The oxytocinergic system is critically involved in the regulation of maternal behavior, which includes maternal aggression. Because
aggression has been linked to anxiety, we investigated the maternal aggression and the role of brain oxytocin in lactating Wistar rats
within the paraventricular nucleus (PVN; HAB, increase; LAB, decrease). Furthermore, oxytocin release was higher within the central
the maternal defense test and local oxytocin release was found in both the PVN and CeA. Using retrodialysis, blockade of endogenous
Enhanced aggressive behavior toward an intruder is part of the
complex pattern of maternal behavior in most mammals (Er-
skine et al., 1978; Numan, 1994; Rosenblatt et al., 1994; Numan
and Insel, 2003). Oxytocin, thought to originate from centrally
projecting paraventricular nucleus (PVN) neurons, is one of the
key neuropeptides regulating the initiation and maintenance of
maternal behavior (Pedersen and Boccia, 2002; Numan and In-
Electrolytic lesions of the PVN reduced maternal aggression
recently reported within the PVN of lactating Wistar rats during
ical manipulation of the oxytocin system within the central nu-
cleus of the amygdala (CeA) or the parvocellular division of the
PVN elicits changes in maternal aggression (Giovenardi et al.,
1998; Johns et al., 1998; Elliott et al., 2001; Lubin et al., 2003).
to anxiety-like behavior (Nyberg et al., 2003; Veenema et al.,
2004), but less is known about the relationship between anxiety
and maternal aggression in lactating females. Therefore, we
sought to reveal whether the dam’s innate level of anxiety deter-
maternal defense test and whether the aggression perceived also
performed on rats selectively bred for extremes in emotionality
[i.e., for high anxiety-related behavior (HAB) and low anxiety-
related behavior (LAB)] (Liebsch et al., 1998; Landgraf and Wig-
ger, 2002). Robust behavioral differences between HAB and LAB
rats are seen not only in emotional (Henniger et al., 2000; Ohl et
al., 2001) but also social (Henniger et al., 2000) and male aggres-
sive (Veenema et al., 2004) behaviors. Importantly, in female
HAB and LAB rats, the differences in inborn anxiety persist in
pregnancy (Neumann et al., 1998) and during lactation (Neu-
oxytocin system of lactating HAB and LAB dams correlates with
the level of maternal aggressive behavior displayed. We therefore
ternal defense test within both the PVN and the CeA using intra-
cerebral microdialysis. The PVN and CeA were examined be-
cause of their roles in emotional and neuroendocrine stress
R. Landgraf for radioimmunological quantification of oxytocin, to Dr. M. Manning for his generous gift of the
oxytocin receptor antagonist, and to Dr. P. Burbach for providing the oxytocin receptor cDNA. We also thank V.
TheJournalofNeuroscience,July20,2005 • 25(29):6807–6815 • 6807
al., 2001; Davis and Whalen, 2001), maternal behavior, and ag-
gression (Insel and Harbaugh, 1989; Elliott et al., 2001; Lubin et
al., 2003; Bosch et al., 2004). We also compared the behavioral
consequences of manipulating local oxytocin actions by retrodi-
alysis of either an oxytocin receptor antagonist or of synthetic
oxytocin. Last, we determined whether differences in local oxy-
observed differences in maternal aggression found in lactating
HAB and LAB dams.
All surgical, sampling, and behavioral protocols were approved by the
Committee on Animal Health and Care of the local governmental ad-
Laboratory Animals by the National Institutes of Health. The bidirec-
have been reviewed recently (Landgraf and Wigger, 2002). Both rat lines
were treated identically in terms of care, mating, and behavioral testing
over the last 10 years.
Virgin female rats (body weight, 250–280 g) selectively bred for HAB
or LAB on the elevated plus-maze were mated, and pregnancy was con-
firmed the next day by the presence of sperm in the vaginal smears
(pregnancy day 1). Pregnant rats were housed in groups of four in stan-
dard rat cages (40 ? 60 ? 20 cm); from day 18 of pregnancy, rats were
8–16 pups, and litters were culled to eight pups. The number of pups
delivered did not differ between HAB and LAB dams. Rats were kept
under standard laboratory conditions (12 h light/dark cycle; lights on at
6:00 A.M.; 22°C; 60% humidity; and ad libitum access to water and
standard rat chow). All behavioral tests were performed on day 3 of
Maternal defense test. To compare maternal aggressive behavior, the
maternal defense test was performed with lactating HAB and LAB resi-
dents and virgin intruders (Wistar rats unselected for anxiety; body
weight, 230–250 g; Charles River, Sulzfeld, Germany) between 10:00
A.M. and 12:00 P.M. The intruder was placed into the resident’s cage for
10 min in the presence of the pups, and the behavior was recorded by a
digital camera. Later, the offensive (attacks, lateral threats, genital sniff-
ing after attack, offensive sniffing), defensive (freezing, immobility after
maternal (nursing, licking/grooming pups, pup carrying) behaviors dis-
played by the resident and the intruder were analyzed every 10 s on a
videotape by an experienced observer blind to the breeding line or treat-
ment (Neumann et al., 2001). After the 10 min testing period, the virgin
intruder was returned to its home cage.
or LAB resident on the anxiety-related behavior of the intruders, lactat-
ing residents and virgin intruders were tested on the elevated plus-maze
compared with a group of naive (i.e., nondefeated) female virgin rats
between the rat’s exploratory drive and its innate fear of open and ex-
posed areas (Pellow et al., 1985; Liebsch et al., 1998). The plus-maze is
built of an elevated (80 cm) plus-shaped platform with two closed (with
40 cm high walls; 20 lux) and two open (each 50 ? 10 cm; 80 lux) arms,
maze was cleaned with water containing a low concentration of a deter-
gent and dried before each rat was tested. The rat was placed in the
were recorded with a video/computer system (Plus-maze version 2.0;
Ernst Fricke) during the 5 min exposure: (1) percentage of entries into
open arms versus total time, (3) latency until first entry into the open
arm, and (4) the number of full entries.
