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Corticotropin-Releasing Factor Receptors Couple to Multiple
G-Proteins to Activate Diverse Intracellular Signaling
Pathways in Mouse Hippocampus: Role in Neuronal
Excitability and Associative Learning
Thomas Blank,
1
Ingrid Nijholt,
1
Dimitris K. Grammatopoulos,
2
Harpal S. Randeva,
2
Edward W. Hillhouse,
2
and
Joachim Spiess
1
1
Department of Molecular Neuroendocrinology, Max Planck Institute for Experimental Medicine, D-37075 Goettingen, Germany, and
2
Sir Quinton Hazell
Molecular Medicine Research Centre, Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom
Corticotropin-releasing factor (CRF) exerts a key neuroregulatory control on stress responses in various regions of the mammalian brain,
including the hippocampus. Using hippocampal slices, extracts, and whole animals, we investigated the effects of human/rat CRF
(h/rCRF) on hippocampal neuronal excitability and hippocampus-dependent learning in two mouse inbred strains, BALB/c and C57BL/
6N. Intracellular recordings from slices revealed that application of h/rCRF increased the neuronal activity in both mouse inbred strains.
Inhibition of protein kinase C (PKC) by bisindolylmaleimide I (BIS-I) prevented the h/rCRF effect only in hippocampal slices from
BALB/c mice but not in slices from C57BL/6N mice. Inhibition of cAMP-dependent protein kinase (PKA) by H-89 abolished the h/rCRF
effect in slices from C57BL/6N mice, withno effect in slices from BALB/c mice. Accordingly, h/rCRF elevated PKA activity in hippocampal
slices from C57BL/6N mice but increased only PKC activity in the hippocampus of BALB/c mice. These differences in h/rCRF signal
transduction were also observed in hippocampal membrane suspensions from both mouse strains. In BALB/c mice, hippocampal CRF
receptors coupled to G
q/11
during stimulation by h/rCRF, whereas they coupled to G
s
,G
q/11
, and G
i
in C57BL/6N mice. As expected on the
basis of the slice experiments, h/rCRF improved context-dependent fear conditioning of BALB/c mice in behavioral experiments, and
BIS-I prevented this effect. However, although h/rCRF increased neuronal spiking in slices from C57BL/6N mice, it did not enhance
conditioned fear. These results indicate that the CRF system activates different intracellular signaling pathways in mouse hippocampus
and may have distinct effects on associative learning depending on the mouse strain investigated.
Key words: neuronal excitability; h/rCRF; PKC; PKA; classical fear conditioning; G-protein; mouse; hippocampus
Introduction
Corticotropin-releasing factor (CRF) is a 41 amino acid neu-
ropeptide that has been implicated in both physiological and
behavioral responses to stress (Spiess et al., 1981; Vale et al.,
1981). During exposure to stress, CRF can be secreted directly
from nerve terminals located in the hippocampus. Specifically,
numerous, large CRF-immunoreactive neurons have been found
in the hippocampal CA1 and CA3 region (Swanson et al., 1983;
Merchenthaler, 1984). Previous studies have shown the modula-
tion of hippocampus-dependent learning and memory by CRF.
Human/rat CRF (h/rCRF) injected directly into the dentate gyrus
consistently enhanced memory retention in rats in a one-way
passive avoidance task (Lee et al., 1993). Injection of h/rCRF into
the dorsal hippocampus shortly before the training enhanced
context- and tone-dependent fear conditioning in BALB/c mice
through CRF receptor 1 (CRFR1) (Radulovic et al., 1999). In
addition to the effects on hippocampal learning tasks, CRF exerts
a profound action on hippocampal neuronal activity. Recent
studies have demonstrated that h/rCRF produces a long-lasting
enhancement of synaptic efficacy in the rat hippocampus in vivo
(Wang et al., 1998, 2000). h/rCRF reversibly increases the spiking
of rat hippocampal pyramidal cells (Aldenhoff et al., 1983) and
enhances the amplitude of CA1 population spikes evoked by
stimulation of the Schaffer collateral pathway (Hollrigel et al.,
1998). We showed recently that application of h/rCRF facilitates
the induction and stability of long-term potentiation (LTP) un-
der defined stimulation conditions in area CA1 of mouse hip-
pocampal slices (Blank et al., 2002).
To examine the signal transduction pathways of h/rCRF in
mouse hippocampus, we studied the G-protein and second-
messenger activation after CRF receptor stimulation in hip-
pocampi of two mouse inbred strains, C57BL/6N and BALB/c.
We chose these two inbred strains because C57BL/6 and BALB/c
mice have repeatedly been found to differ strongly in several
behavioral responses (Oliverio et al., 1973; Peeler and Nowa-
kowski, 1987; Beuzen and Belzung, 1995) and in neurodevelop-
mental and neurochemical parameters (Nowakowski, 1984). For
example, BALB/c mice exhibit stronger anxiety-like responses in
the light– dark choice test (Beuzen and Belzung, 1995), in the
Received May 31, 2002; revised Oct. 25, 2002; accepted Oct. 29, 2002.
This work was supported by the Max Planck Society. D.K.G. is a Wellcome Trust Career Development Fellow. We
thank Dr. Klaus Eckart for the peptide synthesis of [Glu
11,16
] astressin and h/rCRF.
