Cortagine, a specific agonist of corticotropin-releasing factor subtype 1, is anxiogenic and antidepressive in the mouse model

Abstract

Two subtypes of the corticotropin-releasing factor (CRF) receptor, CRF(1) and CRF(2), differentially modulate brain functions such as anxiety and memory. To facilitate the analysis of their differential involvement, we developed a CRF(1)-specific peptidic agonist by synthesis of chimeric peptides derived from human/rat CRF, ovine CRF (oCRF), and sauvagine (Svg). High affinity to the CRF-binding protein was prevented by introduction of glutamic acid in the binding site of the ligand. The resulting chimeric peptide, [Glu(21),Ala(40)][Svg(1-12)]x[human/rat CRF(14-30)]x[Svg(30-40)], named cortagine, was analyzed pharmacologically in cell culture by using human embryonic kidney-293 cells transfected with cDNA coding for CRF(1) or CRF(2), in autoradiographic experiments, and in behavior experiments using male C57BL/6J mice for its modulatory action on anxiety- and depression-like behaviors with the elevated plus-maze test and the forced swim test (FST), respectively. We observed that cortagine was more selective than oCRF, frequently used as CRF(1)-specific agonist, in stimulating the transfected cells to release cAMP. Cortagine's specificity was demonstrated in autoradiographic experiments by its selective binding to CRF(1) of brain sections of the mouse. After injection into the brain ventricles, it enhanced anxiety-like behavior on the elevated plus-maze at a lower dose than oCRF. Whereas at high doses, oCRF injected into the lateral intermediate septum containing predominantly CRF(2) increased anxiety-like behavior as CRF(2)-specific agonists do, cortagine did not. In contrast to its anxiogenic actions, cortagine reduced significantly the immobility time in the FST as described for antidepressive drugs. Thus, cortagine combines anxiogenic properties with antidepressive effects in the FST.

Full text

Cortagine, a specific agonist of corticotropin-
releasing factor receptor subtype 1, is anxiogenic
and antidepressive in the mouse model
Hossein Tezval*, Olaf Jahn*, Cedomir Todorovic, Astrid Sasse, Klaus Eckart, and Joachim Spiess
†
Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, Hermann Rein Strasse 3, D-37075 Go¨ ttingen, Germany
Communicated by Michael G. Rosenfeld, University of California at San Diego, La Jolla, CA, May 6, 2004 (received for review December 16, 2003)
Two subtypes of the corticotropin-releasing factor (CRF) receptor,
CRF
1
and CRF
2
, differentially modulate brain functions such as
anxiety and memory. To facilitate the analysis of their differential
involvement, we developed a CRF
1
-specific peptidic agonist by
synthesis of chimeric peptides derived from human兾rat CRF, ovine
CRF (oCRF), and sauvagine (Svg). High affinity to the CRF-binding
protein was prevented by introduction of glutamic acid in the
binding site of the ligand. The resulting chimeric peptide,
[Glu
21
,Ala
40
][Svg
1–12
]ⴛ[human兾rat CRF
14–30
]ⴛ[Svg
30–40
], named
cortagine, was analyzed pharmacologically in cell culture by using
human embryonic kidney-293 cells transfected with cDNA coding
for CRF
1
or CRF
2
, in autoradiographic experiments, and in behavior
experiments using male C57BL兾6J mice for its modulatory action on
anxiety- and depression-like behaviors with the elevated plus-
maze test and the forced swim test (FST), respectively. We ob-
served that cortagine was more selective than oCRF, frequently
used as CRF
1
-specific agonist, in stimulating the transfected cells to
release cAMP. Cortagine’s specificity was demonstrated in auto-
radiographic experiments by its selective binding to CRF
1
of brain
sections of the mouse. After injection into the brain ventricles, it
enhanced anxiety-like behavior on the elevated plus-maze at a
lower dose than oCRF. Whereas at high doses, oCRF injected into
the lateral intermediate septum containing predominantly CRF
2
increased anxiety-like behavior as CRF
2
-specific agonists do,
cortagine did not. In contrast to its anxiogenic actions, cortagine
reduced significantly the immobility time in the FST as described
for antidepressive drugs. Thus, cortagine combines anxiogenic
properties with antidepressive effects in the FST.
C
orticotropin-releasing factor (CRF), a 41-residue peptide
hormone (1), is the major regulator of the hypothalamus–
pituitary–adrenal axis (2), modulates important brain functions
such as anxiety, learning, food intake, and locomotion, and is
involved in anxiety and mood disorders (3, 4). CRF acts through
two G protein-dependent CRF receptor subtypes, CRF
1
and
CRF
2
, derived from two separate genes (reviewed in ref. 5) and
binds with high affinity to a binding protein (CRFBP), which
serves as a pharmacologically significant reservoir of endoge-
nous CRF (6, 7). Several CRF
1
and CRF
2
splice variants have
been identified (5, 8). In rodents, only the splice variants CRF
1
␣
,
CRF
2
␣
, and CRF
2
␤
are of physiological relevance (5). CRF
1
␣
and
CRF
2
␣
are produced in brain tissue, whereas CRF
2
␤
is mainly
located in blood vessels (9).