Implantation of microdialysis probes
U-shaped microdialysis probe (dialysis membrane; molecular cutoff of
18 kDa; Hemophan; Gambro Dialysatoren, Hechingen, Germany) (for
details, see Neumann et al., 1993) under isoflurane anesthesia and
temperature-controlled conditions using sterile procedures. The micro-
dialysis probes were used either for monitoring local oxytocin release or
oxytocin or an oxytocin receptor antagonist. The target area was either
the PVN [stereotaxic coordinates relative to bregma, 1.6 mm caudal, 1.8
mm lateral to midline, 9.1 mm beneath the surface of the skull; angle of
1998)] or the CeA (2.5 mm caudal, 4.0 mm lateral, 8.5 mm deep). Uni-
lateral implantation was performed for microdialysis studies, and bilat-
eral implantation was performed for retrodialysis studies. Before the
implantation, the probes were flushed and filled with sterile Ringer’s
solution [composed of the following (in mM): 147.1 Na?, 2.25 Ca2?, 4
into the skull. Afterward, the inflow and outflow ends of the probe were
each attached to short PE-20 polyethylene tubing (5 cm long) filled with
Ringer’s solution. After surgery, the rats received a 30 ?l subcutaneous
depot injection of antibiotic (Tardomyocel; Bayer, Leverkusen, Ger-
many). Rats were kept together with their litters in polycarbon observa-
tion cages (38 ? 22 ? 35 cm). The next day, they were handled carefully
to habituate them to the microdialysis procedure and to reduce nonspe-
cific stress responses during the experiment.
Monitoring of oxytocin release within the PVN or CeA and
maternal aggressive behavior
sis probe located within either the PVN or the CeA was connected to a
syringe mounted onto a microinfusion pump via a long piece of PE-20
tubing. The outflow was equipped with a tube holder that allowed direct
sample collection into a 1.5 ml Eppendorf (Eppendorf, Hamburg, Ger-
many) tube (containing 10 ?l of 0.1N HCl) at a distance of 5 cm above
the neck of the animal with a calculated dead space of 12 ?l and a sam-
3.3 ?l/min with sterile Ringer’s solution, pH 7.4, initially for 2 h before
the first sample collection. The following five consecutive 30 min dialy-
sates were collected from the lactating rat: samples 1 and 2 were taken
the maternal defense test was performed for 10 min as described above
with continued microdialysis, after which the intruder was removed.
dialysates were immediately frozen on dry ice and stored at ?20°C until
quantification of oxytocin by radioimmunoassay. In five dams of each
breeding line, maternal aggressive behavior was monitored simulta-
neously with ongoing microdialysis in the PVN or CeA.
Infusion of an oxytocin receptor antagonist into the PVN or the
CeA of HAB dams and monitoring of maternal
Lactating HAB dams were prepared for retrodialysis as described above
with microdialysis probes implanted bilaterally into either the PVN or
the CeA. The probes were perfused at a rate of 1.0 ?l/min with sterile
At 35 min before the 10 min maternal defense test, the inflow tubing
containing the control perfusion solution was exchanged with an inflow
tubing containing the selective oxytocin receptor antagonist (des-Gly-
NH2,d(CH2)5[Tyr(Me)2,Thr4]OVT; 10 ?g/ml dissolved in Ringer’s so-
lution) (Manning et al., 1989) in a concentration that was found to be
effective in previous experiments (Neumann et al., 2000c; Ebner et al.,
2005). The delay for the drug to reach the target site was calculated as 8
min. During the retrodialysis period, an amount of at least 2 ng is ex-
pected to be locally delivered into the surrounding tissue as estimated in
vehicle, exchange of tubings was mimicked to provide identical experi-
mental conditions for both groups. Perfusion with oxytocin receptor
6808 • J.Neurosci.,July20,2005 • 25(29):6807–6815 Boschetal.•BrainOxytocinRegulatesMaternalAggression
antagonist or vehicle was continued during the maternal defense test.
The behavioral performance of the resident rat during the maternal de-
observer unfamiliar with the rat’s treatment.
Infusion of synthetic oxytocin into the PVN of LAB dams and
monitoring of maternal aggressive behavior
Because reduced release of oxytocin within the PVN was found in LAB
residents during the defense of the pups, lactating LAB dams were pre-
pared for retrodialysis within the left and right PVN as described above.
changed to synthetic oxytocin (1 ?g/ml dissolved in Ringer’s solution;
Sigma, Taufkirchen, Germany) or remained unchanged (vehicle con-
trols). The behavior of the resident rat during the 10 min maternal de-
fense test performed during ongoing retrodialysis was monitored every
10 s as described above.
At the end of the microdialysis experiments, rats were killed with an
overdose of isoflurane. The brains were removed, quickly frozen in pre-
verify the placement of the probe in either the PVN or CeA our outside
the targeted region (see Figs. 3, 5, 6), brains were cut into 25?m coronal
cryostat sections and stained with cresyl violet. Data from experimental
subjects were only included in the statistical analysis if the microdialysis
probes were correctly placed in the brain target region.
Radioimmunoassay of oxytocin
Oxytocin content was measured in lyophilized dialysates by a highly
sensitive and selective radioimmunoassay (detection limit, 0.1 pg per
sample; cross-reactivity of the antisera with other related peptides was
?7%) (for details, see Landgraf et al., 1995).
In situ hybridization for oxytocin receptor mRNA
The brains of day 5/6 lactating HAB and LAB dams were removed after
decapitation, frozen in prechilled n-methylbutane on dry ice, and stored
5?-untranslated region of rat oxytocin receptor cDNA was subcloned
into pGEM-7Z. Sense and antisense riboprobes were generated by in
vitro transcription, in the presence of35S-UTP, with SP6- and T7-RNA
polymerase after plasmid linearization with EcoRI or BamHI,
In situ hybridization histochemistry for oxytocin receptor expression.
