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.

Cholinergic signal transduction in the mouse sphenopalatine ganglion.

The sphenopalatine ganglia (SPG) receive their preganglionic innervation from the ventro-lateral reticular formation and nuclei of the caudal pons, and are involved in parasympathetic control of cranial glandular and vascular components including the blood supply to specific brain areas. In 53% of all SPG neurons, a particular member (MOL2.3) of the odorant receptor superfamily is co-expressed with green fluorescent protein (GFP) in MOL2.3 transgenic mouse pups. Choline acetyltransferase and vesicular acetylcholine transporter (VAChT) could be demonstrated in 90% of the GFP-positive, and 60% of the GFP-negative cells, these cells thus representing cholinergic neurons. Some 50% of all SPG neurons were nitrergic at a high rate of VAChT co-expression, the majority of them being GFP-positive. Most SPG neurons received cholinergic innervation as demonstrated by perineuronal VAChT immunoreactive nerve terminals. To characterize cholinergic signal transduction in SPG neurons, calcium imaging experiments were performed in a SPG primary culture system containing GFP-positive and -negative neurons. Ganglionic neurons could repeatedly be activated by cholinergic stimulation in a dose-dependent manner, with calcium entering all cells from the extracellular compartment. Stimulation with specific agonists supported prevalence of nicotinic cholinergic receptors (nAChRs). Inhibition of cholinergically induced intracellular calcium signalling by various omega-conotoxins indicated functional expression of alpha 3 beta 4 and alpha 7 nAChR subtypes in murine SPG cells, which could be supported by RT-PCR analysis of the neonatal mouse SPG. With regard to secondary cholinergic activation, L- but not N-subtype voltage-gated calcium channels might represent a prime target. Nicotinic signal transduction did not prove to be different in GFP-positive as compared to-negative murine SPG neurons.

Characterization of the febrile response induced by fibroblast-stimulating lipopeptide-1 in guinea pigs.

Recently, it has been shown that the Toll-like receptors-2 and -6 agonist fibroblast-stimulating lipopeptide-1 (FSL-1) have the capacity to induce fever and sickness behavior in rats. Since the mechanisms of the fever-inducing effects of FSL-1 are still unknown, we tested the pyrogenic properties of FSL-1 in guinea pigs and assessed a role for TNF-alpha and prostaglandins in the manifestation of the febrile response to this substance. Intra-arterial and intraperitoneal injections of FSL-1 caused dose-dependent fevers that coincided with elevated plasma levels of TNF and IL-6, the intraperitoneal route of administration being more effective than the intra-arterial route. Intra-arterial or intraperitoneal injection of a soluble form of the TNF type 1 receptor, referred to as TNF binding protein (TNFbp), together with FSL-1, completely neutralized FSL-1-induced circulating TNF and reduced fever and circulating IL-6. Intra-arterial or intraperitoneal injection of the nonselective cyclooxygenase (COX)-inhibitor diclofenac depressed fever and FSL-1-induced elevations of circulating PGE2. Circulating TNF and IL-6, however, remained unimpaired by treatment with diclofenac. In conclusion, FSL-1-induced fever in guinea pigs depends, in shape and duration, on the route of administration and is, to a high degree, mediated by pyrogenic cytokines and COX products.