CRF
1
and CRF
2
participate differentially in various biological
functions (3). Thus, activation of the hypothalamus–pituitary–
adrenal axis in response to a stressful stimulus is mainly achieved
through CRF
1
(10). It was demonstrated by gene deletion
experiments that anxiety-like behavior is enhanced predomi-
nantly through CRF
1
(11, 12), whereas it is reduced through
CRF
2
(10, 13). Pharmacological experiments discriminating
between regional actions of CRF
1
and CRF
2
revealed that CRF
2
of the lateral intermediate septum mediates stress-induced en-
hancement of anxiety-like behavior (14), whereas CRF
2
accessed
through the brain ventricles is anxiolytic (13). In contrast, CRF
1
accessed by CRF via the brain ventricles enhances anxiety-like
behavior (15). Several natural CRF-like peptides with different
CRF receptor subtype specificity have been characterized. Thus,
human兾rat CRF (h兾rCRF) and ovine CRF (oCRF) exhibit
preference for CRF
1
, whereas urocortin (Ucn)I (16) is a non-
selective ligand (3, 5). Recently, UcnII (17, 18) and UcnIII (18,
19) were identified on the basis of homology analysis of data
derived from genomic sequence databases and characterized as
highly CRF
2
-selective agonists. In contrast, no natural agonists
with a similar selectivity for CRF
1
have been identified to date,
and no peptidic CRF
1
-specific agonist has been synthesized.
At this time, oCRF is the agonist of choice for the selective
stimulation of CRF
1
in behavioral experiments, because it
preferentially binds to CRF
1
. oCRF’s binding constants for
CRF
1
and CRF
2
differ by two orders of magnitude (20, 21).
However, because of the high local agonist concentration that
often occurs when drugs are directly administered into the
animal brain, the use of oCRF for the stimulation of CRF
1
is
accompanied by unwanted CRF
2
-mediated side effects (C.T.
and J.S., unpublished data). In addition, displacement of endog-
enous ligand from CRFBP (6) by applied CRF agonists may
release CRF-like peptides and thus interfere with the desired
selective stimulation of CRF
1
. To facilitate further elucidation of
the physiological role of CRF
1
, we developed a selective and
potent CRF
1
agonist on the basis of a chimeric peptide strategy.
Methods
Peptide Synthesis. Peptides were synthesized, purified, and char-
acterized as described in refs. 22 and 23.
Binding Assays and Measurement of Intracellular cAMP Accumulation.
Crude membrane fractions were prepared from human embryonic
kidney-293 (HEK-293) cells stably transfected with cDNA encod-
ing either rat CRF
1
␣
(rCRF
1
) or mouse CRF
2
␤
(mCRF
2
␤
) (22). Rat
CRFBP (rCRFBP) was produced by HEK-293 cells stably trans-
fected with cDNA encoding rCRFBP C-terminally fused to a His
6
sequence (23). Ligand binding analysis was performed with scin-
tillation proximity assays (21, 24). For competition binding assays of
rCRF
1
and rCRFBP, [
125
I-Tyr
0
]h兾rCRF was used as radiolabeled
peptide, whereas [
125
I-Tyr
0
]sauvagine (Svg) was used in a binding
assay for mCRF
2
␤
. The HEK-293 cells were plated into 24-well cell
culture plates (25). Intracellular cAMP was determined with the
Biotrak cAMP
125
I scintillation proximity assays system (Amer-
sham Pharmacia Biosciences) according to the manufacturer’s
manual. Stock solutions of peptides were prepared in 10 mM
aqueous acetic acid except for oCRF and cortagine, which were
dissolved in PBS (pH 7.4).
Abbreviations: aSvg-30, antisauvagine-30; CRF, corticotropin-releasing factor; CRF
1
, CRF
receptor subtype 1; CRF
2
, CRF receptor subtype 2; CRFBP, CRF-binding protein; EPM,
elevated plus-maze; FST, forced swim test; h兾rCRF, human兾rat CRF; i.c.v., intracerebroven-
tricularly; mCRF
2
␤
, mouse CRF
2
␤
; oCRF, ovine CRF; rCRF
1
, rat CRF
1
; rCRFBP, rat CRFBP; Svg,
sauvagine; Ucn, urocortin.
*H.T. and O.J. contributed equally to this work.
†
To whom correspondence should be addressed. E-mail: spiess@em.mpg.de.
© 2004 by The National Academy of Sciences of the USA
9468–9473
兩
PNAS
兩
June 22, 2004
兩
vol. 101
兩
no. 25 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0403159101

Determination of Maximum Solubility and Isoelectric Point. After
dissolving the peptides in artificial cerebrospinal fluid (aCSF;
124 mM NaCl兾26.4 mM NaHCO
3
兾10 mM glucose兾3.3 mM
KCl兾2.5 mM CaCl
2
兾2.4 mM MgSO
4
兾1.2 mM KH
2
PO
4
)atpH
7.4, the maximum solubility c
max
was determined by measure-
ment of the peptide concentration in the supernate of a precip-
itate (21). The isoelectric point of the peptides was determined
by isoelectric focusing with a Bio-Rad IEF cell system using
Bio-Rad IEF strips in the pH range 3–10 (21).
Preparation of Peptide Solutions for Behavioral Experiments. All
peptides were dissolved in aCSF except for antisauvagine-30
(aSvg-30) (22), which was initially dissolved in 10 mM aqueous
acetic acid and diluted with 2⫻ aCSF. The final pH of the
peptide solutions was 7.4. The exact peptide concentrations of
the injection solutions were determined by amino acid analysis
as described in ref. 22.
