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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
JPET 320:1023–1029, 2007
Vol. 320, No. 3
113357/3177200
Printed in U.S.A.
5-Hydroxytryptamine2C Receptor Contribution to mChlorophenylpiperazine and N-Methyl-␤-carboline-3carboxamide-Induced Anxiety-Like Behavior and Limbic
Brain Activation
Elizabeth A. Hackler, Greg H. Turner, Paul J. Gresch, Saikat Sengupta, Ariel Y. Deutch,
Malcolm J. Avison, John C. Gore, and Elaine Sanders-Bush
Departments of Pharmacology (E.A.H., P.J.G., A.Y.D., M.J.A., E.S.-B.), Psychiatry (A.Y.D., E.S.-B.), and the Vanderbilt Institute
of Imaging Science (G.H.T., S.S., M.J.A., J.C.G.), Vanderbilt University Medical Center, Nashville, Tennessee
ABSTRACT
Activation of 5-hydroxytryptamine2C (5-HT2C) receptors by the
5-HT2 receptor agonist m-chlorophenylpiperazine (m-CPP)
elicits anxiety in humans and anxiety-like behavior in animals.
We compared the effects of m-CPP with the anxiogenic
GABAA receptor inverse agonist N-methyl-␤-carboline-3-carboxamide (FG-7142) on both anxiety-like behavior and regional
brain activation using functional magnetic resonance imaging
(fMRI) in the rat. We also determined whether the selective
5-HT2C receptor antagonist SB 242084 [6-chloro-2,3-dihydro5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1Hindole-1-carboxyamide dihydrochloride] would blunt m-CPP or
FG-7142-induced neuronal activation. Both m-CPP (3 mg/kg
i.p.) and FG-7142 (10 mg/kg i.p.) elicited anxiety-like behavior
when measured in the social interaction test, and pretreatment
with SB 242084 (1 mg/kg i.p.) completely blocked the behav-
The 5-hydroxytryptamine 2C (5-HT2C) receptor has been
implicated in mood and anxiety disorders and is a target for
development of novel anxiolytic drugs (Wood, 2003). Selective
and nonselective 5-HT2C receptor antagonists reduce anxiety-like behavior in several animal models of anxiety (Stutzmann et al., 1991; Kennett et al., 1995; Griebel et al., 1997).
For example, SB 242084 [6-chloro-2,3-dihydro-5-methyl-N[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1This study was supported by National Institutes of Health Grants T32
GM07628, RO1 MH34007, and R01 EB02326 and a predoctoral fellowship
from the PhRMA foundation (to E.A.H.).
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.113357.
ioral effects of both anxiogenic drugs. Regional brain activation
in vivo in response to anxiogenic drug challenge was determined by blood oxygen level-dependent (BOLD) fMRI using a
powerful 9.4T magnet. Region of interest analyses revealed that
m-CPP and FG-7142 significantly increased BOLD signals in
brain regions that have been linked to anxiety, including the
amygdala, dorsal hippocampus, and medial hypothalamus.
These BOLD signal increases were blocked by pretreatment
with SB 242084. In contrast, injection of m-CPP and FG-7142
resulted in BOLD signal decreases in the medial prefrontal
cortex that were not blocked by SB 242084. In conclusion, the
brain activation signals produced by anxiogenic doses of both
m-CPP and FG-7142 are mediated at least partially by the
5-HT2C receptor, indicating that this receptor is a key component in anxiogenic neural circuitry.
carboxyamide dihydrochloride], a potent and selective
5-HT2C receptor antagonist, is anxiolytic in the social interaction test and the Geller-Seifter conflict test of anxiety (Kennett et al., 1997). Although 5-HT2C receptor antagonism is
anxiolytic, agonist-induced activation of 5-HT2C receptors is
anxiogenic. Activation of 5-HT2C receptors by 5-HT2 receptor
agonists, such as meta-chlorophenylpiperazine (m-CPP) and
6-chloro-2[1-piperazinyl] pyrazine (MK-212), elicits anxiety
in humans and anxiety-like behavior in animals (Charney et
al., 1987; Lowy and Meltzer, 1988; Kahn and Wetzler, 1991;
Gatch, 2003; de Mello Cruz et al., 2005). For example, m-CPP
administration to mice resulted in less time spent on the
open arms of an elevated plus maze (Benjamin et al., 1990),
and in rats, anxiety-like behavior has been reported in the
ABBREVIATIONS: 5-HT2C, 5-hydroxytryptamine2C; m-CPP, m-chlorophenylpiperazine; FG-7142, N-methyl-␤-carboline-3-carboxamide; SB
242084, 6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride; fMRI, functional
magnetic resonance imaging; BOLD, blood oxygen level-dependent; ROI, region of interest; mPFC, medial prefrontal cortex; ANOVA, analysis of
variance; MK-212, 6-chloro-2[1-piperazinyl] pyrazine; AUC, area under the curve; SB 243213, 5-methyl-1-({2-[(2-methyl-3-pyridyl)oxy]-5pyridyl}carbamoyl)-6-trifluoromethylindoline.
