Brain, Behavior, and Immunity 20 (2006) 233–245

www.elsevier.com/locate/ybrbi

Metabotropic glutamate receptors mediate lipopolysaccharide-induced fever and sickness behavior

Tracey J. Weiland ¤, Debra Anthony-Harvey-Beavis, Nicholas J. Voudouris, Stephen Kent

School of Psychological Science, La Trobe University, Bundoora, Vic. 3086, Australia

Received 6 June 2005; received in revised form 20 August 2005; accepted 31 August 2005Available online 20 October 2005

Abstract

Several mechanisms have been proposed for neuroimmune communication supporting the sickness syndrome (fever, anorexia, inactivity,and cachexia) following infection. We examined the role of glutamate as a neurochemical intermediary of sickness behavior induced by intra-peritoneal lipopolysaccharide (LPS). Mice implanted with biotelemetry devices capable of detecting body temperature (Tb) were adminis-tered LPS (50 or 500�g/kg i.p., serotype 0111:B4) with or without i.p. pretreatment with vehicle or broad-spectrum antagonists selective forN-methyl-D-aspartate (NMDA), �-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic (AMPA)/kainite, or metabotropic glutamate (mGlu)receptors. While NMDA and AMPA/kainate receptor antagonism failed to attenuate LPS-induced sickness behavior, antagonism of metab-otropic receptors with L(+)-AP3 reduced the febrile (0–11 h: control: 37.32§0.16 °C, L(+)-AP3: 36.66§0.27), anorexic (control: ¡87§5%,L(+)-AP3: 48§12% scotophase food intake), and cachexic (control: ¡8.9§0.4%, L(+)-AP3: ¡6.1§1.3% body weight) eVects of 500�g/kgLPS, and produced a biphasic Tb eVect in response to 50�g/kg LPS (1 h: ¡0.90§0.26; 6 h: 1.78§0.35 °C relative to baseline). At this dose theTb of L(+)-AP3-treated mice was 1.18 °C lower than controls 2 h post-injection, and 0.68 °C greater that controls 8 h post-injection. Theseresults suggest a role for mGlu receptors in mediating fever, anorexia, and cachexia possibly via activation of extra-vagal pathways, since theattenuating eVect of L(+)-AP3 increased with increasing dosages of LPS. Given the critical role ascribed to mGlu receptors in neurotransmit-ter release and astrocytic processes, it is possible that these observations reXect an L(+)-AP3-induced attenuation of these systems.© 2005 Elsevier Inc. All rights resreved.

Keywords: Fever; Anorexia; Cachexia; LPS; Infection; NMDA; AMPA; Kainate; L(+)-AP3; Glutamate

1. Introduction bi-directional communication pathways between the

During infection and inXammation, the central nervoussystem synchronizes the acute phase reaction (APR), aspectrum of systemic, physiological responses and behav-ioral changes that are crucial for homeostasis and survival(Watkins et al., 1995). The behavioral changes have beencollectively termed “sickness behavior,” and includeanorexia and adipsia (decreased appetite for food andwater), and depressed activity, and are often accompaniedby autonomic responses including fever (Kent et al., 1992).EVective coordination of the APR demands the existence of

* Corresponding author. Present address: Centre for Development ofEmergency Practice, Department of Emergency Medicine St. Vincent’sHealth, 41 Victoria Pde, Fitzroy, Vic. 3065 Australia. Fax: +61 39479 1956.

E-mail address: Tracey.Weiland@svhm.org.au (T.J. Weiland).

0889-1591/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2005.08.007

immune system and the brain. While multiple pathwaysexist, the vagus nerve has been ascribed a critical role inmediating behavioural depression and fever followingintraperitoneally (i.p.) delivered immune activation. Forexample, subdiaphragmatic vagotomy blocks fevers of lowmagnitude (Sehic and Blatteis, 1996), suppression of food-motivated behavior, learned taste aversions, hyperalgesia,and depressed social interaction produced by i.p. proin-Xammatory cytokines, interleukin-1 (IL-1), tumor necrosisfactor-� (TNF-�), and the active fragment of Gram-nega-tive bacteria, lipopolysaccharide (LPS) (Dantzer, 2001;Watkins et al., 1995), which stimulates the production andrelease of pro-inXammatory cytokines (Decker, 1990).

It has become clear that peripherally derived proinXam-matory cytokines such as IL-1, IL-6, and TNF-� communi-cate with the brain (directly or indirectly) to mediate

234 T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245

components of the APR. Several studies have demonstratedthat cytokines are capable of regulating neurotransmitterrelease (Blalock, 2005).

More recently, a constellation of evidence has emergedto suggest a possible role for the neurotransmitter gluta-mate (L-glutamate, glutamic acid) in inXammatory pro-cesses, including the expression of the sickness syndrome.Glutamate acts through several receptor types includingboth directly gated ion channels (ionotropic) and G-proteinmediated second messenger systems (metabotropic recep-tors), and are named after the glutamate analogues thatpreferentially bind them. Ionotropic glutamate receptorsinclude the N-methyl-D-aspartic acid (NMDA), �-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic (AMPA), andkainate receptors. Metabotropic glutamate receptors(mGlu) can be classiWed into three groups that diVer interms of amino acid sequence, agonist pharmacology, andsignal transduction pathways. Group I includes mGlu1 andmGlu5; Group II includes mGlu2 and mGlu3; and GroupIII includes mGlu4 and mGlu6-8 (Nakanishi and Masu,1994). In addition, it is prudent to note that novel groups ofmGlu receptors and their splice variants are still being char-acterized (Kanumilli et al., 2004; Nakanishi and Masu,1994).

Previous evidence suggests a critical role for the vagusnerve in mediating behavioural depression (Dantzer, 2001;Watkins et al., 1995) and febrile responses to low doses ofLPS (Romanovsky et al., 1997). It is possible that glutamateparticipates in the physiology and behavior observed dur-ing the APR. NMDA receptors are present on plasmamembranes of vagal aVerent terminals and their dendritictargets in the rat nucleus tractus solitarius (NTS) (Aicheret al., 1999), and receptor-binding studies have demon-strated an overlapping distribution of NMDA, CCK type2, and dopamine receptors in the NTS of the rat (Qianet al., 1997). Furthermore, at least two studies have foundthat most vagal aVerents are glutamatergic (Sykes et al.,1997; Torrealba and Muller, 1999), thus placing this neuro-transmitter in a good position for vagally mediatedimmune processes.

The vagal glutamatergic system in the NTS has beendemonstrated to be activated by immunoreactive com-pounds. As detected using microdialysis, vagal aVerentrelease of glutamate in the NTS was elevated in response toi.p. LPS at 20 min and again 60 min following injection,whereas IL-1� (4 �g/rat) increased glutamate at 20 and40 min post-injection (Mascarucci et al., 1998). A role forglutamate in systemic inXammation is supported by theobservation that intravenous LPS induces glutamaterelease and c-fos expression in the NTS (Lin et al., 1999).One must remain cognizant of the fact that extracellularglutamate is derived both from nerve terminals and generalmetabolism since glutamate modulates glycolysis in astro-cytes (Pellerin and Magistretti, 1994). Thus, while theseresults support a role for glutamate in processes underlyinginfection the functional signiWcance of extracellular gluta-mate in these studies is unclear. Nonetheless, since subdia-

phragmatic vagotomy attenuates i.p. LPS-induced Fosexpression in the brainstem (Ge et al., 2001; Konsman et al.,2000), a role for glutamate release by vagal terminals dur-ing immune-brain signalling is possible. NMDA-receptorantagonism inhibits LPS-induced c-fos in the paraventricu-lar nucleus of the hypothalamus (PVH), supraoptic nuclei,and A1/A2 regions of the brainstem (Wan et al., 1994). Inaddition, the IL-1�-induced increase in norepinephrine ofthe rat medial pre-frontal cortex was blocked by a competi-tive AMPA receptor antagonist (Kamikawa et al., 1998).

The importance of NMDA and mGlu receptors in medi-ating inXammatory hyperalgesia is already well established(Carlton and Coggeshall, 1999; Lawand et al., 1997). Fur-ther, glutamate administration has been documented toinduce hyperthermia (Singh and Gupta, 1997) and potenti-ate LPS-induced fever (Koulchitsky et al., 1999). Antago-nism of AMPA receptors inhibits locomotor activity (Majet al., 1995), whereas NMDA receptor antagonismincreases feeding (Jahng and Houpt, 2001) and sucroseintake (Bednar et al., 1994). Despite this encouraging col-lection of evidence, the role of this important neurotrans-mitter and its receptors in mediating the expression of thesickness syndrome has not previously been examined.

In light of the anatomical, physiological, and behavioralevidence implicating glutamate in processes of inXamma-tion and behaviors aVected by inXammation and infection,the purpose of the present study was to determine whetherNMDA, AMPA/kainate, or mGlu receptors mediate i.p.LPS-induced fever, anorexia, adipsia, decreased locomotoractivity, and cachexia. Since vagotomy has been demon-strated to block fever (Romanovsky et al., 1997) andanorexia (Sergeev and Akmaev, 2000) in a dose-dependentmanner, we examined the eVects of glutamate receptorantagonism in response to two doses of LPS: 50 and500 �g/kg.

