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PII: S0166-2236(96)01044-2

  1. 1. Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0166 - 2236/97/$17.00 PII: S0166-2236(96)01044-2 TINS Vol. 20, No. 7, 1997 303 46 Vaid, R.R. et al. (1996) Brain Res. 733, 31–40 47 Gonzalez, H.T., De la Cruz, M. and Mantolan, S.B. (1996) J. Histochem. Cytochem. 44, 1399–1413 48 O’Dell, T.J. et al. (1994) Science 265, 542–546 49 Dinerman, J.L. et al. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2414–2418 50 Murphy, K.P.S.J. et al. (1994) Neuropharmacology 33, 1375–1385 51 Bennett, M.R. (1994) Gen. Pharmacol. 25, 1541–1551 52 Hawkins, R.D., Zhuo, M. and Arancio, O. (1994) J. Neurobiol. 25, 652–665 53 Schuman, E.M., Meffert, M.K. and Schulman, H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11958–11962 54 Cummings, J.A., Nicola, S.M. and Malenka, R.C. (1994) Neurosci. Lett. 176, 110–114 55 Hartell, N.A. (1996) Neuron 16, 601–610 56 Son, H. et al. (1996) Cell 87, 1015–1023 57 Kantor, D.B. et al. (1996) Science 274, 1744–1748 58 Norris, P.J., Faull, R.L.M. and Emson, P.C. 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(1995) Neuron 15, 751–754 91 Nosten-Bertrand, M. et al. (1996) Nature 379, 826–829 92 Saucier, D. et al. (1996) Behav. Neurosci. 110, 103–116 93 Hölscher, C., Anwyl, R. and Rowan, M. (1997) NeuroReport 8, 451–454 C. Hölscher – NO – the enigmatic messenger REVIEW Acknowledgements The work was supported by the Irish Health Research Board. The generous support and help of M.J. Rowan is gratefully acknowledged, as well as the help of H.V. Budgey in editing the manuscript. Maturation of the mammalian dorsal root entry zone – from entry to no entry Jon Golding, Derryck Shewan and James Cohen Interfaces between glial cell precursors of the PNS and CNS are established early in development and form the sites where sensory axons enter and motor axons exit the developing CNS.The molecular and cellular interactions that lead to the formation of these glial interfaces are only now becoming apparent. New in-vitro techniques are providing clues as to how the maturation of PNS–CNS glial interfaces generates barriers to regenerating axons. Trends Neurosci. (1997) 20, 303–308 THE INABILITY OF axons of the mature mammalian CNS to regenerate after injury is believed to be due in large part to the combined inhibitory effects of its major macroglial elements: oligodendrocytes and astrocytes. Whilst there is now substantial evidence for an inhibitory role for myelin proteins, synthesized by oligodendrocytes1 , the role of astrocytes remains relatively obscure. Part of the problem is that astro- cytes are heterogeneous, both in their morphology and their interactions with other cell types of the CNS (Ref. 2). Moreover, their antigenic phenotype, es- pecially after injury, has been poorly characterized. Together these problems have made it difficult to study the role of astrocytes in nerve injury. In contrast to the grey and white matter of the CNS, the anatomy of the PNS–CNS interfaces between mammalian spinal nerve roots and the spinal cord is comparatively simple. These sites, where motor and sensory axons, respectively, exit and enter the CNS, contain a unique cellular apposition between astrocytes and Schwann cells. In the dorsal roots, this interface between the CNS and PNS is known as the dorsal root entry zone (DREZ). This region has previously been the focus of studies related to the failure of lesioned sensory axons to reconnect with the spinal cord after injury. Thus, if the dorsal root is damaged in mature animals, the axons of primary afferents regenerate successfully in the Schwann cell-containing PNS portion of the injured roots, but stop abruptly on contacting the DREZ astrocytes. Two important aspects of the DREZ make it an attractive region for studying the role of astrocytes in the failure of nerve repair and regener- ation in the CNS. First, the simplicity of the cellular composition of the DREZ has facilitated studies of interactions between regenerating axons and astro- cytes. Second, Carlstedt showed that, within the first postnatal week in rats, injured sensory axons can regenerate through the DREZ and reconnect within the spinal cord3,4 . This suggests that development of the DREZ deserves closer scrutiny, since it raises the possi- bility that DREZ astrocytes undergo a developmental transition soon after birth in rats and begin to express Jon Golding, Derryck Shewan and James Cohen are at the Dept of Developmental Neurobiology, UMDS-Guy’s Hospital, London, UK SE1 9RT.
