Diancai Cai

Protein Kinase M Maintains Long-Term Sensitization and Long-Term Facilitation in Aplysia

How the brain maintains long-term memories is one of the major outstanding questions in modern neuroscience. Evidence from mammalian studies indicates that activity of a protein kinase C (PKC) isoform, protein kinase Mζ (PKMζ), plays a critical role in the maintenance of long-term memory. But the range of memories whose persistence depends on PKMζ, and the mechanisms that underlie the effect of PKMζ on long-term memory, remain obscure. Recently, a PKM isoform, known as PKM Apl III, was cloned from the nervous system of Aplysia. Here, we tested whether PKM Apl III plays a critical role in long-term memory maintenance in Aplysia. Intrahemocoel injections of the pseudosubstrate inhibitory peptide ZIP (ζ inhibitory peptide) or the PKC inhibitor chelerythrine erased the memory for long-term sensitization (LTS) of the siphon-withdrawal reflex (SWR) as late as 7 d after training. In addition, both PKM inhibitors disrupted the maintenance of long-term (≥24 h) facilitation (LTF) of the sensorimotor synapse, a form of synaptic plasticity previously shown to mediate LTS of the SWR. Together with previous results (Bougie et al., 2009), our results support the idea that long-term memory in Aplysia is maintained via a positive-feedback loop involving PKM Apl III-dependent protein phosphorylation. The present data extend the known role of PKM in memory maintenance to a simple and well studied type of long-term learning. Furthermore, the demonstration that PKM activity underlies the persistence of LTF of the Aplysia sensorimotor synapse, a form of synaptic plasticity amenable to rigorous cellular and molecular analyses, should facilitate efforts to understand how PKM activity maintains memory.

Postsynaptic Regulation of Long-Term Facilitation in Aplysia

Repeated exposure to serotonin (5-HT), an endogenous neurotransmitter that mediates behavioral sensitization in Aplysia[1-3], induces long-term facilitation (LTF) of the Aplysia sensorimotor synapse [4]. LTF, a prominent form of invertebrate synaptic plasticity, is believed to play a major role in long-term learning in Aplysia[5]. Until now, LTF has been thought to be due predominantly to cellular processes activated by 5-HT within the presynaptic sensory neuron [6]. Recent work indicates that LTF depends on the increased expression and release of a sensory neuron-specific neuropeptide, sensorin [7]. Sensorin released during LTF appears to bind to autoreceptors on the sensory neuron, thereby activating critical presynaptic signals, including mitogen-activated protein kinase (MAPK) [8, 9]. Here, we show that LTF depends on elevated postsynaptic Ca2+ and postsynaptic protein synthesis. Furthermore, we find that the increased expression of presynaptic sensorin resulting from 5-HT stimulation requires elevation of postsynaptic intracellular Ca2+. Our results represent perhaps the strongest evidence to date that the increased expression of a specific presynaptic neuropeptide during LTF is regulated by retrograde signals.

The Role of Rapid, Local Postsynaptic Protein Synthesis in Learning-Related Synaptic Facilitation in Aplysia

The discovery that dendrites of neurons in the mammalian brain possess the capacity for protein synthesis stimulated interest in the potential role of local, postsynaptic protein synthesis in learning-related synaptic plasticity. But it remains unclear how local, postsynaptic protein synthesis actually mediates learning and memory in mammals. Accordingly, we examined whether learning in an invertebrate, the marine snail Aplysia, involves local, postsynaptic protein synthesis. Previously, we showed that the dishabituation and sensitization of the defensive withdrawal reflex in Aplysia require elevated postsynaptic Ca(2+), postsynaptic exocytosis, and functional upregulation of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Here, we tested whether the synaptic facilitation that underlies dishabituation and sensitization in Aplysia requires local, postsynaptic protein synthesis. We found that the facilitatory transmitter, serotonin (5-HT), enhanced the response of the motor neuron to glutamate, the sensory neuron transmitter, and this enhancement depended on rapid protein synthesis. By using individual motor neurites surgically isolated from their cell bodies, we showed that the 5-HT-dependent protein synthesis occurred locally. Finally, by blocking postsynaptic protein synthesis, we disrupted the facilitation of the sensorimotor synapse. By demonstrating its critical role in a synaptic change that underlies learning and memory in a major model invertebrate system, our study suggests that local, postsynaptic protein synthesis is of fundamental importance to the cell biology of learning.

Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in Aplysia

Long-term memory (LTM) is believed to be stored in the brain as changes in synaptic connections. Here, we show that LTM storage and synaptic change can be dissociated. Cocultures of Aplysia sensory and motor neurons were trained with spaced pulses of serotonin, which induces long-term facilitation. Serotonin (5HT) triggered growth of new presynaptic varicosities, a synaptic mechanism of long-term sensitization. Following 5HT training, two antimnemonic treatments—reconsolidation blockade and inhibition of PKM—caused the number of presynaptic varicosities to revert to the original, pretraining value. Surprisingly, the final synaptic structure was not achieved by targeted retraction of the 5HT-induced varicosities but, rather, by an apparently arbitrary retraction of both 5HT-induced and original synapses. In addition, we find evidence that the LTM for sensitization persists covertly after its apparent elimination by the same antimnemonic treatments that erase learning-related synaptic growth. These results challenge the idea that stable synapses store long-term memories. DOI: http://dx.doi.org/10.7554/eLife.03896.001

Role of Protein Kinase C in the Induction and Maintenance of Serotonin-Dependent Enhancement of the Glutamate Response in Isolated Siphon Motor Neurons of Aplysia californica

Serotonin (5-HT) mediates learning-related facilitation of sensorimotor synapses in Aplysia californica. Under some circumstances 5-HT-dependent facilitation requires the activity of protein kinase C (PKC). One critical site of PKCs contribution to 5-HT-dependent synaptic facilitation is the presynaptic sensory neuron. Here, we provide evidence that postsynaptic PKC also contributes to synaptic facilitation. We investigated the contribution of PKC to enhancement of the glutamate-evoked potential (Glu-EP) in isolated siphon motor neurons in cell culture. A 10 min application of either 5-HT or phorbol ester, which activates PKC, produced persistent (> 50 min) enhancement of the Glu-EP. Chelerythrine and bisindolylmaleimide-1 (Bis), two inhibitors of PKC, both blocked the induction of 5-HT-dependent enhancement. An inhibitor of calpain, a calcium-dependent protease, also blocked 5-HTs effect. Interestingly, whereas chelerythrine blocked maintenance of the enhancement, Bis did not. Because Bis has greater selectivity for conventional and novel isoforms of PKC than for atypical isoforms, this result implicates an atypical isoform in the maintenance of 5-HTs effect. Although induction of enhancement of the Glu-EP requires protein synthesis (Villareal et al., 2007), we found that maintenance of the enhancement does not. Maintenance of 5-HT-dependent enhancement appears to be mediated by a PKM-type fragment generated by calpain-dependent proteolysis of atypical PKC. Together, our results suggest that 5-HT treatment triggers two phases of PKC activity within the motor neuron, an early phase that may involve conventional, novel or atypical isoforms of PKC, and a later phase that selectively involves an atypical isoform.

Reconsolidation of Long-Term Memory in Aplysia

When an animal is reminded of a prior experience and shortly afterward treated with a protein synthesis inhibitor, the consolidated memory for the experience can be disrupted; by contrast, protein synthesis inhibition without prior reminding commonly does not disrupt long-term memory [1-3]. Such results imply that the reminding triggers reconsolidation of the memory. Here, we asked whether the behavioral and synaptic changes associated with the memory for long-term sensitization (LTS) of the siphon-withdrawal reflex in the marine snail Aplysia californica [4, 5] could undergo reconsolidation. In support of this idea, we found that when sensitized animals were given abbreviated reminder sensitization training 48-96 hr after the original sensitization training, followed by treatment with the protein synthesis inhibitor anisomycin, LTS was disrupted. We also found that long-term (≥ 24 hr) facilitation (LTF) [6], which can be induced in the monosynaptic connection between Aplysia sensory and motor neurons in dissociated cell culture by multiple spaced pulses of the endogenous facilitatory transmitter serotonin (5-HT) [7, 8], could be eliminated by treating the synapses with one reminder pulse of 5-HT, followed by anisomycin, at 48 hr after the original training. Our results provide a simple model system for understanding the synaptic basis of reconsolidation.