2. Method

2.1. Animals

The studies used 72 3–4-month-old, male C57BL/6J mice(mus musculus) obtained from the Central Animal House,La Trobe University. Every eVort was made to age-matchthe animals. At the commencement of the recording period,mice were aged between 91 and 111 days (meanD102§ 2,medianD104).

2.2. Drugs

An immune response was triggered through the i.p.administration of LPS (serotype: 0111:B4; Sigma, Sydney)in doses of 50 �g/kg and 500 �g/kg. Lyophilized LPS wasdissolved in sterile, pyrogen free, isotonic saline solution(Astra Pharmaceuticals, North Ryde, Australia), aliquot-ted, and stored at ¡20 °C in Eppendorf tubes until required.On the morning of experimentation, aliquots were thawedat 4 °C and an appropriate volume of saline was added to

T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245 235

bring the diluent to the correct concentration for injection.Solutions were then raised to room temperature prior toinjection. All LPS injections were administered 1 h follow-ing the onset of the photophase.

Three glutamate receptor antagonists were used in thisstudy: 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxa-line-7-sulphonamide (NBQX), a potent, selective AMPA/kainate receptor antagonist; (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine((+)-MK-801 maleate, hereafter MK-801), a potent, selective NMDAreceptor antagonist which freely crosses the blood–brainbarrier (Vezzani et al., 1989); and L(+)-2-Amino-3-phos-phonoproprionic acid (L(+)-AP3), a broad-spectrum mGlureceptor antagonist. MK-801 was obtained from TocrisCookson (Bristol, UK), and NBXQ and L(+)-AP3 werepurchased from Sigma–Aldrich (St. Louis). The vehicle foreach of these substances was saline.

For each glutamate receptor antagonist, the dose admin-istered was selected based on previous behavioral studies.NBXQ, was administered in a dose of 20 mg/kg, which iswithin the range of doses found to attenuate drug-inducedchanges in locomotion (10–80 mg/kg, i.p.; Vanover, 1998).Although we are unaware of the extent to which this com-pound diVuses across the blood–brain barrier, it has beendemonstrated to aVect centrally mediated events (Vanover,1998) when administered peripherally. MK-801 was admin-istered at a dose of 0.25 mg/kg. This is slightly greater thandoses found to increase feeding (0.1 mg/kg, i.p.; Jahng andHoupt, 2001) and locomotor activity (0.1 mg/kg, i.p.; Hatip-Al-Khatib et al., 2001), but less than that reported to inducestereotypy and impair sensorimotor ability (Irifune et al.,1995), and induce increases in dopamine, norepinephrine,and serotonin (Yan et al., 1997). Available data indicatethat L(+)-AP3 has previously been administered centrallybut not via the i.p. route in behavioral experiments. There-fore, we referred to the less active enantiomer, 2-amino-3-phosphonopropionic acid, for dose information. The doseselected for L(+)-AP3 was 50 mg/kg, which is within therange of i.p. doses of 2-amino-3-phosphonopropionic acidfound to attenuate hyperlocomotion and neurodegenera-tion following ischaemia in the gerbil (Maginn et al., 1995).

Powdered MK-801, NBQX, and L(+)-AP3 were storedat room temperature, according to the manufacturers’instructions. A stock solution of MK-801 was dissolved insaline, aliquotted, and stored at ¡20 °C for up to onemonth after which time it was discarded. On the morning ofinjection, an aliquot of the stock solution was raised toroom temperature and the appropriate volume of salinewas added. Both NBXQ and L(+)-AP3 were dissolved inisotonic saline on the morning of injection.

2.3. Procedure

Throughout experimentation mice were housed individ-ually under a 12:12 light/dark cycle with ambient tempera-ture set at 30§1 °C, which is within the thermoneutral zonefor this species (Oufara et al., 1987). Sawdust and tissue

were provided as bedding. Following a 2-week acclimationperiod, a biotelemetry device (22 mm£ 8 mm; 1.83 g; E-4000, Mini Mitter, Bend, OR) was surgically implanted intothe peritoneal cavity of each animal as described previously(Weiland et al., 2004).

A 7–10 day recovery period followed surgery, afterwhich time mice were housed singly in polypropyleneenclosures (28 cm£ 40 cm£ 17 cm), each of which wasplaced on a receiver. Each biotelemetry device generated acontinuous frequency signal proportional to the animal’sTb (§10¡1 °C). The receiver sampled this frequency at 1-min intervals and this sample was decoded by VitalViewsoftware (Mini Mitter, Bend, OR) and stored electronically.

General locomotor activity was also determined using thissystem. The receiver for each cage was equipped with amatrix of antennas that were continuously signaled by thebiotelemetry device. The receiver scanned the matrix in asequential order to locate the position and orientation of thebiotelemetry device, thus making it possible to detect theglobal activity of the mouse. At 1-min intervals the receivertallied the number of matrices crossed by the mouse and thisinformation was recorded using VitalView software.

Mice were fed pelleted mouse chow (45 mg, A1 precisionfood pellets; P.J. Noyes, Lancaster, NH) released by a verti-cal dispenser located within each cage. Each time the mouseentered the food hopper a photoelectric beam was broken.This generated a signal that was tallied each minute by theVitalView software. One pellet was dispensed on everyeighth entry. Uneaten pellets were removed from the enclo-sure each morning. The number of uneaten pellets was sub-tracted from the total number delivered each day for theWnal data set.

Body weight and water consumption were manuallyrecorded each morning. Body weight was measured to10¡2 g using top-loading scales. Water was dispensed fol-lowing contact with a water nipple fed from a 25 ml verti-cal, polypropylene tube with 0.2 ml graduated markings.

All dependent variables (Tb, activity, body weight, foodhopper entries, and water intake) were monitored during anacclimation period of at least 1 week. Mice were consideredto be adapted to the housing environment when: (a) clearand consistent circadian rhythms in Tb, activity and foodhopper entries were present for at least 3 consecutive days;(b) daily water intake and body weight varied by less than 1ml and 1.5 g, respectively, during this period; and (c) bodyweight was greater than pre-surgical weight. Following suc-cessful acclimation, a 4-day baseline period commencedduring which time all dependent measures were monitored.

Following the 4-day baseline period, each animal wasadministered one of four pretreatments: (1) the NMDAreceptor antagonist, MK-801 (0.25 mg/kg) (2) the AMPA/kainate receptor antagonist, NBQX (20 mg/kg); (3) thebroad-spectrum mGlu receptor antagonist, L(+)-AP3(50 mg/kg), or (4) antagonist vehicle, saline. Each pretreat-ment was followed by one of two doses of LPS (50 or500 �g/kg), or the LPS vehicle, saline. Thus, there were 12groups in total.

236 T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245

All pharmacological manipulations were administeredin a maximum volume of 150�L using 30 gauge needles.Consistent with previous research (Jahng and Houpt, 2001;Maginn et al., 1995; Vanover, 1998), NBQX pretreatmentwas administered 15–20 min prior to treatment, MK-801was administered 15 min prior to treatment, and L(+)-AP3was administered 30 min prior to treatment.

All treatment injections were administered 1 h followingphotophase onset and dependent measures were monitoredfor a further 4 days post-injection.

2.4. Data analyses

Since it was not of interest to compare between doses orantagonists, data for each dose of LPS were analyzed sepa-rately with comparisons made between data for each antag-onist (alone and post-LPS) and that obtained for thepositive control.

Raw data were averaged and means§SEM were calcu-lated for each group. Data for Tb were converted to 2- and12-hourly means. Activity data and food hopper entrieswere converted to 12-hourly means and expressed as per-centage diVerence from baseline.

Group diVerences were determined using univariate ortwo-way (time by pretreatment) analyses of variance(ANOVA), and where appropriate for food and water data,analysis of covariance (ANCOVA) was used. IdentiWcationof potential covariates was made using partial correlationcontrolling for the eVect of pretreatment. Missing data werereplaced with the group mean when using food or waterintake as covariates. To overcome violations of sphericityin the Tb data, the Greenhouse–Geisser statistic is reportedfor within-subjects eVects. Where required, simple maineVects were analyzed using Fisher’s least signiWcant diVer-ence. This statistic is suitable when the number of compari-sons is small (Howell, 1992). When several comparisonswere required, as in the case of Tb data, a Bonferroniadjustment was performed to protect familywise error rate.

To control for pre-existing individual and group diVer-ences, results for LPS-induced diVerences in Tb were ana-lyzed on 2- and 12-hourly mean diVerence from baselinescores. LPS-induced group diVerences for all other depen-dent variables were tested on percentage diVerence fromaverage baseline scores and are presented as such. For Tb,activity, and food intake, data obtained for photophase andscotophase were analyzed separately.

