RNA-binding protein Vg1RBP regulates terminal arbor formation but not long-range axon navigation in the developing visual system.

Local synthesis of ß-actin is required for attractive turning responses to guidance cues of growth cones in vitro but its functional role in axon guidance in vivo is poorly understood. The transport and translation of ß-actin mRNA is regulated by the RNA-binding protein, Vg1RBP (zipcode-binding protein-1). To examine whether Vg1RBP plays a role in axon navigation in vivo, we disrupted Vg1RBP function in embryonic Xenopus laevis retinal ganglion cells by expressing a dominant-negative Vg1RBP and by antisense morpholino knockdown. We found that attractive turning to a netrin-1 gradient in vitro was abolished in Vg1RBP-deficient axons but, surprisingly, the long-range navigation from the retina to the optic tectum was unaffected. Within the tectum, however, the branching and complexity of axon terminals were significantly reduced. High-resolution time-lapse imaging of axon terminals in vivo revealed that Vg1RBP-GFP-positive granules accumulate locally in the axon shaft immediately preceding the emergence a filopodial-like protrusion. Comparative analysis of branch dynamics showed that Vg1RBP-deficient axons extend far fewer filopodial-like protrusions than control axons and indicate that Vg1RBP promotes filopodial formation, an essential step in branch initiation. Our findings show that Vg1RBP is required for terminal arborization but not long-range axon navigation and suggest that Vg1RBP-regulated mRNA translation promotes synaptic complexity.

Neurotrophic actions initiated by proNGF in adult sensory neurons may require peri-somatic glia to drive local cleavage to NGF.

The nerve growth factor (NGF) precursor, proNGF, is implicated in various neuropathological states. ProNGF signals apoptosis by forming a complex with the receptors p75 and sortilin, however, it can also induce neurite growth, proposed to be mediated by the receptor of mature NGF, tyrosine kinase receptor A (TrkA). The way in which these dual effects occur in adult neurons is unclear. We investigated the neurotrophic effects of proNGF on peptidergic sensory neurons isolated from adult mouse dorsal root ganglia and found that proNGF stimulated neurite extension and branching, requiring p75, sortilin and TrkA. Neurite growth rarely occurred in sortilin-expressing neurons but was commonly observed in TrkA-positive, sortilin-negative neurons that associated closely with sortilin-positive glia. ProNGF was unable to induce local trophic effects at growth cones where sortilin-positive glia was absent. We propose that in adult sensory neurons the neurotrophic response to proNGF is mediated by NGF and TrkA, and that peri-somatic glia may participate in sortilin- and p-75 dependent cleavage of proNGF. The potential ability of local glial cells to provide a targeted supply of NGF may provide an important way to promote trophic (rather than apoptotic) outcomes under conditions where regeneration or sprouting is required.

Above-level mechanical hyperalgesia in rats develops after incomplete spinal cord injury but not after cord transection, and is reversed by amitriptyline, morphine and gabapentin.

Spinal cord injury (SCI) is a major cause of persistent neuropathic pain of central origin. Recent evidence suggests neuropathic pain in clinically complete SCI patients correlates with limited sensory function below the lesion (sensory discomplete). On this basis we examined if the onset of mechanical hyperalgesia was different in rodents after a severe incomplete clip-compression SCI versus a complete spinal cord transection at thoracic segment T13. Above-level withdrawal behaviors evoked by forepaw stimulation provided evidence of mechanical hyperalgesia after incomplete but not complete SCI, whereas below-level responses evoked by hindpaw stimulation revealed hypersensitivity after both injuries. The latency of the above-level response was 4-5 wks but was longer after a moderate clip-compression injury. Mechanical hyperalgesia was fully reversed by three analgesic drugs used in treating neuropathic SCI pain, but their duration of action differed significantly, showing a rank order of amitriptyline (24-48 h)≫morphine (6 h)>gabapentin (2 h). Evidence of central sensitization in cervical spinal cord segments that receive sensory projections from the forelimbs was provided by immunohistochemistry for Zif268, a functional marker of neuroplasticity. Zif268-immunoreactive neurons in laminae I/II increased in response to repetitive noxious forepaw stimulation in the incomplete SCI group, and this response was reduced in the complete transection and sham-operated groups. These data are consistent with the hypothesis that neuropathic pain of cord origin is more likely to develop after SCI when there is an incomplete loss of axons traversing the lesion.

Spinal cord compression injury in adult rats initiates changes in dorsal horn remodeling that may correlate with development of neuropathic pain.

