Alterations of the native immune system have been proposed as potential risk factors for multiple neuropsychiatric disorders (Dantzer et al, 2008
; Anisman, 2009
; Maes, 1999
). The mechanisms by which peripheral inflammatory processes modulate behavior are only beginning to be revealed (Dantzer et al, 2008
; Raison et al, 2006
). Behavioral changes triggered by immune system activation in humans overlap with the features of mood disorders linked to disrupted 5-HT signaling, and are treatable with SSRI medications (Yirmiya et al, 2001
; Castanon et al, 2003
). In humans, injection of LPS or interferons produces behavioral changes that resemble aspects of major depression (Dantzer et al, 2008
; van den Biggelaar et al, 2007
). In animal models, such treatments elevate both the circulating and CNS levels of proinflammatory cytokines, including IL-1β (van den Biggelaar et al, 2007
; Qin et al, 2008
), and produce a suite of behavioral alterations often termed as ‘sickness syndrome' (Dantzer et al, 2008
). The degree to which altered 5-HT signaling and/or SERT contributes to these behaviors is unknown. Based on our previous findings that in vitro
treatments of synaptosomes and raphe cells with IL-1β and TNF-α rapidly stimulates SERT activity (Zhu et al, 2006
), we sought to determine whether changes in brain SERT activity could arise from an inflammatory process of peripheral origin.
Peripheral injections of LPS induced a dose- and time-dependent alteration in SERT activity, measured ex vivo
in synaptosomes from both C57BL/6 (inbred) and CD-1 (outbred) mice. The fact that LPS can stimulate SERT in the outbred strain suggests that the effect is robust over a wide variety of mouse haplotypes. LPS treatment appears to increase one or more aspects of SERT catalytic functioning, as we observed no alterations in the total or surface SERT-specific binding in synaptosomes from LPS-treated animals (see also Zhu et al, 2005
). These results are consistent with our findings that in vitro
treatment of synaptosomes with IL1-β increases the apparent affinity of SERT for 5-HT (Zhu et al, 2006
), but does not induce SERT trafficking. Although LPS, injected centrally, produces a number of neurochemical and behavioral alterations (Gottschall et al, 1992
; Plata-Salamán and Borkoski, 1993
), no change in SERT activity occurs when LPS is directly applied to synaptosome preparations, consistent with the need for an LPS-induced factor to stimulate SERT activation. Systemic pretreatment with the p38 MAPK inhibitor SB203580 at a dose without any effects on synaptosomal 5-HT uptake abolishes SERT stimulation induced by LPS. In addition, we found that LPS-induced SERT activation was lost in synaptosomes treated with SB203580, demonstrating a need for local, ongoing synaptic p38 MAPK activation to sustain the enhanced serotonin uptake.
We found that LPS-induced uptake was neurotransmitter selective as synaptosomal GABA and DA transport activities were unaffected by LPS treatment, limiting concerns that nonspecific alterations in thermodynamic factors (eg Na+
gradient and membrane potential) might be responsible for the elevated transport function. Interestingly, we found midbrain NET activity was also stimulated by peripheral LPS. NET, like SERT, can be stimulated in a trafficking-independent manner by p38 MAPK-linked pathways (Apparsundaram et al, 2001
; Zhu et al, 2005
). Peripheral LPS is also known to produce an activation of CNS noradrenergic pathways, and adrenergic receptors (Guo et al, 1996
) have been implicated by an induction of fever from LPS (Bencsics et al, 1995
). At the dose of LPS used in our studies (0.2
mg/kg), no induction of fever was evident over the course of time in our studies. Finally, we extended our in vitro
studies to an in vivo
demonstration that LPS induces enhanced rates of 5-HT clearance in the hippocampus of anesthetized mice. Consistent with these studies, Katafuchi et al (2005
) monitored reductions in extracellular 5-HT in the medial prefrontal cortex using microdialysis, beginning 1–2
h after the systemic immune system stimulation with poly I:C, another inflammatory cytokine inducer and viral mimic. Significantly, in the latter study, the reduction in dialysate 5-HT levels could be reversed by local microinjections of the SERT inhibitors imipramine or fluoxetine. In our preliminary studies with poly I:C, we found that this proinflammatory agent, which produces cytokine stimulation through mechanisms distinct from those seen with LPS, demonstrates similar effects on SERT activity.
Our findings of SERT regulation can be readily integrated into the growing body of literature that links the immune system to 5-HT signaling. For example, peripheral LPS rapidly induces c-fos activation in CNS serotonergic neurons (Hollis et al, 2006
), and alters the 5-HT release and turnover (Lavicky and Dunn, 1994
). With chronic LPS treatments, indoleamine 2,3 dioxygenase (IDO), an enzyme that metabolizes the 5-HT precursor tryptophan, is induced (O'Connor et al, 2009
). SSRIs block the behavioral effects of immune system activation in humans and animal models (Yirmiya et al, 2001
; Castanon et al, 2003
). Interestingly, SERT and 5-HT receptors also contribute to immune system activation (Guo et al, 1996
), reinforcing the idea that 5-HT and immune signaling pathways are reciprocally connected to provide for coordinated responses to environmental challenges.
