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A great deal of effort has been invested in using trophic factors and other bioactive molecules to promote cell survival and axonal regeneration in the adult central nervous system. Far less attention has been paid to investigating potential effects that trophic factors may have that might interfere with recovery. In the visual system, BDNF has been previously reported to prevent regeneration. To test if BDNF is inherently incompatible with regeneration, BDNF was given intraocularly during optic nerve regeneration in the adult goldfish. In vivo imaging and anatomical analysis of selectively labeled axons were used as a sensitive assay for effects on regeneration within the tectum. BDNF had no detectable inhibitory effect on the ability of axons to regenerate. Normal numbers of axons regenerated into the tectum, exhibited dynamic growth and retractions similar to controls, and were able to navigate to their correct target zone in the tectum. However, BDNF was found to have additional effects that adversely affected the quality of regeneration. It promoted premature branching at ectopic locations, diminished the growth rate of axons through the tectum, and resulted in the formation of ectopic collaterals. Thus, although BDNF has robust effects on axonal behavior, it is, nevertheless, compatible with axonal regeneration, axon navigation and the formation of terminal arbors.

Although it is well known that damage to neurons results in release of substances that inhibit axonal growth, release of chemical signals from damaged axons that attract axon growth cones has not been observed. In this study, a 532 nm 12 ns laser was focused to a diffraction-limited spot to produce site-specific damage to single goldfish axons in vitro. The axons underwent a localized decrease in thickness ('thinning') within seconds. Analysis by fluorescence and transmission electron microscopy indicated that there was no gross rupture of the cell membrane. Mitochondrial transport along the axonal cytoskeleton immediately stopped at the damage site, but recovered over several minutes. Within seconds of damage nearby growth cones extended filopodia towards the injury and were often observed to contact the damaged site. Turning of the growth cone towards the injured axon also was observed. Repair of the laser-induced damage was evidenced by recovery of the axon thickness as well as restoration of mitochondrial movement. We describe a new process of growth cone response to damaged axons. This has been possible through the interface of optics (laser subcellular surgery), fluorescence and electron microscopy, and a goldfish retinal ganglion cell culture model.

Retinal explants from embryonic or adult mice were placed on laminin or merosin substrates and the outgrowth of optic fibers was assayed under serum-free conditions. Both substrates strongly promoted outgrowth. A blocking antibody to the [Beta]1/[Beta]3 integrin subunits completely blocked laminin-dependent growth of embryonic optic fibers but had no detectable effect on adult fibers. Similarly, a blocking antibody against the main neurite-promoting region within the globular domain of the E8 fragment of laminin inhibited growth of embryonic fibers but had no effect on adult optic fibers. The [Beta]1 integrin subunit was identified immunohistochemically on both embryonic and adult fibers. These findings indicate that adult fibers have lost the [Beta]1 function which dominates laminin-dependent growth in embryonic fibers but express a receptor for laminin-dependent growth that is not detectable in embryonic fibers. These findings suggest that there are intrinsic differences between embryonic and adult optic fibers that may have implications for regenerative failure in the central nervous system of adult mammals.

When the brain or spinal cord is injured, glial cells in the damaged area undergo complex morphological and physiological changes resulting in the formation of the glial scar. This scar contains reactive astrocytes, activated microglia, macrophages and other myeloid cells, meningeal cells, proliferating oligodendrocyte precursor cells (OPCs), and a dense extracellular matrix. Whether the scar is beneficial or detrimental to recovery remains controversial. In the acute phase of recovery, scar-forming astrocytes limit the invasion of leukocytes and macrophages, but in the subacute and chronic phases of injury the glial scar is a physical and biochemical barrier to axonal regrowth. The signals that initiate the formation of the glial scar are unknown. Both canonical and noncanonical signaling Wnts are increased after spinal cord injury (SCI). Because Wnts are important regulators of OPC and oligodendrocyte development, we examined the role of canonical Wnt signaling in the glial reactions to CNS injury. In adult female mice carrying an OPC-specific conditionally deleted ß-catenin gene, there is reduced proliferation of OPCs after SCI, reduced accumulation of activated microglia/macrophages, and reduced astrocyte hypertrophy. Using an infraorbital optic nerve crush injury, we show that reducing ß-catenin-dependent signaling in OPCs creates an environment that is permissive to axonal regeneration. Viral-induced expression of Wnt3a in the normal adult mouse spinal cord induces an injury-like response in glia. Thus canonical Wnt signaling is both necessary and sufficient to induce injury responses among glial cells. These data suggest that targeting Wnt expression after SCI may have therapeutic potential in promoting axon regeneration. [ABSTRACT FROM AUTHOR]

