Interleukin 1 beta enhances conditioned fear ... - Wiley Online Library

European Journal of Neuroscience, Vol. 18, pp. 1739±1743, 2003

ß Federation of European Neuroscience Societies

Interleukin 1 beta enhances conditioned fear memory in rats: possible involvement of glucocorticoids Cai Song,1 Anthony G. Phillips1 and Brian Leonard2 1 2

Department of Psychiatry, University of British Columbia, 2255 Wesbrook Mall, Vancouver, Canada, V6T 2A1 Brain and Behaviour Research Institute, Academic Hospital Maastricht, University of Maastricht, The Netherlands

Keywords: corticosterone, IL-10, mifepristone (RU486), passive avoidance, PGE2

Abstract Central administration of 15 ng interleukin (IL)-1b in the rat signi®cantly enhanced conditioned fear memory assessed by a passive avoidance task, when retested at 24 and 48 h post-training. Pain threshold was unaffected by 15 ng IL-1b administration. IL-1b treatment also increased serum corticosterone. This increase in serum corticosterone was further enhanced in rats given both IL-1b and footshock. Furthermore, the glucocorticoid receptor antagonist mifepristone blocked IL-1b-induced elevation in corticosterone and also attenuated the enhanced conditioned fear memory. Central administration of IL-1b signi®cantly increased prostaglandin E2 and decreased the anti-in¯ammatory cytokine IL-10 release from whole blood cultures; therefore this treatment appears to be effective in inducing an in¯ammatory response in both the periphery and the brain. The present study con®rms that IL-1b can enhance conditioned fear memory, an effect which is correlated with changes in glucocorticoid function. This facilitation of defensive behaviour could re¯ect adaptive responses which may enhance survival during sickness.

Introduction A growing body of evidence indicates that the immune, endocrine and neurotransmitter systems form a complex interacting network which can have signi®cant effects on behaviour and cognition (Maier & Watkins, 1998). The proin¯ammatory cytokine interleukin (IL)-1b, produced by activated macrophages in the periphery and by microglia and astrocytes in the brain (Johnstone et al., 1999), can induce an in¯ammatory response in the brain, activate the hypothalamic±pituitary±adrenal (HPA) axis and alter brain monoamine metabolism (Song, 2000; Hoozemans et al., 2001). Central effects of IL-1b are mediated in part by IL-1b receptors in the hippocampus and amygdala (Loddick et al., 1998). These effects include changes in sleep pattern, anhedonia, loss of libido and mental disturbance (Hickie & Lloyd, 1995; Krueger et al., 1998), which also may contribute to the cognitive changes in man and `sickness behaviour' in rodents (Dantzer et al., 1998). Intracerebroventricular (i.c.v.) injections of murine IL-1b to rats, or peripheral injection to mice, before training impairs spatial memory (Gibertini et al., 1995; Maier & Watkins, 1998). These behavioural ®ndings are consistent with the presence of IL-1b binding sites in the hippocampus and amygdala (Ericsson et al., 1995; Loddick et al., 1998). IL-1 may also be involved in different types of memory; for example, i.c.v. administration of IL-1 receptor antagonist (RA) blocks the enhancement of conditioned fear produced by inescapable shock (Maier & Watkins, 1995). Selective effects on contextual vs. cued fear conditioning occur following central infusion of IL-1b (10 or 20 ng), with the former dependent on the dorsal hippocampus and the latter on the amygdala (Pugh et al., 1999). IL-1b also plays an important role in neuroendocrine responses to stress, in particular glucocorticoid Correspondence: Dr Cai Song, 1Neuroscience Division, as above. E-mail: [email protected] Received 18 March 2003, revised 11 June 2003, accepted 11 July 2003 doi:10.1046/j.1460-9568.2003.02886.x