Since MK-801 is known to be short acting (Hucker et al.,1983), all analyses were restricted to the Wrst 24 h irrespec-tive of treatment and dependent variable. Other analyseswere conducted as follows: For 2-hourly Tb expressed asdiVerence from baseline, analyses were conducted on theinitial 24 h post-injection period regardless of treatment.For 12-hourly Tb, the Wrst 24 h post-injection were ana-lyzed regardless of treatment. For percentage diVerencefrom baseline activity data, the number of post-injectiondays analyzed was 2 for mice administered 50 and 500 �g/kg LPS, and 1 for saline-treated mice. Regardless of LPS

dose, body weight analyses were conducted on data for theentire post-injection period since bodyweight typicallyfailed to return to baseline. For saline-treated mice, how-ever, body weight data were examined using data collectedduring the Wrst day post-injection. Food pellet consump-tion, expressed as percentage diVerence from baseline, wasanalyzed using separate two-way repeated measures ANO-VAs on the Wrst 24 h for saline treated mice, the Wrst 48 hpost-injection for mice administered 50 �g/kg LPS, and 72 hfor mice administered 500 �g/kg LPS. Water consumptionanalyses were conducted on data obtained during the Wrstday post-injection, irrespective of treatment.

3. Results

The eVects of L(+)-AP3 pretreatment on LPS-inducedsickness are summarized in Table 1. In mice treated with50 �g/kg LPS, pretreatment with L(+)-AP3 had a biphasiceVect. The febrile response was attenuated during the Wrst4 h and enhanced from 6 to 10 h post-injection (Fig. 1A).The clearest group diVerence occurred 2 h post-injectionwhen the Tb of saline-pretreated mice was 1.04§ 0.19 °Cgreater than baseline, and that of L(+)-AP3-pretreated micewas 0.32§0.48 °C less than baseline. ANOVA conductedon 2-hourly photophase data indicated a signiWcant maineVect for time (F(5,40)D14.54, p < .001, �2D .65) but notpretreatment. A signiWcant interaction between time andpretreatment was also found (F(5,40)D7.10, p < .001,�2D .47), and simple main eVects analyses revealed thediVerence between groups at 8–9 h to be signiWcant (F(1,8)D5.30, pD .050, �2D .40).

LPS (50 �g/kg) decreased the locomotor activity of allmice with the largest eVect being observed during the initialscotophase post-injection (Fig. 1B). Compared to positivecontrol mice, L(+)-AP3-pretreated mice were less activethroughout the post-injection period. SigniWcant maineVects for time and pretreatment were detected during theWrst 2 scotophase periods (F(2,18)D 25.65, p < .001, �2D .74;F(1,9)D24.62, p < .001, �2D .73).

The Tb pattern observed for L(+)-AP3-pretreated miceadministered 500 �g/kg LPS, was similar to those adminis-tered L(+)-AP3 prior to 50 �g/kg LPS; Tb decreased com-pared to baseline soon after injection before exceedingbaseline during the middle of the initial photophase(Fig. 1C). The largest decrease in Tb, 0.50§0.20 °C,occurred 1 h following LPS. Following this initial decline,the Tb of L(+)-AP3-pretreated mice was more than 1 °Cgreater than baseline from 4–7 h post-injection, with thepeak increase of 1.61§0.45 °C at 5 h post-LPS. In the sub-sequent scotophase, the Tb of both groups exhibited com-parable decreases relative to baseline. In the later part ofthe scotophase, Tb increased, peaking at 0.70§ 0.13 °Cgreater than baseline before returning to baseline at thestart of photophase 2. During the second photophase, theTb of L(+)-AP3-pretreated mice increased slowly untilpeaking at 1.10§0.14 °C greater than baseline before theonset of the scotophase. The slow increase in photophase 2

T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245 237

was in contrast to the relatively sustained increase observedfor saline-pretreated mice. No treatment eVects wereobserved in scotophase 2.

In the Wrst photophase period post-injection, saline-pre-treated mice produced a greater febrile response comparedto L(+)-AP3-pretreated animals (Fig. 1C). This eVect wasclearest 1–3, 8, and 10 h post-LPS. ANOVA conducted on2-hourly photophase data revealed signiWcant main eVectsfor time (F(5,60)D12.33, p < .001 �2D .51) and pretreatment(F(1,12)D4.86, pD .048 �2D .29), but not their interaction.During the initial scotophase, the Tb responses of saline-and L(+)-AP3-pretreated mice followed a similar patternand only a signiWcant eVect only for time (F(5,60)D28.69,p < .001, �2D .71) was found.

Inspection of 12-hourly means indicated that the Tb ofsaline-pretreated mice was clearly greater than that of L(+)-AP3-pretreated mice during the Wrst 2 photophase periodspost-LPS (0.66 and 0.41 °C, respectively; Fig. 1D). Univari-ate ANOVA indicated a signiWcant group diVerence duringthe Wrst photophase period (F(1, 12)D4.84, pD .048,�2D .29).

The body weight of L(+)-AP3-pretreated mice was lessaVected by 500 �g/kg LPS compared to positive controlsthroughout the post-injection period (Fig. 1E). Maximaldecreases were 8.9§0.4 % (controls) and 6.1§ 1.3% (L(+)-AP3). By the end of the testing period, L(+)-AP3-pretreated

mice, but not saline-pretreated mice, had returned to within2% of baseline body weight. ANOVA revealed a signiWcantmain eVect for pretreatment (F(1, 12)D7.70, pD .017,�2D .39) and time (F(1.53,18.33)D23.28, p < .001, �2D .66),but not their interaction.

Although both groups of LPS-treated (500 �g/kg) micereduced food consumption (Fig. 1F), mice administeredL(+)-AP3 were less aVected. In percentage terms, theanorexic eVects of LPS were greatest during the Wrst photo-phase following LPS. During this period, food consumedby saline and L(+)-AP3-pretreated mice decreased by91§ 4% (14.3§1.6 pellets) and 65§12% (9.2§ 1.3 pellets),respectively. Clear group diVerences were seen during pho-tophase 1 and scotophases 1 and 2 post-LPS. The largestdiVerence was during scotophase 2; post-injection saline-and L(+)-AP3-pretreated mice consumed 87§5% and48§ 12% less than baseline, respectively. For photophasedata, ANOVA indicated a signiWcant main eVect of time(F(1.24, 14.93)D53.74, p < .001, �2D .82) and pretreatment(F(1,12)D6.03, pD .030, �2D .34), but not their interaction.Results for scotophase indicated that food intake variedsigniWcantly as a function of time (F(2, 24)D45.77, p < .001,�2D .79) and pretreatment (F(1,12)D 5.35, pD .039,�2D .31), but not their interaction.

L(+)-AP3 pretreatment was without eVect in micetreated with saline with the exception of activity, which

Table 1The eVects of L(+)-AP3 pretreatment on LPS-induced sickness: summary of results

Treatment Parameter Time eVect Pretreatment eVect Interaction

50 �g/kg LPS Tb (photophase, 2 h) p < .001 NS p < .00150 �g/kg LPS Tb (scotophase, 2 h) p < .001 NS NS50 �g/kg LPS Tb (photophase, 12 h) p < .001 NS NS50 �g/kg LPS Tb (scotophase, 12 h) NS NS NS50 �g/kg LPS Activity (photophase, 12 h) NS NS NS50 �g/kg LPS Activity (scotophase, 12 h) p < .001 p < .001 NS50 �g/kg LPS Body weight p < .001 NS NS50 �g/kg LPS Food intake (photophase, 12 h) p < .001 NS NS50 �g/kg LPS Food intake (scotophase, 12 h) p < .001 NS NS50 �g/kg LPS Water consumption NS

500 �g/kg LPS Tb (photophase, 2 h) p < .001 p < .001 NS500 �g/kg LPS Tb (scotophase, 2 h) p < .001 NS NS500 �g/kg LPS Tb (photophase, 12 h) p < .05500 �g/kg LPS Tb (scotophase, 12 h) NS500 �g/kg LPS Activity (photophase, 12 h) NS NS NS500 �g/kg LPS Activity (scotophase, 12 h) p < .001 NS NS500 �g/kg LPS Body weight p < .001 p < .05 NS500 �g/kg LPS Food intake (photophase, 12hr) p < .001 p < .05 NS500 �g/kg LPS Food intake (scotophase, 12hr) p < .001 p < .05 NS500 �g/kg LPS Water consumption NS

Vehicle Tb (photophase, 2 h) p < .001 NS NSVehicle Tb (scotophase, 2 h) p < .001 NS NSVehicle Tb (photophase, 12 h) NSVehicle Tb (scotophase, 12 h) NSVehicle Activity (photophase, 12 h) p < .05Vehicle Activity (scotophase, 12 h) p < .05Vehicle Body weight p < .001 NS NSVehicle Food intake (photophase, 12 h) NSVehicle Food intake (scotophase, 12 h) NSVehicle Water consumption NS

238 T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245

decreased markedly during scotophases 1-3 (Fig. 1B). ThiseVect was greatest during the initial scotophase when L(+)-AP3-pretreated mice were 32§3% less active compared tobaseline.

During the photophase, the greatest group diVerenceoccurred during the initial photophase when the activity of

saline-pretreated mice was 20§8% less than baseline, andthat of mice administered L(+)-AP3 was 10§7% more thanbaseline. ANOVA indicated a signiWcant eVect due to pre-treatment during the initial photophase (F(1,12)D 7.10,pD .021, �2D .37) and scotophase (F(1,12)D7.49, pD .018,�2D .38).