Correspondence should be addressed to Thomas Blank, Department of Molecular Neuroendocrinology, Max
Planck Institute for Experimental Medicine, Hermann-Rein-Straße 3, D-37075 Goettingen, Germany. E-mail:
blank@em.mpg.de.
Copyright © 2003 Society for Neuroscience 0270-6474/03/230700-08$15.00/0
700 • The Journal of Neuroscience, January 15, 2003 • 23(2):700 –707
open-field paradigm (Oliverio et al., 1973), and in a runway tra-
versal locomotor activity test (Peeler and Nowakowski, 1987).
The impact of h/rCRF on neuronal excitability of CA1 pyramidal
cells was investigated in hippocampal slices from both mouse
inbred strains. Finally, we investigated the effect of h/rCRF on
hippocampus-dependent learning in C57BL/6N and BALB/c
mice.
Materials and Methods
Animals. Experiments were performed with male BALB/c and C57BL/6N
mice (Charles River, Sultzfeld, Germany) 9–12 weeks old. The mice were
housed individually and maintained on a 12 hr light/dark cycle (lights on
at 7:00 A.M.) with access to food and water ad libitum. All experimental
procedures were in accordance with the European Council Directive
(86/609/EEC) and the Animal Section Law under the supervision of the
District Government of Braunschweig (Lower Saxony, Germany).
Electrophysiology. Mice were briefly anesthetized with isoflurane and
then decapitated. In ⬍1 min, the skull was opened, and the brain was
removed and transferred to ice-cold artificial CSF (aCSF) solution of the
following composition (in m
M): 130 NaCl, 3.5 KCl, 1.25 NaH
2
PO
4
, 1.5
MgSO
4
, 2 CaCl
2
, 24 NaHCO
3
, and 10 glucose, pH 7.4 (equilibrated with
95% O
2
–5% CO
2
). Hippocampi were dissected from the chilled brain
hemispheres on ice. Transverse hippocampal slices (400
m) were ob-
tained on a McIlwain tissue chopper (Mickle Laboratory Engineering,
Surrey, UK) and kept submerged (minimum of 1 hr at room temperature
before recordings) in aCSF.
Conventional intracellular recording techniques were used, with glass
microelectrodes filled with 3
M potassium acetate. Microelectrodes were
pulled from borosilicate glass capillaries (World Precision Instruments,
Sarasota, FL) on a horizontal electrode puller (Zeitz-Instrumente, Augs-
burg, Germany). The microelectrode tip resistances ranged from 60 to
100 M⍀ for recordings from mouse hippocampal neurons. Intracellular
signals were recorded with a single-electrode voltage-clamp amplifier
(SEC-05L; NPI Electronics, Tamm, Germany), which performed
current-clamp measurements at high switching frequencies in the range
of 25–30 kHz. Bridge balance was monitored throughout the experiment
and adjusted as required. Traces were stored on a computer using Pulse
7.4 software (Heka, Lambrecht, Germany) for offline analysis. For intra-
cellular recordings, only neurons were included that exhibited over-
shooting action potentials, stable membrane potentials of at least ⫺60
mV, and input resistances of ⱕ35 M⍀. Input resistance was determined
by measuring the voltage deflection at the end of a 100 msec hyperpolar-
ization current step (⫺0.2 nA). Depolarizing current pulses of 3–5 msec
duration were injected through the recording electrode to elicit single
action potentials. Spike frequency adaptation was investigated by inject-
ing each cell with a series of 600 msec depolarizing current pulses (0.2–1
nA; increment, 100 pA). To compare neuronal responses, the membrane
potential of each cell was manually clamped to ⫺65 mV by discontinuous
current injection. In all electrophysiological experiments, n values repre-
sent the number of slices.
Drugs. h/rCRF (Ru¨hmann et al., 1996) and [Glu
11,16
] astressin (Eckart
et al., 2001) were synthesized in our laboratory as described. H-89 and
bisindolylmaleimide I (BIS-I) were obtained from Calbiochem (San Di-
ego, CA). Phorbol 12,13-dibutyrate (PDBu) and 4
␣
-phorbol were both
purchased from Sigma (St. Louis, MO).
Drug treatment. [Glu
11,16
] astressin was dissolved in aCSF to a final
concentration of 280
M. h/rCRF stock solutions were prepared in 10 mM
acetic acid. For cannula injections, dilutions in aCSF to a final concen-
tration of 400 ng/
l were prepared immediately before the experiments.
The final pH of the peptide solution was 7.4. BIS-I was stored as 1 m
M
stock solution in dimethylsulfoxide (DMSO). For injection, the solution
was diluted with aCSF to a final concentration of 0.4 nmol/
l. PDBu and
4
␣
-phorbol were both dissolved in DMSO to 5
g/
l. For injection, the
solutions were diluted with aCSF to a final concentration of 10 ng/
l.
