Recovery of function, peripheral sensitization and sensory neurone activation by novel pathways following axonal injury in Aplysia californica.

Recovery of behavioural and sensory function was examined following unilateral pedal nerve crush in Aplysia californica. Nerve crush that transected all axons connecting the tail to the central nervous system (CNS) eliminated the ipsilateral tail-evoked siphon reflex, whose sensory input travels in the crushed tail nerve (p9). The first reliable signs of recovery of this reflex were observed within 1 week, and most animals displayed tail-evoked siphon responses within 2 weeks. Wide-dynamic-range mechanosensory neurons with somata in the ventrocaudal (VC) cluster of the ipsilateral pleural ganglion exhibited a few receptive fields (RFs) on the tail 3 weeks after unilateral pedal nerve crush, indicating that the RFs had either regenerated or been reconnected to the central somata. These RFs were smaller and sensitized compared with corresponding RFs on the contralateral, uncrushed side. Centrally conducted axon responses of VC sensory neurones to electrical stimulation distal to the nerve crush site did not reappear until at least 10 days after the crush. Because the crush site was much closer to the CNS than to the tail, the failure of axon responses to be restored earlier than the behavioural responses indicates that early stages of reflex recovery are not due to regeneration of VC sensory neurone axons into the tail. Following nerve crush, VC sensory neurones often could be activated by stimulating central connectives or peripheral nerves that do not normally contain the sensory neurone's axons. These results suggest that recovery of behavioral function after nerve injury involves complex mechanisms, including regenerative growth of axotomized VC sensory neurones, sensitization of regenerating RFs and sprouting of VC sensory neurone fibres within the CNS. Furthermore, the rapidity of behavioural recovery indicates that its initial phases are mediated by additional mechanisms, perhaps centripetal regeneration of unidentified sensory neurones having peripheral somata, or transient reconnection of proximal and distal stumps of axotomized VC cells.

Peripheral regeneration and central sprouting of sensory neurone axons in Aplysia californica following nerve injury.

Morphological methods were used to examine injury-induced growth of peripheral and central axons of nociceptive mechanosensory neurones in the ventrocaudal (VC) clusters of the pleural ganglia of Aplysia californica. Pedal nerve crush transected all axons in the nerve while leaving the overlying sheath largely intact. Immunohistochemical staining was performed with an antibody to a sensory-neurone-specific peptide, sensorin-A. Following bilateral crush of pedal nerve p9, which innervates the tail, sensorin-A immunofluorescence was lost distal to the crush site within 2 days. Fine immunopositive fibres began to invade the crush region within 5 days. These fibres arborized in the crush region and gradually extended down the crushed nerve. Immunopositive fibres were found near the tail within 3 weeks. Similar results were obtained after injecting individual sensory neurone somata in the tail/p9 region of the VC cluster with biocytin. Biocytin injections and horseradish peroxidase injections 3 weeks after ipsilateral pedal nerve crush revealed new fibres projecting rostrally from the tail/p9 region of the VC cluster and entering the pleural-cerebral and pleural-abdominal connectives. Such projections were never observed in control, uncrushed preparations. These results demonstrate that nerve injury triggers extensive growth of both peripheral and central processes of the VC sensory neurones.

Axoplasm enriched in a protein mobilized by nerve injury induces memory-like alterations in Aplysia neurons.

Axon regeneration after injury and long-term alterations associated with learning both require protein synthesis in the neuronal cell body, but the signals that initiate these changes are largely unknown. Direct evidence that axonal injury activates molecular signals in the axon was obtained by injecting axoplasm from crushed or uncrushed nerves into somata of sensory neurons with uncrushed axons. Those injected with crush axoplasm behaved as if their axons had been crushed, exhibiting increases in both repetitive firing and spike duration, and a decrease in spike afterhyperpolarization 1 d after injection. Because similar changes occur in the same cells after learning, these data suggest that some of the long-lasting adaptive changes that occur after injury and learning may be induced by common axoplasmic signals. Since the signals in axoplasm must be conveyed to the cell soma, we have begun to test the hypothesis that at least some of these signals are proteins containing a nuclear localization signal (NLS). Axoplasmic proteins at the crush site and those that accumulated at a ligation proximal to the crush were probed with an antibody to an amino acid sequence (sp) containing a NLS that provides access to the retrograde transport/nuclear import pathway. One protein, sp97, displayed properties expected of an axonal injury signal: it responded to injury by undergoing an anterograde-to-retrograde change in movement and, when the ligation was omitted, it was transported to the cell bodies of the injured neurons.