Central nervous action of interleukin-1 mediates activation of limbic structures and behavioural depression in response to peripheral administration of bacterial lipopolysaccharide.

Abstract

Although receptors for the pro-inflammatory cytokine interleukin-1 have long been known to be expressed in the brain, their role in fever and behavioural depression observed during the acute phase response (APR) to tissue infection remains unclear. This may in part be due to the fact that interleukin-1 in the brain is bioactive only several hours after peripheral administration of bacterial lipopolysaccharide (LPS). To study the role of cerebral interleukin-1 action in temperature and behavioural changes, and activation of brain structures during the APR, interleukin-1 receptor antagonist (IL-1ra; 100 microg) was infused into the lateral brain ventricle 4 h after intraperitoneal (i.p.) LPS injection (250 microg/kg) in rats. I.p. LPS administration induced interleukin-1beta (IL-1beta) production in systemic circulation as well as in brain circumventricular organs and the choroid plexus. Intracerebroventricular (i.c.v.) infusion of IL-1ra 4 h after i.p. LPS injection attenuated the reduction in social interaction, a cardinal sign of behavioural depression during sickness, and c-Fos expression in the amygdala and bed nucleus of the stria terminalis. However, LPS-induced fever, rises in plasma corticosterone, body weight loss and c-Fos expression in the hypothalamus and caudal brainstem were not altered by i.c.v. infusion of IL-1ra. These findings, together with our previous observations showing that i.c.v. infused IL-1ra diffuses throughout perivascular spaces, where macrophages express interleukin-1 receptors, can be interpreted to suggest that circulating or locally produced brain IL-1beta acts on these cells to bring about behavioural depression and activation of limbic structures during the APR after peripheral LPS administration.

Full text

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BEHAVIORAL NEUROSCIENCE
Central nervous action of interleukin-1 mediates activation
of limbic structures and behavioural depression in response
to peripheral administration of bacterial lipopolysaccharide
J. P. Konsman,1J. Veeneman,1C. Combe,1S. Poole,2G. N. Luheshi3and R. Dantzer1
1PsychoNeuroImmunologie, Nutrition et Ge ´ne ´tique, CNRS UMR 5526⁄INRA UMR 1286, Universite ´ Victor Se ´galen Bordeaux 2,
Bordeaux 33076, France
2National Institute for Biological Standards and Control, Potters Bar, Herts, UK
3Douglas Hospital Research Center, McGill University, Verdun, QC, Canada
Keywords: amygdala, c-Fos, neuroimmune interactions, rat
Abstract
Although receptors for the pro-inflammatory cytokine interleukin-1 have long been known to be expressed in the brain, their role in
fever and behavioural depression observed during the acute phase response (APR) to tissue infection remains unclear. This may in
part be due to the fact that interleukin-1 in the brain is bioactive only several hours after peripheral administration of bacterial
lipopolysaccharide (LPS). To study the role of cerebral interleukin-1 action in temperature and behavioural changes, and activation of
brain structures during the APR, interleukin-1 receptor antagonist (IL-1ra; 100 lg) was infused into the lateral brain ventricle 4 h after
intraperitoneal (i.p.) LPS injection (250 lg⁄kg) in rats. I.p. LPS administration induced interleukin-1b (IL-1b) production in systemic
circulation as well as in brain circumventricular organs and the choroid plexus. Intracerebroventricular (i.c.v.) infusion of IL-1ra 4 h
after i.p. LPS injection attenuated the reduction in social interaction, a cardinal sign of behavioural depression during sickness, and
c-Fos expression in the amygdala and bed nucleus of the stria terminalis. However, LPS-induced fever, rises in plasma
corticosterone, body weight loss and c-Fos expression in the hypothalamus and caudal brainstem were not altered by i.c.v. infusion
of IL-1ra. These findings, together with our previous observations showing that i.c.v. infused IL-1ra diffuses throughout perivascular
spaces, where macrophages express interleukin-1 receptors, can be interpreted to suggest that circulating or locally produced brain
IL-1b acts on these cells to bring about behavioural depression and activation of limbic structures during the APR after peripheral LPS
administration.
Introduction
Tissue infection provokes local inflammation, fever and depression of
behavioural activity. The discovery that these symptoms are readily
provoked by bacterial lipopolysaccharide (LPS) fragments indicates
that they comprise an organized host-mounted acute phase response
(APR), rather than loss of function due to bacterial proliferation
(Baumann & Gauldie, 1994). The endeavour to identify messengers
mediating the APR led to the cloning of interleukin-1b (IL-1b;
Dinarello, 1984). Subsequent findings showing that IL-1 receptors are
expressed in the brain (Takao et al., 1990; Yabuuchi et al., 1994) and
that IL-1 is bioactive in this organ after peripheral LPS administration
(Fontana et al., 1984; Quan et al., 1994) gave rise to the hypothesis
that it may act in the CNS to bring about fever and behavioural
depression.
The cloning of the interleukin-1 receptor antagonist (IL-1ra;
Dinarello, 1991) enabled testing of this hypothesis by intracerebro-
ventricular (i.c.v.) IL-1ra administration at the time of intraperitoneal
(i.p.) IL-1b injection. In these experimental conditions, i.c.v. IL-1ra
reduces i.p. IL-1b-induced behavioural depression, but not fever (Kent
et al., 1992), indicating that IL-1 can act in the CNS to alter behaviour
during the APR. Although CNS action of peripheral IL-b can be
explained by blood-to-brain transport (Banks et al., 1991), other pro-
inflammatory cytokines capable of provoking fever and behavioural
depression are also detected in blood during systemic bacterial
infection (Torre et al., 1993; Poveda et al., 1999). As peripheral LPS
administration results in increased plasma levels of all pro-inflamma-
tory cytokines (Zuckerman et al., 1989; Zanetti et al., 1992), further
experiments addressed the effects of i.c.v. IL-1ra on i.p. LPS-induced
fever and behavioural depression. Surprisingly, i.c.v. IL-1ra was found
to attenuate i.p. LPS-induced fever (Luheshi et al., 1996; Cartmell
et al., 1999), but not behavioural depression (Bluthe ´ et al., 1992). The
role of brain IL-1b action during the APR is, therefore, far from clear.
The stimulus-dependent effects of i.c.v. IL-1ra administration may
be explained to some extent. Peripheral LPS can act on Toll-like
receptors at the blood–brain interface (Laflamme & Rivest, 2001;
Chakravarty & Herkenham, 2005), where it induces immunoreactive
Correspondence: Dr J. P. Konsman, as above.
