Memory-like alterations in Aplysia axons after nerve injury or localized depolarization

Abstract

Adaptive, long-term alterations of excitability have been reported in dendrites and presynaptic terminals but not along axons. Persistent enhancement of axonal excitability has been described in proximal nerve stumps at sites of nerve section in mammals, but this hyperexcitability is considered a pathological derangement important only as a cause of neuropathic pain. Identified neurons in Aplysia were used to test the hypothesis that either axonal injury or the focal depolarization that accompanies axonal injury can trigger a local decrease in action potential threshold [long-term hyperexcitability (LTH)] having memory-like properties. Nociceptive tail sensory neurons and a giant secretomotor neuron, R2, exhibited localized axonal LTH lasting 24 hr after a crush of the nerve or connective that severed the tested axons. Axons of tail sensory neurons and tail motor neurons, but not R2, displayed similar localized LTH after peripheral depolarization produced by 2 min exposure to elevated extracellular [K(+)]. Neither the induction nor expression of either form of LTH was blocked by saline containing 1% normal [Ca(2+)] during treatment or testing. However, both were prevented by local application of the protein synthesis inhibitors anisomycin or rapamycin. The features of (1) long-lasting alteration by localized depolarization, (2) restriction of alterations to intensely depolarized regions, and (3) dependence of the alterations on local, rapamycin-sensitive protein synthesis are shared with synaptic mechanisms considered important for memory formation. This commonality suggests that relatively simple, accessible axons may offer an opportunity to define fundamental plasticity mechanisms that were important in the evolution of memory.

Figure 1.

Preparations used to examine axonal LTH. p9 or RPAC was threaded through a series of wells. Only the wells containing the stimulated segments of the nerves are shown. The cut nerve ends were always at least 1 cm from the nearest test well. A, Pleural-pedal ganglia with short-segment stimulation configuration illustrated on nerve p9. Intracellular electrodes were used to record evoked action potentials conducted to the somata of tail sensory neurons (SNs) in the pleural ganglion or tail motor neurons (MNs) in the pedal ganglion. The sharp arrow indicates the slot between the wells containing the 2-mm-long tube used to focus test currents flowing from one well to the other on a short nerve segment. The blunt arrow indicates the site of nerve crush, just distal to this test slot. During depolarizing treatment, high-K/low-Ca saline filled the well (6 mm diameter) on each side of the test slot, so that ∼14 mm of the nerve was exposed to the depolarizing solution. Diffusion to other parts of the nerve was prevented by sealing the remaining slots in these two wells with silicone grease. B, Abdominal ganglion with long-segment stimulation configuration illustrated on the RPAC. Evoked spikes were recorded in the soma of giant secretomotor neuron R2 in the abdominal ganglion while applying test currents to a 6 mm segment of the RPAC within a single well, which contained electrodes on each side of the RPAC. In this case, silicone grease sealed both slots of the stimulated well. Note that, in separate experiments, both the short- and long-segment stimulation configurations were used in experiments on tail sensory neurons, tail motor neurons, and R2.

Figure 4.

Sensory neuron axons display localized LTH after high-K treatment of a peripheral nerve segment. A, Localization of axonal LTH to the site of depolarization. Test shocks (50 msec) were delivered between the indicated electrodes set up on nerve p9 in the dual-well configuration for stimulating short nerve segments (∼2 mm) between pairs of wells. Tests were delivered before (pretest) and 24 hr after high-K or sham treatment in the test wells. Asterisks indicate significant differences between ipsilateral test segments revealed by paired t tests. Similar differences were also found between contralateral segments (see Results). B, Test pulses (5 msec) also reveal localized axonal LTH. C, Repetitive firing responses are highly variable after high-K treatment and do not differ significantly from responses in control axons. In B and C, the preparations were the same as in A, but the ganglia and electrodes have been omitted from the figure.

Figure 7.

