Braking News
Authors: Daniel J Goldberg, Peter W Grabham
Journal: Neuron (1999)

Growing axons are guided by cues in the environment: local or released from a distance, positive and negative (8Goodman C.S Annu. Rev. Neurosci. 1996; 19: 341-377Crossref PubMed Scopus (463) Google Scholar). The motile growth cone at the tip of the axon senses and interprets these cues, changing its behavior to alter the direction or speed of growth. Many cues have been identified and their in vivo importance has been verified, but how their binding is transduced into changes in growth cone behavior remains poorly understood. Given the plethora of cues, it would simplify things if common elements of transduction pathways emerged: for example, if stopping of a growth cone at a choice point were always (or, at least, often) caused by elevation of messenger X within the growth cone, whatever the external cue. Recent work points to such key transduction molecules. Moreover, a new paper in Nature literally and figuratively illuminates the inner workings of the growth cone by fluorescently imaging Ca2+ in neurons growing within the developing spinal cord, providing evidence for its importance in vivo in regulating axonal elongation (6Gomez T.M Spitzer N.C Nature. 1999; 397: 350-355Crossref PubMed Scopus (409) Google Scholar). Considering that the response of growth cones to cues is, for technical reasons, typically studied in the reductionist world of the culture dish, this is a noteworthy accomplishment. It has long been known that the growth cone is sensitive to its [Ca2+]i. Large increases caused by neurotransmitters or depolarization cause growth cones in culture to collapse, stopping neuritic elongation (11Kater S.B Mills L.R J. Neurosci. 1991; 11: 891-899Crossref PubMed Google Scholar). Decreases caused by removing Ca2+ from the bathing medium can have similar effects. In some cases, growth cone activity and neuritic elongation can be promoted by elevations of [Ca2+]i over resting levels; focal changes within the growth cone can produce focal protrusive activity appropriate for turning, for example (5Davenport R.W Kater S.B Neuron. 1992; 9: 405-416Abstract Full Text PDF PubMed Scopus (100) Google Scholar). These seemingly discordant results were accommodated by Kater's set-point model, in which substantial deviations in either direction from an optimal [Ca2+]i inhibit motile activity of the growth cone and slow growth (11Kater S.B Mills L.R J. Neurosci. 1991; 11: 891-899Crossref PubMed Google Scholar). This is so reasonable as to be unsurprising; very high or low levels of [Ca2+]i are cytotoxic, presumably one reason that [Ca2+]i is normally tightly buffered. However, there were indications that the deviations from normal did not have to be great to inhibit growth (13Lankford K.L Letourneau P.C Cell Motil. Cytoskel. 1991; 20: 7-29Crossref PubMed Scopus (81) Google Scholar). Microfilaments, so important to the motile activity of the growth cone, are particularly sensitive to [Ca2+]i. The critical unknown was whether signaling through Ca2+ in the growth cone is physiologically relevant. It now seems likely that it is. One line of evidence is its apparent involvement in transducing responses in the growth cone to certain cues that are likely to function in vivo. NI-35 is a growth-inhibitory protein expressed on oligodendrocytes in the CNS (see 1Bandtlow C.E Schmidt M.F Hassinger T.D Schwab M.E Kater S.B Science. 1993; 259: 80-83Crossref PubMed Scopus (247) Google Scholar, for references). Regeneration of injured axons within the spinal cord is promoted by antibodies that neutralize NI-35, attesting to its importance in vivo. NI-35 induces growth cones in culture to collapse. The collapse is associated with a large rise in [Ca2+]i, at least partially due to release from the smooth endoplasmic reticulum (SER) (1Bandtlow C.E Schmidt M.F Hassinger T.D Schwab M.E Kater S.B Science. 1993; 259: 80-83Crossref PubMed Scopus (247) Google Scholar). Blockage of Ca2+ release prevents collapse. Certain positive cues that are likely to function in vivo may also work through Ca2+. NCAM and L1 are growth-promoting members of the immunoglobulin superfamily (20Walsh F.S Doherty P Annu. Rev. Cell Dev. Biol. 1997; 13: 425-456Crossref PubMed Scopus (404) Google Scholar). Disruption of the functioning of either in vivo leads to what appear to be abnormalities in axonal growth (3Cohen N.R Taylor J.S Scott L.B Guillery R.W Soriano P Furley A.J Curr. Biol. 1998; 8: 26-33Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 16Seki T Rutishauser U J. Neurosci. 