Cytomechanics of Neurite Outgrowth from Chick Brain Neurons

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Journal of Cell Science 110, 1179-1186 (1997) 1179 Printed in Great Britain © The Company of Biologists Limited 1997 JCS1398

Cytomechanics of neurite outgrowth from chick brain neurons

Sandeep Chada, Phillip Lamoureux, Robert E. Buxbaum and Steven R. Heidemann* Department of Physiology, Michigan State University, East Lansing, MI 48824, USA *Author for correspondence (e-mail: [email protected])

SUMMARY

Mechanical tension is a direct and immediate stimulus for substantially lower net tensions than peripheral neurons. neurite initiation and elongation from peripheral neurons. This is because, unlike peripheral neurons, there is no We report here that the relationship between tension and minimum threshold tension required for elongation in neurite outgrowth is equally initimate for embryonic chick forebrain neurons; all positive tensions stimulate neurite forebrain neurons. Culture of forebrain neurons was outgrowth. Consistent with this observation, chick unusually simple and reliable, and some of these cells forebrain neurons showed weak retractile behavior in undergo early events of axonal-dendritic polarity. Neurite response to slackening compared to sensory neurons. outgrowth can be initiated de novo by experimental appli- Neurites that were slackened showed only transient elastic cation of tension to the cell margin of forebrain neurons behavior and never actively produced tension, as do chick placed into culture 8-12 hours earlier, prior to spontaneous sensory neurons after slackening. We conclude that tension neurite outgrowth. Experimentally induced neurite is an important regulator of both peripheral and central elongation from these neurons shows the same robust linear neuronal growth, but that elastic behavior is much weaker relationship between elongation rate and magnitude of for forebrain neurons than peripheral neurons from the applied tension as peripheral neurons, i.e. both show a same developing organism. These data have signiﬁcance for ﬂuid-like growth response to tension. Although forebrain the understanding of the morphogenetic events of brain and sensory neurons manifest a similar distribution of development. growth sensitivity to tension (growth rate/unit tension), chick forebrain neurons initiated and elongated neurites at Key words: Cytomechanics, Axonal growth, Brain development