  2. 2. 304 TINS Vol. 20, No. 7, 1997 molecules that repel growing axons. What is the basis for the change in the properties of PNS–CNS glial interfaces, from a conduit for growing axons in devel- opment, to a ‘barrier’ to their reconnection to the mature spinal cord after injury? Here, we first describe what is known of the origins of glial interfaces in early development, and their influence on the formation of axon exit and entry points along the neuraxis. We then review recent progress in identifying the cellular and molecular components of the DREZ that might confer its barrier properties. The formation of PNS–CNS glial interfaces Astrocytes and Schwann cells have different embryo- logical origins in vertebrate development: from neural tube and neural crest, respectively. PNS–CNS glial interfaces arise at the surface of the neural tube, potentially by a tripartite interaction between cells derived from the neural crest, the processes of neuro- epithelial cells, and the intervening basal lamina of the neural tube. Of these, the contribution of neural- crest cells has been the best characterized. In the chick, Le Douarin et al.5 have shown that a subset of neural-crest cells, migrating in the ventrolateral path- way alongside the neural tube, take part in the formation of exit–entry points (Fig. 1A,B). This subset is a late- migrating population that selectively expresses c-cad7, a member of the cadherin family of cell-adhesion mol- ecules, initially in the dorsal midbrain at stage 10, and then progressively further caudally along the neur- axis6 . These c-cad7-positive cells contribute to the formation of ‘boundary caps’, delineating the sites of presumptive exit–entry points (Fig. 1C). Once they adhere, neural-crest cells might breach the basal lam- ina of the neural tube by secreting proteases7,8 , since they are thought to degrade the basal lamina of ecto- derm in this way9 . Although these neural-crest cells are the earliest reported markers of developing exit–entry points, it is possible that restricted domains of the basal lamina of the neural tube define the sites where the migrating c-cad7-expressing neural-crest cells arrest. Thus, the function of c-cad7 might be to aggregate neural-crest cells, ensuring coherent migr- ation to the same loci, but the signals to stop migration at specific points on the neural tube are possibly medi- ated by distinct adhesive mechanisms10 . A further possibility is that specific groups of neuroepithelial cells differentiate at the presumptive exit–entry points and degrade the basal lamina by secreting proteases themselves or by inducing the attachment of neural- crest cells, or both. Thus, a population of chick neuro- epithelial cells at the prospective ventral exit points penetrate the basal lamina of the neural tube at stage 17, when the first motor axons emerge from the ventral exit points11 . In particular, matrix metallopro- teinases (MMPs) specific for glycoprotein substrates of the extracellular matrix (ECM) might be involved, such as the MMP Stromelysin-1, which is expressed by neuroepithelial cells in the chick12 . A recent study further implicates neuroepithelium in determining positional specification of exit–entry points. Niederländer and Lumsden13 excised neural crest from rhombomere (r)3, in which an exit–entry point does not arise, in a stage-10–11 chick host, and transplanted age-matched quail neural crest from r4, in which the exit–entry point of the facial nerve nor- mally develops. However, this failed to generate an inappropriate exit–entry point in r3 of the host, implying that the initial signals for exit–entry point formation are derived from neuroepithelium and not neural crest. Glial interfaces, generated as a result of such cellu- lar interactions, can be grouped into three categories. Most Schwann cell–CNS glial interfaces are segregated into distinct dorsal (sensory-axon) entry points and ventral (motor-axon) exit points (Fig. 1D). However, in some regions of the hindbrain, motor and sensory axons exit or enter the CNS at common sites14 (Fig. 1E), whilst in others, distinct exit points for ventral motor axons are also