James Cohen

Adult Rat Dorsal Root Ganglion Neurons Extend Neurites on Predegenerated But Not on Normal Peripheral Nerves In Vitro

The abilities of embryonic and adult rat sensory neurons to regenerate were compared when cultured on cryostat sections of normal and lesioned sciatic nerve tissues. Differences in neurite growth, visualized by GAP‐43 immunolabelling, were most pronounced on substrata consisting of longitudinal sections of normal versus predegenerated sciatic nerve. Adult dorsal root ganglion (DRG) neurons grew only on the lesioned nerves. Neurites extended along these sections in a characteristically longitudinal orientation, and this growth was not dependent on nerve growth factor. Embryonic DRG neurons extended neurites on sections from both types of nerves. These results highlight important differences in the regenerative abilities of embryonic and adult DRG neurons when grown on physiologically appropriate substrata.

Growth responses of different subpopulations of adult sensory neurons to neurotrophic factors in vitro

Different subpopulations of adult primary sensory neurons in the dorsal root ganglia express receptors for different trophic factors, and are therefore potentially responsive to distinct trophic signals. We have compared the effect of the neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and NT‐3, and of glial cell line‐derived neurotrophic factor (GDNF) on neurite outgrowth in dissociated cultures of sensory neurons from the lumbar ganglia of young adult rats, and attempted to establish subset‐specific effects of these trophic factors. We analysed three parameters of neurite growth (percentage of process‐bearing neurons, length of longest neurite and total neurite length), which may correlate with particular types of axon growth in vivo, and may therefore respond differently to trophic factor presence. Our results showed that percentage of process‐bearing neurons and total neurite length were influenced by trophic factors, whilst the length of the longest neurite was trophic factor independent. Only NGF and GDNF were found to enhance significantly the proportion of process‐bearing neurons in vitro. GDNF was more effective than NGF on small, IB4– neurons, which are known to develop GDNF responsiveness early in postnatal development. NGF, and to a much lesser extent GDNF, enhanced the total length of the neurites produced by neurons in culture. BDNF exerted an inhibitory effect on growth, and both BDNF and NT‐3 could partially block some of the growth‐promoting effects of NGF on specific neuronal subpopulations.

Boundary cap cells constrain spinal motor neuron somal migration at motor exit points by a semaphorin-plexin mechanism

BackgroundIn developing neurons, somal migration and initiation of axon outgrowth often occur simultaneously and are regulated in part by similar classes of molecules. When neurons reach their final destinations, however, somal translocation and axon extension are uncoupled. Insights into the mechanisms underlying this process of disengagement came from our study of the behaviour of embryonic spinal motor neurons following ablation of boundary cap cells. These are neural crest derivatives that transiently reside at motor exit points, central nervous system (CNS):peripheral nervous system (PNS) interfaces where motor axons leave the CNS. In the absence of boundary cap cells, motor neuron cell bodies migrate along their axons into the periphery, suggesting that repellent signals from boundary cap cells regulate the selective gating of somal migration and axon outgrowth at the motor exit point. Here we used RNA interference in the chick embryo together with analysis of null mutant mice to identify possible boundary cap cell ligands, their receptors on motor neurons and cytoplasmic signalling molecules that control this process.ResultsWe demonstrate that targeted knock down in motor neurons of Neuropilin-2 (Npn-2), a high affinity receptor for class 3 semaphorins, causes their somata to migrate to ectopic positions in ventral nerve roots. This finding was corroborated in Npn-2 null mice, in which we identified motor neuron cell bodies in ectopic positions in the PNS. Our RNA interference studies further revealed a role for Plexin-A2, but not Plexin-A1 or Plexin-A4. We show that chick and mouse boundary cap cells express Sema3B and 3G, secreted semaphorins, and Sema6A, a transmembrane semaphorin. However, no increased numbers of ectopic motor neurons are found in Sema3B null mouse embryos. In contrast, Sema6A null mice display an ectopic motor neuron phenotype. Finally, knockdown of MICAL3, a downstream semaphorin/Plexin-A signalling molecule, in chick motor neurons led to their ectopic positioning in the PNS.ConclusionWe conclude that semaphorin-mediated repellent interactions between boundary cap cells and immature spinal motor neurons regulates somal positioning by countering the drag exerted on motor neuron cell bodies by their axons as they emerge from the CNS at motor exit points. Our data support a model in which BC cell semaphorins signal through Npn-2 and/or Plexin-A2 receptors on motor neurons via a cytoplasmic effector, MICAL3, to trigger cytoskeletal reorganisation. This leads to the disengagement of somal migration from axon extension and the confinement of motor neuron cell bodies to the spinal cord.

