Aplysia Neurons Research Articles

Rescue of neurons from undergoing hallmark tau-induced Alzheimer's disease cell pathologies by the antimitotic drug paclitaxel

Through the use of live confocal imaging, electron microscopy, and the novel cell biological platform of cultured Aplysia neurons we show that unfolding of the hallmark cell pathologies induced by mutant-human-tau (mt-human-tau) expression is rescued by 10 nM paclitaxel. At this concentration paclitaxel prevents mt-human-tau-induced swelling of axonal segments, translocation of tau and microtubules (MT) to submembrane domains, reduction in the number of MTs along the axon, reversal of the MT polar orientation, impaired organelle transport, accumulation of macro-autophagosomes and lysosomes, compromised neurite morphology and degeneration. Unexpectedly, higher paclitaxel concentrations (100 nM) do not prevent these events from occurring and in fact facilitate them. We conclude that antimitotic MT-stabilizing reagents have the potential to serve as drugs to prevent or slow down the unfolding of tauopathies.

Role for protein kinase C in controlling Aplysia bag cell neuron excitability

Targeting signalling molecules to ion channels can expedite regulation and assure the proper transition of changes to excitability. In the bag cell neurons of Aplysia, single-channel studies of excised patches have revealed that protein kinase C (PKC) gates a non-selective cation channel through a close, physical association. This channel drives a prolonged afterdischarge and concomitant neuropeptide secretion to provoke reproductive behaviour. However, it is not clear if PKC alters cation channel function and/or the membrane potential at the whole-cell level. Afterdischarge-like depolarizations can be evoked in cultured bag cell neurons by bath-application of Conus textile venom (CtVm), which triggers the cation channel through an apparent intracellular pathway. The present study shows that the CtVm-induced depolarization was reduced by nearly 50% compared to control following dialysis with the G-protein blocker, guanosine-5′-O-2-thiodiphosphate (GDP-β-S), or treatment with either the phospholipase C inhibitor, 1-[6-[[(17β)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), or the PKC inhibitor, sphinganine. Neurons exposed to the PKC activator, phorbol 12-myristate 13-acetate (PMA), displayed depolarization with accompanying spiking, and were found to be far more responsive to depolarizing current injection versus control. Immunocytochemical staining for the two typical Aplysia PKC isoforms, Apl I and Apl II, revealed that both kinases were present in unstimulated cultured bag cell neurons. However, in CtVm-treated neurons, the staining intensity for PKC Apl I increased, peaking at 10 min post-application. Conversely, the intensity of PKC Apl II staining decreased over the duration of CtVm exposure. Our results suggest that the CtVm-induced depolarization involves PKC activation, and is consistent with prior work showing PKC closely-associating with the cation channel to produce the depolarization necessary for the afterdischarge and species propagation.

A Computational Model of the Temperature-dependent Changes in Firing Patterns inAplysiaNeurons

We performed experiments using Aplysia neurons to identify the mechanism underlying the changes in the firing patterns in response to temperature changes. When the temperature was gradually increased from 11℃ to 31℃ the firing patterns changed sequentially from the silent state to beating, doublets, beating-chaos, bursting-chaos, square-wave bursting, and bursting-oscillation patterns. When the temperature was decreased over the same temperature range, these sequential changes in the firing patterns reappeared in reverse order. To simulate this entire range of spiking patterns we modified nonlinear differential equations that Chay and Lee made using temperature-dependent scaling factors. To refine the equations, we also analyzed the spike pattern changes in the presence of potassium channel blockers. Based on the solutions of these equations and potassium channel blocker experiments, we found that, as temperature increases, the maximum value of the potassium channel relaxation time constant, τn(t) increases, but the maximum value of the probabilities of openings for activation of the potassium channels, n(t) decreases. Accordingly, the voltage-dependent potassium current is likely to play a leading role in the temperature-dependent changes in the firing patterns in Aplysia neurons.

