JCB Article Mechanical tension can specify axonal fate in hippocampal neurons Phillip Lamoureux,1 Gordon Ruthel,2 Robert E. Buxbaum,1 and Steven R. Heidemann1 1Department of Physiology, Michigan State University, East Lansing, MI 48824 2Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 Hwould stimulate undifferentiated minor processes of ere we asked whether applied mechanical tension cultured hippocampal neurons to become axons and whether tension could induce a second axon in an already polarized neuron. Experimental tension applied to minor processes produced extensions that demonstrated axonal character, regardless of the presence of an existing axon. Towed neurites showed a high rate of spontaneous growth cone advance and could continue to grow out for 1–3 d after towing. The developmental course of experimental neurites was found to be similar to that of unmanipulated spontaneous axons. Furthermore, the experimentally elon- Introduction Cultured hippocampal neurons have been widely used as a model system for the study of axonal and dendritic develop- ment (Craig and Banker, 1994). These neurons undergo a highly stereotyped sequence of axonal development. Axons develop from among undifferentiated “minor processes,” only one of which typically becomes an axon. Indeed, the rapid outgrowth of one minor process to subsequently become the axon is among the first events in the development of neuronal polarity (Dotti et al., 1988; Craig and Banker, 1994). This suggested to us a possible link between mechanical tension and axonal specification because we have shown that neurite elongation rate is a linear function of the magni- tude of pulling tension, provided either by the growth cone (Lamoureux et al., 1989) or by experimental manipulation (Zheng et al., 1991; Chada et al., 1997; Lamoureux et al., 1997). Axonal specification has been discussed recently in terms of events that break the symmetry of the cell (Andersen and Bi, 2000; Bradke and Dotti, 2000), and experimentally Address correspondence to Steve Heidemann, Dept. of Physiology, Michigan State University, East Lansing, MI 48824-3320. Tel.: (517) 355-6475, ex. 1136. Fax: (517) 355-5125. E-mail: heideman@msu.edu G. Ruthel’s present address is Dept. of Cell Biology and Biochemistry, USAMRIID, 1425 Porter St., Fort Detrick, MD 21702. Key words: cell polarity; axons; biomechanics; hippocampus; develop- mental biology  The Rockefeller University Press, 0021-9525/2002/11/499/10 $5.00 The Journal of Cell Biology, Volume 159, Number 3, November 11, 2002 499–508 http://www.jcb.org/cgi/doi/10.1083/jcb.200207174 gated neurites showed compartmentation of the axonal markers dephospho-tau and L-1 in towed outgrowth after 24 h. Extension of a second axon from an already polarized neuron does not lead to the loss of the spontaneous axon either immediately or after longer term growth. In addi- tion, we were able to initiate neurites de novo that subse- quently acquired axonal character even though spontane- ous growth cone advance began while the towed neurite was still no longer than its sibling processes. This suggests that tension rather than the achievement of a critical neu- rite length determined axonal speciﬁcation. applied tension can break symmetry in several types of cultured neurons by initiating neurites de novo from a rounded cell body (Bray, 1984; Zheng et al., 1991; Chada et al., 1997; Lamoureux et al., 1997). In observations of the earliest events of spontaneous axon initiation in chick sympathetic neurons, tension was again strongly implicated as playing a role in symmetry breaking and neurite initiation (Smith, 1994). Previous work on axonal specification in hippocam- pal neurons has focused on the role of one process attaining a critical length (Dotti and Banker, 1987; Goslin and Banker, 1989) or the role of actin dynamics in the growth cone (Bradke and Dotti, 1999; Ruthel and Hollenbeck, 2000). Both of these aspects of the problem also have links to tension. Neurite lengthening in culture usually involves growth cone advance, which in turn is dependent on the growth cone exerting tension (Lamoureux et al., 1989). The ability of the growth cone to exert tension is widely regarded to depend on the actin cytoskeleton (Lin et al., 1994; Mitchi- son and Cramer, 1996). Thus, the experimental findings on axonal specification combined with cytomechanical results sug- gested to us a simple proximate mechanism for determining the axon: the minor process that first exerts sufficient ten- sion to overcome a threshold and begins rapid elongation becomes an axon. We tested this hypothesis using calibrated glass needles to experimentally apply tension to minor processes of rat 499