Whole brains were sectioned coronally at 15 ?m on a cryostat and thaw
mounted onto clean poly-L-lysine-coated glass microscope slides and
stored at ?70°C. Every fifth section was mounted separately and stained
with toluidine blue (Sigma, Poole, UK) to locate regions of interest.
thawed to room temperature (RT) and immersed in 4% paraformalde-
receptor in a solution mixed with 50% formamide. The probe was ap-
plied to each section at a concentration of 106cpm per slide in 200 ?l of
hybridization solution for 18 h at 55°C in a humidified chamber. Post-
hybridization washes consisted of three 5 min washes in 2? SSC. Sec-
were used to determine the optimal wash temperature for this probe.
Tissue was then dehydrated in a graded series of ethanol containing 300
mM ammonium acetate. The hybridization signal was visualized at the
cellular level by dipping the slides in autoradiographic emulsion (135
5053; Ilford; Agar Scientific, Stansted, UK). Slides were air dried and
stored with desiccant at 4°C for 20 weeks before being developed (D19;
Eastman Kodak, Rochester, NY), counterstained with hematoxylin and
eosin, dehydrated through alcohols to xylene, and finally coverslipped
with DPX (a mixture of distyrene, tricresyl phosphate, and xylene)
mounting medium (Fisher Scientific, Leicestershire, UK). Slides were
examined with a light microscope under bright-field illumination.
riboprobe or pretreatment with RNase-A before hybridization with the
antisense riboprobe, conducted under identical conditions to those for
the antisense probe. There was no detectable hybridization signal with
the sense probe or after RNase-A pretreatment.
Quantification of autoradiograms. Anatomical identification of brain
structures was based on the stereotaxic brain atlas by Paxinos and
Watson (1998). The subdivisions of the PVN into magnocellular and
parvocellular cellular parts were defined after Swanson and Kuypers
(1980). The slides were coded so that the experimenter was unaware of
the treatment of the rats at the time of analysis. Coated section autora-
diograms were evaluated by measuring silver grain density over individ-
ual neurons within the region of interest (40? objective) using a
of background. Background measurements were taken from tissue adja-
cent to the PVN and CeA that exhibited no evident signal. Silver grain
counts were made over 15 randomly chosen labeled neurons per region
Oxytocin receptor binding autoradiography
Brain sections from the same rats used for analysis of oxytocin receptor
situ autoradiography. The oxytocin receptor binding study was per-
formed using a125I-ornithine vasotocin analog (125I-OVTA; vasotocin,
d(CH2)5[Tyr(Me)2,Thr4,Orn8)(125I)Tyr9-NH2]; 2200 Ci/mmol; NEN,
they were fixed in 0.1% paraformaldehyde for 2 min for optimization of
tissue integrity. Sections were then rinsed two times in 50 mM Tris-HCl,
pH 7.4, at RT for 10 min and incubated for 60 min at RT in a solution of
and 50 pM125I-OVTA. They were then washed three times at RT for 5
min in 50 mM Tris-HCl, pH 7.4, with 10 mM MgCl, followed by 30 min
rinse in the same buffer. After dipping the slides in distilled H2O, they
were dried rapidly with cold air. Sections were apposed to Kodak Bi-
oMaxMR film with125I microscale standards for 72 h. Slides from all
groups were processed simultaneously. The autoradiograms were coded
to obscure the identity of the tissue. Brain slices that contained compa-
rable sections of the CeA were measured for each subject to provide
individual means. Autoradiographic125I-receptor binding was quanti-
fied on the film in the CeA as gray density per area minus background
using the NIH Image program (ImageJ 1.31; National Institutes of
Health; http://rsb.info.nih.gov/ij/). Background activity was automati-
For the comparison of behavioral parameters during the maternal de-
fense test (see Figs. 1, 5, 6) and on the elevated plus-maze (see Fig. 2), a
one-way ANOVA was performed. The microdialysis data of each rat was
standardized to the mean of its two basal samples (?100%) and pre-
sented as mean percentage of the group ? SEM (see Fig. 3). To compare
oxytocin levels in dialysates, a two-way (factors, line-by-time) or one-
way (factor, time) ANOVA for repeated measures was used; Newman–
Keuls post hoc analysis was performed after significant interaction. Cor-
relation analysis of offensive behavior and oxytocin release was
performed using simple regression analysis (see Fig. 4). Mann–Whitney
dialysates (see Fig. 3) of the behavioral performance of dams retrodial-
ysed with vehicle, oxytocin, or the oxytocin receptor antagonist outside
the targeted brain area, of oxytocin receptor mRNA expression, and of
oxytocin receptor binding (see Fig. 7). Significance was accepted at p ?
0.05. All statistics were performed using a computer software package
(GB-Stat 6.0; Dynamic Microsystems, Silver Spring, MD).
Boschetal.•BrainOxytocinRegulatesMaternalAggression J.Neurosci.,July20,2005 • 25(29):6807–6815 • 6809
During the maternal defense test, HAB dams displayed signifi-
virgin intruder (F(1,12)? 16.2; p ? 0.002) than LAB dams. The
higher level of maternal aggression in HAB dams was also re-
differences between HAB and LAB dams were observed in the
during the test period.
ior on the elevated plus-maze 10 min after completion of the
maternal defense test to confirm their behavioral extremes. As
maze significantly less as reflected by reduced percentage of en-
tries into the open arms (one-way ANOVA; F(1,16)? 29.7; p ?
0.0001) (Fig. 2a), reduced percentage of time on the open arms
(F(1,16)? 20.9; p ? 0.0003), and less full entries (F(1,16)? 24.8;
p ? 0.0001) when compared with LAB dams.
Virgin intruders defeated by HAB dams displayed significantly
more freezing (one-way ANOVA; F(1,12)? 8.2; p ? 0.014) (Fig.
