502 The Journal of Cell Biology | Volume 159, Number 3, 2002 Figure 5. Spontaneous advance of the growth cone of a stage 3 neurite occurring during the towing process. (A) Experimental neurite (arrowhead) immediately before manipulation. (B–D) Growth cone spontaneously undertakes to change adhesion to the culture dish and extends 48 m over the next 90 min. Bar, 20 m. likelihood of spontaneous growth cone advance, and subse- quent longer term growth. In 10 control experiments, we attached a polylysine- ConA–treated needle to a minor process of a stage 2 neuron and did not exert any tension, i.e., no manipulations of the processes after attachment was achieved. In all 10 trials, the experimental minor process failed to respond in any observ- able manner to attachment only. There were no elongations of the minor processes, no change in motility of their distal ends, and the other unattached, minor processes continued to show their characteristically limited activity. We at- tempted to maintain attachment of each minor process for 3 h, but in half the cases the minor process changed its attach- ment from the needle to the dish and “sat there.” Among those neurons still observable the following day, none of the experimental minor processes subsequently showed sponta- neous elongation into an axon. This unresponsiveness is in sharp contrast to the variety of behaviors stimulated by ma- nipulations of the needle as outlined in the data below. We conclude that attachment and presence of the experimental needle is not itself a growth or motility stimulus to minor processes, rather that tension and deformation is the relevant experimental stimulus imposed by the needle. Experimentally elongated neurites show spontaneous rapid growth Among the earliest indications of spontaneous axon deter- mination in hippocampal neurons is the rapid outgrowth of one minor process (Dotti et al., 1988; Craig and Banker, 1994). During the course of our experiments, we found that neurites that were subjected to alternating periods of tension and relaxation frequently changed their attachment from the needle to the substratum and began elongation guided by its own growth cone motility. Although the growth cone changed attachment and activity to the substratum, the ex- perimental neurite invariably remained attached to the nee- dle, in contrast to the control experiments above. Fig. 5 shows an example of growth past the needle (Fig. 5, B–D) in an early stage 3 neuron. It is noteworthy that rapid growth past the needle first began when the towed neurite was only 60% the length of the original axon, suggesting that match- ing the length of the original axon was not necessary for this behavior. Among 50 experimental neurites (27 stage 2; 23 stage 3), we observed 890 m of spontaneous growth past the needle during a total period of 60 h, comprising individ- ual examples ranging from 5 min to 2 h 20 min. Thus, these serendipitous bouts of spontaneous growth cone advance averaged 15 m/h, although we observed maximum rates of elongation as high as 80 m/h, albeit only during a brief 8-min period. More generally, short term maximum growth rates were in the 20–40 m/h range. During longer term time-lapse observations of axon outgrowth, others reported slower average elongation rates of 6–7 m/h but similar maximum growth rates (Dotti et al., 1988; Ruthel and Banker, 1999). Our higher average rates may reflect the ten- dency for initial axon outgrowth to be more rapid and con- tain fewer pauses than later axon outgrowth (unpublished data). In agreement with Dotti et al. (1988), we observed very little long term change in the lengths of unpulled minor processes ( 1 m/h on average). Experimentally elongated neurites have the same life histories of outgrowth and persistence as do spontaneous early axons If experimentally elongated neurites become axons, we would expect them to remain extended and continue elon- gating in the days after a successful needle removal. In Fig. 2, for example, a minor process of a stage 3 cell was towed and the original axon (left branch) and experimental neurite both showed growth, unlike the remaining minor processes. Fig. 6 shows an example of extensive spontaneous out- growth from an experimental neurite of a stage 2 neuron. After successful needle removal (Fig. 6 B), the experimental neurite grew an additional 225 m during the first 38 h of incubation (Fig. 6 C) and grew a branch 24 h later (Fig. 6 D). Note in these images the near absence in growth of any of the minor processes during this period. After developing the tension-relaxation protocol to encourage growth cone