Membrane-associated guidance cues direct the innervation of forebrain and midbrain by dorsal raphe-derived serotonergic axons

Unlike many neurons that extend an axon precisely to a single target, individual dorsal raphe 5-HT neurons project to multiple brain regions and their axon terminals often lack classical synaptic specializations. It is not known how 5-HT axon collaterals select between multiple target fields, or even if 5-HT axons require specific guidance cues to innervate their targets. Nor is it known how these axon collaterals are restrained within specific innervation target regions. To investigate this, we challenged explants of dorsal raphe with co-explants, or cell membrane preparations of ventral midbrain, striatum or cerebral cortex. We provide evidence for membrane-associated cues that promote 5-HT axon growth into each of these three target regions. The axon growth-promoting activity was heat-, protease- and phosphatidylinositol-phospholipase-C (PI-PLC)-sensitive. Interestingly, 5-HT axons specifically lost the ability to grow in heterotypic explants, or membrane carpets, following contact with ventral midbrain or striatal, but not cortical, explants or membranes. This inductive activity associated with striatal and ventral midbrain membranes was sensitive to both high salt extraction and PI-PLC treatment. By contrast, the activity that inhibited 5-HT axon growth onto heterotypic membranes was sensitive only to high salt extraction. These results provide evidence that a glycosylphosphatidylinositol (GPI)-linked membrane protein promotes 5-HT axon growth, and that short-range membrane-bound, as well as GPI-linked, molecules contribute to the guidance of 5-HT axon collaterals. These findings suggest that 5-HT axon collaterals acquire a target-induced growth-inhibitory response to alternative targets, increasing their selectivity for the newly innervated field.     

Differential regulation of thalamic and cortical axonal growth by hepatocyte growth factor/scatter factor

The initial axonal projections between the cerebral cortex and thalamus are established during embryogenesis. Chemoattractants and repellents are thought to provide specific guidance cues for directional growth of these pathways. Hepatocyte growth factor/scatter factor (HGF/SF) serves as an attractant for developing motor neurons, and its distribution in embryonic pallidum, pallium and thalamus suggests a similar role in forebrain development. We examined the effectiveness of HGF/SF in regulating thalamic and cortical neuronal growth using in vitro assays. HGF/SF increased neurite outgrowth of thalamic, but not cortical neurons, grown in dissociated cultures or as explants. HGF/SF also exhibited a chemoattractant property for thalamic axons, promoting the extension of neurites towards an HGF/SF source. These experiments demonstrate HGF/SF has the capacity to selectively direct thalamocortical projections into an intermediate target, the pallidum, and eventually to their final cortical destination.     

Cerebellar target neurons provide a stop signal for afferent neurite extension in vitro.

The contributions of cell-cell interactions to the establishment of specific patterns of innervation within target brain regions are not known. To provide an experimental analysis of the regulation of afferent axonal growth, we have developed an in vitro assay system, based on the developing mouse cerebellum, in which afferent axons from a brainstem source of mossy fiber afferents, the basilar pontine nuclei, were cocultured with astroglia or granule neurons purified from the cerebellum. In the absence of cells from the cerebellum, pontine explants produced axons that fasciculated and extended rapidly on a culture surface treated with poly-lysine or laminin. When pontine neurites grew onto cerebellar astroglial cells, outgrowth was more abundant than on substrates alone, suggesting that glial cells provide a positive signal for axon extension. Time-lapse video microscopy indicated that the rate of neurite extension increased from less than 50 microns/hr to more than 100 microns/hr when axonal growth cones moved from the culture substratum onto an astroglial-cell surface. Acceleration of neurite extension was also observed as pontine neurites grew onto other pontine neurites. By contrast, when pontine neurites grew on granule neurons, the appropriate targets of mossy fibers, the length of pontine neurites was greatly reduced. As growing axons terminated on granule neurons, the target cells appeared to provide a "stop-growing signal" for axon extension. The length of pontine neurites decreased with increasing granule neuron density. Two lines of evidence suggested that the stop signal was contact mediated. First, video microscopy showed that pontine growth cones stopped extending after contacting a granule neuron. Second, the length of afferent axons was not reduced when pontine neurites grew at a distance from granule neurons. Competition experiments where both astroglia and granule neurons were plated together suggested that the growth arrest signal provided by granule neurons could override the growth-promoting signal provided by astroglial cells. These results suggest that specific cell-cell interactions regulate the growth of pontine afferent axons within their cerebellar target, with axoaxonal and axoglial interactions promoting axon extension and axon-target cell interactions interrupting axon extension.     