1998; 18: 3757-3766Crossref PubMed Google Scholar). The promotion of growth by either in vitro depends on influx of Ca2+ through channels in the plasma membrane, though it is merely assumed these are in the growth cone (20Walsh F.S Doherty P Annu. Rev. Cell Dev. Biol. 1997; 13: 425-456Crossref PubMed Scopus (404) Google Scholar). Laminin is a prominent constituent of the extracellular matrix and is expressed by Schwann cells in peripheral nerve, where it probably promotes growth. It elicits rises in [Ca2+]i in growth cones in culture that may be important in affecting both the rate and direction of growth (12Kuhn T.B Williams C.V Dou P Kater S.B J. Neurosci. 1998; 18: 184-194PubMed Google Scholar; but see below). The aforementioned environmental cues all act as substrate-bound molecules. Molecules released from afar can also influence growth, either attracting or repelling neurites. Here, Ca2+ may also be involved. Netrin-1 is produced by the floor plate of the developing spinal cord, from where it emanates to attract certain axons to, and repel others from, the midline (see 14Ming G.-L Song H.-J Berninger B Holt C.E Tessier-Lavigne M Poo M.-M Neuron. 1997; 19: 1225-1235Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, for references). This Janus-like behavior can be displayed in culture in a particularly intriguing way. A spinal neurite that turns and grows toward a micropipette emitting netrin-1 turns away from that micropipette when the activity of cAMP-dependent protein kinase in the neurite is pharmacologically suppressed (14Ming G.-L Song H.-J Berninger B Holt C.E Tessier-Lavigne M Poo M.-M Neuron. 1997; 19: 1225-1235Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). Neither behavior is seen when [Ca2+]o is drastically reduced, though straight-ahead growth proceeds apace. Similar results are seen with brain-derived neurotrophic factor (BDNF) and acetylcholine. A complementary situation is seen with myelin-associated glycoprotein (MAG), another inhibitory protein expressed by oligodendrocytes (see 19Song H.-J Ming G.-L He Z Lehmann M McKerracher L Tessier-Lavigne M Poo M.-M Science. 1998; 281: 1515-1518Crossref PubMed Scopus (725) Google Scholar, for references). Neurites that are repelled by a distant source of MAG are instead attracted when cAMP is artificially elevated (19Song H.-J Ming G.-L He Z Lehmann M McKerracher L Tessier-Lavigne M Poo M.-M Science. 1998; 281: 1515-1518Crossref PubMed Scopus (725) Google Scholar). Both the repulsion and the attraction disappear in low [Ca2+] medium. Also pointing to a role for Ca2+ in regulating neuritic growth was the discovery that certain growth cones advancing in culture display periodic brief rises in [Ca2+]i (termed waves) that seem to affect the speed of neuritic elongation. 10Gu X Spitzer N.C Nature. 1995; 375: 784-787Crossref PubMed Scopus (471) Google Scholar found, by fluorescence imaging, that frog growth cones spontaneously display about 8 transients per hour in which [Ca2+]i slowly rises to, and decays from, a peak of about double the normal level. Transients occur in the axon and cell body as well, but these are distinct from those in the growth cone. In other words, the Ca2+ wave seems to be generated within the growth cone, though it may spread somewhat into the neurite. The speed of neurite elongation was found to be inversely related to wave frequency up to the normal frequency, so that completely suppressing waves by removing extracellular Ca2+ or by clamping [Ca2+]i to baseline with injected buffer increased the speed 1.6-fold. A key point here is that wave frequency was adjusted by artificially creating wave-like transients in the growth cone: spontaneous waves were suppressed by removing extracellular Ca2+, and then artificial waves were created by puffing on Ca2+-containing medium while depolarizing the cell (to allow influx through voltage-gated Ca2+ channels). These results imply that the Ca2+ waves regulate the speed of growth (Figure 1). Frog spinal neurites growing in culture are somewhat odd. For example, they grow well even on uncoated glass, and the growth cone seems literally to drag the axon forward as it advances (15Okabe S Hirokawa N J. Cell Biol. 1992; 117: 105-120Crossref PubMed Scopus (105) Google Scholar). But, similar results with Ca2+ waves were also seen in chick sensory growth cones in culture (7Gomez T.M Snow D.M Letourneau P.C Neuron. 