INTRODUCTION apparently integrates the complex chemistry underlying axonal elongation to produce a simple fluid-like relationship between Axonal development by peripheral neurons has been shown to a tension input and a growth output. be intimately dependent on mechanical tension. Axons of chick In addition to this fluid-like growth behavior, chick sensory sensory neurons and PC12 cells can be experimentally initiated neurons and PC12 cells show clear evidence of solid, elastic by applying tension to the margin of the cell body (Bray, 1984; behaviors below the tension threshold required for growth. Zheng et al., 1991; Lamoureux et al., 1997), and tension also That is, neurites of these cells behave like springs at low mediates spontaneous initiation of axons by cultured chick tension levels (Dennerll et al., 1988; Lamoureux et al., 1992). sympathetic neurons (Smith, 1994). After initiation, axonal If the neurite is rapidly lengthened by plucking with a needle, elongation occurs visibly over the course of seconds and the increase in force obeys Hook’s law. At longer time scales minutes both from experimentally applied tension (Bray, 1984; (and low tension), the neurites behave like fluid-damped Zheng et al., 1991) and from tension produced by the growth springs, i.e. behave like viscoelastic solids (Dennerll et al., cone (Lamoureux et al., 1989). When axonal elongation is 1989). And neurites of both cultured cell types support a static experimentally stimulated by ‘towing’ with a needle, the axon ‘rest tension’ that does not dissipate with time; i.e. the ‘spring’ can elongate at physiological and far-above-physiological rates of the neurite is normally slightly stretched. for many hours (Bray, 1984; Zheng et al., 1991). Most signif- Van Essen (1997) has proposed that significant aspects of icantly, in our view, the rate of neurite elongation by chick brain morphogenesis are driven by tension, based in part on the sensory neurons and PC12 cells is a simple linear function of reasonable but untested assumption that central neurons behave the applied force above some minimum threshold tension similarly to peripheral neurons in response to mechanical (usually around 100-200 µdynes) under a variety of culture forces. We report here a study of the responses of embryonic conditions and at all elongation rates (Zheng et al., 1991; chick forebrain neurons to experimentally applied tension. Lamoureux et al., 1992, 1997). That is, axonal elongation bears Brain neurons show fluid-like growth responses to tension, a robust mechanical equivalence to the elongation of a entirely similar to that of peripheral neurons from chick Newtonian fluid-mechanical element, i.e. a ‘dashpot’ like the embryos. However, brain neurons show only transient elastic piston on screen doors that prevents slamming. Thus, tension behavior to low tension levels, in contrast to chick sensory 1180 S. Chada and others neurons. We also report that these brain neurons are unusually formaldehyde solution in this same stabilization buffer, all at 37¡C, reliable and convenient to culture. followed by extraction in methanol at −20¡C. The neurites were then incubated with primary antibody to tubulin (kindly provided by Dr David Asai), rinsed, then incubated with fluorescein-labeled MATERIALS AND METHODS secondary antibody. The cells are observed through an Odyssey confocal microscope (Noran Instruments, Middleton, WI) equipped with fluorescent optics. Cell culture The presence or absence of polyribosomes in axon-like and Neurons from the cerebral hemispheres of seven- or eight-day-old dendrite-like neurites extending from chick sensory neurons after 5 chick embryos were cultured essentially by the method of Sensen- days in cultures was determined by transmission electron microscopy. brenner and colleagues (Sensenbrenner et al., 1978; Pettman et al., Fixation, dehydration, embedding and sectioning of cells were carried 1979) with some minor modifications. The principal modification is out by standard methods previously described (Joshi et al., 1986). growth of neurons in L-15 medium supplemented with 0.6% glucose, Sections stained with uranyl acetate and lead citrate were viewed with µ 2 mM glutamine, 100 i.u./ml penicillin, 100 g/ml streptomycin, 100 a Phillips CM-10 transmission electron microscope. ng/ml 7S nerve growth factor (Harlan, IN), and 10% FCS. The use of L-15 medium (Gibco Labs, NY) permits cell growth and microma- nipulation without a CO -supplemented atmosphere. Cells were 2 RESULTS grown at low density (1,500-2,000 cells/cm2) on 60 mm tissue culture dishes pretreated with poly-L-lysine (0.1% in phosphate buffered saline for 30 minutes to 1 hour and rinsed three times with sterile Chick forebrain neurons are exceptionally easy to water). culture and some develop morphologically over the course of 3-5 days Application of mechanical tension to forebrain neurons We cultured brain neurons from the telencephalon of 7- to 8- Force was applied to various regions of chick forebrain neurons with day-old chick embryos by the method of Sensenbrenner and calibrated glass needles as described by Zheng et al. (1991) and colleagues (Sensenbrenner et al., 1978; Pettmann et al., 1979; Lamoureux et al. (1992). Briefly, two needles were mounted in a Louis et al., 1981) with small modifications as described in micromanipulator; one needle was calibrated for its bending constant and used as a pulling needle applied to the cell, while the other needle Materials and Methods. Our principal modification is the use was used as an unloaded reference for bending of the towing needle of supplemented L15 culture medium, which does not require and for possible drift of the micromanipulator system. The bending a CO2 atmosphere for pH control. We find that culture of the constants of the pulling needles were between 3 and 7 µdyne/µm, and forebrain neurons is unusually simple, inexpensive, and needles were pre-treated with concanavalin A (10 mg/ml in reliable, even when compared to the relatively straightforward phosphate-buffered saline) to aid attachment of the cell to the needle. culture of chick sensory neurons (Bray, 1991). The two hemi- All applications of force were recorded by videotape at 24× time lapse spheres of the forebrain are easily identified in the intact for subsequent analysis of neurite length and needle bending, i.e. embryo within the V-shaped notch formed by the two large magnitude of forces applied. eyes and the beak. The hemispheres can be removed without For neurite intiation, chick forebrain neurons without extant recourse to a dissecting microscope. Dissociation by trypsin neurites were attached to calibrated experimental needles at the cell treatment of both hemispheres from a single embryo produces margins. The response of the cell to the application of various tension levels was recorded on videotape and analyzed for tension and neurite a large number of cells, enough for thirty to forty 60 mm length as above. culture dishes. Smaller numbers of dissociated cells can be For determination of the relationship between applied tensions and obtained without trypsin by incubation in Ca2+, Mg2+-free elongation rate, a calibrated needle was attached to the growth cone Hanks’ balanced saline for 30 minutes and mechanical tritura- of a spontaneous neurite, and the neurite was pulled in 30-60 minute tion. Culture on polylysine- or collagen-treated surfaces ‘steps’ of constant force (Zheng et al., 1991). That is, a tension appears to be required for neurite outgrowth. On plain tissue magnitude was chosen, beginning at 10-20 µdyne, and this tension culture plastic, or laminin-treated surfaces, the cells rapidly was held constant for 30-60 minutes by moving the micromanipula- form clumps, most of which do not attach to the surface. As tor to maintain the appropriate deflection of the calibrated needle. previously reported by Sensenbrenner et al., we had most Subsequently, the same technique was used to apply 30-60 minute success with polylysine-treated surfaces. periods of higher tension to the neurite, each level typically 20-50 µdyne higher than the previous value. Neurite lengthening was Fig. 1 shows the developmental course of these cultures over measured from the videotape record of the experiment. For each approx. 5 days. These neurons undergo a stereotyped develop- period of constant force, a rate of elongation was calculated from the mental sequence of neurite outgrowth that resembles that new length of neurite acquired during the period divided by its observed for rat hippocampal neurons in culture (Dotti et al., duration. 1988). Within hours of plating, the margins of cells become highly motile and display the characteristic lamellipodial Observation of cellular organelles activity described for many types of cultured neurons prior to Some tension-induced outgrowths were examined by immunofluor- neurite outgrowth. By 24 hours in culture, most cells have escence microscopy for the presence of microtubules. Following developed short ‘minor’ processes that varied between 10 and initiation or elongation by tension, the distal ends of such neurites 30 µm in length, with a few cells developing a single, long were micromanipulated from the needle back onto the culture neurite of uniform caliber (Fig. 1A). By 2-3 days in culture, substrate. A diamond-tipped ‘objective’ was used to mark the experimental cell by circling the dish beneath. Immunofluorescent staining 25-30% of all cells had developed a single long outgrowth in of microtubules was carried out by a method similar to that of addition to several shorter, ‘minor processes’ extending from Thompson et al. (1984): medium was carefully removed, and the the cell body (Fig. 1B). After 4-5 days in culture, 10-20% of culture was permeabilized in 0.5% Triton X-100 in the microtubule- all cells show a single, long, axon-like projection and several stabilizing buffer described by Thompson et al. (1984), then fixed in shorter, tapered processes that resemble dendrites (Fig. 1C). Brain neuron mechanics 1181