Animals. Nine-week-old male C57BL兾6J mice (Centre D’Elevage
Janvier, Le Genest Saint Isle, France) were individually housed
in Macrolon cages as recommended by the Society for Labora-
tory Animal Science (Hannover, Germany). All experiments
were carried out in accordance with the European Council
Directive (86兾609兾 EEC) with the permission of the District
Government of Braunschweig, State of Lower Saxony, Germany,
which is in full agreement with the American Psychological
Association (Washington, DC) ethical guidelines. All efforts
were made to minimize animal suffering. The number of mice
per group was 9–11.
Autoradiography. Coronal sections (20
␮
m) of CRF
1
⫹/⫹
and
CRF
1
⫺/⫺
mice (provided by Wylie Vale, The Salk Institute for
Biological Studies, La Jolla, CA) were thawed to room temper-
ature and allowed to dry for 20 min. The sections were prein-
cubated for 1 min in incubation buffer (PBS supplemented with
10 mM MgCl
2
兾2 mM EGTA兾0.1% BSA, pH 7.0) and then
incubated for 40 min at room temperature in incubation buffer
containing 200 pM [
125
I-Tyr
0
]Svg, a nonspecific CRF receptor
ligand. Selective displacement was achieved with 1
␮
M cortagine
at CRF
1
or mouse UcnII at CRF
2
. Nonspecific binding was
determined by addition of 1
␮
M Svg. The slides were then
washed for 2 min with ice-cold PBS supplemented with 0.01%
Triton X-100 at pH 7.0 and water. Slides were dried rapidly
under a stream of cold air and exposed to Biomax MR film
(Kodak) for 4 days at ⫺80°C.
Behavioral Experiments. Anxiety-like behavior of C57BL兾6J mice
cannulated in the lateral ventricles or lateral intermediate septal
area (14) was examined 30 min after injection of the CRF
1
agonist under investigation for 5 min in the elevated plus-maze
(EPM) test (26). The CRF
2
-selective antagonist aSvg-30 in aCSF
or aCSF alone was injected 45 min before the EPM test. The
behavior of the mice was recorded by a video camera connected
to a computer and analyzed by the software
VIDEOMOT 2 (TSE,
Bad Homburg, Germany). The time spent, distance crossed, and
number of entries into the open arms, closed arms, and center
were recorded. Shift of preference from the open to the closed
arms was interpreted as an increase of anxiety-like behavior.
Locomotor activity was determined with this test by the distance
traveled.
For the forced swim test (FST), C57BL兾6J mice cannulated in
the lateral ventricles were subjected to swim sessions in individ-
ual glass cylinders (height, 39 cm; diameter, 21.7 cm) containing
water 15-cm deep at 23–25°C. On day 1, all animals were placed
in the cylinder for a preswim session of 15 min. On the test day
24 h later (day 2), the mice were subjected to a test swim session
for 6 min. The water was changed between subjects. All test swim
sessions were recorded by a video camera positioned directly
above the cylinder. A competent observer blind to treatment
scored the videotapes. The behavioral measure scored was the
duration of immobility, defined as time spent still or only using
righting movements to keep the head above water. An increase
in immobility time was interpreted as an increase of depression-
like behavior. In all behavioral experiments, the injections were
carried out bilaterally, and the cannula placement was confirmed
for each mouse by histological examination of the brains after
methylene blue injection (14). The behavioral data are expressed
as mean ⫾ SEM and were analyzed by using two- and one-way
ANOVA, with the Bonferroni–Dunn test applied, post hoc, for
individual between-group comparisons at the P ⬍ 0.05 level of
significance.
Results
Design of Chimeric Peptides and Analysis of Their Affinity and Bio-
logical Potency.
For our chimeric peptide strategy we selected
oCRF and h兾rCRF on the basis of their preferential binding to
CRF
1
and Svg (27) because of its high hydrophilicity and low
isoelectric point (pH 5.1) enhancing solubility in aqueous solu-
tion (21). The sequences of oCRF (compound 1, Fig. 1) and
h兾rCRF (compound 2, Fig. 1) were divided into N-terminal
(residues 1–13), central (residues 14–30), and C-terminal (res-
Fig. 1. Development of cortagine as chimeric peptide derived from oCRF, h兾rCRF, and Svg. The three main building blocks of the chimeric peptides, the
N-terminal, central, and C-terminal domains, are indicated. Sequences derived from h兾rCRF, Svg, and oCRF are underlain in gray, black, and white, respectively.
Z, pyroglutamic acid.
Tezval et al. PNAS
兩
June 22, 2004
兩
vol. 101
兩
no. 25
兩
9469
PHARMACOLOGY

idues 31–40) domains (Fig. 1) on the basis of the recent finding
that CRF contains segregated receptor-binding sites at its N
terminus and C terminus (28). These domains were used as
building blocks and combined with sequences from Svg (com-
pound 3, Fig. 1) to generate different chimeric peptides (Fig. 1).
It was observed that compound 5, but not compound 4, exhibited
low affinity for CRF
2
and high selectivity for CRF
1
(Table 1).
Therefore, it was concluded that residues 14–30 of h兾rCRF were
responsible for a decrease in affinity to CRF
2
. This conclusion
was in agreement with the finding that compound 6 containing
the central domain of h兾rCRF, but not compound 7, was
selective for CRF
1
(Table 1). On the basis of its low affinity to
CRF
2
, compound 6 was selected for further development. An
additional rationale for the selection of compound 6 as lead
compound was its N-terminal pyroglutamic acid derived from
the Svg sequence (Fig. 1). The presence of this cyclic residue may
prevent degradation by major aminopeptidases that require a
free
␣
-amino group for their action (29) and thereby increase the
stability of compound 6 under in vivo conditions.
