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

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

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

RNA from Trained Aplysia

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

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

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

Isoform Specificity of PKMs during Long-Term Facilitation in Aplysia Is Mediated through Stabilization by KIBRA

Persistent activity of protein kinase M (PKM), the truncated form of protein kinase C (PKC), can maintain long-term changes in synaptic strength in many systems, including the hermaphrodite marine mollusk, Aplysia californica Moreover, different types of long-term facilitation (LTF) in cultured Aplysia sensorimotor synapses rely on the activities of different PKM isoforms in the presynaptic sensory neuron and postsynaptic motor neuron. When the atypical PKM isoform is required, the kidney and brain expressed adaptor protein (KIBRA) is also required. Here, we explore how this isoform specificity is established. We find that PKM overexpression in the motor neuron, but not the sensory neuron, is sufficient to increase synaptic strength and that this activity is not isoform-specific. KIBRA is not the rate-limiting step in facilitation since overexpression of KIBRA is neither sufficient to increase synaptic strength, nor to prolong a form of PKM-dependent intermediate synaptic facilitation. However, the isoform specificity of dominant-negative-PKMs to erase LTF is correlated with isoform-specific competition for stabilization by KIBRA. We identify a new conserved region of KIBRA. Different splice isoforms in this region stabilize different PKMs based on the isoform-specific sequence of an α-helix "handle" in the PKMs. Thus, specific stabilization of distinct PKMs by different isoforms of KIBRA can explain the isoform specificity of PKMs during LTF in Aplysia SIGNIFICANCE STATEMENT Long-lasting changes in synaptic plasticity associated with memory formation are maintained by persistent protein kinases. We have previously shown in the Aplysia sensorimotor model that distinct isoforms of persistently active protein kinase Cs (PKMs) maintain distinct forms of long-lasting synaptic changes, even when both forms are expressed in the same motor neuron. Here, we show that, while the effects of overexpression of PKMs are not isoform-specific, isoform specificity is defined by a "handle" helix in PKMs that confers stabilization by distinct splice forms in a previously undefined domain of the adaptor protein KIBRA. Thus, we define new regions in both KIBRA and PKMs that define the isoform specificity for maintaining synaptic strength in distinct facilitation paradigms

Representative 2-DE pattern of gallbladder bile proteins from different phases of cholesterol-phosphatide vesicle and micelle.

<p>The proteins (250 µg) were subjected to 2-DE system (first dimension, IPG strip, pH 3–10 NL, 13 cm; second dimension, 12% SDS-PAGE). Protein spots were visualized by silver staining and analyzed by ImageMaster™ 2-D Platinum software. The spots marked with arrows in the two images indicate the distribution of up-regulated or down-regulated proteins that were successfully identified by MALDI-TOF. The numbers between the two images show the molecular marker (kDa). Annotations in the gels refer to the Spot No. are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054489#pone-0054489-t003" target="_blank">Table 3</a>.</p

Differentially expressed proteins of gallbladder bile between cholesterol cholelithiasis group and control group identified by MALDI-TOF MS.

a)<p>Spot number corresponds to the spot number on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054489#pone-0054489-g002" target="_blank">Fig. 2</a>.</p>b)<p>Protein description refers to the name of each matched protein in NCBInr database.</p>c)<p>Accession number is recorded as a reference for the identification in NCBInr database.</p>d)<p>Theoretical Mr/pI means theoretical molecular weight and iso-electric point of the matched protein.</p>e)<p>Matched peptide refers to the number of peptide matched to the candidate protein.</p>f)<p>Sequence coverage is percent of identified sequence to the complete sequence of the candidate protein.</p>g)<p>Spots were identified by MS/MS analysis and the MASCOT score is indicated.</p

Representative 2-DE profiles of gallbladder bile proteins from cholesterol cholelithiasis group and control group.

<p>The proteins (250 µg) were subjected to 2-DE system (first dimension, IPG strip, pH 3–10 NL, 13 cm; second dimension, 12% SDS-PAGE). Protein spots were visualized by silver staining and analyzed by ImageMaster™ 2-D Platinum software. The spots marked with arrows indicate the distribution of up-regulated or down-regulated proteins that were successfully identified by MALDI-TOF. The numbers between the two images show the molecular marker (kDa). Annotations in the gels refer to the Spot No. are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054489#pone-0054489-t001" target="_blank">Table 2</a>.</p

Differentially expressed proteins between vesicular phase and control micellar phase identified by MALDI-TOF MS.

a)<p>Spot number corresponds to the spot number on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054489#pone-0054489-g003" target="_blank">Fig. 3</a>.</p>b)<p>Protein description refers to the name of each matched protein in NCBInr database.</p>c)<p>Accession number is recorded as a reference for the identification in NCBInr database.</p>d)<p>Theoretical Mr/pI means theoretical molecular weight and iso-electric point of the matched protein.</p>e)<p>Matched peptide refers to the number of peptide matched to the candidate protein.</p>f)<p>Sequence coverage is percent of identified sequence to the complete sequence of the candidate protein.</p>g)<p>Spots were identified by MS/MS analysis and the MASCOT score is indicated.</p