The viral mimetic polyinosinic:polycytidylic acid (poly I:C) induces cellular responses in primary cultures from rat brain sites with an incomplete blood-brain barrier.

Primary microcultures of the organum vasculosum laminae terminalis (OVLT) and the area postrema (AP), brain sites with an incomplete blood-brain barrier, were established from topographically excised rat pup tissue, with cellular identification by marker protein-specific immunocytochemistry. Employing the ratio calcium imaging technique, we showed for the first time that polyinosinic:polycytidylic acid (poly I:C) can induce calcium signalling in single OVLT and AP cells. Poly I:C stimulation caused fast, transient rises in intracellular calcium in about 5% of neurons and astrocytes and some microglial cells. Frequently, the responses of astrocytes and microglial cells showed a shorter onset-latency compared to neurons. In addition, exposure to poly I:C led to a time dependent release of bioactive tumour necrosis factor (TNF) and interleukin-6 (IL-6) into the supernatants of OVLT and AP cultures. The demonstration of direct cellular responses of OVLT- and AP-intrinsic cells to stimulations with poly I:C is in agreement with the discovered existence of Toll-like receptor 3 (TLR3), the cognate receptor for poly I:C, in the brain.

Neurons and glial cells of the rat organum vasculosum laminae terminalis directly respond to lipopolysaccharide and pyrogenic cytokines.

During systemic immune challenge, the organum vasculosum laminae terminalis (OVLT) with its dense vascularization by fenestrated capillaries lacking blood-brain barrier function allows direct access of circulating pyrogens to brain tissue located in close vicinity to the preoptic area. We aimed to analyze direct responses of OVLT cells to exposure to lipopolysaccharide (LPS) and fibroblast-stimulating lipopeptide-1 (FSL-1) or the cytokines tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6. A primary microculture of the OVLT was established from topographically excised rat pup brain tissue, with cellular identification by marker protein-specific immunocytochemistry. Employing the ratio calcium imaging technique, pyrogen-induced calcium signaling in single OVLT cells could be characterized. LPS--as opposed to FSL-1--stimulation caused fast, transient rises in intracellular calcium concentration in 17% of neurons, 9% of astrocytes, and <5% of microglial cells investigated. LPS additionally led to enhanced expression of TNF-α and IL-1ß exclusively in microglial cells, as well as a time-dependent release of TNF-α and IL-6 from OVLT microcultures. TNF-α evoked calcium signals in 11% of neurons, 22% of astrocytes, and 5% of microglial cells tested. A considerable population of neurons (11%) but only few astrocytes and microglial cells responded to IL-6, whereas 8% of microglial cells and 3% of astrocytes or neurons were activated by IL-1ß. The demonstration of direct cellular responses of OVLT-intrinsic cells to stimulations with LPS or cytokines reinforces the suggested role of this brain structure as a responsive brain site to circulating pyrogens.

Tumor necrosis factor-alpha, interleukin-1beta and nitric oxide induce calcium transients in distinct populations of cells cultured from the rat area postrema.

The area postrema (AP) represents the medullary sensory circumventricular organ lacking endothelial blood-brain barrier function at the base of the 4th cerebral ventricle. Administration of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) or the nitric oxide (NO) donor diethylamino-diazenolate-2-oxide (DEA) caused fast transient rises in intracellular calcium concentrations ([Ca(2+)]i) in distinct populations of cells investigated in a primary microculture of the rat AP. TNF-alpha caused rapid elevations of [Ca(2+)]i in 8% of all neurons and astrocytes investigated, with limited responses of microglial cells and no responses of oligodendrocytes. 15% of all neurons investigated responded to IL-1beta, while only 5-7% of the other cell types showed rises in [Ca(2+)]i. The most pronounced effects were caused by treatment with DEA with some 20% responsive astrocytes and oligodendrocytes, 15% neurons and 10% microglial cells. Evidently, the AP can act as a sensor for circulating TNF-alpha and IL-1beta, or for locally produced cytokines and NO during infection and inflammation.

Rat area postrema microglial cells act as sensors for the toll-like receptor-4 agonist lipopolysaccharide.

The area postrema (AP) represents the sensory circumventricular organ lacking endothelial blood-brain barrier function in direct vicinity to the 4th cerebral ventricle. Administration of lipopolysaccharide (LPS), as opposed to muramyldipeptide (MDP) or fibroblast-stimulating lipopeptide-1 (FSL-1), caused fast transient rises in intracellular calcium concentrations in 10-12% of the microglial cells investigated in a primary microculture of the rat AP,with limited responses of neurons, astrocytes and oligodendrocytes. In addition, a marked release of the pro-inflammatory cytokines TNF-alpha and IL-6 was determined in LPS-treated AP microcultures. Pre-incubation of AP microcultures with LPS for 18 h suppressed LPS-induced calcium signaling and attenuated cytokine secretion. Evidently, the AP can act as a sensor for circulating LPS and has the capacity to develop endotoxin-tolerance.