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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" article-type="research-article" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">03896</article-id><article-id pub-id-type="doi">10.7554/eLife.03896</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Neuroscience</subject></subj-group></article-categories><title-group><article-title>Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in <italic>Aplysia</italic></article-title></title-group><contrib-group><contrib contrib-type="author" id="author-16617" equal-contrib="yes"><name><surname>Chen</surname><given-names>Shanping</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16618" equal-contrib="yes"><name><surname>Cai</surname><given-names>Diancai</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16619"><name><surname>Pearce</surname><given-names>Kaycey</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16620"><name><surname>Sun</surname><given-names>Philip Y-W</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="pa1">‡</xref><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-16621"><name><surname>Roberts</surname><given-names>Adam C</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-15430"><name><surname>Glanzman</surname><given-names>David L</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><label>1</label><institution content-type="dept">Department of Integrative Biology and Physiology</institution>, <institution>University of California, Los Angeles</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff><aff id="aff2"><label>2</label><institution content-type="dept">Department of Neurobiology</institution>, <institution>David Geffen School of Medicine at UCLA</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff><aff id="aff3"><label>3</label><institution content-type="dept">Integrative Center for Learning and Memory</institution>, <institution>Brain Research Institute, University of California, Los Angeles</institution>, <addr-line><named-content content-type="city">Los Angeles</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Ramaswami</surname><given-names>Mani</given-names></name><role>Reviewing editor</role><aff><institution>Trinity College Dublin</institution>, <country>Ireland</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>dglanzman@physci.ucla.edu</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn><fn fn-type="present-address" id="pa1"><label>‡</label><p>Mayo Medical School, Rochester, United States</p></fn></author-notes><pub-date publication-format="electronic" date-type="pub"><day>17</day><month>11</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03896</elocation-id><history><date date-type="received"><day>04</day><month>07</month><year>2014</year></date><date date-type="accepted"><day>13</day><month>11</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014, Chen et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Chen et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife03896.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03896.001</object-id><p>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 <italic>Aplysia</italic> 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.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.001">http://dx.doi.org/10.7554/eLife.03896.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03896.002</object-id><title>eLife digest</title><p>Cells called neurons allow information to travel quickly around the body so that we can rapidly respond to any changes that we sense in our environment. This includes non-conscious reactions, such as the knee-jerk reflex in humans.</p><p>Reflexes and other behaviors can be influenced by long-term memory, and it is thought that long-term memory is stored by changes in the synapses that connect neurons to each other. The reflexes of a sea slug known as <italic>Aplysia</italic> are often used to study memory because it has a simple nervous system in which individual sensory neurons (which detect changes) only form synapses with single motor neurons (which control muscles).</p><p>Chen et al. have now studied whether long-term memory is actually stored in these synapses. Sensory neurons and motor neurons removed from <italic>Aplysia</italic> were grown together in Petri dishes and allowed to form synapses. Next, the cells were treated with the hormone serotonin, which promotes long-term memory by, in part, causing the neurons to grow more synapses.</p><p>Afterwards, the cells were given treatments that disrupted long-term memory and also reversed the synaptic growth caused by serotonin. However, it was not only new synapses that retracted: some synapses that had existed before the serotonin treatment were also lost. This apparently random loss of synapses suggests that the memory was not stored in specific synapses. Moreover, long-term memory could be restored after these treatments, which supports that idea that memory does not depend on synapses between the neurons being maintained.