The contribution of microglia to the modulation of neurogenesis under pathological conditions is unclear. Both proand anti-neurogenic effects have been reported, likely reflecting the complexity of microglial activation process. We previously demonstrated that prolonged (72 hr) in vitro exposure to lipopolysaccharide (LPS) endows microglia with a potentially neuroprotective phenotype, here referred as to ''chronic''. In the present study we further characterized the chronic phenotype and investigated whether it might differently regulate the properties of embryonic and adult neural precursor cells (NPC) with respect to the ''acute'' phenotype acquired following a single (24 hr) LPS stimulation. We show that the LPS-dependent induction of the proinflammatory cytokines interleukin (IL)-1a, IL-1b, IL-6, and tumor necrosis factor (TNF)-a was strongly reduced after chronic stimulation of microglia, as compared with acute stimulation. Conversely, the synthesis of the anti-inflammatory cytokine IL-10 and the immunomodulatory prostaglandin E 2 (PGE 2 ) was still elevated or further increased, after chronic LPS exposure, as revealed by real time PCR and ELISA techniques. Acutely activated microglia, or their conditioned medium, reduced NPC survival, prevented neuronal differentiation and strongly increased glial differentiation, likely through the release of proinflammatory cytokines, whereas chronically activated microglia were permissive to neuronal differentiation and cell survival, and still supported glial differentiation. Our data suggest that, in a chronically altered environment, persistently activated microglia can display protective functions that favor rather than hinder brain repair processes. V V C

1These authors contributed equally to this study.Abbreviations used: 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine-methiodide; ACh, acetylcholine; AChE, acetylcholinesterase; ChAT, choline acetyltransferase; dbcAMP, dibutyryl cAMP; DRG, dorsal root ganglia; EGR-1, early growth response factor-1; IP 3 , inositol triphosphate; mAChR, muscarinic acetylcholine receptors; PBS, phosphate-buffered saline; PGE1, prostaglandin E1; PKC, protein kinase C; Ptx, pertussis toxin; QNB, quinuclidinyl benzilate; REST, Repressor Element-1 silencing transcription factor; TCA, trichloroacetic acid; Zn-EGR clones, Zn-EGR transfected clones; Zn-EGR, EGR zinc-finger domain. AbstractNeurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase-transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1-M4 subtypes and the activation of both IP 3 and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor-1 (EGR-1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR-1 induction. The role of EGR-1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR-1. EGR-1 clones show increased neurite extension and a decrease in Repressor Element-1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc-finger domain, a dominant negative inhibitor of EGR-1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR-1 and REST transcription factors.

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Neurotransmitters play important roles in neurogenesis; in particular, acetylcholine and serotonin may regulate neurite elongation. Acetylcholine may also activate transcription factors such as early growth response protein 1 (EGR-1), which plays a role in neurite extension. N18TG2 neuroblastoma cells (which do not produce neurotransmitters and constitutively express muscarinic acetylcholine receptors) were transfected with constructs containing the cDNA for choline acetyltransferase, 5-hydroxytryptamine 1A (5-HT_1A) and 5-HT_2B serotonin receptors to study acetylcholine and serotonin interplay in neurite outgrowth. 5-HT_1A receptor stimulation causes a decrease in EGR-1 levels and inhibition of neurite outgrowth; 5-HT_2B stimulation, however, has no effect. Muscarinic cholinergic stimulation, on the other end, increases EGR-1 levels and fiber outgrowth. Inhibition of EGR-1 binding reduces fiber outgrowth activity. When both cholinergic and 5-HT_1A receptors are stimulated, fiber outgrowth is restored; therefore, acetylcholine counterbalances the inhibitory effect of serotonin on neurite outgrowth. These results suggest that EGR-1 plays a role in the interplay of acetylcholine and serotonin in the regulation of neurite extension during development.