Cannulation. Double guide cannulas (C235; Plastics One, Roanoke,
VA) were implanted using a stereotactic holder during anesthesia with
1.2% avertin (0.02 ml/g, i.p.) under aseptic conditions as described pre-
viously (Stiedl et al., 2000; Blank et al., 2002). Each double guide cannula
with inserted dummy cannula and dust cap was fixed to the skull of the
mouse with dental cement. The cannulas were placed into both lateral
brain ventricles, with anteroposterior (AP) coordinates zeroed at bregma
AP 0 mm, lateral 1 mm, and depth 3 mm or directed toward both dorsal
hippocampi, AP ⫺1.5 mm, lateral 1 mm, and depth 2 mm (Franklin and
Paxinos, 1997). The animals were allowed to recover for 4 –5 d before the
experiments started. On the day of the experiment, bilateral injections
were performed using an infusion pump (CMA/100; CMA Microdialy-
sis, Solna, Sweden) at a constant rate of 0.33
l/min (final volume, 0.25
l per side). Cannula placement was verified post hoc in all mice by
injection of methylene blue. For electrophysiological experiments, dou-
ble guide cannula placement was verified by unilateral methylene blue
injection.
Fear conditioning. The fear conditioning experiments were performed
as described previously (Stiedl et al., 2000; Blank et al., 2002) using a
computer-controlled fear conditioning system (TSE, Bad Homburg,
Germany). Fear conditioning was performed in a Plexiglas cage (36 ⫻
21 ⫻ 20 cm) within a fear conditioning box constantly illuminated (12 V,
10 W halogen lamp, 100 –500 lux). In the conditioning box, a high-
frequency loudspeaker (KT-25-DT; Conrad, Hirschau, Germany) pro-
vided constant background noise [white noise, 68 dB sound pressure
level (SPL)]. The training (conditioning) consisted of a single trial. The
mouse was exposed to the conditioning context (180 sec) followed by a
tone (30 sec, 10 kHz, 75 dB SPL, pulsed 5 Hz). After termination of the
tone, a foot shock (0.7 mA, 2 sec, constant current) was delivered through
a stainless steel grid floor. The mouse was removed from the fear condi-
tioning box 30 sec after shock termination to avoid an aversive associa-
tion with the handling procedure. Under these conditions, the context
served as background stimulus. Background contextual fear condition-
ing but not foreground contextual fear conditioning, in which the tone is
omitted during training, has been shown to involve the hippocampus
(Phillips and LeDoux, 1994). Memory tests were performed 24 hr after
fear conditioning. Contextual memory was tested in the fear condition-
ing box for 180 sec without tone or shock presentation (with background
noise). Freezing, defined as lack of movement except for respiration and
heart beat, was assessed as the behavioral parameter of the defensive
reaction of mice (Blanchard and Blanchard, 1969; Bolles and Riley, 1973;
Fanselow and Bolles, 1979) by a time-sampling procedure every 10 sec
throughout the memory test. In addition, activity-derived measures (in-
activity, mean activity, and exploratory area) were recorded by a photo-
beam system (10 Hz detection rate).
Protein kinase A and protein kinase C assays. cAMP-dependent protein
kinase (PKA) and protein kinase C (PKC) activities were assayed using
the PepTag Assay for nonradioactive detection of PKC or PKA (Promega,
Madison, WI) on the basis of the phosphorylation of fluorescent-tagged
PKC- or PKA-specific peptides. After incubation in either aCSF or 250
n
M h/rCRF for 30 min, hippocampal slices were placed in ice-cold ho-
mogenization buffer [20 m
M Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA,
48 m
M mercaptoethanol, 0.32 M sucrose, and freshly added protease
inhibitor cocktail tablet (Boehringer Mannheim, Mannheim, Germa-
ny)]. The tissue was homogenized with a Teflon-plastic homogenizer
and centrifuged at 100,000 ⫻ g for 30 min in a Beckman Instruments
(Fullerton, CA) XL-80 ultracentrifuge. The resulting supernatant con-
tained the PKA preparation. The pellet was rehomogenized in homoge-
nization buffer and sonicated (four times for 15 sec), incubated for 30
min with Triton X-100 (0.2%), and centrifuged at 100,000 ⫻ g for 30
min. The supernatant contained the membrane-bound PKC prepara-
tion, which was used for the PKC assay. Protein concentrations were
determined with the Bradford assay (Bio-Rad, Munich, Germany). The
assay was performed as described by the manufacturer. An aliquot of the
PKA preparation was incubated for 30 min at 30°C in PepTag PKA 5⫻
reaction buffer (in m
M: 100 Tris-HCl, pH 7.4, 50 MgCl
2
, and 5 ATP) and
0.4
g/
l of the PKA-specific peptide substrate PepTag A1 (L-R-R-A-S-
L-G; Kemptide). The same procedure was used for the PKC preparations
that were incubated in PepTag PKC reaction buffer (in m
M: 100 HEPES,
pH 7.4, 6.5 CaCl
2
, 5 DTT, 50 MgCl
2
, and 5 ATP) containing 0.4
g/
lof
the PKC-specific peptide substrate PepTag C1 (P-L-S-R-T-L-S-V-A-A-
K). The reaction was stopped by heating to 95°C for 10 min. Phosphor-
ylation of the PKA- and PKC-specific substrates was used to measure
Blank et al. •CRF-Mediated Excitability in Mouse Hippocampus J. Neurosci., January 15, 2003 • 23(2):700 –707 • 701
kinase activity. Phosphorylated and unphosphorylated PepTag peptides
were separated on a 0.8% agarose gel by electrophoresis. The gel was
photographed with a transilluminator, and bands indicating substrate
phosphorylated by PKA or PKC were quantified by densitometry (Win-
Cam 2.2; Cybertech, Berlin, Germany). For the PKA and PKC assays, 4.5
and 6.5
g of protein, respectively, were applied.