Selective growth of hippocampal neurites on cryostat sections of rat brain

Dissociated rat hippocampal neurons were cultured on horizontal cryostat sections from neonatal and adult rat brain and their growth patterns visualized by brightfield and interference contrast microscopy. Cells adhered to the sections as individuals or in small clusters and grew extensive neurites. Neurites grew over all areas of neonatal sections without apparent selectivity. For adult sections, however, neurites grew almost exclusively on areas of grey matter: there was no neurite growth on areas of white matter, irrespective of the location of that white matter within the brain. The transition from the neonatal to the adult pattern of growth occurred for sections from animals aged between 14 and 21 days.     

Neurite elongation on chondroitin sulfate proteoglycans is characterized by axonal fasciculation

In the developing or regenerating nervous system, migrating growth cones are exposed to regulatory molecules that positively and/or negatively affect guidance. Chondroitin sulfate proteoglycans (CSPGs) are complex macromolecules that are typically negative regulators of growth cone migration in vivo and in vitro. However, in certain cases, neurites sometimes traverse regions expressing relatively high levels of CSPGs, seemingly a paradox. In our continuing efforts to characterize CSPG inhibition in vitro, we manipulated the ratio of CSPGs to growth-promoting laminin-1 to produce a substratum that supports outgrowth of a subpopulation of dorsal root ganglia (DRG) neurites, while still being inhibitory to other populations of DRG neurons [Exp. Neurol. 109 (1990), 111; J. Neurobiol. 51 (2002), 285]. This model comprises a useful tool in the analysis of mechanisms of growth cone guidance and is particularly useful to analyze how CSPGs can be inhibitory under some conditions, and growth permissive under others. We grew embryonic (E9-10) chicken DRG neurons on nervous system-isolated, substratum-bound CSPGs at a concentration that supports an intermittent pattern of outgrowth, alternating with regions adsorbed with growth-promoting laminin-1 alone, and analyzed outgrowth behaviors qualitatively and quantitatively. A novel finding of the study was that DRG neurites that elongated onto CSPGs were predominantly fasciculated, but immediately returned to a defasciculated state upon contact with laminin-1. Further, cursory inspection suggests that outgrowth onto CSPGs may be initially accomplished by pioneer axons, along which subsequent axons migrate. The outgrowth patterns characterized in vitro may accurately reflect outgrowth in vivo in locations where inhibitory CSPGs and growth-promoting molecules are coexpressed, e.g., in the developing retina where fasciculated outgrowth may be instrumental in the guidance of retinal ganglion cells from the periphery to the optic fissure.     

Age-dependent patterns and rates of neurite outgrowth from CNS neurons on Schwann cell-derived basal lamina and laminin substrata

In the present study, we have examined the growth characteristics of CNS neurons on type I collagen, detergent-treated collagen (dColl), Schwann cell-derived basal lamina (SC-BL), and purified laminin substrata. Neurons from the cerebral cortex, septal basal forebrain, and lumbosacral spinal cord were obtained from embryonic age (E) 15 and E18 rats and grown in vitro as explants on the test substrata. Neurons from either embryonic age displayed radial neurite outgrowth on collagen and dColl substrata. However, pretreatment of collagen with detergents slightly diminished its ability to support neurite outgrowth, as evidence by the 20-40% decrease in the rate of neurite growth on dColl versus the rate calculated for neurons on collagen. In contrast to the similar growth characteristics of E15 and E18 neurons on collagen and dColl, the pattern of neurite outgrowth for CNS neurons on SC-BL and laminin substrata was age dependent. Most E15 neurons grown on SC-BL extended neurites that grew identically to those observed on dColl; these 'non-orienting' neurites maintained a radial orientation to their outgrowth despite encountering interposing channels of SC-BL and grew at rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates to neurons on collagen. In contrast, neurons from E18 rats exhibited neurites that preferentially grew in intimate association with SC-BL channels once contact with the channels was established. These 'orienting' neurites faithfully elongated within the SC-BL and demonstrated a 1.4- to 2.0-fold increase in growth rate compared with the sister cultures of neurons grown on dColl. Furthermore, E18 neurons exhibited a 1.4-fold increase in growth on laminin compared with E18 neurons grown on collagen. A minor population of neurites exhibiting similar characteristics to orienting neurites was also observed in E15 cultures. It is hypothesized that orienting and non-orienting neurites reflect the outgrowth of 'regenerating' and 'developing' neurons, respectively, and may indicate an inherent difference in the ability of regenerating and developing neurons to recognize and respond to the same guidance signals.     