E-mail: jan-pieter.konsman@u-bordeaux2.fr
Received 14 August 2008, revised 8 October 2008, accepted 24 October 2008
European Journal of Neuroscience, Vol. 28, pp. 2499–2510, 2008
doi:10.1111/j.1460-9568.2008.06549.x
ª The Authors (2008). Journal Compilation ª Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience

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IL-1b (van Dam et al., 1992; Nakamori et al., 1994; Konsman et al.,
1999; Goehler et al., 2006), which thus constitutes another source of
brain IL-1b. However, despite the rapid appearance of immunoreac-
tive IL-1b (Hagan et al., 1993; Hillhouse & Mosley, 1993), IL-1 is
only bioactive in the brain 3–6 h after peripheral LPS administration
(Fontana et al., 1984; Quan et al., 1994). Another relevant finding is
that cytokines are rapidly cleared from cerebrospinal fluid (CSF) to the
general circulation (Chen & Reichlin, 1999; Di Santo et al., 1999).
I.c.v. IL-1ra administration at the time of i.p. LPS injection may thus
prevent circulating IL-1b action, while not being present in sufficient
concentrations in the CNS when brain IL-1b is bioactive. In view of
these findings, we studied the role of cerebral IL-1 action in fever and
behavioural depression as well as in activation of brain structures
during the APR by infusing IL-1ra i.c.v. 4 h after i.p. LPS
administration.
Materials and methods
Animals
Pathogen-free male Wistar rats (Charles River, Saint Aubin les Elbeuf,
France) weighing 175–200 g upon arrival were used. Animals were
housed under standard conditions in groups of four in plastic
transparent cages with unrestricted access to food (Extralabo, Provins,
France) and water in a temperature- (21.5–22.5?C) and light-
controlled room (12 h lights on, 12 h lights off) for at least 7 days
before surgery. Juvenile Wistar rats, 25–35 days old, to be used as
social stimuli were housed under the same conditions at 10 per cage.
All animal experiments were conducted in accordance with French
legislation, European Community Council Directive (86⁄609⁄EEC),
and the policies and procedures described by the Canadian Council on
Animal Care in the Guide for the Care and Use of Laboratory Animals
and approved by the Animal Care and Use Committee of the
University of Bordeaux 2.
Surgery
Under ketamine⁄xylazine (61 and 9 mg⁄kg i.p., respectively; Rho ˆne
Me ´rieux, France and Bayer Pharma, Sens, France) anaesthesia, a
23-gauge 7-mm-long stainless steel guide cannula was implanted
using a stereotaxic apparatus so as to terminate just above the roof of
the right lateral cerebral ventricle 2 mm lateral to midline and 0.6 mm
posterior to bregma. Upon completion of surgery, animals were
injected subcutaneously with Rifocine (Marion Merell Dow S.A.,
Levallois-Perret, France). Animals were allowed a 7-day recovery
period in group cages before being housed individually 3–5 days
before the experiments.
Treatments
I.p. injections were made in a volume of 1.0 mL⁄kg to hand-held,
lightly-restrained rats. Rats were injected with saline or 250 lg⁄kg
LPS (Escherichia coli 0127:B8; Sigma, St Fallavier, France) dissolved
in saline. This dose and serotype of LPS was previously shown to
reliably induce behavioural depression and fever (Bluthe ´ et al., 1992;
Konsman et al., 1999). Four hours after i.p. injection of LPS or
vehicle, a 30-gauge injector was lowered through the guide cannula
into the lateral ventricle. A 10-lL Hamilton syringe was used for i.c.v.
infusion of either 3 lL saline or 100 lg recombinant human IL-1
receptor antagonist (rhIL-1ra) kindly provided by AMGEN (Thousand
Oaks, CA, USA) and dissolved in 3 lL of saline. Binding of IL-1ra to
IL-1 receptors displays little, if any, species specificity (Dripps et al.,
1991), thus allowing rhIL-1ra to be used for clearance studies. As i.c.v.
infusion of 100 ng IL-1b readily induces fever, hypothalamo-
pituitary-adrenal (HPA)-axis activation, body weight loss as well as
a reduction in social interaction (Anforth et al., 1998) and a 100- or
1000-fold excess of IL-1ra is needed to block IL-1b effects (Dinarello,
1991), a 100-lg dose of rhIL-1ra was chosen for i.c.v. infusions.
Infusions were given over 3 min after which the injector was left in
place for an additional minute.
Social interaction
Thirty-nine animals were assigned to behavioural experiments. LPS-
induced behavioural depression was assessed by measuring the
reduction in duration of social interaction during 5-min sessions as
previously validated (Bluthe ´ et al., 1992). Animals were trained for
5 min with a juvenile introduced into their home cage 2 days before
the actual experiment. Baseline social interaction was recorded on the
day before the start of the experiment to ensure that animals reliably
displayed social interaction and not sexual or aggressive behaviour.
Social interaction consisted of anogenital sniffing and licking, and
chewing the fur of the juvenile by the experimental animal. Animals
showing less then 50 s of interaction with the juvenile during a 5-min
session were excluded. Behavioural testing was started between 1 and
2 h after lights went off under dimmed red light conditions, and
behaviour was monitored via a video camera. Social interaction was
scored using pre-set keys on a microcomputer (Apple IIE) by an
observer different from the person who injected the rats. Sessions of
social interaction 24 h and 5 min before i.p. injections were used to
calculate baseline social interaction. After i.p. and i.c.v. treatments,
social interaction was measured 5.5 h after i.p. injection, correspond-
ing to 1.5 h after i.c.v. treatment. Different juveniles were presented on
each occasion.
Body temperature
Core body temperature was measured continuously in 30 conscious,
undisturbed, individually housed animals by remote radio-telemetry,
via battery-operated biotelemetry transmitters (Model TA10TA-F40;
Data Sciences, St Paul, MN, USA), which were previously implanted
in the abdominal cavity at the time of brain surgery. The output
frequency (Hz) of each transmitter was monitored by an antenna
mounted in a receiver board situated beneath the cage of each animal,
and channeled to a peripheral processor (Dataquest IV; Data Sciences,
St Paul, MN, USA). Frequencies were sampled at 10-min intervals and
converted to degrees Celsius (?C). Ambient temperature was, as
described above, between 21.5 and 22.5?C to allow for comparison
between experiments. Animals were injected with LPS or saline
between 1 and 2 h after lights went on, as the pyrogenic effects of LPS
are most pronounced during the light phase (Opp & Toth, 1998). Body
temperature was measured for 7.5 h every 10 min, with a brief
interruption at the time of i.c.v. infusion. The mean of three
consecutive measurements for each individual animal was used for
further analysis.
