Male Wistar rats were intraperitoneally (i.p.) injected with bacterial endotoxin (lipopolysaccharide; LPS) at a dose of 100 µg/kg body weight to induce a peripheral inflammatory response. This dose of LPS induces a clear activation of the immune system as indicated by elevated levels of circulating cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 and IL-6 (Linthorst et al., 1997), and increased activity of the HPA axis (Linthorst et al., 1995b). LPS caused a marked increase in hippocampal extracellular levels of 5-HT, reaching maximal levels of about 200-250% of baseline. In addition, LPS induced a rise in extracellular levels of the metabolite 5-HIAA (maximum about 150% of baseline; Linthorst et al., 1995b). Because extracellular 5-HIAA levels are generally considered to reflect the amount of newly synthesized 5-HT (Auerbach et al., 1989; Crespi et al., 1990; Kalén et al., 1988; Linthorst et al., 1994), the LPS-induced increase in extracellular 5-HT in the hippocampus seems to be accompanied by a rise in the synthesis of this indoleamine. Of particular interest in this respect is a recent receptor autoradiography study showing that i.p. administration of LPS caused a prolonged elevation of 5-HT_1A receptors in the hippocampus (Reul et al., unpublished observations). Hence, we hypothesize that the hippocampal serotonergic system ensures a profound and extended activation of 5-HT-regulated postsynaptic events not only by enhanced extracellular concentrations of 5-HT itself, but also by increases in the synthesis of 5-HT and the concentration of 5-HT_1A receptors. Interestingly, i.p. inoculation with LPS had no influence on extracellular levels of 5-HT in the preoptic area, although a small, but delayed rise in 5-HIAA was found in this brain structure (about 120% of baseline; Linthorst et al., 1995a).

Apart from rises in extracellular 5-HT and 5-HIAA levels, i.p. administration of LPS resulted also in moderately elevated levels of NA and its metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG) in the hippocampus (maximal levels reached about 150% and 170% of baseline, respectively; Linthorst et al., 1996). These moderate effects in the hippocampus are in sharp contrast with the dramatic LPS-induced increases in extracellular levels of NA and MHPG in the preoptic area (about 500% and 400% of baseline, respectively; Linthorst et al., 1995a).

Summarizing, we have shown that peripheral inflammation results in distinct neurotransmission responses in the brain. It may therefore be speculated that whereas in the preoptic area predominantly noradrenergic neurotransmission is responsive to peripheral inflammation, in the hippocampus serotonergic neurotransmission seems to be relatively more responding than the noradrenergic system.

It is now generally accepted that peripheral administration of LPS not only evolves in rises in locally produced cytokines, but may also induce the production of cytokines in the brain (see Hopkins and Rothwell, 1995).

Hence, we embarked on a study to elucidate the role of specific cytokines in the LPS-induced increases in hippocampal serotonergic neurotransmission. First, cytokines were singly administered intracerebroventricularly (i.c.v.). It was found that i.c.v. administration of IL-1 and IL-2, but not of TNF-alpha , increased hippocampal extracellular levels of 5-HT and 5-HIAA (Linthorst et al., 1995b; Pauli et al., 1998). Interestingly, the effect of IL-2 on hippocampal 5-HT could be blocked completely by i.c.v. pretreatment with the IL-1 receptor antagonist (IL-1ra), indicating a primary role for IL-1 in regulating serotonergic neurotransmission in the hippocampus (Pauli et al., 1998). These observations prompted us to investigate a putative involvement of brain IL-1 in the LPS-induced activation of hippocampal 5-HT. Indeed, when rats were pretreated i.c.v. with IL-1ra, a 50% reduction of the i.p. LPS-induced increase in 5-HT in the hippocampus was found (Linthorst et al., 1995b). This result strongly suggests that centrally released IL-1 is a principal mediator in the effects of LPS on hippocampal 5-HT. This conclusion was corroborated by experiments in which IL-1 was infused locally into the hippocampus via the microdialysis probe. Local infusion of IL-1 also resulted in elevated levels of 5-HT and 5-HIAA (Linthorst et al., 1994) supporting the notion that IL-1 released or produced locally in the hippocampus may participate in the activation of 5-HT release in this brain structure during peripheral inflammation. Of particular interest in this respect is our recent finding that peripheral administration of LPS caused only changes in 5-HT_1A receptors on the level of the hippocampus, but not in the raphe nuclei. This observation underscores our concept that peripheral inflammation-induced alterations in the raphe-hippocampal serotonergic system are restricted to local actions of cytokines (i.e. IL-1) on the level of the hippocampus.