Fig. 1. (A) Mean (§SEM) hourly Tb (°C) of mice challenged with 50 �g/kg LPS following pretreatment with saline or L(+)-AP3 (n D 5, both groups).Black bars denote scotophase. ¤ denotes groups diVerence, p < .05. (B) Twelve-hourly mean (§SEM) percentage change in locomotor activity of micetreated with saline (n D 7, both groups) or 50 �g/kg LPS following pretreatment with saline (n D 7) or L(+)-AP3 (nD 4). ¤¤¤ denotes signiWcant diVerencebetween LPS-treated groups, p < .001. � denotes signiWcant diVerence between saline-treated groups, p < .05. (C) Mean (§SEM) hourly Tb (°C) of micechallenged with 500 �g/kg LPS following pretreatment with saline or L(+)-AP3 (nD 7, both groups). Black bars denote scotophase. - - - - denotes groupdiVerence, p < .05. (D) Mean (§SEM) 12-hourly Tb (°C) of mice challenged with 500 �g/kg (nD 7, both groups) LPS following pretreatment with saline orL(+)-AP3. (E) Mean (§SEM) percentage change in body weight mice challenged with 500 �g/kg LPS following pretreatment with saline or L(+)-AP3(n D 7, both groups). - - - - - denotes group diVerence, p < .05. (F) 12-hourly mean (§SEM) percentage change in food intake by mice pretreated with saline(n D 5) or L(+)-AP3 (n D 7) and treated with 500 �g/kg LPS. - - - - - denotes group diVerence, p < .05.

-25 -19 -13 -7 0 6 12 180.00

35.00

36.00

37.00

38.00

39.00

Time Relative to Injection (hrs)

Vehicle + 50 ug/kg LPS

L(+)-AP3 + 50 ug/kg LPS *

Bod

y Te

mpe

ratu

re (˚C

)B

ody

Tem

pera

ture

(˚C

)

Bod

y Te

mpe

ratu

re (˚C

)

-25- -14 0-11 24-35 48-59 72-83-100

-80

-60

-40

-20

0

20

40

Time relative to injection (hrs)

Vehicle + Saline

L(+)-AP3 + Saline

Vehicle + 50 ug/kg LPS

L(+)-AP3 +50 ug/kg LPS

*** ***

Diff

eren

ce fr

om b

asel

ine

(%)

Diff

eren

ce fr

om B

asel

ine

(%)

Diff

eren

ce fr

om B

asel

ine

(%)

-25 -19 -13 -7 0 6 12 18 24 30 36 420.00

35.00

36.00

37.00

38.00

39.00

Time Relative to Injection (hrs)

Vehicle + 500 ug/kg LPS

L(+)-AP3 + 500 ug/kg LPS-----------

-25 -13 0 11 23 35 47 59 71 830

35.5

36

36.5

37

37.5

Time Relative to Injection (hrs)

Vehicle + 500 ug/kg LPS

L(+)-AP3 + 500 ug/kg LPS

*

-25- -14 0-11 24-35 48-59 72-83-100

-50

0

50

100

Time Relative to Injection (hrs)

Vehicle + 500 ug/kg LPS

L(+)-AP3 + 500 ug/kg LPS

---------------------------

A B

C

E

D

F

-1 1 2 3 4-14

-12

-10

-8

-6

-4

-2

0

2

4

Time Relative to Injection (days)

Vehicle + 500 ug/kg LPS

L(+)-AP3 + 500 ug/kg LPS

----------------------------------------

α α

T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245 239

The eVects of MK-801 pretreatment on LPS-inducedsickness are summarized in Table 2. A considerable overlapin the pattern of 2-hourly Tb responses occurred during theWrst day post-LPS (50 �g/kg). Despite this, a clear groupdiVerence was observed at 22–23 h post-LPS when the Tbof vehicle- and MK-801-pretreated mice was 37.80§ 0.24and 36.62§0.24 °C (Fig. 2A). ANOVA performed on 2-hourly scotophase data indicated a signiWcant main eVectfor time (F(5, 50)D6.86, p < .001, �2D .41), but not pretreat-ment. A signiWcant time by pretreatment interaction was,however, present (F(5, 50)D2.75, pD .028, �2D .22) with thetwo groups diVering signiWcantly 22–23 h post-injection(F(1,10)D23.75, pD .001, �2D .70).

Compared to positive control mice, MK-801-pre-treated mice were less active relative to baseline. Forscotophase data, an eVect of pretreatment was found(F(1,12)D 5.12, pD .043, �2D .30). MK-801 potentiatedthe decrease in activity induced by LPS (50 �g/kg)during the initial post-injection scotophase whenactivity decreased from baseline by 34§7% for saline-treated mice and 56§ 7% for MK-801-pretreated miceFig. 2B).

Regardless of parameter, there were no signiWcant eVectsdue to MK-801-pretreatment for mice administered 500 �g/kg LPS. For saline-treated mice, however, MK-801 had aslight hyperthermic eVect 0–1 h post-injection with a diVer-

ence of 0.89 °C observed between MK-801-treated mice andsaline controls (Fig. 2C). ANOVA revealed a signiWcantmain eVect for time (F(2.78, 33.35)D 5.31, p < .001, �2D .31)and pretreatment (F(1,12)D5.00, pD .045, �2D .29), but nottheir interaction. During the scotophase, group diVerenceswere apparent 21–23 h post-injection. ANOVA revealed asigniWcant main eVect for time (F(5,60)D 4.58, p < .001,�2D .28) but not pretreatment. The time by pretreatmentinteraction was signiWcant (F(5,60)D 2.42, pD .046,�2D .17), however, simple main eVects revealed no signiW-cant diVerences between pretreatment groups within each2-h time period.

Inspection of 12-hourly means indicated a clear groupdiVerence during the initial post-injection photophase, withthe mean Tb of MK-801-pretreated mice exceeding that ofsaline-pretreated mice by 0.45 °C. ANOVA indicated thisdiVerence to be signiWcant (F(1,12)D 5.58, pD .036,�2D .32), but there were no diVerences during the scoto-phase.

The activity of MK-801-pretreated mice increased mark-edly (53§ 22 %) during the initial photophase, whereas, theactivity of saline-pretreated mice decreased by 20§8%(Fig. 2D). ANOVA indicated the eVect due to pretreatmentto be signiWcant (F(1,12)D9.16, pD .011, �2D .43).

The eVects of NBQX pretreatment on LPS-induced sick-ness are summarized in Table 3. In response to 50�g/kg LPS,

Table 2The eVects of MK-801 pretreatment on LPS-induced sickness: summary of results

Treatment Parameter Time eVect Pretreatment eVect Interaction

50 �g/kg LPS Tb (photophase, 2 h) p < .001 NS NS50 �g/kg LPS Tb (scotophase, 2 h) p < .001 NS p < .00150 �g/kg LPS Tb (photophase, 12 h) NS NS NS50 �g/kg LPS Tb (scotophase, 12 h) NS NS NS50 �g/kg LPS Activity (photophase, 12 h) NS50 �g/kg LPS Activity (scotophase, 12 h) p < .0550 �g/kg LPS Body weight NS50 �g/kg LPS Food intake (photophase, 12 h) NS50 �g/kg LPS Food intake (scotophase, 12 h) NS50 �g/kg LPS Water consumption NS

500 �g/kg LPS Tb (photophase, 2 h) p < .001 NS NS500 �g/kg LPS Tb (scotophase, 2 h) p < .001 NS NS500 �g/kg LPS Tb (photophase, 12 h) NS500 �g/kg LPS Tb (scotophase, 12 h) NS500 �g/kg LPS Activity (photophase, 12 h) NS500 �g/kg LPS Activity (scotophase, 12 h) NS500 �g/kg LPS Body weight NS500 �g/kg LPS Food intake (photophase, 12 h) NS500 �g/kg LPS Food intake (scotophase, 12 h) NS500 �g/kg LPS Water consumption NS

Vehicle Tb (photophase, 2 h) p < .001 p < .05 NSVehicle Tb (scotophase, 2 h) p < .001 NS p < .05Vehicle Tb (photophase, 12 h) p < .05Vehicle Tb (scotophase, 12 h) NSVehicle Activity (photophase, 12 h) p < .05Vehicle Activity (scotophase, 12 h) NSVehicle Body weight NSVehicle Food intake (photophase, 12 h) NSVehicle Food intake (scotophase, 12 h) NSVehicle Water consumption NS

240 T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245

the locomotor activity of both groups decreased during thepost-injection scotophases (Fig. 3A). This decrease wasgreater in NBQX-pretreated mice throughout the entire test-ing period. For photophase data, ANOVA revealed a signiW-cant main eVect for pretreatment (F(1,12)D8.15, pD .014,�2D .41), but not time or their interaction. For scotophasedata a signiWcant main eVect was found for time (F(1,12)D25.35, p < .001, �2D .68), pretreatment (F(1,12)D12.99,pD .004, �2D .52), and time by pretreatment interaction(F(1,12)D5.01, pD .045, �2D .39). Simple main eVects analy-ses revealed the activity of NBQX-pretreated mice to be sig-niWcantly less than saline-pretreated mice during the initialscotophase (F(1,12)D14.54, pD .002, �2D .55).

