Ž .Journal of Neuroimmunology 121 2001 32–39www.elsevier.comrlocaterjneuroin

IL-1 receptor type I gene expression in the amygdala of inflammatorysusceptible Lewis and inflammatory resistant Fischer rats

Patrick Frost a, Ruth M. Barrientos b, Shinya Makino c, Ma-Li Wong a,), Esther M. Sternberg b

a UCLA Neuropsychiatric Institute and Brain Research Institute, 3357A Gonda Center, 695 Charles Young Dr., So., Los Angeles, CA 90095-1761, USAb National Institute of Mental Health, National Institutes of Health, Bldg. 10, Room 2D-46, 10 Center DriÕe, Bethesda, MD 20892-1284, USA

c Second Department of Internal Medicine, Kobe UniÕersity School of Medicine, Kobe, Japan

Received 12 June 2001; received in revised form 15 August 2001; received in revised form 14 September 2001; accepted 19 September 2001

Abstract

Ž . Ž .Lewis LEWrN and Fischer F344rN rats have different responses to inflammatory and behavioral stressors due to differences inŽ .hypothalamus–pituitary–adrenal HPA axis function. For example, LEWrN rats are more sensitive to restraint, inflammation and

experimentally induced autoimmunity due to decreased HPA activity. The HPA axis response to peripheral inflammation is mediated, atŽ .least in part, by IL-1b and its receptor, IL-1 type I IL-1RI . Here, we studied the distribution of IL-1RI mRNA in the brains of LEWrN

Ž .and F344rN rats, and demonstrated that IL-1RI mRNA expression has significantly increased in the basolateral nucleus BLA of theamygdala of LEWrN rats. These findings suggest that strain-specific HPA axis responses may be mediated by extrahypothalamicpathways. q 2001 Elsevier Science B.V. All rights reserved.

Keywords: IL-1 receptor type I; Amygdala; Lewis rat; Fischer rat; HPA axis

1. Introduction

The bi-directional regulatory feedback loop controllingŽ .hypothalamus–pituitary–adrenal HPA axis and immune

function is relevant to the physiological and psychologicalresponse to certain stressors. For example, LPS-mediatedactivation of the peripheral immune system stimulates

w Ž .inflammatory e.g. interleukin-1 beta IL-1b , IL-6, tumorŽ .x wnecrosis factor alpha TNF-a , and anti-inflammatory e.g.

Ž . xIL-1 receptor antagonist IL-ra , IL-10, IL-13 cytokinegene expression by astrocytes, microglia and neurons

Žwithin the rat brain Ban et al., 1992; Benveniste, 1992;Schobitz et al., 1993; Tchelingerian et al., 1993; Buttini

.and Boddeke, 1995; Wong et al., 1997 . Cytokines, in turn,affect the expression of neuroendocrine factors, including

Ž . Žcorticotropin-releasing hormone CRH Bernardini et al.,. Ž1990; Rivest and Rivier, 1994 , arginine vasopressin Hil-

. Ž .lhouse, 1994 , neuropeptide Y Schwartz et al., 1995 ,Ž . Žadrenocorticotropic hormone ACTH Lee and Rivier,

. Ž1994, 1995 , and inducible nitric oxide synthase Okuda et

) Corresponding author.Ž .E-mail address: mali@ucla.edu M.-L. Wong .

.al., 1995; Romero et al., 1996 . Completing this regulatoryloop, HPA axis neuroendocrine factors downregulate theimmunerinflammatory response by stimulating potent im-

Ž . Žmunosupressive glucocorticosteroid GC expression Be-.sedovsky et al., 1986 .

