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168 Although convergence of different channels can destroy the place-coding scheme, connections between different neurons within the same array are obviously necessary at some stage for computation using place codes. Such connections can transform the placecoding scheme, for example, from a simple linear system to a complex nonlinear one, by using the distributed patterns of excitation of the array neurons. Contrast enhancement by lateral inhibition is an example of using distributed neuronal activities to improve the place-coding scheme. In fact, the excitatory receptive field of a spacespecific neuron is surrounded by a large inhibitory area ts, which appears to be mediated by inhibitory synapses on that neuron t9. Similarly, the stimulus selectivity of some neurons in the cat visual and somatosensory cortices depends upon inhibitory interactions2°'~1.

Projection of centrally synthesized maps The presumed map of interaural phase differences in the laminar nucleus is a centrally synthesized map. It projects topographically onto its target area, which in turn projects, with appropriate transformation (i.e. the columnar arrangement), onto the external nucleus of the inferior colliculus. Space-specific neurons offer an even more striking example of topographical projection. They send their axons to the optic tectum, where many neurons respond to both visual and auditory stimuli. For such cells the best areas of their visual and auditory receptive fields are in register, except in the most peripheral regions of space. Moreover, 'the auditory and visual maps of space have the same orientation, positions, magnification factors, and termination coordinates at the anterior and dorsal edges of the tectum '22. It should be emphasized that two maps designed on different principles, one centrally synthesized the other peripherally generated, are aligned. These findings suggest that the two types of map differ from one another only in the production of the first map in the system. In other words, the difference between the two types lies in the first site of transformation to place codes. Once a map is generated, the same rules of projection and interactions among the constituent neurons apply, whether the map is peripherally or centrally generated. Hence, there may exist general rules for processing using place codes.

The maps of sensory space may be a logical consequence of the use of place codes. The rules for separating and combining place codes for different signals and for computation using place codes may be important determinants of the anatomical and physiological characteristics of all brain maps.

Acknowledgements I thank C. E. Cart, W. E. Sullivan, T. Takahashi, S. Volman, Van Essen and H. Wagner for critically reading and correcting the manuscript. C. E. Carr (Figs 3 and 7) and W.E. Sullivan (Figs 5 and 6) kindly prepared the illustrations. This work was supported by NIH grant NS14619.

Selected references 1 Knudsen,E.I.andgonishi, M.(1978)Science 200, 795-797 2 Suga, N. and O'Neill, W. E. (1979) Science 206, 351-353 3 Hubel, D. H., Wiesel, T. N. and Stryker, M. P. (1978) J. Comp. Neurol. 177,361-380 4 Konishi, M. and Knudsen, E. I. (1982) in Cortical Sensory Organization (Vol. 3, Multiple Auditory Areas) (Woolsey, C. N., ed.), pp. 219-229, Humana Press 5 Knudsen, E. I. and Konishi, M. (1979) J. Comp. Physiol. 133, 13-21 6 Moiseff, A. and Konishi, M. (1981) Ju Neurosci. 1, 40-48

7 Knudsen, E. [., Konishi, M. and Pettigrew, 1. D. (1977) Science 198, 1278-1280 8 Knudsen, E. I. (1983)J. Comp. Neurol. 218, 174-186 9 Takahashi, T., Moiseff, A. and Konishi. M. (1984) J. Neurosci. 4, 1781-1786 10 Sullivan, W. E. and Konishi, M. (1984) J. Neurosci. 4, 1787-1799 II Lavine, R. A. (1971) J. Neurophysiol. 34, 467-4-83 12 Jhaveri, S. and Morest, K . D . (19821 Neuroscience 7,837-853 13 Jeffress, L. A. ( 1 9 4 8 ) J . Comp. Physiol. Psychol. 41, 35-39 14 Sullivan, W. E. and KonishL M. Proc. Nad Acad. Sci. (in press) 15 Young, S. R. and Rubel, E . W . (1983) J. Neurosci. 3, 1373-1378 16 Van Essen, D. C. and Maunsell, 1. H. R. (1983) Trends Neurosci. 6, 370-375 17 Newsome, W. T., Wintz, R. H., Dunstelen. M. R. and Mikami, A. (1985) J. Neurosci. 5~ 825-840 18 Knudsen, E. I. and Konishi, M. (1978) Science 202, 778-780 19 Moiseff, A. (1985) Soc. Neurosci. Abstr. 1t, 735 20 Hicks, T. P. and Dykes, R. W. (1983) Brain Res. 274, 160-164 21 Gilbert, C. D. (1985) Trends Neurosci. 8,160165 22 Knudsen, E. 1. (1982)J. Neurosci. 2, 11771194