On the basis of the finding that neither Glu-2 of the N-terminal
domain nor the central residues Ala-22, Arg-23, and Glu-25 of
h兾rCRF have a significant influence on receptor selectivity (21,
30), only the C-terminal residues 38, 39, and 41 were considered
for amino acid replacements. A comparison of the sequences of
oCRF, h兾rCRF, and Svg revealed that oCRF binding preferen-
tially to CRF
1
shares residues Leu-38 and Asp-39 with the
nonselective Svg in equivalent positions (Fig. 1). It was therefore
hypothesized that Ala-41 of oCRF may contribute to the binding
preference of this ligand. This hypothesis was first tested by the
synthesis and characterization of [Ala
41
]h兾rCRF. In comparison
with h兾rCRF, [Ala
41
]h兾rCRF showed an increase in CRF
1
selectivity by a factor of 3 and thus a similar selectivity as oCRF
(data not shown). As expected on the basis of this result, a
peptide highly selective for CRF
1
was obtained when the same
replacement was carried out for compound 6 to generate com-
pound 8 (Fig. 1). In comparison with compound 6, compound 8
exhibited a ⬎5-fold increase in affinity to CRF
1
, whereas only a
slight increase of affinity to CRF
2
was found (Table 1).
The high affinity of compound 8 to CRFBP (Table 1) was
removed by employing the recently reported single amino acid
switch concept determining the affinity to CRFBP (21). Ac-
cordingly, Ala-21 of compound 8 was replaced by a Glu residue,
an exchange that has been shown to decrease the affinity of
h兾rCRF to CRFBP by two orders of magnitude (21). Compound
9 (Fig. 1), obtained by this change, bound with high affinity to
CRF
1
, whereas the affinity to CRFBP was abolished (Table 1).
Replacement of Met-20 with norleucine to prevent the forma-
tion of methionine sulfoxide, a modification that is known to
abolish the bioactivity of CRF-like peptides (2), resulted in a
significant decrease of affinity to CRF
1
(data not shown) and was
therefore not introduced. We named compound 9, the final
product of our development, cortagine. The overall enhanced
specificity of cortagine over oCRF was indicated by the ratio of
the binding affinities to CRF
1
and CRF
2
[IC
50
(mCRF
2
␤
)兾
IC
50
(rCRF
1
); Table 1]. For cortagine and oCRF, values of 208
and 89, respectively, were found.
The biological potency of cortagine and oCRF was evaluated
by the determination of the EC
50
values for intracellular accu-
mulation of cAMP in transfected HEK-293 cells. In agreement
with the binding data, the biological potencies of cortagine and
oCRF were high at rCRF
1
and about one to two orders of
magnitude lower at mCRF
2
␤
(Table 2). The enhanced selectivity
of cortagine was reflected by the ratios of the biological poten-
cies [EC
50
(mCRF
2
␤
)兾EC
50
(rCRF
1
); Table 2]. Values of 89 and
19 were found for cortagine and oCRF, respectively.
By the analysis of the maximal solubility of cortagine, it was
determined that cortagine, like oCRF, was soluble at a concen-
tration of up to 1,000
␮
M (Table 2), so that there was no
limitation for behavioral experiments in view of the agonist
doses typically used.
Binding of Cortagine to Native CRF
1
. Autoradiography of mouse
brain sections from CRF
1
⫹/⫹
and CRF
1
⫺/⫺
mice was performed
to demonstrate cortagine’s selectivity for native CRF
1
(Fig. 2).
The [
125
I-Tyr
0
]Svg-binding patterns in the brains of CRF
1
⫺/⫺
mice (Fig. 2b) and of CRF
1
⫹/⫹
mice after treatment with
cortagine (Fig. 2d) did not significantly differ. CRF receptor
visible in the choroid plexus of the brain sections of these animals
(Fig. 2 b and d) was identified as CRF
2
by displacement with
UcnII (Fig. 2c).
Modulation of Anxiety-Like Behavior by Cortagine. The behavioral
effects of cortagine were determined in the EPM test and the
FST. It has been demonstrated earlier that activation of CRF
1
accessed through the brain ventricles enhances anxiety-like
behavior in the EPM test (21), the most frequently used rodent
model of anxiety, and suppresses locomotor activity (31) under
various conditions. Therefore, we performed a series of behavior
experiments with the EPM test. A two-way ANOVA with
treatment and dose as between-subject factors indicated signif-
icant treatment and dose main effects and significant interaction
after administration of peptides intracerebroventricularly (i.c.v.)
