Neuroscience 140 (2006) 1415–1434
NEURAL–IMMUNE INTERFACE IN THE RAT AREA POSTREMA
L. E. GOEHLER,* A. ERISIR AND R. P. A. GAYKEMA
Program in Sensory and Systems Neuroscience, Department of Psy-
chology and Neuroscience Graduate Program, University of Virginia,
Charlottesville, VA 22904, USA
Abstract—The area postrema functions as one interface be-
tween the immune system and the brain. Immune cells within
the area postrema express immunoreactivity for the pro-in-
flammatory cytokine, interleukin-1␤ following challenge with
immune stimulants, including lipopolysaccharide (from bac-
terial cell walls). As a circumventricular organ, the area pos-
trema accesses circulating immune-derived mediators, but
also receives direct primary viscerosensory signals via the
vagus nerve. Neurons in the area postrema contribute to
central autonomic network neurocircuitry implicated in brain-
mediated host defense responses. These experiments were
directed toward clarifying relationships between immune
cells and neurons in the area postrema, with a view toward
potential mechanisms by which they may communicate. We
used antisera directed toward markers indicating microglia
(CR3/CD11b; OX-42), resident macrophages (CD163; ED-2),
or dendritic cell-like phenotypes (major histocompability
complex class II; OX-6), in area postrema sections from li-
popolysaccharide-treated rats processed for light, laser
scanning confocal, and electron microscopy. Lipopolysac-
charide treatment induced interleukin-1␤-like immunoreactiv-
ity in immune cells that either associated with the vasculature
(perivascular cells, a subtype of macrophage) or associated
with neuronal elements (dendritic-like, and unknown pheno-
type). Electron microscopic analysis revealed that some im-
mune cells, including interleukin-1␤-positive cells, evinced
membrane apposition with neuronal elements, including den-
drites and terminals, that could derive from inputs to the area
postrema such as vagal sensory fibers, or intrinsic area pos-
trema neurons. This arrangement provides an anatomical
substrate by which immune cells could directly and specifi-
cally influence individual neurons in the area postrema, that
may support the induction and/or maintenance of brain re-
sponses to inflammation. © 2006 IBRO. Published by Elsevier
Ltd. All rights reserved.
Key words: interleukin-1, lipopolysaccharide, microglia, den-
dritic cell, perivascular cell, macrophage.
The discovery of neuropeptides and similar “neuroactive”
substances in immune cells initiated a new perspective on
the potential ways that immune and neural cells commu-
*Correspondence to: L. E. Goehler, Department of Psychology, P.O.
Box 400400, University of Virginia, Charlottesville, VA 22904, USA.
Tel: ⫹1-434-243-3547; fax: ⫹1-434-982-4785.
E-mail address: goehler@virginia.edu (L. E. Goehler).
Abbreviations: ABC, avidin– biotin peroxidase complex; CR3, comple-
ment receptor 3; DAB, 3,3=-diaminobenzidine; GFAP, glial fibrillary
protein; IL-1␤, interleukin-1␤; IR, immunoreactivity; LPS, lipopolysac-
charide; MHC, major histocompability complex; PB, phosphate buffer;
PBS, phosphate-buffered saline; PBS-T, phosphate-buffered saline with
0.5% Triton X-100; TH, tyrosine hydroxylase; TLR, Toll-like receptor.
0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.neuroscience.2006.03.048
1415
nicate (Blalock, 1994; Salzet et al., 2000). Subsequent
findings revealed a close functional relationship between
the immune and nervous systems, indicating that these
two systems are acting in concert to regulate neural, en-
docrine, and immune functioning. Notably, immune activa-
tion can provoke dramatic changes in nervous system
functioning, leading to an adaptive reorganization (Hart,
1988) that include the familiar symptoms of sickness, in-
cluding fever and social withdrawal (Elmquist et al., 1997b;
Dantzer, 2004), as well as alterations in mood (anxiety or
depression) and cognition (Maier and Watkins, 1998;
Dantzer, 2004). As yet, potential mechanisms that underlie
these brain-mediated effects continue to fuel debate.
The brain has traditionally been considered as an im-
mune-privileged structure, based on the lack of a well-
defined lymphatic system or dedicated immune structures
such as lymph nodes, and the presence of barrier func-
tions (blood– brain and blood– cerebrospinal fluid barriers)
that exclude most hydrophilic and large blood-borne mol-
ecules from access. This belies the fact that the brain is
host to rich and diverse populations of immune cells, in-
cluding microglia within the brain parenchyma (Gehrmann
et al., 1995), perivascular cells associated with blood ves-
sels throughout the brain parenchyma (Williams et al.,
2001), as well as the macrophages and dendritic-like cells
that populate the choroid plexus, meninges, and circum-
ventricular organs “outside” the blood– brain barrier (Mc-
Menamin, 1999; McMenamin et al., 2003). These myeloid-
derived cells contribute to a variety of functions, including
immune surveillance, as well as the production of neuro-
trophic factors supporting development, wound repair, and
plasticity (Batchelor et al., 2002; Boulanger and Shatz,
2004; Roumier et al., 2004). In addition, a subpopulation of
brain immune cells, including perivascular cells as well as
other, less well-characterized cells (macrophage/micro-
glia-like) responds to early phases of peripheral immune
activation with the expression of pro-inflammatory media-
tors, such as prostaglandins (Elmquist et al., 1997a;
Schiltz and Sawchenko, 2002) and cytokines (Nakamori et
al., 1994; Konsman et al., 1999; Van Dam et al., 1995,
2000). Thus, these cells may play an immune sensory role,
i.e. contribute to the induction and modulation of brain-
mediated host defense responses (as above).
Whereas brain-derived prostaglandins seem to play a
role in the induction of thermogenic and neuroendocrine
responses (Ericsson et al., 1997; Roth et al., 2002), among
the array of signals generated in response to immune
activation or inflammation, the induction of IL-1␤ within the
brain is believed to constitute a critical point in the cascade
of events leading to behavioral responses including social
withdrawal and psychomotor retardation associated with
1416 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434
behavioral depression (Kent et al., 1992; Bluthe et al.,
1995; Nadjar et al., 2005). Within the timeframe during
which behavioral responses become evident (one to two
hours after immune challenge), immune cells associated
with the meninges, choroid plexus, and circumventricular
organs are the predominant, if not only, sources of inter-
leukin-1␤ (IL-1␤) in the brain (Nakamori et al., 1994; Van
Dam et al., 1995, 2000; Vernet-der Garabedian et al.,
2000). Taken together, these findings indicate that CNS
sources of IL-1␤ that are effectively outside the blood–
brain barrier appear critical for the induction and/or main-
tenance of sickness behaviors such as social withdrawal.
In circumventricular organs (e.g. subfornical organ, or-
ganum vasculosum lamina terminalis, area postrema), un-
like other barrier-associated tissues, immune cells coexist
with neurons. This arrangement might allow for a potential
direct and specific (cell-to-cell) interaction between im-
mune cells and neurons, in contrast to the paracrine-type
action by which cytokines may act via volume transmis-
sion. However, evidence for such an interaction is lacking.
The demonstration that cells apparently resident in these
structures respond to immune activation with the induction
of immunoreactivity (IR) for the pro-inflammatory cytokine,
IL-1␤ or the uncleaved pro-IL-1␤ (Konsman et al., 1999;
Van Dam et al., 2000) and IL-1␤ mRNA (Quan et al., 1998,
1999) provided strong evidence that these cells are playing
a sensory role, but detailed information regarding specific
cell types expressing (pro-)IL-1␤ IR, or their physical rela-
tionships to neurons is as yet, lacking. Whereas Konsman
et al. (1999) demonstrated colocalization of IL-1␤ IR in
cells with isolectin B4 (indicating macrophage/microglia
lineage) in the area postrema, isolectins nonspecifically
label various cell types, including the central processes of
peripheral nerves. From this work it is not possible to know
whether IL-1␤ IR derives from microglia, perivascular cells
or other subtype of macrophages, dendritic-like cells, or
other cells. Such information would be valuable as specific
populations of ED2-positive perivascular and meningeal
macrophages express distinct cell membrane proteins
such as mannose receptors that can be specifically tar-
geted in drug therapy (Polfliet et al., 2001). Further, it is not
known what type of signaling mechanisms (paracrine vs.
direct, cell-cell) might obtain. Thus, the precise mecha-
nisms by which immune cells in circumventricular organs
signal neurons are unknown. Nonetheless, this previous
work has underlined a potentially important immune sen-
sory role for cells in circumventricular organs.
The objectives of the studies reported here were to
explore potential mechanisms by which brain-derived
IL-1␤ can access or influence neuronal circuits that may
mediate sickness behavior, by 1) identifying phenotypes of
the immune cells cohabiting the area postrema that ex-
press IL-1␤ IR upon immune challenge, and 2) determining
whether direct interactions, via physical contact, occur be-
tween IL-1-expressing, or other immune cells, and neu-
rons. Double-label light, confocal laser-scanning, and
transmission electron microscopy were used to distinguish
different immune cell types and their relationships to neu-
rons, in brain sections from rats challenged in vivo with i.p.
lipopolysaccharide (LPS). Based on ultrastructural charac-
teristics and expression of cell marker proteins, we were
able to identify both perivascular and dendritic-like immune
cells that produce IL-1␤ following immune challenge, and
demonstrate that some of these cells make direct physical
contact with neuronal elements within the area postrema.
EXPERIMENTAL PROCEDURES
Animals
The animals used for these studies were 48 Sprague–Dawley rats
of either sex weighing between 300 and 400 g (Taconic Labora-
tories, Germantown, NY, USA). The animals were housed in pairs
in polypropylene cages within a barrier cage rack (Allentown
Caging Smart Bio-pak, Allentown, NJ, USA) under standard hous-
ing conditions (12-h h light/dark cycle, lights on at 7 AM) with food
and water available ad libitum. All experiments and procedures
were carried out in accordance with the National Institutes of
Health Guide for the Care and Use of Laboratory Animals (NIH
Publications No. 80 –23; revised 1996) and were in accordance
with protocols approved by the University of Virginia Institutional
Animal Care and Use Committee. All efforts were made to mini-
mize number of animals used and their suffering.
Injection of LPS
Expression of the cytokine IL-1␤ within 2– 4 h following peripheral
pro-inflammatory challenge is a hallmark of cerebral innate im-
mune cells that may serve a sentinel function in the brain. Be-
cause immunohistochemically detectable IL-1␤ protein is reliably
induced by a pro-inflammatory stimulus, e.g. bacterial endotoxin
(LPS) derived from gram-negative bacteria, animals received ei-
ther i.p. injection of LPS (n⫽25) or saline/no injection (n⫽23) prior
to kill and tissue dissection. LPS (from E. coli, serotype 0111:B4,
Sigma, St. Louis, MO, USA) was dissolved in sterile 0.9% NaCl
and injected i.p. at doses of either 0.1 (n⫽20) or 0.4 (n⫽5) mg/kg.
The animals were deeply anesthetized either 120 (n⫽6), 150
(n⫽2), or 240 (n⫽10) minutes later with an i.p. injection of Nem-
butal (60 mg/kg) and perfused transcardially briefly with Tyrode’s
solution followed by fixative containing 4% paraformaldehyde,
15% (v/v) saturated picric acid, and with 0.25% glutaraldehyde
added, in 0.1 M sodium phosphate buffer (PB; pH 7.4). The brains
were removed and kept in cold fixative without glutaraldehyde
overnight, and stored the next day in cold PB containing 0.1%
sodium azide as preservative.
Tissue processing
Antibodies. Non-neural cells in the area postrema were
labeled using antibodies directed toward proteins expressed by
different cell phenotypes. For myeloid immune-derived cells we
assessed IR (1) for major histocompability complex (MHC) class II, a
marker for antigen presenting cells, most notably, but not exclusively,
dendritic cells (Banchereau and Steinman, 1998), using the mouse
monoclonal OX-6, (2) for microsialin (CD68), a marker for peripheral
and meningeal macrophages, using the monoclonal ED1, (3) for the
scavenger receptor CD163-like antigen, using ED2, and (4) for mi-
croglia, using the OX-42 monoclonal directed toward complement
receptor 3 (CR3 or CD11b). These monoclonals were purchased
from Biosource (OX-6: cat. # ARU0111, ED1: cat. # ARU0151),
BD-Pharmingen (biotinylated OX-6: cat. # 554927, OX-42: cat. #
554859) and Serotec (ED2: cat. # MCA342R). We also labeled
astrocytes using a rabbit polyclonal antibody directed toward glial
fibrillary protein (GFAP, from Chemicon, cat. # AB5804), because of
their ramified morphology similar to microglia and some reports
regarding their ability to express IL-1␤ (Dong and Benveniste,
2001). Polyclonal affinity-purified anti-rat IL-1␤ antibodies, ob-
 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434
tained from Abcam (raised in rabbit, cat. # ab9787, lot 80734) and
from R&D Systems (raised in goat, cat. # AF-501-NA, lot
YR052091) were used to identify LPS-responsive cells in the area
postrema in endotoxin-challenged rats. Finally, we used a rabbit
polyclonal raised against the catecholamine synthesizing enzyme
tyrosine hydroxylase (TH), from Chemicon (cat. # AB152), to label
a major subset of area postrema neurons in conjunction with
double labeling with OX-6. The pan-neuronal marker PGP9.5
(Chemicon, cat. # AB1761) was applied in combination with the
anti-IL-1 antibodies to explore possible neuronal origin of IL-1␤
expression. The anti-IL-1␤ antibodies do not show cross-reactivity
to other cytokines (GM-CSF, IL-1␣, IL-2, IL-4, IL-10, TNF-␣) ac-
cording to the vendors’ specifications. The specificity of the anti-
IL-1␤ antisera was further assessed by preabsorption of the incu-
bation solution with the immunogen recombinant mature rat IL-1␤
(R&D Systems, cat # 501-RL) and by Western blotting of rat brain
(area postrema) tissue as described below.
General pre-incubation treatments. The brainstems con-
taining the area postrema were dissected and sectioned on a
vibratome at a thickness of 50 ␮m. Sections were collected seri-
ally in ice-cold PB (pH 7.4) containing 0.1% sodium azide. Sec-
tions containing the area postrema were then selected (approxi-
mately six to eight per rat) and either assigned to procedures for
electron microscopic analysis or for light microscopic analysis.
Sections for electron microscopy underwent the following treat-
ments prior to any of the various immunohistochemical detec-
tion protocols as described below. First, the sections were
immersed in 20% sucrose in PB, flash-frozen on dry ice-chilled
isopentane while flattened in aluminum weighing boats, and
subsequently warmed back to room temperature. This freeze–
thaw procedure was repeated one more time to improve antibody
penetration without seriously compromising ultrastructural fea-
tures. Second, sections were transferred to phosphate-buffered
saline (PBS; 0.145 M NaCl, 0.01 M PB), and treated with freshly
dissolved sodium borohydrate (0.5%) for 30 min, followed by three
rinses in PBS. Thereafter the sections were treated with 0.3%
hydrogen peroxide and 0.1% sodium azide in PBS for 30 min to
quench endogenous peroxidase activity. This latter procedure
was omitted if sections were not subjected to any peroxidase
staining procedure later on. The third treatment involved the avi-
din– biotin blocking procedure using Vector’s blocking kit, leading
to a drastically reduced “noise” in staining methods utilizing biotin-
assisted detection. Sections were incubated in avidin and biotin
(four drops per ml PBS) for 1 h each interspersed with rinses in
PBS. Fourth, nonspecific antibody binding was reduced by incu-
bating the sections overnight in Fab= fragments of anti-rat IgG or
anti-mouse IgG (1:200, raised in either goat or donkey to match
normal serum and secondary antibody host), diluted in incubation
buffer modified for EM (PBS containing 0.1% sodium azide and
3% normal serum, of which the host is the same as the secondary
antibody utilized, e.g. goat or donkey). All incubations lasting
overnight or longer were kept at low temperature (4 °C) under
gentle agitation. The sections selected for light and confocal laser
scanning microscopy were treated as described above with the
following notable exception: instead of undergoing the flash freeze–
thaw procedure they were to incubation buffer to which 0.5%
Triton X-100 was added for all pre-incubations (e.g. avidin– biotin
block, normal sera and Fab= anti-rat/mouse IgG). For IL-1␤ immu-
nostaining, non-specific background was reduced by pre-incubat-
ing anti-IL-1␤ antisera overnight with brain sections from rats that
received saline injections or were untreated (which tissue was not
further used in these experiments), before being applied to tissue
sections as described above.
Immunoperoxidase histochemistry for light microscopy. Fol-
lowing the pre-embedding steps, selected sections were washed and
incubated in one of the following primary antibodies: the mono-
clonals OX-6, OX-42, ED1, ED2 (1:300 –1:500), rabbit anti-GFAP
1417
(1:1000), rabbit anti-IL-1␤ (1:2000), goat anti-IL-1␤ (1:2000) over-
night at room temperature (RT). The next day the slides were
washed and incubated in biotin-conjugated secondary antibody
(goat anti-mouse IgG or goat anti-rabbit IgG (1:500; overnight at
4 °C). All antibodies were diluted in phosphate-buffered saline with
0.5% Triton X-100 (PBS-T) containing 0.1% sodium azide and
10% NGS. Finally, sections were incubated in avidin– biotin per-
oxidase complex (ABC, Vector Elite kit, 1:100 in PBS-T, 2 h;
Vector Laboratories, Burlingame, CA, USA) and rinsed in Tris–
HCl (pH 7.6). Peroxidase staining was performed using 3,3=-
diaminobenzidine (DAB, Sigma) as a chromogen dissolved in
Tris–HCl (40 mg/100 ml), glucose oxidase (Sigma; type V-S,
0.02%) and ␤-D-glucose (0.1% w/v) to generate hydrogen perox-
ide (modified after Shu et al., 1988), yielding a reddish– brown