Western blotting. Hippocampi of C57BL/6N or BALB/c mice were dis-
sected out and homogenized in TBS (10 m
M Tris, pH 7.6, and 150 mM
NaCl), 10% sucrose, and a protease inhibitor cocktail tablet (Boehringer
Mannheim). The homogenate was centrifuged at 20,000 ⫻ g for 30 min
at 4°C. The supernatant was removed, and the membrane pellet was
resuspended in a second identical wash step and centrifuged again at
20,000 ⫻ g for 30 min at 4°C. The supernatant was removed, and the
membrane pellet was resuspended in TBS, 1 m
M EDTA, and 1% sodium
cholate and incubated for 60 min with constant mixing at 4°C. By cen-
trifugation at 155,000 ⫻ g for 60 min (4°C), the supernatant containing
soluble membrane proteins was obtained. Protein concentrations were
determined with a Bradford assay (Bio-Rad). Equal amounts of protein
for each group were separated on a 10% SDS gel and transferred to an
Immobilon-P membrane (Millipore, Bedford, MA) using a semidry
transfer apparatus. The blot was probed using an anti-G
q/11
␣
subunit
antibody (1:4000; Calbiochem), an anti-G
s
␣
subunit antibody (1:1000;
NEN, Boston, MA), or an antibody directed against G
␣
i-1,2,3
-protein
(1:200; Calbiochem). These antibodies were detected by secondary anti-
bodies conjugated to alkaline phosphatase. CDP-Star (Tropix, Bedford,
MA) was used as a chemiluminescence substrate. During dephosphory-
lation, the substrate decomposed, producing a prolonged emission of
light that was imaged on photographic film (Fuji Super RX; Fujifilm,
Tokyo, Japan). The relative density of the bands was measured by densi-
tometry using the software WinCam 2.2 for Windows.
Preparation of hippocampal membranes. Membranes were prepared as
described previously (Grammatopoulos et al., 2001). Hippocampi of
C57BL/6N or BALB/c mice were homogenized in Dulbecco’s PBS (ex-
traction buffer) containing 10 m
M MgCl
2
,2mM EGTA, 1.5 gm/l bovine
serum albumin (BSA) (w/v), 0.15 m
M bacitracin, and 1 mM phenylmeth-
ylsulfonylfluoride (PMSF), pH 7.2, at 22°C. The homogenate was centri-
fuged at 1500 ⫻ g for 30 min at 4°C. The pellet was discarded, and the
supernatant was spun at 45,000 ⫻ g for 60 min at 4°C. Using the homog-
enizer, the final pellet was resuspended in 10 ml of the described extrac-
tion buffer. The protein concentration of the membrane suspension was
determined using the bicinchoninic acid method (Smith et al., 1985)
with BSA as a standard.
Synthesis of
32
P-GTP-
␥
-azidoanilide and photolabeling of G
␣
subunits.
32
P-GTP-
␥
-azidoanilide (
32
P-GTP-AA) was synthesized as described
previously (Schwindinger et al., 1998). Mouse hippocampal membranes
were incubated in a darkroom with or without h/rCRF (100 n
M) for 5
min at 30°C before the addition of 5
Ci of
32
P-GTP-AA in 120
lof50
m
M HEPES buffer, pH 7.4, containing 30 mM KCl, 10 mM MgCl
2
,1mM
benzamidine, 5
M GDP, and 0.1 mM EDTA. After incubation for 3 min
at 30°C, membranes were collected by centrifugation and resuspended in
100
l of the above buffer containing 2 mM glutathione, placed on ice,
and exposed to UV light (254 nm) at a distance of 5 cm for 5 min.
G-protein immunoprecipitation.
32
P-GTP-AA-labeled G-proteins
were precipitated by centrifugation and solubilized in 120
l of 2% SDS.
Then, 360
lof10mM Tris-HCl buffer, pH 7.4, containing 1% (v/v)
Triton X-100, 1% (v/v) deoxycholate, 0.5% (w/v) SDS, 150 m
M NaCl, 1
m
M DTT, 1 mM EDTA, 0.2 mM PMSF, and 10
g/ml aprotinin was added,
and insoluble material was removed by centrifugation. Solubilized mem-
branes were divided into 100
l aliquots, and each aliquot was incubated
with 10
l of undiluted G-protein antiserum at 4°C. Subsequently, 50
l
of protein A-Sepharose beads (10% w/v in the above buffer) was added,
and the incubation was continued at 4°C overnight. The beads were
collected by centrifugation, washed twice, and dried under vacuum. The
immune complexes were dissociated from protein A by reconstitution in
Laemmli’s buffer (100
l) and boiling in a water bath for 5 min. Samples
were subjected to gel electrophoresis. The gels were stained with
Coomassie blue, dried, and exposed to Fuji x-ray film at ⫺70°Cfor
2–5 d. The relative density of the bands was measured by optical
density scanning using the software Scion Image-

3b for Windows
(Scion, Frederick, MD).
Statistics. Statistical comparisons were made by using Student’s t test
and ANOVA. Data were expressed as mean ⫾ SEM. Significance was
determined at the level of p ⬍ 0.05.