into the lateral ventricles of male C57BL兾6J mice 30 min before
testing. The values for the percent time spent in the open arms
[F
(1, 80)
⫽ 16.57, P ⬍ 0.05 for treatment; F
(4, 80)
⫽ 36.68, P ⬍ 0.05
for dose; and F
(4, 80)
⫽ 5.38; P ⬍ 0.05 for interaction] (Fig. 3a)
and number of open arm entries [F
(1, 80)
⫽ 10.66, P ⬍ 0.05 for
treatment; F
(4, 80)
⫽ 28.27, P ⬍ 0.05 for dose; and F
(4, 80)
⫽ 6.21,
P ⬍ 0.05 for interaction] (Fig. 3b) revealed a significantly higher
anxiogenic potency of cortagine than of oCRF (Bonferroni–
Table 1. Binding affinites of oCRF, h/rCRF, Svg, and their
chimeric analogs
Compound
IC
50
,nM
rCRF
1
mCRF
2
␤
rCRFBP*
1 1.8 (1.1–2.4) 160 (120–200) 450 (420–480)
2
†
1.6 (1.3–1.9) 42 (25–59) 0.54 (0.38–0.71)
3
†
0.52 (0.29–0.74) 0.9 (0.72–1.1) 57 (45–70)
4 0.47 (0.18–0.77) 0.69 (0.45–0.93) ND
5 2.0 (0.80–3.1) 330 (140–530) ND
6 9.5 (4.8–14) 700 (490–910) ND
7 1.8 (0.75–2.8) 0.98 (0.59–1.4) ND
8 1.8 (1.4–2.1) 400 (360–450) 1.9 (1.8–2.0)
9
‡
2.6 (1.6–3.4) 540 (480–590) ⬎1,000
IC
50
values are the mean of at least four experiments performed in dupli-
cate; 95% confidence intervals are given in parentheses. ND, not determined.
*The intermediate compounds of the agonist development were not tested
for their affinity to rCRFBP.
†
Binding data taken from Eckart et al. (21).
‡
Cortagine.
Table 2. Comparison of the pharmacological and
physicochemical properties of cortagine and oCRF
Peptide
Biological potency EC
50
,nM
c
max
,
␮
M pI*rCRF
1
mCRF
2
␤
Cortagine 0.18 (0.10–0.26) 16 (11–20) ⬎1,000 4.8
oCRF 0.47 (0.14–0.80) 8.8 (6.0–12) ⬎1,000 6.4
EC
50
values are the mean of at least four experiments performed in dupli-
cate; 95% confidence intervals are given in parentheses.
*Isoelectric points were determined by isoelectric focusing.
9470
兩
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0403159101 Tezval et al.

Dunn test, P ⬍ 0.05, for percent time spent and number of open
arm entries of the EPM). The significant interaction was due to
differences between the 30-ng doses of cortagine and oCRF in
modulating anxiety-like behavior in the EPM test as confirmed
by analyses of simple main effects of dose. In particular, 30 ng
of cortagine but not of oCRF significantly decreased the percent
time spent in the open arms [F
(1, 18)
⫽ 15.37, P ⬍ 0.05] and
number of open arm entries [F
(1, 18)
⫽ 10.89, P ⬍ 0.05] of the
EPM test. Thus, cortagine was more potent than oCRF under
these conditions. Interestingly, the peptides tested did not differ
in their ability [F
(1, 80)
⫽ 0.85, P ⬎ 0.05] to reduce locomotor
activity (Fig. 3c).
We also investigated the specificity of cortagine by monitoring
the EPM behavior of C57BL兾6J mice after injection of cortagine
into the lateral intermediate septum, which predominantly con-
tains CRF
2
(32). Administration of 100 ng (21 pmol) of oCRF,
but not of 100 ng (23 pmol) of cortagine into the lateral septum,
30 min before testing in the EPM exerted a profound anxiogenic
effect as indicated by a decreased percent time spent in the open
arms [F
(3, 31)
⫽ 7.32, P ⬍ 0.05] (Bonferroni–Dunn test, P ⬍ 0.05
vs. aCSF) and number of open arm entries [F
(3, 31)
⫽ 6.34; P ⬍
0.05] (Bonferroni–Dunn test, P ⬍ 0.05 vs. aCSF) (Fig. 4 a and
b) without affecting the locomotor activity [F
(3, 31)
⫽ 1.16; P ⬎
0.05] (Fig. 4c) in the EPM. Thus, the differences between the
intrinsic in vitro activities of cortagine and oCRF to activate
CRF
2
(Table 2) were confirmed by the behavioral observations.
When 400 ng (110 pmol) of the CRF
2
-selective antagonist
aSvg-30 was injected intraseptally 15 min before the application
of 100 ng of oCRF, the anxiogenic action of oCRF in the EPM
test was completely prevented. In view of the specificity of
aSvg-30 selectively blocking CRF
2
(14), it was concluded that the
anxiogenic action of oCRF was mediated by septal CRF
2
.
Modulation of the Immobility in the FST. Previous studies have
demonstrated that antagonism of CRF
1
decreases the immobil-
ity time in the FST, a rodent model of depression-like behavior
(33, 34). To examine the effect of selective activation of CRF
1
on
the immobility time, C57BL兾6J mice were injected with cortag-
ine and tested in the FST. One group of mice was injected i.c.v.
with 300 ng (68 pmol) of cortagine or 300 ng (64 pmol) of oCRF
30 min before the preswim session (day 1) and examined in the
test swim session 24 h later (day 2). The second group of mice
was exposed to the preswim session without injection (day 1).