The cachexic eVects of 50 �g/kg LPS were greatest onday 1 post-injection. Mice pretreated with NBQX lost5.2§0.7% of body weight, and returned to baseline by day4 post-injection. Similar changes were observed in saline-pretreated mice. ANOVA revealed a main eVect for time(F(3,36)D 58.50, p < .001, �2D .83), but not pretreatment.The time by pretreatment interaction was, however, signiW-cant (F(3,36)D 4.60, pD .008, �2D .28), but simple main

eVects indicated there were no signiWcant group diVerencesfor any of the post-injection days.

In response to 50�g/kg LPS, both groups of mice exhibiteda decrease in food intake. No diVerences in food intake wereobserved during the initial day post-injection. However, dur-ing the second photophase, consumption by NBQX-pre-treated mice returned to pre-injection levels, whereas saline-treated animals consumed 28§9% less than at baseline. ThisdiVerence, while signiWcant, is only equivalent to 5 pellets or9% of total daily food intake. ANOVA conducted on photo-phase data indicated a signiWcant main eVect for time(F(1,12)D29.48, p<.001, �2D .71), pretreatment (F(1,12)D4.75,pD .050, �2D .28), but not for their interaction.

With the exception of water consumption, NBQX waswithout eVect on Tb, activity, body weight, and food intakein mice treated with 500 �g/kg LPS or saline. Followingsaline-injection, water consumption by mice pretreatedwith saline remained within 5% of baseline. In contrast,water consumption by mice administered NBQX prior tosaline decreased by 18§ 3% on day 1 following injection,and tended towards baseline thereafter (Fig. 3B). ANOVA

Fig. 2. (A) Mean (§SEM) hourly Tb (°C) of mice administered 50 �g/kg LPS following pretreatment with saline (n D 5) or MK-801 (n D 7). Black bardenotes scotophase. (B) 12-hourly mean (§SEM) percentage change in locomotor activity of mice challenged with 50 �g/kg LPS following pretreatmentwith saline or MK-801, (nD 7, both groups). ¤ denotes group diVerence, p < .05. (C) Mean (§SEM) hourly Tb (°C) of mice administered saline followingpretreatment with saline or MK-801 (n D 7, both groups). Black bars denote scotophase. - - - - denotes group diVerence, p < .05. (D) 12-hourly mean(§SEM) percentage change in locomotor activity of mice treated with saline following MK-801 or antagonist vehicle (n D 7, both groups). � denotesgroup diVerence, p < .05.

-25 -19 -13 -7 0 6 12 180.00

35.00

36.00

37.00

38.00

39.00

0.0035.00

36.00

37.00

38.00

39.00

Time Relative to Injection (hrs)

-25 -19 -13 -7 0 6 12 18Time Relative to Injection (hrs)

Vehicle + 50 ug/kg LPS

MK-801 + 50 ug/kg LPS

Bod

y Te

mpe

ratu

re (˚C

)B

ody

Tem

pera

ture

(˚C

)

-25- -14 0-11 24-35 48-59 72-83-100

-80

-60

-40

-20

0

20

40

60

Time relative to injection (hrs)

Vehicle + 50 ug/kg LPS

MK-801 + 50 ug/kg LPS

*

Diff

eren

ce fr

om b

asel

ine

(%)

Diff

eren

ce fr

om b

asel

ine

(%)

Vehicle + Saline

MK-801 + Saline---------------

-25- -14 0-11 24-35 48-59 72-83-100

-80

-60

-40

-20

0

20

40

60

80

Time relative to injection (hrs)

Vehicle + Saline

MK-801 + Saline

A B

C D α

τ

T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245 241

indicated a signiWcant main eVect due to pretreatment(F(1,9)D 11.23, pD .008, �2D .56).

4. Discussion

In examining the potential involvement of glutamatereceptor activation in LPS-induced behavioral depres-sion and fever, the present study revealed that (1)

NMDA receptor antagonism exacerbated scotophasehypoactivity elicited by 50 �g/kg LPS i.p., (2) AMPA/kainite receptor antagonism exacerbated the fever andhypoactivity induced by 50 �g/kg LPS i.p., and (3) antag-onism of mGlu receptors resulted in a biphasic eVect onTb following 50 �g/kg LPS i.p., and attenuated thefebrile, anorexic, and cachexic eVects of a 10-fold largerdose of LPS.

Table 3The eVects of NBQX-801 pretreatment on LPS-induced sickness: summary of results

Treatment Parameter Time eVect Pretreatment eVect Interaction

50 �g/kg LPS Tb (photophase, 2 h) p < .001 NS NS50 �g/kg LPS Tb (scotophase, 2 h) p < .001 NS NS50 �g/kg LPS Tb (photophase, 12 h) NS50 �g/kg LPS Tb (scotophase, 12 h) NS50 �g/kg LPS Activity (photophase, 12 h) NS p < .05 NS50 �g/kg LPS Activity (scotophase, 12 h) p < .001 p < .01 p < .0550 �g/kg LPS Body weight p < .001 NS p < .0150 �g/kg LPS Food intake (photophase, 12 h) p < .001 p D .05 NS50 �g/kg LPS Food intake (scotophase, 12 h) p < .001 NS NS50 �g/kg LPS Water consumption NS

500 �g/kg LPS Tb (photophase, 2 h) p < .05 NS NS500 �g/kg LPS Tb (scotophase, 2 h) p < .05 NS NS500 �g/kg LPS Tb (photophase, 12 h) NS500 �g/kg LPS Tb (scotophase, 12 h) NS500 �g/kg LPS Activity (photophase, 12 h) NS NS NS500 �g/kg LPS Activity (scotophase, 12 h) p < .001 NS NS500 �g/kg LPS Body weight p < .001 NS NS500 �g/kg LPS Food intake (photophase, 12 h) p < .001 NS NS500 �g/kg LPS Food intake (scotophase, 12 h) p < .001 NS NS500 �g/kg LPS Water consumption NS

Vehicle Tb (photophase, 2 h) p < .001 NS NSVehicle Tb (scotophase, 2 h) p < .001 NS NSVehicle Tb (photophase, 12 h) NSVehicle Tb (scotophase, 12 h) NSVehicle Activity (photophase, 12 h) NSVehicle Activity (scotophase, 12 h) NSVehicle Body weight p < .001 NS NSVehicle Food intake (photophase, 12 h) NSVehicle Food intake (scotophase, 12 h) NSVehicle Water consumption p < .01

Fig. 3. (A) 12-hourly mean (§SEM) percentage change in locomotor activity of mice challenged with 50 �g/kg LPS following pretreatment with saline orNBQX (n D 7, both groups). ¤ denotes group diVerence, p < .05. ¤¤ denotes group diVerence, p<.01. (B) Mean (§SEM) percentage change in averagewater consumption of mice administered saline following pretreatment with saline (n D 5) or NBQX (n D 6). ¤¤ denotes group diVerence, p < .01.

-25- -14 0-11 24-35 48-59 72-83-100

-80

-60

-40

-20

0

20

40

Time relative to injection (hrs)

Vehicle + 50 ug/kg LPS

NBQX + 50 ug/kg LPS

**

* *

-1 1 2 3 4-30

-20

-10

0

10

20

30

Time Relative to Injection (days)

Vehicle + Saline

NBQX + Saline

**

Diff

eren

ce fr

om b

asel

ine

(%)

Diff

eren

ce fr

om b

asel

ine

(%)

A B

242 T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245

Clearly, the most interesting Wndings arising from thisstudy are those obtained in relation to mGlu receptors.While previous research has indicated a role for mGlureceptors in mediating inXammatory hyperalgesia (seeSchöepp et al., 1999 for review) the present study demon-strates for the Wrst time an involvement of mGlu receptorsin mediating the febrile and behavioral changes associatedwith infection. The mGlu receptor family comprises threebroad subgroups (groups I–III) with each group havingmultiple subtypes and splice variants (Nakanishi andMasu, 1994). The mGlu antagonist employed in this studyis considered to be a broad-spectrum antagonist. However,like other antagonists of this nature (Schöepp et al., 1999),its activity at all the known receptor clones has not yet beenreported (Cartmell and Schöepp, 2000). Since mGlu recep-tor subtypes can have opposing functions (Bruno et al.,1997; Mukhin et al., 1996), determination of the precisereceptor subtype in mediating LPS-induced sickness, is ofobvious importance. In the interim, however, the possiblepathways and mechanisms by which mGlu receptors mayparticipate in LPS-mediated responses deserve speculation.

The extent to which L(+)-AP3 penetrates the central ner-vous system is unknown. Previous studies have, however,demonstrated that AP-3, the less active enantiomer ofL(+)-AP3, exerts centrally mediated eVects when adminis-tered peripherally (Maginn et al., 1995). Since informationregarding the BBB penetrability of L(+)-AP3 is lacking, it ispossible that the eVects observed were mediated centrally,peripherally, or both.