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Received September 6, 2006; accepted November 28, 2006
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Hackler et al.
Materials and Methods
Drugs. m-CPP (Tocris Bioscience, Ellisville, MO) was administered at a dose of 3 mg/kg i.p. FG-7142 complexed with hydroxypropyl-␤-cyclodextrin (Sigma-Aldrich, St Louis, MO) was injected at a
dose of 10 mg/kg i.p. SB 242084, a generous gift from SmithKline
Beecham (Harlow, UK), was administered at a dose of 1 mg/kg i.p.
m-CPP and FG-7142 were dissolved in saline (0.9%). SB 242084 was
sonicated into a suspension using 10% Tween 80 in saline.
Social Interaction Paradigm. Anxiety-like behavior of rats was
measured using a modified social interaction paradigm (Varlinskaya
and Spear, 2002). The social interaction paradigm is a test that
measures the total social interactions of a drug-treated rat paired
with a control rat that is unfamiliar to the drug-treated rat. Six
groups, each consisting of six adult male Sprague-Dawley rats (250 –
300 g), were used: vehicle, m-CPP, FG-7142, SB 242084, SB 242084/
m-CPP (pretreatment 10 min before m-CPP administration), or SB
242084/FG-7142 (pretreatment 10 min before FG-7142 administration). The day before testing, each of the manipulated (drug-treated)
rats was familiarized to the social interaction chamber for approximately 30 min under low-light conditions. On the day of testing, rats
were injected with drug or vehicle and, 30 min later, placed into a
45 ⫻ 30 ⫻ 20-cm clear acrylic chamber bisected by a clear central
partition containing a 9 ⫻ 7-cm opening. Each drug-treated familiarized rat was paired with a nonfamiliarized control rat. Behavioral
testing was conducted during the dark cycle between 7:00 PM and
11:00 PM under low-light conditions. Behavior was recorded by
video/camcorder for future scoring by observers blind to the treatment conditions. The total social interactions of the drug-treated rat
were quantified by counting the frequency of the following: social
investigation (sniffing), contact behavior (crawling over and/or
crawling under), and play-fighting during a 10-min test session. The
data were analyzed using one-way ANOVA with Tukey’s multiple
comparison post hoc test.
Animal Surgery and Preparation. All procedures were approved by the Vanderbilt University Medical Center Institutional
Animal Care and Use Committees and were conducted according to
the NIH Guide for the Care and Use of Laboratory Animals. Adult
male Sprague-Dawley rats (n ⫽ 6 per group) were housed on a
12:12-h light/dark cycle, with lights off at 6:00 PM. On the day of
surgery, rats were anesthetized with 2% isoflurane and tracheotomized (14-gauge, 3 cm long; Johnson & Johnson, New Brunswick,
NJ). The femoral artery was cannulated (P50 polyethylene tubing;
Braintree Scientific, Braintree, MA) for measurement of blood gases
(Scanley et al., 2001). A P50 catheter was inserted into the intraperitoneal cavity for drug administration. Anesthetized rats were
connected to a mechanical ventilator (Kent Scientific, Litchfield, CT)
delivering a 33:67% O2/N2O gas mixture at a respiratory rate of 48 to
52 breaths/min and inspiration pressure of 14 to 18 cm of H2O. The
rat’s head was immobilized in a custom-built Plexiglas stereotaxic
apparatus (Ken Wilkins, Vanderbilt University Institute of Imaging
Science, TN) to reduce the likelihood of motion artifacts. Subdermal
electrocardiograph electrodes were implanted into the forepaws to
monitor heart rate, and a rectal probe was inserted to monitor body
temperature. A respiration pillow sensor (SA Instruments, Inc.,
Stony Brook, NY) was positioned underneath the abdomen of each
rat. Heart rate, respiration rate, and temperature were measured
using a magnetic resonance-compatible monitoring and gating system, which controlled a thermocoupled heating unit to warm the
animal while inside the scanner (SAM-PC; SA Instruments, Encinitas, CA).