Axonal path-finding is important in the development of the nervous system, nerve repair and nerve regeneration. The behaviour of the growth cone at the tip of the growing axon determines the direction of axonal growth and migration. We have developed an optical-based system to control the direction of growth of individual axons (nerve fibres) using laser-driven spinning birefringent spheres. One or two optical traps position birefringent beads adjacent to growth cones of cultured goldfish retinal ganglion cell axons. Circularly polarized light with angular momentum causes the trapped bead to spin. This creates a localized microfluidic flow generating an estimated 0.17 pN shear force against the growth cone that turns in response to the shear. The direction of axonal growth can be precisely manipulated by changing the rotation direction and position of this optically driven micromotor. A physical model estimating the shear force density on the axon is described. [ABSTRACT FROM AUTHOR]

Purpose: Growing axons express a number of proteins associated with axonal growth which are thought to be critical for regeneration and sprouting. Whether these proteins are expressed during injury-induced axonal remodeling is tested in this paper. Methods: The posterior half of the adult goldfish tectum was removed leaving the anterior half intact. This causes optic fibers from nasal retina, which project to posterior tectum, to displace temporal fibers from the anterior remnant and form a compressed retinotopic projection of the entire retina onto the anterior tectum. Immunohistochemistry using an antibody shown here to recognize growing and regenerating fibers in goldfish was used to monitor optic fibers. Results: As expected, surgery induced reactivity in the axotomized nasal axons peaking at 1 month which returned to normal at 2 months when compression was completed. Unexpectedly, axons from temporal retina showed no detectable reactivity even though they were induced to grow anteriorly by the invading nasal fibers. Conclusions: Extensive axonal remodeling and synaptic rearrangement can occur without reentering the growth state associated with axonal growth and regeneration. [ABSTRACT FROM AUTHOR]

Neurons may possess activity-dependent homeostatic mechanisms that permit them to globally alter synaptic strength as activity varies. We used the retinotectal projection of goldfish to test this idea in the intact adult CNS. We first altered tectal neuron activity by selectively manipulating excitatory input. When excitatory synaptic drive to tectal neurons was eliminated by blocking optic fibers, current evoked at optic synapses increased by 183% within 90 min. With partial activity blockade, the increase in synaptic strength scaled with the magnitude of activity depression. This silence-induced potentiation was also rapidly reversible. Conversely, an increase in optic input was followed by a decrease in evoked synaptic current. When optic drive was not altered and tectal neuronal activity was instead increased or decreased pharmacologically via GABAA receptors, synaptic strength again changed inversely with activity, indicating that synaptic strength changed in response to neuronal activity and not excitatory drive. Furthermore, altered synaptic strength tended to return ongoing activity to baseline. Changes in synaptic strength could also be detected in heterosynaptic pathways, indicating a global response. Finally, changes in synaptic strength were associated with corresponding changes in ongoing and evoked firing rates, indicating that the responsivity of tectal neurons was altered. Thus, tectal neurons exhibit archetypical homeostasis, one of the first robust examples in the intact adult CNS. [ABSTRACT FROM AUTHOR]

Axonal regeneration can occur within hours of injury, the first step being the formation of a new growth cone. For sensory and retinal axons, regenerative ability in vivo correlates with the potential to form a new growth cone after axotomy in vitro. We show that this ability to regenerate a new growth cone depends on local protein synthesis and degradation within the axon. Axotomy in vitro leads to a fourfold to sixfold increase in ³H-leucine incorporation in both neurones and axons, starting within 10 min and peaking 1 h after axotomy. Application of protein synthesis inhibitors (cycloheximide and anisomycin) to cut axons, including axons whose cell bodies were removed, or proteasome inhibitors (lactacystin and N-acetyl-Nor-Leu-Leu-Al) all result in a reduction in the proportion of transected axons able to reform growth cones. Similar inhibition of growth cone formation was observed on addition of target of rapamycin (TOR), p38 MAPK (mitogen-activated protein kinase), and caspase-3 inhibitors. Comparing retinal and sensory axons of different developmental stages, levels of ribosomal protein P0 and phosphorylated translation initiation factor are high in sensory axons, lower in embryonic axons, and absent in adult retinal axons. Conditioning lesions, which increase the regenerative ability of sensory axons, lead to increases in intra-axonal protein synthetic and degradative machinery both in vitro and in vivo. Collectively, these findings suggest that local protein synthesis and degradation, controlled by various TOR-, p38 MAPK-, and caspase-dependent pathways, underlie growth cone initiation after axotomy. [ABSTRACT FROM AUTHOR]