GAP-43 dependency defines distinct effects of netrin-1 on cortical and spinal neurite outgrowth and directional guidance

Growth-associated protein-43 (GAP-43) is a major nervous system protein whose phosphorylation by protein kinase C regulates growth cone responses to extracellular guidance cues via F-actin. GAP-43 is essential for axon pathfinding in both cortical afferents and efferents: when it is genetically deleted, somatosensory, auditory and visual somatotopic maps fail to form, and telencephalic commissural axons fail to cross the midline. Here we investigated whether the midline guidance cue netrin-1 depends on GAP-43 for its functions in neurite growth and guidance. We used 3-dimensional collagen gel co-cultures to show that both endogenous netrin-1, expressed by the spinal cord floor plate, and recombinant netrin-1, expressed by transfected COS7 cells, stimulate neurite outgrowth and chemotropic guidance of neocortical callosal axons. In contrast both were significantly inhibited in GAP-43 (−/−) neocortical callosal axons, mimicking the in vivo phenotype. Conversely, neither netrin-1-stimulated neurite outgrowth nor guidance of dorsal spinal cord commissure axons were affected when GAP-43 was absent, again consistent with in vivo phenotype but suggesting fundamental differences in how neocortical and spinal cord axons respond to netrin-1. In addition, differences in GAP-43 dependency also distinguished how ventrolateral cortical efferents respond to netrin-1: in contrast to callosal neurites, in which netrin-1 required GAP-43 in order to stimulate both outgrowth and guidance, in ventrolateral efferents, netrin-1 required GAP-43 only to stimulate outgrowth, but not guidance. Moreover, netrin-1 increased the numbers of both types of cortical, but not spinal neurites. The results demonstrate previously unappreciated diversity in how different classes of neurons respond to the same guidance cue.     

Tissue sections from the mature rat brain and spinal cord as substrates for neurite outgrowth in vitro: extensive growth on gray matter but little growth on white matter

The failure of axons to regenerate within the brain and spinal cord of mature mammals has been attributed to the absence of growth-promoting substances, especially extracellular matrix components, or to the presence of growth-inhibiting substances, particularly components associated with CNS myelin. The ability of mature mammalian CNS tissue to support neurite regeneration was tested by growing explants of embryonic chick lumbar sympathetic ganglia on fresh frozen sections of the mature rat brain and spinal cord. The extent of neurite outgrowth was quantified using morphometric analysis for explants grown on sections that included most of the major anatomical divisions of the CNS. Extensive, but variable, regeneration was present on gray matter regions, whereas major white matter tracts showed poor support, if any, for neurite growth. The results are consistent with the presence of growth-inhibiting factors associated with CNS white matter but also indicate that most gray matter regions of the mature mammalian brain and spinal cord will support axonal regeneration in tissue culture in spite of the absence of known extracellular matrix components.     

Neurite guidance by non-neuronal cells in culture: preferential outgrowth of peripheral neurites on glial as compared to nonglial cell surfaces