Secretomotor neuron R2 displays LTH of its giant axon after nerve crush but not high-K treatment. A, Axonal LTH (monitored as a decrease in spike threshold during 100 msec test pulses) is not observed 24 hr after either 2 or 4 min depolarization of a segment of the RPAC with high-K saline containing 1% normal [Ca²⁺]. B, LTH of the axon of R2 is produced by crushing the RPAC in the low-Ca solution. C, Crushing the RPAC in normal saline induces LTH of the axon of R2 that requires local protein synthesis. All tests were performed in low-Ca saline. Anisomycin (10 μm; Aniso) or rapamycin (20 nm; Rapa) blocked crush-induced LTH. Asterisks indicate differences relative to the crush (Cr) group. Anisomycin and rapamycin also significantly increased spike threshold compared with the thresholds in uncrushed (Uncr), sham-treated axons (see Results). D, Example of repetitive firing recorded in the soma of R2 during weak 1 sec test pulses (one times the 100 msec pretest threshold) in low-Ca saline before and 1 d after RPAC crush in the test well. E, Repetitive firing in axons expressing LTH (as shown by decreased axonal spike threshold) is highly variable. Each column shows the entire range of repetitive firing responses for each group before and 24 hr after crush of the RPAC in the test well. The top of each box indicates the 75th quartile, the bottom of the box indicates the 25th quartile, and the horizontal line in the middle is the median response. The ends of the vertical lines projecting above and below each box show the maximum and minimum responses in each group.

Figure 2.

Low-Casaline containing 1% normal [Ca²⁺] blocks release of neuromodulators in the nervous system. A, Low-Ca saline eliminates hyperexcitability of the soma of pleural sensory neurons observed 1-2 min after p9 shock. Top, Example of responses from a single sensory neuron tested before (Pre) and 1 min after (Post) p9 shock in normal saline and in low-Ca saline. Bottom, Summary of soma excitability effects from 10 sensory neurons tested in both normal and low-Ca saline. ^**p < 0.05; paired t test. In this figure and the others, error bars indicate SEM (unless otherwise indicated). B, Low-Ca saline eliminates hyperpolarizing responses of pleural sensory neurons evoked by the 50 msec pulse to nerve p9. No evoked slow responses (hyperpolarizing or depolarizing) were detected in any sensory neurons tested in low-Ca saline (n = 6). C, Low-Ca saline (right column) eliminates detectable EPSPs in a pedal motor neuron evoked by a 50 msec pulse to nerve p9. No evoked EPSPs were detected in any motor neurons tested in low-Ca saline (n = 40).

Figure 3.

Sensory neuron axons display localized LTH after peripheral nerve injury, and this LTH depends on local protein synthesis. A, Responses recorded in the soma while testing axon spike threshold and repetitive firing before and 24 hr after crushing nerve p9. B, Nerve crush in normal saline significantly decreased axon spike threshold for 50 msec test pulses delivered close to (0.5-1.5 mm away) but not distant from (2-4 mm) the crushed (Cr) site. Asterisk indicates significant difference (Dunnett's test) relative to uncrushed (Uncr) control group. C, Nerve crush in low-Ca saline also decreased spike threshold. D, Crush-induced (in low-Ca saline) decrease in spike threshold was prevented by local application during the crush of protein synthesis inhibitors, anisomycin (10 μm; Aniso) or rapamycin (20 nm; Rapa). In both C and D, asterisks indicate comparisons with the crushed (Cr) group in C (Dunnett's tests).

Figure 5.

LTH of sensory neuron axons induced by peripheral high-K treatment requires local protein synthesis. Local application of the protein synthesis inhibitors anisomycin (10 μm; Aniso) or rapamycin (20 nm; Rapa) blocked the 24 hr decrease in axonal spike threshold. Asterisks indicate statistically significant differences from the high-K-treated group. Untreated nerves did not receive the sham wash with ASW.

Figure 6.

Tail motor neuron axons display localized LTH after high-K treatment, which depends on local protein synthesis. During high-K treatment and all excitability tests, the nerve segment was bathed in low-Ca saline. Local application of anisomycin (10 μm; Aniso) blocked the 24 hr decrease in spike threshold of the motor axons. Asterisks indicate statistically significant differences from the high-K-treated group.