Loss of the limbic mineralocorticoid receptor
impairs behavioral plasticity
Stefan Berger*, David P. Wolfer, Oliver Selbach, Heike Alter*, Gitta Erdmann*, Holger M. Reichardt*,
Aisa N. Chepkova, Hans Welzl, Helmut L. Haas, Hans-Peter Lipp, and Gunther Schutz*
*German Cancer Research Center, Division Molecular Biology of the Cell I, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany; Institute of Anatomy,
University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland; and Department of Neurophysiology, Heinrich-Heine-University,
40001 Dusseldorf, Germany
Edited by Bruce S. McEwen, The Rockefeller University, New York, NY, and approved November 10, 2005 (received for review May 10, 2005)
Corticosteroid action in the brain is mediated by the mineralocor-
ticoid (MR) and the glucocorticoid (GR) receptor. Disturbances in
MR- and GR-mediated effects are thought to impair cognition,
behavior, and endocrine control. To assess the function of the
limbic MR in these processes, we inactivated the MR gene in the
forebrain of the mouse using the CreloxP-recombination system.
We screened the mice with a limbic MR deficiency in various
learning and exploration tests. The mutant mice show impaired
learning of the water-maze task and deficits in measures of
working memory on the radial maze due to behavioral persever-
ance and stereotypy. They exhibit a hyperreactivity toward a novel
object but normal anxiety-like behavior. The behavioral changes
are associated with abnormalities of the mossy fiber projection and
an up-regulation of GR expression in the hippocampus. Adult
mutant mice show normal corticosterone levels at circadian trough
and peak. This genetic model provides important information
about the consequences of a permanently altered balance between
limbic MR and GR, with implications for stress-related neuroendo-
crine and neuropsychiatric diseases.
behavior neuroendocrine synaptic plasticity
C orticosteroids influence neuronal excitability and plasticity,
neurogenesis and neuronal death, and neuroendocrine con-
trol and behavioral responses (1, 2). Their actions are mediated
by two ligand-activated transcription factors, the mineralocor-
ticoid (MR) and the glucocorticoid (GR) receptors. The MR
binds corticosterone with a 10-fold higher affinity than the GR
and, in addition, binds the mineralocorticoid aldosterone with
equal affinity (3). The GR is expressed throughout the brain, in
neurons and glial cells, and is most abundant in hypothalamic
neurons producing corticotropin-releasing hormone. The MR is
expressed in neurons only, with highest abundance in the limbic
system, in particular within the hippocampus (4). The limbic MR
strongly responds to corticosterone, because the prereceptor
protection mechanism achieving aldosterone selectivity in tight
epithelia is not present (5). Thus, MR and GR, whose DNA
binding domains are almost identical, form a binary response
system for corticosterone that is able to regulate differentially
two overlapping networks of target genes (3). The adrenal
release of corticosterone is controlled by the hypothalamic
pituitaryadrenal (HPA) axis that shows circadian activity, with
low activity at the onset of the resting phase and high activity at
the onset of the activity phase, and a strong increase in activity
after stress. The high-affinity MR is always substantially occu-
pied, even at basal levels of HPA-axis activity. High concentra-
tions of corticosterone, at circadian peak or after stress, pro-
gressively saturate the GR (3).
Lesioning and electrical stimulation studies suggest an overall
inhibitory influence of the hippocampus on HPA-axis activity
(6), whereas the amygdala appears to have an excitatory influ-
ence (7). Intracerebroventricular or intrahippocampal adminis-
tration of a MR antagonist in rats elevates basal trough levels and
the diurnal rise of plasma corticosterone (810) suggesting that
www.pnas.orgcgidoi10.1073pnas.0503878102
the hippocampal MR is involved in the maintenance of basal
HPA-axis activity throughout the circadian cycle. Intrahip-
pocampal application of a GR antagonist suppresses adrenal
corticosterone release under conditions in which MR antago-
nists enhance HPA-axis activity (10). Such effects are in agree-
ment with electrophysiological data showing opposite effects of
MR and GR activation on excitability and excitatory outflow of
the hippocampus (11). Electrophysiological studies at the cellu-
lar level in the hippocampus on Ca2 influx and responsiveness
to transmitters revealed that conditions of predominant MR
activation are implicated in the maintenance of excitability, so
that steady excitatory input to the hippocampal CA1 area results
in considerable excitatory hippocampal output (11). Synaptic
strength is enhanced [long-term potentiation (LTP)] when MR
is almost completely activated and GR is only partially activated.