Ten minutes before the start of each scan, rats were paralyzed
with pancuronium bromide (2 mg/kg i.p.), and isoflurane was reduced to 1% for the duration of the experiment. To monitor blood
gases (oxygen and carbon dioxide) and pH, 300 ␮l of blood was
withdrawn and analyzed both before and after each scan. The mean
(⫾ S.E.M.) prescan values for all 36 rats used in the fMRI studies
were pH ⫽ 7.46 ⫾ 0.02, pCO2 ⫽ 36.7 ⫾ 1.57 mm Hg, and pO2 ⫽
158 ⫾ 4.18 mm Hg. The postscan values were pH ⫽ 7.38 ⫾ 0.02,
pCO2 ⫽ 48.7 ⫾ 3.17 mm Hg, and pO2 ⫽ 138 ⫾ 6.40 mm Hg.
Statistical analyses (one-way ANOVA with Tukey’s multiple comparison post hoc test) indicated that there was no significant difference between drug treatment groups for either the prescan or the
postscan period.
Magnetic Resonance Imaging. A 20-mm dual transmit-receive
radio frequency surface coil (Varian Instruments, Palo Alto, CA) was
secured to the dorsal surface of the rat’s head. The anesthetized rat
was placed in a 9.4 Tesla magnet with a 21-cm bore. The MRI system
was controlled by a Varian Inova console running VnmrJ 6.1D software (Varian). Before drug administration, a full set of high-resolution fast spin-echo structural magnetic resonance images was collected to guide placement of the image slices for fMRI (TR ⫽ 4000 ms,
echo spacing ⫽ 10 ms, average ⫽ 2, matrix ⫽ 256 ⫻ 128, thickness ⫽
2 mm) and for subsequent coregistration of the fMRI data sets. Drugs
(vehicle, m-CPP, FG-7142, or SB 242084 alone or in combination
with m-CPP or FG-7142) were administered via the i.p. catheter
after a 20-min baseline period. For antagonist pretreatment experi-
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light-dark box, the open-field test, and the social interaction
test (Bilkei-Gorzo et al., 1998; Bagdy et al., 2001; Martin et
al., 2002; Campbell and Merchant, 2003). The 5-HT2C receptor antagonist SB 242084 blocks the anxiogenic effects of
m-CPP in rats (Bagdy et al., 2001; Campbell and Merchant,
2003). In addition, indirect activation of 5-HT2C receptors
contributes to the anxiogenic effects following acute treatment with selective serotonin reuptake inhibitors (Bagdy et
al., 2001). Taken together, these studies suggest that 5-HT2C
receptor activation is an important component of anxiogenesis.
Recent advances in functional magnetic resonance imaging
(fMRI) technology allow the study of pharmacological-induced regional activation in the living rat brain (Chen et al.,
2005; Ireland et al., 2005; Steward et al., 2005). Blood oxygen
level-dependent (BOLD) fMRI is a method that has distinct
advantages over other in vivo imaging techniques. A prominent advantage of fMRI is the ability to collect temporal data
of a drug’s effect in different brain regions in a single animal.
BOLD-induced brain activation has been used to examine the
effects of pharmacological agents, such as amphetamine
(Dixon et al., 2005), cocaine (Febo et al., 2005), and citalopram (Steward et al., 2005).
m-CPP has been advanced as a useful pharmacological
probe for fMRI studies of regional activation induced by
5-HT2C receptor activation (Houston et al., 2001; Anderson et
al., 2002). However, m-CPP has significant affinities for several serotonin receptors in addition to the 5-HT2C receptor,
clouding the interpretation of in vivo studies (Hamik and
Peroutka, 1989; Campbell and Merchant, 2003). In the current report, we used SB 242084, a selective 5-HT2C receptor
antagonist (Kennett et al., 1997), to evaluate the contribution
of the 5-HT2C receptor to m-CPP-induced anxiety-like behavior and regional BOLD signal changes within the amygdala,
hippocampus, hypothalamus, and the medial prefrontal
cortex. To determine whether a 5-HT2C receptor-dependent
process contributes to drug-induced anxiety-like behavior,
in general, animals were injected with the GABAA receptor inverse agonist N-methyl-␤-carboline-3-carboxamide
(FG-7142), a potent anxiogenic agent (Dorow et al., 1983).
The pattern of regional activation produced by FG-7142 was
compared with that observed in animals that were pretreated with the 5-HT2C receptor antagonist SB 242084.