Results
In hippocampal slices from both C57BL/6N and BALB/c mice,
stable intracellular recordings were obtained from CA1 pyrami-
dal neurons. The resting membrane potentials of pyramidal neu-
rons from C57BL/6N mice (⫺68.4 ⫾ 0.9 mV; n ⫽ 38) and
BALB/c mice (⫺69.6 ⫾ 1.2 mV; n ⫽ 33) did not differ signifi-
cantly, nor did the membrane input resistance of CA1 cells of
C57BL/6N mice (56.5 ⫾ 3.5 M⍀; n ⫽ 38) and BALB/c mice
(58.7 ⫾ 3M⍀; n ⫽ 33) differ significantly from each other.
Likewise, there were no significant differences between the spike
amplitudes, with values of 63.4 ⫾ 1.2 mV (n ⫽ 19) found for
C57BL/6N mice and 62.2 ⫾ 1.3 mV (n ⫽ 13) for BALB/c mice.
When mouse CA1 pyramidal cells of either strain were excited
by prolonged depolarizing current pulses, they responded with
prolonged spiking (Fig. 1A). The discharge rate was highest at the
beginning of the current pulse (1 nA) and declined to a steady rate
during the course of the depolarizing pulse (Fig. 1B). Increasing
stimulus intensities elicited enhanced neuronal spiking. In re-
sponse to strong depolarizing current pulses (1 nA, 600 msec),
C57BL/6N and BALB/c mouse pyramidal cells fired 18.7 ⫾ 2.5
(n ⫽ 12) and 18.6 ⫾ 7.3 (n ⫽ 7) spikes, respectively (Fig. 1C).
h/rCRF was applied to mouse hippocampal slices to investi-
gate the effects on the neuronal spiking behavior. The number of
spikes elicited by a 600 msec depolarizing current pulse was in-
creased by 88 ⫾ 24% (n ⫽ 7; p ⬍ 0.05) in C57BL/6N (data not
shown) mice and by 87 ⫾ 39% (n ⫽ 8; p ⬍ 0.05) in BALB/c mice
after addition of 250 n
M h/rCRF (Fig. 2A). After 30 min of wash-
ing in aCSF, spiking was still elevated by 85 ⫾ 21% (n ⫽ 7; p ⬍
0.05) in C57BL/6N (data not shown) mice and by 86 ⫾ 35% (n ⫽
8; p ⬍ 0.05) in pyramidal cells from BALB/c mice (Fig. 2A).
Within 90 min, the firing rate returned to control values and was
no longer significantly different from the firing rate before
h/rCRF application in C57BL/6N mice (2 ⫾ 6%; n ⫽ 7) and
Figure 1. A, Representative intracellular recordings from CA1 pyramidal neurons in hip-
pocampal slices from C57BL/6N mice and BALB/c mice showing responses to 600 msec depo-
larizing current pulses. B, Number of spikes elicited in 100 msec fragments during a single
depolarizing (depol.) current pulse (600 msec, 1 nA). C, Plot of the number of spikes elicited by
a 600 msec depolarizing pulse versus stimulus (stim.) intensity.
702 • J. Neurosci., January 15, 2003 • 23(2):700 –707 Blank et al. •CRF-Mediated Excitability in Mouse Hippocampus
BALB/c mice (4 ⫾ 7%; n ⫽ 8) (Fig. 2A). In CA1 hippocampal
neurons from both mouse strains, the h/rCRF effect was an-
tagonized by the CRF receptor antagonist [Glu
11,16
] astressin
(Fig. 2B).
In subsequent experiments, we investigated the underlying
second-messenger pathways activated by h/rCRF to increase the
neuronal excitability in mouse hippocampus. When slices were
preincubated with the selective and cell-permeable PKA inhibitor
H-89, the firing rate of hippocampal neurons from C57BL/6N
mice was not significantly enhanced by h/rCRF (7 ⫾ 5%; n ⫽ 6;
p ⫽ NS) (Fig. 3A). In contrast, after the H-89 treatment, h/rCRF
still enhanced the neuronal activity of hippocampal neurons
from BALB/c mice by 55 ⫾ 10% (n ⫽ 5; p ⬍ 0.05) (Fig. 3B).
When hippocampal slices from BALB/c mice were preincubated
with BIS-I, a highly selective cell-permeable PKC inhibitor, sub-
sequent h/rCRF application did not significantly increase the
neuronal firing rate (4 ⫾ 2%; n ⫽ 5; p ⫽ NS) (Fig. 3D). In
contrast, after BIS-I treatment, h/rCRF application still enhanced
neuronal spiking in CA1 cells from C57BL/6N mice by 52 ⫾ 9%
(n ⫽ 6; p ⬍ 0.05) (Fig. 3C). In these mice, bath application of the
potent PKC activator PDBu increased the spiking behavior of
hippocampal neurons by 79 ⫾ 24% (n ⫽ 5; p ⬍ 0.05) (Fig. 3E).