Subsequent complete occupation of GR (and MR), e.g., after
stress, decreases the hippocampal output and the synaptic effi-
cacy in the CA1 region (12).
Chronic absence or chronic overexposure to corticosterone
results in structural changes of the hippocampus, indicating that
corticosterone-dependent gene expression is crucial for hip-
pocampal integrity (1). The dentate gyrus shows neurogenesis
also during adulthood, which can be blocked by chronic stress
and adrenal steroids (13).
The use of receptor-specific antagonists suggests that MR- and
GR-mediated effects are important for cognitive and behavioral
processes as well. Memory in spatial and avoidance tasks is
impaired in the absence of GR activation after the learning task
(1416), whereas MR modulates ongoing behavioral activity.
MR antagonists are only effective during, but not after, the
training session (15, 16). Thus, activation of MR is thought to be
implicated in memory acquisition (appraisal of information and
response selection), whereas activation of GR is thought to be
related to consolidation of acquired information (17).
NEUROSCIENCE
The role of the limbic MR in neuronal excitability and
plasticity as well as in HPA-axis regulation and behavior has so
far been investigated by pharmacological means only; that is, by
changing ligand accessibility using MR antagonists or adrenal-
ectomy in combination with low corticosterone substitution that
should predominantly occupy MR. The conclusions drawn from
such studies depend on antagonist specificity and application of
appropriate corticosterone doses. We used a genetic approach to
inactivate the MR gene in the forebrain of the mouse to
investigate whether a permanently altered balance between MR
and GR will disturb basal circadian HPA-axis activity and
behavior. Indeed, mice lacking limbic MR were impaired in
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: GR, glucocorticoid receptor; HPA, hypothalamicpituitaryadrenal; LTP,
long-term potentiation; MR, mineralocorticoid receptor; Pn, postnatal day n; PVN, nucleus
paraventricularis.
To whom correspondence should be addressed. E-mail: g.schuetz@dkfz.de.
2005 by The National Academy of Sciences of the USA
PNAS January 3, 2006 vol. 103 no. 1 195200
hippocampus-dependent learning procedures and showed mas-
sively increased object exploration. However, they showed nor-
mal anxiety-like behavior and normal basal circadian HPA-axis
activity.

Materials and Methods

Generation of Conditional Mutant Mice. To flank exon 3 of the
mouse Nr3c2 gene encoding the MR by loxP sites, we used
homologous recombination in embryonic stem cells. For more
details, see Supporting Methods, which is published as supporting
information on the PNAS web site. For the conditional inacti-
vation of the MR in the forebrain, we used CaMKCre-transgenic
mice (18). The MRf lox/f loxCaMKCre mice (MRCaMKCre) and
their control (MRf lox/f lox) littermates were obtained by breeding
MRf lox/f lox with MRf lox/wtCaMKCre mice. The genetic back-
ground comprised a mix of C57BL6, 129Ola, and FVBN. The
use of Cre deleter mice (19) to recombine the MRf lox allele in the
germ line and the breeding of the obtained MRnull allele to
homozygosity (MRnull/null mice) resulted in postnatal lethality
and a phenotype identical to MR knockout mice (data not
shown).