cytoplasm of the dendrite-like processes contained abundant polysomes throughout their length and a lower density of microtubules and neurofilaments than the axon-like processes (Fig. 2A). In contrast, the long axon-like processes of forebrain neurons (Fig. 2B) showed a complete absence of polysomes in regions 50-100 µm from the soma and a similar density of microtubules and neurofilaments to that observed previously in the axon-like neurites of chick sensory neurons (Baas et al., 1987). However, compartmentation of MAP-2 into the soma- todendritic compartment, a commonly used marker for axonal/dendritic polarity in rat neurons (Craig and Banker, 1994), did not occur in chick forebrain neurons even after 9 days in culture (immunofluorescence data not shown). In rat hippocampal neurons, differential compartmentation of MAP2 is a relatively late event, requiring approximately 7 days for complete compartmentation (Caceres et al., 1986) while axonal elongations can be identified within 2-3 days after plating at which time ribosomal compartmentation has occurred (Deitch and Banker, 1993). Chick brain neurons initiate neurites in response to experimentally applied tension As prevously described for chick sensory neurons (Zheng et al., 1991), forebrain neurons in culture were able to initiate and elongate neurites in response to mechanical tension. Eight hours after plating, initiation of neurites from cultured chick forebrain neurons was induced by experimentally applying tension to random locations along the margin with calibrated glass needles (Figs 3 and 4). In approximately half the cases, a uniform caliber cytoplasmic process formed with its distal tip attached to the needle (Fig. 3). Initiation in which the pulling needle was distal to the neurite required quite small forces. In 14 experiments, the mean initiating tension was 31±6 (s.e.m.) µdynes, with the minimum tension of 5 µdynes and a maximum of 80 µdynes. This is substantially less than the forces typically required for experimentally induced neurite initiation from chick sensory neurons (100-200 µdynes; Zheng et al., 1991) or PC12 cells (300-1,000 µdynes; Lamoureux et Fig. 1. Development of spontaneously initiated neurites from al., 1997). Within the experimentally initiated forebrain cultured chick forebrain neurons. A series of phase micrographs neurites, immuno-cytochemistry using fluorescently labeled taken from a circled region of a culture dish at various intervals β following plating. Stages in development are as in Dotti et al. (1988). antibodies against -tubulin demonstrated the presence of (A) After one day in culture, one neuron has extended a rapidly intact microtubule arrays (Fig. 3D). The intensity of staining growing neurite (stage 3) while most other neurons have extended within experimentally initiated neurites was entirely similar to only ‘minor processes’ (stage 2) or no processes at all (stage 1). that of spontaneously initiated (growth cone-mediated) (B) At 2.5 days following plating, both cells in the left hand corner neurites. The remaining half of experimentally initiated have extended axon-like neurites (the neurite from the left-most cell neurites elongated with the needle remaining attached to the extends to the right and beneath the neighboring cell), while both cell body and the distal tip of the elongation remaining attached cells in the right hand side of the panel remain in stages 1 and 2. to the dish (Fig. 4). As shown in Fig. 4, a growth cone was (C) After 4.5 days in culture, both neurons on the left upper corner observed at the distal tip attached to the dish, which showed show extensive outgrowth of a branching axon, and dendrite-like processes have become longer. Cells on the right side remain in normal motile activity. Curiously, such ‘proximal initiations’ stages 1 and 2. Bar, 50 µm. (needle on the cell-body side of the neurite) required much higher forces than distal initiations (needle at the distal end of the outgrowth as shown in Fig. 3). The mean initiating force The majority of cells, however, do not develop to this stage in 14 ‘proximal initiations’ was 127±26 µdynes, with a (stage 4 of Dotti et al., 1988), and a substantial fraction of cells minimum of 35 µdynes and a maximum of 330 µdynes. never even develop a single long neurite (Fig. 1B). In those cells that do develop axon-like and dendrite-like processes, Elongation rate is directly proportional to applied ultrastructural examination of the two types of processes tension showed two well-described cytoplasmic differences character- We determined the quantitative relationship between neurite istic of axonal/dendritic polarity (Deitch and Banker, 1993; elongation and mechanical tension by a method similar to that Craig and Banker, 1994). In all five neurons examined, the described by Zheng et al. (1991) for peripheral neurons from 1182 S. Chada and others