However, 300 ng (68 pmol) of cortagine or 300 ng (64 pmol) of
oCRF was administered 30 min before the test swim session 24 h
later (day 2). Interestingly, a two-way ANOVA with treatment
and order (prepreswim vs. pretest swim injection) as between-
subject factors revealed significant treatment and order main
effects and treatment ⫻ order interaction for immobility time
during the preswim session [F
(2, 41)
⫽ 10.24, P ⬍ 0.05 treatment;
F
(1, 41)
⫽ 19.52, P ⬍ 0.05 order; and F
(2, 41)
⫽ 5.63, P ⬍ 0.05
treatment ⫻ order] and test swim session [F
(2, 41)
⫽ 19.93, P ⬍
0.05 treatment; F
(1, 41)
⫽ 34.07, P ⬍ 0.05 order; and F
(1, 41)
⫽
10.65, P ⬍ 0.05 treatment ⫻ order] in the FST. Bonferroni–Dunn
post hoc analysis showed that prepreswim or pretest swim
treatment with cortagine or oCRF significantly decreased the
immobility time during the subsequent swim sessions in com-
parison with the aCSF treatment (P ⬍ 0.05 vs. aCSF) (Fig. 5 a
and b). Similarly, prepreswim injection of the two peptides
significantly decreased the immobility time during the preswim
session (P ⬍ 0.05 vs. test swim session), whereas the pretest swim
Fig. 2. Autoradiography of CRF receptor subtypes in the mouse brain. (a and
b)[
125
I-Tyr
0
]Svg-binding (200 pM) on coronal sections at the level of the
hippocampus (Hipp) of WT and CRF
1
⫺/⫺
mice. (c and d) Selective displacement
of CRF
2
or CRF
1
with UcnII or cortagine, respectively, on brain sections from WT
mice. Amg, amygdala; CP, choroid plexus; Ctx, cortex.
Fig. 3. Enhancement of anxiety-like behavior by cortagine. i.c.v. adminis-
tered 30 ng (6.8 pmol), 100 ng (23 pmol), and 300 ng (68 pmol) of cortagine
produced increased anxiety-like behavior and reduced locomotor activity as
indicated by the time spent on the open arms (a), number of entries into the
open arms (b), and total distance traveled (c) on the EPM. i.c.v. administered
100 ng (21 pmol) and 300 ng (64 pmol) of oCRF also significantly decreased the
time spent on the open arms (a), number of entries into the open arms (b), and
total distance traveled (c) on the EPM. Statistically significant differences:
Bonferroni–Dunn test;
*
, P ⬍ 0.05 vs. control (aCSF injection); a, P ⬍ 0.05 vs.
oCRF at respective dose.
Tezval et al. PNAS
兩
June 22, 2004
兩
vol. 101
兩
no. 25
兩
9471
PHARMACOLOGY

injection exerted a similar effect during the test swim session
(P ⬍ 0.05 vs. preswim session) (Fig. 5 a and b). Analysis of simple
main effects of treatment revealed that injection of cortagine but
not oCRF before the preswim session resulted in a significantly
reduced immobility time during the test swim session 24 h later
[F
(2, 22)
⫽ 5.94, P ⬍ 0.05] (Bonferroni–Dunn test, P ⬍ 0.05 vs.
oCRF-injected group) (Fig. 5b). No such difference was ob-
served between cortagine and oCRF-pretreated mice during the
preswim session [F
(2, 22)
⫽ 9.65, P ⬍ 0.05] (Bonferroni–Dunn
test, P ⬎ 0.05 cortagine- vs. oCRF-injected group) (Fig. 5a).
It had to be considered that the different effects of cortagine
and oCRF could be explained by an increased half-life time of
cortagine. This possibility was tested by i.c.v. injecting 300 ng (68
pmol) of cortagine and 300 ng (64 pmol) of oCRF, respectively
(day 1), 24 h before the test swim session (day 2) (Fig. 5c). As
an additional control, the same mice were injected i.c.v. 24 h later
with 300 ng of cortagine or oCRF and subjected to a 6-min retest
swim 30 min after injection (Fig. 5c). A two-way ANOVA with
treatment as between-subject factor and time (day 2 vs. day 3) as
within-subject factor revealed significant treatment [F
(2, 21)
⫽
7.98, P ⬍ 0.05] and time [F
(1, 21)
⫽ 41.51, P ⬍ 0.05] main effects,
and treatment ⫻ time interaction [F
(2, 21)
⫽ 92.24; P ⬍ 0.05]. The
significant interaction was produced by the fact that the groups
did not differ on day 2 [F
(2, 21)
⫽ 0.98, P ⬎ 0.05], but differences
appeared on day 3 [F
(2, 21)
⫽ 25.08, P ⬍ 0.05] (Bonferroni–Dunn
test, P ⬍ 0.05 vs. aCSF). These results excluded the possibility
that the prolonged action of cortagine was responsible for its
differential effects on immobility time in FST.
Discussion
By combining sequences from Svg, h兾rCRF, and oCRF, cortag-
ine was developed (Fig. 1). Cortagine’s specificity and potency
were initially established by its selective and high affinity to
CRF
1
of transfected HEK-293 cells and its potency to release
cAMP from these cells. Cortagine’s selective binding to native
CRF
1
of brain sections of the mouse was demonstrated in
autoradiographic experiments with CRF
1
-deficient mice and
their WT littermates (Fig. 2).
Because cortagine did not exhibit high affinity binding to
CRFBP, it was excluded that the effective dose of cortagine was
decreased by binding to CRFBP and diluted by endogenous
CRF-like peptide released from CRFBP.
It has to be considered that rat CRF
1
and mouse CRF
2
␤
produced by transfected cells and not mouse CRF
1
and CRF
2
␣
,
the predominant splice variant of the rodent brain (9), were used
for the pharmacological characterization of cortagine. However,
the high affinity of cortagine for mouse CRF
1
was demonstrated
by its potency to enhance anxiety-like behavior after injection
i.c.v. (Fig. 3). It has been earlier established that CRF
1
activated
by oCRF or h兾rCRF injected i.c.v. mediates the enhancement of
anxiety-like behaviors (31). The selectivity of cortagine was
Fig. 4. Absence of significant interaction of cortagine with CRF
2
of the
mouse brain. Cortagine (100 ng, 23 pmol) or oCRF (100 ng, 21 pmol) were
applied intraseptally 30 min before EPM. aCSF or aSvg-30 (400 ng, 110 pmol)
were administered 45 min before EPM. Time spent on the open arms (a),
number of open arm entries (b), and distance traveled (c) during the test are
depicted. Statistically significant differences: Bonferroni–Dunn test;
*
, P ⬍
0.05 vs. control (aCSF兾aCSF).