5-HT2C Receptor Contribution to Anxiety
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Results
m-CPP and FG-7142-Induced Behavioral Changes
Are Blocked by SB 242084. Acute administration of both
m-CPP and FG-7142 significantly reduced the total amount
of time spent in social interaction compared with vehicleinjected animals (Fig. 1A; ANOVA: F(2,15) ⫽ 130.9, p ⬍
0.0001). The doses used were chosen based upon previous
experiments performed using m-CPP and FG-7142 in the
social interaction test as a behavioral measure for anxietylike behavior in rats (Short and Maier, 1993; Rex et al.,
2004). Pretreatment with SB 242084 significantly blocked
the anxiogenic effects of m-CPP in the social interaction test
(SB 242084/m-CPP; Fig. 1B; ANOVA: F(3,20) ⫽ 27.23, p ⬍
0.0001). The dose of SB 242084 was chosen based upon previous rodent behavioral experiments (Martin et al., 2002;
Campbell and Merchant, 2003; Knapp et al., 2004). Post hoc
analysis revealed that the m-CPP treatment group was significantly different from vehicle, SB 242084/m-CPP, and SB
242084 alone. In addition, pretreatment with SB 242084 10
min before FG-7142 injection completely blocked the anxiogenic effects of FG-7142 as indicated in Fig. 1C (SB 242084/
FG-7142; ANOVA: F(3,20) ⫽ 19.75, p ⬍ 0.0001). Post hoc
analysis revealed that the FG-7142 treatment group was
significantly different from vehicle, SB 242084/FG-7142, and
SB 242084 alone.
m-CPP-Induced BOLD Signal Changes in Limbic
Brain Regions. m-CPP administration increased BOLD signals in limbic brain regions associated with anxiety: the
amygdala, hippocampus, and hypothalamus (Table 1). Temporal changes in the BOLD signal following m-CPP injection
are illustrated in Fig. 2A, which shows activation maps in a
series of 5-min increments for the entire 40-min postinjection
period. The maximal intensity of the BOLD signal increases
was observed approximately 15 min postinjection.
Fig. 1. SB 242084 pretreatment blocks anxiety-like behavior resulting
from m-CPP or FG-7142. A, i.p. injections of vehicle, m-CPP (3 mg/kg), or
FG-7142 (10 mg/kg; FG) were administered to rats 30 min before behavioral testing (n ⫽ 6 per treatment group). Both drugs produced a marked
decrease in social interactions. ⴱⴱⴱ indicates p ⬍ 0.001 compared with
vehicle. B, m-CPP significantly decreased social interactions compared
with vehicle. Pretreatment (10 min before m-CPP) with SB 242084 (1
mg/kg) blocked the anxiogenic effects of m-CPP (SB/m). m-CPP-induced
reduction in social interactions was significantly different from vehicle
(indicated by ⴱ, p ⬍ 0.001), SB/m (indicated by †, p ⬍ 0.001), and SB
242084 alone (SB, indicated by ‡, p ⬍ 0.001). SB alone and SB/m were not
different from vehicle. C, pretreatment (10 min before FG-7142) with SB
(1 mg/kg) blocked the anxiogenic effects of FG-7142 (SB/FG). FG-7142induced reduction in social interactions was significantly different from
vehicle (indicated by ⴱ, p ⬍ 0.001), SB/FG (indicated by †, p ⬍ 0.001), and
SB alone (indicated by ‡, p ⬍ 0.001). SB alone and SB/FG are not different
from vehicle.
Injection of m-CPP resulted in activation of the amygdala,
with increases in the BOLD signal within 1 min after m-CPP
challenge that continued to rise over the next 15 min until a
plateau was reached (Fig. 3A). The ability of m-CPP to activate the amygdala was completely blocked by pretreatment
with SB 242084 (Fig. 3B; ANOVA: F(3,20) ⫽ 15.40, p ⬍
0.0001). BOLD signal increases induced by m-CPP are significantly different from those observed from vehicle, SB
242084/m-CPP, and SB 242084 alone.
m-CPP also elicited BOLD signal increases in the hippocampus and hypothalamus that were blocked by pretreatment with SB 242084. BOLD signal increases within the
hippocampus (Fig. 4A) and hypothalamus (Fig. 5A) were
observed 1 min after m-CPP injection and plateaued within
15 min. Statistical analyses demonstrated that m-CPP-induced BOLD signal increases in dorsal hippocampus were
blocked by pretreatment with SB 242084 (Fig. 4B; ANOVA:
F(3,20) ⫽ 7.680, p ⫽ 0.0013), as were BOLD signal increases in
the medial nuclei of the hypothalamus (Fig. 5B; ANOVA:
F(3,20) ⫽ 17.22, p ⬍ 0.0001) and the paraventricular nuclei of
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ments, SB 242084 was injected after the baseline period, and m-CPP
or FG-7142 was injected 10 min later. Functional images were acquired using a fast gradient-echo sequence [TR (repetition time) ⫽
200 ms, TE (echo time) ⫽ 12 ms, ␣ ⫽ 20°, NEX (number of excitations) ⫽ 2, 64 ⫻ 64 ⫻ 8 matrix, 30 ⫻ 30 ⫻ 2 mm FOV (field of view)].