Immunohistochemistry and Timm Staining. Adult (4 months old)

and 12-day-old mice were transcardially perfused with 4%
paraformaldehyde. The dissected brains were postfixed over-
night at 4C in 4% paraformaldehyde and cut at a thickness of
50 m on a vibratome (Leica). At postnatal day (P) 0 and P6 the
brains were dissected without prior paraformaldehyde perfu-
sion. The floating sections were processed for immunohisto-
chemical detection by using the VECTASTAIN ABC system
(Vector Laboratories) and diaminobenzidine (Sigma) incuba-
tion. We used the following primary antibodies: polyclonal
anti-MR (4), polyclonal anti-Cre (20), polyclonal anti-GR (Santa
Cruz Biotechnology), and polyclonal anti-CRH (Abcam).
Hippocampal mossy fiber synaptic fields were visualized by
Timm staining (21). Adult mice (4 months old) were perfused
transcardially with phosphate-buffered 1% sodium sulfide and
3% glutaraldehyde. After postfixation and cryoprotection, Fig. 1. Generation of mice lacking MR in the forebrain. (a) Organization of
40-m cryosections were obtained and developed in a solution the mouse MR wild-type locus around exon 3. This exon was flanked with loxP
containing Arabic gum, hydroquinone, citric acid, and silver sites by homologous recombination in embryonic stem cells. Transient expres-
nitrate. sion of Cre recombinase in embryonic stem cells resulted in removal of the
selection cassette (neomycin resistancethymidine kinase), generating the
Corticosterone Measurement. One week before the experiment, MRflox allele. This allele encodes an active MR protein but is recombined in any
adult mice (4 months old) were separated and housed individ- cell expressing Cre recombinase. Recombination results in deletion of exon 3
and thereby in inactivation of this allele (MRnull allele). The scheme shown
ually (light on 08:0020:00). To determine the plasma cortico-
depicts the wild-type locus, the targeting vector, and the resulting alleles.
sterone level at circadian trough and peak, blood sampling was Triangles represent loxP sites, the filled box represents exon 3, open boxes
performed in the morning (09:0010:00) and the evening represent the probes used for Southern blot analysis, and the arrows represent
(19:0020:00) by bleeding after decapitation, with the time from primers used for genotyping by PCR. E, EcoRV; S, SpeI. (bp) MR protein
first handling of the animal to completion of bleeding not expression in control animals (b, e, h, k, and n) and Cre expression (d, g, j, m,
exceeding 45 s (n 78 per genotype, gender, and time point). and p) and the corresponding loss of MR expression (c, f, i, l, and o) in
For evaluation of the stress release of corticosterone, blood MRCaMKCre mice as revealed by immunohistochemistry on vibratome sections
samples were taken in the morning (09:0010:00) immediately of 12-day-old animals. Depicted are the regions that show highest expression
after 40 min of restraint stress for which animals were placed in of MR in controls: lateral septum and indusium griseum (bd), CA1 (eg), CA2
and CA3 (hj), dentate gyrus (km), and central amygdala (np).
50-ml conical tubes (plastic conical tube with the bottom re-
moved) (n 1012 per genotype and gender). Plasma cortico-
sterone was measured by using a commercially available RIA kit
the EthoVision system (Noldus Information Technology) at an
(MP Biomedicals). Results are expressed as mean SEM. The
image frequency of 4.2 per s. Raw data were transferred to
measurements were analyzed by using unpaired, two-tailed
WINTRACK 2.4 (www.dpwolfer.chwintrack) for off-line analysis.
Students t test.
For statistical analysis, see Supporting Methods.
Behavioral Studies. One week before the experimental period,
Results
male and female mice (47 months old) were transferred to
standard single-mouse cages, maintained at a 12:12-h inverted Generation of Mice Lacking MR in the Forebrain. Using homologous
cycle (light on 20:0008:00), and tested during the dark period. recombination in mouse embryonic stem cells, we generated a
The home cage rack was brought to the test room at least 30 min modified MR allele (MRf lox allele), in which exon 3, encoding
before each experiment. The behavioral tests were performed as the first zinc finger of the MR DNA binding domain, was flanked
described in refs. 22 and 23. During all of the tests, except the by loxP sites (Fig. 1a). To generate mice lacking MR in the
conditioned taste aversion, animals were video-tracked by using forebrain, we crossed mice harboring the MRf lox allele to

196 www.pnas.orgcgidoi10.1073pnas.0503878102 Berger et al.