Formation of Essential Ultrastructural Interface between Cultured Hippocampal Cells and Gold Mushroom-Shaped MEA- Toward ?IN-CELL? Recordings from Vertebrate Neurons

Using cultured Aplysia neurons we recently reported on the development of a novel approach in which an extracellular, non-invasive multi-electrode-array system provides multisite, attenuated, intracellular recordings of subthreshold synaptic potentials, and action potentials (APs), the so called “IN-CELL” recording configuration (to differentiate it from intracellular recordings). Because of its non-invasive nature, the configuration can be used for long term semi intracellular electrophysiological monitoring of APs and synaptic potentials. Three principals converge to generate the IN-CELL configuration: (a) engulfment of approximately 1 μm size gold mushroom-shaped microelectrodes (gMμE) by the neurons, (b) formation of high seal resistance between the cell’s plasma membrane and the engulfed gMμE, and (c), autonomous localized increased conductance of the membrane patch facing the gMμE. Using dissociated rat hippocampal cultures we report here that the necessary morphological and ultrastructural relationships to generate the IN-CELL recording configuration are formed between hippocampal cells and the gMμEs. Interestingly, even <1 μm thin branches expand and engulf the gMμE structures. Recordings of spontaneous electrical activity revealed fast ∼2 ms, 0.04–0.75 mV positive monophasic APs (FPMP). We propose that the FPMP are attenuated APs generated by neurons that engulf gMμEs. Computer simulations of analog electrical circuits depicting the cell–gMμE configuration point out the parameters that should be altered to improve the neuron–gMμE electrical coupling.

A presynaptic role for FMRP during protein synthesis–dependent long-term plasticity in Aplysia

Loss of the Fragile X mental retardation protein (FMRP) is associated with presumed postsynaptic deficits in mouse models of Fragile X syndrome. However, the possible presynaptic roles of FMRP in learning-related plasticity have received little attention. As a result, the mechanisms whereby FMRP influences synaptic function remain poorly understood. To investigate the cellular locus of the effects of FMRP on synaptic plasticity, we cloned the Aplysia homolog of FMRP and find it to be highly expressed in neurons. By selectively down-regulating FMRP in individual Aplysia neurons at the sensory-to-motor neuron synapse reconstituted in co-cultures, we demonstrate that FMRP functions both pre- and postsynaptically to constrain the expression of long-term synaptic depression induced by repeated pulses of FMRF-amide. In contrast, FMRP has little to no effect on long-term synaptic facilitation induced by repeated pulses of serotonin. Since other components of signaling pathways involved in plasticity appear to be conserved between Aplysia and mammalian neurons, our findings suggest that FMRP can participate in both pre- and postsynaptic regulation of enduring synaptic plasticity that underlies the storage of certain types of long-term memory.

Serotonin stimulation of cAMP-dependent plasticity in Aplysia sensory neurons is mediated by calmodulin-sensitive adenylyl cyclase.

Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neurons (SNs) in Aplysia has been proposed as a molecular coincidence detector during conditioning. We identified four putative ACs in Aplysia CNS. CaM binds to a sequence in the C1b region of AC-AplA that resembles the CaM-binding sequence in the C1b region of AC1 in mammals. Recombinant AC-AplA was stimulated by Ca(2+)/CaM. AC-AplC is most similar to the Ca(2+)-inhibited AC5 and AC6 in mammals. Recombinant AC-AplC was directly inhibited by Ca(2+), independent of CaM. AC-AplA and AC-AplC are expressed in SNs, whereas AC-AplB and AC-AplD are not. Knockdown of AC-AplA demonstrated that serotonin stimulation of cAMP-dependent plasticity in SNs is predominantly mediated by this CaM-sensitive AC. We propose that the coexpression of a Ca(2+)-inhibited AC in SNs, together with a Ca(2+)/CaM-stimulated AC, would enhance the associative requirement for coincident Ca(2+) influx and serotonin for effective stimulation of cAMP levels and initiation of plasticity mediated by AC-AplA.

Clustering of excess growth resources within leading growth cones underlies the recurrent “deposition” of varicosities along developing neurites

Varicosities (VRs) are ubiquitous neuronal structures that are considered to serve as presynaptic structures. The mechanisms of their assembly are unknown. Using cultured Aplysia neurons, we found that in the absence of postsynaptic targets, VRs form at the leading edge of extending neurites when anterogradely transported organelles accumulate within the palm of the growth cone (GC) at a rate that exceeds their utilization by the GC machinery. The aggregation of excess organelles at the palm of the GC leads to slowdown of the GC's advance. As the size of the organelle clusters increases, the rate of organelle sequestration diminishes and the supply of building blocks to the GC resumes. The GCs' advance is re-initiated, “leaving behind” an organelle-loaded nascent VR. These mechanisms account for the recurrent “deposition” of almost equally spaced VRs by advancing GCs. Consistent with the view that VRs serve as “ready-to-go” presynaptic terminals, we found that a short train of action potentials leads to exocytosis of labeled vesicles within the varicosities. We propose that the formation and spacing of VRs by advancing GCs is the default outcome of the balance between the rate of supply of growth-supporting resources and the usage of these resources by the GC's machinery at the leading edges of specific neurites.