Spinal cord injury commonly causes chronic, neuropathic pain. The mechanisms are poorly understood but may include structural plasticity within spinal and supraspinal circuits. Our aim was to determine whether structural remodeling within the dorsal horn rostral to an incomplete injury differs from a complete spinal cord transection. Four immunohistochemical populations of primary afferent C-fibers, and descending catecholamine and serotonergic projections, were examined in segments T9-T12 at 2 and 12 weeks after a T13 clip-compression injury in adult male rats. Dorsal root ganglia were also examined. Two weeks after injury, fibers immunoreactive for calcitonin gene-related peptide (CGRP) or GDNF-family receptors (GFRalpha1, GFRalpha2, GFRalpha3) showed distinct injury responses within the superficial dorsal horn. CGRP fibers decreased, but GFRalpha1, GFRalpha2 and GFRalpha3 fibers did not change. In contrast, all groups were decreased by 12 weeks after injury. Catecholamine fibers showed a decrease at 2 weeks followed by an increase in density at 12 weeks, whereas serotonergic fibers showed a decrease (restricted to deep dorsal horn) at 12 weeks. These results show that the dorsal horn of the spinal cord undergoes substantial structural plasticity rostral to a compression injury, with the most profound effect being a prolonged and possibly permanent loss of primary afferent fibers. This loss was more extensive and more prolonged than the loss that follows spinal cord transection. Our results provide further evidence that anatomical reorganization of sensory and nociceptive dorsal horn circuits rostral to an injury could factor in the development or maintenance of spinal cord injury pain.

Acute and chronic changes in dorsal horn innervation by primary afferents and descending supraspinal pathways after spinal cord injury.

Sprouting of peptidergic nociceptive and descending supraspinal projections to the dorsal horn following spinal cord injury (SCI) has been proposed as a mechanism of neuropathic pain. To identify structural changes that could initiate or maintain SCI pain, we used a complete transection model in rats to examine how structural remodeling in the dorsal horn rostral to the lesion relates to distance from injury, laminar region, and duration of injury. The major classes of C-fiber primary afferents differed greatly in their susceptibility to structural and chemical changes and their ability to undergo plasticity. Peptidergic primary afferents showed a widespread loss throughout the dorsal horn of segments approaching the injury site. Some of this loss may have been due to decreased neuropeptide expression. The reduction in peptidergic fibers was transient, indicating compensatory sprouting and perhaps also increased neuropeptide expression within the cord. Nonpeptidergic afferents expressing GFRalpha1 were largely unaffected by SCI. In contrast, in GFRalpha2-expressing nonpeptidergic afferents SCI caused a permanent loss of dorsal horn innervation. Unexpectedly, GFRalpha2 was transiently induced throughout deeper laminae but this was not due to upregulation of GFRalpha2 in dorsal root ganglia. We also observed permanent sprouting of catecholamine terminals of supraspinal origin. This was restricted to the superficial laminae. Our results show that SCI caused a loss of sensory input as well as structural remodeling such that the balance of nociceptive inputs and descending modulation was permanently altered. These changes may contribute to mechanisms rostral to the site of SCI that trigger and maintain neuropathic pain.

Purine release from spinal cord microglia after elevation of calcium by glutamate.

The propagation of Ca2+ waves in a network of microglial cells, after its initiation by glutamate, is mediated by purinergic transmission. In this study, we investigated the mechanisms by which glutamate releases ATP from cultured spinal cord microglia. The 4-fold increase in ATP release from microglia in response to glutamate (0.5 mM) was blocked by alpha-aminohydroxy-5-methyl-isoxazole-4-proprionate (AMPA)/kainate receptor antagonist 6-cyano-7-nitroguinoxaline-2,3-dione and specific AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) but not by N-methyl-d-aspartic acid or metabotropic glutamate receptor antagonists. Glutamate acting on AMPA receptors evoked an ATP release that was blocked by antagonizing the rise in intracellular Ca2+ as a result of its release from internal stores as well as by antagonizing protein kinase C with chelerythrine. Glutamate-stimulated ATP release was significantly antagonized by the cystic fibrosis transmembrane conductance regulator (CFTR) blockers flufenamic acid and glibenclamide. A role for the CFTR was further confirmed using microglia from CFTR knockout mice, which released significantly less ATP than microglia from control wild-type mice in response to glutamate. Use of 6-methoxy-1-(3-sulfopropyl)quinolinium fluorescence assay revealed functional CFTR in microglia. These observations suggest that glutamate acted on microglial AMPA receptors to stimulate release of Ca2+ from intracellular stores as well as a Ca2+-dependent isoform of protein kinase C, which then acts to trigger release of ATP with the CFTR acting as a regulator of the ATP release process, perhaps through another channel or transporter.