produced (for example, cranial nerves VI and XII) (Fig. 1F). This pattern is also evident in the cervical spinal cord, where common dorsal exit–entry points are produced transiently during development, while distinct ventral motor-axon exit points also arise15,16 (Fig. 1F). Development of PNS–CNS glial interfaces and interactions with axons Although the changes in antigenic phenotype asso- ciated with the maturation of CNS glial cells and Schwann cell precursors have been well documented, few studies have focused specifically on developing glia at PNS–CNS interfaces. Boundary-cap cells in the trunk region of the mouse are the first neural-crest cells at this axial level to express the transcription fac- tor Krox20 [at embryonic day (E)10.5] and subse- quently to become positive for S-100, a marker of dif- ferentiated Schwann cells, by E12.5 (Ref. 17). Early phenotypic changes specific to CNS glia at such inter- faces have not been reported. However, in the rat spinal cord, the only definitive astrocytic marker, the cytoskeletal protein GFAP, is first detected in the distal processes of radial glia at the margins of the ventral, then dorsal spinal cord, at E16 and E19 re- spectively18 , coincident with the growth of the final cohort of axons through the glial interfaces in these regions. Could these studies imply that the Schwann cells and CNS glia that populate the interfaces mature before glia elsewhere in the nervous system? Precocious development might be required for appro- priate interactions to take place between the earliest arriving axons and the glial cells at PNS–CNS inter- faces. Thus, it is known that in the case of primary afferents, there occurs a protracted ‘waiting period’ at the surface of the dorsal grey matter, in the vicinity of the DREZ. Recent work suggests that this stalling of axons is regulated by the local expression of SemIII (D), a member of the semaphorin family19–21 . In the mouse, neurotrophin 3 (NT3)-dependent muscle sensory afferents are the first to grow into the grey matter of the spinal cord at E14.5, to reach their ventral motoneurone targets, coinciding with the age at which the growth of their axons becomes insensi- tive to the repulsive effects of SemD in tissue culture. In contrast, NGF-dependent small-diameter afferents, whose growth continues to be inhibited by SemD in vitro, grow into their target fields within the super- ficial laminae of the dorsal horn at E17.5, only after expression of SemD mRNA in the spinal cord has receded ventrally. In addition, another semaphorin, SemA, is expressed in the nerve roots of the mouse trunk between E12.5 and E14.5, and thus by analogy, might also be involved in confining the growth of sensory afferents22 . J. Golding et al. – Axon growth and regeneration across PNS–CNS glial interfacesREVIEW
  3. 3. TINS Vol. 20, No. 7, 1997 305 Postnatal changes at the DREZ generate a barrier to axon growth Shortly after birth, changes occur in the organiz- ation of the mammalian DREZ that might be correlated with the inability of regenerating primary sensory axons to re-enter the spinal cord after injury. Astrocytes extend processes up to 100 ␮m into the dorsal roots between basal lamina tubes of Schwann cells, and gaps are present in this intervening basal lamina at the DREZ (Ref. 23). This organization is not only thought to confer mechanical strength on the DREZ (Ref. 24), but also increases the surface area of direct contact between Schwann cells and astrocytes, generating a unique environment at the interface25 . Moreover, this cellular organization ensures that astrocytic processes are the first CNS elements that are encountered by regenerating primary sensory axons. In key experiments carried out by Carlstedt3,4 on the influence of age on the ability of injured rat sensory afferents to reconnect with the spinal cord, a ‘critical period’ was identified, between birth and one week, when significant numbers of injured axons were able to regenerate into the cord. In older animals, regener- ating labelled axons were observed stopping at, or turning back from, the DREZ. Electron microscopic studies in adult rats have shown that