Growing axons in the peripheral nervous system (PNS) encounter a variety of cellular and extracellular substrates. Since it is difficult to sort out the possible contributions of these diverse components of the extracellular environment to axonal guidance in vivo, I have developed an in vitro system to study neurite outgrowth on two classes of cells which may provide as substrates for growing axons during development or regeneration: glial cells, e.g., astrocytes and Schwann cells, and nonglial cells, e.g., fibroblasts. Although neurites from sympathetic and spinal sensory ganglia explants grew onto preformed monolayers of both glial and nonglial cells, glial cells were a markedly better substrate. On the glial cells the neurites extended at a rate of 25 to 30 micron/hr and traveled singly or in fine fascicles; their growth cones displayed long filopodia and migrated on the upper surface of the monolayer cells. Conditioned media experiments suggested that neurite outgrowth on glial cell monolayers was not mediated by soluble secreted factors. These results indicate that the glial cell surface is an attractive substrate for neurite outgrowth. In contrast, on nonglial cells the rate of outgrowth was only 10 to 15 micron/hr, large neurite fascicles were common, and the growth cones migrated beneath the monolayer cells in contact with the underlying artificial substrate. This location of the growth cone, coupled with the observation that conditioned medium from these cells promoted neurite outgrowth only when bound to artificial substrates, suggests that secreted substrate-associated components may be an important determinant of neurite outgrowth on nonglial cell monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of Specific Efferent Connections in Organotypic Slice Cultures from Rat Visual Cortex Cocultured with Lateral Geniculate Nucleus and Superior Colliculus

Cells in the cerebral cortex project to many distant regions in the brain. Each cortical target receives input from a specific population of cells which have a characteristic morphology and which are located in a distinct cortical layer. In an attempt to learn about the mechanisms by which this stereotypic output pattern is generated during development, we have studied the formation of cortical projections in an in vitro system. Slices from developing rat visual cortex were cocultured with slices from the superior colliculus, the major target of cells in layer 5, and the lateral geniculate nucleus, the major target of cells in layer 6. Cortical neurons which established connections with tectal and thalamic explants were retrogradely labelled with fluorescent dyes. It was found that, in vitro , different populations of neurons project to these two targets, and that the laminar position and cellular morphology of the projecting cells were similar to their in vivo counterparts. These specific connections were established when the target explants were placed either next to the white matter or next to the pial side of cortical slice cultures. The axons of cells projecting to ectopic positioned explants reoriented their trajectories and grew through the cortical grey matter directly towards their targets. Thus subcortical targets exert an orienting effect specifically on their innervating cells and attract growing axons of the appropriate cells at a distance. These results suggest that different targets release different molecules that act selectively on specific populations of neurons. Therefore, chemotropic guidance is likely to play a significant role in the development of specific connections between cortical neurons and their target areas.     

Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection.

The thalamocortical axon (TCA) projection originates in dorsal thalamus, conveys sensory input to the neocortex, and has a critical role in cortical development. We show that the secreted axon guidance molecule netrin-1 acts in vitro as an attractant and growth promoter for dorsal thalamic axons and is required for the proper development of the TCA projection in vivo. As TCAs approach the hypothalamus, they turn laterally into the ventral telencephalon and extend toward the cortex through a population of netrin-1-expressing cells. DCC and neogenin, receptors implicated in mediating the attractant effects of netrin-1, are expressed in dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors implicated in repulsion, are not. In vitro, dorsal thalamic axons show biased growth toward a source of netrin-1, which can be abolished by netrin-1-blocking antibodies. Netrin-1 also enhances overall axon outgrowth from explants of dorsal thalamus. The biased growth of dorsal thalamic axons toward the internal capsule zone of ventral telencephalic explants is attenuated, but not significantly, by netrin-1-blocking antibodies, suggesting that it releases another attractant activity for TCAs in addition to netrin-1. Analyses of netrin-1 -/- mice reveal that the TCA projection through the ventral telencephalon is disorganized, their pathway is abnormally restricted, and fewer dorsal thalamic axons reach cortex. These findings demonstrate that netrin-1 promotes the growth of TCAs through the ventral telencephalon and cooperates with other guidance cues to control their pathfinding from dorsal thalamus to cortex.     