Arachidonic acid metabolites are known to play an important role in the actions of cytokines released after an immune challenge (Gottschall et al., 1992; Rothwell, 1991). Systemic administration of LPS induces the release of prostaglandins in the brain (Sirko et al., 1989; Smith et al., 1994) and are involved in the IL-1-induced release of ACTH (Katsuura et al., 1988).

We have found that prostaglandins also play an important role in neurotransmission responses during inflammation. I.p. pretreatment of rats with indomethacin (10 mg/kg body weight) largely but not completely prevented LPS-induced increases in hippocampal serotonergic and noradrenergic and in preoptic noradrenergic neurotransmission (Linthorst et al., 1995a, 1996). The indomethacin pretreatment, however, was able to entirely block the fever, tachycardia and sickness behavior responses (Linthorst et al., 1995a). Our data, therefore, suggest that additional mediators appear to be involved in LPS-induced neurotransmitter responses. Nitric oxide may be a likely candidate because several studies have shown an involvement of this gaseous transmitter in brain responses to inflammation (Karanth et al., 1993; Raber and Bloom, 1994; Rivier and Shen, 1994).

Although characterization of neurotransmission responses to peripheral inflammation is of high interest, the next step should be the elucidation of the functional implications of these changes. Using the multidisciplinary approach as described under 1., we have studied the temporal correlates of the rises in NA in the preoptic area during peripheral inflammation. Peripheral administration of LPS was found to induce a biphasic fever and tachycardia response. Moreover, levels of free corticosterone rose and the rats showed clear signs of sickness behavior, such as profound immobility and a curled-up body posture (Linthorst et al., 1995a,b). The LPS-induced elevation in preoptic NA ran in parallel with the first rise in body temperature and the increase in free corticosterone levels. Based on this fact and the observation that maximum NA levels are reached about 60 min earlier than the peak in body temperature and corticosterone concentrations, we have postulated a role for preoptic NA in inflammation-induced fever and HPA axis activation (Linthorst et al. 1995a).

Next, we aimed to clarify the functional implications of hippocampal serotonergic changes in response to peripheral administration of LPS. Based on the contemplations described below, we hypothesize that the increased levels of 5-HT in the hippocampus are instrumental in (some aspects of) sickness behavior. It is thought that the raphe-hippocampal serotonergic system plays a key role in sensory information processing and the modulation of stress-induced behavioral and neuroendocrine responses. A positive relationship between behavioral activity of the animal on the one hand, and the firing rate of serotonergic neurons and extracellular levels of 5-HT in the terminal areas on the other hand has been found by several groups (Jacobs and Azmitia, 1992; Kalén et al., 1989; Linthorst et al., 1994). The most pronounced correlation between extracellular 5-HT and behavioral activity was observed in the hippocampus, although this correlation was also present in the neocortex and the preoptic area (Linthorst et al., 1995a, 1995b). Moreover, until now it was demonstrated that various stressors always increase hippocampal extracellular levels of 5-HT in parallel with behavioral activation (Jacobs and Azmitia, 1992; Kalén et al., 1989; Rueter and Jacobs, 1996). The situation during immune stress is, however, exceptional. Peripheral inflammation caused a rise in hippocampal 5-HT levels which was accompanied by a decrease in behavioral activity (sickness behavior). This effect seems to be located at the level of the hippocampus, without a concomitant increase in the firing rate of the serotonergic cell bodies. Hence, a situation seems to have been realized in which sensory information processing in the hippocampus is modulated by 5-HT without a parallel increase in behavioral activity due facilitation of motor output via an action of 5-HT at the level of the brain stem (Reul et al., 1998). In fact, facilitation of motor output is clearly a maladaptive response during inflammation. The observation that behavioral lethargy during immune stimulation is always accompanied by enhanced hippocampal levels of 5-HT (for instance in the case of LPS, IL-1ß, IL-2) underpins a role for hippocampal 5-HT in sickness behavior. In contrast, TNF-alpha at doses that induced fever and activation of the HPA axis, neither generated a reduction in behavioral activity nor increased hippocampal 5-HT concentrations (Pauli et al., 1998). Recent results derived from experiments on long-term i.c.v. corticotropin-releasing hormone-infused rats provided an additional argument for our hypothesis. It was found that chronic elevation of brain corticotropin-releasing hormone evolves in an attenuated rise in hippocampal 5-HT levels during peripheral inflammation together with a delayed development of sickness behavior (Linthorst et al., 1997).