Ž .The histocompatible similar Lewis LEWrN and Fis-Ž .cher F344rN rats exhibit marked differences in HPA

axis responses to immune and behavioral stressors, such asexperimental autoimmune encephalomyelitis and strepto-

Ž .coccal cell wall peptidoglycan polysaccharide SCW in-duced arthritis, and serve as a model to study the role of

ŽimmunerHPA axis interactions Sternberg et al., 1989,.1990, 1992 . LEWrN rats, for example, have attenuated

responses to restraint, enhanced exploration in an openfield, and two-fold higher responses to acoustic startle thanF344rN rats. Furthermore, plasma ACTH and GC levelsinduced by specific immune and behavioral stressors have

Ždecreased in amplitude and duration in LEWrN rats Grota.et al., 1997; Sternberg et al., 1989 . Finally, the inflam-

matory response of LEWrN rats can be inhibited bytreatment with exogenous dexamethasone, while the GCreceptor antagonist, RU-486, can induce SCW-mediated

Ž .autoimmunity in F344rN rats Sternberg et al., 1989 .These findings suggest that neuroendocrine dysregulation,

0165-5728r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.Ž .PII: S0165-5728 01 00440-4

( )P. Frost et al.rJournal of Neuroimmunology 121 2001 32–39 33

and not innate differences in immune response, plays animportant role in the strain-specific differences in inflam-matory and disease susceptibility.

Ž .Interleukin-1b IL-1b mediates HPA response to awŽwide range of inflammatory and behavioral stressors Di-

. xnarello, 1996 for review . The action of IL-1b is signaledŽ .through the type I receptor IL-1RI . IL-1RI has been

identified in brain in the choroid plexus, hippocampus,cerebellum, vascular and peri-vascular structures. The ab-sence of IL-1RI mRNA expression and binding of labeledIL-1 in the paraventricular nucleus of the hypothalamusŽ .PVN has been surprising. The function of the PVNneurons may be modulated by multiple sources, includinginputs from limbic system-associated regions, such as thehippocampus and amygdala, which express IL-1RI. Thus,we hypothesized that IL-1RI mRNA levels may be differ-entially expressed in the hypothalamus or CNS regionsthat modulate hypothalamic functions of LEWrN andF344rN rats, which could provide insights into the poten-tial mechanisms underlying the differential IL-1b-mediatedstimulation of HPA function in these rats. To test thathypothesis, we used in situ hybridization histochemistryŽ .ISHH to study the distribution of IL-1RI in the brains ofLEWrN and F344rN rats.

2. Materials and methods

Female virus- and antibody-free LEWrN and F344rNŽ . Ž .rats ns16 Harlan Sprague–Dawley, Indianapolis, IN

were housed three to four per cage at 24 8C in a humidity-Žcontrolled room with 12-h lightrdark cycle lights-on at

.0600 h and off at 1800 h . Standard rat chow and waterwere available ad libitum throughout the experiment. Theprocedures were approved by the Animal Care and UseCommittee of the National Institute of Mental HealthŽ .NIMH . Rats were allowed to acclimatize for 1 weekprior to the decapitation and collection of the brain tissue.Rats were decapitated between 1000 and 1100 h, corre-sponding to 4–5 h after lights-on, and their brains wereimmediately frozen on dry ice, stored at y70 8C, andserially sectioned and processed for in situ hybridization

Ž .histochemistry ISHH .

2.1.1. In situ hybridization histochemistry

Frozen brain tissue was cut coronally in 15-mm thicksections for ISHH at the level of the bed nucleus of the

Ž .stria terminalis BNST , PVN, and central amygdaloidŽ .nucleus CeA . Sections of the BNST were taken at bregma

y0.26 to y0.40 mm; PVN at bregma y1.60 to y1.90mm; and CeA at bregma y2.60 to y2.80 mm. Thesections were thaw-mounted and air dried on gelatin-coatedslides, and stored at y70 8C prior to ISHH. Ribonu-

cleotide probes directed against the reported sense andantisense IL-1RI sequence were generated from rat IL-1RI

ŽcDNA generously provided by Dr. Ronald H. Hart, Rut-. Ž .gers University, Newark, NJ Hart et al., 1993 . Tran-

scription of probes was carried out using the RiboprobeŽ .System Promega Biotech, Madison, WI in the presence