1b) and more overall defensive behavior (F(1,12)? 11.1; p ?
0.006) compared with intruders defeated by LAB dams, whereas
cage exploration (F(1,12)? 13.0; p ? 0.004) and overall explor-
ative behavior were significantly higher in virgins defeated by
LAB dams (F(1,12)? 13.1; p ? 0.004).
The anxiety-related behavior of virgin intruders (commer-
cially obtained and unselected for emotionality) on the elevated
plus-maze was dependent on the resident exposed to. Virgin in-
truders who were defeated by the more aggressive HAB dams
were significantly more anxious, because they entered the open
arms less frequently (F(2,28)? 10.4; p ? 0.0004) (Fig. 2b) and
spent less time in the open arms (F(2,28)? 7.7; p ? 0.002) com-
pared with virgin intruders defeated by a LAB dam and to non-
defeated virgin controls.
The oxytocin content in dialysates sampled under basal condi-
lactating HAB and LAB rats (one-way ANOVA; factor, line;
F(1,12)? 2.24; p ? 0.16) (Fig. 3a, inset). Oxytocin release within
the PVN significantly differed between lactating HAB and LAB
residents (two-way ANOVA for repeated measures; factor, line;
F(1,52)? 6.20; p ? 0.03) (Fig. 3a), although it did not change
significantly over time (factor, time; F(4,44)? 0.53; p ? 0.72).
However, when performing a one-way ANOVA for repeated
measures separately on data from each group, a significant time
effect could be found in both HAB (F(4,56)? 8.91; p ? 0.0001)
and LAB (F(4,56)? 13.0; p ? 0.0001) dams. Oxytocin release
within the PVN increased in HAB ( p ? 0.01 vs sample 2) but
decreased in LAB residents ( p ? 0.01 vs sample 2) during expo-
sure to the maternal defense test. Differences in local oxytocin
release were also reflected by significant differences in ? values
tantly, oxytocin content in microdialysates sampled outside the
PVN remained at basal levels during exposure to maternal de-
fense, at least in LAB dams (Fig. 3a).
The amount of offensive behavior displayed by the HAB and
(r2? 0.56; p ? 0.005) (Fig. 4a).
Under basal conditions, oxytocin content in the dialysates from
the CeA did not significantly differ between lactating HAB and
LAB residents (one-way ANOVA; factor, line; F(1,10)? 0.75; p ?
0.41) (Fig. 3b, inset). Oxytocin release within the CeA changed
factor, time; F(4,36)? 4.11; p ? 0.008) (Fig. 3b) but was not
different between lactating HAB and LAB residents (factor, line;
F(1,44)? 1.05; p ? 0.33). During the maternal defense test, the
oxytocin content increased significantly in dialysate sample 3
comparedwithsample2ofbothHAB( p?0.001)andLAB( p?
0.05) dams. When performing statistics on the oxytocin content
in sample 3, there was a trend toward higher oxytocin content in
HAB compared with LAB rats during maternal defense ( p ?
0.06) (Fig. 3b, inset). Oxytocin content in microdialysates sam-
pled outside the CeA during maternal defense was unchanged
compared with basal levels (Fig. 3b).
The amount of offensive behavior displayed by the HAB and
LAB dams did correlate with the oxytocin release within the CeA
(r2? 0.67; p ? 0.004) (Fig. 4b).
anxiety (b) during the 10 min maternal defense test. a, The occurrence of attacks and total
tored in HAB and LAB residents on day 3 of lactation. b, The occurrence of total offensive,
n ? 7 per group. Data are expressed as mean ? SEM. **p ? 0.01; *p ? 0.05 versus LAB
Behavior of HAB and LAB residents (a) and their virgin intruders unselected for
percentage of time spent on the open arms of the maze, as well as the total number of full
controls is included. The numbers in parentheses indicate group size. Data are expressed as
6810 • J.Neurosci.,July20,2005 • 25(29):6807–6815 Boschetal.•BrainOxytocinRegulatesMaternalAggression
In HAB dams, oxytocin release within the PVN was found to be
elevated during exposure to the maternal defense (see above).
Retrodialysis of the oxytocin receptor antagonist bilaterally into
the PVN resulted in a significant reduction in the level of the
overall offensive behavior compared with vehicle-treated HAB
dams (factor, treatment; F(1,11)? 5.22; p ? 0.04) (Fig. 5a). The
amount of defensive (F(1,11)? 1.59; p ?
0.23) (Fig. 5a), exploratory, and maternal
behaviors (data not shown) were un-
changed. In contrast, in LAB dams in
which local oxytocin release was found to
be reduced during the maternal defense
test, local administration of the oxytocin
receptor antagonist did not alter any be-
havioral measure (Fig. 5a).
oxytocin receptor antagonist closely out-
side the left and right PVN (n ? 3) (Fig.
5d) did not affect the behavioral perfor-
treated with vehicle (n ? 2) outside the
target area with respect to the parameters
number of attacks, offensive, and defen-
sive behavior (data not shown).
the PVN was found to be reduced during
maternal defense. Retrodialysis of syn-
thetic oxytocin bilaterally into the PVN
did not significantly affect the total
amount of offensive behavior (factor,
treatment; F(1,13)? 2.60; p ? 0.13) (Fig.
6). However, there was a tendency toward
an increase in the offensive parameter lat-
eral threat (F(1,13)? 4.01; p ? 0.06).
Bilateral retrodialysis application of
oxytocin closely outside the left and right
behavioral performance of LAB dams
target areas with respect to the parameters number of attacks,
lateral threat, and offensive and defensive behavior (data not
Oxytocin release was more pronounced within the CeA of HAB
compared with LAB dams during exposure to the maternal de-
fense (see above). In lactating HAB rats, retrodialysis of the oxy-
tocin receptor antagonist bilaterally into the CeA resulted in a
significant decrease in the total amount of attacks (factor, treat-
ment; F(1,14)? 5.65; p ? 0.03) (Fig. 5b) and offensive behavior
(F(1,14)? 8.40; p ? 0.012) compared with vehicle-treated HAB
dams. The defensive, exploratory, and maternal behaviors dis-
the oxytocin receptor antagonist into the CeA did not signifi-
cantly change any of the behavioral parameters monitored
Furthermore, when the oxytocin receptor antagonist was ap-
plied closely outside the left and right CeA by retrodialysis (Fig.