Plasma corticosterone assay
Trunk blood was collected from 26 animals 5.5 h after LPS or saline
injection in chilled tubes containing ethylenediaminetetraacetic acid
(Sigma, St Fallavier, France) and centrifuged at 1800 g for 15 min at
4?C. Plasma was collected and stored at )80?C. Plasma corticosterone
2500J. P. Konsman et al.
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was measured following ethanol extraction by a radiocompetitive
binding assay using rhesus monkey transcortin (Murphy, 1967).
Tritiated corticosterone was used as a tracer and dextran-coated
charcoal as an absorbant for unbound corticosterone. The assay
sensitivity was 15.6 pg⁄tube. Intra- and inter-assay variation coeffi-
cients were 5.2% and 3.6%, respectively.
Body weight
Animals assigned to behavioural experiments were weighed on a
precision balance (0.1 g; Precisa France, Poissy, France) before i.p.
and i.c.v. administration of drugs, as well as 2 h after i.c.v. infusion,
which corresponds to 6 h after i.p. injection. Food intake was not
assessed, as repeated testing of social interactions was found to disturb
spontaneous feeding behaviour.
Plasma and CSF cytokine assays
Trunk blood was collected at 5.5 and 7.5 h after LPS or saline
injection during the light phase in chilled tubes containing ethylene-
diaminetetraacetic acid and centrifuged at 1800 g for 15 min. Plasma
was collected and stored at )80?C until cytokine assays. CSF was
collected by cisterna magna puncture and stored at )80?C until assays,
except for those samples containing traces of blood that were
discarded. Rat IL-1b concentrations were determined using rat-
specific enzyme-linked immunosorbent assay (ELISA; National
Institute of Biological Standards and Control, South Mimms, UK),
as previously described (Safieh-Garabedian et al., 1995; Rees et al.,
1999). Clearance of i.c.v.-infused rhIL-1ra from the brain into the
systemic circulation was assessed using a human IL-1ra-specific
sandwich ELISA (R&D Systems Europe, Lille, France). Assay
sensitivitiesfor rhIL-1ra and
and 4.6 pg⁄mL, respectively. According to the manufacturer, the
rhIL-1ra-recognizing antibodies used for ELISA detection did not
show significant cross-reactivity with 50 ng⁄mL rat recombinant
IL-1ra. Given that plasma levels of endogenous IL-1ra 4 h after i.p.
administration of doses of LPS similar to those used in the present
study are below 50 ng⁄mL (Fofie et al., 2005), rhIL-1ra-specific
ELISA was not expected to detect any endogenous rat IL-1ra.
IL-1b
inplasma were 3.9
Tissue processing
Rats assigned to behavioural experiments were killed 7.5 h after LPS
treatment with an overdose of sodium pentobarbital administered i.p.
Once the hind paw reflex upon plantar pinching had disappeared,
brains were fixed by intracardiac perfusion of saline via the ascending
aorta followed by 4% paraformaldehyde in 0.1 m borate buffer (pH
9.5 at 10?C). Brains were post-fixed for 4 h, and then cryoprotected in
30% sucrose in 0.1 m phosphate buffer (pH 7.4) for 48 h. A series of
one in six frontal 30-lm cryostat sections through the whole brain
were collected in cold cryoprotectant (0.05 m phosphate buffer, 30%
sucrose, 30% ethylene glycol) and stored at )20?C until immuno-
cytochemical processing.
Immunohistochemistry
Immunocytochemistry for c-Fos and IL-1b was performed as previ-
ously described (Konsman et al., 1999). A commercially available
antiserum to c-Fos (Santa Cruz Biotechnology, Santa Cruz, CA, USA)
diluted 1 : 2000 was used. The c-Fos antiserum was raised against a
synthetic peptide corresponding to amino acids 3–16 at the N-terminal
of human and mouse c-Fos, and did not cross-react with Fos-related
antigens (manufacturer’s specifications). For IL-1b immunocyto-
chemistry, a sheep antiserum generated to recombinant rat IL-1b
(Dr S. Poole, NIBSC, Potters Bar, UK) was used at a dilution of
1 : 1000. This IL-1b antiserum has previously been shown to
specifically recognize the pro- and mature form of IL-1b in microglia
(Chauvet et al., 2001).
After washing off the cryoprotectant solution, immunohistochem-
ical processing was performed on free-floating sections using the
streptavidin-biotin-immunoperoxidase technique. Briefly, non-specific
binding sites were blocked by a 1-h incubation of sections in Tris-
buffered saline (pH 7.4; TBS) containing 0.3% Triton X-100 and 0.2%
casein. The first antibody was added for 60 h at 4?C in the same
buffer. After four rinses in TBS, sections were treated for 30 min in
0.3% (v⁄v) hydrogen peroxide followed by rinses in TBS. Sections
were incubated for 2 h at room temperature with biotinylated donkey
anti-sheep⁄goat or biotinylated donkey anti-rabbit immunoglobulins
G (1 : 1000; Amersham, Les Ulis, France) depending on the first
antibody, and stained using the ABC (1 : 1000; Vectastain Elite,
Vector Laboratories, Burlingame, CA, USA) with diaminobenzidine
nickel-enhanced glucose oxidase as a chromogene.
Microscopy and image analysis
Stained sections were examined through a 10· objective mounted on a
transmitted light microscope, and images were captured using a high-
resolution CCD video camera and fed into a personal computer. Image
processing was performed using a Quantimet Q-600HR computerized
image analysis system (Leica, Rijswijk, The Netherlands) on grey
images by defining brightness and the surface above which labelling
was to be taken into account. Once established, these parameters
remained unchanged. The image was then converted to a binary image
and the number of Fos-immunoreactive cells was measured in at least
four sections of the nucleus of the solitary tract, the ventromedial
preoptic area, the parvocellular part of the paraventricular nucleus of
the hypothalamus, the central nucleus of the amygdala, the dorsolat-
eral part of the bed nucleus of the stria terminalis and in three sections
of the area postrema. These structures have previously been shown to
contain Fos-positive cells after i.p. injection of LPS (Konsman et al.,
1999). The mean number of c-Fos-positive cells was then determined
for each structure in each animal and used for further analysis.
Photomicrographs for illustration were taken with the same setup, and
Adobe Photoshop was used to adjust contrast and brightness and to
apply labels.
Data presentation and statistical analysis
All data are reported as means ± SEM. The temperature for each
animal was expressed as difference in degrees Celsius compared
with body temperature 30 min before i.p. injection and cumulated
over the 4-h period between i.p. injection and i.c.v. infusion, and
over a 2-h period after i.c.v. infusion. Before i.c.v. infusion, body
temperature data were analysed by a one-way repeated-measures
anova (i.p. injection as between-subject factor, time as within-
subject factor). After i.c.v. infusion body temperature was analysed
by a two-way repeated-measures anova (i.p. injection and i.c.v.
infusion as between-subject factors, time as within-subject factor).