</p><p>This work offers hope that it might be possible to develop treatments that help to restore long-term memory in people suffering from Alzheimer's disease and other conditions that affect long-term memory.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.002">http://dx.doi.org/10.7554/eLife.03896.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>long-term memory</kwd><kwd>synaptic structure</kwd><kwd>memory reconsolidation</kwd><kwd>protein kinase Mζ</kwd><kwd>epigenetics</kwd><kwd><italic>Aplysia californica</italic></kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>other</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000065</institution-id><institution content-type="university">National Institute of Neurological Disorders and Stroke</institution></institution-wrap></funding-source><award-id>R01 NS029563</award-id><principal-award-recipient><name><surname>Glanzman</surname><given-names>David L</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000025</institution-id><institution content-type="university">National Institute of Mental Health</institution></institution-wrap></funding-source><award-id>R01 MH096120</award-id><principal-award-recipient><name><surname>Glanzman</surname><given-names>David L</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000154</institution-id><institution>Division of Integrative Organismal Systems</institution></institution-wrap></funding-source><award-id>IOS 1121690</award-id><principal-award-recipient><name><surname>Glanzman</surname><given-names>David L</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>Storage of long-term memory in <italic>Aplysia</italic> does not require stable synapses.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>There is significant empirical support for the idea, proposed by Ramón Y Cajal more than a century ago (<xref ref-type="bibr" rid="bib15">Cajal, 1894</xref>), that long-term memories are expressed in the brain, in part, by changes in synaptic connectivity. A corollary of this idea, accepted by many, if not most, modern neuroscientists, is that memories are maintained by persistent molecular and cellular alterations in synaptic structures themselves (<xref ref-type="bibr" rid="bib6">Bailey and Kandel, 2008</xref>; <xref ref-type="bibr" rid="bib40">Kandel et al., 2014</xref>). Here, we have tested the idea that long-term memory (LTM) is stored at synapses using the marine mollusk <italic>Aplysia californica</italic>. The relatively simple behavior and nervous system of this model invertebrate organism offer several major advantages for understanding memory at the level of modifications of individual synapses (<xref ref-type="bibr" rid="bib39">Kandel, 2001</xref>). A form of learning in <italic>Aplysia</italic> whose cellular and molecular substrates are particularly well understood is sensitization of the gill- and siphon-withdrawal reflex (<xref ref-type="bibr" rid="bib16">Carew et al., 1971</xref>; <xref ref-type="bibr" rid="bib12">Brunelli et al., 1976</xref>; <xref ref-type="bibr" rid="bib3">Antonov et al., 1999</xref>; <xref ref-type="bibr" rid="bib39">Kandel, 2001</xref>; <xref ref-type="bibr" rid="bib23">Glanzman, 2010</xref>). Sensitization of the withdrawal reflex exhibits a long-term (≥24 hr) form (<xref ref-type="bibr" rid="bib61">Pinsker et al., 1973</xref>) due, in part, to long-term facilitation (LTF) of the monosynaptic connection between the sensory and motor neurons that mediate the reflex (<xref ref-type="bibr" rid="bib21">Frost et al., 1985</xref>). Importantly, the monosynaptic sensorimotor connection can be reconstituted in dissociated cell culture, and LTF of the in vitro synapse can be induced by training with pulses of serotonin (5HT), the monoaminergic neurotransmitter that mediates sensitization in <italic>Aplysia</italic> (<xref ref-type="bibr" rid="bib12">Brunelli et al., 1976</xref>; <xref ref-type="bibr" rid="bib26">Glanzman et al., 1989</xref>; <xref ref-type="bibr" rid="bib51">Marinesco and Carew, 2002</xref>). Cellular and molecular analyses of this form of long-term synaptic plasticity have provided major mechanistic insights into long-term memory in <italic>Aplysia</italic> (<xref ref-type="bibr" rid="bib27">Goelet et al., 1986</xref>; <xref ref-type="bibr" rid="bib18">Dash et al., 1990</xref>; <xref ref-type="bibr" rid="bib7">Bartsch et al., 1995</xref>; <xref ref-type="bibr" rid="bib52">Martin et al., 1997</xref>), insights that have generalized to learning and memory in other organisms, including mammals (<xref ref-type="bibr" rid="bib81">Yin et al., 1994</xref>, <xref ref-type="bibr" rid="bib80">1995</xref>; <xref ref-type="bibr" rid="bib20">Frey and Morris, 1997</xref>; <xref ref-type="bibr" rid="bib42">Kogan et al., 1997</xref>; <xref ref-type="bibr" rid="bib38">Josselyn et al., 2001</xref>). Accordingly, we used the in vitro sensorimotor synapse in initial experiments to determine whether LTM is stored at synapses.