Fig. 2. Altered mossy fiber projections and hippocampal GR up-regulation
in adult MRCaMKCre mice. (a and b) Timm stain labeling of the Zn2-containing
mossy fiber terminals. Compared with control animals (a), MRCaMKCre mice
showed an aberrant layout of mossy fiber projections (b): variable excess of
supragranular boutons in the dentate gyrus (single arrowheads), extension of
the infrapyramidal mossy fiber field to the tip of CA3 and beyond into CA2
(double arrowheads), and a fuzzy distal boundary of the suprapyramidal field
with boutons spreading across the pyramidal cell layer into CA2 and CA1
(asterisk). (c and d) Hippocampal GR expression as revealed by immunohisto-
chemistry. GR is up-regulated in the cornu ammonis of adult MRCaMKCre mice
(d) compared with controls (c), most apparently in CA2 and CA3.
BAC-transgenic mice expressing Cre recombinase under the
control of the regulatory elements of the mouse CaMKII gene
(18) to obtain MRf lox/f loxCaMKCre (MRCaMKCre) mice. The MR
protein is detectable as early as embryonic day 16.5. During pre-
and postnatal development, as well as during adulthood, MR
protein detection in the forebrain of the mouse is restricted to
the limbic system (4). We found that the Cre transgene we used
is already expressed in the limbic system at embryonic day 16.5
(see Fig. 6a, which is published as supporting information on the
PNAS web site). Therefore, we checked the loss of MR immu-
noreactivity in MRCaMKCre mice at P0, P6, and P12. This analysis
revealed that MR protein expression is already reduced at P0 and
is almost lost at P6 (see Figs. 6b and 7, which are published as
supporting information on the PNAS web site). No immunore-
activity is found in the limbic system of MRCaMKCre mice neither
at P12 (Fig. 1 bp) nor in the adult (data not shown). The careful
examination of the tissue sections on high power did not reveal
any MR expressing neurons that might have escaped from
recombination.
In rats, expression of MR is also found in the hypothalamus
(24). Because the nucleus paraventricularis (PVN) is an impor-
tant site for feedback inhibition of the HPA axis, we analyzed this
region for expression of MR. We could detect neither the MR
protein nor the MR mRNA in the PVN of the mouse (see Fig.
8, which is published as supporting information on the PNAS
web site). Nevertheless, the PVN shows high expression of Cre
recombinase and therefore recombination of the MR gene is
expected.
Inactivation of the MR gene in the forebrain did not impair
survival. MRCaMKCre mice were visually indistinguishable from
control littermates and performed normally when tested for
sensory (acoustic startle profile and prepulse inhibition) and
motor (rotarod) function (data not shown). In adult MRCaMKCre
mice, a gross survey of the cornu ammonis and the dentate gyrus
of the hippocampus by using Nissl staining showed no conspic-
uous change in cell number and density (data not shown).
However, analysis of the projection of granule cell axons to the
CA3 field of the hippocampus revealed an aberrant layout of the
mossy fibers in MRCaMKCre mice compared with control animals
(Fig. 2 a and b). Determination of the hippocampal GR expres-
Berger et al.
Fig. 3. Normal basal circadian HPA-axis activity in MRCaMKCre mice. (a and b)
Plasma corticosterone levels of adult male (a) and female (b) MRCaMKCre mice
and their control littermates at diurnal trough (morning) and peak (evening),
as well as after 40 min of restraint stress, showed no significant differences
between both genotypes.
sion by immunohistochemistry revealed an up-regulation of GR
in the cornu ammonis of MRCaMKCre mice compared with
controls (Fig. 2 c and d). In the PVN, a site without detectable
MR expression, the expression of GR is not changed (Fig. 8).