5d), the behavioral performance (attacks, offensive, and defen-
sive behavior) of HAB (n ? 3) or LAB (n ? 2) dams was not
altered compared with dams treated with vehicle (HAB, n ? 2;
LAB, n ? 2) outside the target areas (data not shown).
conditions (samples 1 and 2). During the third dialysis sampling period, a virgin intruder rat was placed into the cage of the
lactating resident for 10 min (maternal defense) with continued microdialysis (insider). Also shown are individual oxytocin
Oxytocin release within the PVN (a) and the CeA (b) of lactating HAB and LAB residents in response to maternal
Boschetal.•BrainOxytocinRegulatesMaternalAggression J.Neurosci.,July20,2005 • 25(29):6807–6815 • 6811
The expression of oxytocin receptor
mRNA either in the magnocellular ( p ?
0.20) or the parvocellular ( p ? 0.58) part
of the PVN or in the CeA ( p ? 0.42) did
not differ significantly between HAB and
LAB dams (Fig. 7a). Oxytocin receptor
binding in the CeA did not differ between
lactating HAB and LAB dams ( p ? 0.37)
(Fig. 7b). However, comparison of the
CeA of all lactating HAB and LAB dams
with data pooled from both virgin HAB
and LAB rats revealed a significant in-
crease of oxytocin receptor mRNA in lac-
tating dams ( p ? 0.03) (Fig. 7a).
We have shown that maternal aggression
in lactating rats is dependent on inborn
emotionality and correlates with oxytocin
nal defense test, HAB residents were more
aggressive than LAB residents, which was
also reflected by more defensive postures
and an elevated level of anxiety of virgin
intruders defeated by HAB dams. In addi-
tion, oxytocin release increased within
both the PVN and the CeA of lactating
whereas it decreased or only slightly in-
creased in LABs. Local infusion of an oxy-
tocin receptor antagonist into either the
PVN or the CeA of lactating HAB resi-
dents, but not closely outside these target
regions, revealed that locally released, en-
dogenous oxytocin supports the high level
of maternal aggressive behavior. Likewise,
oxytocin infusion into the PVN tended to
increase maternal aggression in LAB resi-
dents. Oxytocin receptor mRNA expres-
sion and binding in the PVN or CeA did
not differ between HAB and LAB dams.
Thus, differences in the dynamics of oxy-
of oxytocin receptors, underlies maternal
Our results from the elevated plus-maze
demonstrate that the extremes in anxiety-
related behavior of HAB and LAB dams
persist in lactation even after exposure to
the maternal defense test. Importantly, al-
are significantly more protective of their
offspring by being more offensive than
LAB dams. This is in line with studies on
their maternal behavior in which HAB
dams spend more time on the nest and
collect their pups faster during the pup re-
(insider) the paraventricular nucleus of lactating LAB residents. a, The amount of attacks and lateral threats and of offensive,
Behavioral consequences of application of synthetic oxytocin or vehicle infused via retrodialysis bilaterally into
6812 • J.Neurosci.,July20,2005 • 25(29):6807–6815Boschetal.•BrainOxytocinRegulatesMaternalAggression
trieval test, whereas the occurrence of licking and grooming the
offspring is similar in HAB and LAB dams (Neumann et al.,
2005). Similarly, mice selected for HAB (Kro ¨mer et al., 2005)
show a comparable style of maternal behavior during lactation,
including more pup-directed behavior and enhanced maternal
aggression toward an intruder during the maternal defense test
(A. H. Veenema and I. D. Neumann, unpublished observation)
man primates, mothers who show frequent behavioral signs of
anxiety scored higher in maternal protectiveness, suggesting a
link between protective parenting style and emotionality (Mae-
stripieri, 1999). However, our finding of high maternal aggres-
sion in rats with high innate level of anxiety-related behavior is
contrary to reports in (nonselected, genetically similar) lactating
mice that demonstrated the least anxious mice being the most
aggressive ones (Maestripieri and D’Amato, 1991; Parmigiani et
al., 1999). Similarly, in rats, manipulations that resulted in en-
hanced emotionality consequently reduced maternal aggressive
behavior (Lonstein et al., 1998; Boccia and Pedersen, 2001). In
not affect the rats’ fearfulness (Hansen and Ferreira, 1986; Fer-
reira et al., 1987), and separate neuronal regulation of maternal
and Insel, 2003).
The level of maternal aggression significantly influenced the
behavior of the virgin intruder rats. Intruders exposed to HAB
dams not only displayed more defensive behavior during the de-
fense test compared with those exposed to LAB dams but also
became more anxious on the plus-maze immediately after the
exposure to an aggressive male resident results in enhanced anx-
iety (Heinrichs et al., 1992; Liebsch et al., 1995; Haller et al.,
2003). However, this is the first study demonstrating that expo-
sure to an offensive dam results in the same behavioral phenom-
enon in virgin female rats and that the increase in anxiety seems
to be strictly dependent on the level of aggressive behavior
defense test correlated with the rise in local oxytocin release both
aggression seen in the anxious HAB dams
during maternal defense was found to be
accompanied by a significant increase in
oxytocin release into the extracellular
space of the PVN, whereas local oxytocin
dams. Together with the finding that
blockade of oxytocin receptors within the
PVN by local administration of a selective
oxytocin receptor antagonist reduced ma-
ternal aggression in HABs (Fig. 5), our re-
sults support the hypothesis that oxytocin
release in the PVN is necessary for the dis-
play of maternal aggressive behavior. In-
terestingly, the same treatment did not al-
ter maternal aggressive behavior in LAB
dams, which showed a reduced release of
ing pup defense. However, when oxytocin
concentrations in the PVN of LAB dams
were artificially increased by local retrodi-
alysis of the synthetic neuropeptide, the animals tended to be
more aggressive (Fig. 6), further demonstrating a role for oxyto-
cin in the PVN to mediate maternal aggression.