1995; 14: 1233-1246Abstract Full Text PDF PubMed Scopus (120) Google Scholar). None of the foregoing actually demonstrated that Ca2+ is used to regulate neuritic growth in vivo, where the environment is complex and largely uncharted. This is why the new paper from 6Gomez T.M Spitzer N.C Nature. 1999; 397: 350-355Crossref PubMed Scopus (409) Google Scholar is a real advance. They were able not only to image changes in [Ca2+] within growth cones in the intact frog spinal cord but also to suppress or induce waves by photorelease of caged compounds and determine the effects on growth. The results are gratifyingly similar to those in culture. Growth cones tend to exhibit few if any waves when the axon is growing rapidly, but 5–15 waves per hour during stalled or slow growth. Suppression of waves in a stalled growth cone by photorelease of a caged Ca2+ chelator starts elongation; induction of waves in a rapidly advancing growth cone by photorelease of caged Ca2+ slows elongation markedly. Stalling or slowing is stereotypically seen at certain points in an axon's growth trajectory, such as when a turn onto a new path must be made. Gomez and Spitzer were able to observe one such point and found that the stalling was associated with the appearance of multiple Ca2+ transients, which disappeared when the turn was made and the axon had accelerated on a fascicle. Periodic Ca2+ transients have been observed in many types of cells, often in response to an agonist. Generally, they are thought to involve Ca2+-induced Ca2+ release from the SER (2Berridge M.J Neuron. 1998; 21: 13-26Abstract Full Text Full Text PDF PubMed Scopus (1747) Google Scholar). This may be the case in the growth cone. There is abundant SER in growth cones (4Dailey M.E Bridgman P.C J. Neurosci. 1989; 9: 1897-1909Crossref PubMed Google Scholar), and it can release considerable Ca2+ (7Gomez T.M Snow D.M Letourneau P.C Neuron. 1995; 14: 1233-1246Abstract Full Text PDF PubMed Scopus (120) Google Scholar). The amplitudes of the spontaneous transients in chick sensory growth cones in culture are decreased by ryanodine (7Gomez T.M Snow D.M Letourneau P.C Neuron. 1995; 14: 1233-1246Abstract Full Text PDF PubMed Scopus (120) Google Scholar), which blocks one of the channels through which Ca2+ is released from the SER. But, for both the chick and the frog growth cones, a significant contributor to the transients is influx through channels in the plasma membrane. Growth cones are rich in conventional voltage-gated Ca2+ channels (9Gottmann K Lux H.D Perspect. Dev. Neurobiol. 1995; 2: 371-377PubMed Google Scholar), yet blockage of these does not affect waves (7Gomez T.M Snow D.M Letourneau P.C Neuron. 1995; 14: 1233-1246Abstract Full Text PDF PubMed Scopus (120) Google Scholar). The general Ca2+ channel blockers, La3+ and Co2+, do suppress the waves, suggesting that entry of Ca2+ into the growth cone through an uncharacterized channel is important. Why transients rather than sustained changes in [Ca2+]i are used remains a question for many cell types, not only growth cones. A single wave slows elongation of chick sensory neurites in culture, an effect that substantially outlasts the wave itself (7Gomez T.M Snow D.M Letourneau P.C Neuron. 1995; 14: 1233-1246Abstract Full Text PDF PubMed Scopus (120) Google Scholar). The effect wanes within minutes, however, so repeated waves are needed for sustained slowing. The more frequent the waves, then, the more frequent the bouts of slow growth and the greater the overall slowing or the more sustained the stalling. In this case, the overall effect is proportional to wave frequency. Why not, however, simply have a rise in [Ca2+]i to the level seen at the peak of a transient but then sustained for as long as needed? Gradation of effect could be achieved by varying the amplitude of the increase. Several explanations have been offered: to prevent prolonged exposure to destructively high [Ca2+]i, to prevent adaptation to the desired effect of high [Ca2+]i, and to allow a higher signal-to-noise ratio (the