Ž35 . Žof S UTP )1000 Cirmmol, DuPontrNEN, Boston,. ŽMA . ISHH was conducted as previously described Wong

. 5et al., 1997 . Briefly, 5=10 cpm of radiolabeled anti-sense or sense control riboprobe per 25 ml of hybridizationbuffer was used; 25 ml of hybridization buffer was appliedon each section, which was then covered by a coverslip.Hybridization was carried out at 52 8C for 18 h. After

Žhybridization, slides were treated with RNase A Sigma, St.Louis, MO , washed sequentially up to 0.1X SSC at 60 8C,

dehydrated, and dried at room temperature.

2.1.2. Analysis and quantification

In the analysis of IL-1RI mRNA, the slides and 14C-Žstandards of known radioactivity American Radiochemi-

.cals, St. Louis, MO were placed in X-ray cassettes ex-35 Žposed to S-sensitive film Hyperfilm-bMax, Amersham,

Fig. 1. Localization of baseline IL-1RI mRNA expression in the brains ofŽ . ŽF344rN first column, A to C and LEWrN rats second column, D to

.F . A series of film autoradiographs show the regional localization ofIL-1RI mRNA expression at the level of the bed nucleus of the stria

Ž .terminalis bregma y0.26 to y0.40 mm, A and D , paraventricularŽ .nucleus of the hypothalamus bregma y1.60 toy1.90 mm, B and E ; and

Ž .central nucleus of the amygdala bregma y2.60 to y2.80 mm, C and F .Ž . Ž .Inset panel C shows IL-1RI sense probe control. Arrow panel F

indicates IL-1RI mRNA localized in basolateral nucleus of amygdala.Bars1.2 mm.

( )P. Frost et al.rJournal of Neuroimmunology 121 2001 32–3934

.Arlington Heights, IL for 14 days. Films were developedŽ .D19, Eastman Kodak, Rochester, NY for 5 min at 20 8C.To determine the anatomical localization of probe at thecellular level, the sections were dipped in NTB-2 nuclear

Ž .emulsion Eastman Kodak and exposed for 8 weeks.Ž .Slides were developed D19, Eastman Kodak for 2 min at

16 8C and counterstained with cresyl violet. The amount ofprobe hybridized in the BNST, PVN, and CeA was mea-sured as regional optical densities of autoradiographic filmimages with a computerized image analysis system com-posed of a light box, a solid state video camera, andMacintosh II-based IMAGE software developed by WayneRasband, Research Service Branch, National Institute of

Ž .Mental Health Rasband and Bright, 1995 . Optical densi-ties for each region were obtained in two consecutivesections per rat. Values were converted to disintegration

Ž .per minute per milligram dpmrmg of rat brain tissueusing a standard curve generated by 14C standards. Statisti-cal significance between the F344rN and LEWrN groupswas determined by unpaired Student’s t-test. A P value of-0.05 was considered significant.

3. Results

The baseline expression of IL-1RI mRNA in LEWrNand F344rN rats is shown in Fig. 1. The pattern ofexpression included the hippocampus, choroid plexus,

Ž .meninges, amygdala and vasculature Fig. 1 . As controlfor our findings, we examined the levels of IL-1RI mRNAexpression in the PVN and BNST. There was no signifi-cant difference in IL-1RI mRNA expression in either the

Ž . ŽPVN of the hypothalamus Fig. 1B and E 222.22"21.29dpmrmg LEWrN brain tissue versus 209.17"32.64

. Ždpmrmg F344rN brain tissue; Ps0.46 or BNST Fig.. Ž1A and D 71.07"15.35 dpmrmg LEWrN brain tissue

versus 68.61"7.51 dpmrmg F344rN brain tissue; Ps.0.60 , for either strain. Interestingly, the IL-1RI expression

in the amygdala of LEWrN rats was significantly higherŽthan F344rN rats 290.44"29.44 dpmrmg LEWrN brain

tissue versus 228.26"34.03 dpmrmg F344rN brain tis-. Ž .sue; P-0.001 Fig. 1C and F . Hybridization with IL-1RI

sense riboprobes resulted in a low signal that was barelyŽ .visible on film inset in Fig. 1C .