One possible mechanism of oxytocin action in the PVN in
cerebral injection of CRH was shown recently to inhibit aggres-
sion in lactating mice, and inhibition of CRH might be necessary
for maternal aggression (Gammie et al., 2004). In support of this
hypothesis, brain oxytocin inhibits the activity of CRH neurons
(Windle et al., 2004), and, specifically, oxytocin released within
the PVN inhibits the activity of the hypothalamo–pituitary–
adrenal axis (Neumann et al., 2000a), which is driven mainly by
lesions of the PVN reduced various components of maternal ag-
gression. In contrast, ibotenic acid lesions of the parvocellular
part of the PVN or local inhibition of oxytocin synthesis resulted
in increased bite frequency of rat dams against a male intruder
(Giovenardi et al., 1998), although in this context the contribu-
tion of locally released oxytocin in the PVN or central projection
sites remains unknown.
Our results also provide evidence for increased oxytocin re-
defense. This is remarkable, because there is only sparse oxyto-
cinergic innervation and low concentrations of oxytocin in the
release has found to be difficult, and oxytocin was only recently
reported to be detectable in CeA dialysates (Bosch et al., 2004;
Ebner et al., 2005). Oxytocin release within the CeA appeared
ternal defense, and local administration of the oxytocin receptor
antagonist significantly attenuated aggressive behavior in HAB
dams only, whereas it was without effect in LABs. Therefore, we
conclude that oxytocin released within the amygdala also con-
tributes to maternal aggression. Interestingly, in lactating Wistar
rats unselected for anxiety-related behavior, oxytocin release
within the CeA remained unchanged during maternal defense,
but they displayed a low level of offensive behavior (unselected,
14%; HAB, 44%; LAB, 25%) (Bosch et al., 2004). Thus, a direct
correlation between the level of maternal aggression and the
(virg) female HAB and LAB rats versus pooled lactating HAB and LAB rats (gray columns) (a) and OXT-R binding in the CeA of
Boschetal.•BrainOxytocinRegulatesMaternalAggression J.Neurosci.,July20,2005 • 25(29):6807–6815 • 6813
repeated administration of oxytocin in the CeA resulted in en-
hanced aggression toward the male intruder. In contrast, local
infusion of an oxytocin receptor antagonist 4 h before testing
increased the frequency of attacks against a male intruder rat
(Lubin et al., 2003). These results may not be in conflict, because
blockade of oxytocin receptors might trigger a rebound effect
and, thus, induce even more local oxytocin release after 4 h
(Landgraf and Neumann, 2004). Overall, our findings on brain
oxytocin as a mediator of aggression are supported by studies on
pared with wild-type mice (Young et al., 1998).
Oxytocin also regulates anxiety-related behavior (McCarthy et
al., 1996; Windle et al., 1997; Neumann et al., 2000b; Mantella et
al., 2003), an effect that could be localized within the amygdala
(Bale et al., 2001; Neumann, 2002). Thus, oxytocin is likely to
promote maternal aggressive behavior indirectly via transient
regulation of fear and anxiety possibly by regulation of the activ-
ity of the brain CRH system (Windle et al., 2004). As another
possible mechanism, oxytocin inhibits the release of GABA
dams (Neumann and O. J. Bosch, unpublished observation).
the availability of inhibitory or excitatory amino acids within the
CeA (Ebner et al., 2005; Huber et al., 2005), the activity of the
brain CRH system (Liebsch et al., 1995), the state of anxiety, and
the level of maternal aggression (Gammie et al., 2004).
In contrast to differences in local oxytocin release between
HAB and LAB females, the expression and density of oxytocin
also been reported for male rats (Wigger et al., 2004). Enhanced
firmed in lactating HAB and LAB dams (pooled) of this study,
and binding (Insel, 1990; Freund-Mercier et al., 1994) in limbic
brain areas of lactating rats may, in general, contribute to their
complex behavioral adaptations, including an enhanced level of
aggression. However, our data suggest that local oxytocin release
patterns within the PVN and the CeA, rather than differences at
the receptor level, determine the expression of maternal aggres-
Bale TL, Davis AM, Auger AP, Dorsa DM, McCarthy MM (2001) CNS
anxiety and sex behavior. J Neurosci 21:2546–2552.
Boccia ML, Pedersen CA (2001) Brief vs. long maternal separations in in-
fancy: contrasting relationships with adult maternal behavior and lacta-
tion levels of aggression and anxiety. Psychoneuroendocrinology
Bosch OJ, Kro ¨mer SA, Brunton P, Neumann ID (2004) Release of oxytocin
defence. Neuroscience 124:439–448.
Consiglio AR, Lucion AB (1996) Lesion of hypothalamic paraventricular
nucleus and maternal aggressive behavior in female rats. Physiol Behav
DavisM,WhalenPJ (2001) Theamygdala:vigilanceandemotion.MolPsy-
Ebner K, Bosch OJ, Kro ¨mer SA, Singewald N, Neumann ID (2005) Release
of oxytocin in the rat central amygdala modulates stress-coping behav-
iour and the release of excitatory amino acids. Neuropsychopharmacol-
Elliott JC, Lubin DA, Walker CH, Johns JM (2001) Acute cocaine alters
oxytocin levels in the medial preoptic area and amygdala in lactating rat
dams: implications for cocaine-induced changes in maternal behavior
and maternal aggression. Neuropeptides 35:127–134.