Consistent with a role for glutamate in vagally mediatedresponses to infection and inXammation, is the Wnding thatboth LPS and IL-1� increase glutamate concentration inthe NTS in a timeframe consistent with that required for arapid inXammatory response (Mascarucci et al., 1998). TheNTS is a key site of vagal aVerent termination and mostvagal aVerents are known to be glutamatergic (Sykes et al.,1997; Torrealba and Muller, 1999), although it is not yetknown whether they possess mGlu receptors. Pharmaco-logical studies have, however, indicated the presence of allthree subgroups of mGlu receptors in the rat NTS (Joneset al., 1998, 1999; Matsumura et al., 1999). Although othersubgroups remain to be studied, mGlu receptors of thegroup II subtype are present within the gastrointestinal sys-tem (Larzabal et al., 1999). Thus, although further anatomi-cal evidence is wanting, it is feasible, that glutamate maymediate some of the eVects of LPS and IL-1� by actingeither in the periphery at the level of the vagus nerve, orcentrally at the NTS.

Interestingly, however, L(+)-AP3 pretreatment attenu-ated the anorexia and cachexia induced by the high, but notthe low, dose of LPS, and was more eVective in diminishingthe febrile eVect of the high dose of LPS. The vagus nerve isknown to be a critical pathway when fever is induced bylow doses of immune activators (Hansen and Krueger,1997; Romanovsky et al., 1997). Subdiaphragmatic VGXwas recently demonstrated to block anorexia induced bylow but not high doses of LPS (Sergeer and Akmaev, 2000).

The eVectiveness of L(+)-AP3 in attenuating the febrileeVects of LPS increased with increasing dose, and foranorexia, was restricted to the highest dose of LPS. Thus,one may speculate that mGlu receptors participate in theproduction and/or maintenance of fever when extra-vagalpathways are recruited, as may occur in more pronouncedimmune responses. Whether this is in fact the case remainsto be determined.

Although the distribution of mGlu receptors within theCNS is, to a large extent, still being characterized, they areknown to be located within thalamic and hypothalamicnuclei, in addition to the NTS (Chen et al., 2002; Matsum-ura et al., 1999; Van den Pol et al., 1995) which is critical inthe integration of messages from the periphery. Thus, cen-tral mGlu receptors are suitably located for an involvementin the behavioral eVects of LPS.

The L(+)-AP3-induced attenuation of multiple indices ofillness (fever, cachexia, anorexia) is suggestive of a role formGlu receptors in mediating the initiation of sickness. Inaddition to their localization on neurons, mGlu receptorsare found on glial cells including microglia and astrocytes(Biber et al., 1999), which are known to synthesize inXam-matory mediators including IL-1, IL-6 (Xie et al., 2003),and prostaglandins (Pistritto et al., 2000). Interestingly,astrocytic mGlu receptor activation results in an increase inintracellular calcium, which results in the release of prosta-glandin E2 in vitro, which, in turn, is thought to give rise tocalcium oscillations or “calcium waves” (Zonta et al., 2003),a putative means of astrocyte-astrocyte or astroctye-neuronsignalling (Winder and Conn, 1996). Thus, mGlu antago-nism may retard this signalling and thereby reduce the neu-ral changes initiated by peripheral immune activation.Alternatively, mGlu receptor activation may directly aVectproinXammatory cytokine release. Activation of mGluR3enhanced the release of IL-6 in cultured human astrocytes,an eVect that was prevented by selective group II mGluRantagonism (Aronica et al., 2005). Within the CNS, glialcells vastly outnumber neurons and astrocytes are consid-ered fundamental for both the synthesis of glutamatethrough the production of glutamine, and the maintenanceof low levels of extracellular glutamate through their abilityto accumulate extracellular glutamate released by neurons(Ye and Sontheimer, 1998). Thus, although the precise roleof astrocytic mGlu receptors is still being determined, andis likely to vary with diVering receptor subtypes, their acti-vation may signiWcantly aVect the activity of neurons, andthereby inXuence the initiation and maintenance of sicknessbehavior and the febrile response.

The ubiquitous distribution of glutamate throughout theCNS, together with the abundance of glutamatergic path-ways, renders the interaction between this neurotransmitterand others almost unavoidable. In addition to mediatingglutamatergic functions, the synaptic release of glutamatecan regulate non-glutamate transmitter-containing neuronsby way of excitatory glutamatergic inputs to these cells. IneVect then, glutamate can act on post-synaptic non-gluta-matergic neurons. Due to the relatively recent provision of

T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245 243

compounds selective for mGlu receptors, the regulation ofneurotransmitter release by activation of mGlu receptors isjust beginning to be clariWed (see Cartmell and Schöepp,2000 for detailed review). While little is known about theinteractions between glutamate and other neurochemicalsspeciWcally linked to inXammatory states, mGlu receptorshave been reported to regulate the release of GABA fromthe thalamus (Salt and Eaton, 1995, 1998), dopamine in thestriatum (Verma and Moghaddam, 1998) and nucleusaccumbens (Taber and Fibiger, 1995), hippocampal CCK(Breukel et al., 1998), and substance P from trigeminalnucleus slices (Cuesta et al., 1999). It is possible then thatglutamate may participate in the regulation of inXamma-tory states via mGlu-mediated interaction with other neu-rochemical systems.

Thermoregulatory eVectors were not examined as partof the present study, consequently it is not possible toexclude the Tb results obtained were due to changes in sym-pathetic nervous activity. This is relevant since intrahypo-thalamic glutamate modulates the activity of the nervesthat innervate the interscapular brown adipose tissue(Yoshimatsu et al., 1993). Nonetheless, it is encouraging tonote that mGlu antagonism is without eVect on AVP-induced hypothermia, and associated changes in brownadipose tissue, blood pressure, and heart rate (Paro et al.,2003). Moreover, in the present study no diVerences wereobserved between control mice pretreated with L(+)-AP3and those pretreated with vehicle.

The failure of NMDA and AMPA/kainate receptorantagonism to alleviate the behavioral and metaboliceVects of LPS is somewhat unexpected. NMDA receptorsare present on cell bodies of vagal aVerents (Lewis et al.,1987), and NMDA and AMPA/kainite receptors are pres-ent in the NTS (Aicher et al., 1999; Van den Pol et al., 1994)and the hypothalamus (Zhang and MiZin, 1998). Animportant role for NMDA receptors is suggested by thefact that MK-801 blocks i.p. LPS-induced c-fos expressionin the PVH, supraoptic nuclei, and A1/A2 regions of thebrainstem (Wan et al., 1994). Furthermore, both MK-801and NBQX blocked the TNF-�-induced c-fos expression inthe NTS 90 min post-injection (Emch et al., 2001). How-ever, since the physiological and/or behavioral relevance ofthis neuronal activation is unknown, it is possible thationotropic glutamate receptor activation may be necessaryfor inXammatory processes other than the components ofsickness behavior examined here.

Successful antagonism of the NMDA receptor was indi-cated by the pronounced increase in locomotor activity inmice administered saline following MK-801, a commonobservation with this antagonist (Bednar et al., 1994).Given the strength of this eVect, it is particularly surprisingthat the hypoactivity induced by LPS was not attenuated,but rather was exacerbated, by MK-801. Additionally, thisis the Wrst report of a brief, rapid increase in Tb of miceadministered saline following MK-801 pretreatment.Whether this is a product of the concomitant increase inlocomotor activity, however, remains unknown. MK-801 is

known to diVuse freely across the blood–brain barrier (Vez-zani et al., 1989). Although not directly assessed in the pres-ent study, the locomotor eVects observed here aresuggestive of a centrally mediated eVect.

Consistent with the exacerbation of LPS-induced hypo-activity seen with MK-801, antagonism of the AMPA/kai-nate receptors by NBQX potentiated the hypoactivity inmice treated with 50 �g/kg LPS. Antagonism of AMPA/kainite receptors, using a similar dose to that of the presentstudy, has previously been demonstrated to decrease spon-taneous locomotion (Mead and Stephens, 1998). This doesnot, however, account for the eVects observed here since thehourly locomotor activity of control animals was not sig-niWcantly aVected by NBQX. This discrepancy may be dueto the fact that Mead and Stephens (1998) examined loco-motion over several 20-min time periods using an appara-tus that permitted forward movements only, and was novelto the animals. This is particularly important to the inter-pretation of their results since AMPA/kainate antagonismis known to elicit anxiogenic behavior (Karcz-Kubicha andLiljequist, 1995). It should be noted that others have foundno eVect of NBQX on locomotion (Boldry et al., 1993),including those employing the same dose used here (Filliatet al., 1998). Unexpectedly, NBQX-pretreated mice weresigniWcantly adipsic following injection. Whether this eVectis speciWc to AMPA/kainate blockade or results from a sec-ondary eVect of NBQX is unknown. NBQX, has, however,been reported to decrease sucrose intake in food-deprivedand non-food-deprived rats (Zheng et al., 2002). In sum, thepresent study fails to provide conclusive evidence indicatingsuccessful antagonism of AMPA/kainate receptors byNBQX. There is, however, no reason to suggest that NBQXwas ineVective. Further, although information regardingthe penetrability of this compound across the blood–brainbarrier is lacking, we have no evidence to suggest that thisdid not occur in the present study, particularly since thedose employed is comparable to studies demonstrating cen-trally mediated eVects of NBQX following peripheraladministration of this compound (Vanover, 1998).

In summary, the present study indicates a role for mGlureceptors in mediating the Tb, anorexic, and cachexicresponses to 500�g/kg LPS. Antagonism of NMDA andAMPA/kainite receptors, however, did not diminish theresponses elicited by LPS, but in contrast exacerbated thelocomotor eVects of 50 �g/kg LPS.