To obtain evidence for the role of SERT in LPS-induced behavioral changes, we used two antidepressant-sensitive measures, the TST and FST, that monitor the animal's willingness to sustain struggling in the face of an inescapable stressor. We found that mice injected with a dose of LPS that does not induce overt motor effects or fever produced enhanced immobility in the TST and FST. LPS increased immobility in both the inbred (C57BL/6) and outbred (CD-1) strains. Moreover, the time dependence and dose–response of LPS's effects on TST immobility closely paralleled with those found for SERT activation. SB203580, only at doses that blocked LPS-induced synaptosomal SERT activation, abolished LPS-induced immobility, potentially illuminating a novel pharmacological mechanism by which behavioral despair might be overcome. The effect of LPS on immobility was not seen in SERT KO mice. All of these findings are consistent with the requirement for and involvement of SERT in the observed despair behavior induced by this proinflammatory stimulus. As antidepressants that block SERT have effects on the immune system, one might argue that the SERT KO background limited the ability of LPS to induce CNS cytokines in these animals. However, we observed that these knockout mice displayed an LPS-induced stimulation of midbrain NET activity similar to that observed in wild-type mice, suggesting that the lack of a behavioral effect in the knockout was more likely due to the inability of LPS-stimulated cytokines to increase in SERT activity in the mutant's CNS. In addition, although many antidepressants target both SERT and NET together, or NET alone, the normal response of NET to LPS treatment in these mice would suggest that LPS-induced behavioral despair cannot be induced by reductions in central noradrenergic neurotransmission alone, and points to the changes in 5-HT neurotransmission as a critical event in immune-related depressive behavior.
LPS is known to increase CNS levels of IL-1β, TNFα, and IL-6, and we have shown that both IL-1β and TNFα can stimulate synaptosomal SERT activity in a p38 MAPK-dependent manner. In this study, we demonstrate that the LPS-induced increase in SERT activity is dependent on the expression of the IL-1R receptor. Similarly, we found that LPS was unable to induce immobility in the IL-1R knockout mouse at a dose that was effective in the wild-type mouse. Consistent with these findings, Minor et al (2006)
have reported that i.c.v. administration of the IL-1 receptor antagonists (IL-1ra) blocks LPS-induced immobility in the FST. Additionally, multiple groups have shown that IL-1ra blocks LPS-induced reductions in social interaction tests (Konsman et al, 2008
; Arakawa et al, 2009
), mimicking the effects of antidepressant medications in these paradigms (Yirmiya, 1996
; Castanon et al, 2001
). Taken together, our findings are consistent with the suggestion that LPS was unable to induce midbrain IL-1R-mediated stimulation of SERT in the IL-1R knockouts, and that lack of this central p38 MAPK-dependent SERT activation limited the expression of behavioral despair in response to LPS in these animals.
We do not presume that changes in SERT activity alone are sufficient to induce the full spectrum of depression traits, nor that our animal model can reproduce all the elements of a complex neuropsychiatric disorder. Furthermore, it is clear that activation of IL-1R can be only one component of a complex interaction of neuronal systems involved in the expression of depressive-like behavior in animal models. We focused here on an acute LPS treatment paradigm to constrain the number of variables underlying the biochemical and behavioral changes. Nonetheless, we were able to identify the mechanisms that could be viewed as precipitating events in a chain of circumstances involved in depressive illness. If sustained, augmented, or autonomously engaged, such events could support more profound behavioral changes. Indeed, the relatively rapid changes in SERT activity we observe following immune system activation could serve as triggers for long-lasting, cytokine-related reductions in extracellular 5-HT levels that are later supported by elevations in SERT mRNA and protein expression (Ramamoorthy et al, 1995
, Katafuchi et al, 2006
), and depletion in tryptophan levels through induction of metabolic pathways (Capuron and Dantzer, 2003
). In this regard, IL-1R activation has been implicated in depressive behavior induced through chronic stress (Koo and Duman, 2008
; Goshen et al, 2008
) and learned helplessness paradigms (Maier and Watkins, 1995
), consistent with the idea that depression can arise as a perturbation of affective CNS networks by endogenous cytokines released in response to uncontrollable stress. Additional studies that focus on the long-term effects of immune system activation on SERT and SERT-dependent behaviors are needed to provide a more complete understanding of the relationships between immune system activation, central IL-1 receptor activation, p38-mediated activation of SERT, and the elaboration of depressive behavior in animal models. In addition, a more comprehensive evaluation of genetic variation in this pathway might suggest sources of genetic risk for affective disorders (Müller and Schwarz, 2007
; Koo and Duman, 2009
), and lead to improved options for treating human depressive illness.