Activation of protein kinase A contributes to the expression but not the induction of long-term hyperexcitability caused by axotomy of Aplysia sensory neurons.

Nociceptive sensory neurons (SNs) in Aplysia provide useful models to study both memory and adaptive responses to nerve injury. Induction of long-term memory in many species, including Aplysia, is thought to depend on activation of cAMP-dependent protein kinase (PKA). Because Aplysia SNs display similar alterations in models of memory and after nerve injury, a plausible hypothesis is that axotomy triggers memory-like modifications by activating PKA in damaged axons. The present study disproves this hypothesis. SN axotomy was produced by (1) dissociation of somata from the ganglion [which is shown to induce long-term hyperexcitability (LTH)], (2) transection of neurites of dissociated SNs growing in vitro, or (3) peripheral nerve crush. Application of the competitive PKA inhibitor Rp-8-CPT-cAMPS at the time of axotomy failed to alter the induction of LTH by each form of axotomy, although the inhibitor antagonized hyperexcitability produced by 5-HT application. Strong activation of PKA in the nerve by coapplication of a membrane-permeant analog of cAMP and a phosphodiesterase inhibitor was not sufficient to induce LTH of either the SN somata or axons. Furthermore, nerve crush failed to activate axonal PKA or stimulate its retrograde transport. Therefore, PKA activation plays little if any role in the induction of LTH by axotomy. However, the expression of LTH was reduced by intracellular injection of the highly specific PKA inhibitor PKI several days after nerve crush. This suggests that long-lasting activation of PKA in or near the soma contributes to the maintenance of long-term modifications produced by nerve injury.

Intrinsic injury signals enhance growth, survival, and excitability of Aplysia neurons.

Neurons undergo extensive changes in growth and electrophysiological properties in response to axon injury. Efforts to understand the molecular mechanisms that initiate these changes have focused almost exclusively on the role of extrinsic signals, primarily neurotrophic factors released from target and glial cells. The objective of the present investigation was to determine whether the response to axonal injury also involves intrinsic axoplasmic signals. Aplysia neurons were removed from their ganglia and placed in vitro on a substratum permissive for growth, but in the absence of glia and soluble growth factors. Under these conditions, neurites emerged and grew for approximately 4 d. Once growth had ceased, the neurites were transected. In all, 46 of 50 cells regenerated, either by resorbing the remaining neurites and elaborating a new neuritic arbor or by merely replacing the neurites that had been severed. Cut cells also exhibited enhanced excitability and, paradoxically, prolonged survival, when compared with uninjured neurons. These findings indicate that axons contain intrinsic molecular signals that are directly activated by injury to trigger changes underlying regeneration and compensatory plasticity.

Retrograde transport of plasticity signals in Aplysia sensory neurons following axonal injury.

Following injury to their peripheral branches, mechanosensory neurons in Aplysia display long-term plasticity that is expressed as soma hyperexcitability, synaptic facilitation, and neurite outgrowth. To investigate the nature of signals that convey information about distant axonal injury, we have investigated the development of injury-induced soma hyperexcitability in two in vitro preparations. In isolated ganglia, proximal nerve crush caused hyperexcitability to appear sooner than did distal crush, and the difference in development of hyperexcitability indicated that the injury signal moved at a rate (36 mm/d) similar to previously reported rates of retrograde axonal transport in this animal. This hyperexcitability was not due to interruption of continuous retrograde transport of trophic substances (a negative signal) because inhibitors of axonal transport applied to uncrushed nerve segments did not induce hyperexcitability. Indeed, inhibitors of axonal transport blocked crush-induced hyperexcitability, indicating that positive injury signals are involved. Crush-induced hyperexcitability was unaffected by bathing the nerve in tetrodotoxin or the ganglion in Cd2+, suggesting that the retrograde signals depend upon neither spike activity in the nerve nor synaptic transmission in the ganglion. Close excision of sensory neuron somata (which largely eliminated delays attributable to axonal transport) produced soma hyperexcitability that was expressed after 10 hr and lasted at least 17 d. These data indicate that axonal injury mobilizes signal molecules that are conveyed by retrograde axonal transport into the soma and possibly the nucleus, where they induce long-term plasticity similar to that expressed by these cells during learning and memory.