2 receptors may act in an opposing manner
(Skelton et al., 2000). It has been suggested that
CRH2 receptors are involved in "coping"
with stress rather than in the direct behavioral response to stress,
which is hypothesized to be mediated by CRH1
receptors (Liebsch et al., 1999). The present results, however,
indicate that CRH1 and
CRH2 receptors play similar or parallel roles in the regulation of stress-related behavior, and that this regulation may occur through different brain regions. It may be that the CRH
receptor subtypes are differentially affected by long-term drug
treatment but that their roles in mediating acute stress-induced behavioral effects are similar.
The present finding that acute blockade of
CRH2 receptors within the LS decreases
stress-induced freezing is not surprising if one considers the
electrophysiological role of CRH in the LS and the role of the LS in
regulating defensive behaviors. Single-unit recordings from the LS
indicate that within this structure, CRH is inhibitory (Siggins et al.,
1985); the LS is thought to provide a tonic inhibition over the
expression of defensive behaviors (Albert and Walsh, 1982, 1984;
Graeff, 1994). Thus, stimulation of CRH receptors within the LS
(perhaps through stress-induced CRH or urocortin release) might be
expected to disinhibit defensive behaviors such as freezing. Antagonism
of these receptors would prevent this disinhibition and would maintain
the tonic inhibition over defensive behaviors, thereby reducing the
expression of freezing.
The precise circuitry through which CRH1 and
CRH2 receptors interact to control
stress-related behaviors remains to be determined. The LS is important
in modulating defensive behaviors (Graeff, 1994), and the CeA regulates
the expression of fear-related responses (Davis and Shi, 1999). The
present studies indicate that antagonism of
CRH2 receptors within the LS or
CRH1 receptors within the CeA decreases the
expression of fear-induced defensive behavior. In rats, both the LS and
CeA receive CRH- or urocortin-containing terminals (Swanson et al.,
1983; Sakanaka et al., 1988; Kozicz et al., 1998; Bittencourt et al.,
1999). In addition, anatomical tract-tracing studies indicate
reciprocal connections between these two structures (Risold and
Swanson, 1997) and also indicate that both structures project to the
periaqueductal gray (PAG; Rizvi et al., 1991; Risold and Swanson,
1997), a midbrain structure that, when stimulated, elicits defensive
behaviors such as freezing (Bandler et al., 1985; Behbehani, 1995).
Because stress-induced freezing is a defensive behavior necessary for
an organism's survival, it may be adaptive to have parallel systems
involving the two CRH receptor subtypes that can either independently
or interactively modulate the expression of this critical form of
behavioral inhibition. It is possible that information from the
CRH2-LS system and the
CRH1-CeA system converges within the PAG to
regulate the expression of freezing. Although further studies must be
performed to test this hypothesis, the present findings provide a
valuable starting point for identifying the specific role of CRH
receptor subtypes in the neuroanatomical circuitry subserving
stress-related defensive behaviors.
Because increased CRH activity has been hypothesized to play a role in
mediating anxiety and depressive disorders, there has been much
emphasis on developing new antidepressant or anxiolytic drugs that
antagonize CRH receptors (McCarthy et al., 1999).
CRH1 receptor antagonists have shown some
preliminary success in an open-label clinical trial as a novel class of
antidepressants (Zobel et al., 2000). Anxiety disorders and depression
have been conceptualized as an aberrant manifestation of adaptive
stress- or fear-related defensive behaviors (Bakshi and Kalin, 2002). The present results indicate that acute antagonism of
CRH2 receptors reduces stress-induced
defensive behavior. This receptor subtype may thus play an important
role in mediating aberrant expressions of fear-related responses that
become dysregulated in stress-related disorders such as anxiety or
depression. Thus, in addition to the CRH1
receptor, attention should also be focused on the development of
nonpeptide CRH2 receptor antagonists, which
could be useful as therapeutic agents for stress-related psychopathology.
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FOOTNOTES |
Received May 23, 2001; revised Jan. 14, 2002; accepted Jan. 16, 2002.
This work was supported by National Institutes of Health (NIH)
Grant MH-40855 (N.H.K.), the HealthEmotions Research Institute, and
Meriter Hospital. V.P.B. was supported by NIH Grant MH-12360. We thank
Dr. Brian Baldo for thoughtful comments on the project and this manuscript.
Correspondence should be addressed to Dr. Vaishali P. Bakshi,
Department of Psychiatry, University of Wisconsin, 6001 Research Park
Boulevard, Madison, WI 53719. E-mail: vbakshi{at}facstaff.wisc.edu.
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TOP
ABSTRACT
INTRODUCTION
