Figure 10. De novo initiation of short experimental neurite and subsequent spontaneous lengthy elongation during 4 d of incubation. (A) Neurite of stage 2 cell immediately before needle attachment. Note the presence of landmark cells around the experimental neuron. (B) After the 20 min required for needle attachment and 3 min of applied tension, a short neurite has been initiated. (C) 3 h later at the end of towing, the experimental neurite has elongated 30 m. Note that the experimental neurite remains shorter than its sibling neurites. (D) Immediately after needle detachment, which slightly elongated and deformed the experimental axon. (E) 3 d after D (and a 90 counterclockwise rotation of the optical field), the experimental neurite (arrowheads) has elongated extensively, whereas the remaining minor processes have remained short. Bar, 50 m. ever, we obtained 14 examples, including both stage 2 and stage 3 neurons, where experimental tension caused axonal specification in experimental neurites only towed a short dis- tance, so they were shorter than their sibling minor processes at the end of towing. Most of these came from experiments that exploited the capacity to initiate neurites de novo (and thus begin at zero length) by applying mechanical tension to the cell body. The initiation protocol was similar to that de- scribed previously for other cultured neurons (Zheng et al., 1991; Chada et al., 1997; Lamoureux et al., 1997). Attach- ment of the needle to the margin of the hippocampal cell body and subsequent towing causes initiation of a uniform caliber neurite (Figs. 10 and 11). Given the required deli- cacy of towing to initiate a neurite, the initiation protocol al- lowed a 2–3-h period of towing that ceased while the experi- mental neurite was still shorter than its sibling neurites. Among 14 initiation experiments in which the towed neu- Axonal specification by tension | Lamoureux et al. 505 rite remained shorter than its siblings (and the needle was successfully removed), 5 out of 14 showed no growth of any of the neurites and 9 out of 14 subsequently grew into long, axon-like extensions. Fig. 10 shows an example of the latter category allowed to continue outgrowth for several days hav- ing clearly become the axon. Fig. 11 shows an example in which the initiated neurite was fixed and stained for dephos- pho-tau after 24 h of outgrowth. This neurite showed nearly complete compartmentation of the antigen to the axon, al- though not a gradient in this instance. In addition to neu- rites initiated de novo, there were five examples of axonal specification from experiments in which towing of an exist- ing minor processes ceased before the experimental neurite became longer than sibling neurites. The short experimental neurites that became axons had 51 sibling minor processes among them at the end of towing, and only 2 of these showed any subsequent elongation. Clearly, specification of the experimental neurites was due to towing and not a ran- dom specification by chance. In the two cases of additional minor process outgrowth, the experimentally induced neu- rite persisted and so minor process elongation formed neu- rons with two apparent axons after several days of growth. Discussion Mechanical tension applied to hippocampal neurons can initiate and elongate processes into uniform-caliber neurites of considerable length. We and many others have observed that the neurites of hippocampal neurons at early times in culture fit into two clearly distinct categories, axon or minor process (Dotti et al., 1988; Craig and Banker, 1994). Thus, the neurites we elongated experimentally can only be axons or long minor processes. Four well-established markers indi- cate that our experimentally extended neurites differentiated into axons and were not simply long minor processes. These four indicators of axonal character are rapid growth cone ad- vance rate, continued persistence and elongation similar to that of spontaneous axons, and axonal enrichment of de- phospho-tau and of L1. Rapid growth cone advance is one of the earliest and most frequently cited indicators of axonal specification in hippo- campal neurons (Dotti et al., 1988; Craig and Banker, 1994). We found that if we applied intermittent force with a towing needle to mimic the periodic force that normally underlies axonal elongation by growth cones (Lamoureux et al., 1998), then the towed neurite developed a growth cone that rou- tinely (re)attached to the substratum and advanced rapidly without the further aid of experimentally applied tension. We regard this unusually rapid growth cone activity to be a particularly convincing indicator of axonal character for sev- eral reasons. First, it provides evidence that tension stimulates the growth cone, the usual agent of neurite lengthening. That is, the rapid extension of one minor process to become an axon indicates that one growth cone has become more active than those of the sibling neurites, and neurite towing pro- duced just the sort of substantial and routine stimulation of growth cone activity (Fig. 5) expected of axonal specification. Further, the towed neurite “volunteered” this growth cone activity insofar as the distal end of the neurite was initially otherwise engaged by attachment to the needle. Finally, no