Developing Neurons Form Transient Nanotubes Facilitating Electrical Coupling and Calcium Signaling with Distant Astrocytes

Despite the well-documented cooperation between neurons and astrocytes little is known as to how these interactions are initiated. We show here by differential interference contrast microscopy that immature hippocampal neurons generated short protrusions towards astrocytes resulting in tunneling nanotube (TNT) formation with an average lifetime of 15 minutes. Fluorescence microscopy revealed that all TNTs between the two cell types contained microtubules but 35% of them were F-actin negative. Immunolabeling against connexin 43 showed that this gap junction marker localized at the contact site of TNTs with astrocytes. Using optical membrane-potential measurements combined with mechanical stimulation, we observed that ∼35% of immature neurons were electrically coupled with distant astrocytes via TNTs up to 5 hours after co-culture but not after 24 hours. Connexin 43 was expressed by most neurons at 5 hours of co-culture but was not detected in neurons after 24 hours. We show that TNTs mediated the propagation of both depolarization and transient calcium signals from distant astrocytes to neurons. Our findings suggest that within a limited maturation period developing neurons establish electrical coupling and exchange of calcium signals with astrocytes via TNTs, which correlates with a high neuronal expression level of connexin 43. This novel cell-cell communication pathway between cells of the central nervous system provides new concepts in our understanding of neuronal migration and differentiation.