Serotonin-Induced Cleavage of the Atypical Protein Kinase C Apl III in Aplysia

A constitutively active kinase, known as protein kinase Mζ (PKMζ), is proposed to act as a long-lasting molecular memory trace. While PKMζ is formed in rodents through translation of a transcript initiating in an intron of the protein kinase Cζ (PKCζ) gene, this transcript does not exist in Aplysia californica despite the fact that inhibitors of PKMζ erase memory in Aplysia in a fashion similar to rodents. We have previously shown that, in Aplysia, the ortholog of PKCζ, PKC Apl III, is cleaved by calpain to form a PKM after overexpression of PKC Apl III. We now show that kinase activity is required for this cleavage. We further use a FRET reporter to measure cleavage of PKC Apl III into PKM Apl III in live neurons using a stimulus that induces plasticity. Our results show that a 10 min application of serotonin induces cleavage of PKC Apl III in motor neuron processes in a calpain- and protein synthesis-dependent manner, but does not induce cleavage of PKC Apl III in sensory neuron processes. Furthermore, a dominant-negative PKM Apl III expressed in the motor neuron blocked the late phase of intermediate-term facilitation in sensory-motor neuron cocultures induced by 10 min of serotonin. In summary, we provide evidence that PKC Apl III is cleaved into PKM Apl III during memory formation, that the requirements for cleavage are the same as the requirements for the plasticity, and that PKM in the motor neuron is required for intermediate-term facilitation.

Role of protein synthesis and DNA methylation in the consolidation and maintenance of long-term memory in Aplysia

Previously, we reported that long-term memory (LTM) in Aplysia can be reinstated by truncated (partial) training following its disruption by reconsolidation blockade and inhibition of PKM (Chen et al., 2014). Here, we report that LTM can be induced by partial training after disruption of original consolidation by protein synthesis inhibition (PSI) begun shortly after training. But when PSI occurs during training, partial training cannot subsequently establish LTM. Furthermore, we find that inhibition of DNA methyltransferase (DNMT), whether during training or shortly afterwards, blocks consolidation of LTM and prevents its subsequent induction by truncated training; moreover, later inhibition of DNMT eliminates consolidated LTM. Thus, the consolidation of LTM depends on two functionally distinct phases of protein synthesis: an early phase that appears to prime LTM; and a later phase whose successful completion is necessary for the normal expression of LTM. Both the consolidation and maintenance of LTM depend on DNA methylation. DOI: http://dx.doi.org/10.7554/eLife.18299.001

RNA from Trained Aplysia Can Induce an Epigenetic Engram for Long-Term Sensitization in Untrained Aplysia

Visual Abstract The precise nature of the engram, the physical substrate of memory, remains uncertain. Here, it is reported that RNA extracted from the central nervous system of Aplysia given long-term sensitization (LTS) training induced sensitization when injected into untrained animals; furthermore, the RNA-induced sensitization, like training-induced sensitization, required DNA methylation. In cellular experiments, treatment with RNA extracted from trained animals was found to increase excitability in sensory neurons, but not in motor neurons, dissociated from naïve animals. Thus, the behavioral, and a subset of the cellular, modifications characteristic of a form of nonassociative long-term memory (LTM) in Aplysia can be transferred by RNA. These results indicate that RNA is sufficient to generate an engram for LTS in Aplysia and are consistent with the hypothesis that RNA-induced epigenetic changes underlie memory storage in Aplysia.

Effects of axotomy on cultured sensory neurons of Aplysia: long-term injury-induced changes in excitability and morphology are mediated by different signaling pathways.

To facilitate an understanding of injury-induced changes within the nervous system, we used a single-cell, in vitro model of axonal injury. Sensory neurons were individually dissociated from the CNS of Aplysia and placed into cell culture. The major neurite of some neurons was then transected (axotomized neurons). Axotomy in hemolymph-containing culture medium produced long-term hyperexcitability (LTH-E) and enhanced neuritic sprouting (long-term hypermorphogenesis [LTH-M]). Axotomy in the absence of hemolymph induced LTH-E, but not LTH-M. Hemolymph-derived growth factors may activate tyrosine receptor kinase (Trk) receptors in sensory neurons. To examine this possibility, we treated uninjured (control) and axotomized sensory neurons with K252a, an inhibitor of Trk receptor activity. K252a depressed the excitability of both axotomized and control neurons. K252a also produced a distinct pattern of arborizing outgrowth of neurites in both axotomized and control neurons. Protein kinase C (PKC) is an intracellular signal downstream of Trk; accordingly, we tested the effects of bisindolylmaleimide I (Bis-I), a specific inhibitor of PKC, on the axotomy-induced cellular changes. Bis-I blocked LTH-E, but did not disrupt LTH-M. Finally, because Trk activates the extracellular signal regulated kinase pathway in Aplysia sensory neurons, we examined whether this pathway mediates the injury-induced changes. Sensory neurons were axotomized in the presence of U0126, an inhibitor of mitogen-activated/extracellular receptor-regulated kinase. U0126 blocked the LTH-M due to axotomy, but did not impair LTH-E. Therefore distinct cellular signaling pathways mediate the induction of LTH-E and LTH-M in the sensory neurons.