Stimulating New Turns

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it then, this finding may be the first genetic evidence in support of the “handshake hypothesis.” Bolstering this sentiment is the finding of Hevner et al. that connections between midline thalamic nuclei and cingulate cortex, a cortical region distinct from neocortex, do form in the Tbr1 mutant; these projections arise from different parts of thalamus than those to neocortex and take a distinct pathway, one through the external capsule rather than internal capsule, and therefore would likely not depend upon subplate axons for their pathfinding. Although Tbr1 is not expressed in the derivatives of the medial or lateral ganglionic eminences within which the internal capsule forms, it is expressed in the eminentia thalami, the entopeduncular nucleus, and the caudal ganglionic eminence, structures positioned near the more proximal part of the path of thalamocortical axons to the neocortex. Therefore, the aberrant thalamocortical projection in the Tbr1 mutant may be due, at least in part, to the loss of potential guidance cues that may normally be associated with these structures. In closing, the study of Hevner et al. has clearly shown that Tbr1 is essential for several critical early events in cortical development, including proper lamination and formation of cortical input and output axonal projections. In addition, this study has set the stage for important future work in defining the genes regulated by Tbr1, and in sorting out which neurons require cell-autonomous expression of Tbr1 and which neurons require Tbr1 nonautonomously for their proper development. Studies to be done in the coming years should introduce further order to the chaotic cortex created by the loss of Tbr1. Noelle D. Dwyer and Dennis D. M. O’Leary Molecular Neurobiology Laboratory The Salk Institute La Jolla, California 92037 Selected Reading Braisted, J.E., Catalano, S.M., Stimac, R., Kennedy, T.E., TessierLavigne, M., Shatz, C.J., and O’Leary, D.D.M. (2000). J. Neurosci. 20, 5792–5801. Bulfone, A., Smiga, S.M., Shimamura, K., Peterson, A., Puelles, L., and Rubenstein, J.L. (1995). Neuron 15, 63–78. Bulfone, A., Wang, F., Hevner, R., Anderson, S., Cutforth, T., Chen, S., Meneses, J., Pedersen, R., Axel, R., and Rubenstein, J.L. (1998). Neuron 21, 1273–1282. Caviness, V.S., Crandall, J.E., and Edwards, M.A. (1988). In Cereb. Cortex, Vol. 7, A. Peters and E.G. Jones, eds. (New York: Plenum Press), pp. 59–84. Gleeson, J.G., and Walsh, C.A. (2000). Trends Neurosci. 23, 352–359. Hevner, R.F., Shi, L., Justice, N., Hsueh, Y.-P., Sheng, M., Smiga, S., Bulfone, A., Goffinet, A.M., Campagnoni, A.T., and Rubenstein, J.L.R. (2001). Neuron 29, this issue, 353–366. Kawano, H., Fukuda, T., Kubo, K., Horie, M., Uyemura, K., Takeuchi, K., Osumi, N., Eto, K., and Kawamura, K. (1999). J. Comp. Neurol. 408, 147–160. Koester, S.E., and O’Leary, D.D.M. (1994). J. Neurosci. 14, 6608– 6620. McConnell, S.K., Ghosh, A., and Shatz, C.J. (1994). J. Neurosci. 14, 1892–1907. Miyashita-Lin, E.M., Hevner, R., Wassarman, K.M., Martinez, S., and Rubenstein, J.L.R. (1999). Science 285, 906–909. Molnar, Z., and Blakemore, C. (1995). Trends Neurosci. 18, 389–397.

Tuttle, R., Nakagawa, Y., Johnson, J.E., and O’Leary, D.D.M. (1999). Development 126, 1903–1916. Zhou, C., Qiu, Y., Pereira, F.A., Crair, M.C., Tsai, S.Y., and Tsai, M.J. (1999). Neuron 24, 847–859.