secretion, which is through the activation of prostaglandin (PG) E2 receptor gene expression and increase in PGE2 synthesis (Zhang & Rivest, 1999; Ek et al., 2000). Both IL-1RA and a cyclooxygenase inhibitor can block IL-1b-induced increase in the secretion of CRF and glucocorticoid (Shintani et al., 1995; Ericsson et al., 1997). In part, disruptive effects of IL-1b on cognition may be mediated by glucocorticoid receptors in the hippocampus (Oitzl et al., 1998; Brabham et al., 2000). Contextual fear conditioning also depends on normal function of the hippocampus and can be facilitated by glucocorticoids (Cordero et al., 1998). This in turn raises the possibility that IL-1b may facilitate fearrelated memory function while exerting disruptive effects on spatial learning and memory mediated by the hippocampal formation. Yirmiya et al. (2002) partially con®rm this hypothesis in their recent report that i.c.v. administration of human IL-1b enhanced passive avoidance in rats. The present study extended these ®ndings in several new directions. First, IL-1b was administered i.c.v. subchronically (3±4 days) to better mimic the pathological condition over a course of in¯ammation or a short-term of illness. Second, pain threshold was measured to rule out the possibility that the effect of IL-1b on passive avoidance conditioning may re¯ect increased sensitivity to noxious stimuli. Third, the role of glucocorticoids in modulating fear-related memory by IL-1b was assessed by coadministration of the glucocorticoid antagonist mifepristone (RU486). Fourth, given the important role of PGE2 in IL-1binduced increase in glucocorticoid secretion, following behavioural tests, serum concentrations of corticosterone and the release of PGE2 were measured in rats given central injections of IL-1b. Fifth, IL-10, produced by T-helper 2 cells (Th2), was also assayed because this antiin¯ammatory cytokine antagonizes the effect of proin¯ammatory cytokines (such as IL-1b) produced by Th1 and macrophages (Lucey et al., 1996). Further justi®cation for measuring the release of IL-10 from blood culture comes from reports that an imbalance between proand anti-in¯ammatory cytokines is associated with several psychiatric disorders (Schwarz et al., 2001).

1740 C. Song et al.

Materials and methods

corticosterone measurement. Intra- and interassay coef®cients of variation were 6.8% and 5.6%, respectively (Song et al., 1998).

Animals Male Wistar rats (250±280 g, Charles River, Quebec, Canada) were maintained at 21  1 8C with a 12-h light±dark cycle. Food and water were available ad libitum. Animals were housed two per cage and handled daily. The research protocol was approved by the Animal Care Committee of the University of British Columbia, Canada.

Histological determination

Surgery

Statistical analysis

Animals were anaesthetized with ketamine (100 mg/kg) and xylazine (2 mg/kg) and placed in a stereotaxic apparatus. Guide cannulae (24gauge), for i.c.v. administration, were located stereotaxically over the left lateral ventricle (AP 1, L 1.6, 1 mm depth) as described in detail elsewhere (Strausbaugh & Irwin, 1992).

Behavioural data from the passive avoidance test were assessed by two-way (IL-1b  footshock) repeated-measures ANOVA. Biochemical data were analysed using two-way ANOVA. Both data sets were then analysed with the Scheffe post hoc test. Signi®cance was set at a value of P < 0.05. Results are expressed as mean  SEM.

Reagents and i.c.v. injection

Procedure

Rat recombinant IL-1b was obtained from NIBSC, Potters Bar, UK (biological activity 317 IU/mg) and dissolved in sterile, pyrogen-free saline at a dose of 15 ng/10 mL/rat. The glucocorticoid receptor antagonist RU486 (Sigma, Canada), was dissolved in absolute ethanol and then diluted with sterile saline for administration to rats at a dose of 100 ng in 10 mL/rat. The ®nal concentration of ethanol injected was 1% (Calvo & Volosin, 2001). In the third experiment, 1% ethanol saline served as a vehicle injection for control rats. Rats were gently held in a soft cotton towel every day for 2 weeks before the injection. On the injection day, rat IL-1b, RU486 or saline were taken into a needle (4.2 mm length, 26-guage) which was connected to a PE 50 polyethylene tube. After unscrewing the cap, the needle was gently inserted into the guide cannula and drugs or saline was slowly infused into the rat brain (in 30 s). The needle remained in place for 1 min. In experiment 3, rats treated with both IL1 and RU486 i.c.v. received these drugs sequentially via the same injection needle.