Masakazu Konishi is at the Division of Biology, 216-76, California Institute of Technology, Pasadena, CA 91125, USA.

Neu ron-astre alla interactk)ns in vitro and in Wvo Mary E. Hatten and Carol A. Mason The role of astroglia in providing a structural framework for neuronal development has long been recognized, but the influence of neurons on astroglial development remains largely unexplored. An in-vitro model system has been devised to test the cellular and molecular mechanisms of neuronal migration. This model system has opened new possibilities for examining the effects of neurons on ustroglial differentiation and proliferation, and how these effects relate to disturbances in glial morphology and neuronal migration in mutant cerebella. Although an obligatory association between growing axons and astroglia has not yet been demonstrated, the means by which glia might foster axon outgrowth, and conversely, axons might trigger glial differentiation, are discussed. In recent years, it has become evident that we must understand neuron-glia interactions in order to understand fully the development and plasticity of the mammalian nervous system. The seminal work of Kuffler 1 and Rakic 2~ has sparked an interest in glia, yet we remain ignorant of their functions, their diversity among brain regions and their differentiation. All of these aspects are likely to be important in CNS repair and tumorigenesis. In the adult brain, astroglia are presently seen as acolytes to the

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neurons, providing a favorable ionic environment, modulating extracellular levels of neurotransmitters and serving as spacers that organize the neurons. In the developing mammalian brain, astroglia play an important role in the genesis of basic tissue structure. In the young brain two important features of astroglial cells are that their form seems to fit the architectonic needs of the neurons (elongated forms guide migration 4 and stellate forms compartmentalize mature neuronsS), and that their number is coordinated

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with the number of neurons. Thus the mechanisms that control both glial morphological differentiation and proliferation need to be understood. The studies by Levitt and Rakic6 suggest that the lineages of neurons and glia diverge quite early in brain development. Raft and his colleagues, using immunocytochemical studies to trace the lineage of gila in the developing optic nerve, have demonstrated that there are at least two families of astrocytes in the optic nerve, and that one of them (the type II astrocyte) shares a precursor with oligodendrocytes7. From their in-vitro studies, they have proposed that there are cues in the cellular environment that direct glial precursors to become either oligodendrocytes or astrocytess. The number of families of astrocytes, as determined by their distribution or their functional interactions with neurons, has yet to be studied. By extrapolation from the clear role for astroglia in guiding neuronal migration, many have proposed that glia organize the emerging intermediate zones and guide axon growth through the young developing axon tracts. These studies have been hampered by the fact that, unlike radial glia, which can be visualized easily by Golgi and immunocytochemical staining, immature glial forms are hard to demonstrate in primitive axon tracts. Our laboratories have focused on three aspects of neuron-giia interactions: the cellular and molecular mechanisms of neuronal migration along glia; the control of glial differentiation and proliferation; and the nature and relationship of neurons to glia in immature axon tracts. We have addressed the first two issues with an in-vitro model system developed in our laboratory, and the third has been analysed in vivo. For most of these questions, we have turned to the mouse cerebellar cortex; the mouse because it is a species with a host of neurological mutants with developmental defects, and the cerebellum because its cells and incoming axons are the best characterized of any brain region as to their time of origin, pathways of migration and patterns of connectivity. We studied granule neuron migration, because it is the paradigm for 'glial guidance' of neuronal migration, with granule ceils moving along the radially aligned processes of the Bergmann glia2. To develop a model system for cellcell interactions between neurons and astroglia, we first defined a micro-