Hallmark cellular pathology of Alzheimer’s disease induced by mutant human tau expression in cultured Aplysia neurons

The mechanisms underlying neurodegenerative diseases are the outcome of pathological alterations of evolutionary conserved molecular and cellular cascades. For this reason, Drosophila and C. elegans serve as useful model systems to study various aspects of neurodegenerative diseases. Here, we introduce the advantageous use of cultured Aplysia neurons (which express over 100 disease-related gene homologs shared with mammals), as a platform to study cell biological processes underlying the generation of tauopathy. Using live confocal imaging to follow cytoskeletal elements, autophagosomes, lysosomes, anterogradely and retrogradely transported organelles, complemented with electron microscopy, we demonstrate that the expression of mutant human tau in cultured Aplysia neurons leads to the development of hallmark Alzheimer disease (AD) pathologies. These include a reduction in the number of microtubules and their redistribution, impaired organelle transport, a dramatic accumulation of macro-autophagosomes and lysosomes, compromised neurite morphology and degeneration. Our study demonstrates the accessibility of the platform for long-term live imaging and quantification of subcellular pathological cascades leading to tauopathy. Based on the present study, it is conceivable that this system can also be used to screen for reagents that alter the pathological cascades.

Ribosomal Protein S6 Kinase Is a Critical Downstream Effector of the Target of Rapamycin Complex 1 for Long-term Facilitation in Aplysia

Long-term facilitation (LTF) in Aplysia is a leading cellular model for elucidating the biochemical mechanisms of synaptic plasticity underlying learning. In Aplysia, LTF requires translational control downstream of the target of rapamycin (TOR) complex 1 (TORC1). The major known downstream targets of TORC1 are 4E binding protein (4E-BP) and S6 kinase (S6K). By removing the site within these regulators required for their interaction with TORC1, we have generated dominant negative proteins that disrupt specific pathways downstream of TORC1. Expression of dominant negative S6K, but not dominant negative 4E-BP, in Aplysia sensory neurons (SNs) blocked 24-h LTF. TORC1 is directly activated by the small GTP-binding protein, Ras homologue enriched in brain (Rheb). To determine the effects of TORC1 activation on translation in Aplysia neurons, we have examined the effects of expressing a constitutively active form of the Aplysia orthologue of Rheb, ApRheb (ApRheb(Q63L)). Expression of ApRheb(Q63L) increased 4E-BP phosphorylation and the level of general, cap-dependent translation within the SN cell soma in a rapamycin-sensitive manner. This increase in cap-dependent translation was blocked neither by dominant negative 4E-BP nor dominant negative S6K. Thus, we demonstrate that S6K is an important downstream target of TORC1 in Aplysia and that it is necessary for 24-h LTF, but not for TORC1-mediated increases in somatic cap-dependent translation.

Transglutaminase participates in the blockade of neurotransmitter release by tetanus toxin: evidence for a novel biological function

Inhibition of neuroexocytosis by tetanus neurotoxin (TeNT) involves VAMP-2/synaptobrevin-2 cleavage. However, deletion of the TeNT activity does not completely abolish its inhibitory action. TeNT is a potent activator of the cross-linking enzyme transglutaminase 2 (TGase 2) in vitro. The role of the latter mechanism in TeNT poisoning was investigated in isolated nerve terminals and intact neurons. TeNT-induced inhibition of glutamate release from rat cortical synaptosomes was associated with a simultaneous activation of neuronal transglutaminase (TGase) activity. The TeNT-induced blockade of neuroexocytosis was strongly attenuated by pretreatment of either live Aplysia neurons or isolated nerve terminals with specific TGase inhibitors or neutralizing antibodies. The same treatments completely abolished the residual blockade of neuroexocytosis of a non-proteolytic mutant of TeNT light chain. Electrophysiological studies indicated that TGase activation occurs at an early step of TeNT poisoning and contributes to the inhibition of transmitter release. Bioinformatics and biochemical analyses identified synapsin I and SNAP-25 as potential presynaptic TGase substrates in isolated nerve terminals, which are potentially involved in the inhibitory action of TeNT. The results suggest that neuronal TGase activity plays an important role in the regulation of neuroexocytosis and is one of the intracellular targets of TeNT in neurons.