The duration of social investigation after i.p. and i.c.v. treatments
was expressed as percentage of baseline interaction and analysed
using a two-way anova (i.p. injection and i.c.v. infusion as
Brain interleukin-1 activates limbic structures2501
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between-subject factors). Changes in body weight were expressed in
grams and analysed by a one-way anova until i.c.v. infusion and by
a two-way anova after i.c.v. infusion (i.p. injection and i.c.v.
infusion as between-subject factors). Plasma corticosterone and
cytokine concentrations were expressed in ng⁄mL and pg⁄mL,
respectively, and analysed using two-way anovas (i.p. injection and
i.c.v. infusion as between-subject factors). The number of c-Fos-
immunoreactive cells in brain structures was also analysed using a
two-way anova (i.p. injection and i.c.v. infusion as between-subject
factors). Non-parametric tests were used when data were not
normally distributed or did not show equal variance. Significant
anova effects were further analysed by the Newman–Keuls and
Tukey post hoc tests. In all cases, a level of P < 0.05 was considered
as statistically significant.
Results
One animal was excluded from further analysis because of incorrect
cannula placement. Two other animals were excluded from analysis
because they did not reach the 50-s limit of social interaction during
the 5-min baseline session.
I.p. LPS-induced IL-1b in brain circumventricular organs
and choroid plexus
Ten to 12 days after intracortical implantation of a stainless steel guide
cannula, we observed IL-1b-immunoreactive cells around the cannula
track regardless of i.p. and i.c.v. treatments (not shown). In addition to
the cortex and corpus callosum penetrated by the guide cannula and
injector, we observed IL-1b-positive cells in the ipsilateral ventral
hippocampal commissure (Fig. 1C). No other parenchymal sites of
IL-1b expression were observed in the brains of animals that received
an i.p. injection of saline. In contrast to our previous study in naı ¨ve
animals (Konsman et al., 1999), we observed IL-1b-immunoreactive
cells associated with the walls of the ipsilateral lateral (Fig. 1B) and
third ventricle, as well as within the meninges of saline-injected
animals bearing intracortical cannulas. However, with the exception of
the meninges, other brain structures lacking a functional blood–brain
barrier (BBB), including the choroid plexus (Fig. 1D) and circum-
ventricular organs (Fig. 1A, C and E), were devoid of IL-1b-positive
cells after i.p. administration of saline.
I.p. injection of LPS (250 lg⁄kg) induced IL-1b-immunoreactive
cells in circumventricular organs and adjacent parenchyma, including
the organum vasculosum of the lamina terminalis (Fig. 1F), the
Fig. 1. IL-1b-immunoreactive cells in rat brain circumventricular organs, choroid plexus and ventricular walls 7.5 h after i.p. injection of saline or 250 lg ⁄ kg
lipopolysaccharide (LPS). Besides the ipsilateral cortex and corpus callosum penetrated by the guide cannula, parenchymal IL-1b expression in animals that received
an i.p. injection of saline was observed only in the ventral hippocampal commissure (C). At CSF–tissue interfaces, IL-1b-immunoreactive cells were associated with
the walls of the lateral ventricle ipsilateral to the cannula (B), the third ventricle and the meninges in saline-injected animals. However, with the exception of the
meninges, other brain structures lacking a functional BBB, including the choroid plexus (D) and circumventricular organs (A, C and E), were devoid of
IL-1b-immunoreactive cells after i.p. administration of saline. I.p. injection of LPS (250 lg ⁄ kg) did not increase IL-1b expression in the ventral hippocampal
commissure (H) or in the ventricle walls (G, L), but induced IL-1b-immunoreactive cells in circumventricular organs, including the organum vasculosum of the
lamina terminalis (F), the subfornical organ (H) and the area postrema (J), and also in the choroid plexus (I). Labelled cells often contained cellular extrusions
encompassing blood vessels (K, M and O), while the shape of IL-1b-positive cells in adjacent parenchyma resembled that of microglia (* in K). Note the presence of
some smaller IL-1b-immunoreactive cells that were not associated with blood vessels in the choroid plexus, but occurred over the free surface of the choroid plexus
(* in N). AP, area postrema; cc, central canal; ChP, choroid plexus; LPS, lipopolysaccharide; lv, lateral ventricle; oc, optic chiasm; OVLT, organum vasculosum of the
lamina terminalis; sal, saline; SFO, subfornical organ; vhc, ventral hippocampal commissure. Scale bars: 100 lm (A–J); 25 lm (K–O). Rectangles represent zones in
(F–J) shown in higher magnification in (K–O).
2502 J. P. Konsman et al.
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subfornical organ (Fig. 1H) and the area postrema (Fig. 1J), as well as
in the choroid plexus (Fig. 1I) 7.5 h later. This distribution is similar to
that previously reported 8 h after i.p. LPS administration in animals
not bearing cannulas and not subject to behavioural testing (Konsman
et al., 1999). Within circumventricular organs, labelled cells often
contained cellular extrusions encompassing blood vessels, while the
shape of IL-1b-positive cells in adjacent neural parenchyma resembled
that of ramified microglia (Fig. 1K, M and O), as previously reported
(Konsman et al., 1999). Some smaller IL-1b-immunoreactive cells
were not associated with blood vessels in the choroid plexus and
occurred over the free surface of the choroid plexus (Fig. 1N). The
localization and shape of these cells correspond to those of epiplexus
cells described by Ling et al. (1998), and may represent intraventric-
ular macrophages. In spite of the presence of IL-1b-positive cells that
were seemingly in contact with the CSF, no immunoreactive IL-1b
was detected in the CSF after i.p. LPS administration (data not
shown). I.c.v. infusion of IL-1ra did not affect LPS-induced IL-1b
immunoreactivity in the brain.
I.c.v.-infused rhIL-1ra was detected in systemic circulation
1.5 and 3.5 h later
Previous studies have shown that after i.c.v. infusion of IL-1b, this
cytokine can be detected in systemic circulation for several hours (Di
Santo et al., 1999). In accordance with these data, two-way anovas
showed significant increases in plasma rhIL-1ra concentrations 1.5
and 3.5 h after i.c.v. infusion of 100 lg rhIL-1ra (rhIL-1ra:
F1,17= 2754; P < 0.001 and F1,17= 168; P < 0.001, respectively),
but no effect of LPS injection or an interaction between LPS and rhIL-
1ra treatments (Fig. 2A and C). Mean plasma rhIL-1ra concentrations
Fig. 2. Clearance of intracerebroventricular (i.c.v.)-infused 100 lg recombinant human interleukin-1 receptor antagonist (rhIL-1ra) into systemic blood (A, C), and
effects on plasma interleukin-1b (IL-1b) levels (B, D) 1.5 and 3.5 h later. sal–sal, animals injected i.p. with saline and i.c.v. with saline; sal–IL-1ra, animals receiving
an i.p. injection of saline followed by an i.c.v. infusion of rhIL-1ra; LPS–sal, rats injected i.p. with lipopolysaccharide (LPS) and i.c.v. with saline; LPS–IL-1ra, rats
receiving an i.p. injection of LPS followed by an i.c.v. infusion of rhIL-1ra. **P < 0.01, LPS–sal vs. LPS–IL-1ra. Experimental groups consisted of five to seven
animals.