Fig. 2. Ultrastructure of two neurite types from a single chick forebrain neuron at day 5 of culture. (A) A thin section from a short, tapering neurite showing the frequent occurrance of polysomes, some of which are denoted by arrows. (B) A thin section from the single, long, uniform caliber neurite approx. 100 µm from cell body. No polysomes are evident but both microtubules and 10 nm filaments are abundant and at higher density than in dendrite-like processes. Bar, 0.5 µm. embryonic chicks. In one experimental series, experimentally elongation rate per unit tension) is quite similar to that of chick initiated neurites from cells plated 8 hours previously (as in sensory neurons (compare to Fig. 3; Zheng et al., 1991). For Fig. 3) were tethered by their distal end to a calibrated glass both peripheral and central neurons the sensitivity ranged needle and subjected to a step function protocol of towing. between 0.5 and 5 µm per hour per µdyne of applied force, Each tension step was maintained at constant magnitude for with the majority of cells having a tension sensitivity around 1 30-60 minutes by appropriate adjustments of the micromanip- µm/hour per µdyne . However, a significant difference between ulator, and a sequence of 3-6 steps was applied to single peripheral neurons and these central neurons was the absence neurite, each step being 20-50 µdynes greater in magnitude of a clear positive threshold tension required for elongation, than the previous step. As previously observed for chick which was typically between 100 and 200 µdynes for sensory sensory neurons, the neurites did not equilibrate their length to neurons. Rather, as shown in Fig. 5, most cells showed a zero- a given force; rather they elongated continuously in response growth-rate intercept very close to zero tension. In one case, to experimentally applied tension. Indeed, the elongation rate the analysis of the data suggested a small elongation rate under was a linear function of applied tension (Fig. 5a). A similar compression (left-most graph, Fig. 5b). As suggested by these protocol of applying steps of tension to spontaneously initiated analytical results, we observed that chick forebrain neurons (growth cone-mediated) neurites after 24 hours in culture also elongated continuously at even the smallest applied tensions produced a linear relationship between elongation rate and we could measure, i.e. the neurites elongated with any pull on applied tension (Fig. 5b). Fig. 6 shows one such experiment in the neurite. which a spontaneously initiated neurite was towed and then subsequently processed for immunocytochemistry for micro- Retraction behavior of forebrain neurites tubules, demonstrating that the tension-induced length of The absence of a positive threshold tension for neurite neurite had a normal array and density of neuritic microtubules. elongation and the low net tensions at which neurite elonga- Quantitatively, the tension-growth relationship of forebrain tions were observed suggested that forebrain neurons show less neurons showed two similarities to that of chick sensory elastic behavior than chick sensory neurons. We found it neurons and one important difference that were consistent for impossible to analyze elastic behavior of forebrain neurons by both spontaneously- and experimentally-initiated neurites: the the ‘plucking’ technique previously used for chick sensory first similarity is that the linearity of the relationships was neurons and PC12 cells (Dennerll et al., 1988; Lamoureux et robust: in 10 of 11 such experiments the correlation coefficient al., 1992) because of the growth habit of cultured forebrain between tension and elongation rate was 0.9 or greater. Second, neurons. Sensory neurons and PC12 cells are attached to the the frequency distribution of the tension sensitivities of neurite culture surface only at the cell body and growth cone, allowing elongation (Fig. 7, the values of the slopes of the lines, i.e. the the unattached neurite to be plucked like a guitar string. In Brain neuron mechanics 1183

