Interleukin-1 receptor activation by systemic lipopolysaccharide induces behavioral despair linked to MAPK regulation of CNS serotonin transporters

Abstract

Serotonin (5-hydroxytryptamine, 5-HT) has long been implicated in regulation of mood. Medications that block the neuronal 5-HT transporter (SERT) are used as major pharmacological treatment for mood disorders. Conversely, stimuli that enhance SERT activity might be predicted to diminish synaptic 5-HT availability and increase the risk for 5-HT-related CNS disorders. We have shown that the inflammatory cytokines enhance brain SERT activity in cultured serotonergic cells and nerve terminal preparations in vitro. In this study, we establish that intraperitoneal injection of the cytokine-inducer lipopolysaccharide (LPS) stimulates brain SERT activity, acting at doses below those required to induce overt motor suppression. SERT stimulation by LPS is paralleled by increased immobility in both the tail suspension test (TST) and the forced swim test (FST); antidepressant-sensitive alterations are thought to model aspects of behavioral despair. Both the stimulation of SERT activity and induced immobility are absent when LPS is administered to interleukin-1 receptor (IL-1R)-deficient mice and in the presence of SB203580, an inhibitor of IL-1R-stimulated p38 MAPK. Moreover, the ability of LPS to enhance immobility in TST is lost in SERT knockout mice. These findings reveal an ability of peripheral inflammatory stimuli to enhance brain SERT activity through IL-1R and p38 MAPK pathways in vivo and identify a requirement for SERT expression in immune-system-modulated despair behaviors. Our studies identify IL-1R- and p38 MAPK-dependent regulation of SERT as one of the mechanisms by which environmentally driven immune system activation can trigger despair-like behavior in an animal model, encouraging future analysis of the pathway for risk factors in neuropsychiatric disorders.

Figure 1

Peripheral injection of LPS induces 5-HT uptake in mouse brain. (a) Course of time of LPS effects on midbrain 5-HT uptake. At the indicated times following i.p. injection of 0.2 mg/kg LPS, midbrain synaptosomes from C57BL/6 mice (n=3 for each time point) were assessed for 5-HT uptake as described in Materials and methods. (b) Dose–response of LPS on midbrain 5-HT uptake, assayed 60 min after i.p. LPS at the indicated doses (n=3 for each dose). (c) Saturation kinetic analysis of LPS stimulation. C57BL/6 mice were injected with saline or LPS (0.2 mg/kg i.p.) and 5-HT uptake was conducted 1 h after treatment (n=6). Vmax for saline- and LPS-treated animals were 48.9±4.1 and 44.4±1.8 fmol/min per mg protein, respectively, and the Km for saline and LPS treated mice were 412.7±64.4 and 195.6±19.8 nM, respectively. ^*p<0.05 saline vs LPS. (d) Effect of LPS on 5-HT uptake in different brain regions, assayed 60 min after LPS (0.2 mg/kg i.p.). FC=frontal cortex, Hipp=hippocampus, ST=striatum, MB=midbrain (n=3 for each condition). (e) Neurotransmitter transport specificity of peripheral LPS assayed using midbrain synaptosomes (n=3 for each condition). (f) Lack of effect of direct application of LPS on midbrain 5-HT transport. IL-1β (10 ng/ml) or LPS (0.1–10 μg/ml) was incubated for 10 min at 37°C before 5-HT transport assays (n=3). ^*p<0.05; ^**p<0.01 vs vehicle control.

Figure 2

Evaluation of p38 MAPK and IL-1R in basal and LPS-stimulated 5-HT transport. (a) Loss of LPS (0.2 mg/kg, 60 min) effect on 5-HT, but not NE uptake in SERT knockout mice (n=3 for each condition). (b) SB203580 (doses as indicated) was injected i.p. in C57BL/6 mice followed by midbrain synaptosomal 5-HT uptake assays 60 min after injection (n=3). (c) Vehicle or SB203580 (50 μg/kg i.p.) was administered to mice 30 min before LPS injection. 5-HT transport was monitored 60 min after LPS (0.2 mg/kg) treatment (n=3). (d) Synaptosomes from LPS-treated C57BL/6 mice were preincubated with SB203580 (2 μM) for 15 min followed by a 5-min 5-HT uptake assay (n=3 for each condition). (e) Wild-type (C57BL/6) and IL-1R knockout mice were injected with vehicle or LPS (0.2 mg/kg). Midbrain synaptosomal 5-HT uptake assay was conducted 1 h after injection (n=3 for each). (f) Synaptosomes were prepared from midbrains of wild-type or IL-1R knockout mice. Vehicle or IL-1β (10 ng/ml) was incubated with synaptosomes for 10 min at 37°C, followed by a 5-min ³H-5HT uptake assay (n=3). ^*p<0.05; ^**p<0.01 vs vehicle control; ^#p<0.05 vs LPS.

Figure 3

Effect of LPS (0.2 mg/kg) on 5-HT clearance time in mouse hippocampus. (a) Representative recording of hippocamal 5-HT clearance pre saline and post saline injection (i.p.). (b) Representative recording of hippocampal 5-HT clearance pre and post LPS injection (0.2 mg/kg, i.p.). (c) Time dependence of saline or LPS (0.2 mg/kg i. p.) effects on hippocampal 5-HT clearance. Points represent mean±SEM of results from nine animals (saline) and six animals (LPS). ^*p<0.05 vs saline.

Figure 4

SERT, IL-IR, and p38 MAPK-dependent behavioral effects of LPS. (a) C57BL/6 mice were injected with saline (n=8) or LPS (0.2, 1.0, and 5.0 mg/kg i.p., n=6 for each dose). Total activity was monitored during the indicated time. Total activity of mice tested 20 min before treatment served as the control condition. Total activity at 24, 48, and 72 h time points was measured for 20 min. ^*p<0.05; ^**p<0.01 vs saline control. (b) Time-response of LPS in TST. Saline or LPS (0.2 mg/kg i.p.) was injected to C57BL/6 mice and the TST was conducted at the indicated time points (n=6 for each condition). (c) C57BL/6 mice were injected with saline, LPS (0.2 mg/kg), SB203580 (50 μg/kg), or SB203580 (50 μg/kg) 10 min before LPS (0.2 mg/kg). TST was conducted 60 min after the last injection (n=6–10). (d) C57BL/6 wild-type or IL-1R intact or IL-1R knockout mice (all with C57BL/6 background) were administered saline or LPS (0.2 mg/kg i.p.). TST was conducted 60 min after the last injection (n=6 for each condition). (e) C57BL/6 wild-type or SERT knockout (KO) mice were administered saline or LPS (0.2 mg/kg i.p). TST was conducted 60 min after the last injection (n=6 for each condition). (f) C57BL/6 mice were injected with saline, LPS (0.2 mg/kg), SB203580 (50 μg/kg), or SB203580 (50 μg/kg) 10 min before LPS (0.2 mg/kg). FST was conducted 60 min after the last injection (n=6 for each condition).