culture system that promotes rapid astrogiial process outgrowth and reforming of contacts with neurons. We then used cellular antigen markers the giial filament protein for astroglia and the NILE glycoprotein for neurons - to visualize neuronal associations with astroglia in vitro 9. Three important findings emerged from our initial in-vitro studies. First, astroglia provide a template for the positioning of cerebellar neurons in

vitro 9. Second, neuronal associations with astrnglia /n vitro are developmentally regulated, occurring during the period of glial-guided granule n e u r o n migration /n vivo, but not at embryonic stages prior to granule neuron migration along Bergmann glia. Finally, embryonic astrnglial cells differentiate into complex shapes and interact with neurons in vitro on a schedule that is roughly equivalent to that seen in vivo t°.

Fig. 1. Cerebellar neurons behave differently on different forms of astroglia. After 48 h in vitro, microcultures of cerebellar cells harvested at postnatal day 7 were stained with antibodies to glial filament protein. (a) Several dozen unstained neurons nestle at or near branch points of the processes of stained stellate astroglia (,4). Migrating neurons are not seen on stellate astrocytes. Neurons (mn ) migrate on glial cells ( B) that have longer processes (gp). At higher magnification (b),the thickened, leading process of the cell resembling a migrating neuron (mn) contacts the stained astroglial process (gp). (rm ) = resting neuron. (a and c) x 348; (b) x 868. (Taken, with permission, from Ref. 11.)

170 We then combined PAP antibody localization methods with time-lapse video microscopy and electron microscopy to analyse astroglial morphology and the behavior of neurons that are on astroglia of different shapes. Two forms of astroglial cells were seen in the cultures and each interacted differently with the neuronslL The predominant form, resembling astrocytes of the internal granular layer, had a stellate shape and clustered a large number of neurons among its arms. The second form, resembling Bergmann glia, had longer arms and bound far fewer neurons along its processes. The areas of

7 7 N S -- April 1980

contact between the neurons and glia had three notable features - the membranes of both were ruffled, p u n c t a adherentia were common, and coated vesicles were seen in the glial processes. Thus these sites of contact resembled attachment points rather than gap junctions11 The most exciting finding was the direct demonstration of neuronal migration along the arms of the Bergmann-like glia, but not along the stellate form of astroglia in culture. Neurons moved bidirectionally in vitro (at about the same speed that Rakic has calculated from in-vivo studies) by extending a thickened leading process

along and spiraling about the glial arm. This study thus confirmed the glial guidance theory put forward by Ramon y Cajal and by Rakic and Sidman. Recently we have used the technique of Allen to enhance the Nomarski optical view of migrating cells with a combi-nation of video and computer image analysis methods. This has allowed us to visualize, at very high resolution, the contacts of the leading process with the glial arm, and to study in detail the effects on these events of various antisera directed against adhesive proteins. In contrast, neurons do not migrate along the arms of the stellate astroglial

Fig. 2. (a) Electron microscopy o f the apposition between a cerebeUar neuron and a Bergmann-like astroglial process (go). The thin rim o f cytoplasm and heterochromatin in the nucleus identities this as a granule cell. (b) A t higher po wer, the glial process on which the neuron sits contains intermediate filaments (f) and a coated vesicle (cv). Note ruffling o f both the neuronal and glial membranes (r). (a) x 5000; (b) × 15 000. (Taken, with permission, from Ref. 11.)

