activity, as described above, 31 of 38 experimental neurites (20 stage 2 and 11 stage 3 neurons) that were successfully detached from the needle grew or at least remained extended for 24 h after experimental intervention. 23 out of 38 grew at least an additional 50% beyond their towed length; 12 of these 23 more than doubled their towed length. Five more grew slightly (15–25%), whereas three remained ex- tended but did not change length significantly. Only 7 out of 38 neurites failed to persist; generally the towed neurite retracted and no axon formed at all; in two cases a different neurite became an axon. We wished to determine whether the extent of axonal growth and its stability among experimental neurites was similar to that of spontaneously specified axons at early times in stage 3. We followed the fate of 45 selected neurons that had spontaneously developed to stage 3 (one neurite 75 m) on the day after plating. Thus, these cells were in culture for the same period and showed a similar extent of development as our experimental neurons, except that here we did not use stage 2 neurons. These controls were sub- jected to similar insults as experimental neurons (same changes of medium, several hours on the heated microscope stage, same perturbations of the medium incident to trans- port, etc.) but without needle interventions of any kind. Of these 45 early stage 3 neurons, 30 maintained the initially specified neurite as an axon by remaining extended or grow- ing. Of these, 5 out of 30 remained extended with no addi- tional growth, 5 out of 30 grew slightly, 8 grew more than 50%, and 6 more than doubled their length, whereas 5 grew an apparent second axon, and 1 became tripolar. Impor- tantly, in the remaining 15 cases, the originally specified axon retracted, and a different neurite subsequently became the axon. Thus, the extent of outgrowth and stability of specification among spontaneously initiated axons was simi- lar to that seen among experimentally manipulated neurites. On an anecdotal level, our observations of these control neu- rons were noteworthy because we saw that unmanipulated axons undergo many of the same growth behaviors that ma- Axonal specification by tension | Lamoureux et al. 503 Figure 6. Longer term spontaneous growth subsequent to towing neurite of stage 2 cell. (A) Neurite of stage 2 cell immediately after needle attachment. (B) Neuron at end of towing process after needle detachment. (C) Same neuron 38 h after B. The towed neurite has undergone 225 m of spontaneous elongation. (D) 1 d after C, the towed neurite has now grown a branch. Bar, 20 m. Figure 7. Immunofluorescent localization of dephospho-tau in experimental neurite of stage 2 neuron after 1 d of incubation. (A) Needle is attached to one of the smallest minor processes of a stage 2 neuron. (B) After slightly more than 2 h of towing with alternating periods of tension and relaxation (see Results), the experimental neurite has elongated 25 m. At this point, the growth cone advanced slightly beyond the needle, and this opportunity was taken to remove the needle and the culture replaced into the incubator. (C) By the next day, the experimental neurite had elongated 90 m. (D) 2 h later, the neurite had elongated an additional 20 m and was fixed and processed for dephospho-tau localization. As in this example, a clear compartmentation and gradient of dephospho-tau was seen in experimental neurites allowed to continue elongation for 1 d after towing. (Those experimental neurites fixed immediately after towing showed compartmentation but no gradient [not depicted].) Bar, 20 m.