precipitate. In some instances, nickelous ammonium sulfate was
added (1.50 mg/ml) to the DAB (20 mg/100 ml) solution to inten-
sify the staining yielding a purplish– black color. One series of
sections was processed as above except that the primary anti-
body was omitted from the incubation buffer (omission controls).
The slides were dried overnight, cleared in ethyl alcohol and
Histoclear, and coverslipped with DPX prior to viewing with an
Olympus BX-51 brightfield microscope. Images were collected
with a Magnafire digital camera (Optronics) coupled to an Apple
PowerMac G4 equipped. The obtained micrographs were slightly
adjusted for brightness and contrast and labeled using Adobe
Photoshop CS2 (version 9, MacOS 10.3). Except for these minor
adjustments, the images were not altered.
Double labeling immunoperoxidase. Selected sections were
stained for two different antigens by following above-described pro-
cedure twice in succession, the first reacted in nickel-enhanced
DAB to yield a blue/purplish– gray to black staining, the second
reacted in regular DAB to yield a reddish– brown staining. Follow-
ing the first reaction, sections were treated with the hydrogen
peroxidase–sodium azide mix (0.3% and 0.1%) again to eliminate
residual peroxidase activity before starting the second procedure.
The following combinations of primary antibodies were applied: (1)
OX-6 and TH to assess the fine anatomical relationship between
the MHC class II-expressing cells and the abundant TH-positive
AP neurons; (2) OX-42 and OX-6 to address the possible co-
expression of both immune cell markers; and (3) IL-1␤ and OX-6
to determine whether the constitutive MHC class II-expressing
immune cells are among those that respond to LPS challenge with
the production of IL-1␤ protein. Care was taken to avoid intense
precipitation of either reaction product as that hinders the reliable
detection of both in the cells. Colocalization was indicated by
blending of the gray and reddish– brown colors or by the presence
of both colors in different compartments of the same cell. Although
this method carries limitations due to possible visual interference
due to the nature of the reaction products, it has proven to be
superior in showing the fine morphological features of the cells
and their fine ramifications at the light microscopic level.
Because OX-6 and OX-42 are both mouse monoclonal anti-
bodies, we adapted the protocol to avoid or minimize cross-
reactivity. First sections were incubated in OX42, followed by
biotinylated Fab= fragment of goat anti-mouse IgG (1:500; Jack-
son Immunoresearch) and ABC. After staining with nickel-en-
hanced DAB, sections were treated with the avidin– biotin blocking kit
and subsequently incubated overnight in biotinylated OX-6 (Pharm-
ingen, 1:400), followed by ABC and staining in regular DAB.
Double-labeling immunofluorescence. The phenotypic char-
acteristics of the cells in the area postrema that express IL-1␤
protein in response to systemic endotoxin challenge were estab-
lished by concurrent immunofluorescent detection of IL-1␤ protein
and either PGP9.5, a pan-neuronal marker, or each of the innate
immune cell markers detectable with the monoclonals ED2, OX-
42, and OX-6. All antibodies were diluted in incubation buffer
containing 0.5% Triton X-100, 0.1% sodium azide, and 3% normal
1418 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434
donkey or goat serum (host for serum is the same as that for the
secondary antibody). One selection of sections was incubated in
goat anti-rat IL-1␤ for 24 – 48 h, followed by Cy3-conjugated don-
key anti-goat IgG (Jackson Immunoresearch) overnight at 4 °C.
Subsequently the sections were incubated in either of the follow-
ing antibodies diluted in the same incubation buffer: PGP9.5 (1:
1000), OX-6 (1:500), OX-42 (1:250), and ED2 (1:400) for 24 h
followed by FITC-conjugated F(ab)2 fragment of donkey anti-rab-
bit or mouse IgG (1:200) overnight. Finally the sections were
mounted and coverslipped in Vectashield anti-fading medium
(Vector Laboratories). Another selection of sections was incu-
bated in rabbit anti-rat IL-1␤ antiserum followed by Alexa Fluor
488-labeled goat anti-rabbit IgG (1:200). Antibodies were diluted
in incubation buffer containing 3% normal goat serum), with incu-
bation times as described above. The following sequence involved
either ED2, OX-6, or OX-42 (dilution as above) followed by Alexa
Fluor 546-conjugated goat anti mouse IgG (1:200). These sec-
tions were mounted on microscope slides from PBS and cover-
slipped in glycerol/PBS mixture. The immunofluorescence was
confirmed using an Olympus BX51 equipped with epi-fluores-
cence filter sets (Chroma) specific for each of the fluorochromes.
In order to obtain sharp optical focus at different levels through the
sections, colocalization of the fluorescent markers was estab-
lished with the aid of a laser scanning confocal microscope (Olym-
pus Fluoview B2111) with 40⫻ objectives. Krypton/argon lasers of
488 nm (green) and 543 nm (red) wavelengths were used in
combination with barrier filters, set to excite the specimen with
only one laser line at a time. Optical sections were generated at a
resolution of 1024⫻1024 pixels for each fluorochrome by separate
scans of the specimens in subsequent planes that were either 2 or
3 ␮m apart, which is well within the diameter of most cells. The
excitation emitted from the specimens was captured and visual-
ized using FluoView FV300 software version 3.3 (Olympus Optical
Co., Ltd.). For semi-quantitative assessment of colocalization of
IL-1␤ with each of the immune cell phenotypes labeled with ED2,
OX-6 and OX-42, one section per rat through the rostral third of
the area postrema was selected for each combination of double
labeling from the four LPS-treated rats used (0.4 mg/kg i.p., two at
2.5 and two at 4 h). The sections were scanned at multiple z axis
levels separated by 3 ␮m and the cells fluorescing for ED2, OX-6
or OX-42 were counted, as well as those that show IL-1␤ immu-
nofluorescence, and those co-expressing both fluorophores. From
these numbers the percentage of colocalization for each rat was
calculated. As no differences were noted between the two time
points, data from all four animals were pooled. Sections from a
fifth saline-treated rat were included and showed fluorescence for
the ED2, OX-6 and OX-42 indistinguishable from the endotoxin
treated animals, but completely lacked immunofluorescence for
IL-1␤.
Electron microscopy
Single immunolabeling of innate immune cell markers and
IL-1␤ for electron microscopy. Selected sections through the
area postrema were stained for the three immune cell markers
detected by ED2, OX-42 and OX-6 using a pre-embedding immu-
nogold and silver enhancement protocol. Immunogold labeling
combined with silver enhancement provides a sensitive technique
for both light and electron microscopy in particular for antigens
present on the outer cell surface, which is the case for those
identified with the ED2, OX-6 and OX-42 monoclonal antibodies
(phenotypical markers for resident tissue macrophages, putative
dendritic sentinel cells, and microglia, respectively). All incuba-
tions were done at 4 °C. Sections were incubated for 24 – 48 h in
either ED2, OX-6, or OX-42 diluted 400⫻ in incubation solution
adapted for EM (PBS containing 3% NGS and 0.1% sodium azide;
Triton X-100 was omitted). Following three rinses in PBS, the
sections were transferred to and incubated overnight in solution
containing goat anti-mouse IgG conjugated with 1 nm size gold
particles (British Biocell international, Ted Pella, Inc.) or 4 nm
colloidal gold particles (Jackson ImmunoResearch). The sections
were rinsed in PBS, fixed in 1% glutaraldehyde for 5 min, and
transferred to sodium citrate buffer (0.1 M, pH 6.5) prior to silver
enhancement. Silver enhancement was carried out according to
the manufacturer’s instructions (IntenSE kit, Amersham), and the
development was terminated upon visual confirmation of staining
in the light microscope. The sections were washed in citrate
buffer, transferred to 0.1 M PB before osmification and resin
embedding.
Another selection of sections through the area postrema un-
derwent single immunoperoxidase staining for rat IL-1␤ with DAB
as the chromogen. Sections passed through the following incuba-
tions rabbit anti-IL-1␤ (1:2000, diluted in EM incubation buffer with
3% NGS for 24 – 48 h), biotinylated goat anti-rabbit IgG (1:500;
overnight at 4 °C), and ABC (Vector, 1:500 in PBS). The sections
were rinses in Tris–HCl, and reacted in DAB. Alternatively, sec-
tions were incubated in goat anti IL-1␤ (1:2000, in incubation
solution containing NDS) and biotinylated donkey anti-goat IgG
(1:500) before proceeding to ABC and DAB. Following completion
of the staining, sections underwent osmification and resin embed-
ding as described below.
Dual immunogold/peroxidase labeling for electron microscopy.
Sections through the area postrema were first processed for IR for
OX-6, ED2, and OX-42 with immunogold labeling using the pro-
cedure described above. Following the silver intensification, sec-
tions were washed in PBS and subjected to a second, immuno-
peroxidase staining procedure involving immunoperoxidase stain-
ing for TH to label catecholaminergic neurons that are abundant in
the area postrema. The second staining sequence included incu-
bation in the primary antibody for 24 – 48 h at 4 °C, followed by
biotinylated goat anti-rabbit IgG (overnight), and ABC (4 h, over-
night). The sections were then stained with DAB. Thereafter the
sections were transferred to 0.1 M PB and prepared for osmifica-
tion and resin embedding.
Osmification, embedding and ultrathin sectioning for EM.
The sections designated for EM examination were postfixed with
osmium tetroxide and embedded in resin using routine protocols.
In short, the sections were fixed in 1% osmium tetroxide (in 0.1 M
PB) for 1 h, dehydrated in increasing concentrations of ethyl
alcohol, during which they were incubated en bloc in 4% uranyl
acetate (in 70% ethyl alcohol) for increased contrast for a mini-
mum of 2 h. Then the sections were transferred into 90 and 100%
ethyl alcohol, rinsed in acetone twice, and left in a 1:1 mixture of
acetone and EPON resin (EMBED 812; EMS) overnight, and
transferred to full resin. Sections were flat embedded between two
sheets of Aclar, and the resin allowed to polymerize overnight at
60 °C. Small pieces containing the area postrema were excised
and laid flat on the inner surfaces of BEEM capsule caps. The
capsules were filled with EPON resin and polymerized overnight
at 60 °C. Ultrathin sections were cut at a thickness of 90 –100 nm
on an ultramicrotome (Leica), placed on copper mesh grids, and
examined on a JEOL 1010 transmission electron microscope.
Electron micrographs were captured on film (Kodak 4489) that
was developed with Kodak D-19 developer and commercial fixer
(Eastman Kodak Co., Rochester, NY, USA). Each negative was
then digitized using an optical scanner (Epson Perfection 1200
Photo) at a pixel density of 600 d.p.i. In addition, images were
captured with the use of a SIA-12C digital camera (siacam.com).
Images were taken using a 2⫻2 binning yielding a density of
2048⫻2048 pixels. The digitized images were imported with the
MaxIm DL CCD software and stored as eight bit TIFF files after
“stretch” procedure to yield optimal brightness and contrast
properties.
Omission and pre-absorption controls. Because non-spe-
cific or spurious staining can emerge especially when using poly-
 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434
clonal antibodies (Saper and Sawchenko, 2003), we applied sev-
eral procedures to test the specificity of the anti-IL-1b antibodies in
the brain tissue used in this study. Selected sections through the
area postrema were subjected to pre-incubation treatments to
reduce non-specific immunolabeling as described above. These
sections, from both saline- and LPS-treated rats were then incu-
bated in non-specific rabbit or mouse IgG to replace the primary
antisera. In addition, sets of consecutive sections were incubated
in two solutions with the anti-rat IL-1␤ antisera (1:2000), of which
one was first adsorbed overnight at 4 °C with an excess of the
immunogen rat recombinant IL-1␤ (R&D Systems, 2 ␮g/0.5 ␮g
primary antibody). The sections were then processed for immu-
noperoxidase labeling in an identical way as described above for
light microscopy.
Western blot analysis. Two male rats were used for this
purpose, one of which was challenged with LPS (0.4 mg/kg i.p.)
and the other with sterile saline. Four hours later, they were
perfused transcardially with Tyrode’s solution, after which the
brains and pituitary glands were rapidly removed. Selected brain
regions were dissected on an ice-cold surface, weighed, and
flash-frozen in liquid nitrogen after which they were stored at
⫺80 °C until use. The dorsal medulla, including the area pos-
trema, and pituitary glands (weighing 10 –12 mg) were homoge-
nized in 25 ␮l 20 mM HEPES buffered saline (pH 7.8) containing
1 mM EDTA, 1 mM EGTA, and protease inhibitors, and centri-
fuged at 14,000 r.c.f. The supernatant was derived from each
sample and mixed with Laemmli loading buffer (Bio-Rad) and 5%
␤-mercaptoethanol, brought to 100 °C for 2 min and loaded onto
a polyacrylamide minigel (Bio-Rad) together with recombinant rat
IL-1␤ (10 ng/lane), and a prestained precision protein ladder for
the estimation of protein size. Proteins were separated at 150 V
and transferred onto a PVDF membrane (Bio-Rad) overnight at 30
V. The membrane was subsequently incubated in blocking solu-
tion (5% nonfat milk in PBS and 0.1% Tween-20) for 30 min, in
rabbit anti-IL-1␤ (1:5000, previously pre-absorbed with normal
brain sections, as above for immunohistochemistry) overnight at
4 °C, followed by biotinylated goat anti-rabbit (1:1000) for 1 h and
streptavidin–alkaline phosphatase (Jackson ImmunoResearch,
1 ␮g/ml Tris-buffered saline, pH 8.0). The membrane was stained
with a BCIP/NBT substrate kit (Vector).
RESULTS
The experiments reported here were directed toward iden-
tifying subtypes of immune cells resident in the area pos-
trema, and determining whether, given the cohabitation of
immune cells and neurons in this structure, any direct
interactions occur (appositions with possible synaptic spe-
cializations in cell membranes). The results revealed mul-
tiple immune cell phenotypes, some of which abutted neu-
ronal elements, and respond to peripheral immune chal-
lenge by inducing expression of IL-1␤-like IR.
Expression patterns of immune cell markers in the
area postrema
Light microscopic analysis revealed several, slightly over-
lapping, populations of immune cells, which differed pri-
marily in their distribution throughout the area postrema
(Fig. 1), the type of marker they expressed, and their
response to peripheral immune challenge with LPS.
OX-6: MHC class II. Cells immunoreactive for the
MHC class II protein (labeled with OX-6) were distributed
throughout the area postrema, but were particularly nu-
merous along the dorsal aspect, and enriched in the mid-
1419
to-dorsal central region (Fig. 1A), and, albeit fewer in num-
ber, along the lateral borders with the subjacent nTS. The
MHC class II-IR cells were morphologically highly hetero-
geneous. Most cells were quite large with somata ranging
from 15 ␮m to 30 ␮m. These cells exhibited two to three
dendritic-like processes and were either spindle shaped,
with prominent processes on either end, or shaped rather
like a rocket (Fig. 2A) with a tuft of processes emanating
from one end and a single, sometimes branching process
extending opposite. Other cells exhibited a marked pyra-
midal shape (Fig. 2A). The dendritic processes varied in
length and number of ramifications, and some cells pos-
sessed an extensive network of branches. A few cells were
oval or round and lacking obvious processes, and others,
more rarely, were highly ramified with a smaller soma, and
resembled microglia in morphology (Fig. 2B). The number
of MHC class II-immunoreactive cells varied among indi-
vidual rats and their distribution was often skewed toward
the dorsal aspect of the area postrema in some cases and
along the lateral border in others. Cells labeled constitu-
tively with OX-6 were not encountered within the nTS, but
consistently present in adjacent choroid plexus and, when
still attached, also on the leptomeninges (pia mater).
OX-42: CD11b (CR3). As they do within the brain
parenchyma, the OX-42-labeled cells were distributed
throughout the area postrema in a more or less uniform
fashion that was virtually the same among the different
animals (Fig. 1B). In contrast to the rather idiosyncratic
distribution and morphological features of the cells labeled
with OX-6, those expressing CR3/CD11b evinced classic
microglial morphology, with small oval or elongated so-
mata and numerous branching processes that showed a
classic ramified pattern (Fig. 2D).
ED2: CD163-like antigen (scavenger receptor). ED2-
positive cells generally showed an even distribution
throughout the entire area postrema with no apparent pref-
erence to certain portion (Fig. 1C). In addition they were
encountered adjacent to the fourth ventricle, and also oc-
cupied meninges. ED2, like OX-6, labeled a population of
cells characterized by large somata. A ramified appear-
ance was rare among ED2-positive cells, but otherwise
these cells were morphologically similar to the spindle,
rocket and pyramidal shaped OX-6 positive cells (Fig. 2E).
However, the branches appear to be generally shorter and
lack the extensive ramification characteristic for many
OX-6 labeled cells. A microglia-like morphology was never
seen among ED2-positive cells. In contrast to cells ex-
pressing OX-6 and OX-42, ED2-positive cells associate
almost exclusively with vasculature, and often curve into
crescent-moon-like shapes as they wrap around the ves-
sels. In this way, ED-2 positive cells were highly similar to
perivascular macrophages, or perivascular cells.
GFAP. The GFAP antiserum labeled classic astro-
cytic cells with very small somata from which multiple long
smooth and delicate processes emanate. These cells were
numerous and uniformly distributed in both the area pos-
trema and brainstem parenchyma (Fig. 1D). The morphol-
1420 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434

