Multiaxonal neurons mature and form neuronal networks. (A–E) Maturation of taxol-treated neurons. To unequivocally identify the origin of axons in the dense neuronal network neurons establish, we used cultures of mouse hippocampal neurons in which a subset of cells expressed EGFP under the control of a ubiquitously active promoter (Okabe et al., 1997). Mixed wild-type/GFP cultures containing 1–3% of neurons expressing GFP allow individual neurons to be followed in dense networks (A and B). Taxol (final concentration 3 μm) was added to the medium of the mixed culture after 1 DIV, neurons were further grown in the presence of the drug, fixed after 11 DIV, and immunostained for GFP (green) and the presynaptic marker synapsin 1 (blue). (A and B) Taxol-treated neurons have formed multiple axons (arrows) in 81.3 ± 2.2% of the cases (n > 120 neurons). (C–E) Higher magnifications of the regions marked in A and B. The multiaxonal GFP-positive neurons cluster the presynaptic marker synapsin 1 in their axons. Magnifications of the regions boxed in C–E are shown as insets to better visualize the localization of synapsin 1 on GFP-positive axons (arrows). Bars: (A and B) 100 μm; (C–E) 20 μm.