Fig. 5. Immobility time after cortagine application in the FST. (a) Cortagine
(300 ng, 68 pmol) or oCRF (300 ng, 64 pmol) was administered i.c.v. 30 min
before the 15-min preswim on day 1. The test swim was performed 24 h later
(day 2). (b) Mice subjected to the preswim on day 1 without injection were
injected on day 2 with the two peptides (doses as in a) and subjected to the 15
min test swim 30 min later. (c) After injection of the two peptides (doses as
above), the mice were subjected to the test swim 24 h later (day 2). On day 3,
the two peptides were injected 30 min before a retest swim. Injections are
indicated by arrows. Statistically significant differences: Bonferroni–Dunn
test;
*
, P ⬍ 0.05 vs. control (aCSF); #, P ⬍ 0.05 vs. oCRF-injected group.
9472
兩
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0403159101 Tezval et al.

confirmed when it was injected into the lateral intermediate
septum, which contains predominantly CRF
2
␣
(32) (Fig. 4). At
a dose of 21 pmol, oCRF enhanced anxiety-like behavior,
whereas cortagine did not. The data on binding and biopotency,
as well as on the modulation of anxiety-like behavior, are in
agreement with earlier pharmacological analyses indicating that
the splice variants of CRF
2
do not differ significantly in their
pharmacological profile (5, 19).
The enhancement of anxiety-like behavior by activation of
CRF
1
with cortagine and oCRF was accompanied by a large
reduction in locomotor activity. The involvement of CRF
1
in
locomotor activity has also been demonstrated with CRF
1
-
deficient mice, which exhibit hyperlocomotion in the open field
(12). However, the differential anxiogenic effects of cortagine
and oCRF after intraseptal and intraventricular injections did
not correlate with locomotion differences (Figs. 3 and 4).
Therefore, we assumed that the locomotor effect represented an
independent behavior variable. Such an assumption is consistent
with factor analyses of mouse behavior in the EPM test, where
indices of anxiety and locomotor activity were loaded on sepa-
rate factors (35).
Potential effects of cortagine and oCRF on depression-like
behaviors were investigated by using the FST, a common animal
model of depression, using the immobility time as measure of
depression-like behavior (36) (Fig. 5). Surprisingly, we found
that both cortagine and oCRF significantly decreased the im-
mobility time when applied immediately before the preswim or
the test swim session. Moreover, administration of cortagine, but
not oCRF, before the preswim session resulted in a decrease of
the immobility time during the test swim session 24 h later.
Opposite effects were expected on the basis of the reported
findings that antagonism to CRF
1
decreases depression-like
behavior in rodents as determined by FST (33, 34), as well as in
humans (37). It is suggested that the cortagine-induced CRF
1
activation decreased the depression-like behavior independently
of its possible effects on general activity. Thus, the data pre-
sented here confirm the involvement of CRF
1
in anxiety- and
depression-like behaviors. However, the surprising cortagine
effect combining anxiogenic and antidepressive potencies indi-
cates that CRF
1
-involving processes of anxiety and depression
formation are not necessarily positively correlated.
Thomas Zeyda and Milos Zarkovic are gratefully acknowledged for
helpful discussions. We thank Yu-Wen Li (Bristol-Myers Squibb) for
helping to set up autoradiography and Lars van Werven, Cathrin Hippel,
Thomas Liepold, and Bodo Zimmermann for expert technical help.
1. Spiess, J., Rivier, J., Rivier, C. & Vale, W. W. (1981) Proc. Natl. Acad. Sci. USA
78, 6517–6521.
2. Vale, W. W., Spiess, J., Rivier, C. & Rivier, J. (1981) Science 213, 1394–1397.
3. Eckart, K., Jahn, O., Radulovic, J., Radulovic, M., Blank, T., Stiedl, O., Brauns,
O., Tezval, H., Zeyda, T. & Spiess, J. (2002) Recept. Channels 8, 163–177.
4. Reul, J. M. & Holsboer, F. (2002) Curr. Opin. Pharmacol. 2, 23–33.
5. Perrin, M. H. & Vale, W. W. (1999) Ann. N.Y. Acad. Sci. 885, 312–328.
6. Behan, D. P., Heinrichs, S. C., Troncoso, J. C., Liu, X. J., Kawas, C. H., Ling,
N. & De Souza, E. B. (1995) Nature 378, 284–287.
7. Karolyi, I. J., Burrows, H. L., Ramesh, T. M., Nakajima, M., Lesh, J. S., Seong,
E., Camper, S. A. & Seasholtz, A. F. (1999) Proc. Natl. Acad. Sci. USA 96,
11595–11600.
8. Hillhouse, E. W., Randeva, H., Ladds, G. & Grammatopoulos, D. (2002)
Biochem. Soc. Trans. 30, 428–432.
9. Lovenberg, T. W., Chalmers, D. T., Liu, C. & De Souza, E. B. (1995)
Endocrinology 136, 4139–4142.