Engelmann M, Ludwig M, Landgraf R (1992) Microdialysis administration
of vasopressin and vasopressin antagonists into the septum during pole-
jumping behavior in rats. Behav Neural Biol 58:51–57.
Erskine MS, Barfield RJ, Goldman BD (1978) Intraspecific fighting during
late pregnancy and lactation in rats and effects of litter removal. Behav
Feldman S, Weidenfeld J (1995) Neural mechanisms involved in the corti-
costeroid feedback effects on the hypothalamo-pituitary-adrenocortical
axis. Prog Neurobiol 45:129–141.
Ferreira A, Dahlof LG, Hansen S (1987) Olfactory mechanisms in the con-
trol of maternal aggression, appetite, and fearfulness: effects of lesions to
cortex. Behav Neurosci 101:709–717, 746.
Ferris CF, Foote KB, Meltser HM, Plenby MG, Smith KL, Insel TR (1992)
Oxytocin in the amygdala facilitates maternal aggression. Ann NY Acad
Freund-Mercier MJ, Stoeckel ME, Klein MJ (1994) Oxytocin receptors on
oxytocin neurones: histoautoradiographic detection in the lactating rat.
J Physiol (Lond) 480:155–161.
Gammie SC, Negron A, Newman SM, Rhodes JS (2004) Corticotropin-
releasing factor inhibits maternal aggression in mice. Behav Neurosci
Giovenardi M, Padoin MJ, Cadore LP, Lucion AB (1998) Hypothalamic
paraventricular nucleus modulates maternal aggression in rats: effects of
ibotenic acid lesion and oxytocin antisense. Physiol Behav 63:351–359.
GrayTS,CarneyME,MagnusonDJ (1989) Directprojectionsfromthecen-
tral amygdaloid nucleus to the hypothalamic paraventricular nucleus:
possible role in stress-induced adrenocorticotropin release. Neuroendo-
HallerJ,LevelekiC,BaranyiJ,MikicsE,BakosN (2003) Stress,socialavoid-
ance and anxiolytics: a potential model of stress-induced anxiety. Behav
Hansen S, Ferreira A (1986) Food intake, aggression, and fear behavior in
the mother rat: control by neural systems concerned with milk ejection
and maternal behavior. Behav Neurosci 100:64–70.
Heinrichs SC, Pich EM, Miczek KA, Britton KT, Koob GF (1992)
defeated rats via direct neurotropic action. Brain Res 581:190–197.
Henniger MS, Ohl F, Holter SM, Weissenbacher P, Toschi N, Lorscher P,
Wigger A, Spanagel R, Landgraf R (2000) Unconditioned anxiety and
related behaviour. Behav Brain Res 111:153–163.
HermanJP,CullinanWE (1997) Neurocircuitryofstress:centralcontrolof
Huber D, Veinante P, Stoop R (2005) Vasopressin and oxytocin excite dis-
Insel TR (1990) Regional changes in brain oxytocin receptors post-partum:
time-course and relationship to maternal behaviour. J Neuroendocrinol
InselTR,HarbaughCR (1989) Lesionsofthehypothalamicparaventricular
nucleus disrupt the initiation of maternal behavior. Physiol Behav
Johns JM, Noonan LR, Zimmerman LI, McMillen BA, Means LW, Walker
CH, Lubin DA, Meter KE, Nelson CJ, Pedersen CA, Mason GA, Lauder
JM (1998) Chronic cocaine treatment alters social/aggressive behavior
in Sprague-Dawley rat dams and in their prenatally exposed offspring.
Ann NY Acad Sci 846:399–404.
Pu ¨tz B, Deussing JM, Holsboer F, Landgraf R, Turck CW (2005) Iden-
tification of glyoxalase-I as a protein marker in a mouse model of ex-
tremes in trait anxiety. J Neurosci 25:4375–4384.
Landgraf R, Neumann ID (2004) Vasopressin and oxytocin release within
the brain: a dynamic concept of multiple and variable modes of neu-
ropeptide communication. Front Neuroendocrinol 25:150–176.
Landgraf R, Wigger A (2002) High vs low anxiety-related behavior rats: an
animal model of extremes in trait anxiety. Behav Genet 32:301–314.
Landgraf R, Neumann I, Holsboer F, Pittman QJ (1995) Interleukin 1-?
6814 • J.Neurosci.,July20,2005 • 25(29):6807–6815Boschetal.•BrainOxytocinRegulatesMaternalAggression
stimulatesbothcentralandperipheralreleaseofvasopressinandoxytocin Download full-text
in the rat. Eur J Neurosci 7:592–598.
Liebsch G, Landgraf R, Gerstberger R, Probst JC, Wotjak CT, Engelmann M,
HolsboerF,MontkowskiA (1995) ChronicinfusionofaCRH1receptor
antisense oligodeoxynucleotide into the central nucleus of the amygdala
reduced anxiety-related behavior in socially defeated rats. Regul Pept
LiebschG,MontkowskiA,HolsboerF,LandgrafR (1998) Behaviouralpro-
behaviour. Behav Brain Res 94:301–310.
Lonstein JS, Simmons DA, Stern JM (1998) Functions of the caudal periaq-
ueductal gray in lactating rats: kyphosis, lordosis, maternal aggression,
and fearfulness. Behav Neurosci 112:1502–1518.
Lubin DA, Elliott JC, Black MC, Johns JM (2003) An oxytocin antagonist
infused into the central nucleus of the amygdala increases maternal ag-
gressive behavior. Behav Neurosci 117:195–201.
Maestripieri D (1999) The biology of human parenting: insights from non-
human primates. Neurosci Biobehav Rev 23:411–422.
