Action Potentials Increase Myelination via an LIF-Dependent Mechanism with Astrocytes Providing the Major Source of LIF in Response to 2MeSATP Treatment(A) LIF neutralizing antibody blocked both the 2MeSATP and action potential-mediated increase in myelination (ANOVA, p = 0.43, n = 68), suggesting that action potentials and 2MeSATP increase myelination via the same LIF-dependent mechanism. Error bars represent SEM. (B) The release of LIF after 24 hr electrical stimulation of DRG axons in coculture with oligodendrocytes was blocked by stimulation in the presence of 30 U/ml apyrase, an enzyme that rapidly degrades extracellular ATP (p < 0.0001, ANOVA among all four groups; p < 0.05 elect. Stim. versus elect. Stim. + apyrase). This is consistent with the hypothesis that ATP released from axons firing action potentials in turn induces release of LIF by a mechanism involving activation of P2 purinergic receptors. Treatment with apyrase alone had no effect on LIF release. Error bars represent SEM. (C) Astrocytes release LIF in response to 2MeSATP treatment in a concentration-dependent manner, with a maximal response between 100 and 300 μM. Error bars represent SEM. (D) To identify the cellular source of LIF in co-cultures, astrocytes, DRG neurons, and oligodendrocytes were cultured separately and treated with 100 μM 2MeSATP for 1-3 days. In contrast to the large amount of LIF released by 2MeSATP treatment of astrocytes, monocultures of DRG neurons or oligodendrocytes released little LIF. The concentration of LIF in treated versus untreated cultures of astrocytes was significantly different (0.247 6 0.055 versus 7.21 6 0.049 ng/ml LIF in control versus 2MeSATP; p < 0.0001). The small amount of LIF released in oligodendrocyte cultures could derive from contaminating GFAP-positive cells, which increase in oligodendrocyte cultures from 3% to 8% by 10 days in culture. Double asterisks, p < 0.001. Error bars represent SEM.