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forms, but rather stay in place and extend growth cones. These studies suggest that neuronal behavior relates to astrogiial morphology - neurons migrate on Bergmann-like giia and are immobilized on astrocyte-like gila u. A central question yet unanswered is whether the cellular properties that underly the architectonic roles of gila (especially their shape and cell surface components), are intrinsic or are induced by interactions with particular neurons. When cells were cultured from the neurological mutant weaver, a mouse that suffers failure of granule neuron migration in concert with abnormalities Fig. 4. Morphology of ustroglia cultured in the absence of neurons. After 48 h in vitro, cells from fraction la were stained with antisera to gllal ]~ilamentprotein. The predominant form of stained cell has a flattened morphology and a dense, perinuclear bundle of filaments (a and b). Some cells have a more elongated form (c) with stained ~laments and enlarged 'end feet'. A few stained cells are multipolar (d) with flattened endings. Phase-contrust microscopy x 475. (Taken, with permission, from Ref. 14.)

Fig. 3. Neuronal migration on astroglial processes in vitro. After 24 h in culture, cells taken from mouse cerebellum on postnatal day 7 were observed with time-lapse video microscopy. A phase-bright neuron (arrow) with a thickened leading process (lp) is seen moving along a long glial process. In this field, the migrating neuron pushes beneath and subsequently pusses by a stationary neuron also attached to the glial arm. (a) Time zero, Co) after 30 min, (c) after 60 min, (d) after 90 min. Photographs were taken from the video monitor. Phase-contrast microscopy × 1290. (Taken, with permission from Ref. 11.)

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I Fig. 5. Effect of neurons on astroglial differentiation. In the absence of neurons (-N), cerebellar astroglia have aflattened, undifferentiated morphology and, with time, proliferate rapidly. When granule neurons are added ( + N), astroglial proliferation ceases, the glia differentiate into shapes commonly seen in vivo and in vitro and specific neuron-glia associations occur. Most striking is that neurons (arrows) migrate on elongated forms.

in the shape and alignment of the Bergmann glia 12'13, three findings emerged. Very few granule cells survived and little neurite outgrowth was seen, the morphology of the astroglia was abnormal and n e u r o n glia associations were mostly absent. In addition, lectin agglutination studies showed a defect in the cell surface elements of granule neurons, providing evidence that the weaver gene is normally expressed in granule neurons t4. These studies led us to consider whether it is the mutant gene or the scarcity of neurons per se that is responsible for the abnormalities in astroglial differentiation in weaver mice. To test this, we developed a method to separate rapidly (within minutes) neurons and astroglia into highly purified cellular fractions. We then recombined the purified neurons and glia from normal or mutant cerebella in varying ratios, and analysed astroglial morphology is. In the absence of neurons, normal astroglia lost the complex shapes commonly seen in our cultures and had a very flat morphology. When a neuronal fraction rich in granule neurons and devoid of astroglia was added, the astroglia rapidly differentiated into complex forms. The unexpected and exciting finding from these

studies was that astroglial morphology and proliferation were reciprocally regulated by the neurons. In the absence of neurons, the glia proliferated rapidly. When neurons were added, cell division stopped and the glia differentiated xs. Recent evidence indicates that the effects of neurons on glia are contact-mediated 16. 'Mixing and matching' neurons and astroglia purified from weaver and normal mice has recently demonstrated that weaver neurons fail to migrate on wild type astroglial processes in vitro, and that they impair astroglial differentiation. In contrast, normal neurons associate with weaver astroglia, inducing their differentiation and forming tight appositions seen in migrating neurons in vivo (M. E. Hatten, R. K. H. Liem and C . A . Mason, unpublished observations). These studies suggest that the granule neuron is a primary site of action of the weaver gene and that neuron-glia interactions regulate astroglial differentiation ~7. It should now be possible to use this method to recombine cells from different brain regions or points of development, and to test the cellular regulation of neuron-glia relationships. These in-vitro studies have thus led us to the view that during CNS development, neurons and astroglia