regenerating axons stop growing precisely at the astrocytic processes within the DREZ (Ref. 26). Growth cones that contact DREZ astrocytes exhibit ultrastructural features that are more reminiscent of presynaptic end- ings than advancing growth cones26 . These studies suggested that contact with mature DREZ astrocytes might activate a ‘physiological stop pathway’26 within the neurones, which is proposed to be analogous to the mechanisms whereby growth cones stop at their appropriate targets27,28 . Further evidence for the role of mature astrocytes in preventing regenerating axons (and possibly Schwann cells) from crossing the DREZ is also provided by experiments in which the dorsal spinal cord of the rat is depleted of glia by X-irradiation soon after birth. This treatment generates gaps in the glial limitans through which Schwann cells migrate ectopically into the spinal cord along astrocyte- and oligodendrocyte- free sensory afferents29 . Invasion by Schwann cells is limited to the glia-depleted areas of the CNS and the few remaining astrocytes appear to block the further migration of Schwann cells. Examination of dorsal root lesions that had been generated two weeks after X-irradiation revealed that several regenerating sensory axons entered the spinal cord through the astrocyte-free DREZ (Ref. 30). What is known of the identity of the molecules that might contribute to the DREZ barrier? Tenascin and sulphated proteoglycans have been implicated as inhibitors of axon growth that are expressed by DREZ astrocytes31 and in other regions of the developing CNS (Refs 32,33). Thus, Silver et al. reported that both tenascin and sulphated proteoglycans become con- centrated at the CNS side of the rat DREZ towards the end of the critical period31 , although a later study indi- cated that tenascin mRNA and protein are strongly expressed by rat DREZ astrocytes from birth34 . Following injury to the dorsal root in rats older than the critical age, these molecules become highly con- centrated at the DREZ and within proximal regions of the dorsal horn31 , in association with extensively branched ‘reactive’ astrocytes35 . Reactive astrocytes are a major component of the glial scars that are formed around CNS lesions in adult rats, and through which axons fail to regenerate1,36 . In vitro, myelin-free plasma membranes isolated from glial scars in lesioned brains of adult rats have been shown to inhibit the growth of neurites from dorsal root ganglia, and septal and hippocampal J. Golding et al. – Axon growth and regeneration across PNS–CNS glial interfaces REVIEW A C D E F B Neural tube Basal lamina Developing sensory ganglion Developing exit points Neural-crest cell c-cad7-expressing neural-crest cell Neuroepithelial cell Motor neurone Hindbrain or cervical spinal cord HindbrainTrunk spinal cord Fig. 1. Development of PNS–CNS glial interfaces. (A,B) Schematic transverse sections through the neural tube that illustrate the formation of the PNS–CNS glial interface. The earliest known marker of prospective PNS–CNS glial interfaces is the cadherin c-cad7. A subpopulation of c-cad7-expressing neural-crest cells migrate as a group from the dorsal surface of the neural tube (A) to form boundary caps, the sites where PNS–CNS glial interfaces ultimately develop (B). Neural-crest cells, neuroepithelium, the basal lamina of the neural tube and axons (some projecting only transiently, dotted line) can all contribute to the formation of glial interfaces, as summarized in B. (C) In a transverse section through the chick hindbrain at stage 16, in-situ hybridization identifies a c-cad7-expressing subpopulation of neural-crest cells that are forming boundary caps. Scale bar, 50 ␮m. Photo courtesy of C. Niederländer. (D) In the trunk regions, glial interfaces are segre- gated into distinct dorsal (sensory-axon) entry and ventral (motor- axon) exit points. (E,F) However, at some levels of the hindbrain, common dorsal exit–entry points for branchial motor and sensory axons are formed (E) whilst at other hindbrain levels and in the cervical spinal cord, common dorsal exit–entry points as well as ventral exit points are formed (F).