</p><p>Current evidence supports the idea that LTM can be modified or even eliminated under certain circumstances. One of these goes under the rubric of reconsolidation blockade. Here, a stimulus is delivered to an animal that serves to reactivate the LTM for a previous learning experience. If, immediately after delivery of this reminder, the animal is treated with an inhibitor of protein synthesis (<xref ref-type="bibr" rid="bib59">Nader et al., 2000</xref>), or subjected to electroconvulsive shock (<xref ref-type="bibr" rid="bib56">Misanin et al., 1968</xref>), the LTM will be apparently eliminated. Based on this evidence, it has been proposed that the reminder stimulus returns the LTM to a labile state in which new protein synthesis is required to reconsolidate the memory; and that inhibition of protein synthesis during this period of reconsolidation can erase the original memory (<xref ref-type="bibr" rid="bib58">Nader and Hardt, 2009</xref>) (but see <xref ref-type="bibr" rid="bib43">Lattal and Abel, 2004</xref>). Another manipulation that can apparently erase LTM permanently is inhibition of the constitutively active catalytic fragment of the atypical protein kinase Cζ; the ongoing activity of this catalytic fragment, named PKMζ, appears to be required for the maintenance of several forms of LTM in mammals (<xref ref-type="bibr" rid="bib66">Sacktor, 2011</xref>); inhibition of PKMζ, in the absence of a reminder, has been reported to abolish consolidated memory (but see <xref ref-type="bibr" rid="bib44">Lee et al., 2013</xref>; <xref ref-type="bibr" rid="bib76">Volk et al., 2013</xref>).</p><p>We, and others, have shown that the synaptic memory for LTF of in vitro sensorimotor connections can be apparently eliminated when sensorimotor cocultures are treated with a protein synthesis inhibitor following reminder training (<xref ref-type="bibr" rid="bib14">Cai et al., 2012</xref>; <xref ref-type="bibr" rid="bib45">Lee et al., 2012</xref>; <xref ref-type="bibr" rid="bib37">Hu and Schacher, 2014</xref>). In addition, inhibiting the activity of PKM Apl III, the constitutively active fragment of the <italic>Aplysia</italic> atypical protein kinase C (PKC Apl III) (<xref ref-type="bibr" rid="bib75">Villareal et al., 2009</xref>; <xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>; <xref ref-type="bibr" rid="bib10">Bougie et al., 2012</xref>), also erases consolidated LTF (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>). LTF is mediated partly by the growth of new sensory neuron varicosities (<xref ref-type="bibr" rid="bib25">Glanzman et al., 1990</xref>; <xref ref-type="bibr" rid="bib6">Bailey and Kandel, 2008</xref>). Here we investigated whether blocking the reconsolidation of the memory for LTF, or inhibiting PKM Apl III, altered this long-term change in presynaptic structure. We found that the synaptic growth induced by LTF training was reversed by these two memory-disrupting manipulations; however, although the overall number of presynaptic varicosities reverted to the original, pretraining level, the resultant morphological pattern of sensorimotor synapses differed significantly from the original one. These results imply that the persistence of memory does not require the stability of particular synaptic connections.</p><p>We provide additional support for this idea with data from behavioral experiments in which we show that LTM can be reinstated in intact <italic>Aplysia</italic> following its apparent disappearance due to reconsolidation blockade or PKM inhibition. Because these two antimnemonic manipulations not only disrupt the behavioral expression of LTM, but also eliminate the synaptic changes—both electrophysiological (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>; <xref ref-type="bibr" rid="bib45">Lee et al., 2012</xref>) and morphological (present data)—closely associated with LTM in <italic>Aplysia</italic> (<xref ref-type="bibr" rid="bib5">Bailey and Chen, 1983</xref>, <xref ref-type="bibr" rid="bib4">1988</xref>; <xref ref-type="bibr" rid="bib21">Frost et al., 1985</xref>; <xref ref-type="bibr" rid="bib25">Glanzman et al., 1990</xref>), our results challenge the idea that the synapse is a cellular site for long-term memory storage in <italic>Aplysia</italic>. In other behavioral experiments we show that both the disruption of LTM through inhibition of PKM Apl III and LTM induction require epigenetic changes. These results point to the nucleus of neurons as the potential locus of the engram in <italic>Aplysia</italic>.