Fig. 1. Distribution of different immune cell populations in the area postrema characterized by their expression of MHC class II antigen labeled with
OX-6 (A), CD11b/CR3 labeled with OX-42 (B), CD163-like antigen labeled with ED2 (C), GFAP (D), a cytoplasmic lysosomal antigen (CD68) labeled
with ED1 (E), and IL-1␤ IR (F, G). IL-1␤ IR in F was induced by LPS challenge (0.4 mg/kg, 4 h), as the area postrema in saline-treated rats did not
show any IR for IL-1␤ (G). Scale bar⫽100 ␮m in A (applies to all panels).

ogy of these cells was quite distinct from those expressing
abovementioned immune-related markers (Fig. 2F) and
those stained for IL-1␤ (see below). For this reason, GFAP
was not further included in the analysis. Processes of
GFAP-positive cells in the area postrema encircle blood
vessels and were particularly prominent at the margins of
the area postrema and subjacent nTS.
ED1: CD68-like antigen (macrosialin). ED1-positive
cells were extremely sparse in the area postrema com-
pared with those expressing other immune markers, and
were not seen in the brainstem parenchyma (Fig. 1E). A
few somata were seen within the meninges and interface
with the fourth ventricle, as well as numerous processes
stained in a grainy fashion. Most labeled cells were round
or oval. IR for ED1 was irregularly distributed within the
cytoplasm of these cells (Fig. 2C), a feature also seen in
ED1-positive putative macrophages in other tissues, in-
cluding peripheral nerve (Goehler et al., 1999).
IL-1␤. The two different polyclonal antibodies gave
excellent staining for rat IL-1␤ IR in the area postrema of
rats that were treated with bacterial endotoxin (LPS). In
animals treated i.p. with 0.4 mg/kg LPS, strong IL-1␤-like
 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434 1421