Under basal conditions, PKA activity, as measured by the
phosphorylated state of a PKA-specific target peptide, was lower
in hippocampal brain slices from C57BL/6N mice than in hip-
pocampal brain slices from BALB/c mice. After h/rCRF treat-
ment, PKA activity in hippocampal slices from C57BL/6N mice
was increased, whereas it was decreased in hippocampal slices
from BALB/c mice compared with the corresponding PKA activ-
ities in control slices (Fig. 4A). Because membrane translocation
of PKC is considered to be an indicator of PKC activation (Kraft
and Anderson, 1983), we assayed PKC activity in the membrane-
bound fraction of hippocampal slice homogenates. After h/rCRF
incubation of slices, PKC activity was apparent only in hip-
pocampal slices of BALB/c mice (Fig. 4B), with no detectable
PKC activity in hippocampal slices of C57BL/6N mice. The ob-
served differences in the activation of second-messenger path-
ways after h/rCRF application can be attributed to variations in
the abundance of G-proteins. However, using immunoblots, we
Figure 2. Effect of h/rCRF on neuronal spiking of BALB/c mouse CA1 pyramidal cells elicited
by 600msec depolarizing currentpulses. A, Traces were sampled before, during, and30 and 90
minafter h/rCRF (250n
M,10 min) application.B,Recordings were madebeforeand 20 minafter
coapplicationof h/rCRF(250n
M,10 min)and[Glu
11,16
]astressin (1
M)over aperiodof10 min.
Pulse intensity was kept constant during each experiment; holding potential, ⫺65 mV.
Figure 3. Effectof thePKC inhibitor BIS-I and ofthe PKAinhibitor H-89 on h/rCRF-mediated
modulation ofexcitability. Representative recordingsin CA1 pyramidal cells from C57BL/6N (A,
C ) and BALB/c (B, D) mice showing the effect of 250 n
M h/rCRF applied over a period of 20 min
after preincubation with BIS-I (1.2
M, 1 hr) or H-89 (10
M, 3 hr). E, Spiking behavior of CA1
pyramidal cells from C57BL/6N before and during bath application of PDBu (100 n
M). Pulse
intensity was kept constant during each experiment.
Figure 4. PKA and PKC activity in hippocampal slices of C57BL/6N and BALB/c mice. Hip-
pocampal slices were incubated in either 250 n
M h/rCRF (30 min) or aCSF (30 min, as control).
Partially purified homogenates of these slices (n ⫽ 11) from six animals were tested for the
ability tophosphorylate a PKA-specific(L-R-R-A-S-L-G; Kemptide) (A) ora PKC-specific (P-L-S-
R-T-L-S-V-A-A-K) ( B)peptidic substrate ina nonradioactive assay.Identical amountsofprotein
were used for each sample.
Blank et al. •CRF-Mediated Excitability in Mouse Hippocampus J. Neurosci., January 15, 2003 • 23(2):700 –707 • 703
did not observe any significant differences in the abundance
of G
s
-, G
I
-, and G
q/11
-proteins (Fig. 5
A). In subsequent ex-
periments, we analyzed CRF receptor-mediated activation of
G-proteins in hippocampal membrane suspensions. After
h/rCRF application, the nonhydrolyzable GTP analog
32
P-
GTP-AA binds to the GTP-binding site of activated G-protein
␣
-chains and forms a stable complex, which can be identified
with specific G
␣
antibodies (Offermanns et al., 1991). Thus, spe
-
cific activation of individual G-proteins can be demonstrated. In
hippocampal membranes of C57BL/6N mice, h/rCRF induced
activation of G
s
,G
i
, and G
q/11
with an order of potency G
s
⬎
G
q/11
⬎ G
i
, whereas in hippocampal membranes of BALB/c mice,
only stimulation of G
q/11
was detectable after h/rCRF treatment
(Fig. 5B,C).
To further delineate the impact of the observed different
h/rCRF-mediated signaling pathways on learning and memory,
mice were subjected to contextual fear conditioning, a
hippocampus-dependent associative learning paradigm (Kim
and Fanselow, 1992; Phillips and LeDoux, 1992, 1994). When
BALB/c mice received a bilateral h/rCRF injection intracerebrov-
entricularly (n ⫽ 7) (Fig. 6A) and were trained 2 hr after the
injection, contextual fear was significantly enhanced compared
with naive ( p ⬍ 0.05; n ⫽ 9) (Fig. 6A) and vehicle-treated ( p ⬍
0.01; n ⫽ 30) animals (Fig. 6 A). This h/rCRF effect was prevented
by either [Glu
11,16
] astressin (n ⫽ 7) or BIS-I (n ⫽ 7). Both
compounds had no effect when applied alone (Fig. 6 A). To ex-
clude the possibility that h/rCRF was acting via a brain structure
that has projections to the hippocampus, h/rCRF and BIS-I were
administered locally into the dorsal hippocampus. Contextual
fear was also significantly elevated when h/rCRF was injected
intrahippocampally ( p ⬍ 0.05; n ⫽ 6) (Fig. 6A). BIS-I had no
effect when administered intrahippocampally alone (n ⫽ 5) but
abolished the h/rCRF-mediated enhancement of conditioned
fear (n ⫽ 6; p ⫽ NS) (Fig. 6 A). In C57BL/6N mice, freezing was
not significantly changed when h/rCRF was injected 2 hr (n ⫽ 15;
p ⫽ NS) (Fig. 6B) before the training session. However, injection
of PDBu 2 hr before the training (n ⫽ 9) significantly enhanced
contextual fear compared with the contextual fear of naive ( p ⬍
0.05; n ⫽ 9) (Fig. 6B) and vehicle-treated ( p ⬍ 0.05; n ⫽ 27)
animals (Fig. 6B). There was no significant change of contextual
fear after injection of the inactive isomer 4
␣
-phorbol ( p ⫽ NS;
n ⫽ 4) (Fig. 6B).