</p></sec><sec sec-type="results" id="s2"><title>Results</title><sec id="s2-1"><title>Effect of blockade of reconsolidation of long-term synaptic memory on synaptic structure</title><p>In electrophysiological experiments involving 5HT-induced LTF of sensorimotor synapses in dissociated cell culture, we previously showed that a ‘reminder’ stimulus—a single, 5-min pulse of 5HT—could trigger the apparent reconsolidation of synaptic memory, as indicated by the vulnerability of consolidated LTF to disruption by administration of a protein synthesis inhibitor (anisomycin) following the reminder. Specifically, treatment with a single pulse of 5HT and anisomycin 24 hr or more after the induction of LTF reversed the synaptic facilitation (<xref ref-type="bibr" rid="bib14">Cai et al., 2012</xref>; see also; <xref ref-type="bibr" rid="bib45">Lee et al., 2012</xref>; <xref ref-type="bibr" rid="bib37">Hu and Schacher, 2014</xref>). Here we asked whether the blockade of the reconsolidation of LTF also reverses the synaptic growth that underlies LTF (<xref ref-type="bibr" rid="bib25">Glanzman et al., 1990</xref>).</p><p>Sensory neurons (SNs) of established sensorimotor cocultures were labeled with dextran fluorescein, and motor neurons (MNs) with dextran rhodamine, via pressure injection; the neurons were then imaged using laser scanning confocal fluorescence microscopy, and the presynaptic varicosities in contact with a postsynaptic structure (either a motor neurite or the postsynaptic soma) were quantified (<xref ref-type="bibr" rid="bib25">Glanzman et al., 1990</xref>) (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>). After initial imaging some cocultures received five spaced 5-min pulses of serotonin (5X5HT training, 100 µM). 24 hr later the cocultures were reimaged and the varicosities requantified. As previously reported (<xref ref-type="bibr" rid="bib25">Glanzman et al., 1990</xref>), the 5X5HT training produced a significant increase in the number of presynaptic varicosities contacting the MN (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). After reimaging, two groups of the trained cocultures received a single 5-min pulse of 5HT (1X5HT, 100 µM) to reactivate the synaptic memory induced by 5X5HT training (<xref ref-type="bibr" rid="bib14">Cai et al., 2012</xref>). Immediately following the reminder pulse of 5HT, one group (5X5HT-1X5HT-Aniso group) received anisomycin (10 µM) treatment for 2 hr. Two other groups of 5X5HT-trained cocultures did not get the reminder pulse of 5HT; one of these groups (5X5HT-Aniso) received the anisomycin treatment, whereas the other group (5X5HT) received vehicle solution instead. A final group of cocultures (Controls) did not receive either 5HT or anisomycin; instead, the Controls were treated with vehicle solution at the experimental times that the 5X5HT training and anisomycin treatment were administered to other groups. At 48 hr after the original imaging session all of the cocultures were imaged and the varicosities once more quantified.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.003</object-id><label>Figure 1.</label><caption><title>Blockade of memory reconsolidation reverses 5HT-induced synaptic growth.</title><p>(<bold>A</bold>) Experimental protocol. The vertical blue bars represent pulses of 5HT, and the horizontal red bar represents anisomycin/vehicle treatment. A reminder pulse of 5HT (single blue bar) was delivered to the 5X5HT-1X5HT-Aniso cocultures prior to the anisomycin. (<bold>B</bold>) Sample confocal micrographs of a Control coculture. Blue arrowheads, presynaptic varicosities present at 0 hr; red arrowheads, new varicosities that appeared during 0–24 hr; and yellow arrowheads, varicosities formed during the 24–48 hr period. Scale bar, 20 μm. (<bold>C</bold>) Mean normalized varicosity number at 24 hr and 48 hr in the Control (<italic>n</italic> = 21), 5X5HT (<italic>n</italic> = 21), 5X5HT-Aniso (<italic>n</italic> = 19), and 5X5HT-1X5HT-Aniso (<italic>n</italic> = 26) groups. The number of varicosities measured at 24 hr and 48 hr was normalized to the number present at 0 hr (Note that differences in the data for the 5X5HT and 5X5HT-1X5HT groups were not significant, and two groups have been grouped together in the graph. The separate data for the 5X5HT-1X5HT group can be found in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). A repeated-measures ANOVA indicated that there was a significant group × time interaction (F<sub>[3,83]</sub> = 9.5, p < 0.0001). Planned comparisons using one-way ANOVAs indicated that the group differences at 24 and 48 hr were highly significant (F<sub>[3,83]</sub> = 8.6 for 24 hr and 12.5 for 48 hr; p < 0.0001 for the results of each ANOVA). SNK posthoc tests on the 24 hr data showed that the mean normalized varicosity number in the 5X5HT group (192.8 ± 12.7%), 5X5HT-Aniso group (180.9 ± 11.6%), and 5X5HT-1X5HT-Aniso group (179.5 ± 14.6%) was each significantly greater than that in the Control group (110.3 ± 8.5%; p < 0.001 for all comparisons). The increase in varicosity number persisted to 48 hr in the 5X5HT group (197.9 ± 12.2%, p < 0.001) and 5X5HT-Aniso group (191.6 ± 13.2%, p < 0.001), but not in the 5X5HT-1X5HT-Aniso group (124.4 ± 10.9%), when compared to the Control group (108.9 ± 15.2%). The difference between the 5X5HT-Aniso and 5X5HT-1X5HT-Aniso groups was highly significant (p < 0.001). Asterisks indicate a significant difference for comparisons with the Control group; plus signs indicate a significant difference for comparisons with the 5X5HT-1X5HT-Aniso group. Here and in subsequent figures one symbol indicates p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001. Error bars represent ±SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.003">http://dx.doi.org/10.7554/eLife.03896.003</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03896.