Analysis of HPA-Axis Activity and Hippocampal Synaptic Plasticity.
Lesions of the hippocampus raise resting levels of glucocorti-
coids and stress-induced responses in glucocorticoid secretion
(6). Studies using MR antagonists suggest that the hippocampal
MR is involved in the maintenance of basal circadian HPA-axis
activity (810). Therefore, we determined the basal HPA-axis
activity in MRCaMKCre mice by measuring the corticosterone
levels at the diurnal trough in the morning and at the diurnal
peak in the evening. In addition, we determined the release of
corticosterone after restraint stress. Males and females were
analyzed separately, because female mice display higher plasma
corticosterone levels than male mice. The corticosterone mea-
surements in adult mice revealed no significant differences
between MRCaMKCre mice and littermate control animals, nei-
ther at diurnal trough and peak nor after restraint stress (Fig. 3
a and b). In the PVN, an important site for feedback inhibition
of the HPA-axis activity after stress, MRCaMKCre mice showed
under basal conditions no change in CRH mRNA (Fig. 8).
The CA1 region is the major source of hippocampal output
conferring inhibitory constraints on HPA-axis activity (11). The
analysis of the basic properties of synaptic transmission in the
CA1 region of hippocampal slices from MRCaMKCre and control
mice revealed no significant differences in either stimulus input
output relations of synaptic responses or paired pulse facilitation
NEUROSCIENCE
between both groups (see Fig. 9 a and b, which is published as
supporting information on the PNAS web site). High- and
low-frequency stimulation of the Schaffer collateral axons in the
CA1 region of hippocampal slices from MRCaMKCre mice reliably
induced LTP and long-term depression, respectively, of synaptic
transmission not significantly different from that obtained from
control mice (Fig. 9 c and d).
Impaired Learning Behavior of MRCaMKCre Mice. To investigate the
learning abilities of the MRCaMKCre mice, we tested two inde-
pendent cohorts of animals matched for genotype, gender, and
age (in total 64 animals) in several learning paradigms. We
assessed the spatial learning of MRCaMKCre mice in the Morris
water maze in which mice must use visual cues to find a platform
that is hidden in a constant location beneath the surface of
opaque water. Mice were trained for 18 trials for a first platform
location (place acquisition) followed by 12 trials with a new
platform location in the opposite quadrant (place reversal), with
trial 19 serving as probe trial. In this water-maze task, the
MRCaMKCre mice showed a clear acquisition deficit. They showed
reduced swim speed and an increased use of inappropriate
PNAS January 3, 2006 vol. 103 no. 1 197
strategies, such as passive floating and aimless swimming. Over-
all escape performance was impaired in MRCaMKCre mice ac-
cording to all evaluated measures (see Table 1, which is pub-
lished as supporting information on the PNAS web site). Despite
reduced average performance, they reached the same perfor-
mance as controls at the very end of training (Fig. 4a). Upon
relocation of the platform, both groups displayed a transient
increase of escape latencies, indicating that spatial learning had
occurred. MRCaMKCre mice showed normal retention of spatial
memory in the probe trial, also according to the annulus
crossings of the trained quadrant, the most stringent measure of
spatial selectivity (Fig. 4 b and c). During the reversal phase,
MRCaMKCre mice were more strongly affected by the relocation
of the platform and again performed inferior to controls,
reaching performance levels similar to controls only at the very
end (Fig. 4a and Table 1). During reversal training, a few
MRCaMKCre mice showed such intense perseverative searching
for the old goal site that this was directly evident to the observer
(Fig. 4d).