Fig. 2. Micrographs at high magnification showing the cell morphology features of the different cells encountered in the area postrema. (A) OX-6 recognizing
MHC class II antigen strongly labels a variety of cells with pyramidal (p), rocket (r), and spindle (s) appearance. Some cell bodies emanate fine cilia-like
protrusions (asterisk). (B) OX-6-stained cells with extensively ramifying arborizations giving them a microglia-like appearance. (C) ED1 staining of cytoplasmic
lysosomal antigen (CD68) in a subpopulation of immune cells. (D) OX-42 (CR3/CD11b)-positive microglial cells with extensive ramifications. Although they
resemble those in the brain parenchyma proper, the ones in the area postrema appear to have larger somata. (E) ED2 (CD163)-labeled cells with spindle
and crescent appearance. Many are closely associated with vasculature. (F) GFAP IR reveals smooth ramifications emanating from small somata,
characteristic for astroglia. (G) IL-1␤ IR (4 h after 0.4 mg/kg LPS i.p.) in cells with spindle and crescent appearance, some of which are associated with
vasculature (asterisk). (H) Inset, lower magnification image of the area postrema showing total elimination of staining for IL-1 IR when primary antiserum was
preadsorped with recombinant rat IL-1␤ (4:1 ratio w/w). Scale bar⫽25 ␮m in E (applies to panels A—G); 25 ␮m in H.