Fig. 4. De novo neurite initiation by application of tension proximal to the neurite. (A) Approx. 8 hours following plating, tension is applied to the margin of a forebrain neuronal cell body with a calibrated glass needle. (B) One hour later, the cell body has firmly attached to the needle and a neurite begins to be initiated from the soma by pulling. In this example, and approximately 50% of all tension-induced initiation, the margin of the cell remains attached to the dish while the soma is pulled free and is towed. (C) Later (157 Fig. 3. De novo neurite initiation by application of tension distal to minutes after B), the neurite has elongated some 90 µm. Bar, 10 µm. the neurite. (A) Approx. 8 hours following plating, tension is applied to the margin of a forebrain neuronal cell body with a calibrated glass needle. The reference needle can be seen to the right, out of focus (needle distance measurements/tension measurements were neurites peeled from their dish attachment: they elongated sig- taken every 5 minutes by a through focus to image both calibrated nificantly during such micromanipulations and formed flaccid, and reference needles within 5 seconds of each other). (B) Forty roughly sinusoidal elongations totally unsuitable for plucking. minutes later, a uniform caliber process has formed with the Consequently, we analyzed the tendency of such flaccid, calibrated needle attached to the distal end of the process, where a sinusoidal processes to contract and shorten. We previously growth cone appears to have formed. (C) At 140 minutes following found that chick sensory neurites rapidly take up slack when frame b, the neurite had reached a final length of approx. 75 µm and was manipulated onto the dish surface. (D) An immunofluorescent the needle attached to the growth cone is moved toward the cell image of the same neuron shown in c, fixed, lysed, processed and body; i.e. after the micromanipiulator is ‘backed up.’ In ‘stained’ for microtubules with a monoclonal antibody to β-tubulin. response to such sudden declines of static tension, chick Bars, 10 µm. sensory neurites contracted to rapidly take up the sinusoids that form initially, and in about 2/3 of the cases the neurites strongly contracted to acheive tension levels substantially greater than contrast, chick forebrain neurites are attached all along their the initial tension prior to slackening of the neurite (Dennerll length on surfaces that support cell adhesion and neurite et al., 1989). This robust contractile/elastic behavior was not outgrowth such as polylysine-treated and collagen-treated observed in chick forebrain neurons. Rather, as shown in Fig. culture dishes. (On plain tissue culture plastic, and plastic 8, slackening of forebrain neurites after peeling the neurite treated with laminin or fibronectin, the cells adhere poorly, from the dish was followed by complex behavior with weak form floating clumps, and generally fail to extend neurites.) recovery of tension accompanied by neurite elongation at the Nor could these elastic measurements be made on forebrain recovered tension. First, the sinusoids clearly straightened out 1184 S. Chada and others

Fig. 6. Immunofluorescent micrograph of the microtubule array in a chick forebrain neurite subjected to experimental ‘towing’ to elongate the neurite. A chick forebrain neuron at 1 day of culture was tethered by its growth cone to a glass needle and towed to produce elongation (length at start of towing shown by vertical bar near cell body) . Subsequently, the distal end was manipulated back onto the dish surface and the cells were lysed, fixed and processed for immunofluorescence localization of microtubules as described in Materials and Methods. The microtubule array in the experimentally elongated region is similar in appearance to the spontaneously elongated region of the same neurite and to the spontaneosly elongated neurites of surrounding cells. Horizontal calibration bar, 10 µm.

again initially recovers but rapidly declines without interven- tion and the neurite again lengthens. In general, it was not possible to stabilize neurite length for even several minutes at the smallest positive tension we can conﬁdently measure with our needles (approx. 10 µdynes). This is an easy experimental manipulation for both chick sensory neurons and PC12 cells.