smaller signals in an amplitude-modulated system being subject to disappearance in the background noise). Another possibility is that Ca2+ serves as a switch in the cascade leading to slowing of growth; a threshold [Ca2+]i must be attained, but further increases are superfluous. At present, we don't know. Of perhaps more interest is the question of how Ca2+ slows growth. If the basic slowing effect is produced by a single wave, as suggested above, then we need primarily consider the effect that this single rise in [Ca2+]i has on growth cone activity, without being concerned with its periodicity. Increases of [Ca2+]i in the range reported for waves seem capable of reducing actin polymer in the peripheral region of the growth cone and the protrusive activities dependent on it, thereby slowing growth (Figure 1, left). Massive increases in [Ca2+]i decimate the actin network, causing collapse of the growth cone, probably at least partly by activating proteases like calpain (17Song D.K Malmstrom T Kater S.B Mykles D.L J. Neurosci. Res. 1994; 39: 474-481Crossref PubMed Scopus (45) Google Scholar). The more modest increase in a wave is probably insufficient for this but might activate actin-severing proteins like gelsolin. However, growth cones pausing at choice points typically spread and send out even more protrusions, which might aid in sampling the environment. Protrusions contacting and perhaps adhering to lateral structures in the environment might, in fact, impede growth. Given that focal increases in [Ca2+]i can cause filopodia and veils to protrude (5Davenport R.W Kater S.B Neuron. 1992; 9: 405-416Abstract Full Text PDF PubMed Scopus (100) Google Scholar), we should consider the possibility that the wave is actually promoting the formation of protrusions and this slows forward progress (Figure 1, center). Clearly, the key here is determining the behavior of the growth cone and the distribution and activity of the actin cytoskeleton during a wave. Is Ca2+ strictly a brake for the growth cone? Even as such, it could be used by growth promoters as well as inhibitors. If the brake is normally partially on, a cue could accelerate growth by releasing it—that is, by suppressing transients, as 6Gomez T.M Spitzer N.C Nature. 1999; 397: 350-355Crossref PubMed Scopus (409) Google Scholar suggest for laminin. The data do not yet neatly fit this scheme. Other results suggest that laminin speeds growth by increasing [Ca2+]i in the growth cone (12Kuhn T.B Williams C.V Dou P Kater S.B J. Neurosci. 1998; 18: 184-194PubMed Google Scholar). Influx of Ca2+, though not detectably changing [Ca2+]i, seems an essential step in the transduction pathway by which NCAM and L1 promote growth (20Walsh F.S Doherty P Annu. Rev. Cell Dev. Biol. 1997; 13: 425-456Crossref PubMed Scopus (404) Google Scholar). Perhaps small increases (not easily detected) promote, while larger changes (of the sort seen in a wave or during collapse induced by NI-35) inhibit. But still there is the puzzling role of Ca2+ in turning responses to distant diffusible cues. Here, there is no clear-cut association with growth toward or away from the cue; neither occurs without Ca2+ in the bathing medium, though growth continues. One might hypothesize that the lack of Ca2+ renders the growth cone incapable of turning in response to a cue, perhaps deficient in forming the filopodia that may be important in guidance. Yet, the growth cone remains capable of turning normally toward other cues (18Song H.-J Ming G.-L Poo M.-M Nature. 1997; 388: 275-279Crossref PubMed Scopus (520) Google Scholar). Perhaps relevant here is an older finding that raising cAMP in the growth cone can lower high [Ca2+]i, thereby mitigating the latter's inhibitory effect on growth (13Lankford K.L Letourneau P.C Cell Motil. Cytoskel. 1991; 20: 7-29Crossref PubMed Scopus (81) Google Scholar). Ca2+ could potentially regulate many proteins in the growth cone, so to confine it neatly to one predominant role in mediating effects of cues on the growth cone may be unrealistic. But the case for its importance is solidifying, and with the recent evidence that cyclic nucleotides can control the sign of the response (attraction or repulsion) to diffusible cues, we may be seeing the emergence of key downstream molecules on which signaling pathways converge to regulate the growth cone.