are highly interdependent. Neurons appear to induce astroglial differentiation, which, in turn, facilitates neuronal migration and positioning. Disruptions in cell-cell interactions between neurons and glia, as is the case in the weaver mouse, apparently leads to a cascade of missed cues ending in the abnormal differentiation and poor survival of both neurons and glia. The underlying aim of our studies has been to provide a functional assay for the molecules that regulate neuronal migration. Recently we have raised several polyclonal antisera against whole cerebellar cells that block neuron-glia associations in culture. One of these, named GAM-1 for glia association molecule 1, concomitantly disrupts neuron-glia association and impairs astroglial differentiation is. Thus, in the presence of Fab fragments of the immune serum, neurons are randomlY distributed and gliat processes are short and stunted. By immunocytochemical localization using light and electron microscopes, the GAM-1 antibody was found to bind both neuronal and glial cell surfaces with heavy labelling at neuron-glia junctions. We are now in the process of identifying the antigens for these antisera and purifying them. By immunoblotting and immunopreeipitation, five or more glyeoproteins have been recognized. Absorption of the antiserum with PC12 cells (neurons that do not bind to CNS glia), leaves a major glycoprotein band, present as a smear in SDS-PAGE gels between 85 and 95 kDa. It remains to be proven whether monospecific probes against this band will block neuron-glia association in vitro. From another perspective, others have asked whether glial cells regulate the morphological differentiation of neurons. Prochiantz and his colleagues have shown that neuritic arbors of mesencephalic neurons express different shapes when plated with glia taken from the striatum versus the mesencephalon 19. Recent studies have examined cytoskeletal elements and have indicated that the different neuronal shapes induced by cell-cell contacts with different types of astroglia might relate to the construction of axonal as opposed to dendritic arborizations (A. Prochiantz, pers. commun.). In other work, Mudge has shown that neurons in the peripheral nervous system also rely on their interactions with glia for their morphological differentiation z~. Thus the differentiation of neurons and

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glia appears to be reciprocally regulated during brain development, presumably by the molecules that govern their cell-cell contacts and by factors that influence their cytoskeletal organization. Although it is widely assumed that astroglia in axon tracts guide axons directionality, there is as yet no case in which this has been proven conclusively. The strongest evidence for a role for glial cells in axon guidance comes from invertebrate systems, where pioneer neurons contact particular nonneuronal cells in their pathway2t'22. In vertebrate systems, it is hard to visualize such contacts, both because immature glia are difficult to identify immunochemically and because of the proportionately larger number of young axons present. For mouse optic nerve23 and amphibian spinal cord 24, glia and the openings or 'channels' they bind have been proposed as the guides for the initial routing of axons23-25. Once the axons are in the optic nerve, the ratio of axons to glia becomes very large (especially in higher mammals), and the question arises of whether specific contacts, either axon-axon or axon-glia, are really necessary; alternatively the axons could find their way indiscriminately,touching either axons or gila without making a 'choice'. The latter view is supported by recent studies on the developing optic nerve of mouse26 and of monkey27, showing that axons grow along other axons as well as along immature glia, and that there is not a one-to-one relationship between axons and glia in the optic tract. In this system, it appears that during axon outgrowth the broad lamellopodia of the growth cones of axons, rather than glial elements, wrap around groups of axons. In addition, axons do not preserve a fiber-fiber projection along the tract, but rather freely exchange neighbors as they wend their way to the chiasm27. In the developing cerebellum and optic nerve, we have shown that quite immature forms of glia are present in the emerging white matter during neurite extension, and that their predominant cytoskeletal element shifts from vimentin to glial filaments only after growing axons pass through the tract 28,29. In both systems, the cessation of axon outgrowth appears to signal further differentiation of astroglia, and astroglial process outgrowth commences with extension and branching of glial arms. It is at this point when astroglial processes wrap themselves

around axon bundles26. The time course of glial differentiation in the axon pathway raises the question of whether axons trigger glial differentiation, as we have seen with granule neurons and cerebellar astroglia in