This study consisted of four experiments. In the ®rst experiment, animals were divided into four groups of nine subjects. Two groups received i.c.v. saline or IL-1b (15 ng) without footshock and the other groups received identical treatments with footshock. During the ®rst habituation trial, animals were injected with vehicle or IL-1b and 50 min later were permitted to explore the apparatus for 2 min. Latency to enter the dark chamber was recorded for each animal. On day 2 (the acquisition trial), 50 min after IL-1b or saline administration, rats received either footshock or no footshock after they entered the dark chamber. The latency to enter the dark chamber was again recorded. On day three (the retention trial, 24 h after footshock), 50 min after IL1b administration, each rat was placed into the white compartment and the latency to enter the dark compartment was recorded during a 5-min period (Song et al., 1997). Rats were then killed, and trunk blood was collected for corticosterone assays. The second experiment included the same four groups (n ˆ 9) as experiment one and incorporated retest trials at 24 h and 48 h after the acquisition trial with the footshock. On day four (the second retention trial, 48 h after footshock), 50 min after IL-1b administration, the rat was placed in the white compartment again and the latency to enter the dark compartment recorded during a 5-min period. Animals were then anaesthetized with halothane and blood samples were obtained by cardiac puncture for the determination of PGE2 and IL-10. In the third experiment, animals were assigned to four groups (n ˆ 8) and received saline, IL-1, RU486 or RU486 combined with IL-1, respectively. The procedure for passive avoidance was the same as the ®rst experiment, while all animals received the footshock. On day 3 (retention day), following the test for passive avoidance, animals were killed and trunk blood was taken for the measurement of serum corticosterone levels. The fourth experiment measured pain threshold in two groups (n ˆ 6), 50 min after infusion of saline or 15 ng IL-1b.

Behavioural studies A `step-through' passive avoidance test was used to establish a conditioned fear memory. The apparatus consisted of a wooden box divided into two compartments of equal dimensions (24  16  20 cm). A white chamber, illuminated by one 40-W incandescent light bulb, was connected to a rear black chamber equipped with a grid ¯oor. The two chambers were separated by a guillotine door. Footshock (1.5 mA for 1 s) was delivered in the dark box (Song et al., 1997). Pain threshold was measured in the dark box of the apparatus. The electronic current was increased from 0 mA in 0.1-mA steps with a 30-s intertest interval until the animal showed a ¯inch response. The footshock was increased further until animals showed a slight jump. The current levels for both responses were recorded and the corresponding mean current values de®ned the `¯inch' and `jump' thresholds.

After decapitation, brains were rapidly removed, placed on an ice block and the location of the cannulae and injection site was con®rmed in coronal section of the brain. The data were excluded from all animals with injection sites outside the lateral ventricular.

Biochemical assays Heparinized blood were diluted 1 : 10 with RPMI-1640 medium containing 1% penicillin. For mitogen stimulation, phytohaemoglutinin: (5 mg/mL) and lipopolysaccharide (20 mg/mL) (stimulated) were used. All samples were incubated for 72 h in a humidi®ed atmosphere incubator with 5% CO2 at 37 8C. The supernatant was removed and stored at 70 8C for subsequent assays (Song et al., 1998). The release of IL-10 and PGE2 were measured by ELISA (Biosource International, CA, USA) and enzyme immunoassay (EIA) (Assay Designs, Inc., Ann Arbor, USA), respectively, as described previously (Song et al., 1998). A commercial radioimmunoassay kit (Immuchem corticosterone RIA kit for rats, ICN Biochemical, Costa Mesa, CA, USA) was used for the

