imentally initiated, short neurites also developed into axons as shown by continued outgrowth and compartmentation of dephospho-tau (Figs. 10 and 11). Another common theme in models of axon specification is the existence of an unidentified inhibitor that prevents more than one neurite from becoming an axon (Craig and Banker, 1994; Andersen and Bi, 2000; Bradke and Dotti, 2000). This is meant to explain the finding that hippocampal neu- rons normally develop only one axon, and when the axon is transected to the length of its siblings, each minor process (including the transected axon) has an equal chance of be- coming the new axon (Dotti and Banker, 1987; Goslin and Banker, 1989). Surprisingly, tension-mediated elongation of minor processes and neurite initiation de novo were no more difficult to achieve with stage 3 neurons, which already pos- sessed an axon, than it was with stage 2 neurons. In fact, ex- perimental neurite behavior was indistinguishable among stage 2 and stage 3 neurons in 1–2-d-old cultures. That is, presence of an axon did not affect ease of needle attachment, forces required for elongation, tendency for detachment while under tow, likelihood of spontaneous growth cone advance, subsequent longer term growth, or high levels of dephospho-tau immunofluorescence. (Rather, the only in- stances in which experimental elongations of minor processes were problematic were in stage 2 neurons from 3–4-d-old cultures. We presume this reflects a general incapacity for neurite elongation in these neurons. That is, if the cell had not extended an axon by 3–4 d, then it appeared to be inca- pable of supporting substantial elongation either spontane- ously or experimentally.) However the formation of multiple axons might normally be inhibited, our results on stage 3 neurons indicate that such inhibition is easily overcome by the stimulus of mechanical tension. In this regard, we found among individually identified neurons in early, spontaneous stage 3 that formation of a second axon and of axonal respeci- fication is more frequent than we expected. As noted earlier, Ruthel and Hollenbeck (2000) reported that 4% of individu- ally identified stage 2 neurons developed two axons during 14 h of observation. Also, Bradke and Dotti (2000) reported that even well-differentiated dendrites of hippocampal neu- rons could become axons and that actin depolymerization could induce a high frequency of multiple axons. The pre- sumptive inhibitory influence of an axon on the growth of other neurites may be less robust than once thought. The results of this study establish that mechanical tension is a proximate stimulus to break symmetry in a neuron and specify which process becomes the axon. In the uniform en- vironment of a culture dish, this occurs as a stochastic choice among minor processes as first suggested by Craig and Banker (1994). That is, during the growth and retraction of minor processes that occurs in stage 2 neurons the first growth cone that happens to exert enough tension to begin rapid and sustained elongation becomes the axon. In the an- imal, the outcome of this stochastic “tug of war” (Bradke and Dotti, 2000) is undoubtedly affected by the environ- ment surrounding the cell. The growth cones of the minor processes probe their environment for chemical signals that stimulate a particular growth cone to exert more force and thus lead to axon specification (Brittis and Silver, 1995; Esch et al., 1999). Regardless of whether or not environ- Axonal specification by tension | Lamoureux et al. 507 mental cues are present, there must be proximate chemical stimuli that underlie the generation of mechanical tension. Effective tension is ultimately dependent on a variety of in- tracellular conditions, presumably including actin dynamics and myosin function. For example, recent studies suggest that axonal specification and growth rate is sensitively de- pendent on expression of proteins expected to increase actin turnover (Meberg and Bamburg, 2000; Kunda et al., 2001). In our view, the current study complements biochemical in- vestigations by showing that there is a clear physical basis for the development of a minor process into an axon, which in turn is dependent on biochemical dynamics. Materials and methods Culture of hippocampal neurons Hippocampal neurons were cultured from embryonic rats (E18 or E19) based on the method of Goslin and Banker (1991) with several modifica- tions. After trituration of cells, cells were frozen in commercial cell culture freezing medium (11101–011; Life Technologies, GIBCO-BRL) in aliquots that each represented the cells obtained from a single hippocampus. Ali- quots were stored in liquid N2 until use. As reported previously, hippo- campal neurons cultured after cryopreservation were indistinguishable from fresh cultures (Mattson and Kater, 1988; Ruthel and Hollenbeck, 2000). After thawing, cells were plated onto poly lysine–treated tissue cul- ture dishes and initially cultured in a 5% CO2 atmosphere for 2–4 h in glia-conditioned (Goslin and Banker, 1991) MEM medium (61100–053; GIBCO-BRL) containing 2 mM glutamine, 0.6% glucose, 10% horse se- rum, 100 U/ml of penicillin, and 100 g/ml streptomycin. After this initial culture period in bicarbonate-buffered medium, cells were transferred to glia-conditioned CO2-independent medium (18045–088; Life Technolo- gies) containing 4 mM glutamine, 1 mM pyruvate, 0.6% glucose, 10% horse serum, 100 U/ml of penicillin, 100 g/ml streptomycin, and several defined growth supplements, including insulin, transferrin, estradiol, etc., as previously detailed for the culture of chick brain neurons (Heidemann et al., 2001). These additions dramatically improve the frequency of axonal development in embryonic chick brain neurons, and we found that they also promote the development of rat hippocampal neurons. Application of experimental tension to minor processes Force was applied to hippocampal neurons with calibrated glass needles using a method that has been described in detail elsewhere (Heidemann et al., 1999). Briefly, two needles were mounted in a micromanipulator; one needle was calibrated for its bending constant and used as a pulling needle applied to the cell, whereas the other needle was used as an unloaded ref- erence for bending of the towing needle and for assessing movements of the system imposed through the micromanipulator. The bending constants of the pulling needles were between 3–9 dyne (30–90 pN)/ m, and nee- dles were pretreated with poly lysine (0.1% in PBS) and subsequently with concanavalin A (10 mg/ml in PBS) to aid attachment of the cell to the nee- dle. All applications of force were recorded by videotape at 48–120 time lapse for subsequent analysis of neurite length and needle bending (a mea- sure of force magnitude). To determine the relationship between applied tension and elongation rate, a calibrated needle was attached to the growth cone of a spontaneous neurite, and the neurite was pulled in 60–100-min “steps” of constant force (Zheng et al., 1991). That is, a tension magnitude was chosen, begin- ning at 30–50 dyne, and this tension was held constant for 1 h by mov- ing the micromanipulator to maintain the appropriate deflection of the calibrated needle. Subsequently, the same technique was used to apply 60–100-min periods of higher tension to the neurite, each level typically 20–50 dyne higher than the previous value. Neurite elongation was measured from the videotape record of the experiment. For each period of constant force, a rate of elongation was calculated from the change in neu- rite length divided by the period of time that force was applied. After most towing experiments, attempts were made to manipulate the distal end of the neurite off the needle and back onto the surface of the dish for observation of independent growth by the towed neurite or for im- munofluorescent analysis. Needle removal was more difficult for hippo- campal neurons than for other cultured neurons with which we have worked owing to the much greater tendency of hippocampal neurites to break and for the cell body to detach in response to the forces needed to