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were 8559 ± 187 and 3134 ± 191 pg⁄mL at 1.5 and 3.5 h after i.c.v.
rhIL-1ra infusion, respectively. Given that 250 g Wistar rats contain
17 mL blood (Lee & Blaufox, 1985), plasma concentrations 1.5 and
3.5 h after rhIL-1ra i.c.v. infusion can be calculated to correspond to
0.145% and 0.053% of the amount administered.
I.c.v. rhIL-1ra attenuated i.p. LPS-induced plasma IL-1b 3.5 h
later
In view of the presence of rhIL-1ra in systemic blood and the known
stimulatory effect of peripheral IL-1b on its own synthesis (Pa ´ez
Pereda et al., 1996; Hansen et al., 1998), it was of interest to analyse
the effects of i.c.v. rhIL-1ra infusion on plasma levels of IL-1b. A two-
way anova on plasma IL-1b levels 1.5 h after i.c.v. infusion showed
the expectedsignificantincrease
F1,21= 14,8; P < 0.001), but no effect of i.c.v. rhIL-1ra infusion or
any interaction between LPS and rhIL-1ra treatments (Fig. 2B). The
same analysis 3.5 h after i.c.v. infusion did, however, reveal an
interaction between LPS and rhIL-1ra treatments (LPS · rhIL-1ra:
F1,20= 4.95; P < 0.05) in addition to the significant effect of i.p. LPS
injection (LPS: F1,20= 6.16; P < 0.05). Post hoc analysis showed that
at this time point plasma IL-1b levels were still elevated in LPS-
treated rats, but that this response was significantly reduced by i.c.v.
rhIL-1ra infusion (P < 0.01; Fig. 2D).
after LPSinjection (LPS:
I.c.v. rhIL-1ra attenuated i.p. LPS-induced reduction in social
interaction
One of the cardinal signs of behavioural depression displayed by a
rodent after peripheral LPS injection is the reduction of social
interactions (Bluthe ´ et al., 1992). A two-way anova on the
duration of social interaction with a conspecific juvenile after
treatments compared with baseline showed the expected significant
reduction after i.p. LPS injection (LPS: F1,30= 78.0; P < 0.001). In
addition, this analysis revealed an effect of i.c.v. rhIL-1ra infusion
(rhIL-1ra: F1,30= 5.59; P < 0.05) and an interaction between LPS
and rhIL-1ra treatments (LPS · rhIL-1ra: F1,30= 5.96; P < 0.05).
Post hoc analysis showed that the duration of social interactions
was reduced in animals that received LPS (P < 0.001), and this
effect was attenuated by i.c.v. treatment with rhIL-1ra (P < 0.05;
Fig. 3A).
I.c.v. rhIL-1ra did not affect i.p. LPS-induced fever
Previous studies have shown that i.p. administration of the same dose
and serotype of LPS results in a biphasic fever response starting 2 h
later and lasting up to 8 h after injection (Konsman et al., 1999). A
one-way repeated-measures anova on body temperature before i.c.v.
infusion showed that, as expected, body temperature was significantly
increased in i.p. LPS-treated rats (LPS: F1,25= 13.6; P < 0.01;
Fig. 3B), and revealed a significant interaction between time and
LPS administration (LPS · time: F8,208= 20.2; P < 0.001). Post hoc
analysis showed that body temperature was significantly elevated from
2.5 to 4 h after i.p. LPS injection compared with animals that were
given saline (not shown). A two-way repeated-measures anova
analysis on body temperature after i.c.v. infusion revealed that body
temperature was still elevated in LPS-treated animals (LPS:
F1,24= 12.2; P < 0.01), even though it started to drop over time
(LPS · time: F6,150= 3.0; P < 0.01). However, i.c.v. IL-1ra infusion
did not significantly alter body temperature at any time point
(P > 0.10; not shown).
I.c.v. rhIL-1ra did not alter i.p. LPS-induced rises in plasma
corticosterone
As activation of the hypothalamo-pituitary-adrenal axis resulting in
corticosterone release plays an important role in energy mobilization
necessary to raise body temperature (Scho ¨bitz et al., 1994), plasma
levels of this hormone were studied. Plasma levels of corticosterone in
saline-treated animals were slightly elevated 5.5 h after i.p. injection
compared with basal levels occurring normally 6 h after switching off
the lights (De Boer & Van der Gugten, 1987). This may be due to the
i.c.v. infusion that had taken place 1.5 h earlier (Kim et al., 1998). In
spite of this, a two-way anova did show the expected significant
increase of plasma corticosterone after i.p. LPS injection (LPS:
F1,21= 21.8; P < 0.001), but did not reveal an effect of i.c.v. rhIL-1ra
infusion or an interaction between LPS and rhIL-1ra treatments
(Fig. 3C).
I.c.v. rhIL-1ra did not modify i.p. LPS-induced weight loss
Mobilization of energy stocks to mount a fever response after i.p. LPS
administration results in weight loss. As variances of body weight
changes before i.c.v. infusion were not equal between saline and LPS-
treated animals, a non-parametric test was performed. A Kruskal–
Wallis analysis on ranks of changes in body weight during the 4 h
following i.p. injection showed the expected significant loss of body
weight after i.p. LPS administration (LPS: H = 20.1; P < 0.001).
A two-way anova analysis of changes in body weight after i.c.v.
infusion between 4 and 6 h after i.p. injection confirmed the effect of
LPS treatment (LPS: F1,29= 4.2; P < 0.05), but did not reveal any
effect of i.c.v. rhIL-1ra infusion on body weight or an interaction
between LPS and IL-1ra treatments (Fig. 3D).
I.c.v. rhIL-1ra inhibited i.p. LPS-induced c-Fos expression in
limbic structures, but not in caudal brainstem or hypothalamus
C-Fos immunoreactivity was low in most of the studied brain
structures of saline-treated rats. I.p. injection of 250 lg⁄kg LPS
followed 4 h later by an i.c.v. infusion of sterile saline resulted in Fos
induction 3.5 h later in the caudal brainstem, the preoptic and
neuroendocrine hypothalamus and limbic structures.
