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-1β (IL-1β), 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-1β 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-1β. 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.

Research Highlights
► OVLT cells respond to LPS stimulation with a rapid increase of intracellular Ca2+.
► OVLT microglial cells produce cytokines in response to LPS stimulation.
► TNF-α, IL-β, and IL-6 evoke Ca2+ signals in distinct populations of OVLT cells.
► The responses to LPS and IL-6 are unique compared to other CVOs.
► The role of the OVLT as a rapid sensor of circulating pyrogens is reinforced.