Results IL-1b administration enhanced conditioned fear memory in a passive avoidance test but did not change the pain threshold In experiment 1, on days 1 and 2, before footshock was experienced, saline- and IL-1b-treated rats did not differ in their latency to enter the dark compartment (day 1: saline, 7.04  1.24 s; IL-1b, 6.57  0.67 s; day 2: saline, 8.3  0.63 s; IL-1b, 7.17  0.89 s). On the retention trial (day 3), an ANOVA analysis con®rmed that footshock (24 h before) signi®cantly increased latencies to enter the dark compartment when compared to the rats which did not receive the footshock

ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1739±1743

IL-1b enhances conditioned fear

1741

took signi®cantly longer to enter the dark compartment than rats given footshock plus saline treatment (F3,28 ˆ 15.65, P < 0.01; Fig. 1B). A two-way ANOVA revealed that coadministration of RU486 blocked the effect of IL-1b on conditioned fear memory (F3,28 ˆ 10.73, P < 0.05; Fig. 1B). In the fourth experiment, no signi®cant difference in pain threshold was observed between rats which received saline or IL-1b administration (¯inch: saline, 0.908  0.028; IL-1b, 0.932  0.023; jump: saline, 1.40  0.034; IL-1b, 1.43  0.027).

(F3,32 ˆ 41.42, P < 0.0001) (saline, 10.34  1.56; saline plus footshock, 159.74  22.72; IL-1, 9.63  1.28; IL-1 plus footshock, 273.29  27.82). ScheffeÂ's post hoc test indicated that the comparisons between two groups given saline with or without footshock, or between IL-1b with and without footshock, were signi®cant (P < 0.01). A two-way ANOVA con®rmed a signi®cant interaction between the footshock and IL-1b (F3,32 ˆ 19.28, P < 0.01). The step-through latencies of rats receiving IL-1b and footshock were signi®cantly greater than those receiving saline and footshock (P < 0.01, ScheffeÂ's post hoc test). In the second experiment, similar results as in experiment 1 were observed. On day 1 and day 2, IL-1b or saline treatment without footshock did not signi®cantly change the latency to enter the dark compartment (Fig. 1A). Twenty-four hours after footshock (on day 3), a signi®cant increase in the latency was recorded in groups given footshock (ANOVA: F3,32 ˆ 31.42, P < 0.0001). The Scheffe comparisons between the groups treated with saline alone and saline plus footshock, or between the groups treated with IL-1b alone and IL-1b plus footshock, were signi®cant (P < 0.01). The two-way ANOVA also revealed an interaction between IL-1 and footshock (F3,32 ˆ 19.75, P < 0.01; Fig. 1A). The Scheffe comparison indicated that a signi®cant increase in the step-through latency occurred in the group given both IL-1 and footshock when compared to the group which received saline plus footshock (P < 0.05). Forty-eight hours after the initial training with footshock, step-through latencies of rats in the footshock groups and control group still differed signi®cantly (F3,32 ˆ 24.25, P < 0.001). Two-way ANOVA con®rmed an interaction between footshock and IL1b which was also signi®cant (F3,32 ˆ 8.913, P < 0.05; Fig. 1A). In the third experiment, similar results were observed in rats treated with IL-1b and footshock. On the 24-h retention test, these animals

In experiment 1, i.c.v. administration of IL-1b signi®cantly elevated corticosterone secretion in the blood with or without footshock (in experiment 1, F3,32 ˆ 8.97, P < 0.01; Fig. 2A). Signi®cant increases in the levels of this hormone were also found in rats which received footshock with saline injection in the passive avoidance apparatus (F3,32 ˆ 34.70, P < 0.0001). Two-way ANOVA con®rmed an interaction between IL-1b and fear memory; corticosterone concentration was further increased in rats which had received both footshock and IL-1b when compared to rats trained with footshock alone or given IL-1b alone (F3,32 ˆ 5.65, P < 0.05; Fig. 2A). In the analysis of results from experiment three, the ANOVA indicated a signi®cant effect of IL-1b administration (F3,28 ˆ 7.24, P < 0.01). The Scheffe comparison between groups revealed that the level of corticosterone was signi®cantly increased in the group given IL-1b and footshock when compared to the group which received saline and footshock (P < 0.05). Central RU486 infusion also had a signi®cant effect on the concentration of this hormone (F3,28 ˆ 7.57, P < 0.01). The Scheffe comparison between groups revealed that RU486

