Axonal specification by tension | Lamoureux et al. 501 Figure 3. Elongation by towing produces neurites with a high density array of microtubules in stage 2 and stage 3 neurons. (A) At the beginning of towing of a stage 2 neuron. (B) At the end of towing 4:30 later. Immediately after needle removal, the cell was fixed, lysed, and processed for microtubule immunofluorescent localization (C). Bar, 20 m. (A’) At the beginning of towing of a stage 3 neuron. (B’) At the end of towing 9 h later. (C’) Immediately after needle removal, the cell was fixed, lysed, and processed for microtubule immunofluorescent localization. Bar, 20 m. pocampal neurites produced extensive elongation that main- tained a uniform caliber (Figs. 1 and 2) and contained a high density array of microtubules (Fig. 3). Also similar to all other cultured neurons investigated, the elongation rate of hippocampal neurites was a robust linear function of ex- perimentally applied tension (Fig. 4). That is, each neurite shows a linear relationship of elongation rate with respect to the tension levels applied to it, but the slopes (tension sensi- tivity) and x-intercepts (threshold for growth) of the linear relationships differ from cell to cell and vary over a range. For example, the top line in Fig. 4 comes from a stage 3 neuron whose spontaneous axon was pulled, whereas the bottom line is the result of towing a minor process of a stage 2 neuron. The minor processes and axons of hippo- campal neurons showed qualitative and quantitative charac- teristics quite similar to that of chick forebrain neurons (Chada et al., 1997; Heidemann et al., 2001). Like chick forebrain neurons, rat hippocampal neurites have a low threshold, 20–40 dynes to begin elongation as shown in Fig. 4. Peripheral neurons, in contrast, do not typically elongate their neurites until threshold forces of 100–200 dynes are reached (Zheng et al., 1991; Lamoureux et al., 1997). Like chick forebrain neurons, hippocampal neurons show rapid growth (100 m/h) at tensions of 100 dynes. Like the two types of neurites shown in Fig. 4, all hippocampal neurites show an increase in growth rate of 1–3 m/h for each additional dyne of tension applied. This degree of growth responsiveness to tension is similar to that found for chick sensory neurons, chick forebrain neu- rons, and PC12 cells (Zheng et al., 1991; Chada et al., 1997; Lamoureux et al., 1997). Thus, hippocampal neu- rites, minor processes, or axons grew in response to tension very much as do other cultured neurons (Heidemann et al., 1995). More generally, we noted no systematic difference in experimental neurite behavior between stage 2 and stage 3 neurons in 1–2-d-old cultures or between axons and minor processes. That is, all neurites behaved over a similar range with respect to ease of needle attachment, forces required for elongation, tendency for detachment while under tow, Figure 4. Growth rate of rat hippocampal neurites as a function of applied tension. Two neurites from two neurons were subjected to a step-function protocol of experimental tension, where each step (data point) represents towing at constant tension for 60–90 min. Growth rate was calculated from the increased length of the neurites divided by the elapsed time under tow at that tension. The top line was from a spontaneous axon of a stage 3 neuron, and the bottom line was from a minor process of a stage 2 neuron.