Functional images following drug administration were superimposed on anatomical images taken before drug injection to identify
regions of interest (ROI), i.e., areas that show significant changes in
the BOLD signal.
Data Analysis. All fMRI data analyses were performed using
MATLAB software (version 7.0.4; MathWorks, Inc., Natick, MA).
Activation maps were generated for each data set using a sliding t
test, comparing the time-averaged data over a period of 300 s (5 min)
from the postinjection period to a comparable preinjection period.
For each animal, the effects of baseline drift and high-frequency
noise in the fMRI signal were removed by polynomial curve fitting
and low-pass data filtering, respectively. To assess changes in specific brain regions, ROI analyses were performed using a rat brain
atlas as a guide for structural identification (Paxinos and Watson,
1982). ROIs were drawn to include the dorsal hippocampus, the
amygdaloid complex, the hypothalamus (at the level of medial and
paraventricular nuclei), and the medial prefrontal cortex (mPFC).
For group analyses, ROI BOLD signal intensities (⌬S/So) from individual animals were averaged across animals. Area under the curve
(AUC) was calculated for the postinjection period of each animal in
each drug treatment group. The values from both the left and right
side of the brain were averaged, and data were analyzed using
one-way ANOVA with Tukey’s multiple comparison post hoc test.
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Hackler et al.
TABLE 1
Summary of m-CPP and FG-7142-induced BOLD signal changes
The symbols (⫹) or (⫺) in the 2nd and 3rd column indicate m-CPP and FG-7142-induced BOLD signal increases or decreases, respectively, relative to vehicle (p-values listed
for both m-CPP and FG-7142). Yes (Y) or No (N) in the 4th and 5th column indicates whether or not SB 242084 pretreatment blocks m-CPP and FG-7142-induced BOLD
signal changes in the ROIs measured.
Region of Interest
Amygdala
Dorsal hippocampus
Medial nuclei of hypothalamus
Paraventricular nuclei of hypothalamus
Medial prefrontal cortex
m-CPP-Induced BOLD
Signal Changes
⫹,
⫹,
⫹,
⫹,
⫺,
P
P
P
P
P
⬍
⬍
⬍
⬍
⬍
FG-7142-Induced BOLD
Signal Changes
0.001
0.05
0.001
0.001
0.05
⫹,
⫹,
⫹,
⫹,
⫺,
P
P
P
P
P
⬍
⬍
⬍
⬍
⬍
0.001
0.01
0.01
0.001
0.01
SB 242084 Pretreatment
Blocks m-CPP-Induced BOLD
Signal Changes
SB 242084 Pretreatment
Blocks FG-7142-Induced
BOLD Signal Changes
Y
Y
Y
Y
N
Y
Y
Y
N
N
Fig. 4. m-CPP-induced BOLD signal increases in the hippocampus are
blocked by SB 242084. A, averaged BOLD signal changes (⌬S/So, n ⫽ 6
per treatment group) for vehicle (⽧), m-CPP (3 mg/kg, f), SB 242084 (1
mg/kg) pretreatment before m-CPP (3 mg/kg, SB/m, F), and SB 242084
alone (1 mg/kg, SB, Œ). The time of drug injection is 0 on the x-axis. B,
AUC analysis of ROI data from part A. m-CPP-induced BOLD signal
changes were significantly different from vehicle (indicated by ⴱ, p ⬍
0.05), SB/m (indicated by †, p ⬍ 0.001), and SB alone (indicated by ‡, p ⬍
0.05).
Fig. 3. m-CPP-induced BOLD signal increases in the amygdala are
blocked by SB 242084. A, averaged BOLD signal changes (⌬S/So, n ⫽ 6
per treatment group) for vehicle (⽧), m-CPP (3 mg/kg, f), SB 242084 (1
mg/kg) pretreatment before m-CPP (3 mg/kg, SB/m, F), and SB 242084
alone (1 mg/kg, SB, Œ). The time of drug injection is 0 on the x-axis. B,
AUC analysis of ROI data from part A. m-CPP-induced BOLD signal
changes were significantly different from vehicle (indicated by ⴱ, p ⬍
0.001), SB/m (indicated by †, p ⬍ 0.001), and SB alone (indicated by ‡, p ⬍
0.001).
the hypothalamus (data not shown; ANOVA: F(3,20) ⫽ 28.99,
p ⬍ 0.0001). m-CPP-induced BOLD signals are significantly
different from those of vehicle, SB 242084/m-CPP, and SB
242084 alone in both areas. Although the combination of SB
242084/m-CPP appeared to produce a decrease in the BOLD
signal (Figs. 4 and 5), statistical analysis revealed that this
effect was not different from vehicle or SB 242084 alone.