The mechanisms and possible pathways involved inmediating the eVects of L(+)-AP3 on responses to LPS arepresently unknown but it is possible that they are non-vagal. Future investigations into this matter will no doubtbeneWt from further characterization of mGlu receptor sub-types, their distribution, and diVerences in their pre- andpost-synaptic functions. An examination of the eVects ofLPS following administration with selective antagonists forthe various subgroups and subtypes of mGlu receptors isstrongly warranted, as is further examination of the eVectsof mGlu receptor activation on other neurochemical sys-tems and on glial functioning during inXammatory states.

244 T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245

References

Aicher, S.A., Sharma, S., Pickel, V.M., 1999. N-methyl-D-aspartate recep-tors are present in vagal aVerents and their dendritic targets in thenucleus tractus solitarius. Neuroscience (Oxford) 91, 119–132.

Aronica, E., Gorter, J.A., Rozemuller, A.J., Yankaya, B., Troost, D., 2005.Activation of metabotropic glutamate receptor 3 enhances interleukin(IL)-1b-stimulated release of IL-6 in cultured human astrocytes. Neu-roscience (Oxford) 130, 927–933.

Bednar, I., Qian, M., Quershi, G.A., Kallstrom, L., Johnson, A.E., Carrer,H., Sodersten, P., 1994. Glutamate inhibits behavior. J. Neuroendocri-nol. 6, 403–408.

Biber, K., Laurie, D.J., Berthele, A., Sommer, B., Tolle, T.R., Gebicke-Har-ter, P.J., van Calker, D., Boddeke, H.W., 1999. Expression and signalingof group I metabotropic glutamate receptors in astrocytes and microg-lia. J. Neurochem. 72, 1671–1680.

Blalock, J.E., 2005. The immune system as the sixth sense. J. Intern. Med.257, 126–138.

Boldry, R., Kelland, M., Engbar, T., Chase, T., 1993. NBQX inhibitsAMPA-induced locomotion after injection into the nucleus accum-bens. Brain Res. 600, 331–334.

Breukel, A.I.M., Weigant, V.M., Lopes da Silva, F.H., Ghijsen, W.E.J.M.,1998. Presynaptic modulation of cholecystokinin release by proteinkinase C in the rat hippocampus. J. Neurochem. 70, 341–348.

Bruno, V., Sureda, F.X., Storto, M., Casabona, G., Caruso, A., Knopfel, T.,Kuhn, R., Nicoletti, F., 1997. The neuroprotective activity of group-IImetabotropic glutmate receptors requires new protein syntheses andinvolves a glial–neuronal signalling. J. Neurosci. 17, 1891–1897.

Carlton, S.M., Coggeshall, R.E., 1999. InXammation-induced changes inperipheral glutamate receptor populations. Brain Res. 820, 63–70.

Cartmell, J., Schöepp, D., 2000. Regulation of neurotransmitter release bymetabotropic glutamate receptors. J. Neurochem. 75, 889–907.

Chen, C.Y., Ling Eh, E.H., Horowitz, J.M., Bonham, A.C., 2002. Synaptictransmission in nucleus tractus solitarius is depressed by Group II andIII but not Group I presynaptic metabotropic glutamate receptors inrats. J. Physiol. 538, 773–786.

Cuesta, M.C., Arcaya, J.L., Cano, G., Sanchez, L., Maixner, W., Suarez-Roca, H., 1999. Opposite modulation of capsaicin-evoked substance Prelease by glutamate receptors. Neurochem. Int. 35, 471–478.

Dantzer, R., 2001. Cytokine-induced sickness behavior: mechanisms andimplications. Ann. NY Acad. Sci. 933, 222–234.

Decker, K., 1990. Biologically active products of stimulated liver macro-phages (kupVer cells). Eur. J. Biochem. 192, 245–261.

Emch, G., Hermann, G.E., Rogers, R.C., 2001. TNF-�-induced c-fos gener-ation in the nucleus of the solitary tract is blocked by NBQX and MK-801. Am. J. Physiol., R1394–R1400.

Filliat, P., Pernot-Marino, I., Baubichon, D., Lallement, G., 1998. Behav-ioral eVects of NBQX, a competitive antagonist of the AMPA recep-tors. Pharmacol. Biochem. Behav. 59, 1087–1092.

Ge, X., Yang, Z., Duan, L., Rao, Z., 2001. Evidence for involvement of theneural pathway containing the peripheral vagus nerve, medullary vis-ceral zone and central amygdaloid nucleus in neuroimmunomodula-tion. Brain Res. 914, 149–158.

Hansen, M.K., Krueger, J.M., 1997. Subdiaphragmatic vagotomy blocksthe sleep- and fever-promoting eVects of interleukin-1�. Am. J. Physiol.273, R1246–R1253.

Hatip-Al-Khatib, I., Mishima, K., Iwasaki, K., Fujiwara, M., 2001.Microdialysates of amines and metabolites from core nucleus accum-bens of freely moving rats are altered by dizocilpine. Brain Res. 902,108–118.

Howell, D.C., 1992. Statistical Methods for Psychology, third ed. Kent,Boston, PWS.

Hucker, H.B., Hutt, J.E., White, S.D., Arison, B.H., Zacchei, A.G., 1983.Disposition and metabolism of (+)-5-methyl-10,11-dihydro-5H-dib-enzo[a,d] cyclohepten-5,10-imine in rats, dogs, and monkeys. DrugMetab. Dispos. 11, 54–58.

Irifune, M., Shimizu, T., Nomoto, M., Fukuda, T., 1995. Involvement of N-methyl-D-aspartate (NMDA) receptors in noncompetitive NMDA

receptor antagonist-induced hyperlocomotion in mice. Pharmacol.Biochem. Behav. 51, 291–296.

Jahng, J.W., Houpt, T.A., 2001. MK801 increases feeding and decreasesdrinking in nondeprived, freely feeding rats. Pharmacol. Biochem.Behav. 68, 181–186.

Jones, N.M., Monn, J.A., Beart, P.M., 1998. Type I and II metabotropicglutamate receptors regulate the outXow of [3H]D-aspartate and[14C]gamma-aminobutyric acid in rat solitary nucleus. Eur. J. Pharma-col. 353, 43–51.

Jones, N.M., Beart, P.M., Monn, J.A., Widdop, R.E., 1999. Type I and IImetabotropic glutamate receptors mediate depressor and bradycardicactions in the nucleus of the solitary tract of anaesthetized rats. Eur. J.Pharmacol. 380, 129–135.

Kamikawa, H., Hori, T., Nakane, H., Aou, S., Tahiro, N., 1998. IL-1�increases noradrenaline levels in rat frontal cortex: involvement ofprostanoids, NO, and glutamate. Am. J. Physiol. 275, R803–R810.

Kanumilli, S., Toms, N.J., Roberts, P.J., 2004. Novel metabotropic gluta-mate receptor negatively coupled to adenylyl cyclase in cultured ratcerebellar astrocytes. Glia 46, 1–7.

Karcz-Kubicha, M., Liljequist, S., 1995. EVects of post-ethanol administra-tion of NMDA and non-NMDA receptor antagonists on the develop-ment of ethanol tolerance in C57B1 mice. Psychopharmacol. (Berl.)120, 49–56.

Kent, S., Bluthé, R.-M., Kelley, K.W., Dantzer, R., 1992. Sickness behavioras a new target for drug development. Trends Pharmacol. Sci. 13, 24–28.

Konsman, J.P., Luheshi, G.M., Bluthe, R.M., Dantzer, R., 2000. The vagusnerve mediates behavioural depression, but not fever, in response toperipheral immune signals; a functional anatomical analysis. Eur. J.Neurosci. 12, 4434–4446.

Koulchitsky, S.V., Leukovets, V.S., Tchitchkan, D.N., Soltan, V.V., Kul-chitsky, V.A., 1999. Role of the solitary tract nucleus and caudal ven-trolateral medulla in temperature responses in endotoxemic rats. LifeSci. 64, 37–43.

Larzabal, A., Losada, J., Mateos, J.M., Benitez, R., Garmilla, I.J., Khun, R.,Grandes, P., Sarria, R., 1999. Distibution of the group II metabotropicglutamate receptor (mGluR2/3) in the enteric nervous system of therat. Neurosci. Lett. 276, 91–94.

Lawand, N.B., Willis, W.D., Westlund, K.N., 1997. Excitatory amino acidreceptor involvement in peripheral nociceptive transmission in rats.Eur. J. Pharmacol. 324, 169–177.

Lewis, S.J., Cincotta, M., Verbene, A.J.M., Farrot, B., Lodge, D., Beart,P.M., 1987. Receptor autoradiography with [3H]L-glutamate reveals thepresence and axonal transport of glutamate receptors in vagal aVerentneurones of the rat. Eur. J. Pharmacol. 144, 413–415.

Lin, H.C., Wan, F.J., Kang, B.H., Wu, C.C., Tseng, C.J., 1999. Systemicadministration of lipopolysaccharide induces release of nitric oxideand glutamate and c-fos expression in the nucleus tractus solitarii(NTS) of rats. Hypertension 33, 1218–1224.