DISCUSSION

The principal result of our investigation is that brain neurons Fig. 5. Growth rate of chick forebrain neurites as a function of of chick embryos respond to mechanical tension with growth applied tension. (a) Data from two different neurons subjected to responses qualititively similar to that previously reported for experimentally induced neurite initiation, as in Fig. 4, after about 8 peripheral neurons from the same source. Microtubule-rich hours in culture. (b) Data from three different neurons with spontaneously initiated (growth cone-mediated) neurites (‘minor cytoplasmic processes can be initiated de novo by applying processes’) after approx. 24 in culture. For both experimentally and spontaneously initiated neurites, elongation rate is a linear function of applied tension. Note that neurite elongation occurred at essentially all positive tensions, i.e. chick forebrain neurons show no clear minimum tension required for growth.

but the tension recovery was substantially less than expected for a Hookean spring with no evidence for active contraction (tension overshoots compared to initial value). In the experiment shown in Fig. 8, for example, a neurite of 250 µm at an initial tension of 120 µdynes (following detachment and a 30 minute period to reach mechanical equilibrium) was subjected to four small reductions in length (‘back ups’) over the course of about an hour. The net reduction in length totalled approx. 50 µm or 20% of the initial length, but tension was reduced to approx. half the initial value. Upon tension recovery, the neurite began to lengthen. For example, between the second Fig. 7. Frequency distribution of tension sensitivities of towed and third slackening shown in Fig. 8 (15-30 minutes) the neurite growth. Tension sensitivity is given as the growth rate in needle was stabilized at the peak of the spontaneous tension µm/hour per µdyne of tension for 11 experimental neurites (different recovery (lower trace), causing the neurite to lengthen (upper cells) subjected to step-function protocol of towed elongation (as in trace). Following the third slackening (30-50 minutes), tension Fig. 5). Brain neuron mechanics 1185