The caudal brainstem contains the dorsal vagal complex, made up
of the area postrema, nucleus of the solitary tract and the dorsal
motor nucleus of the vagus nerve, which can rapidly activate
forebrain structures after i.p. LPS injection (Marvel et al., 2004). The
area postrema contains IL-1 receptor mRNA (Ericsson et al., 1995)
and expresses c-Fos after LPS administration (Konsman et al.,
1999), while the adjacent nucleus of the solitary tract is an important
relay structure between the viscera and the hypothalamus and limbic
system (Konsman et al., 2000a). Two-way anovas on the number of
c-Fos-positive nuclei in the area postrema and nucleus of the solitary
tract did indeed show significant increases after i.p. LPS adminis-
tration (LPS: F1,25= 14.8; P < 0.001 and LPS: F1,25= 20.3;
P < 0.001, respectively), but did not reveal any effect of i.c.v.
rhIL-1ra infusion or an interaction between LPS and rhIL-1ra
treatments (Fig. 4A).
The ventromedial preoptic area and paraventricular nucleus of the
hypothalamus are known to express c-Fos and to be involved in the
fever and HPA-axis activation after peripheral LPS administration
(Horn et al., 1994; Elmquist et al., 1997; Caldwell et al., 1998). In the
ventromedial preoptic area and paraventricular nucleus of the
hypothalamus, two-way anovas on the number of c-Fos-positive
nuclei showed the expected significant increase after LPS injection
2504J. P. Konsman et al.
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European Journal of Neuroscience, 28, 2499–2510

Page 7

(LPS: F1,27= 42.0; P < 0.001 and LPS: F1,29= 15.0; P < 0.001,
respectively), but did not reveal any effect of i.c.v. rhIL-1ra infusion or
an interaction between LPS and rhIL-1ra treatments (Fig. 4B).
The central amygdala and dorsolateral bed nucleus have previously
been shown to express c-Fos after peripheral LPS administration
independently of hypotension that can be induced by high doses of
LPS (Tkacs et al., 1997). In the dorsolateral bed nucleus of the stria
terminalis, a two-way anova on the number of c-Fos-positive nuclei
did indeed show an increase after i.p. LPS administration (LPS:
F1,26= 60.5; P < 0.0001). In addition, this analysis revealed a
significant effect of i.c.v. rhIL-1ra infusion (rhIL-1ra: F1,26= 40.9;
P < 0.0001), as well as a significant interaction between LPS and
rhIL-1ra (LPS · rhIL-1ra: F1,26= 48.4; P < 0.0001). Post hoc anal-
ysis showed that i.p. LPS significantly increased the number of c-Fos-
positive cells in the bed nucleus (P < 0.001), and that this effect was
prevented by i.c.v. rhIL-1ra infusion (P < 0.001; Figs 4C and 5). A
two-way anova on the number of c-Fos-positive nuclei in the central
amygdala revealed a significant effect of i.c.v. rhIL-1ra infusion
(rhIL-1ra: F1,28= 6.24; P < 0.05), and a significant interaction
between LPS and rhIL-1ra treatments (LPS · rhIL-1ra: F1,28= 7.32;
P < 0.05). According to post hoc analysis, the i.p. LPS-induced
increase in the number of c-Fos-immunoreactive cells in the central
amygdala (P < 0.05) was prevented by i.c.v. rhIL-1ra infusion
(P < 0.01; Figs 4D and 6).
Fig. 3. Effects of i.p. injection of 250 lg ⁄ kg lipopolysaccharide (LPS) or saline followed 4 h later by an i.c.v. infusion of recombinant human interleukin-1 receptor
antagonist (rhIL-1ra) or saline on social interaction (A), body temperature changes (B), plasma corticosterone (C) and body weight changes (D). (A) Baseline social
interaction with a juvenile conspecific was measured for 5 min 24 h and just before i.p. injection. Social interaction was assessed again 5.5 h after i.p. injection,
corresponding to 1.5 h after i.c.v. infusion, and expressed as percentage of baseline social interaction. (B) Cumulated changes in body temperature over the 4 h
between i.p. injection and i.c.v. infusion are shown in light grey, while those during the 2 h following i.c.v. infusion are depicted in dark grey. (C) Blood for plasma
corticosterone assays was withdrawn 5.5 h after i.p. injection, corresponding to 1.5 h after i.c.v. infusion. (D) Changes in body weight over the 4 h between i.p.
injection and i.c.v. infusion are shown in light grey, while those occurring during the 2 h following i.c.v. infusion are represented in dark grey. *P < 0.05, LPS–sal vs.
LPS–IL-1ra. Experimental groups consisted of six to 11 animals. Abbreviations, see Fig. 2.
Brain interleukin-1 activates limbic structures2505
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Page 8

Discussion
The present study shows that i.c.v. infusion of rhIL-1ra 4 h after i.p.
LPS injection attenuated the reduction in social interaction, one of the
cardinal signs of behavioural depression during the APR, and c-Fos
expression in the central amygdala and bed nucleus of the stria
terminalis. However, LPS-induced fever and c-Fos expression in the
caudal brainstem and hypothalamus were not altered by i.c.v. rhIL-1ra
infusion. These results show for the first time that IL-1 acts in the brain
to induce behavioural depression during the APR mounted by the host
upon detection of bacterial LPS fragments, and suggest that limbic
structures are activated by its action.
Brain and CSF IL-1b-immunoreactivity
Several studies have shown that corpus callosum-penetrating intra-
hippocampal cannulas induce IL-1-immunoreactivity in distant brain
regions and exacerbate peripheral LPS-induced IL-1 production
(Tchelingerian et al., 1993; Gabellec et al., 1999; Holguin et al.,
2007). To reduce IL-1 production in distant brain regions, we
implanted guide cannulas into the cortex 10–12 days prior to
experiments and penetrated the corpus callosum for i.c.v. administra-
tion only 3.5 h before the end of the experiment. After this procedure,
we observed IL-1b-immunoreactive cells in the cortex around the
cannula, in the corpus callosum, ventral hippocampal commissure,
ventricular walls and meninges, but not in other brain structures of
saline-injected animals. Our observations are in accordance with
previous findings on IL-1a (Tchelingerian et al., 1993), and indicate
that hippocampus-sparing intracortical cannulas do not provoke IL-1
production in the parenchyma of distant brain structures.