Fig. 1. (A) The effect of i.c.v. administration IL-1b (15 ng/rat) on conditioned fear memory in the passive avoidance test. P < 0.01 vs. saline or IL-1b group without fear; #P < 0.05 vs. saline with fear, n ˆ 9. (B) The effect of central RU486 (100 ng/rat) administration on the enhancement of conditioned fear memory induced by IL-1b on passive avoidance. P < 0.05 vs. IL-1 with fear group; #P < 0.01 vs. saline with fear, n ˆ 8.

Fig. 2. (A) The effect of i.c.v. administration IL-1b (15 ng/rat) and fear on serum concentrations of corticosterone. P < 0.01 vs. saline control group; # P < 0.05 vs. saline with fear group, n ˆ 9. (B) The effect of central RU486 administration on the elevation of corticosterone concentrations induced by IL1b and fear. P < 0.05 vs. saline with fear; #P < 0.05 vs. IL-1 with fear, n ˆ 8.

Changes in corticosterone concentrations following IL-1b alone or with footshock

ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1739±1743

1742 C. Song et al. signi®cantly attenuated the elevation of corticosterone level induced IL-1b and stress (P < 0.05; Fig. 2B). IL-1b-induced changes in the release of PGE2 and IL-10 In experiment 2, IL-1b (15 ng) signi®cantly increased PGE2 release from whole blood culture following mitogen stimulation (F3,32 ˆ 8.288, P < 0.01; Fig. 3A). The Scheffe post hoc test revealed that the PGE2 concentration was signi®cantly increased in the group which received IL-1b when compared to the group which received saline (P < 0.05). The PGE2 concentration was slightly increased in the nonstimulated blood of rats receiving IL-1b when compared to the saline treatment group (saline, PGE2 30.63  3.34 mg/mL; IL-1b, PGE2 39.11  3.68 mg/mL, F3,32 ˆ 3.36, P ˆ 0.074). Shock alone did not signi®cantly change PGE2 release (Fig. 3A). IL-10 release from whole blood culture without mitogen stimulation was undetectable. A reduction in IL-10 release by IL-1b administration was found after mitogen stimulation (F3,32 ˆ 15.24, P < 0.01). Foot shock alone did not signi®cantly affect IL-10 release (Fig. 3B).

Discussion In the present study, i.c.v. administration of IL-1b (15 ng), 50 min prior to training and retention trials, signi®cantly enhanced a passive avoidance response in rats re-exposed to a test environment in which they had received footshock upon entering the dark compartment 24 or 48 h earlier. Maier & Watkins (1995) reported that i.c.v. administration of IL-1RA blocks the enhancement of conditioned fear produced by inescapable shock. Delayed facilitation of passive avoidance behaviour was observed 5 days after post-training injection of human IL-1b to rats (Yirmiya et al., 2002). These investigators also found a disruption of conditioned fear memory 8 days after post-training injection of IL-1RA. Previous studies have demonstrated that IL-1b can disrupt those types of memory dependent on hippocampal functions (Gibertini et al., 1995; Maier & Watkins, 1995; Pugh et al.,

Fig. 3. The effect of central administration of IL-1b on mitogen-stimulated release of PGE2 (A) and the release of IL-10 (B) in the supernatant of whole blood culture. P < 0.01 vs. saline group, n ˆ 9.