Next, we tested the MRCaMKCre mice on the eight-arm radial
maze. At the beginning of a working memory procedure all arms
were baited and the mice were permitted to collect and consume
all baits. Reentering a previously visited arm was counted as an
error. Aborted choices and bait neglect errors were as infrequent
in MRCaMKCre mice as in controls, indicating normal adaptation
to the test situation. As judged by the number of correct choices
during the first eight arm visits, the average performance of
MRCaMKCre mice was significantly impaired (Fig. 4e). Counting
reentry errors during the entire session revealed that unlike
controls, MRCaMKCre mice were unable to overcome their ten-
dency to visit the same arms over time (Fig. 4f ). As in the water
maze, MRCaMKCre mice showed an increased tendency to be
passive. Control animals made, as typically observed in mice,
strong use of regular response patterns to increase their effi-
ciency. MRCaMKCre mice did this to a lesser degree and showed
less consistent choice patterns (Table 1).
We analyzed the performance of MRCaMKCre mice in associa-
tive learning tests involving aversive stimuli. In the two-way
avoidance test, mice are placed in a two-chamber box and taught
to avoid an electric shock preceded by a warning light by running
into the opposite compartment. In this test, MRCaMKCre mice
were indistinguishable from control animals and showed the
same response latency and locomotor activity (data not shown).
Fig. 4. Impaired learning of MRCaMKCre mice in a water-maze place navigation
In the conditioned taste aversion test, mice learn to associate a task with reversal and in a working memory procedure on the radial maze. (a)
novel taste with nausea, and as a consequence avoid drinking Place navigation: Training performance (six trials per day, blocks of two trials,
fluid with this specific taste. During the saccharin-water choice cumulative search error in ms). Despite reduced average performance, MRCaMKCre
tests after conditioning, MRCaMKCre and control mice showed mice (n 33, controls 31) showed significant learning at indistinguishable rates
indistinguishable strong saccharin avoidance and similar subse- compared with controls during place acquisition as well as during place reversal.
quent extinction (data not shown). During subsequent cue navigation, the tested subset of MRCaMKCre mice (n 16,
controls 16) outperformed the control mice (which showed a transient wall-
MRCaMKCre Mice Show Increased Reactivity to a Novel Object. We hugging response) during the first trial block, but the performances became
indistinguishable during the second block. (b) Place navigation: Probe trial (%
assessed the exploratory behavior of MRCaMKCre mice in five
time in quadrant). Both groups spend significantly more time in the trained
tests differing in the overall degree of novelty and aversiveness quadrant. (c) Place navigation: Probe trial. Both groups crossed the trained goal
of the environment. In all tests, zones can be defined within the annulus significantly more often than control annuli in the adjacent quadrants.
arenas that allow the quantification of exploratory activity and (d) Place navigation: Path plots of trials 19 30 (days 4 and 5, place reversal). A
fear-related behavior. MRCaMKCre mouse showing perseverative searching for the old goal site. Plots of
In the open field, MRCaMKCre mice and controls showed a representative control mouse are shown for comparison. Filled square, actual
identical levels of activity and rates of habituation within and goal; open square, previous goal; filled dot, begin; open dot, end of path. (e)
across days (data not shown). Both groups strongly avoided the Radial maze: Number of correct choices in the first eight. Both groups (n 27,
center field and preferred the wall zone to the same degree (Fig. controls 30) showed indistinguishable learning rates and performed above
chance, but the average performance of MRCaMKCre mice was clearly inferior. ( f)
5a). On the elevated O maze, MRCaMKCre and control mice
Radial maze: Reentry errors. Whereas controls showed significant learning, no
strongly avoided the open sectors. Again, sector preferences significant reduction of errors could be observed in MRCaMKCre mice. Filled circles
were not affected by the genotype (Fig. 5b). In the lightdark columns, MRCaMKCre; open circlescolumns, control.
box, both groups preferred the dark to the lit compartment to the
same degree (Fig. 5c). Although MRCaMKCre mice were slightly
less active than controls on the O maze and in the light-dark box activity and rates of habituation in the emergence test (Fig. 5d).
test (Table 2, which is published as supporting information on In an object exploration test, both groups showed a clear
the PNAS web site), both groups showed identical levels of approach response toward a novel object that was introduced

198 www.pnas.orgcgidoi10.1073pnas.0503878102 Berger et al.