In responsetoi.p. LPS
IL-1b-immunoreactive cells in brain circumventricular organs and
the choroid plexus, known to express Toll-like receptor 4 (Laflamme
& Rivest, 2001; Chakravarty & Herkenham, 2005). Their number and
administration,weobserved
Fig. 4. C-Fos expression in the nucleus of the solitary tract (A), paraventricular nucleus of the hypothalamus (B), dorsolateral bed nucleus of the stria terminalis (C)
and lateral central amygdala (D) after i.p. injection of 250 lg ⁄ kg lipopolysaccharide (LPS) or saline followed 4 h later by an i.c.v. infusion of recombinant human
interleukin-1 receptor antagonist (rhIL-1ra) or saline. Brains were harvested 7.5 h after i.p. injection, corresponding to 3.5 h after i.c.v. infusion. **P < 0.01,
LPS–sal vs. LPS–IL-1ra. Experimental groups consisted of five to 11 animals. Abbreviations, see Fig. 2.
2506J. P. Konsman et al.
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European Journal of Neuroscience, 28, 2499–2510

Page 9

morphology were similar to those of IL-1b-immunoreactive cells
previously observed in naı ¨ve animals (Nakamori et al., 1994;
Konsman et al., 1999; Goehler et al., 2006), indicating that cannula
implantation and i.c.v. drug administration did not modify LPS-
induced IL-1b expression in these organs. Although CSF-contacting
IL-1b-positive cells were observed in the choroid plexus, no
immunoreactive IL-1b was detected in CSF. Taken together, our
findings indicate that brain circumventricular organs, along with
blood-to-brain transport, rather than CSF, are the main sources of brain
IL-1b after i.p. LPS administration.
I.c.v. IL-1ra can reduce i.p. LPS-induced IL-1b plasma levels
Although IL-1 and rhIL-1ra rapidly reach the circumventricular
organs, hypothalamus and amygdala after their i.c.v. infusion (Plotkin
et al., 1996; Konsman et al., 2000b), only ?1% of the amount
administered is still present in the brain 2 h later (Di Santo et al.,
1999). Our present observations that 0.15% and 0.05% of
i.c.v.-administered rhIL-1ra were detected in plasma 1.5 and 3.5 h
later are similar to the amounts of i.c.v.-infused IL-1b that occur in
systemic circulation at these time points (Chen & Reichlin, 1999;
Di Santo et al., 1999). I.c.v.-administered IL-1b is indeed rapidly
cleared from the brain, and results in higher and longer-lasting plasma
and liver levels than its intravenous injection (Chen & Reichlin,
1999; Di Santo et al., 1999). Along with its presence in blood, rhIL-
1ra can, therefore, be expected to have reached the liver in the present
study. As IL-1b induces its own synthesis monocytes (Pa ´ez Pereda
et al., 1996) and in the liver (Hansen et al., 1998), the presence of
rhIL-1ra in the blood and liver may reduce i.p. LPS-induced increases
in IL-1b. Given that plasma IL-1b concentrations after i.p. LPS were
not found to be altered 1.5 h after i.c.v. rhIL-1ra infusion, any change
in behaviour or physiology observed 1.5 h after i.c.v. rhIL-1ra was
most probably the consequence of rhIL-1ra action in the brain.
However, 3.5 h after i.c.v. infusion we observed that rhIL-1ra reduced
i.p. LPS-induced plasma levels of IL-1b, suggesting that rhIL-1ra
prevented IL-1b-induced IL-1b production in liver macrophages and
blood monocytes.
Brain IL-1 action does not contribute to LPS-induced fever,
increased plasma corticosterone or brainstem and hypothalamic
c-Fos expression
Our observation that i.c.v. rhIL-1ra attenuated i.p. LPS-induced rises
in plasma IL-1b 3.5 h later may explain some apparent discrepancies
between the present and other published studies. Indeed, earlier
studies, in which doses of 50–500 lg rhIL-1ra were administered i.c.v.
twice before fever onset, reported blunted fever responses after i.p.
LPS administration (Luheshi et al., 1996; Cartmell et al., 1999). In
contrast, in the present study a single i.c.v. infusion of 100 lg rhIL-1ra
4 h after i.p. LPS injection did not affect fever. To explain this
discrepancy, we propose that plasma rhIL-1ra after repeated i.c.v.
infusion interfered with the induction of the prostaglandin-synthesiz-
ing enzyme cyclooxygenase-2 at the BBB, either directly by
interfering with IL-1b action on receptors expressed by cerebral
endothelial cells (Konsman et al., 2004) or indirectly by reducing
plasma IL-1b levels as observed in the present study. As prostaglandin
synthesis and action are essential in febrile responses to i.p. LPS
administration (Li et al., 1999; Oka et al., 2003), brain-to-blood
clearance may explain the attenuating effect of repeated i.c.v. rhIL-1ra
infusions on fever. It is important to point out that another study has
shown that cerebral overexpression of IL-1ra sufficient to attenuate
febrile responses to i.c.v. administration of IL-1b does not alter i.p.
LPS-induced fever (Lundkvist et al., 1999). Our present observations
thus extend previous findings from our laboratories using i.p. IL-1b as
a stimulus (Kent et al., 1992), and indicate that IL-1b action in the
brain does not play a major role in fever during the APR provoked by
peripheral LPS administration.
Activation of the HPA-axis resulting in corticosterone release plays
an important role in energy mobilization necessary to raise body
Fig. 5. LPS-induced c-Fos expression in the dorsolateral bed nucleus of the
stria terminalis depends on central nervous action of IL-1. After i.p. injection of
saline, few c-Fos-positive cells were observed, irrespective of whether these
animals received an i.c.v. infusion of saline or rhIL-1ra (A, B). Extensive c-Fos
expression occurred 7.5 h after i.p. LPS injection in animals that received a
subsequent i.c.v. infusion of saline (C), and this effect was prevented by i.c.v.
rhIL-1ra administration (D). ac, anterior commissure; lv, lateral ventricle. Scale
bar: 100 lm.
Fig. 6. LPS-induced c-Fos expression in the central amygdala depends on
central nervous action of IL-1. After i.p. injection of saline, few c-Fos were
observed (A), although i.c.v. infusion of rhIL-1ra tended to increase c-Fos
expression as compared with i.c.v. infusion of saline (B). C-Fos expression
increased 7.5 h after i.p. LPS injection in animals that received a subsequent
i.c.v. infusion of saline (C), and this effect was prevented by i.c.v. rhIL-1ra
administration (D). Scale bar: 100 lm.