It should be emphasized that our hypothesis on the functional implications of peripheral inflammation-induced neurotransmitter changes is based on correlative evidence. Additional experiments using specific noradrenergic and serotonergic antagonists, lesion paradigms, antisense oligodeoxynucleotide treatments and mutant animals will be needed to further increase our insight into the role of neurotransmission changes in brain-mediated responses to peripheral inflammation.

1. Auerbach, SB, Minzenberg, MJ, and Wilkinson, LO (1989) Extracellular serotonin and 5-hydroxyindoleacetic acid in hypothalamus of the unanesthetized rat measured by in vivo dialysis coupled to high-performance liquid chromatography with electrochemical detection: dialysate serotonin reflects neuronal release. Brain Research, 499:281-290.
2. Crespi, F, Garratt, JC, Sleight, AJ, and Marsden, CA (1990) In vivo evidence that 5-hydroxytryptamine (5-HT) neuronal firing and release are not necessarily correlated with 5-HT metabolism. Neuroscience, 35:139-144.
3. Dantzer, R, Bluthé, RM, Kent, S, and Kelley, KW (1991) Behavioural effects of cytokines. In N Rothwell and R Dantzer (Eds.), Interleukin-1 in the brain (pp. 135-150). Oxford: Pergamon Press.
4. Gottschall, PE, Komaki, G, and Arimura, A (1992) Interleukin-1 activation of the central nervous system. In N Rothwell and R Dantzer (Eds.), Interleukin-1 in the brain (pp. 27-49). Oxford: Pergamon Press.
5. Hart, BL (1988) Biological basis of the behavior of sick animals. Neuroscience and Biobehavioral Reviews, 12:123-137.
6. Hopkins, SJ, and Rothwell, NJ (1995) Cytokines and the nervous system I: expression and recognition. Trends in Neurosciences, 18:83-88.
7. Jacobs, BL, and Azmitia, EC (1992) Structure and function of the brain serotonin system. Physiological Reviews, 72:165-229.
8. Kalén, P, Strecker, RE, Rosengren, E, and Björklund, A (1988) Endogenous release of neuronal serotonin and 5-hydroxyindoleacetic acid in the caudate-putamen of the rat as revealed by intracerebral dialysis coupled to high-performance liquid chromatography with fluorimetric detection. Journal of Neurochemistry, 51:1422-1435.
9. Kalén, P, Rosegren, E, Lindvall, O, and Björklund, A (1989) Hippocampal noradrenaline and serotonin release over 24 hours as measured by the dialysis technique in freely moving rats: correlation to behavioural activity state, effect of handling and tail-pinch. European Journal of Neuroscience, 1:181-188.
10. Karanth, S, Lyson, K, and McCann, SM (1993) Role of nitric oxide in interleukin 2-induced corticotropin-releasing factor release from incubated hypothalami. Proceedings of the National Academy of Sciences of the United States of America, 90:3383-3387.
11. Katsuura, G, Gottschall, PE, Dahl, RR, and Arimura, A (1988) Adrenocorticotropin release induced by intracerebroventricular injection of recombinant human interleukin-1 in rats: possible involvement of prostaglandin. Endocrinology, 122:1773-1779.
12. Linthorst, ACE, Flachskamm, C, Holsboer, F, and Reul, JMHM (1994) Local administration of recombinant human interleukin-1 beta in the rat hippocampus increases serotonergic neurotransmission, hypothalamic-pituitary-adrenocortical axis activity, and body temperature. Endocrinology, 135:520-532.