IR appeared in cells with the area postrema at both 2 and
4 h (Fig. 1F). Cells stained for IL-1␤ IR were consistently
absent in the area postrema of rats that were untreated or
were injected with sterile saline (Fig. 1G). In LPS-treated
rats, IL-1␤-immunoreactive cells were evenly distributed
across the area postrema (Fig. 1F). IL-1␤ IR appeared
primarily in spindle- and pyramid-shaped cells, as well as
in some with a rocket-like morphology (Fig. 2G), similar to
those stained with ED2 and OX-6. Weak IL-1␤ IR was
seen in few cells with microglia-like morphology (highly
ramified with small somata), but never in round cells. The
morphology of IL-1␤-positive cells was very different from
those staining for GFAP IR, and was inconsistent with
possible localization within astrocytes. Many, but not all,
IL-1␤-immunoreactive cells were closely associated with
the vasculature (see also Fig. 3H, and below), some of
which exhibiting the typical crescent moon-shapes as seen
with the ED2-positive cells.
Dual OX-6 and TH staining. Double-labeling for both
MHC-II and TH revealed a dense population of TH-positive
neurons in the area postrema among which the MHC-II-
labeled cells intermingle (Fig. 3A, B). Closer examination
at the light microscopic level indicates that many branches
emanating from the MHC-II-labeled cells appear to form
appositions with the TH-positive neuronal elements includ-
ing their dendrites and somata (Fig. 3C, D).
Codistribution of OX-6 and OX-42 labeling. Based on
morphological similarities between some of the MHC-II
(OX-6)-labeled cells and the CR3/CD11b (OX-42) -positive
ones (see Fig. 2B, C), we investigated whether these
immune cell markers can be co-expressed. Double-label
immunoperoxidase labeling nevertheless clearly re-
vealed separate populations of OX-6- and OX-42-la-
beled cells (Fig. 3E), even when OX-6-labeled cells
appear in a strongly ramified microglia-like appearance
(Fig. 3F). Caution, however, is needed as a light staining
can be masked by the intensity of the other. Attempts to
address this issue using dual labeling with fluoro-
chromes unfortunately failed due to methodological
problems.
1422 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434

Fig. 3. Dual immunoperoxidase staining for MHC class II (with OX-6) and for either TH (in A–D), CD11b/CR3 (with OX-42, in E and F), or IL-1␤
(in F–H). Nickel-enhanced and regular DAB as chromogens yielded distinct purple– gray to black and reddish– brown staining. (A) Characteristic
distribution in the area postrema of constitutive MHC class II expression (stained black) against the background of reddish– brown TH-positive
neurons. (B–D) OX-6 stains cells with multiple dendritic ramifications (black arrow B), many of which make appositions with TH-labeled neuronal
somata and dendrites (arrows in C and D). (E) OX-42 and OX-6 label distinct populations of myeloid cells in the area postrema. Whereas OX-42
reveals the extensively ramified microglia (black arrows), OX-6 labels other cells that are spindle- and rocket-shaped (red arrows). (F) Staining
for IL-1␤ (in black) and OX-6 (reddish– brown) in the AP reveals dendriform and spindle-shaped cells that express either IL-1␤ IR alone (black
arrows) or both IL-1␤ and MHC-II IR (a mixing of gray and brown, indicated with both short black and red arrows). (G–I) Laser scanning confocal
imaging of neuronal elements in the area postrema labeled with Alexa Fluor 488 (green) for PGP9.5 and a totally separate population of cells
emitting Alexa Fluor 546 fluorescence (red) indicative of IL-1␤ IR. Many, but not all, IL-1␤-labeled elements are located in perivascular space, and none
were found to be double-labeled for PGP9.5. The arrow points at the location of an IL-1␤-labeled cell embedded in between PGP9.5-labeled
neurons. In panels F, H, and I, IL-1␤-like IR was induced 4 h after 0.4 mg/kg endotoxin challenge, and occurred in cells with a variety ramifications
and spindle-like extensions. Scale bars⫽100 ␮m in A, 20 ␮m in B, D (applies also to panel C), E, and F; 100 ␮m in I (applies also to panels G
and H).

Double-labeling of IL-1␤ and neurons. Fluorescent
double labeling for IL-1␤ and the pan-neuronal marker
PGPG9.5 occurred in strictly separated groups of cells in the
area postrema without any colocalization (Fig. 3G–I). The
PGP9.5 antibody labeled a dense collection of round and
oval cell bodies, and a dense plexus of fibers and punctate
labeling indicative of dendritic, axonal and dendritic labeling.
In contrast, IL-1␤ immunofluorescence highlighted distinct
cells that were spindle- and rocket-shaped. The latter distrib-
uted mainly within the perivascular spaces apparent in the
double-labeled image (Fig. 3H), although several were em-
bedded within the neuronal plexus (see arrow in Fig. 3H, I).
 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434 1423

Fig. 4. Laser scanning confocal imaging of cells in the area postrema labeled with Alexa Fluor 488 (green) for IL-1␤-like IR and with Alexa FLuor 546
(red) for IR to one of the three immune cells markers: CD163 (ED2 clone, in A–C), MHC class II (OX-6 clone, in D–F), and CR3/CD11b (OX-42 clone,
in G–I). The images were compiled from stacking 12–15 optical scans 3 ␮m apart). All sections were from the same representative male rat treated
with 0.4 mg/kg endotoxin 2.5 h prior to kill. (A–C) Double-labeling reveals that virtually all CD163-positive cells strongly express IL-1␤ (see arrows
aimed at some of them appearing yellow in C). Although there is a high degree of colocalization, there is a portion of IL-1␤-expressing cells that do
not express the CD163 antigen. The asterisk indicates one CD163-positive cell expressing no IL-1␤ IR that is positioned just outside the AP and within
the nTS, associated with a blood vessel. (D–F) Of the MHC-II-positive cells labeled with OX-6, only a subpopulation expresses IL-1␤ (indicated with
arrows), and with varying intensity. (G–I) The OX-42-labeled population of microglia barely overlaps with the IL-1␤-positive cells, only a small number
of double-labeled cells were encountered (the arrow points to one). Double-labeled cells generally showed weak fluorescence for IL-1␤. Scale
bar⫽100 ␮m in A (applies to all panels).