Stimulating New Turns In this issue of Neuron, a paper by Ming et al. (2001) shows that stimulation of action potentials can dramatically alter the turning responses of growth cones to attractive and repulsive guidance factors in culture. Turning up a gradient of netrin-1 is enhanced, myelinassociated glycoprotein (MAG)–induced repulsive turning is converted to attraction, and Sema3A-induced repulsive turning is converted to full growth cone collapse. This study continues a line of investigation that opened with the remarkable 1997 discovery from Muming Poo’s laboratory that attraction can be changed to repulsion in cultured Xenopus spinal neurons by changing cAMP levels pharmacologically (Song et al., 1997). In a series of subsequent papers, Poo and colleagues investigated the basis of this effect (Song and Poo, 1999). This has led to a balanced model of turning that divides attractants and repellents into two classes based on whether they are influenced by cAMP or cGMP levels. In this model, diffusible factors induce both polymerization (P) and depolymerization (D) of actin. The P/D ratio determines whether a factor is attractive or repulsive. Attractants generally stimulate P over D, and repellents stimulate D over P. But the P/D ratio is also influenced by baseline levels of cAMP (class 1) or cGMP (class 2). In the class 1 system, protein kinase A, stimulated by high cAMP, phosphorylates effectors of the P/D machine and pushes the P/D ratio toward P. In low cAMP, D is favored. Thus, growth cones turn toward a gradient of netrin-1 when cAMP is high and away when cAMP is low. MAG is a class 1 repellent that in normal cAMP levels stimulates D more than P, yet when cAMP is raised to high levels in the growth cone, the P/D ratio increases and MAG becomes attractive. Sema3A is a class 2 repellent that is affected by cGMP levels in a similar way. This model explains much of growth cone turning in vitro as influenced by pharmacological perturbations of cAMP and cGMP and raises the following questions. What normally causes changes in the levels of cyclic nucleotides in growth cones? Do these changes have an affect on guidance in vivo? Hopker et al. (1999) were the first to show that a natural substrate molecule, laminin, decreases cAMP in retinal growth cones, thereby switching netrin-induced attraction to repulsion and providing a mechanism for how these axons turn away from the laminin-rich surface of the optic fiber layer into the netrin-1-rich optic nerve head to exit the retina (Ho¨pker et al., 1999). The Ming et al. (2001) study here provides another possible natural mechanism for modulating cAMP since calcium, which enters the growth cone in response to impulse activity, is known to stimulate certain adenylate cyclases and raise cAMP levels (Song and Poo,

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1999). In this case, inhibiting the increase in either calcium or cAMP levels abolishes the dependence of growth cone turning responses on electrical stimulation. Do calcium rises induced by action potentials occur naturally in growth cones during pathfinding? Using calcium imaging, Spitzer and colleagues observed short single spikes of calcium in the growth cones of cultured Xenopus spinal neurons as a result of spontaneous action potentials invading the growth cone and opening voltage-gated calcium channels (Gu et al., 1994). These calcium spikes occur at a spontaneous rate of about three per hour and do not lead to changes in the rate of outgrowth (Gomez and Spitzer, 2000). Ming et al. use higher frequency electrical stimulation of the cell body (ten pulses at 2 Hz) to produce a more sustained increase in growth cone calcium. Low-frequency stimulation does not lead to an alteration in growth cone turning just as the rather isolated spontaneous spikes do not affect axon growth. Spitzer and colleagues have also shown that slower spontaneous calcium transients, called waves, occur in growth cones in vitro and in vivo and that the rate of growth cone advancement is inversely proportional to the frequency of these transients (Gomez and Spitzer, 1999, 2000). However, these waves are the result of intracellular release from calcium stores and are not dependent on impulse activity (Gu et al., 1994). Thus, one might wonder if these axons really use impulse activity in growth cone guidance during normal development. While the literature clearly shows that electrical activity influences synapse formation and terminal arborization (Goodman and Shatz, 1993; Catalano and Shatz, 1998; Dantzker and Callaway, 1998), there is little indication that impulse activity in the developing brain is needed for the formation of normal axon tracts or pathways. Indeed, in the absence of all normal impulse activity throughout development, the axonal connections in the nervous system look remarkably good (Harris, 1980). Because Ming et al. use a growth cone turning assay, it is tempting to assume that this behavior is relevant only for axon pathfinding. However, cultured neurons are limited in the behaviors they can express, and so it is possible that the “read out” seen as a directional turn may, in fact, represent something different such as an aspect of target innervation or terminal arbor formation. If bursts of spontaneous axon potentials in growing neurons were coordinated with target innervation, something that is not known (although there are suggestions that this might be so), the results could help explain target selection errors in growing axons deprived of activity. Finally, the potential significance of this work to axon regeneration should not be overlooked. It has been proposed that repulsion to molecules like MAG at injury sites plays a role in preventing damaged CNS axons from regenerating, because their growth cones, unlike those of embryonic axons, have low levels of cAMP (Cai et al., 1999). Thus, stimulation of injured CNS axons might return cAMP levels to more embryonic levels and thus help regenerating axons across repulsive barriers and make the correct pathfinding decisions that they did when they were young. The recent work of Al-Majed et al. (2000) suggests that electrical stimulation of motor nerves increases the speed and accuracy of peripheral regeneration, but in this case the effect is mediated through activity of the cell body and not the growth cone.