measure with our needles. In addition, forebrain neurons showed substantially weaker recovery of tension following slackening than chick sensory neurons. Unlike sensory neurons, chick forebrain neurons showed no capacity to actively generate tension in the neurite shaft following slackening. Although chick forebrain neurons showed clear evidence of short-term elastic recovery following slackening, these neurites were unable to maintain a given force over longer periods (20-30 minutes) without lengthening and reduction of tension. That is, forebrain neurites generally behave as viscoelastic fluids; they show some immediate recovery of tension in response to slackening (elastic behavior) but behave as a fluid over longer time scales. In contrast, neurites of peripheral neurons behave principally as viscoelastic solids at low tensions, showing fluid behavior only in providing a damped approach to an otherwise spring-like length-tension equilibrium. These differences in elastic behaviors between forebrain and sensory neurons is unlikely to be due to microtubule or neurofilament differences because both cell types show similar cytoskeletal arrays in axon-like processes. Further, the elastic properties of neurons are due principally to the actin cytoskeleton of the neurite shaft (Dennerll et al., 1988), which is poorly understood in terms of density, arrangement and functional differences. The mechan- Fig. 8. Neurite tension following experimentally induced neurite ical behavior of forebrain neurites would appear to be unusual slackening. An axonal neurite of a cell after approx. 2 days in culture was tethered at its distal end by a calibrated glass needle and the in that many decades of work on cytomechanics has shown that neurite was peeled from its attachment to the dish. Following a wide variety of cell types respond to small forces or defor- detachment, the neurite was held above the dish surface and put mations as viscoelastic solids (e.g. Chambers and Fell, 1931; under easily measurable tension (filled triangles near zero time) Hiramoto, 1963; Harris et al., 1981; Pasternak and Elson, 1985; which caused immediate lengthening (open triangles near zero time). Elgsaeter et al., 1986; Albrecht-Buehler, 1987; Hudspeth, This was halted by rapid slackening of the neurite by moving the 1989; Harold, 1990; Ingber and Folkman, 1988). Thus, micromanipulator toward the cell body and the neurite length (upper cultured chick forebrain neurons show an exaggerated fluid- trace) and neurite tension (lower trace) were recorded. Slackening like growth response to tension, even when compared to was introduced four times during the course of this experiment sensory neurons from the same source. (arrows). See Results for fuller explanation. The attractive proposal of Van Essen (1997) for brain morphogenesis is based primarily on the assumption that neuronal processes are under sustained tension. This entails that the tension to the margin of the cell body. Like peripheral neurons, tissue show macroscopic elasticity for extended periods. Van brain neurons also show a simple, robust, Newtonian-fluid-like Essen also invokes other elastic properties, e.g. the mechani- relationship between neurite elongation rate and tension mag- cal compliance of different brain regions should depend upon nitudes. Thus, we suggest that increases in axonal, and local anisotropies in cellular architecture. Although our results possibly, dendritic length that accompany the growth of the do not provide direct support for Van Essen’s hypothesis, brain following synapse formation (Van Essen, 1997) occur by neither do they constitute strong negative evidence against the the same tensile ‘towed growth’ mechanism long postulated to hypothesis. First, we do not wish to minimize our clear obser- allow peripheral axons to accommodate skeletal (overall) vations of transient elastic behavior in forebrain neurites, growth of animals (Weiss, 1941). measured on a time scale of minutes. The failure to demon- Indeed, our data suggest that the response to tension by chick strate sustained tension over a longer time scale might reflect forebrain neurons is more purely growth-related than with insensitivity of our needle methodology, which applies peripheral neurons. These brain neurons initiate and elongate minimum forces in the 10 µdyne range and might be too crude neurites at substantially lower net tensions than are required for these sensitive neurons. That is, forebrain neurons may for chick sensory neurons, routinely growing in response to have a substantially lower growth threshold than sensory tensions <100 µdynes. This sensitivity to low tensions is not neurons, which above their threshold also show little evidence the result of greater intrinsic growth sensitivity to tension; both of long-term elastic behavior. A difference in growth threshold sensory and forebrain neurons increase their growth rate at seems plausible given the substantially greater ‘delicacy’ of roughly 1 µm/hour for each additional µdyne of tension (Fig. forebrain tissue compared to dorsal root ganglion tissue, the 7). Rather, chick forebrain neurons in culture show substan- source of chick sensory neurons. It also seems plausible that tially less elastic behavior at low tensions than do chick sensory under normal in vivo conditions, chick cerebral neurons might neurons. In sharp contrast to chick sensory neurons or PC12 have higher growth thresholds and generate substantial tensile neurites, chick forebrain neurons show no clear evidence of a forces that do