The area postrema (AP) represents the sensory circumventricular organ in direct vicinity to the 4th cerebral ventricle lacking a tight endothelial blood-brain barrier. During the process of bacterial infection, neurons / glial cells of the AP are therefore accessible to circulating, bacteria-related "pathogen-associated molecular patterns" (PAMPs) such as lipopolysaccharide (LPS) derived from gram-negative bacteria, muramyldipeptide (MDP) from gram-positive bacteria or fibroblast-stimulating lipopeptide-1 (FSL-1) from Mycoplasma. Due to the known expression of the respective Toll-like receptors (TLRs) for these PAMPs within the AP, direct activation of its cellular elements by a given PAMP seems possible. To study specific PAMP-mediated cellular activation, an AP primary microculture was established from topographically excised brain tissue of 5–6 days old rat pups. The Fura-2 ratio imaging technique was used to quantify PAMP-induced calcium signalling in AP micro-culture chambers superfused with oxygenated HEPES buffer containing LPS, MDP, FSL-1, glutamate or potassium. Subsequent immunolabeling with antisera directed against cell-specific marker proteins allowed identification of neurons, astrocytes, oligodendrocytes and microglial cells (dormant and activated). Additionally, concentrations of proinflammatory cytokines (TNF-a, IL-6) released into the supernatant of LPS-stimulated AP microcultures were determined by use of specific bioassays. Bath application of LPS in induced fast, transient rises in intracellular calcium concentration in 4% of neurons, 2.5% of astrocytes, < 1% of oligodendrocytes but 10% of microglial cells investigated. Only very few AP cells responded to stimulations with MDP or FSL-1. In the supernatants of LPS-treated AP-microcultures, significant amounts of bioactive TNF-a and a moderate to strong increase of IL-6 were detected, and immunocyto-/histochemical analysis suggests activation of microglial cells in parallel. Pre-incubation of AP microcultures with LPS for 18 hours caused a complete abrogation of LPS-induced calcium signaling and a strong attenuation of LPS-induced cytokine formation by AP-cells. The demonstration of direct cellular responses of AP-intrinsic microglia cells to LPS-stimulation raises the intriguing possibility that the AP can act as a sensor for circulating PAMPs, mainly the TLR4-agonist LPS. Thereby, a rapid induction of brain-controlled illness responses prior to formation of endogenous secondary mediators within the brain seems possible. The marked induction of cytokine formation by activated microglial cells upon LPS-stimulation may cause delayed inflammatory symptoms in vivo. Finally, the lack of response to LPS by pre-exposure of AP cultures with this PAMP suggests that there is a brain-intrinsic development of a phenomenon termed "endotoxin tolerance".

Supported by Deutsche Forschungsgemeinschaft (DFG)(GE 649/6–1).