Thus, we are left with a tantalizing quandary: the result is exciting but the in vivo relevance awaits resolution. Bill Harris and Christine Holt Department of Anatomy Cambridge University Downing Street Cambridge CB2 3DY United Kingdom Selected Reading Al-Majed, A.A., Neumann, C.M., Brushart, T.M., and Gordon, T. (2000). J. Neurosci. 20, 2602–2608. Cai, D., Shen, Y., De Bellard, M., Tang, S., and Filbin, M.T. (1999). Neuron 22, 89–101. Catalano, S.M., and Shatz, C.J. (1998). Science 281, 559–562. Dantzker, J.L., and Callaway, E.M. (1998). J. Neurosci. 18, 4145– 4154. Gomez, T.M., and Spitzer, N.C. (1999). Nature 397, 350–355. Gomez, T.M., and Spitzer, N.C. (2000). J. Neurobiol. 44, 174–183. Goodman, C.S., and Shatz, C.J. (1993). Cell 72 (suppl.), 77–98. Gu, X., Olson, E.C., and Spitzer, N.C. (1994). J. Neurosci. 14, 6325– 6335. Harris, W.A. (1980). J. Comp. Neurol. 194, 303–317. Ho¨pker, V.H., Shewan, D., Tessier-Lavigne, M., Poo, M., and Holt, C. (1999). Nature 401, 69–73. Ming, G.-l., Henley, J., Tessier-Lavigne, M., Song, H.-j., and Poo, M.-m. (2001). Neuron 29, this issue, 441–452. Song, H.J., Ming, G.L., and Poo, M.M. (1997). Nature 388, 275–279. Erratum: Nature 389 (6649), 1997. Song, H.J., and Poo, M.M. (1999). Curr. Opin. Neurobiol. 9, 355–363.

CAK␤/Pyk2 Activates Src: Another Piece in the Puzzle of LTP Induction The fundamental role of NMDA receptors in the induction of many forms of long-term potentiation (LTP) is very well established. A general model is that Ca2⫹ entry through activated NMDA receptors initiates biochemical cascades that lead to an increase in AMPA receptor– mediated current and thus results in long-lasting potentiation of synaptic efficacy (Bliss and Collingridge, 1993). Molecular events that can enhance or diminish NMDA receptor responses are therefore likely to have profound implications for the induction of LTP. For example, it is known that the nonreceptor tyrosine kinases Src and Fyn can upregulate NMDA receptors currents. Subsequently, it was shown that Src phosphorylates the NR2 subunits and that the Src-mediated enhancement of NMDA receptor responses is a required step in LTP induction (Salter, 1998). There remain, as ever, many issues that need to be resolved. One question is how Src-mediated phosphorylation of NMDA receptors is regulated in the neuron, and more specifically, what is the signal for Src activation during induction of LTP? Many pathways have been shown to lead to Src activation, but the sequence of events responsible for Src-mediated phosphorylation of