  4. 4. 306 TINS Vol. 20, No. 7, 1997 neurones of embryonic rat, whilst membranes isolated from uninjured brains supported neurite outgrowth37 . This growth-inhibitory effect could be removed by pretreatment with proteoglycan-degrading enzymes37 . Furthermore, purified chondroitin-sulphate proteo- glycans38 and tenascin39 have been shown to act as barriers to growth of CNS neurites in vitro when pre- sented as sharp substrate boundaries. However, the increased expression of inhibitory molecules is only one possible mechanism that might account for the failure of axon growth at the mature DREZ. Another possibility is that maturation of astrocytes, both in vivo and in vitro, might downregulate the production of cell-adhesion molecules that promote axon growth40 , in tandem with the upregulation of barrier molecules.This raises the question of whether matu- rational changes that are intrinsic to normal astro- cytes are themselves responsible for the acquisition of barrier properties at the DREZ. Culture model systems enable the interaction of axons with the DREZ to be studied in novel ways To test whether maturing astrocytes acquire the ability to inhibit axon growth, it is necessary to con- front growing primary sensory axons with the unin- jured DREZ, a scenario that is impossible in vivo. However, this has been made possible by adapting an in-vitro cryoculture approach41–43 . In this technique, sensory neurones are cultured on thin cryostat sec- tions of nerve tissue, an environment that closely rep- resents the one that they would encounter in vivo. The ECM and cell-surface molecules are pre- served, whilst cells within the frozen tissue are non-viable and are no longer able to secrete soluble factors. This allows the differential effects of substrate and soluble factors to be studied. By preparing longitudinal cryostat sections of the dorsal spinal cord of the rat, it has been possible to incorporate the DREZ and attached dorsal roots and use these as substrates for cultures of dissociated neurones44 of the dorsal root ganglia (Fig. 2). Neurones that adhere to the dorsal roots extend neurites along the basal lamina tubes of Schwann cells towards the DREZ, as they would in vivo, where they might grow across to the spinal cord, stop, or turn back along the dorsal root. A major advantage of this approach is that DREZ from various developmental stages, both before and after the hypothesized critical period, can be employed as substrates, enabling us to study more closely how the development of this region influences neurite growth. Thus, we have found that neurites growing from neurones of neonatal dorsal root ganglia cross the newborn (postnatal day 0, P0) DREZ more readily than P6 or adult DREZ (Fig. 3A,B). This supports the idea that inhibitors of axon growth appear at the DREZ during a critical period within the first postnatal week3,4 that is independent of injury-induced responses by glial cells. This might indicate that the sulphated proteoglycans that accumulate at the DREZ by P6 are sufficient to halt growing primary sensory axons, but the possibility remains that other, as yet uncharacterized, molecules might also be involved. Significantly, neurite outgrowth on the spinal cord or dorsal root, immediately central or peripheral to the DREZ, was similar at different ages that encompassed the critical period, suggesting that changes intrinsic to the DREZ are initially responsible for developing a barrier to regenerating axons. By adulthood, both the DREZ and the CNS adjacent to it were poor substrates for outgrowth of neurites of the neonatal dorsal root ganglia, suggesting that after the critical period ad- ditional inhibitory molecules become expressed generally within the CNS. Influence of age of neurones on their interactions with the DREZ By testing a range of ages of neurones in cryocul- ture, we were also able to analyse separately the influ- ence of development of neurones of the dorsal root ganglia on the ability of neurites to cross the DREZ. We found that neurites from early embryonic neur- ones were less sensitive to the P6 DREZ inhibitors44 (Fig. 3C) than neurites from more mature neurones, suggesting that, in tandem with changes at the DREZ J. Golding et al. – Axon growth and regeneration across PNS–CNS glial interfacesREVIEW A Cryoculture B C D Cryosection Plane of section Dorsal roots Dorsal root entry zone (DREZ) Dissociated DRG neurones Cryosection Neonatal DRG neurone on adult DREZ cryosection after immunolabelling Rostral Grey matter Ventral root Fig. 2. Axon interactions with the dorsal root entry zone (DREZ) can be modelled in vitro, using cryoculture. In this technique, uninjured spinal cord, taken from rats of various ages (A), is cut into thin (8–10 ␮m) longitudinal sections on a cryostat (B). These sections are transferred to sterile glass coverslips and are used as substrates for the growth of neurites from dissociated neurones of rat dorsal root ganglia (DRG) (C). After 18 h, the cultures are fixed and stained (D) with antibodies against: laminin to label the basal lamina tubes of the Schwann cells of the dorsal roots (in green), GFAP to label astrocytes within the CNS (in blue) and GAP-43 to label growing neurites and their cell bodies (in red). Neurites from neurones of postnatal DRG grow well along the dorsal roots, but stop at the PNS–CNS interface of the adult DREZ, mimicking closely the behaviour of regenerating axons after a prior lesion in vivo. Abbreviation: P0, new- born, postnatal day 0.