more complex with the recent finding that neural retinal cells release complexes of proteins and glycosaminoglycans called adherons37'3s, which stimulate cell adhesion and neurite extension. Glaser and his coworkers have suggested that a 170000 kDa surface vitro. Little is known about the organ- antigen C1H3 on neural retinal cells ization and disposition of glia in other interacts with this complex to mediate brain regions, especially at early phases neurite growth 39'4°. Lander et al. 41 of development. It will be important to have also isolated an outgrowth proanalyse this in detail before general- moting factor from conditioned izations about the role of glia in medium that is an aggregate of heparan directing and organizing fiber projec- sulfate proteoglycan, laminin and entactin. The cellular source - neuronal tions can be made. The central issue in these studies has or glial - of these extracellular been whether astroglia affect axon adhesion-promoting complexes must growth by contact or by trophic be resolved. interactions. In-vitro studies have been It has also been found that laminin used to test the adhesive specificity of and fibronectin, extracellular molneurites for glia as opposed to other cell ecules that mediate cell-cell adhesion types, and to begin to sort out which outside the nervous system, can serve cell surface components promote neu- as substrates for neurite outgrowth. rite extension. Several groups have Letourneau and others have shown shown that neurons prefer astroglial that both PNS and CNS neurites cells to fibroblasts as substrates for adhere to laminin substrates in culture neurite growth 3°-32. and that some neurites grow well on a A number of groups have used in- fibronectin-coated surface42-44. There vitro methods to assess what sorts of is mounting evidence that growth cones molecules are released by non-neu- have receptors for laminin and, in some ronal cells into the culture medium or cases, fibronectin. Recently CSAT, a onto the culture substratum to promote 140 kDa surface antigen present on neurite growth. Varon and others have growth cones45, has been shown to serve isolated neuronotropic factors as a receptor for both laminin and (NETs) 33"34, substrate-bound neurite fibronectin (A. F. Horwitz, pers. promoters (NPFs) 34 and nerve growth commun.). This dual receptor has been factor (NGF)-like proteins33'36 from proposed to be a transmembrane link medium conditioned by normal and that connects the cytoskeleton with the transformed astroglial cell lines in extracellular matrix. In-vitro studies culture. These studies have become with the CSAT antigen indicate that it

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Fig. 6. Model for defects in weaver (wv/wv) neuron--glio interactions. Normal neurons form appositions typical of migrating cells on either normal or weaver astroglia. Weaver neurons fail to migrate on either type o f astroglial cell. In all cases, astroglial differentiation is enhanced by the presence of normal neurons and is impaired by weaver neurons. (Taken, with permission, from Ref. 17.)

174 is involved in both the extension of neurites and the modulation of neurite bundling46. An important question is whether fibronectin and laminin are present in the developing brain, and whether glia produce them. Both fibronectin and laminin have been extremely difficult to visualize in vivo. Our laboratory and that of Pearlman have reported the transient expression of fibronectin in very discreet, limited CNS areas at early embryonic periods 47'48, but others have disputed these findings. Rogers and Letourneau have shown that laminin is present in early dorsal and ventral roots of the chick spinal cord (P. C. Letourneau, pers. commun.). Recently Liesi has demonstrated that astrocytes produce laminin in vitro 49 and that laminin is prominent in glial scars seen after CNS injury5°. Thus astrocytes seem prime candidates as sources of laminin in vivo. A further question is whether gila express molecules thought to mediate axon-axon interactions. Rutishauser has shown that antibodies against NCAM 51 disrupt axon-axon interactions needed for optic nerve outgrowth s2. Silver and Rutishauser have recently suggested that N-CAM is present on the endfeet of radial neuroepithelial cells and that this provides a preformed adhesive pathway for growing optic axons. Astroglia also appear to make glycosaminoglycans in vitro 53, raising the question as to whether they are produced in emerging axon tracts. Antibodies and other specific probes for chondroitin sulfate proteoglycans and hyaluronic acid have recently been developed 54-56 and have been used to demonstrate the presence of these glycosaminoglycans in the developing cerebellar axon tract; this material then disappears from the extracellular space after the tract is established. In-vitro studies by Carbonetto suggest that neurons do not grow on purified glycosaminoglycanss6. Thus, rather than provide a substrate per se, complex carbohydrates in axon tracts are more likely to provide a corridor filled with highly extended and hydrated molecules that can be penetrated easily by growing axons and can, in some cases, serve as a binding site for neurite adhesion molecules. At present it is difficult to argue that gila play an instructive role in axon growth. The data suggest that glia provide growth factors and a 'menu' of substrates, primarily into the extra-