In contrast to the other anxiety-related sites, m-CPP
caused BOLD signal decreases in the mPFC, which were not
blocked by pretreatment with SB 242084 (data not shown;
ANOVA: F(3,20) ⫽ 5.923, p ⫽ 0.0046). Although vehicle, mCPP, and SB 242084 alone were significantly different from
each other, there was no significant difference between the
m-CPP and SB 242084/m-CPP treatment conditions.
FG-7142-Induced BOLD Signal Changes in Limbic
Brain Regions. FG-7142 administration resulted in BOLD
signal increases in limbic brain regions associated with anxiety: the amygdala, hippocampus, and hypothalamus (Table
1). Temporal analyses of BOLD signal increases following
FG-7142 injection are illustrated as activation maps in a
series of 5-min increments for the 40-min postinjection period
(Fig. 6). The intensity of the BOLD signal increases remained
near maximum throughout the 40-min scan, indicating a
long duration of action (Fig. 6). Like m-CPP, injection of
FG-7142 generated BOLD signal increases in the amygdala,
hippocampus, and medial hypothalamus. The BOLD signal
increases observed 15 min after FG-7142 injection appeared
less intense than those for the same time point following
m-CPP injection, with the exception of the hippocampal area,
where FG-7142-induced BOLD signal increases were more
robust.
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Fig. 2. m-CPP-induced BOLD signal increases in rat brain. A, temporal
activation maps following m-CPP injection. Representative activation
maps showing BOLD signal increases from 5 to 40 min following m-CPP
injection. A p-value gradient on the right illustrates color-coordinated
levels of BOLD signal significance. B, anatomical section contains the
following regions of interest: amygdala (white boxes), hippocampus (blue
box), medial hypothalamus (red box).
5-HT2C Receptor Contribution to Anxiety
Fig. 7. FG-7142-induced BOLD signal increases in the amygdala are
blocked by SB 242084. A, averaged BOLD signal changes (⌬S/So, n ⫽ 6
per treatment group) for vehicle (⽧), FG-7142 (10 mg/kg, FG, f), SB
242084 (1 mg/kg) pretreatment before FG-7142 (10 mg/kg, SB/FG, F),
and SB 242084 alone (1 mg/kg, SB, Œ). The time of drug injection is 0 on
the x-axis. B, AUC analysis of ROI data from part A. FG-7142-induced
BOLD signal changes were significantly different from vehicle (indicated
by ⴱ, p ⬍ 0.001), SB/FG (indicated by †, p ⬍ 0.001), and SB alone
(indicated by ‡, p ⬍ 0.001).
Fig. 6. Temporal activation maps following FG-7142 injection. Representative activation maps showing BOLD signal increases from 5 to 40 min
following FG-7142 injection. A p-value gradient on the right illustrates
color-coordinated levels of BOLD signal significance.
ROI analyses of amygdala following injection of vehicle,
FG-7142, SB 242084/FG-7142, or SB 242084 alone are shown
in Fig. 7. BOLD signal increases were observed within 1 min
following drug injection (Fig. 7A). Unlike m-CPP, BOLD signal increases following FG-7142 injection did not plateau
during the 40-min postinjection period, and this was observed in all of the ROIs examined. Longer scans were performed with FG-7142 in an additional three rats, and the
BOLD signal increases did not plateau within a 60-min
postinjection period (data not shown), indicating different
pharmacokinetic profiles for m-CPP and FG-7142. Statistical
analysis of amygdala data demonstrates that the FG-7142
BOLD signal increases were blocked by pretreatment with
SB 242084 (Fig. 7B; ANOVA: F(3,20) ⫽ 19.63, p ⬍ 0.0001).