  5. 5. TINS Vol. 20, No. 7, 1997 307 during the critical period, there are corresponding maturational changes in the expression of axonal receptors for ligands that influence growth. A parallel approach in vivo involves transplanting allografts of embryonic dorsal root ganglia into adult rats. In these animals some immature axons were found to have entered the spinal cord of the adult host45 , although it is unclear whether they actually grew through the DREZ. Both studies are consistent with an emerging general principle that immature neurones are better able to extend axons within the mature CNS environ- ment46–50 , possibly as a result of their lack of receptors that recognize inhibitory ligands. The complementary findings that have been obtained from both in-vivo and in-vitro studies on maturation of the DREZ are summarized in Fig. 4. J. Golding et al. – Axon growth and regeneration across PNS–CNS glial interfaces REVIEW DR injury site Reactive gliosis DREZ Some injured DRG axons regenerate through the DREZ Regenerating DRG axons fail to cross the DREZ In vivo Cryoculture model Neonatal rhizotomy DR injury site Mature rhizotomy P P E E Oligodendrocytes Postnatal (P) or embryonic (E) DRG neurones CNS DRDREZ CNS DRDREZ Neonatal cryosection Mature cryosection Astrocyte (growth permissive) Astrocyte processes extend into DR, increasing the width of the DREZ, and become growth inhibitory DRG DRG Fig. 3. An in-vitro model of the dorsal root entry zone (DREZ) repli- cates in-vivo observations. The critical period for regeneration of pri- mary sensory axons across the DREZ in vivo might be dependent upon developmental changes that occur both at the DREZ and within neur- ones of dorsal root ganglia (DRG). Cryosections of uninjured spinal cord, incorporating dorsal roots and DREZ from newborn (P0) (A) or postnatal day 6 (P6) (B) rats were used as substrates for the growth of neurites from DRG neurones of the P0 rat. Neurites (labelled red with anti-GAP-43 antibody and arrowed in A) consistently cross the P0 DREZ more successfully than the P6 DREZ (dotted line, between anti-laminin- stained endoneural tubes of Schwann cells and the spinal cord), while their growth on P0 and P6 spinal cord adjacent to the DREZ is similar. Because the DREZ substrates are taken from uninjured animals, this suggests that during the first postnatal week, changes intrinsic to the DREZ generate an environment that is inhibitory for axon growth. Although the P6 DREZ inhibits neurite crossing by postnatal DRG neur- ones, neurites from embryonic (E15) DRG neurones are less sensitive to this barrier (arrow in C), suggesting that developmental changes in neuronal receptors are also important for the generation of a barrier to growing axons at the DREZ. Scale bars, 70 ␮m (A and B), 50 ␮m (C). Fig. 4. In vivo and in vitro experimental models of the rat dorsal root entry zone (DREZ). Both approaches demonstrate a critical neonatal period, between birth and 1 week post- natally, during which regenerating primary sensory axons of dorsal root ganglia (DRG) can cross the DREZ. This might be due to maturational or injury-induced changes at the DREZ, or both. As the rat matures postnatally, astrocytic processes (but not oligodendrocytes) extend into the dorsal root (DR) (bottom panels), expanding the region of contact between astrocytes and Schwann cells (which defines the DREZ). Molecules that inhibit axon growth might become concentrated at these contact sites within the DREZ, as indicated by the change in the DREZ astrocyte surface from green (growth permissive) to red (growth inhibitory). A conse- quence of DR injury (rhizotomy) after the critical age is a marked gliosis at the DREZ. Molecules that inhibit axon growth might become associated with these reactive glia (top panels). By using uninjured spinal cord cryosections to model the DREZ in vitro, it is possible to examine how DRG axons behave at the neonatal and adult DREZ in the absence of reactive gliosis. As with the in-vivo studies, postnatal DRG neurones (P) fail to grow neurites across the mature DREZ, suggesting that maturational changes are primarily involved in generating a barrier to axon growth at the end of the critical period. When embryonic DRG neurones (E) are applied to the cryosections, they are able to extend neurites across both the neonatal and mature DREZ, suggesting that they have yet to acquire functional receptors for the inhibitory DREZ ligands.