Fluorescent voltage imaging of Aplysia neuron assisted by potassium channel blocker

Fluorescent voltage imaging of Aplysia neuron assisted by potassium channel blocker

Long-Lasting Synaptic Potentiation Induced by Depolarization Under Conditions That Eliminate Detectable Ca2+ Signals

Activity-dependent alterations of synaptic transmission important for learning and memory are often induced by Ca(2+) signals generated by depolarization. While it is widely assumed that Ca(2+) is the essential transducer of depolarization into cellular plasticity, little effort has been made to test whether Ca(2+)-independent responses to depolarization might also induce memory-like alterations. It was recently discovered that peripheral axons of nociceptive sensory neurons in Aplysia display long-lasting hyperexcitability triggered by conditioning depolarization in the absence of Ca(2+) entry (using nominally Ca(2+)-free solutions containing EGTA, "0Ca/EGTA") or the absence of detectable Ca(2+) transients (adding BAPTA-AM, "0Ca/EGTA/BAPTA-AM"). The current study reports that depolarization of central ganglia to approximately 0 mV for 2 min in these same solutions induced hyperexcitability lasting >1 h in sensory neuron processes near their synapses onto motor neurons. Furthermore, conditioning depolarization in these solutions produced a 2.5-fold increase in excitatory postsynaptic potential (EPSP) amplitude 1-3 h afterward despite a drop in motor neuron input resistance. Depolarization in 0 Ca/EGTA produced long-term potentiation (LTP) of the EPSP lasting > or = 1 days without changing postsynaptic input resistance. When re-exposed to extracellular Ca(2+) during synaptic tests, prior exposure to 0Ca/EGTA or to 0Ca/EGTA/BAPTA-AM decreased sensory neuron survival. However, differential effects on neuronal health are unlikely to explain the observed potentiation because conditioning depolarization in these solutions did not alter survival rates. These findings suggest that unrecognized Ca(2+)-independent signals can transduce depolarization into long-lasting synaptic potentiation, perhaps contributing to persistent synaptic alterations following large, sustained depolarizations that occur during learning, neural injury, or seizures.

Changing gears from chemical adhesion of cells to flat substrata toward engulfment of micro-protrusions by active mechanisms

Microelectrode arrays increasingly serve to extracellularly record in parallel electrical activity from many excitable cells without inflicting damage to the cells by insertion of microelectrodes. Nevertheless, apart from rare cases they suffer from a low signal to noise ratio. The limiting factor for effective electrical coupling is the low seal resistance formed between the plasma membrane and the electronic device. Using transmission electron microscope analysis we recently reported that cultured Aplysia neurons engulf protruding micron size gold spines forming tight apposition which significantly improves the electrical coupling in comparison with flat electrodes (Hai et al 2009 Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices J. R. Soc. Interface 6 1153–65). However, the use of a transmission electron microscope to measure the extracellular cleft formed between the plasma membrane and the gold-spine surface may be inaccurate as chemical fixation may generate structural artifacts. Using live confocal microscope imaging we report here that cultured Aplysia neurons engulf protruding spine-shaped gold structures functionalized by an RGD-based peptide and to a significantly lesser extent by poly-l-lysine. The cytoskeletal elements actin and associated protein cortactin are shown to organize around the stalks of the engulfed gold spines in the form of rings. Neurons grown on the gold-spine matrix display varying growth patterns but maintain normal electrophysiological properties and form functioning synapses. It is concluded that the matrices of functionalized gold spines provide an improved substrate for the assembly of neuro-electronic hybrids.

Persistent Ca2+Current Contributes to a Prolonged Depolarization inAplysiaBag Cell Neurons

Neurons may initiate behavior or store information by translating prior activity into a lengthy change in excitability. For example, brief input to the bag cell neurons of Aplysia results in an approximate 30-min afterdischarge that induces reproduction. Similarly, momentary stimulation of cultured bag cells neurons evokes a prolonged depolarization lasting many minutes. Contributing to this is a voltage-independent cation current activated by Ca(2+) entering during the stimulus. However, the cation current is relatively short-lived, and we hypothesized that a second, voltage-dependent persistent current sustains the prolonged depolarization. In bag cell neurons, the inward voltage-dependent current is carried by Ca(2+); thus we tested for persistent Ca(2+) current in primary culture under voltage clamp. The observed current activated between -40 and -50 mV exhibited a very slow decay, presented a similar magnitude regardless of stimulus duration (10-60 s), and, like the rapid Ca(2+) current, was enhanced when Ba(2+) was the permeant ion. The rapid and persistent Ca(2+) current, but not the cation current, were Ni(2+) sensitive. Consistent with the persistent current contributing to the response, Ni(2+) reduced the amplitude of a prolonged depolarization evoked under current clamp. Finally, protein kinase C activation enhanced the rapid and persistent Ca(2+) current as well as increased the prolonged depolarization when elicited by an action potential-independent stimulus. Thus the prolonged depolarization arises from Ca(2+) influx triggering a cation current, followed by voltage-dependent activation of a persistent Ca(2+) current and is subject to modulation. Such synergy between currents may represent a common means of achieving activity-dependent changes to excitability.