Fig. 5. MRCaMKCre mice (n 31, controls 33) were hyperreactive in an object
exploration task but showed normal anxiety- and exploration-related behav-
iors in other tests. (a) Open-field. Zone preferences were not affected by the
genotype. Chance levels for percent time depended on relative zone size and
are shown by a dotted line. (b) Elevated O maze. Sector preferences were not
affected by the genotype. Chance levels for percent time depended on
relative zone size and are shown by a dotted line. (c) Light dark box (% time
in dark). Both groups preferred the dark box over the lit compartment to the
same degree. (d) Emergence test (distance moved in mmin). The level of
activity and the rate of habituation were not affected by the genotype. (e)
Object exploration test. MRCaMKCre mice displayed more than two times
greater horizontal exploratory activity toward the object (mmin). (f ) Object
exploration test. MRCaMKCre mice showed nearly three times as much estimated
vertical exploratory activity toward the novel object (xmin) as did controls.
into an otherwise familiar arena, but MRCaMKCre mice displayed
increased horizontal and vertical exploratory activity toward the
object compared to controls (Fig. 5 e and f ). This strong increase
of exploratory activity toward a novel object was not associated
with a general increase of locomotor activity (Table 2).
To find an association between the massively increased ex-
ploration of a novel object by MRCaMKCre mice and their
performance deficit in the water maze and the radial maze, we
performed a factor analysis of behavioral variables. For this
analysis, two representative factorial variables were chosen from
the object exploration task: the place navigation test in the water
maze, and the radial maze working memory procedure (Table 3,
which is published as supporting information on the PNAS web
site). The analysis revealed a strong association between the
mutation effect on reentry errors in the radial maze task and
spatial perseverance during place reversal in the water maze. In
another subset of animals, radial maze reentry errors were more
associated with the hyperreactivity in the novel object test.
To rule out that the Cre-transgene accounts for the
behavioral phenotype of the MRCaMKCre mice, we compared
MRwt/wtCaMKCre mice and their respective littermate con-
trols (MRwt/wt mice) in the water maze, the radial maze, and
the object exploration task. This analysis revealed that the
Berger et al.
Cre-transgene has no effect on its own in these behavioral
tasks (Table 4, which is published as supporting information on
the PNAS web site).
Discussion
We report the generation of a forebrain-specific inactivation of
the mouse MR gene that allows the analysis of MR function in
neurons of the limbic system. Whereas the germ line inactivation
(knockout) of the mouse MR gene results in postnatal lethality
due to renal loss of sodium and water (25), the forebrain-specific
inactivation did not impair survival. Therefore, we were able to
characterize the role of limbic MR in hippocampal development,
HPA-axis regulation, cognitive and behavioral processes, and
synaptic plasticity in the CA1 region.
Loss of Limbic MR Results in Aberrant Hippocampal Mossy Fiber
Layout and GR Up-Regulation. In contrast to an earlier study showing
that the loss of CREB protein starts at P6 and is complete 2 weeks
after birth (18), we show by using the same Cre transgenic mouse
line that the MR protein is already reduced at P0, and the loss of
MR is almost complete at P6. We suspect that this discrepancy
reflects a different turnover of the two proteins and not a different
timing of the recombination of the two floxed alleles.
The early perinatal onset of the absence of MR induces
adaptive changes, like the observed hippocampal GR up-
regulation and sprouting of mossy fibers. Thus, the phenotype we
describe is showing the consequences of a permanently disturbed
balance of GR and MR signaling. The power of the genetic
approach we used, in contrast to pharmacological approaches
using receptor antagonists, is the high selectivity of interference
with and the completeness of inactivation of the respective
receptor-mediated signaling pathway. The continuous absence in
a developing organism allows separating redundant from non-
redundant functions. To determine whether the actual behav-
ioral phenotype described here is the consequence of the altered
hippocampal development or the lack of MR in the adult brain,
we currently develop transgenic mice expressing an inducible
Cre-fusion protein (26) in the forebrain that allows the inacti-
vation of the MR gene in the adult brain only.