Adult olfactory ensheathing glia promote the long-distance growth of adult retinal ganglion cell neurites in vitro

In vivo, transplanted adult olfactory ensheathing glia (OEG) and adult Schwann cells (SC) can support the regrowth of at least some transected axons within adult CNS neuropil. In the present study, we developed an in vitro adult rat retinal explant model to explore the influence of primary adult SC and OEG on retinal ganglion cell (RGC) neurite regrowth in the presence of glial cells endogenous to the retina. Retinal quadrants were plated RGC-side down onto aclar hats coated with either pure collagen (type 1), collagen with OEG, collagen with SCs, or collagen coated with both OEG and SCs. Regrowing retinal neurites extended onto the pure collagen substrate, largely in association with astrocytes that migrated out from the explants (mean number of neurites: 144+/-65 SEM). The additional presence of OEG (669+/-122), but not SCs (97+/-41), supported the regrowth of significantly greater numbers of RGC neurites. Furthermore, this OEG-stimulated regeneration was over significantly greater distances; >68% of neurites extended >500 microm from the explant, compared with explants plated onto SCs or collagen alone (15% and 29%, respectively). When OEG and SCs were co-cultured the number of regenerating neurites was reduced (397+/-81) compared with the pure OEG treatment. Analysis of explants on pure collagen substrates fed with media conditioned by purified OEG or SC showed no increase in neurite outgrowth compared with control treatments, suggesting that the enhanced growth in the presence of OEG is a contact-mediated effect. The observed differences between the abilities of OEG and SC to support the growth of CNS-derived fibers in the presence of astrocytes support the suggestion that OEG may be better suited for direct transplantation into CNS neuropil following injury.     

Neuronal age influences the response to neurite outgrowth inhibitory activity in the central and peripheral nervous systems.

Axonal regeneration is abortive in the central nervous system (CNS) of adult mammals, but readily occurs in the injured peripheral nervous system (PNS). Recent experiments indicate an important role for both intrinsic neuronal features and extrinsic substrate properties in determining the propensity for axonal regrowth. In particular, certain components of adult mammalian CNS myelin have been shown to exert a strong inhibitory influence on neurite outgrowth. To determine whether the potent neurite outgrowth inhibitory activity found in CNS myelin may also be present in PNS myelin and to study the influence of neuronal age on neurite outgrowth, we used a cryoculture assay in which dissociated rat dorsal root ganglion (DRG) neurons of different ages were challenged to extend neurites on fractionated myelin and cryostat sections from the PNS (sciatic nerve and myelin-free degenerated sciatic nerve) and CNS (optic nerve) of adult rats. The CNS environment of the optic nerve did not support E17 to P8 DRG neurite adhesion or outgrowth. E17 DRG neurons, unlike their older counterparts, however, were able to attach and extend neurites onto normal sciatic nerve and onto purified PNS myelin. In contrast, a vigorous neurite outgrowth response from all the ages tested was observed on the myelin-free degenerated sciatic nerve. These results indicate that PNS myelin is a potent inhibitor of neurite outgrowth and that DRG neuronal age plays an important role in determining the propensity for neurite outgrowth and regenerative response on inhibitory PNS and CNS substrata.     

Retinal neurite growth on astrocytes is not modified by extracellular matrix, anti-L1 antibody, or oligodendrocytes.

Two factors that may influence the course of axonal regeneration in the central nervous system (CNS) are extracellular matrix (ECM) and cell surface molecules that may enhance or inhibit neurite outgrowth. Whereas cultured astrocytes have been reported to be a good substratum for neurite outgrowth, there is recent evidence that cultured oligodendrocytes are inhibitory. To test the influences of 1) ECM components, 2) the L1 adhesion molecule, and 3) the inhibitory potential of mature oligodendrocytes in the astrocytic environment, we have utilized a culture system in which neurites from embryonic rat retina grow vigorously on astrocyte monolayers. The major ECM components were assembled in neonatal rat cortical astrocyte-retina co-cultures only when the medium contained serum. In electron microscopic studies of serum containing cultures, retinal neurites were seen to be related to astrocyte surfaces but rarely were found in contact with ECM; in serum-free medium the association between neurites and astrocytes was similar. In addition, the growth of neurites was vigorous whether ECM was present or absent. Presence of antibodies against the cell surface adhesion molecule L1 did not inhibit retinal neurite elongation on glial fibrillary acidic protein-positive astrocytes. When oligodendrocytes from adult rat spinal cord were combined with the astrocytes, retinal neurites grew as well on the mixed glial population as on astrocytes alone. Immunostaining for galactocerebroside showed many oligodendrocyte processes to be aligned in the direction of neurite growth, suggesting association between the two cell types. This association was verified by electron microscopy. Furthermore, retinal explants extended neurites among myelin basic protein-positive oligodendrocytes cultured without astrocytes. Thus, the astrocyte surface is a strong promoter of neurite growth from embryonic rat retina. This growth did not depend upon either ECM or the L1 adhesion molecule. Because neurites grew on astrocytes in the presence of mature oligodendrocytes or among oligodendrocytes alone, we conclude that oligodendrocytes do not inhibit neurite growth under certain conditions.     