Paclitaxel induces axonal microtubules polar reconfiguration and impaired organelle transport: implications for the pathogenesis of paclitaxel-induced polyneuropathy

In differentiated axons almost all microtubules (MTs) uniformly point their plus ends towards the axonal tip. The uniform polar pattern provides the structural substrate for efficient organelle transport along axons. It is generally believed that the mass and pattern of MTs polar orientation remain unchanged in differentiated neurons. Here we examined long-term effects of the MTs stabilizing reagent paclitaxel (taxol) over MTs polar orientation and organelle transport in cultured Aplysia neurons. Unexpectedly, we found that rather than stabilizing the MTs, paclitaxel leads to their massive polar reconfiguration, accompanied by impaired organelle transport. Washout of paclitaxel does not lead to recovery of the polar orientation indicating that the new pattern is self-maintained. Taken together the data suggest that MTs in differentiated neurons maintain the potential to be reconfigured. Such reconfiguration may serve physiological functions or lead to degeneration. In addition, our observations offer a novel mechanism that could account for the development of peripheral neuropathy in patients receiving paclitaxel as an antitumor drug.

Local calcium-dependent mechanisms determine whether a cut axonal end assembles a retarded endbulb or competent growth cone

The transformation of a cut axonal end into a growth cone (GC), after axotomy, is a critical event in the cascade leading to regeneration. In an earlier series of studies we analyzed the cellular cascades that transform a cut axonal end into a competent GC. We found that axotomy of cultured Aplysia neurons leads to a transient elevation of the free intracellular Ca 2+ concentration ([Ca 2+] i), calpain activation and localized proteolysis of submembranal spectrin. These events are associated with the formation of distinct microtubule (MT) based vesicle traps that accumulate anterogradely transported vesicles that fuse with the spectrin free plasma membrane in support of the growth process (Erez, H., Malkinson, G., Prager-Khoutorsky, M., De Zeeuw, C.I., Hoogenraad, C.C., and Spira, M.E. 2007. Formation of microtubule-based traps controls the sorting and concentration of vesicles to restricted sites of regenerating neurons after axotomy. J. Cell Biol. 176: 497–507.; Erez, H., and Spira, M.E. 2008. Local self-assembly mechanisms underlie the differential transformation of the proximal and distal cut axonal ends into functional and aberrant growth cones. J. Comp. Neurol. 507: spc1.). Here we report that under conditions that limit calcium influx into the cut axonal end, axotomy leads to the formation of endbulbs (EBs) rather than to competent GCs. Under these conditions typical MT based vesicle traps are not formed, and Golgi derived vesicles concentrate at the very tip of the cut axon. Since under these conditions the spectrin barrier is not cleaved, vesicle fusion with the plasma membrane and actin polymerization are retarded and growth processes are impaired. We conclude that the immediate assembly of competent GC or an EB after axotomy is the outcome of autonomous local events that are shaped by the magnitudes of the [Ca 2+] i gradients at the site of injury.

Insights into a molecular switch that gates sensory neuron synapses during habituation in Aplysia

This review focuses on synaptic depression at sensory neuron-to-motor neuron synapses in the defensive withdrawal circuit of Aplysia as a model system for analysis of molecular mechanisms of sensory gating and habituation. We address the following topics: 1. Of various possible mechanisms that might underlie depression at these sensory neuron-to-motor neuron synapses in Aplysia, historically the most widely-accepted explanation has been depletion of the readily releasable pool of vesicles. Depletion is also believed to account for synaptic depression at long interstimulus intervals in a variety of other systems. 2. Multiple lines of evidence now indicate that vesicle depletion is not an important contributing mechanism to synaptic depression at Aplysia sensory neuron-to-motor neuron synapses. More generally, it appears that vesicle depletion does not contribute substantially to depression that occurs with those stimulus patterns that are typically used in studying behavioral habituation. 3. Recent evidence suggests that at these sensory neuron-to-motor neuron synapses in Aplysia, synaptic depression is mediated by an activity-dependent, but release-independent, switching of individual release sites to a silent state. This switching off of release sites is initiated by Ca 2+ influx during individual action potentials. We discuss signaling proteins that may be regulated by Ca 2+ during the silencing of release sites that underlies synaptic depression. 4. Bursts of 2–4 action potentials in presynaptic sensory neurons in Aplysia prevent the switching off of release sites via a mechanism called “burst-dependent protection” from synaptic depression. 5. This molecular switch may explain the sensory gating that allows animals to discriminate which stimuli are innocuous and appropriate to ignore and which stimuli are more important and should continue to elicit responses.