Peripheral, but not central, axotomy induces neuropilin-1 mRNA expression in adult large diameter primary sensory neurons

Neuropilin‐1 (NP‐1) is a component of the receptor for semaphorin3a (Sema3a), a member of a large family of molecules with widespread expression and demonstrable influence (via their ability to repel growing axons) on nervous system development. Recent studies have shown that some types of adult mammalian neurons retain the capacity to respond to Sema3a, particularly in relation to neuronal injury and regeneration. Although variations in expression of Sema3a mRNA have been revealed in neurons in both the central and peripheral nervous systems in this context, relatively little is known about NP‐1 expression patterns. In this study we investigated the expression of NP‐1 mRNA in adult dorsal root ganglion (DRG) neurons in intact and lesioned animals. We compared the effect of unilateral lesioning of the sciatic nerve or unilateral dorsal rhizotomy at lumbar levels L4/5, and bilateral dorsal funiculus lesioning at thoracic levels T10/11 on NP‐1 mRNA expression in the cell bodies of lumbar DRGs. A significantly increased level of NP‐1 mRNA expression was detected only following sciatic nerve lesioning (P < 0.001), but not after rhizotomy or dorsal funiculus lesioning. Furthermore, this upregulation was mainly confined to large diameter neurons of DRGs at lumbar levels L4/5, which provide the main sensory contribution to the sciatic nerve. These results suggest a role for NP‐1 in the axonal response to peripheral nerve injury, which may be specific to a particular subset of primary sensory neurons. J. Comp. Neurol. 423:492–499, 2000.

Behaviour of DRG sensory neurites at the intact and injured adult rat dorsal root entry zone: Postnatal neurites become paralysed, whilst injury improves the growth of embryonic neurites

The dorsal root entry zone is a PNS‐CNS junction between Schwann cells and astrocytes, defining the site where dorsal root ganglia (DRG) axons enter the adult mammalian spinal cord. Following dorsal root injury (rhizotomy), DRG axons regenerate within the PNS environment of the root but stop at the DREZ and fail to re‐enter the spinal cord. We have used an in vitro model to compare how neurites growing from embryonic (E13) and postnatal (P0 and adult) DRG neurons behave at the uninjured and rhizotomized adult rat DREZ. We find that both freshly dissected and conditioned‐lesioned postnatal DRG neurons seldom grow neurites across cryosections of the uninjured or rhizotomized DREZ. However, embryonic DRG neurons more readily grow neurites across cryosections of the uninjured and 7‐day post‐lesion (dpl) DREZ and are dramatically better able to cross the 21 dpl DREZ. This enhanced growth was abolished by co‐incubation with a function‐blocking antiserum to β1‐integrin receptors, whilst immunoreactivity for some β1‐integrin ligands (tenascin‐C and fibronectin) increased at the DREZ by 21 dpl, suggesting that β1‐integrin ligands may stimulate the growth of embryonic neurites across the 21 dpl DREZ. Fluorescence time‐lapse video‐microscopy was used to record the behaviour of dye‐labelled postnatal DRG neurites as they encounter the uninjured adult DREZ in vitro. Neurites rarely turned around at the DREZ, but instead became paralysed. Of a variety of chemical modifications to uninjured DREZ cryosections, only treatment with methanol, chloroform, or the protease inhibitor D‐phe‐pro‐arg chloromethylketone hydrochloride (PPACK, 100 μM) caused any increase in the proportion of postnatal neurites which crossed the DREZ. GLIA 26:309–323, 1999.

Influence of laminin-2 on Schwann cell-axon interactions.

The dy/dy mouse suffers from a form of muscular dystrophy caused by a substantial reduction in laminin α2‐chain protein, a major component of both muscle and Schwann cell basal laminae. This article examines the effect of laminin α2 deficiency on Schwann cell–axon interactions both in vivo at varying intervals after nerve crush, and in vitro, in cocultures of neurons and Schwann cells. The morphological spectrum of aberrant Schwann cell–axon associations seen in uncrushed dy/dy sciatic nerves was recapitulated during regeneration: myelination of regenerating axons was delayed compared with the process in unaffected mice and the relatively few myelin sheaths which were formed in dy/dy distal nerve stumps were often uncompacted. In vitro, Schwann cells dissociated from adult dy/dy sciatic nerves predictably failed to express detectable laminin α2‐chain and displayed an unusual multipolar morphology. Branching of neurites, in terms both of numbers of terminal branches and of complexity of branching, from dorsal root ganglia neurons grown on dy/dy Schwann cells, was significantly less extensive than that seen when neurons were cocultured with Schwann cells from unaffected littermates, but this effect was reversed by exogenous laminin‐2. Our results lend strong support to the view that laminin‐2 is essential for establishing and/or maintaining Schwann cell–axon interactions, in normal and in regenerating nerves. GLIA 32:109–121, 2000.