Ž . Ž .Fig. 2. A series of bright and darkfield photomicrographs arranged to show the localization of IL-1RI mRNA A to F in LEWrN first column, A to CŽ .and F344rN rats second column, D to F in the basolateral nucleus of the amygdala. Bright field low magnification photomicrographs are shown in the

Ž . Ž .first row A and D , corresponding to the darkfield photomicrographs presented in the second row B and E . High magnification bright fieldŽ . Ž . Ž . Žphotomicrographs are shown in the last row C and F . Note that regions containing high concentrations of mRNA are white in B and E . Black dots C

. Ž . Ž . Ž . Ž . Ž . Ž .and F over the cells represent silver grains overlying mRNA. Bars450 mm for A , B , D , and E ; 30 mm for C and F .

( )P. Frost et al.rJournal of Neuroimmunology 121 2001 32–39 35

Light and darkfield photomicrographs of cresyl violet-stained sections confirmed that IL-1RI expression located

Ž .within the basolateral nucleus BLA of the amygdala inŽ .LEWrN rats Fig. 2B was higher than in F344rN rats

Ž .Fig. 2E . Furthermore, the distribution of mRNA signalwas diffused and associated primarily with light-staining

Ž .neuronal cell bodies Fig. 2C and F . Localization ofŽ .IL-1RI mRNA in both the hippocampus Fig. 3 , as well as

Ž .meninges and vasculature regions Fig. 4 was similar inboth strains of rat, and compatible with the distribution in

ŽSprague–Dawley rats Ericsson et al., 1995; Yabuuchi et.al., 1994; Wong and Licinio, 1994 . Light and darkfield

photomicrographs demonstrate that the expression of IL-1RI was localized diffusely throughout the dentate gyrusŽ . Ž .Fig. 3C and G and the CA1 regions Fig. 3D and H , andwas primarily associated with light-staining neuronal cellbodies. Baseline levels of IL-1RI were also observed within

Ž .the choroid plexus Fig. 4D and H . IL-1RI mRNA wasobserved in the vascular regions of both F344rN and

Ž .LEWrN rats Fig. 4A, B, C, E, F, and G . The observed

Ž .Fig. 3. A series of bright and darkfield photomicrographs arranged to show the localization of IL-1RI mRNA in LEWrN first column, A to D andŽ . Ž .F344rN rats second column, E to H in the hippocampus. Bright field low magnification photomicrographs are shown in the first row A and E ,

Ž .corresponding to the darkfield photomicrographs presented in the second row B and F . High magnification bright field photomicrographs are of dentateŽ . Ž . Ž . Ž .gyrus C and G and the CA1 region D and H is shown. Note that regions containing high concentrations of mRNA are white in B and F . Black dots

Ž . Ž . Ž . Ž . Ž . Ž . Ž . Ž . Ž .C, D, G, and H over the cells represent silver grains overlying mRNA. Bars450 mm for A , B , E , and F ; 30 mm for C , D , G , and H .