FG-7142 is significantly different from vehicle, SB 242084/
FG-7142, and SB 242084 alone. SB 242084/FG-7142 appeared to decrease the BOLD signal; however, this was not
significantly different from vehicle or SB 242084 alone. FG7142 generated strong BOLD signal increases within the
hippocampus (Fig. 8A) and medial hypothalamus (Fig. 9A)
that were evident within 1 min. Statistical analysis demonstrated that FG-7142-induced BOLD signal increases in hippocampus were blocked by pretreatment with SB 242084
(Fig. 8B; ANOVA: F(3,20) ⫽ 9.678, p ⫽ 0.0004), as were BOLD
Fig. 8. FG-7142-induced BOLD signal increases in the hippocampus are
blocked by SB 242084. A, averaged percent BOLD signal changes (⌬S/So,
n ⫽ 6 per treatment group) for vehicle (⽧), FG-7142 (10 mg/kg, FG, f),
SB 242084 (1 mg/kg) pretreatment before FG-7142 (10 mg/kg, SB/FG, F),
and SB 242084 alone (1 mg/kg, SB, Œ). The time of drug injection is 0 on
the x-axis. B, AUC analysis of ROI data from part A. FG-7142-induced
BOLD signal changes were significantly different from vehicle (indicated
by ⴱ, p ⬍ 0.01), SB/FG (indicated by †, p ⬍ 0.001), and SB alone (indicated
by ‡, p ⬍ 0.01).
signal increases in the medial nuclei of the hypothalamus
(Fig. 9B; ANOVA: F(3,20) ⫽ 11.87, p ⫽ 0.0001), but not in the
paraventricular nuclei of hypothalamus (data not shown).
FG-7142-induced BOLD signal increases in both hippocampus and hypothalamus differed from vehicle, SB 242084/FG7142, and SB 242084 alone. Although SB 242084/FG-7142
and SB 242084 appeared to decrease the BOLD signal in
hypothalamus, these were not significantly different from
vehicle or each other.
As with m-CPP, BOLD signal decreases were observed in
the mPFC following injection of FG-7142 (data not shown;
ANOVA: F(3,20) ⫽ 4.563, p ⫽ 0.0011). Although vehicle, mCPP, and SB 242084 alone were significantly different from
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Fig. 5. m-CPP-induced BOLD signal increases in the hypothalamus are
blocked by SB 242084. A, averaged BOLD signal changes (⌬S/So, n ⫽ 6
per treatment group) for vehicle (⽧), m-CPP (3 mg/kg, f), SB 242084 (1
mg/kg) pretreatment before m-CPP (3 mg/kg, SB/m, F), and SB 242084
alone (1 mg/kg, SB, Œ) within the hypothalamus. The time of drug
injection is 0 on the x-axis. B, AUC analysis of ROI data from part A.
m-CPP-induced BOLD signal changes were significantly different from
vehicle (indicated by ⴱ, p ⬍ 0.001), SB/m (indicated by †, p ⬍ 0.001), and
SB alone (indicated by ‡, p ⬍ 0.001).
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Hackler et al.
each other, there was no significant difference between the
FG-7142 and SB 242084/FG-7142 treatment conditions in
the mPFC.
Discussion
The present results point to the 5-HT2C receptor as integral to the anxiety-like behavior and limbic brain activation
produced by two anxiogenic drugs with different mechanisms
of action: m-CPP and FG-7142. In fMRI studies, we focused
on brain regions that are key elements in a distributed brain
network subserving anxiety: amygdala, hippocampus, medial
hypothalamus, paraventricular nucleus, and the medial prefrontal cortex. This is the first study to characterize the
5-HT2C receptor contribution to m-CPP-induced BOLD signal changes. Because the 5-HT2C receptor has been suggested to modulate GABA neurotransmission (HuidobroToro et al., 1996; Feng et al., 2001; Serrats et al., 2005), we
also determined whether pharmacological blockade of the
5-HT2C receptor would modify BOLD signal changes induced
by another anxiogenic drug, FG-7142, which targets the
GABAA receptor.
In agreement with previous studies (Short and Maier,
1993; Rex et al., 2004), we found that m-CPP and FG-7142
were both anxiogenic in the social interaction test. In addition, we found that pretreatment with a selective 5-HT2C
receptor antagonist blocked the behavioral effects of m-CPP
and FG-7142. Although previous studies have reported that
SB 242084 blocks m-CPP-induced behaviors (Kennett et al.,
1997; Bagdy et al., 2001; Campbell and Merchant, 2003), our
observation that SB 242084 blocks FG-7142-elicited anxietylike behavior is novel and underscores the possible central
role for the 5-HT2C receptor in anxiety. Although our behavioral study did not reveal an anxiolytic effect of SB 242084,
the experimental conditions were optimized for demonstrating a reduction in social interaction (anxiogenic-like behavior), rather than an increase (anxiolytic-like behavior).