10. Bale, T. L., Contarino, A., Smith, G. W., Chan, R., Gold, L. H., Sawchenko,
P. E., Koob, G. F., Vale, W. W. & Lee, K. F. (2000) Nat. Genet. 24, 410–414.
11. Smith, G. W., Aubry, J. M., Dellu, F., Contarino, A., Bilezikjian, L. M., Gold,
L. H., Chen, R., Marchuk, Y., Hauser, C., Bentley, C. A., et al. (1998) Neuron
20, 1093–1102.
12. Timpl, P., Spanagel, R., Sillaber, I., Kresse, A., Reul, J., Stalla, G. K., Blanquet,
V., Steckler, T., Holsboer, F. & Wurst, W. (1998) Nat. Genet. 19, 162–166.
13. Kishimoto, T., Radulovic, J., Radulovic, M., Lin, C. R., Schrick, C., Hoosh-
mand, F., Hermanson, O., Rosenfeld, M. G. & Spiess, J. (2000) Nat. Genet. 24,
415–419.
14. Radulovic, J., Ru¨hmann, A., Liepold, T. & Spiess, J. (1999) J. Neurosci. 19,
5016–5025.
15. Koob, G. F. (1999) Biol. Psychiatry 46, 1167–1180.
16. Vaughan, J., Donaldson, C., Bittencourt, J., Perrin, M. H., Lewis, K., Sutton,
S., Chan, R., Turnbull, A. V., Lovejoy, D., Rivier, C., et al. (1995) Nature 378,
287–292.
17. Reyes, T. M., Lewis, K., Perrin, M. H., Kunitake, K. S., Vaughan, J., Arias,
C. A., Hogenesch, J. B., Gulyas, J., Rivier, J., Vale, W. W. & Sawchenko, P. E.
(2001) Proc. Natl. Acad. Sci. USA 98, 2843–2848.
18. Hsu, S. Y. & Hsueh, A. J. W. (2001) Nat. Med. 7, 605–611.
19. Lewis, K., Li, C., Perrin, M. H., Blount, A., Kunitake, K., Donaldson, C.,
Vaughan, J., Reyes, T. M., Gulyas, J., Fischer, W., et al. (2001) Proc. Natl. Acad.
Sci. USA 98, 7570–7575.
20. Behan, D. P., Grigoriadis, D. E., Lovenberg, T., Chalmers, D., Heinrichs, S.,
Liaw, C. & De Souza, E. B. (1996) Mol. Psychiatry 1, 265–277.
21. Eckart, K., Jahn, O., Radulovic, J., Tezval, H., van Werven, L. & Spiess, J.
(2001) Proc. Natl. Acad. Sci. USA 98, 11142–11147.
22. Ru¨hmann, A., Bonk, I., Lin, C. J. R., Rosenfeld, M. G. & Spiess, J. (1998) Proc.
Natl. Acad. Sci. USA 95, 15264–15269.
23. Jahn, O., Eckart, K., Sydow, S., Hofmann, B. A. & Spiess, J. (2001) Peptides
(Tarrytown, NY) 22, 47–56.
24. Hofmann, B. A., Sydow, S., Jahn, O., van Werven, L., Liepold, T., Eckart, K.
& Spiess, J. (2001) Protein Sci. 10, 2050–2062.
25. Sydow, S., Radulovic, J., Dautzenberg, F. M. & Spiess, J. (1997) Mol. Brain Res.
52, 182–193.
26. Radulovic, J., Kammermeier, J. & Spiess, J. (1998) Behav. Brain Res. 95,
179–189.
27. Montecucchi, P. & Henschen, A. (1981) Int. J. Pept. Protein Res. 18, 113–120.
28. Beyermann, M., Rothemund, S., Heinrich, N., Fechner, K., Furkert, J., Dathe,
M., Winter, R., Krause, E. & Bienert, M. (2000) J. Biol. Chem. 275, 5702–5709.
29. Saido, T. C., Iwatsubo, T., Mann, D. M. A., Shimada, H., Ihara, Y. &
Kawashima, S. (1995) Neuron 14, 457–466.
30. Brauns, O., Brauns, S., Zimmermann, B., Jahn, O. & Spiess, J. (2002) Peptides
(Tarrytown, NY) 23, 881–888.
31. Dunn, A. J. & Berridge, C. W. (1990) Brain Res. Rev. 15, 71–100.
32. Chalmers, D. T., Lovenberg, T. W. & De Souza, E. B. (1995) J. Neurosci. 15,
6340–6350.
33. Griebel, G., Simiand, J., Steinberg, R., Jung, M., Gully, D., Roger, P., Geslin,
M., Scatton, B., Maffrand, J. P. & Soubrie, P. (2002) J. Pharmacol. Exp. Ther.
301, 333–345.
34. Bale, T. L. & Vale, W. W. (2003) J. Neurosci. 23, 5295–5301.
35. Lister, R. G. (1987) Psychopharmacology (Berlin) 92, 180–185.
36. Porsolt, R. D., Le Pichon, M. & Jalfre, M. (1977) Nature 266, 730–732.
37. Zobel, A. W., Nickel, T., Kunzel, H. E., Ackl, N., Sonntag, A., Ising, M. &
Holsboer, F. (2000) J. Psychiatr. Res. 34, 171–181.
Tezval et al. PNAS
兩
June 22, 2004
兩
vol. 101
兩
no. 25
兩
9473
PHARMACOLOGY