( )P. Frost et al.rJournal of Neuroimmunology 121 2001 32–3936

Ž .Fig. 4. A series of bright and darkfield photomicrographs arranged to show the localization of IL-1RI mRNA in LEWrN first column, A to D andŽ .F344rN rats second column, E to H in the choriod plexus and vasculature. Bright field low magnification photomicrographs are shown in the first row

Ž . Ž .A and E , corresponding to the darkfield photomicrographs presented in the second row B and F . High magnification bright field photomicrograph ofŽ . Ž . Ž . Ž .vasculature C and G and choriod plexus D and H are shown. Note that regions containing high concentrations of mRNA are white in B and F . Black

Ž . Ž . Ž . Ž . Ž . Ž . Ž . Ž . Ž .dots C, D, G, and H over the cells represent silver grains overlying mRNA. Bars45 mm for A , B , E , and F ; 30 mm for C , D , G , and H .

distribution of IL-1RI mRNA in our study is compatiblewith the previous reports of IL-1RI expression in the CNS

Žof Sprague–Dawley rats Ericsson et al., 1995; Yabuuchi.et al., 1994; Wong and Licinio, 1994 .

4. Discussion

Previous studies showed that the expression of IL-1RImRNA expression pattern in outbred Sprague–Dawley

rats included hippocampus, vasculature, choriod plexus,Žmeninges, amygdala and cerebellum Ericsson et al., 1995;

.Yabuuchi et al., 1994; Wong and Licinio, 1994 . In thepresent study, both LEWrN and F344rN rats have similarIL-1RI mRNA expression patterns when compared toSprague–Dawley, except for significantly higher basal lev-els of IL-RI mRNA in the BLA of LEWrN rats. Further-more, we have previously reported that stimulated HPAaxis responses in LEWrN and F344rN rats differ, butbasal hypothalamic PVN CRH mRNA levels and basal

( )P. Frost et al.rJournal of Neuroimmunology 121 2001 32–39 37

pituitary–adrenal function were similar, despite the pro-found differences in the susceptibility to inflammatory

Ž .disease and behavioral responses Sternberg et al., 1989 .Our findings that LEWrN rats have significantly greaterbasal levels of IL-1RI mRNA in the BLA of the amygdala,but not the hypothalamus, suggest that some of the HPAactivation differences between LEWrN and F344rN ratsmay be due to differences in IL-1b-mediated signaling inextrahypothalamic areas.

The amygdala is a complex of heterogenous nucleilocated at the medial edge of the temporal lobe and playsan important role in the organized neural system thatintegrates the brain’s response to psychological and physi-

Žologic responses to stress Herman et al., 1996; Xu et al.,.1999 . The BLA consists of the lateral, basal and acces-

sory basal nuclei, which are significant in mediating fearand memory. For example, recent studies have demon-strated that the amygdala, in particular the CeA and medialŽ .MeA nucleus of the amygdala, may be involved inactivating the HPA axis in response to a variety of stimuli,

Žincluding inflammatory cytokines and restraint Herman et.al., 1996; Day et al., 1999 . The extensive and highly

organized network of intra-amygdaloid projections sug-gests that after entering the amygdala, a stimulus will havemultiple parallel representations. It also suggests that eachamygdaloid nucleus or its division carries out a differentfunction established by its specific representation of thestimulus attributes. Therefore, the fact that no direct pro-jection has been described between the BLA and thehypothalamus implies that any modulatory role of the BLAmay have in the HPA axis is indirect. The BLA can

Ž .influence the HPA function through two projections: 1other amygdaloid nuclei, specially the CeA, which is theprimary output nucleus for amygdaloid projections to the

Ž . Žhypothalamus and 2 the hippocampus BLA projects to.hippocampus CA1-3 subfields . The CeA receives input

from several amydaloid nuclei, including the BLA, andprojects directly to the PVN via a small number of cells, aswell as, indirectly, by way of the BNST. Additionally, the

Ž .CeA projects to the nucleus tractus solitarius NTS andŽ .the ventrolateral medulla VLM . Catecholaminergic pro-

jections from the BNST, NTS and VLM constitute theŽ .primary afferent projections to the PVN McDonald, 1998 .