Discussion
In this study, we provide evidence that signal processing of
h/rCRF in mouse hippocampus was mediated through two dif-
ferent signal transduction pathways. Slice experiments revealed
that h/rCRF increased CA1 hippocampal neuronal activity via
PKC in the hippocampus of BALB/c mice and via PKA in the
hippocampus of C57BL/6N mice. Hippocampus-dependent
learning evaluated by context-dependent fear conditioning was
improved only in BALB/c mice after h/rCRF injection but not in
C57BL/6N mice. Western blots from mouse hippocampal mem-
brane proteins showed identical amounts of the relevant
G-protein subunits in both mouse strains. However, application
of h/rCRF induced activation of G
q/11
in the hippocampus of
BALB/c mice and G
s
,G
q/11
, and G
i
in the hippocampus of
C57BL/6N mice. h/rCRF increased neuronal excitability in the
hippocampus of both mouse strains but improved fear condi-
tioning only in BALB/c and not in C57BL/6N mice. Thus, it
might be concluded that the h/rCRF-induced increase in neuro-
nal activity is not sufficient to enhance fear conditioning but that
the stimulation of specific intracellular signaling cascades is also
required. In support of this hypothesis, we observed recently that
inhibition of hippocampal Ca
2⫹
/calmodulin-dependent kinase
II (CaMKII) prevents stress-mediated facilitation of fear condi-
tioning with no effect on primed hippocampal LTP (Blank et al.,
2002). This observation implies that facilitation of neuronal ac-
tivity was necessary along with activation of CaMKII to enhance
fear conditioning.
In mouse hippocampus, CRFR1 was reported to be the pre-
dominant CRF receptor subtype (Van Pett et al., 2000). However,
we cannot conclude whether the differences in G-protein activa-
tion result from the different coupling of a single receptor sub-
Figure 5. h/rCRF-induced activation of G
s
-, G
I
-, and G
q/11
-proteins. A, Basal levels of G
s
,G
i
,
and G
q/11
in hippocampal membrane fractions from C57BL/6Nand BALB/c mice. The bar graph
summarizesWestern blot data(mean⫾ SEM)of three independentexperimentseachwithfive
animals per mouse strain. B, Autoradiograph of h/rCRF-induced photolabeling of G
␣
subunit
subtypes from hippocampal membranes of C57BL/6N (n ⫽ 30) and BALB/c (n ⫽ 30) mice.
Membranes were incubated with
32
P-GTP-AA in the presence and absence of h/rCRF (100 nM),
followed by UV cross-linking and immunoprecipitation of the G
␣
subunit subtypes using spe-
cific antibodies. Proteins were resolved by SDS-PAGE, followed by autoradiographic visualiza-
tion. C, Bar graph summarizing autoradiograph data. *p ⬍ 0.05 indicates statistically signifi-
cant differences.
704 • J. Neurosci., January 15, 2003 • 23(2):700 –707 Blank et al. •CRF-Mediated Excitability in Mouse Hippocampus
type or the different coupling in combination with differences in
the distribution profile of CRF receptor subtypes in the hip-
pocampus of both mouse strains.
All of the known effects of CRF in the rat hippocampus in-
volve receptor-coupled activation of G
s
and adenylate cyclase and
an increase in cellular levels of cAMP (Chen et al., 1986; Battaglia
et al., 1987; Pihoker et al., 1992; Haug and Storm, 2000). This is in
agreement with the activation of G
s
in hippocampi of C57BL/6N
mice. However, it was reported that h/rCRF also activates the
phospholipase C (PLC)–PKC-pathway in rat Leydig cells (Ulisse
et al., 1990), in cultured rat astrocytes (Takuma et al., 1994), in rat
cerebellum (Miyata et al., 1999), and in rat cerebral cortex
(Grammatopoulos et al., 2001). In addition, Malenka et al.
(1986) reported that activation of PKC markedly reduces accom-
modation of neuronal spiking in rat hippocampal pyramidal
cells. Both aspects together are in agreement with our conclusion
that, in BALB/c mice, G
q/11
-dependent PKC activation mediated
the h/rCRF-induced increase of neuronal activity. Surprisingly,
PKA activity was reduced in hippocampal slices from BALB/c
mice during application of h/rCRF. This effect might be initiated
by G
q/11
stimulation, which has been shown to be associated with
an increase of the abundance of G-protein
␥
subunits. These
subunits inhibit type I adenylyl cyclase and thereby decrease PKA
activity (Taussig et al., 1993; Chen et al., 1997). Activation of
G
q/11
,G
s
, and G
i
, as observed in hippocampi of C57BL/6N mice,
synergistically stimulates adenylyl cyclase type 2 (Lustig et al.,
1993), thus also increasing the cAMP formation. In the
membrane-bound fraction of hippocampal slice homogenates
prepared from C57BL/6N mice, no PKC activity was detected
after h/rCRF application. We did not detect any significant con-
tribution of PKC to the h/rCRF-induced increase in neuronal
spiking behavior of CA1 pyramidal cells in C57BL/6N mice.