not occur in the particular in vitro conditions minimum tension required for growth; forebrain neurons reported here. In this regard, we note that the intact embryonic elongated with the smallest pulling force we could confidently cerebral hemispheres show clear evidence of macroscopic elas- 1186 S. Chada and others ticity, which would be unlikely if none of their constituent Harris, A. K., Stopak, D. and Wild, P. (1981). Fibroblast fraction as a neurons were under tension. Additional experiments using mechanism for collagen morphogenesis. Nature 290, 249-251. different sources of central neurons and possibly lower Hiramoto, Y. (1963). Mechanical properties of sea urchin eggs. Exp. Cell Res. 32, 59-75. tensions will be needed for a more incisive test of the role of Hudspeth, A. J. (1989). How the ear’s works work. Nature 341, 397-404. tension in brain morphogenesis. Ingber, D. E. and Folkman, J. (1988). Tension and compression as basic determinants of cell form and function. Utilization of a cellular tensegrity This work was supported by a grant from the NSF (IBN 96--03460). mechanism. In Cell Shape, Determinants, Regulation and Regulatory Role We thank David Van Essen for stimulating conversations concerning (ed. W. D. Stein and F. Bronner), pp. 3-31. Academic Press, San Diego. the role of tension in brain morphogenesis, for sharing his work prior Joshi, H. C., Baas, P., Chu, D. T. and Heidemann, S. R. (1986). The to publication, and for reading and commenting on the manuscript. cytoskeleton of neurites after microtubule depolymerization. Exp. Cell Res. 163, 233-245. Lamoureux, P., Buxbaum, R. E. and Heidemann, S. R. (1989). Direct evidence that growth cones pull. Nature 340, 159-162. REFERENCES Lamoureux, P., Zheng, J., Buxbaum, R. E. and Heidemann, S. R. (1992). A cytomechanical investigation of neurite growth on different culture surfaces. Albrecht-Buehler, G. (1987). Role of cortical tension in fibroblast shape and J. Cell Biol. 118, 655-661. movement. Cell Motil. Cytoskel. 7, 54-67. Lamoureux, P., Altun-Gultekin, Z. F., Lin, C., Wagner, J. A. and Baas, P. W., Sinclair, G. I. and Heidemann, S. R. (1987). Role of Heidemann, S. R. (1997). Rac is required for growth cone function but not microtubules in the cytoplasmic compartmentation of neurons. Brain Res. neurite assembly. J. Cell Sci. 110, 635-641. 420, 73-81. Louis, J. C., Pettmann, B., Courageot, J., Rumigny, J. F., Mandel, P. and Bray, D. (1984). Axonal growth in response to experimentally applied tension. Sensenbrenner, M. (1981). Developmental changes in cultured neurones Dev. Biol. 102, 379-389. from chick embryo cerebral hemispheres. Exp. Brain Res. 42, 63-72. Bray, D. (1991). Isolated chick neurons for the study of axonal growth. In Pasternak, C. and Elson, E. L. (1985). Lymphocyte mechanical response Culturing Nerve Cells (ed. G. Banker and K. Goslin), pp. 119-135. MIT triggered by cross-linking surface receptors. J. Cell Biol. 100, 860-872. Press, Cambrige, MA. Pettman, B., Louis, J. C. and Sensenbrenner, M. (1979). Morphological and Caceres, A., Banker, G. A. and Binder, L. (1986). Immunocytochemical biochemical maturation of neurons cultured in the absence of glial cells. localization of tubulin and microtubule-associated protein 2 during the Nature 281, 378-380. development of hippocampal neurons in culture. J. Neurosci. 6, 714-722. Sensenbrenner, M., Maderspach, K., Latzkovits, L. and Jaros, G. G. Chambers, R. and Fell, H. B. (1931). Micro-operations of cells in tissue (1978). Neuronal cells from chick embryo cerebral hemispheres cultivated cultures. Proc. Roy. Soc. Lond. B 109, 380-403. on polylysine-coated surfaces. Dev. Neurosci. 1, 90-101. Craig, A. M. and Banker, G. A. (1994). Neuronal polarity. Annu. Rev. Smith, C. (1994). Cytoskeletal movements and substrate interactions during Neurosci. 17, 267-310. initiation of neurite outgrowth by sympathetic neurons in vitro. J. Neurosci. Deitch, J. S. and Banker, G. A. (1993). An electron microscopic analysis of 14, 384-398. hippocampal neurons developing in culture: early stages in the emergence of Thompson, W. C., Asai, D. J. and Carney, D. H. (1984). Heterogeniety polarity. J. Neurosci. 13, 4301-4315. among microtubules of the cytoplasmic microtubule complex detected by a Dennerll, T. J., Joshi, H. C., Steel, V. L., Buxbaum, R. E. and Heidemann, S. monoclonal antibody to alpha tubulin. J. Cell Biol. 98, 1017-1025. R. (1988). Tension and compression in the cytoskeleton, II Quantitative Van Essen, D. C. (1997). A tension-based theory of morphogenesis and measurements. J. Cell Biol. 107, 665-674. compact wiring in the central nervous system. Nature 385, 313-318. Dennerll, T. J., Lamoureux, P., Buxbaum, R. E. and Heidemann, S. R. Weiss, P. (1941). Nerve pattern, the mechanics of nerve growth. Growth (Suppl. (1989). The cytomechanics of axonal elongation and retraction. J. Cell Biol. Third Growth Symp.) 5, 163-203. 108, 3073-3083. Zheng, J., Lamoureux, P., Santiago, V., Dennerll, T., Buxbaum, R. E. and Dotti, C. G., Sullivan, C. A. and Banker, G. A. (1988). The establishment of Heidemann, S. R. (1991). Tensile regulation of axonal elongation and polarity by hippocampal neurons in culture. J. Neurosci. 8, 1454-1468. initiation. J. Neurosci. 11, 1117-1125. Elgsaeter, A., Stokke, B. T., Mikkelsen, A. and Branton, D. (1986). The molecular basis of erythrocyte shape. Science 234, 1217-1225. Harold, F. M. (1990). To shape a cell, an inquiry into the causes of morphogenesis of microorganisms. Microbiol. Rev. 54, 381-431. (Received 15 January 1997 Ð Accepted 11 March 1997)