TINS- April 1980 cellular space, for axon extension. Alternatively gila in the axon tract play the roles traditionally assigned to gila in the adult brain - keepers of the ionic environment and spacers for the neuronal elements. The present challenges are to find the molecules necessary for neuronal migration along astroglia, and to determine whether these or other molecules regulate astroglial differentiation and proliferation. In addition, we must carefully analyse the cellular relationships of glia with axons in axon tracts in vivo, study the extracellular molecules made by astroglia to see which, if any, are critical to axon development, and test whether axons induce astroglial differentiation. Selected references 1 Kuffler, S. W. and Nicholls, J. G. (1966) Ergeb. Physiol. Biol. Chem. Exp. Pharmalkol. 57, 1-90 2 Rakic, P. (1971) J. Comp. Neurol. 141,283-312 3 Rakic, P. (19721J. Comp. Neurol. 145, 61-89 4 Rakic, P., Stensaas, L. J., Sayre, E. P. and Sidman, R. L. (1974) Nature 250, 31-34 5 Palay, S. L. and Chan-Palay, V. (1974) Cerebellar Cortex, Cytology and Organization, pp. 289-316, Springer-Verlag 6 Levitt, P., Cooper, M.L. and Rakie, P. (19811 J. Neurosci. 1, 27-39 7 Raft, M., Miller, R. and Noble, M. 0983) Nature 303, 390-396 8 Temple, S. and Raft, M. (19851 Nature 313, 223-225 9 Hatten, M. E. and Liem, R. K. H. (1981) J. Cell Biol. 90, 622-630 l0 Hatten, M. E. (1984) Dev. Brain Res. 13, 309--313 II Hanen, M. E., Liem, R. K, H. and Mason, C. A. (1984) J. Cell Biol. 98, 193-204 12 Rakic, P. and Sidman, R. L. (1973)J. Comp. NeuroL 152, 103-132 13 Sotelo, C. and Changeaux, J-P. (1974) Brain Res. 77, 484-491 14 Hatten, M. E., Liem, R. K. H. and Mason, C. A. (1984)J. Neurosci. 4, 1163-1172 15 Hatten, M. E. (1985) J. Cell Biol. 100, 384396 16 Hatten, M. E., Woods, M., Sanchez, J., Liem, R. K. H. and Mason, C. A. Neurosci. Abstr. l l (in press) 17 Hatten, M. E., Liem, R. K. H. and Mason, C. A. (1986)J. Neurosci. (inpress) 18 Edmondson, J. L. and Hatten, M. E. (19841 Soc. Neurosci. Abstr. 10, 759 19 Denis-Donini, S., Glowinski, J. and Prochiantz, A. (1984) Nature 307, 641-643 20 Mudge, A. (1984) Nature 309, 367-369 21 Berlot, J. and Goodman, C.S. (1984) Science 223, 493-495 22 Nardi, J. B. (19831 Dev. Biol. 95, 163-174 23 Silver, J, and Sidman, R. L. (1980) J, Comp. Neur. 189, 101-112 24 Singer, M., Nordlander, R. H. and Egar, M. (19791 J, Comp. Neur. 185, 1-22 25 Nordlander, R. H. and Singer, M. (1982) Devel. Br. Res. 4, 181-193 26 Bovolenta. P. and Mason, C . A . Soc.