However, the treatment of hippocampal slices from C57BL/6N
mice with BIS-I and the H-89 treatment of slices from BALB/c
mice showed the tendency to reduce the spiking rate compared
with controls. This observation suggests that, in both mouse
strains, neuronal activity is sensitive to changes in PKA and PKC
activity.
Our observation that only PKA was ac-
tivated in hippocampal slices of
C57BL/6N mice during application of
h/rCRF might be because receptors with
dual signaling properties often stimulate
different pathways with different effica-
cies. A
3
adenosine receptors, for example,
interact with G
i
-proteins and, to a lesser
extent, with G
q/11
-proteins in CHO cells
(Palmer et al., 1995). These receptors were
shown to inhibit adenylyl cyclase in all cell
types tested, whereas stimulation of PLC
was cell type dependent. Although acti-
vated CRF receptors coupled to G
q/11
in
hippocampal membranes of C57BL/6N
mice, h/rCRF neither activated PKC in
hippocampal slices nor enhanced the con-
ditioned fear response. This result is sur-
prising because, in experiments using
PDBu, we demonstrated that hippocam-
pal neuronal excitability and conditioned
fear of C57BL/6N mice was enhanced by
activation of PKC. In contrast, h/rCRF
also stimulated G
q/11
in hippocampal
membranes of BALB/c mice and im-
proved hippocampus-dependent learning via activation of PKC
in this mouse strain. Similar results were reported by Fordyce et
al. (1985), who found that stimulation of hippocampal PKC ac-
tivity enhances contextual learning, as determined by the fear
conditioning task in DBA mice. In the hippocampus of C57BL/6J
mice, a PKA-dependent period for contextual memory consoli-
dation develops between 1 and 3 hr after training (Bourtchou-
ladze et al., 1998). Considering the activation of the PKA system
in the hippocampus of C57BL/6N mice during h/rCRF applica-
tion, it is surprising that h/rCRF did not facilitate contextual fear
conditioning in C57BL/6N mice. In a recent study, the crucial
temporal relationship between PKA inhibition and training nec-
essary to produce impairment of the consolidation of fear mem-
ory was demonstrated (Bourtchouladze et al., 1998). A narrow
time window exists for PKA inhibition before the training. When
mice are treated with PKA inhibitor 20 –30 min before contextual
conditioning, they show dramatic amnesia. However, inhibition
of PKA 3 hr before training does not affect retention 24 hr after
training. Thus, in the present study, h/rCRF might have had no
effect on long-term contextual memory in C57BL/6N mice be-
cause PKA was not activated within the decisive time window.
To summarize, we demonstrated that h/rCRF activated at
least two different signaling cascades in mouse hippocampus, the
PLC–PKC pathway (via interaction with G
q/11
) and the cAMP–
PKA pathway (via interaction with G
s
,G
q/11
, and G
i
). Future
experiments will have to determine whether hippocampal CRF
receptors can switch their coupling between different G-protein
subunits triggered by the activation of specific signaling events
such as protein phosphorylation (Lawler et al., 2001). Alterna-
tively, the observed multisignaling activity of h/rCRF might be
caused by the activation of different types of CRF receptors cou-
pling to G
s
and to G
q/11
to initiate independent activation of
adenylyl cyclase and PLC. These findings suggest a possible inter-
mediary role for differential CRF receptor coupling in determin-
ing distinct endocrine and behavioral stress responses. In support
of this hypothesis, both mouse strains are differentially respon-
sive to neurogenic, psychogenic, and systemic stress, with a
greater stress reactivity and adrenal glucocorticoid release in
Figure 6. Effect of h/rCRF on context-dependent fear conditioning of BALB/c ( A) and C57BL/6N ( B) mice injected with aCSF,
h/rCRF, [Glu
11,16
] astressin,PDBu, or 4
␣
-phorbol 2hr before the training asindicated. For combinedtreatment, [Glu
11,16
] astres
-
sin and BIS-I were given 15 min before h/rCRF application. Freezing was measured in the retention test performed 24 hr after
training. Injectionswere performedintracerebroventricularly (i.c.v.)or intrahippocampally(i.h.) asindicated. *p ⬍ 0.05 indicates
statistically significant differences versus vehicle-injected animals and naive animals.
Blank et al. •CRF-Mediated Excitability in Mouse Hippocampus J. Neurosci., January 15, 2003 • 23(2):700 –707 • 705
BALB/cByJ mice than in C57BL/6ByJ mice (Anisman et al.,
2001). Our results add to the existing data showing that the ge-
netic background can affect the behavioral phenotypes of genet-
ically modified mice generated for elucidating the molecular basis
of learning and memory (McNamara et al., 1998; Dobkin et al.,
2000; Dockstader and van der Kooy, 2001). In view of the contri-
bution of the hippocampus to numerous forms of learning (for
review, see Kesner et al., 2000; Kim and Baxter, 2001; Maren,
2001) and the fact that h/rCRF represents an early signal in the
neuroendocrine response to stress (Koob and Bloom, 1985), our
present findings may represent an important step toward under-
standing the cellular and molecular processes underlying inter-
strain variability concerning the impact of stress on learning and
memory (Brush et al., 1988; Francis et al., 1995; Palmer and
Prinz, 1999).
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• 23(2):700 –707 • 707