Maj, J., Rogoz, Z., Skuza, G., Jaros, T., 1995. Some behavioral eVects ofCNQX and NBQX, AMPA receptor antagonists. Pol. J. Pharmacol.Pharm. 47, 269–277.

Maginn, M., Caldwell, M., Kelly, K.P., Leonard, B.E., 1995. The eVect of 2-amino-3-phosphonoproprionic acid (AP-3) in the gerbil model of cere-bral ischaemia. Eur. J. Pharmacol. 282, 259–262.

Mascarucci, P., Perego, C., Terrazzino, S., De Simoni, M.G., 1998. Glu-tamic acid release in the nucleus tractus solitarius induced but periphe-ral endotoxin and interleukin-1. Neuroscience (Oxford) 86, 1285–1290.

Matsumura, K., Tsuchihashi, T., Kagiyama, S., Abe, I., Fujishima, M.,1999. Subtypes of metabotropic glutamate receptors in the nucleus ofthe solitary tract of rats. Brain Res. 842, 461–468.

Mead, A.N., Stephens, D.N., 1998. AMPA-receptors are involved in theexpression of amphetamine-induced behavioral sensitisation, but notthe expression of amphetamine-induced conditioned activity in mice.Neuropharmacol. 37, 1131–1138.

Mukhin, A., Fan, L., Faden, A.I., 1996. Activation of metabotropic gluta-mate receptor subtype mGluR1 contributes to post-traumatic neuro-nal injury. J. Neurosci. 16, 6012–6020.

T.J. Weiland et al. / Brain, Behavior, and Immunity 20 (2006) 233–245 245

Nakanishi, I., Masu, S., 1994. Molecular diversity and functions of gluta-mate receptors. Annu. Rev. Biophys. Biomol. Struct. 23, 319–348.

Oufara, S., Barre, H., Rouanet, J.L., Chatonnet, J., 1987. Adaptation toextreme ambient temperatures in gerbils and mice. Am. J. Physiol. 253,R39–R45.

Paro, F.M., Almeida, M.C., Carnio, E.C., Branco, L.G., 2003. Role of L-gluta-mate in systemic AVP-induced hypothermia. J. App. Physiol. 94, 271–277.

Pellerin, L., Magistretti, P.J., 1994. Glutamate uptake into astrocytes stim-ulates aerobic glycolysis: a mechanism coupling neuronal activity toglucose utilization. Proc. Natl. Acad. Sci. USA 91, 10625–10629.

Pistritto, G., Ciabattoni, G., Mancuso, C., Tringali, G., Preziosi, P., Nava-rra, P., 2000. Signaling pathways involved in lipopolysaccharide stimu-lation of prostaglandin production by rat hypothalamic astroglial cells.J. Endotoxin Res. 6, 307–311.

Qian, M., Johnson, A.E., Kallstrom, L., Carrer, H., Sodersten, P., 1997.Cholecystokinin, dopamine D2 and N-methyl-D-aspartate binding sitesin the nucleus of the solitary tract of the rat: possible relationship toingestive behaviour. Neuroscience (Oxford) 77, 1077–1089.

Romanovsky, A.A., Simons, C.T., Székely, M., Kulchitsky, V.A., 1997. Thevagus nerve in the thermoregulatory response to systemic inXamma-tion. Am. J. Physiol. 273, R407–R413.

Salt, T.E., Eaton, S.A., 1995. Distinct presynaptic metabotropic receptorsfor L-AP4 and CCG-1 on GABAergic terminals: pharmacological evi-dence using novel �-methyl derivative mGluR antagonist, MAP4 andMCCG, in the rat thalamus in vivo. Neuroscience (Oxford) 65, 5–13.

Salt, T.E., Turner, J.P., 1998. Modulation of sensory inhibition in the ventro-basal thalamus via activation of group II metabotropic receptors by 2R,4R-aminopyrrolidine-2,4-dicarboxylate. Exp. Brain Res. 121, 181–185.

Schöepp, D.D., Jane, D.E., Monn, J.A., 1999. Pharmacological agents act-ing at subtypes of metabotropic glutamate receptors. Neuropharma-cology 38, 1431–1476.

Sehic, E., Blatteis, C.M., 1996. Blockade of lipopolysaccharide-inducedfever by subdiaphragmatic vagotomy in guinea pigs. Brain Res. 726,160–166.

Sergeer, V.G., Akmaev, I.G., 2000. EVects of vagotomy and bacterial lipo-polysaccharide on food intake and expression of cyclooxygenase-2mRNA in rat brain vessels. Bull. Exp. Biol. Med. 129 (6), 553–555.

Singh, J., Gupta, M.C., 1997. EVect of aspartate and glutamate on nocicep-tion, catelepsy, and core temperature in rats. Indian J. Physiol. Pharma-col. 41, 123–128.

Sykes, R.M., Spyer, K.M., Izzo, P.N., 1997. Demonstration of glutamateimmunoreactivity in vagal sensory aVerents in the nucleus tractus soli-tarius of the rat. Brain Res. 762, 1–11.

Taber, M.T., Fibiger, H.C., 1995. Electrical stimulation of the prefrontalcortex increases dopamine release in the nucleus accumbens of the rat:modulation by metabotropic glutamate receptors. J. Neurosci. 15,3896–3904.

Torrealba, F., Muller, C., 1999. Ultrastructure of glutamate and GABAimmunoreactive axon terminals of the rat nucleus solitarius, with anote on infralimbic cortex aVerents. Brain Res. 820, 20–30.

Van den Pol, A.N., Hermans-Borgmeyer, I., Hofer, M., Ghosh, P., Hein-mann, S., 1994. Ionotropic glutamate-receptor gene expression inhypothalamus: localization of AMPA, kainate, and NMDA receptorRNA with in situ hybridization. J. Comp. Neurol. 343, 428–444.

Van den Pol, A.N., Romano, C., Ghosh, P., 1995. Metabotropic glutamatereceptor mGluR5 subcellular distribution and developmental expres-sion in hypothalamus. J. Comp. Neurol. 362, 134–150.

Vanover, K.E., 1998. EVects of AMPA receptor antagonists on dopamine-mediated behavior in mice. Psychopharmacology (Berlin) 136, 123–131.

Verma, A., Moghaddam, B., 1998. Regulation of striatal dopamine releaseby metabotropic glutamate receptors. Synapse 28, 220–226.

Vezzani, A., SeraWni, R., Stasi, M.A., Caccia, S., Conti, I., Tridico, R.V.,Samanin, R., 1989. Kinetics of MK-801 and its eVect on quinolinicacid-induced seizures and neurotoxicity in rats. J. Pharmacol. Exp.Ther. 249 (1), 278–283.

Wan, W., Wetmore, L., Sorensen, C.M., Greenberg, A.H., Nance, D.M.,1994. Neural and biochemical mediators of endotoxin and stress-induced c-fos expression in the rat brain. Brain Res. Bull. 34, 7–14.

Watkins, L.R., Maier, S.F., Goehler, L.E., 1995. Cytokine-to-brain commu-nication: a review and analysis of alternative mechanisms. Life Sci. 57,1011–1026.

Weiland, T.J., Voudouris, N.J., Kent, S., 2004. The role of CCK2 receptorsin energy homeostasis: insights from the CCK2 receptor-deWcientmouse. Physiol. Behav. 82, 471–476.

Winder, D.G., Conn, P.J., 1996. Roles of metabotropic receptors in glialfunction and glial-neuronal communication. J. Neurosci. Res. 46, 131–137.

Xie, Z., Morgan, T.E., Rozovsky, I., Finch, C.E., 2003. Aging and glialresponses to lipopolysaccharide in vitro: greater induction of IL-1 andIL-6, but smaller induction of neurotoxicity. Exp. Neurol. 82, 135–141.

Yan, Q.S., Reith, M.E., Jobe, P.C., Dailey, J.W., 1997. Dizocilpine (MK-801) increases not only dopamine but also serotonin and norepineph-rine transmission in the nucleus accumbens as measured by microdialy-sis in freely moving rats. Brain Res. 765, 149–158.

Ye, Z.C., Sontheimer, H., 1998. Astrocytes protect neurons from neuro-toxic injury by serum glutamate. Glia 22, 237–248.

Yoshimatsu, H., Egawa, M., Bray, G.A., 1993. Sympathetic nerve activityafter discrete hypothalamic injections of L-glutamate. Brain Res. 601,121–128.

Zhang, J., MiZin, S.W., 1998. DiVerential roles for NMDA and non-NMDA receptor subtypes in baroreceptor aVerent integration in thenucleus of the solitary tract of the rat. J. Physiol. 511, 733–745.

Zheng, H., Patterson, C., Berthoud, H., 2002. Behavioral analysis ofanorexia produced by hindbrain injections of AMPA receptor antago-nist NBQX in rats. Am. J. Physiol. 282, R147–R155.

Zonta, M., Sebelin, A., Gobbo, S., Fellin, T., Pozzan, T., Carmignoto, G.,2003. Glutamate-mediated cytosolic calcium oscillations regulate apulsatile prostaglandin release from cultured rat astrocytes. J. Physiol.553, 407–414.