Double-labeling of IL-1␤ and immune cell markers.
IL-1␤-like immunofluorescence was encountered in loosely
distributed cells across the entire area postrema in LPS-
treated rats, but was completely absent in saline-treated
rats. Cells with IL-1␤-like immunofluorescence evinced a
morphology highly similar to cells that stain with OX-6 and
ED2. In accordance with this highly similar appearance,
confocal laser scanning microscopy revealed that virtually
all of the ED2-labeled cells residing within the borders of
the area postrema (Fig. 4A–C) and also about half of the
OX-6-positive cells (Fig. 4D–F) showed IL-1␤-like immu-
nofluorescence (see also Fig. 5A). ED2-positive cells that
were occasionally found outside the area postrema (i.e. in
the subjacent nTS) were not double-labeled. In contrast,
microglia identified with OX-42 rarely co-localized immu-
nofluorescence for IL-1␤ (Fig. 4G–I). In general, the pop-
1424 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434
Fig. 5. (A) Bar graphs showing the rate of colocalization with IL-1␤ of
the three immune cell populations identified by labeling with ED2
(CD163 antigen), OX-6 (MHC class II antigen) and OX-42 (CR3/
CD11b antigen) as observed in the laser scanning confocal micro-
scope. Data from four male rats were pooled (2.5 [n⫽2] and 4 h [n⫽2]
following 0.4 mg/kg endotoxin) as no differences were apparent be-
tween the time points. Whereas almost all ED2-positive cells express
IL-1␤, about half of the OX-6-positive cells and a small fraction (7%) of
the OX-42-labeled microglia do so. (B) Among the population of en-
dotoxin-responsive cells expressing IL-1␤, many of them show the
ED2 phenotype, but only a small portion of them are OX-6 and OX-
42-positive. A significant portion of these IL-1␤-positive cells ex-
presses neither marker.
ulation of generally bright IL-1␤-labeled cells is very dis-
tinct from the plexus of OX-42-labeled microglia. Despite
the high incidence of colocalization of IL-1␤ within the ED2
and OX6-labeled populations, a smaller but significant por-
tion of cells showing bright immunofluorescence for IL-1␤
expressed neither marker (Fig. 5B), indicating another
group of LPS-responsive cells that express neither MHC
class II nor CD163-like protein recognized by respectively
OX-6 and ED2.
Ultrastructural features of immune cells in the area
postrema
Cells expressing immune-related markers share many
highly similar ultrastructural features. Particularly charac-
teristic are the nuclei, which are irregularly shaped and
contain large patches of highly condensed chromatin
(Figs. 6A, B). At rest (in saline-treated rats) these cells
appear remarkably similar, with the most notable distinc-
tion being their location within the area postrema, and
response to peripheral endotoxin challenge.
OX-6: MHC class-II IR
Membrane-associated MHC-II IR was occasionally de-
tected in perivascular cells (Fig. 6B), but most frequently
occurred in cells distributed among the neurons and astro-
cytes within the parenchymal area postrema (Fig. 6A). The
latter type shared characteristics with true dendritic cells,
including high levels of constitutive MHC-II expression,
which is dramatically upregulated following systemic en-
dotoxin treatment (Fig. 6C, F). MHC-II positive cells con-
tained a plethora of tubules in their dendritic extensions,
and contained numerous mitochondria and smooth endo-
plasmic reticulum. These cells invariably were surrounded
by extracellular space (Fig. 6A), that is, they did not appear
to be anchored to extracellular matrix. Appositions be-
tween MHC-II-IR cells and neurons were frequently en-
countered (Fig. 6C–F). In sections processed for both
MHC-II and TH-IR, MHC-II-laden ramifications apposed
neuronal elements that were TH-positive (Fig. 7), as well
as some that were not. Appositions occurred on cell so-
mata (Fig. 6C) and dendrites (Figs. 6 D, E, 7) and some
were observed on neuronal axons and terminals as well. A
remarkable aspect of many appositions was the absence
of any MHC-II label along the outer cell membrane lining
up with the neuronal cell membrane (Fig. 6C).
OX-42: CR3/CD11b IR
Silver-enhanced gold particles lining the outer membranes
indicating CR3/CD11b IR were most frequently observed
along processes and around somata (Fig. 8). Occasionally
CR3/CD11b IR occurred in perivascular-like cells encir-
cling the endothelium (Fig. 8A). The somata typically were
small, with little perinuclear cytoplasm (Fig. 8C). OX-42-
labeled profiles and especially the distal ends were often
observed in the vicinity of neuronal processes, which they
sometimes contacted (Fig. 8D–F).
ED2: CD163-like antigen IR
CD163-like IR was found in the outer membranes of cells
associated with the vasculature (Fig. 9). These cells con-
tained many tubules and a relatively electron-dense cyto-
plasm, often including opaque inclusions (possibly lyso-
somes). Many of these cells encircled the endothelium, or
lurked within the perivascular space, extending one or two
processes to contact astrocytic or endothelial cells (Fig.
9B, C) These processes were characteristically very small
in diameter, and sometimes resemble cilia as they were
short and curved slightly. One larger and longer apical
process usually protruded from the ED-2-labeled cell wrap-
ping around the endothelial cell. Although ED-2-labeled
cells were usually positioned closely to endothelial cells
forming the wall of blood vessels, most were in direct
contact with small-diameter extensions of astroglia inter-
spersed in between (Fig. 9A, B, D).
IL-1␤-like IR
IR for IL-1␤ was found in two types of cells. One type asso-
ciated with the vasculature (Fig. 10), and evinced character-
istics similar to the ED-2 positive cells (perivascular-like cells).
These cells encircled the vessels (Fig. 10A, C), often making
contacts with endothelial cells or astrocytes. IL-1 positive
perivascular-like cells contained many inclusions of various
sizes indicative of phagocytic activity. The other type of IL-
1␤-positive cell closely associated with neurons (Fig. 11),
including somata (Fig. 11A, E) and dendrites (Fig. 11B–D). In
some cases, IL-1␤-like immunoreactive processes entirely
encircled neuronal dendrites (Fig. 11B, C). This type of inter-
action appeared to be rather specific, in that IL-1␤ positive
processes contacted only a subset of neuronal dendrites in
their vicinity. Astrocytes were typically also closely associated
 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434 1425

Fig. 6. Electron micrographs of immune cells in the area postrema constitutively expressing the MHC class II antigen, visible by the black granular
deposits along the outer membranes. (A, B) Low magnification power images of OX-6-labeled cells associated with neurons (in A) and vasculature
(in B). Note the characteristically large extracellular space that surrounds these cells in both instances. The cell nuclei of OX-6-labeled immune cells
[indicated with Nu (i)] contain patches of dense chromatin very distinct from neuronal cell nuclei [indicated with Nu (n)]. Arrows in A indicate the outer
membrane dotted with the granular silver deposits. (C) High power electron micrograph showing an OX-6-labeled dendritic profile (indicated with
asterisk) that abuts a neuronal soma at the junction of a neuronal dendrite to the left. The outer membrane lining along the neuronal soma is clear
of OX-6 label (indicated with arrows). (D, E) OX-6-positive profile (indicated with asterisks) with scattered granular label along the outer membrane
(arrows in D) in close proximity to a neuronal dendrite, which is shown in panel E at higher magnification. (F) OX-6-labeled dendritic profile (indicated
with asterisks) running along a neuronal soma (arrows). Sections were derived from endotoxin-treated male rats (0.1 mg/kg, 2 h: A, B, C; 4 h: F) and
saline-treated male (D, E). Scale bars⫽1 ␮m in A, B, and D; 500 nm in C, E, and F.

with both the neuronal immune cells processes sometimes 11D). In line with observations with light and confocal laser
interjected between them (Fig. 11C). IL-1␤ positive pro- scanning microscopy, ultrathin sections through the area
cesses were particularly evident in the neuropil along the postrema from rats that received i.p. injections of sterile
lateral borders of the area postrema, adjacent to the nTS (Fig. saline lacked any discernable IL-1␤ IR.
1426 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434

Fig. 7. Close associations between the OX-6-labeled cells and TH positive neuronal elements in the area postrema (2 h following 0.1 mg/kg LPS i.p.).
OX-6 positive ramifications (indicated with asterisks) were identified with silver granule deposits along the outer cell membranes (some of them are
indicated with concave arrows in A), and form close anatomical associations with TH-immunoreactive neuronal dendrites (TH⫹ d) visualized with the
electron-dense DAB precipitate. The close proximity of both profiles is also indicated with arrows in B and C. The membrane apposing the TH-positive
profile often lack OX-6 label (as in A and B) but not always (as in C). D indicates a “threesome” of an OX-labeled profile, a TH-positive, and an
unlabeled neuronal d. Scale bars⫽500 nm in all panels.