Brain interleukin-1 activates limbic structures2507
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European Journal of Neuroscience, 28, 2499–2510

Page 10

temperature (Scho ¨bitz et al., 1994). A previous study has shown that
i.c.v. IL-1ra infusion attenuates i.p. IL-1b-, but not i.p. LPS-, induced
rises in plasma corticosterone, when molecules are administered
within a 30-min time window (Dunn, 2000). However, i.c.v.-
administered IL-1ra around the time of i.p. LPS injection may not
be present in sufficient concentrations to prevent IL-1b action when it
is bioactive in the CNS several hours later. We, therefore, studied the
effect of i.c.v. rhIL-1ra infusion 4 h after i.p. LPS injection on plasma
corticosterone levels 1.5 h after i.c.v. administration. Our findings
show that even in these conditions i.c.v. rhIL-1ra does not alter i.p.
LPS-induced rises in plasma corticosterone.
Given that the brain structures and circuits involved in fever and
activation of the HPA-axis are well known, we also studied CNS
expression of the cellular activation marker c-Fos after i.c.v. rhIL-1ra
and i.p. LPS administration. Lesion studies have shown that the
preoptic area and paraventricular nucleus of the hypothalamus are
crucial in LPS-induced fever (Horn et al., 1994; Caldwell et al., 1998),
while noradrenergic brainstem projections to the paraventricular
nucleus of the hypothalamus are involved in rises in plasma
corticosterone after i.p. LPS injection (Bienkowski & Rinaman,
2008). In corroboration with our observations that i.c.v. rhIL-1ra did
not affect fever or rises in plasma corticosterone after LPS adminis-
tration, we did not find any effect of i.c.v. rhIL-1ra on i.p. LPS-
induced caudal brainstem or hypothalamic c-Fos expression.
Brain IL-1 contributes to i.p. LPS-induced behavioural
depression
Behavioural depression and adoption of a ‘curled-up’ position can be
considered adaptive in response to an acute infection as they reduce
spreading of bacteria and heat loss (Hart, 1988). In contrast to the lack
of effect of i.c.v. rhIL-1ra on febrile and endocrine responses to LPS,
we found that i.c.v. infusion of rhIL-1ra 4 h after peripheral LPS
injection attenuated the reduction in social interaction 1.5 h later.
Earlier attempts to block brain IL-1b action by infusing 100 lg rhIL-
1ra i.c.v. at the time of peripheral LPS injection have been without
effect on the reduction of social interaction (Bluthe ´ et al., 1992),
probably because rhIL-1ra was no longer present in sufficient
concentrations when IL-1b was bioactive in the brain.
After its i.c.v. infusion, rhIL-1ra spreads along preferential diffusion
pathways formed by perivascular spaces (Konsman et al., 2000b). I.p.
LPS-induced IL-1b production in brain circumventricular organs and
adjacent neural parenchyma, as found in our present and previous
study (Konsman et al., 1999), may diffuse throughout these spaces,
while circulating IL-1b may enter perivascular spaces after active
transport across the BBB (Banks et al., 1991). Interestingly, these
spaces are occupied by macrophages expressing IL-1 type 1 receptors
that are activated by i.p. LPS administration (Konsman et al., 2004).
Moreover, our most recent findings show that elimination of brain
perivascular macrophages bearing IL-1 receptors attenuated decreased
locomotor activity after i.p. LPS injection (Chaskiel & Konsman,
unpublished findings). These observations indicate that IL-1 action on
perivascular macrophages mediates at least some LPS-induced signs
of behavioural depression.
Brain IL-1b mediates i.p. LPS-induced c-Fos expression
in limbic structures
As a first approach to identify potential neural substrates underlying
i.p. LPS-induced brain IL-1-dependent behavioural changes, we
studied CNS c-Fos expression in animals used for behavioural testing.
Previous work, including our own, has shown that c-Fos-immunore-
activity in the brain is still reliably observed between 4 and 8 h after
peripheral LPS injection (Elmquist et al., 1993; Konsman et al.,
1999). As c-Fos immunoreactivity is maximal ?2 h after application
of a focal stimulus (Sharp et al., 1991), such as i.c.v. infusion, we
reasoned that its detection 3.5 h later reflected brain activation
corresponding to the time of behavioural testing, 1.5 h after i.c.v.
rhIL-1ra infusion and 5.5 h after i.p. LPS injection.
I.c.v. infusion of rhIL-1ra 4 h after i.p. LPS injection prevented
c-Fos-immunoreactivity in the central amygdala and dorsolateral bed
nucleus of the stria terminalis. Even though the brainstem dorsal
vagal complex can rapidly activate these limbic structures after i.p.
LPS injection (Marvel et al., 2004), i.c.v. rhIL-1ra did not alter i.p.
LPS-induced c-Fos expression in the dorsal vagal complex. Our
findings therefore indicate that IL-1 acts in the forebrain to sustain
activation of the central amygdala and dorsolateral bed nucleus of
the stria terminalis. As mentioned, forebrain perivascular macro-
phages bear IL-1 type 1 receptors and express cyclooxygenase-2
after i.p. LPS injection (Konsman et al., 2004). However, cyclo-
oxygenase inhibitors do not alter i.p. LPS-induced Fos expression in
the central amygdala and bed nucleus of the stria terminalis (Lacroix
& Rivest, 1997), suggesting that prostaglandin synthesis by forebrain
perivascular macrophages is not involved in brain IL-1-dependent
activation of limbic structures. In addition to prostaglandins, brain
perivascular cells also produce NO after peripheral LPS administra-
tion (Wong et al., 1996). As NO induces c-Fos mRNA in the central
amygdala and bed nucleus (Chikada et al., 2000), it may constitute
an intermediate between central IL-1b action on perivascular
macrophages and i.p. LPS-induced c-Fos expression in limbic
structures.
Conclusion
In conclusion, the present study shows that IL-1b acts in the forebrain
to sustain reduced social interaction, a cardinal sign of behavioural
depression during the APR, and c-Fos expression in the central
amygdala and bed nucleus of the stria terminalis after peripheral LPS
administration. The sites of central IL-1b action and the role of limbic
structures in behavioural depression during the APR remain to be
determined in future experiments.
Acknowledgements
Rat ELISA reagents were produced within the context of the European
Community BIOMED 2 (CT97-2492) and TMR (CT97-0149) projects.
AMGEN (Thousand Oaks, CA, USA) kindly supplied rhIL-1ra. We thank
J. Keijser (University of Groningen, the Netherlands) for assistance with image
analysis. The authors declare that they do not have any financial or other
involvement that might bias the present work.
Abbreviations
APR, acute phase response; BBB, blood–brain barrier; CSF, cerebrospinal
fluid; ELISA, enzyme-linked immunosorbent assay; HPA-axis, hypothalamo-
pituitary-adrenal-axis;i.c.v., intracerebroventricular;
IL-1ra, interleukin-1 receptor antagonist; IL-1b, interleukin-1b; LPS, lipopoly-
saccharides; rh, recombinant human; TBS, Tris-buffered saline.
i.p.,intraperitoneal;
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