Anin VitroModel of the Rat Dorsal Root Entry Zone Reveals Developmental Changes in the Extent of Sensory Axon Growth into the Spinal Cord

The dorsal root entry zone (DREZ) forms the junction between the dorsal roots of the peripheral nervous system and the spinal cord. In rats older than 1 week, lesioned primary sensory axons regenerate within the dorsal roots but stop at the DREZ, and are thus unable to reconnect with the spinal cord. To analyze the causes of this failure, we have developed a culture model of the interaction of sensory axon growth cones with the intact DREZ, whereby dissociated dorsal root ganglion neurons from rats of various ages are grown on longitudinal cryosections of rat spinal cord, incorporating the DREZ and attached dorsal roots, from neonatal, 1-week-old (P6), or adult animals. Neurites of all ages grew along the roots to the DREZ, where their ability to cross into the spinal cord depended on both their age and that of the spinal cord substrate. Neurites from neonatal neurons failed to cross either the P6 or adult DREZ, but a substantial proportion crossed the immature neonatal DREZ. Early embryonic neurites exhibited substantial crossing on both immature and adult DREZ. These findings strongly suggest that soon after birth, the normal mammalian DREZ acquires growth inhibitory activity that is recognized by the axons of postnatal but not early embryonic sensory neurons.

Embryonic optic nerve tissue fails to support neurite outgrowth by central and peripheral neurons in vitro

The failure of axon regeneration in the injured mammalian central nervous system has been ascribed, in part, to the inhibitory effects of myelin proteins. To investigate the influence of myelination on neurite growth and regeneration by both central nervous system and peripheral nervous system neurons, isolated rat neonatal retinal ganglion cells and adult and neonatal dorsal root ganglion neurons were cultured on cryostat sections of both immature unmyelinated and mature fully myelinated adult rat optic nerve. In agreement with earlier studies using neonatal peripheral neurons, the adult optic nerve failed to support neurite outgrowth from any of the neurons tested. A new finding was that tissue sections from unmyelinated optic nerve (aged embryonic days 18 and 20, and postnatal days 1–3), also failed to support the growth of neurites from neonatal retinal ganglion cells and both neonatal and adult dorsal root ganglion neurons. Neonatal retinal ganglion cells also failed to extend neurites on sections of pre‐degenerated sciatic nerve, a tissue shown in our previous work to be a good substratum for supporting neurite growth for both neonatal and adult DRG neurons. These results suggest that cells in the immature optic nerve either express widely acting axon growth inhibitory molecules unrelated to previously described myelin proteins, or do not synthesize appropriate axon growth promoting molecules. They also reveal that, for axon regeneration, central nervous system and peripheral sensory neurons require distinct substratum interactions.

PREFERENTIAL GROWTH OF NEONATAL RAT DORSAL ROOT GANGLION CELLS ON HOMOTYPIC PERIPHERAL NERVE SUBSTRATES IN VITRO

Developing sensory neurons interact with molecular signals in the local environment to generate stereotypic nerve pathways. Regenerating neurons seem to lose the ability to reinnervate their original sites in the targets, resulting in abnormal sensory input and consequent clinical pathophysiology. The specificity of reinnervation of peripheral targets by regenerating axons is thus crucial for normal recovery of function. In this study, we have examined evidence for selectivity of interactions between primary afferent neurons from identified levels of the spinal cord and different peripheral nerve environments by culturing these neurons on sections of nerves to muscle and viscera. We have compared the growth of a population of sensory afferents normally innervating somatic targets (dorsal root ganglion cells from L4 and L5) with populations containing many afferents innervating visceral targets (L6 and S1 dorsal root ganglia and nodose ganglion). These neurons, from newly born rats, were cultured on unfixed cryostat sections of normal and prelesioned gastrocnemius nerve, pelvic spinal nerve and vagus nerve from adult rats. Normal muscle nerve was seen to support the regeneration of a significantly greater proportion of somatic neurons, with longer neurites, than the visceral nerves. Similarly, much higher proportions of the‘visceral’population of afferent neurons were seen to extend neurites on the normal visceral nerve substrates, with longer neurites, than on the muscle nerve substrate. The selectivity displayed by the sensory neurons for their normal nerve substrates was abolished when they were cultured on prelesioned nerve substrates subjected to Wallerian degeneration, which was apparent from the equivalent and increased proportions of growing neurons having comparable neurite lengths, on all the nerve substrates. We conclude that sensory neurons recognize and respond to substrate‐specific and substrate‐bound molecules present in normal adult peripheral nerves, and that these differences are lost in prelesioned nerves following Wallerian degeneration.