Characterization of the anti IL-1␤ antibodies
Selected sections through the area postrema from both
saline- and LPS-treated rats that were incubated in non-
specific rabbit or mouse IgG to replace the primary anti-
bodies showed no positive staining. Preabsorption with the
immunogen recombinant rat IL-1␤ completely eradicated
the staining for IL-1␤-like IR otherwise seen in the area
postrema from LPS-treated rats (Fig. 2H). No IL-1␤-like
staining appeared in sections through the area postrema
from saline-treated rats irrespective of preabsorption.
Western blot analysis (SDS-PAGE) of IL-1␤ IR when pre-
viously pre-absorbed with normal rat brain sections re-
vealed a single band with an approximate molecular
weight of 31 kDa in the lanes loaded with supernatant of
homogenates from dorsal medulla of the LPS-treated, but
not the saline-treated rat (Fig. 12). This observation indi-
cates the presence of pro-IL-1␤ prior to post-translational
splicing into mature IL-1␤ (17 kDa), as no 17 kDa band
(detectable in the lane loaded with the rat recombinant
IL-1␤) was present, indicating that the IL-1␤-like IR in the
area postrema described here predominantly represents
newly synthesized pro-hormone in response to LPS treat-
ment.
DISCUSSION
The results of these studies have revealed an intimate
relationship between immune cells and neuronal ele-
ments in the area postrema, providing potential for a
novel, direct influence of immune cells on neurons within
the CNS. Following peripheral immune challenge with
bacterial endotoxin, robust IR for the pro-inflammatory
cytokine IL-1␤ was induced in many innate immune
 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434 1427

Fig. 8. Electron micrographs of OX-42 labeled immune cells (microglia) in the area postrema. Immunoreactivity for the CD11b antigen as silver
granules distributes along the outer cell membranes of the OX-42-labeled cells and their profiles (indicated with asterisks in all panels. Similar staining
was observed following saline (A–C) and endotoxin (D–F) treatment. (A) An OX-42-labeled profile curves along the vasculature. (B) The cytoplasm
of the OX-42-positive ramifications is rich in endoplasmic reticulum, mitochondria and small vesicular inclusions. (C) OX-42-labeled soma containing
a cell nucleus (Nu) with electron-dense patches of chromatin. (D, E) OX-42-labeled ramifications come in close proximity with neuronal dendrites (d),
however do not form close membrane-to-membrane associations. (F) Extensive ramifications in distal ends of microglia. Scale bars⫽1 ␮m in all
panels.

cells, which associated either with the vasculature or
with neuronal elements. The findings suggest multiple
roles for immune cells in the area postrema, and provide
a potential mechanism by which brain-derived cytokines
might directly influence constituents of the neurocircuitry
that mediates brain responses to infection and infla-
mmation.
Complexity of identifying immune cell phenotypes
Characterization of immune cell populations in the brain
has been confusing and controversial. The varying mor-
phology and expression of functional markers such as
MHC-II (OX-6) imply that these cells may correspond to
different activation states of the same cell, or they may
1428 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434

Fig. 9. ED2-labeled perivascular immune cells showing silver-gold immunoprecipitate along the outer cell membranes. The cells exhibit nuclei (Nu)
contain characteristic electron-dense patches of chromatin. Although the cells are associated with the vasculature, astrocytes (as) often intersperse.
Very thin and short cilia-like ramifications (asterisks in B–D) emanating from the soma are another characteristic feature of these ED2-labeled cells
that often make close contacts with as (arrows in B and C). Electron-dense inclusions seen in the somata in panels A and D indicate phagocytic activity.
Other abbreviations: bv, blood vessel; endo, endothelial cell. Scale bars⫽1 ␮m in all panels.

represent cells of separate lineage. In the area postrema,
subpopulations of cells can be described based on the 1)
differential expression of CD163, CR3/CD11b, and MHC-
II, 2) on morphological differences, and 3) on location
within the area postrema. Nevertheless, it is likely that
these are overlapping populations, and cells may co-ex-
press two or all of these markers. However, based on
morphological appearance and distribution characteristics
(as shown in Figs. 1 and 2), co-expression of CD163
(ED-2) and CR3/CD11b (OX42) seems unlikely, based on
morphological and tissue distribution differences. Some
overlap between CD163 (ED-2) and MHC-II (OX-6) is likely
based on OX-like IR seen very occasionally on perivascu-
lar cell membranes, but limited, as the overall distribution
of MHC-II-expressing (OX-6) cells differs from that of the
CD163-positive (ED-2) ones. That CD163 (ED2) and
MHC-II (OX-6) expression occurs in largely separate pop-
ulations in the area postrema appears to be in line with
similar observations in dura mater, leptomeninges, and
choroid plexus isolated from rat brain (McMenamin, 1999).
Similarly, CR3/CD11b (OX-42) and MHC-II (OX-6) may
colocalize in a small subset of cells, as we observed that a
small proportion of MHC-II-positive cells displays a mor-
phology highly similar to those CR3/CD11b-labeled cells
characteristic of microglia. Although the populations of
immune cells we describe may not represent truly separate
 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434 1429

Fig. 10. IL-1␤-like immunoreactivity in perivascular immune cells following endotoxin treatment (0.4 mg/kg i.p., 4 h). The DAB precipitation
indicative of IL-1␤ IR substantially darkens the cytoplasm. (A) Perivascular IL-1␤-positive cell with two distal ends of the nucleus (Nu) and a
cilia-like extension (indicated with asterisk). Although the cell wraps around the endothelial cell (endo), it is partly interspersed by an astrocytic
ramification (as), a feature also characteristic for ED-2 positive cells. The cell is rich in organelles including mitochondria. (B) IL-1␤-labeled soma
with multiple thin extensions (asterisk). (C, D) IL-1␤-positive profiles (distal somata) associated with blood vessels (bv). (E) An IL-1␤-positive
cell positioned within the area postrema parenchyma that shows partially extruding vesicles that are densely stained (arrow). Scale bars⫽1 ␮m
in all panels.

phenotypes, assessment of the distribution of the different
markers provides information as to the potential functional
roles of each immune cell phenotype (see Guillemin and
Brew, 2004) and provides information relevant for potential
intervention with e.g. targeted immunolesion techniques.
Immune cell subpopulations: substrate for immune
sensory specialization?
The results from these studies confirm and extend earlier
reports regarding the myeloid immune-like identity of IL-
1␤-immunoreactive cells in circumventricular organs. Na-
kamori et al. (1994) described IL-1␤ expression in macro-
phages exclusively restricted to the OVLT (a forebrain
circumventricular organ) in rabbits, whereas IL-1␤ expres-
sion in cells associated with the vasculature of the brain
parenchyma was attributed to microglia. Konsman et al.
(1999) described colocalization of IL-1-like IR with lectin-
binding cells, also supporting a myeloid immune cell-like
identity. However, lectin-binding does not distinguish be-
tween subtypes (e.g. macrophage, microglial or dendritic-
like) of immune cells. Under the conditions during our
study, that is, peripherally administered endotoxin at mod-
1430 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434

Fig. 11. IL-1␤-immunoreactive cells associated with neurons in the area postrema following endotoxin treatment (0.4 mg/kg i.p., 2 h: D, E and 4 h:
A–C). (A) Cell soma and proximal extension showing IL-1␤ IR in close proximity to a neuronal cell body. The neuron is characterized by its lighter
stained nucleus [Nu(n)] and the embedded nucleolus. The insets a and b show higher magnification of the image areas indicated with asterisks and
labeled a and b. Here the IL-1␤-labeled profile directly opposes a neuronal dendrite (d in a), and the neuronal soma (n in b). (B) An IL-1␤-labeled profile
(asterisk) wraps around a neuronal d. Dense immunoreactive label along the outer membrane is visible juxtaposing the neuronal membrane (arrow).
(C) An extensively ramified IL-1␤-positive profile that wraps itself around two neuronal d, the larger of the two is completely wrapped (two arrows
indicate the “closure”). The lower arrows and the asterisk indicate a darkening of the neuronal membranes, which could indicate possible released and
bound IL-1␤. (D) A IL-1␤-IR profile (asterisk) in tight association with a neuronal d in the border region of the area postrema and nucleus of the solitary
tract. (E) A distal IL-1␤-IR ramification runs along the surface of a neuronal soma with nucleus [Nu(n)]. Scale bars⫽1 ␮m in A and E; 500 nm in B–D
and insets a, b.