Both m-CPP and FG-7142 activated the amygdala, hip-
Downloaded from jpet.aspetjournals.org at ASPET Journals on May 6, 2017
Fig. 9. FG-7142-induced BOLD signal increases in the hypothalamus are
blocked by SB 242084. A, averaged percent BOLD signal changes (⌬S/So,
n ⫽ 6 per treatment group) for vehicle (⽧), FG-7142 (10 mg/kg, FG, f),
SB 242084 (1 mg/kg) pretreatment before FG-7142 (10 mg/kg, SB/FG, F),
and SB 242084 alone (1 mg/kg, SB, Œ) within the hypothalamus. The time
of drug injection is 0 on the x-axis. B, AUC analysis of ROI data from part
A. FG-7142-induced BOLD signal changes were significantly different
from vehicle (indicated by ⴱ, p ⬍ 0.01), SB/FG (indicated by †, p ⬍ 0.001),
and SB alone (indicated by ‡, p ⬍ 0.001).
pocampus, and the medial and paraventricular nuclei of the
hypothalamus, as measured by fMRI. The m-CPP-induced
BOLD signal increases are consistent with the recent report
by Stark et al. (2006). m-CPP-induced BOLD signal increases
in each area plateaued ⬃15 min after injection. An anxiolytic
dose of SB 242084 blocked BOLD signal increases generated
by m-CPP in each of the ROIs. Taken together, these data
suggest that m-CPP-induced activation of the amygdala, the
hippocampus, and the hypothalamus is mediated by the
5-HT2C receptor. FG-7142 produced a BOLD signal pattern
that was similar, but not identical, to that seen after m-CPP
challenge, with increases observed in the amygdala, hippocampus, and hypothalamus. However, unlike m-CPP, the
BOLD signal produced by FG-7142 did not plateau within
the 40-min period following drug administration, probably
reflecting a difference in pharmacokinetics between the two
anxiogenic agents. This is the first fMRI study to characterize FG-7142-induced BOLD signal changes, and in general,
the pattern of FG-7142-induced BOLD signal increases is
consistent with a previous c-Fos study (Singewald et al.,
2003). A key, novel finding is that SB 242084 pretreatment
blocked FG-7142-induced BOLD signal increases in all but
two of ROIs, suggesting that the 5-HT2C receptor is integral
to FG-7142-elicited activation of limbic brain regions that
subserve anxiety. Both m-CPP and FG-7142 elicited BOLD
signal decreases in the mPFC, suggesting that the BOLD
signal activation is region-specific and not an overall nonspecific effect. Although blockade of the BOLD activation with a
5-HT2C receptor-selective antagonist suggests that the
5-HT2C receptor is a key component of m-CPP and FG-7142induced brain activation, it would be important to use multiple 5-HT receptor antagonists to investigate the possible
role of additional 5-HT receptors.
Our studies were performed in rats lightly anesthetized
with isoflurane. Because movement-associated artifacts
would preclude any analysis of BOLD signal data (Hajnal et
al., 1994), it was not possible to perform these experiments in
the unanesthetized rat. It is likely that isoflurane dampened
brain metabolic activity. Both inhalation and injectable anesthetics have been shown to alter cerebral glucose metabolism (Dudley et al., 1982; Ori et al., 1986). Use of anesthesia
may also alter synaptic transmission, resulting in an overall
increase in inhibitory transmission throughout the brain
(Richards, 1995). Despite the potential confounds associated
with isoflurane, the increases that we observed in BOLD
signals are consistent with previous c-Fos studies reporting
anxiety-induced activation of the amygdala and other regions
(Singewald et al., 2003). Because heart rate and blood gases
remained stable throughout the fMRI-scanning period, regardless of the experimental treatment, it is unlikely that
changes in these parameters resulted in any BOLD signal
artifacts.
In summary, the findings of this study indicate that the
brain activation that accompanies a behavioral state of anxiety critically involves the 5-HT2C receptor. Consistent with
this conclusion, SB 243213, an inverse agonist at the 5-HT2C
receptor, is anxiolytic in rodent behavioral tests and blocks
anxiety-like behavior in ethanol-withdrawn rats (Knapp et
al., 2004; Overstreet et al., 2003). Interestingly, SB 243213
shows no evidence of tolerance or dependence (Kennett et al.,
1997; Wood et al., 2001). Because tolerance and withdrawal
symptoms are inherent problems with long-term benzodiaz-
5-HT2C Receptor Contribution to Anxiety
epine use (Bateson, 2002), 5-HT2C receptor antagonists may
prove to be superior anxiolytic agents. The current evidence
that a 5-HT2C receptor antagonist blocks the anxiety-like
behavior elicited by anxiogenic agents with different mechanisms of action provides additional evidence for 5-HT2C receptor antagonists as potential targets for treatment of
anxiety disorders.
Acknowledgments
We thank Dr. Jason M. Williams for helpful advice and technical
assistance. We also thank Jarrod True and Jennifer Begtrup of the
Vanderbilt University Institute of Imaging Science for technical
support.
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