Autaptic Excitation Elicits Persistent Activity and a Plateau Potential in a Neuron of Known Behavioral Function

Synaptic connections from a neuron onto itself (autapses) are not uncommon, but their contributions to information processing and behavior are not fully understood. Positive feedback mediated by autapses could in principle give rise to persistent activity, a property of some neurons in which a brief stimulus causes a long-lasting response. We have identified an autapse that underlies a plateau potential causing persistent activity in the B31/B32 neurons of Aplysia. The persistent activity is essential to the ability of these neurons to initiate and maintain components of feeding behavior. Persistent activity in B31/B32 arises from a voltage-dependent muscarinic autapse and from pharmacologically identical network-based positive feedback. Depolarization via the autapse begins later than network-driven excitation, and the effect of the autapse is therefore overshadowed by the earlier network-based depolarization. In B31/B32 neurons isolated in culture only the autapse is present, and the autapse functionally replaces the missing network-based feedback. Properties of B31/B32 provide insight into a possible general function of autapses. Autapses might function along with synapses from presynaptic neurons as components of feedback loops.

Activity-dependent calpain activation plays a critical role in synaptic facilitation and post-tetanic potentiation

Synaptic facilitation and post-tetanic potentiation (PTP) are believed to necessitate active regeneration of the release machinery and supply of synaptic vesicles to a ready-releasable site. The prevailing hypothesis assumes that synapsins play pivotal roles in these processes. Using a cholinergic synapse formed between cultured Aplysia neurons (B2 and MCn), we demonstrate here that the calcium-activated protease-calpain serves as a major regulating element in the cascade that links electrical activity, elevation of the free intracellular calcium concentration, and short-term synaptic enhancements such as facilitation and PTP. Our study revealed that calpain inhibitors (calpeptin and MG132) transform a facilitating synapse into a depressing one, and reduce its PTP by 80.6%. Inhibition of CaM kinases, PKA, and MAPK also reduced PTP at this synapse. When inhibitors of these kinases were applied together with calpeptin, tetanic stimuli led to synaptic depression. We concluded that at this synapse facilitation and PTP are mediated mainly by the calpain-dependent processes and to a smaller extent by the CaMKs/PKA/MAPK-dependent cascades.

Long-Lasting Hyperexcitability Induced by Depolarization in the Absence of Detectable Ca2+Signals

Learning and memory depend on neuronal alterations induced by electrical activity. Most examples of activity-dependent plasticity, as well as adaptive responses to neuronal injury, have been linked explicitly or implicitly to induction by Ca(2+) signals produced by depolarization. Indeed, transient Ca(2+) signals are commonly assumed to be the only effective transducers of depolarization into adaptive neuronal responses. Nevertheless, Ca(2+)-independent depolarization-induced signals might also trigger plastic changes. Establishing the existence of such signals is a challenge because procedures that eliminate Ca(2+) transients also impair neuronal viability and tolerance to cellular stress. We have taken advantage of nociceptive sensory neurons in the marine snail Aplysia, which exhibit unusual tolerance to extreme reduction of extracellular and intracellular free Ca(2+) levels. The axons of these neurons exhibit a depolarization-induced memory-like hyperexcitability that lasts a day or longer and depends on local protein synthesis for induction. Here we show that transient localized depolarization of these axons in an excised nerve-ganglion preparation or in dissociated cell culture can induce short- and intermediate-term axonal hyperexcitability as well as long-term protein synthesis-dependent hyperexcitability under conditions in which Ca(2+) entry is prevented (by bathing in nominally Ca(2+) -free solutions containing EGTA) and detectable Ca(2+) transients are eliminated (by adding BAPTA-AM). Disruption of Ca(2+) release from intracellular stores by pretreatment with thapsigargin also failed to affect induction of axonal hyperexcitability. These findings suggest that unrecognized Ca(2+)-independent signals exist that can transduce intense depolarization into adaptive cellular responses during neuronal injury, prolonged high-frequency activity, or other sustained depolarizing events.