  6. 6. 308 TINS Vol. 20, No. 7, 1997 Future prospects Current knowledge on the provenance of exit and entry points of the neural tube is limited, but recent work suggests that cells of neuroepithelial origin, rather than of the neural crest, determine the sites where these points form in development. Further information on the molecular and structural proper- ties of these immature glial environments should enlighten us as to the optimal conditions for axon growth across such interfaces. The mechanisms that underlie the failure of injured axons to regenerate across mature interfaces remain equally elusive. Complementary in-vivo and in-vitro studies that focus on the DREZ offer the prospect of circumventing some of the complexity encountered elsewhere in the CNS, and provide new leads as to the identity of the molecules responsible. Until recently, the most likely mechanism involved changes in the composition of the ECM in the vicinity of the DREZ, which were proposed to be instrumental in blocking the reconnection of lesioned primary afferent axons with the spinal cord. However, the spatiotemporal pattern of expression of SemD in the developing spinal cord, and its selective repulsive effects on ingrowth of sensory afferents, have been invoked more recently to explain the patterning of primary afferent inner- vation20 . The effects of injuries to the mature CNS on possible re-expression of SemD within the DREZ and dorsal horn, and of its cognate receptor on sensory axons, remain to be determined, but might also be linked to the failure of afferent re-innervation. Similar possibilities are raised by the recent demonstration of the key role played by the Eph-receptor family and their ligands in regulating the establishment of topo- graphic maps in CNS development51 , again mediated by selective repulsive effects on growing axons. The continuing elucidation of complementary expression patterns of Eph receptors and their ligands through- out the nervous system52,53 might help to provide some of the answers as to why PNS–CNS interfaces change from entry to no-entry zones. Selected references 1 Schwab, M.E. and Bartholdi, D. (1996) Physiol. Rev. 76, 319–370 2 Wilkin, G.P., Marriot, D.R. and Cholewinski, A.J. (1990) Trends Neurosci. 13, 43–46 3 Carlstedt, T. et al. (1987) Neurosci. Lett. 74, 14–18 4 Carlstedt, T. (1988) J. Neurocytol. 17, 335–350 5 Le Douarin, N.M. et al. (1992) in Sensory Neurons: Diversity, Development, and Plasticity (Scott, S.A., ed.), pp. 143–170, Oxford University Press 6 Nakagawa, S. and Takeichi, M. 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(1990) Neuroscience 37, 301–315 50 Wictorin, K. and Björklund, A. (1992) NeuroReport 3, 1045–1048 51 Cheng, H-J. et al. (1994) Cell 82, 371–381 52 Gale, N.W. et al. (1996) Neuron 17, 9–19 53 Kolodkin, A.L. (1996) Trends Neurosci. 19, 507–513 J. Golding et al. – Axon growth and regeneration across PNS–CNS glial interfacesREVIEW Acknowledgements We are grateful to Christianne Niederländer for Fig. 1C and thank Anthony Graham and Kuldip Bedi for their helpful comments on the manuscript. This work was supported by Action Research and the Medical Research Council. Letters to the Editor Letters to the Editor concerning articles published recently in TINS are welcome. Please mark clearly whether they are intended for publication; the authors of the article referred to are given an opportunity to respond to any of the points made in the Letter. Maximum length: 500 words. The Editor reserves the right to edit Letters for publication. 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