Thus, the BLA may modulate the activity of the PVNthrough its projections to other nuclei of the amygdala andthrough its projection to the hippocampus. Certain stereo-typic behaviors are affected differently by distinct amyg-daloid nuclei. For instance, stimulation of the CeA andlateral nuclei suppresses defensive rage behavior and facil-itates predatory attack behavior, while the opposite out-come occurs with the stimulation of the anterior, basome-

Ždial and medial amygdaloid nuclei Smith and Flynn,.1980; Shaikh and Siegel, 1994 .

Our previous findings that basal HPA activity andimmune responses in F344rN and LEWrN rats are simi-lar strongly suggest that differences must exist upstream of

Ž .these systems Sternberg et al., 1989, 1990, 1992 . There-fore, it is reasonable to hypothesize that the relative hy-poresponsiveness of the HPA system in LEWrN rats isrelated to increased IL-1b-mediated signals originatingfrom the other parts of the brain not present in F344rNrats. Supporting this hypothesis, we observed significantlygreater baseline expression of IL-1RI in the BLA ofLEWrN rats, but not in other related regions of the brainŽ .e.g. PVN of the hypothalamus . Increased IL-1RI expres-sion would be expected to result in an overall higherresponse to IL-1b in the amygdala, perhaps resulting indifferential regulation of HPA neuroendocrine function.On the other hand, it is also possible that the upregulatedbaseline levels of IL-1RI-mediated signaling in response toIL-1b contributes to a decrease in the sensitivity of thehypothalamus to inflammatory stimuli, thus limiting HPAaxis activation and subsequent GC-mediated immune sup-pression. In either case, the functional role of differencesin amygdala IL-1RI expression on behavioralrautoimmun-ity susceptibility differences in F344rN and LEWrN ratsremains to be shown.

While it has been shown that glucocorticoid administra-tion increases the level of CRH receptor mRNA in the

Ž .hypothalamus of outbred rats Makino et al., 1995 , therehave been no previous studies of IL-1RI expression in theCNS of either F344rN or LEWrN rat strain. Thus, theseexperiments are important in complementing the HPArim-mune system axis loop, by examining the receptor targetsof IL-1b in the brain. However, whether the higher basalIL-1RI mRNA levels in the amygdala of LEWrN rats aredirectly related to the decreased HPA axis activation re-mains to be determined. In this regard, the specific role ofthe IL-1RI, the amygdala and the extrahypothalamic CRHsystems in regulating HPA axis function and behaviorrequires more study, including direct evidence of differen-tial signaling.

In summary, we have shown that LEWrN and F344rNrats show small, but significant basal level differences inthe BLA expression of IL-1RI, but not in the PVN orBNST. IL-1b exerts a positive effect on the HPA axiswhen it is applied directly into the PVN. The amygdala, inparticular the extrahypothalamic CRH system, may have

Žan inhibitory effect on the HPA axis Gomez et al., 1999;.Makino et al., 1999 . For example, Fawn–Hooded rats,

which have abnormal HPA axis responses, were recentlyshown to have augmented expression of CRH in the CeA

Ž .compared to Sprague–Dawley rats Gomez et al., 1999 .Thus, while the increased baseline levels of IL-1RI mRNAin the amygdala of LEWrN rats is unlikely to result inover-stimulation of the HPA, it is possible that the differ-ences in amygdala IL-1RI expression described here con-tribute to stress andror fear-related behaviors and immuneresponses mediated through the amygdala. However, theexact functional consequences of increased IL-1RI expres-sion in the BLA of LEWrN rats remain to be defined.Therefore, future studies should test the hypothesis that

( )P. Frost et al.rJournal of Neuroimmunology 121 2001 32–3938

HPA axis differences in LEWrN and F344rN rats, whichcan be detected following perturbation of this system byinflammatory triggers and other potent stimuli, may be dueto distinctive modulation of the HPA axis.

Acknowledgements

This work was partially support by the National Al-liance for Research on Schizophrenia and Depression and

Ž .by NIH Grant P 50 AT00151-0 M-L.W. . We thank Ms.Anne Cabrinha for her assistance in preparing themanuscript.

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