13. Linthorst, ACE, Flachskamm, C, Holsboer, F, and Reul, JMHM (1995a) Intraperitoneal administration of bacterial endotoxin enhances noradrenergic neurotransmission in the rat preoptic area: relationship with body temperature and hypothalamic-pituitary- adrenocortical axis activity. European Journal of Neuroscience, 7:2418-2430.
14. Linthorst, ACE, Flachskamm, C, Müller-Preuss, P, Holsboer, F, and Reul, JMHM (1995b) Effect of bacterial endotoxin and interleukin-1 beta on hippocampal serotonergic neurotransmission, behavioral activity, and free corticosterone levels: an in vivo microdialysis study. Journal of Neuroscience, 15:2920-2934.
15. Linthorst, ACE, Flachskamm, C, Holsboer, F, and Reul, JMHM (1996) Activation of serotonergic and noradrenergic neurotransmission in the rat hippocampus after peripheral administration of bacterial endotoxin: involvement of the cyclo-oxygenase pathway. Neuroscience, 72:989-997.
16. Linthorst, ACE, Flachskamm, C, Hopkins, SJ, Hoadley, ME, Labeur, MS, Holsboer, F, and Reul, JMHM (1997) Long-term intracerebroventricular infusion of corticotropin-releasing hormone alters neuroendocrine, neurochemical, autonomic, behavioral, and cytokine responses to a systemic inflammatory challenge. Journal of Neuroscience, 17:4448-4460.
17. Pauli, S, Linthorst, ACE, and Reul, JMHM (1998) Tumour necrosis factor-alpha and interleukin-2 differentially affect hippocampal serotonergic neurotransmission, behavioural activity, body temperature and hypothalamic-pituitary-adrenocortical axis activity in the rat. European Journal of Neuroscience, 10:868-878.
18. Raber, J, and Bloom, FE (1994) IL-2 induces vasopressin release from the hypothalamus and the amygdala: Role of nitric oxide-mediated signaling. Journal of Neuroscience, 14:6187-6195.
19. Reul, JMHM, Labeur, MS, Wiegers, GJ, and Linthorst, ACE (1998) Altered neuroimmunoendocrine communication during a condition of chronically increased brain corticotropin-releasing hormone drive. Annals of the New York Academy of Sciences, 840:444-455.
20. Rivier, C, and Shen, GH (1994) In the rat, endogenous nitric oxide modulates the response of the hypothalamic-pituitary-adrenal axis to interleukin-1 beta, vasopressin, and oxytocin. Journal of Neuroscience, 14:1985-1993.
21. Rothwell, NJ (1991) Functions and mechanisms of interleukin 1 in the brain. Trends in Pharmacological Sciences, 12:430-436.
22. Rueter, LE, and Jacobs, BL (1996) A microdialysis examination of serotonin release in the rat forebrain induced by behavioral environmental manipulations. Brain Research, 739:57-69.
23. Sirko, S, Bishai, I, and Coceani, F (1989) Prostaglandin formation in the hypothalamus in vivo: effect of pyrogens. American Journal of Physiology, 256:R616-24.
24. Smith, T, Hewson, AK, Quarrie, L, Leonard, JP, and Cuzner, ML (1994) Hypothalamic PGE(2) and cAMP production and adrenocortical activation following intraperitoneal endotoxin injection - In vivo microdialysis studies in Lewis and Fischer rats. Neuroendocrinology, 59:396-405.