1999). A possible mechanism whereby IL-1b modulates these speci®c forms of learning and memory comes from the evidence that IL-1b gene expression is increased in the hippocampus during long-term potentiation (LTP) of synaptic transmission, a procedure considered to underlie certain aspects of learning and memory (Schneider et al., 1998). Furthermore, IL-1RA administration results in a reversible impairment of LTP maintenance (Schneider et al., 1998). Previous studies have reported that i.c.v. administration of IL-1b at doses of 1±10 ng/rat produce analgesia or have no effect, whereas lower doses (10±100 pg/rat) produce hyperalgesia (Adams et al., 1993; Yabuuchi et al., 1996). In the present study, we demonstrated that i.c.v. administration of 15 ng IL-1b had no effect on pain threshold in the rat. This ®nding indicates that IL-1b-induced enhancement in conditioned fear memory cannot be attributed to increased sensitivity to footshock. In the present passive avoidance study, serum concentrations of corticosterone were lowest in the saline-treated controls and highest in the IL-1b plus fear conditioning group, values for the other treatment groups being intermediate. Rats with the highest corticosterone concentrations (IL-1b plus fear) also had the longest retention latencies. Therefore, the facilitation of fear-related memory by IL-1b may be related to enhanced release of corticosterone relative to rats given IL1b alone or saline plus re-exposure to the apparatus in which they had received footshock. This hypothesis was supported by the observation that the corticosterone antagonist RU486 blocked both the enhancement of fear-related memory by IL-1b and the increase in serum corticosterone levels produced by IL-1b in combination with reexposure to a fear-inducing environment. Elevated glucocorticoid concentrations have previously been shown to be positively correlated with enhanced aversive or fear-conditioned memory (Cordero et al., 1998). Furthermore, exogenous administration of corticosterone enhanced conditioned fear memory (Rudy & Pugh, 1998). Decreased serum corticosterone concentration and reduction in the stress effects of corticosterone following the administration of the glucocorticoid receptor antagonist RU486 have also been reported by others (Goujon et al., 1995; Breivik et al., 2000). Because both PGE2 and IL-10 may cross the blood±brain barrier in either direction (Engblom et al., 2002; Aharoni et al., 2000), and have opposite roles in an in¯ammatory response, the present study measured their changes following IL-1b administration. The co-occurance of a decrease in the release of anti-in¯ammatory cytokine IL-10 and an increase in the release of PGE2 from blood cultures suggests that the central administration of IL-1b not only induces changes in brain function but also initiates in¯ammatory changes in the periphery. Similar changes in PGE2 and IL-10 have also recently been found in several brain regions following central IL-1b administration (our unpublished observation). It is known that peripheral administration of IL-1b can induce cyclooxygenase 2 activity in the brain, thereby increasing PGE2 synthesis (Koyama et al., 1999). PGE2 action in the ventrolateral medulla is an important step in activation of the HPA axis during in¯ammatory responses (Engblom et al., 2002). The cellular signal of an in¯ammatory response in the brain may be carried by PGE2 to the periphery. Although it has been reported that PGE2 plays an important role during in¯ammation-induced stress (Engblom et al., 2002), the stress associated with footshock alone in our study did not affect PGE2 release. There was also no interaction between footshock and IL-1b on PGE2 release. Comparable changes in PGE2 releases were observed following IL-1b treatment alone or in combination with prior footshock. Therefore, it is unlikely that elevated PGE2 levels could account for enhanced memory for fear-related events observed in the present study.

ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1739±1743

IL-1b enhances conditioned fear It has been previously reported that IL-1b-induced changes in neurotransmission and glucorcoticoid secretion are exacerbated by stressors (Song et al., 1999; Anisman & Merali, 2003). However, the mechanism by which IL-1b and footshock act synergistically to enhance glucocorticoid release cannot be speci®ed at present. Nevertheless the enhancement of conditioned fear memory by IL-1b may have adaptive signi®cance by lessening exposure to potential threats during sickness or injury.

Acknowledgements The authors thank Mr Fred Lepiane and Ms Xuwen Li for technical assistance. This study was supported by a grant from Laxdale Ltd, Scotland, UK.

Abbreviations ELISA, quantitative enzyme-linked immunosorbent assay; HPA, hypothalamic±pituitary±adrenal; i.c.v., intracerebroventricular; IL, interleukin; PGE2, prostaglandin E2; RA, receptor antagonist; RU486, mifepristone.

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ß 2003 Federation of European Neuroscience Societies, European Journal of Neuroscience, 18, 1739±1743