The early postnatal period was shown to be critical for the
development of the hippocampal mossy fiber layout (27). The
early perinatal onset of the MR deficiency in MRCaMKCre mice
results in an aberrant mossy fiber layout that is comparable with
the altered mossy fiber projection in adult salt-rescued MR
knockout mice (28), in which the MR gene is inactivated in all
cells from gestation on. However, in contrast to the salt-rescued
MR knockout mice, the altered mossy fiber projection in MR-
NEUROSCIENCE
CaMKCre mice was not associated with a conspicuous decreased
density of granule cells in the upper blade of the dentate gyrus.
Limbic MR Deficiency Changes Neither Basal Circadian HPA-Axis
Activity Nor Basic Synaptic Properties in the CA1 Region. Adult
MRCaMKCre mice showed normal basal corticosterone levels as
well as normal CRH mRNA and GR protein expression in the
PVN. In addition, we could not detect MR expression in the
PVN. Therefore, we conclude that the limbic MR is either
dispensable for the maintenance of the basal HPA-axis activity
or can be completely substituted. The hippocampal GR up-
regulation might reflect such a compensatory mechanism.
Electrophysiological analysis of the basic synaptic properties and
LTP induced by high-frequency stimulation revealed no differences
between MRCaMKCre mice and controls. Whereas some studies have
shown that the induction of LTP can be affected by stress and
hormonal manipulations (12, 29), others indicate that induction of
LTP is less sensitive than primed-burst potentiation (PBP) to stress
and hormonal manipulations (30, 31). A more extensive electro-
physiological evaluation of LTP and PBP in the CA1 region and
other hippocampal subregions, e.g., mossy fiberCA3 pathway, in
PNAS January 3, 2006 vol. 103 no. 1 199
combination with hormonal manipulations, is required to precisely
define the electrophysiological correlates of the behavioral pheno-
type delineated in this study.
Loss of Limbic MR Impairs Learning and Induces Behavioral Stereo-
typy. The behavioral screen of MRCaMKCre mice in various
learning and exploration tests revealed that the genetic inacti-
vation of MR in the limbic system causes a mixed behavioral
syndrome with distinct cognitive deficits. Several but not all
behavioral changes correspond to a septo-hippocampal malfunc-
tion syndrome. Among them are the impaired learning of the
water-maze task and deficits in measures of working memory on
the radial maze. Changes that are not related to learning include
the hyperreactivity toward a novel object and the behavioral
perseverance, two typical signs of septal andor hippocampal
lesions in rats (32).
On the other hand, the MRCaMKCre mice showed no improve-
ment of two-way avoidance, another classic hippocampal lesion
syndrome in rats (33), and showed clearly intact spatial memory.
Conditioned taste aversion, a learning task less dependent on
hippocampal integrity but dependent on amygdalar modulation
(34), was not affected in MRCaMKCre mice. Learning deficits were
also reported in studies using MR antagonists in rats. In contrast
to our study, the acute and continuous intracerebroventricular
administration of a MR antagonist led to impaired retention of
spatial memory (16, 35), whereas on the eight-arm radial maze,
MR antagonists only led to an increase in reentry errors when
applied in combination with GR antagonists (36).
The loss of MR in MRCaMKCre mice is not restricted to the
septum and hippocampus. It is possible that the loss of MR in the
amygdala contributes to the observed behavioral phenotype of
MRCaMKCre mice.
Limbic MR Deficiency Spares Anxiety-Related Behavior but Induces
Abnormal Exploratory Behavior Toward a Novel Object. Studies in
rats using MR antagonists suggested that MR is involved in the
regulation of anxiety-like behavior (37, 38). This involvement
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