Neurosci. Abstr. 11 (in press) 27 Williams, R. and Rakic, P. (1985) Proc. Natl Acad. Sci. USA 82, 3906-39111 28 Bovolenta, P., Liem, R. K. H. and Mason. C. A. (1984) Devel. BioL 102~ 248-259 29 Bovolenta, P., Liem, R. K. H. and Mason, (7. A. (19841 Soc. Neurosci. Abstr. 10, 427 30 Noble, M., Fok-Seang, J. and Cohen. J. (1984)J. Neurosci. 4, 1892-1903 31 Fallon, J. R. (19851J. Cell Biol. 100,198--207 32 Mallat, M., Moura Neto, V., Gros, F., Glowinski, J. and Prochiantz. A. Dev. Br. Res. (in press) 33 Varon, S. and Bunge, R. P. (1978) Annu. Rev. Neurosci. 1,327-362 34 Adler, R. and Varon, S. (1981) Dev. Biol. 8, 1-11 35 Muller, H. W., Beckh, S. and Seifert, W. (19841 Proc. Natl Acad. Sci. USA 81, 12481252 36 Monard, D., Solomon, F., Reutsch, M. and Gysin, R. (19731 Proc. Natl Acad. Sci. USA 70, 1894-1897 37 Schubert, D., LaCorbiere, M., Klien, F. G. and Birdwell, C. (19831J. Cell Biol. 96,990998 38 Schubert, D. and LaCorbiere, M. (19851 J. Cell Biol. 1130,56-63 39 Cole, G. J. and Glaser, L. (19841J. Cell Biol. 99, 1605-1612 40 Cole, G. J., Schubert, D. and Glaser, L. (1985) J. Cell Biol. 100, 1192-1199 41 Lander, A. D., Fuji, D, K. and Reichardt, I.. F. (19851 Proc. Natl Acad. Sci. USA 82, 2183-2187 42 Manthorpe, M., Engvall, E., Ruoslati, E., Longo, F. M., Davis, G. E. and Varon, S. (1983) J. Cell Biol. 97, 1882-1890 43 Rogers, S. L., Letourneau, P.C., Palm, S. L., McCarthy, J. and Furcht, L. (19831 Dev. Biol. 98, 212-220 44 Rogers, S. L., McCarthy, J. B., Palm, S. L., Furcht, L. T. and Letourneau, P. C. (1985) J. Neurosci. 5, 369--378 45 Damsky, C., Knudsen, K., Bradley, D., Buck, C. and Horwitz, A. F. (1984) J. Cell Biol. 100, 1528--1539 46 Bozyczko, D. and Horwitz, A. F.J. Neurosci. (in press) 47 Hatten, M. E., Furie, M. B. and Rifkin, D.B. (1982) J. Neurosci. 2, 1195-1206 48 Pearlman, A. L., Stewart, G. R. and Cohen, J. P. (1984) Soc. Neurosci. Abstr. 10, 39 49 Liesi, P., Dahl, D. and Valeri. A. (19831 J. Cell Biol. 96, 920--924 50 Leisi, P., Kaakkola, S., Dahl, D. and Vaheri, A. (19841 E M B O J. 3, 683-686 51 Rutishauser, U. (1984) Nature 310, 549-554 52 Thanos, S., Bonhoeffer, F. and Rutishauser, U. (19841 Proc. Natl Acad. Sci. USA 81, 1906-19111 53 Aquino, D. A., Margolis, R . U . and Margolis, R. K. (1984)J. Cell Biol. 99, 1130 54 Ripellino, J. A., Klinger, M, M., Margolis, R . U . and Margolis, R. K. J. Histochem. Cytochem. (in press) 55 Knudson, C. B. and Toole, B. P. (19851 J. Cell. Biol. 100, 1753-1758 56 Carbonetto, S., Gruver, M. M. and Turner. D. C. (19831 J. Neurosci. 3, 2324-2335 Mary E. Hatten and Carol A. Mason are at the Department of Pharmacology, New York University School of Medicine, 550 First Avenue, New York, N Y I0016, U S A