Maestripieri D, D’Amato FR (1991) Anxiety and maternal aggression in
house mice (Mus musculus): a look at interindividual variability. J Comp
Manning M, Kruszynski M, Bankowski K, Olma A, Lammek B, Cheng LL,
KlisWA,SetoJ,HaldarJ,SawyerWH (1989) Solid-phasesynthesisof16
potent (selective and nonselective) in vivo antagonists of oxytocin. J Med
MantellaRC,VollmerRR,LiX,AmicoJA (2003) Femaleoxytocin-deficient
mice display enhanced anxiety-related
McCarthy MM, McDonald CH, Brooks PJ, Goldman D (1996) An anxio-
lytic action of oxytocin is enhanced by estrogen in the mouse. Physiol
NeumannI,RussellJA,LandgrafR (1993) Oxytocinandvasopressinrelease
within the supraoptic and paraventricular nuclei of pregnant, parturient
and lactating rats: a microdialysis study. Neuroscience 53:65–75.
Neumann ID (2002) Involvement of the brain oxytocin system in stress
coping: interactions with the hypothalamo-pituitary-adrenal axis. Prog
Brain Res 139:147–162.
NeumannID (2003) Brainmechanismsunderlyingemotionalalterationsin
the peripartum period in rats. Depress Anxiety 17:111–121.
Neumann ID, Wigger A, Liebsch G, Holsboer F, Landgraf R (1998) In-
creased basal activity of the hypothalamo-pituitary-adrenal axis during
pregnancy in rats bred for high anxiety-related behaviour. Psychoneu-
Neumann ID, Wigger A, Torner L, Holsboer F, Landgraf R (2000a) Brain
oxytocin inhibits basal and stress-induced activity of the hypothalamo-
pituitary-adrenal axis in male and female rats: partial action within the
paraventricular nucleus. J Neuroendocrinol 12:235–243.
Neumann ID, Torner L, Wigger A (2000b) Brain oxytocin: differential in-
hibition of neuroendocrine stress responses and anxiety-related behav-
iour in virgin, pregnant and lactating rats. Neuroscience 95:567–575.
NeumannID,Kro ¨merSA,ToschiN,EbnerK (2000c) Brainoxytocininhib-
involvement of hypothalamic and limbic brain regions. Regul Pept
Neumann ID, Toschi N, Ohl F, Torner L, Kro ¨mer SA (2001) Maternal de-
fence as an emotional stressor in female rats: correlation of neuroendo-
J Neurosci 13:1016–1024.
Neumann ID, Wigger A, Kro ¨mer S, Frank E, Landgraf R, Bosch OJ (2005)
Differential effects of periodic maternal separation on adult stress coping
in a rat model of extremes in trait anxiety. Neuroscience 132:867–877.
Numan M (1994) Maternal behaviour. In: The physiology of reproduction,
Ed 2 (Knobil E, Neill JD, eds), pp. 221–301. New York: Raven.
NumanM,InselTR (2003) Hormones,brainandbehavior.Theneurobiol-
NybergJM,VekovischevaO,SandnabbaNK (2003) Anxietyprofilesofmice
selectively bred for intermale aggression. Behav Genet 33:503–511.
OhlF,ToschiN,WiggerA,HennigerMS,LandgrafR (2001) Dimensionsof
emotionality in a rat model of innate anxiety. Behav Neurosci
Parmigiani S, Palanza P, Rogers J, Ferrari PF (1999) Selection, evolution of
hav Rev 23:957–969.
Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates, Ed 4.
Pedersen CA, Boccia ML (2002) Oxytocin links mothering received, moth-
ering bestowed and adult stress responses. Stress 5:259–267.
PellowS,ChopinP,FileSE,BrileyM (1985) Validationofopen:closedarms
entries in an elevated plus-maze as a measure of anxiety in the rat. J Neu-
rosci Methods 14:149–167.
Rosenblatt JS, Factor E, Mayer AD (1994) Relationship between maternal
aggression and maternal care in the rat. Aggress Behav 20:243–255.
N (2004) Neurobiological correlates of high (HAB) versus low anxiety-
related behavior (LAB): differential Fos expression in HAB and LAB rats.
Biol Psychiatry 55:715–723.
SwansonLW,KuypersHG (1980) Theparaventricularnucleusofthehypo-
retrograde fluorescence double-labeling methods. J Comp Neurol 194:
Veenema AH, Torner L, Maloumby R, Neumann ID (2004) Correlates of
male aggressive behavior with stress coping in different animal models.
World J Biol Psychiatry 5 [Suppl 1]:43.
Vyas A, Bernal S, Chattarji S (2003) Effects of chronic stress on dendritic
arborization in the central and extended amygdala. Brain Res
Wigger A, Sanchez MM, Mathys KC, Ebner K, Frank E, Liu D, Kresse A,
NeumannID,HolsboerF,PlotskyPM,LandgrafR (2004) Alterationsin
central neuropeptide expression, release, and receptor binding in rats
bred for high anxiety: critical role of vasopressin. Neuropsychopharma-
Windle RJ, Shanks N, Lightman SL, Ingram CD (1997) Central oxytocin
administration reduces stress-induced corticosterone release and anxiety
behavior in rats. Endocrinology 138:2829–2834.
Windle RJ, Kershaw YM, Shanks N, Wood SA, Lightman SL, Ingram CD
(2004) Oxytocin attenuates stress-induced c-fos mRNA expression in
specific forebrain regions associated with modulation of hypothalamo–
pituitary–adrenal activity. J Neurosci 24:2974–2982.
Young LJ, Muns S, Wang Z, Insel TR (1997) Changes in oxytocin receptor
mRNA in rat brain during pregnancy and the effects of estrogen and
interleukin-6. J Neuroendocrinol 9:859–865.
Young III WS, Shepard E, DeVries AC, Zimmer A, LaMarca ME, Ginns EI,
Amico J, Nelson RJ, Hennighausen L, Wagner KU (1998) Targeted re-
duction of oxytocin expression provides insights into its physiological
roles. Adv Exp Med Biol 449:231–240.
Boschetal.•BrainOxytocinRegulatesMaternalAggressionJ.Neurosci.,July20,2005 • 25(29):6807–6815 • 6815