erate doses, with two to four hours’ survival, the most
robust induction of IL-1␤-like IR in the area postrema was
contributed by CD163 (ED2)-labeled tissue macrophages/
perivascular cells. MHC-II (OX-6)-positive dendritic-like
cells, and another morphologically similar group of cells
expressing neither marker also responded. Although some
CR3/CD11b (OX42)-positive cells with a microglia-like
morphology were also IL-1␤-positive, they were far fewer
in number, at least at the earlier time points chosen in this
study. In addition, at these early time points, neither neu-
ron nor astroglial cells were found IL-1␤-positive. Thus,
these findings demonstrate differential responsivity to pe-
ripheral LPS challenge among cells within the area pos-
trema.
The differences in responses to peripheral LPS chal-
lenge as revealed by IL-1␤ expression between the im-
mune phenotypes of cells in the area postrema may derive
from functional requirements. A picture is emerging from
recent studies (Chakravarty and Herkenham, 2005; Val-
lieres and Sawchenko, 2003) that indicate a dichotomy
 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434
Fig. 12. Western blot of tissue supernatant from the dorsomedial
medulla of a LPS-treated rat (right lane) showing immunoreactive
protein of 31 kDa size as compared with the ladder in the left column,
likely to be the pro-IL-1␤. The middle column shows recombinant rat
IL-1␤ (10 ng) at approximately 17.5 kDa.
between “resident” (microglia) and “immigrant” immune
cells in the brain. Microglia enter the brain during prenatal
and early postnatal development, seem to turn over slowly
and are not readily replenished (Vallieres and Sawchenko,
2003). The immigrants, which are primarily perivascular
cells associated with the brain vasculature and (presum-
ably) macrophages and dendritic cells outside the blood–
brain barrier (meninges and choroids plexus) or where it is
weak (circumventricular organs), show, in contrast, a
higher rate of turnover and readily repopulate from hema-
topoietic precursors (Hickey et al., 1992; Hess et al.,
2004). The apparent bias of earlier and more robust IL-1␤
expression toward “immigrant” type cells observed in our
study implies that these cells constitute the principal immune
sensory cell in the brain. Indeed, CD163 (ED2)-positive
perivascular cells have previously been implicated in im-
mune-to-brain signaling (Schiltz and Sawchenko, 2002)
based on their responses to systemic IL-1␤ administration.
MHC-II expression has been previously described in
immune cells of the area postrema (Pedersen et al., 1997),
however the findings here represent the first report implicat-
ing these cells in contributing to IL-1␤ production. Whereas
the function of contact between MHC-II (OX-6)-immunoreac-
tive cells and neurons in the area postrema cannot be deter-
mined from this morphological study, the apparently specific
targeting of immune cells and TH-IR neurons (Fig. 3A–D)
suggests immune influence on area postrema functions,
such as emesis (Miller and Leslie, 1994; Jovanovich-Micic et
al.,1995) or relay to rostral autonomic regions, notably the
parabrachial nucleus (Yamamoto et al., 2003).
Potential mechanisms of immune-neural interaction
in the area postrema
The finding that IL-1␤-like IR cells are differentially distrib-
uted within the area postrema suggests multiple mecha-
1431
nisms by which IL-1␤ can influence area postrema func-
tions. IL-1␤-IR cells that contact neurons could directly
influence specific neural circuits (e.g. those involved in
emesis vs. those involved in viscerosensory relay to the
forebrain). Such a direct action would likely serve to in-
crease the fidelity of immune-neural signaling, by enabling
more rapid and consistent interaction of IL-1␤ with recep-
tors. In contrast, perivascular cells are positioned to influ-
ence functions associated with the vasculature, including
immune cell trafficking (Ching et al., 2005), and could
release IL-1␤ into the abundant extracellular space of the
area postrema to influence neurons, astrocytes, or other
immune-like cells via a paracrine-like mechanisms. Fur-
ther, given the observation that astrocytes were typically
interspersed around and between the IL-1␤-IR immune
processes and the neuronal dendrites and axons, IL-1␤
may also modulate astrocytic functions.
Evidence for physiological effects of IL-1␤ on neurons
derives from reports that IL-1␤ influences intracellular cal-
cium levels in cortical synaptosomes (Campbell and
Lynch, 2000) and cultured hippocampal neurons (Viviani et
al., 2003). A similar effect of IL-1␤ on neurons in the area
postrema would be consistent with the findings of Desson
and Ferguson (2003) showing that in another circumven-
tricular organ, the subfornical organ, IL-1␤ influences neu-
ronal excitability. The effects of IL-1␤ on neurons in the
area postrema are likely mediated by the type 1 IL-1 re-
ceptors, which have been reported to be expressed by
several other neuronal subtypes, including sensory neu-
rons (Ek et al., 1998), as well as neurons in the hypothal-
amus (Diana et al., 1999), hippocampus (French et al.,
1999), and amygdala (Ericsson et al., 1995). Thus, find-
ings from studies using both physiological and anatomical
approaches support the idea that IL-1␤ can exert its ac-
tions in the brain via direct effects on neurons.
In addition to a potential immunosensory role, im-
mune–neural contact in the area postrema may serve
other functions. For instance, these immune cells may
provide trophic factors specific for neuronal subpopula-
tions, and/or may play a role in synaptic plasticity, as do
microglia in other brain regions (Roumier et al., 2004). At
any rate, immune cells likely play multiple roles in the area
postrema, related to different challenges of developmental
and/or immune status (Boulanger and Shatz, 2004).
The area postrema as an immune sensory structure
Evidence of an immunosensory function of the area pos-
trema derives from multiple lines of evidence. Neurons in
the area postrema consistently express c-Fos-IR (an acti-
vation marker) following both central and peripheral im-
mune challenge (Brady et al., 1994; Goehler et al., 2005).
Toll-like receptors (TLR), which are immune sensory re-
ceptors (Beutler et al., 2003), are expressed in the area
postrema, including TRL4 (LaFlamme and Rivest, 2001)
and TLR9 (Sako et al., 2005). Further, the area postrema
is one of the structures identified in the rat to express IL-1
receptor type I mRNA (Cunningham et al., 1992; Ericsson
et al., 1995), providing a substrate by which IL-1␤ pro-
duced within the area postrema (see also Quan et al.,
1432 L. E. Goehler et al. / Neuroscience 140 (2006) 1415–1434
1998; Konsman et al., 1999; Van Dam et al., 2000) likely
influences neurons cohabiting the area postrema, and
from there on to neuronal targets in other parts of the brain
(see below).
A functional role of the area postrema in brain-medi-
ated host defense responses is supported by findings that
lesions of the area postrema can attentuate HPA axis
responses or hypothalamic norepinephrine increase to
systemic immune activation (Ishizuka et al., 1997; Lee et
al., 1998), and inactivation of the dorsal vagal complex,
including the area postrema, blocks sickness-induced be-
havioral depression and c-Fos expression in the brain
(Marvel et al., 2004). On the other hand, Ericsson et al.
(1997) did not find impairment of either HPA response or
medullary and hypothalamic c-Fos protein expression fol-
lowing i.v. IL-1␤ administration. The reasons for this dis-
crepancy are not evident, but underline the fact that im-
mune signals influence the brain via multiple pathways and
mechanisms that are as yet incompletely understood.
The area postrema is well suited for a sensory role sig-
naling both peripheral immune activation as well as inflam-
mation within the nervous system (e.g. meningitis). In addi-
tion to possessing a weak blood– brain barrier, the area pos-
trema protrudes into the fourth ventricle, allowing it access to
substances (including pathogens) in cerebrospinal fluid not
normally found in brain parenchyma. Circumventricular or-
gans contain abundant extracellular space, which results in
fluid dynamics that favor slower throughput of substances,
facilitating their function as sensory structures (Gross, 1991).
Moreover, the area postrema is distinct from other circum-
ventricular organs in that it receives direct input from periph-
eral sensory nerves, via terminations of vagal sensory fibers
within its lateral margins (Beckstead and Norgren, 1979;
Chernicky et al., 1984; Strain et al., 1990; Sykes et al., 1997;
Zheng et al., 2002). Vagal sensory fibers have been impli-
cated as immunosensory conduits (Watkins et al., 1995; Ek
et al., 1998; Gaykema et al., 1998; Goehler et al., 1998) that
likely play a role in signaling immune activation in internal
visceral structures, such as the gut (Dantzer, 2004; Goehler
et al., 2005). Targets of the area postrema projections are
restricted to specific components of the ascending visceros-
ensory pathway, notably the nTS and external lateral division
of the lateral parabrachial nucleus of the pons (Shapiro and
Miselis, 1985; Herbert et al., 1990; Rogers and McCann,
1993; Cunningham et al., 1994). Previously reported findings
that neurons of the area postrema reliably respond to both
peripherally and centrally administered immune stimulants
(Wan et al., 1993; Elmquist et al., 1996; Konsman et al.,
1999) or live bacteria (Goehler et al., 2005) support the idea
that the area postrema indeed contributes to brain responses
to peripheral immune activation. Taken together with the
findings presented here, the close relationships between cy-
tokine-producing immune cells and neurons observed in the
area postrema are therefore consistent with a mechanism by
which circulating pathogens or pathogen products, via influ-
ence upon specific neural circuits originating in the area
postrema, may induce, modulate, or maintain brain-mediated
host defense responses.
CONCLUSION
The use of immunoelectron and confocal microscopy has
revealed multiple phenotypes of immune cells in the brain-
stem area postrema and demonstrated that some of these,
including some that express IL-1␤-like IR following periph-
eral immune challenge, make significant physical contact
with neurons. Thus the resolution afforded by this tech-
nique has revealed a potentially direct mechanism by
which neurons and immune cells may interact within brain
circumventricular organs.
The diversity of immune cells’ phenotypes in the area
postrema, compared with the normal brain parenchyma,
may well follow from the fact that circumventricular organs
occupy a position at the interface between the CNS and
the periphery. Although the blood– brain barrier is weak in
these structures, they contain neurons typical of brain
parenchyma rather than those associated with the periph-
eral nervous system. This arrangement allows for the po-
tential for interaction of immune sentinel cells with brain
neurons, either directly via membrane apposition, or via
local paracrine-type mechanism, and supports the idea
that circumventricular organs serve as immune sensory
structures.
Acknowledgments—The authors would like to thanks Ms. Bonnie
Shepard for excellent and invaluable technical assistance. This
work was supported by NIH grants MH 64648, MH 68834 and
EY12138.
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APPENDIX
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.neuroscience.2006.03.048.