Growth of regenerating goldfish axons is inhibited by rat oligodendrocytes and CNS myelin but not but not by goldfish optic nerve tract oligodendrocytelike cells and fish CNS myelin

Encounters of regenerating goldfish retinal axons with oligodendrocytes and CNS myelin of mammals and fish were monitored in in vitro assays. Upon contact with highly branched rat oligodendrocytes, goldfish axons collapsed or grew around but never crossed these cells. However, in the presence of the antibody IN-1 against the oligodendrocyte-associated growth-inhibitory proteins, axons did grow over highly branched oligodencrocytes. In contrast to the mammalian oligodendrocytes, goldfish optic nerve/tract-derived oligodendrocytelike cells allowed the growth of axons across their surface and even along their processes. The fish growth cones avoided entering the region of rat CNS myelin applied to polylysine/laminin-coated coverslips or failed to elongate on this substrate. They were, however, able to pass over CNS myelin of fish. When exposed to rat CNS myelin as the sole substrate, axonal outgrowth from fish retinal explants was inhibited almost entirely. However, outgrowth on fish CNS myelin was substantial, but many more axons extended on fish or rat brain membranes that were depleted of myelin. Thus, goldfish retinal axons are sensitive to the axon-growth-inhibiting cell-surface molecules of mammalian oligodendrocytes as well as CNS myelin. Fish optic nerve oligodendrocytelike cells and fish CNS myelin lack these inhibitory properties and are growth permissive. These in vitro experiments suggest that the success of axonal regeneration in the fish optic nerve is causally related to the presence of growth-permissive properties and to the absence of growth inhibitors on fish optic nerve/tract oligodendrocytelike cells.     

Nondirected axonal growth on basal lamina from avian embryonic neural retina

The vitreous surface of the embryonic avian retinal neuroepithelium was isolated by mechanical disruption of the retina mounted between 2 adhesive substrata. The 200-micron-thick sheath covered an area of up to 1 cm2 and consisted of the vitreal basal lamina with a lamina densa, 2 laminae rarae, and a carpet of ventricular cell endfeet on top of the lamina. The vitreal endfeet were removed by detergent treatment and an extracellular basal lamina was obtained. The laminae were further characterized by immunohistochemistry and immunoblotting. A 190 kDa laminin protein was detected in laminae with and without vitreal endfeet, whereas the membrane-bound neural cell adhesion molecule (N-CAM) was detectable only on the endfeet of the ventricular cells and was absent in the detergent-treated basal laminae. Neither immunoblotting nor immunostaining revealed fibronectin in these preparations. Explants of retina, sensory ganglia, and cerebellum from chick, quail, and mouse were cultured on the basal lamina as a substratum. In all cases axonal outgrowth was excellent, with a growth rate similar to that in situ. Outgrowing axons from sensory ganglia and cerebellar explants were accompanied by migratory cells, which, in the case of sensory ganglia, were flat cells and, in the case of cerebellar explants, resembled granular neurons. Optic axons grew on the laminae in an asymmetric, explant-inherent pattern specific for the position of origin of the explant. On detergent-treated basal laminae, as well as on laminin, the retinal axons grew in a clockwise orientation. This axonal growth pattern was specific for retinal tissue and was not observed with axons from other neural explants. In spite of the excellent substrate properties provided by the substratum, cues for growing axons (toward or away from the optic disk) were not detectable in the basal lamina preparations.     

Rapid initiation of neurite outgrowth onto laminin from explants of adult mouse retina induced by optic nerve crush

One optic nerve of an adult mouse was crushed in the orbit. After 8 days, both retinas were explanted onto laminin-coated coverslips. Within 24 h, neurites grew out onto this substrate from explants with prior crush and by 48 h two-thirds of explants had neurites. On polylysine or plain glass, outgrowth was generally not seen until 48 h and this was significantly reduced compared to laminin. Explants without optic nerve crush did not extend neurites until about 5 days later. We suggest that axotomy induces a time-dependent regenerative response in adult mammalian retinal ganglion cells which includes the expression of laminin responsiveness.     

Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina

Despite evidence that glial cell surfaces and components of the extracellular matrix (ECM) support neurite outgrowth in many culture systems, the relative contributions of these factors have rarely been compared directly. Specifically, it remains to be determined which components of peripheral nerve support growth of central nerve fibers. We have directly compared neurite outgrowth from embryonic day 15 rat retinal explants placed onto beds of (1) Schwann cells without ECM, (2) Schwann cells expressing ECM (including a basal lamina), (3) cell-free ECM prepared from neuron-Schwann cell cultures, (4) nonglial cells (fibroblasts), and (5) 2 isolated ECM components, laminin and type I collagen. From the first day in culture, retinal explants extended neurites when placed on Schwann cells without ECM. Outgrowth on Schwann cells expressing ECM was also extensive, but not obviously different form that on Schwann cells alone. Ultrastructural study revealed that 95% of retinal neurites in ECM-containing cultures contacted other neurites and Schwann cell surfaces exclusively. On cell-free ECM prepared from neuron-Schwann cell cultures, neurite extension was poor to nonexistent. No neurite outgrowth occurred on fibroblasts. Retinal explants also failed to extend neurites onto purified laminin and ammoniated type I collagen substrata; however, growth was rapid and extensive on air-dried type I collagen. In cultures containing islands of air-dried type I collagen on a laminin-coated coverslip, retinal explants attached and extended neurites on collagen, but these neurites did not extend off the island onto the laminin substratum. We conclude from these experiments that neurite extension from embryonic rat retina is supported by a factor found on the surface of Schwann cells and that neither organized nor isolated ECM components provide this neurite promotion. These findings are discussed in relation to possible species differences in growth requirements for retinal ganglion cell neurites and to the specificity of response of different CNS neurites to ECM substrata.     

Specific target-directed axonal outgrowth from transplanted embryonic rodent retinae into neonatal rat superior colliculus

We have investigated whether there is evidence of target-directed growth of retinal axons to the tectum, and whether the surface of the rostral brainstem is an obligatory substrate for growing optic axons by transplanting embryonic mouse retinae to the cerebral aqueduct of neonatal rats. Such retinae emit axons that grow dorsally through the brain parenchyma to reach the superficial layers of the superior colliculus either by running dorsally along the midline, or by following a dorsally directed, radially arching course through the brain parenchyma. Studies of the early outgrowth pattern from transplanted retinae, and comparison studies of the axonal outgrowth from similarly placed cortical grafts suggest that outgrowth of retinal axons is target-directed and specific to optic axons.     

Cotransplantation of embryonic mouse retina with tectum, diencephalon, or cortex to neonatal rat cortex

Retinae from embryonic mice were transplanted to the occipital cortex of neonatal rats together with their normal target regions, tectum or diencephalon, from embryonic mice or rats. In control experiments, retinae were cotransplanted with embryonic rat occipital cortex. In over 80% of the experimental animals, both transplants differentiated and grew. Ganglion cells in the retinae cotransplanted close to tectum or diencephalon survived for at least 15 weeks. Their survival was associated with the development of a distinct optic fiber layer and outgrowth of axons from the transplanted mouse retina. Specific innervation of distinct patches within the cotransplanted rat tectum or diencephalon was demonstrated by the use of an anti-mouse antibody. The innervated regions, which could be as far away as 1.3 mm from the retinae, were correlated with cytological features of the cotransplanted tectum or diencephalon. By contrast, the host cortex was never innervated by the transplanted retinae. In the control animals in which the retinae were cotransplanted with occipital cortex and in four animals in which the cotransplants lay more than 2.7 mm apart, no ganglion cells were identified and there was no evidence of an optic fiber layer, outgrowth of axons, or innervation. These results support the idea that in order to survive, retinal ganglion cells need to innervate an appropriate target region. Further, the specific innervation of regions within the cotransplanted tectum or diencephalon suggests that these target regions are able to exert a tropic influence on the axons of retinal ganglion cells, even in the absence of many of the normal structure cues.