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Graphs presenting the normalized mean varicosity numbers for the three 5HT-trained groups not subjected to reconsolidation blockade.</title><p>The mean normalized varicosity number at 24 hr was 192.7 ± 15.0% in the 5X5HT group (<italic>n</italic> = 18), 193.3 ± 17.6% in the 5X5HT-1X5HT group (<italic>n</italic> = 3), and 180.9 ± 11.6% in the 5X5HT-Aniso group (<italic>n</italic> = 19). At 48 hr the mean normalized varicosity number was 192.9 ± 13.7% in the 5X5HT group, 228.3 ± 17.4% in the 5X5HT-1X5HT group, and 191.6 ± 13.2% in the 5X5HT-Aniso group. A repeated-measures ANOVA indicated that neither the interaction (F<sub>[2,37]</sub> = 0.8, p = 0.45), nor the overall effects of group (F<sub>[2,37]</sub> = 0.3, p = 0.73), was significant.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.004">http://dx.doi.org/10.7554/eLife.03896.004</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896fs001"/></fig></fig-group></p><p>The overall increase in the number of presynaptic varicosities induced by 5X5HT training on Day 1 persisted through the third imaging session in the 5X5HT and 5X5HT-Aniso groups of cocultures (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). (Notice that because there were no significant differences between the 5X5HT and 5X5HT-1X5HT groups, these two groups have been combined into a single group [5X5HT] in <xref ref-type="fig" rid="fig1">Figure 1C</xref>. However, the data for the 5X5HT-1X5HT group are presented separately in the graph in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). The reminder pulse of 5HT coupled with inhibition of protein synthesis caused the number of varicosities to revert to the pretraining (0 hr) value in the 5X5HT-1X5HT-Aniso group. This structural result parallels the electrophysiological results previously reported for <italic>Aplysia</italic> sensorimotor cocultures (<xref ref-type="bibr" rid="bib14">Cai et al., 2012</xref>; <xref ref-type="bibr" rid="bib45">Lee et al., 2012</xref>; <xref ref-type="bibr" rid="bib37">Hu and Schacher, 2014</xref>), and provides additional support for the notion that 1X5HT reactivated the synaptic memory induced by the 5X5HT training.</p><p>Besides quantifying changes in overall varicosity number, we tracked the fate of each SN varicosity in every coculture over the course of the experiments. The varicosities were put into one of three categories: ‘original’ varicosities, that is, the varicosities present at 0 hr; ‘5HT-induced’ varicosities, the varicosities that appeared during the 24 hr after 5HT treatment (this category pertained only to the groups given 5X5HT training); and ‘new’ varicosities, varicosities formed during the 24–48 hr period. For the analysis of varicosity fate the three groups of cocultures that received the 5X5HT training without reconsolidation blockade—that is, the 5X5HT, 5X5HT-1X5HT and 5X5HT-Aniso groups—were consolidated into a single group labeled ‘5HT-No reconsolidation/No blockade’ in <xref ref-type="fig" rid="fig2">Figure 2</xref>. Inspection of the fates of individual varicosities in this group yielded surprising results. First, many of the 5HT-induced SN varicosities in the 5HT-No reconsolidation/No blockade group did not persist until the final imaging session (<xref ref-type="fig" rid="fig2 fig3">Figures 2A and 3A</xref>); instead, there was significant retraction of the 5HT-induced varicosities between 24 and 48 hr in this group. Second, there was also retraction of the original varicosities during this period (<xref ref-type="fig" rid="fig2 fig3">Figures 2B and 3A</xref>). Varicosities in the 5X5HT-1X5HT-Aniso group—the group of trained cocultures subjected to reconsolidation blockade—exhibited a similar pattern of retraction of 5HT-induced and original varicosities, but the amount of retraction was significantly greater than that observed in the 5HT-No reconsolidation/No blockade cocultures (<xref ref-type="fig" rid="fig2 fig3">Figures 2A,B and 3B</xref>). A third unexpected finding was the substantial growth of new varicosities in all trained groups between 24 and 48 hr; the amount of the growth was significantly greater, however, in the 5-HT-No reconsolidation/No blockade cocultures than in the 5X5HT-1X5HT-Aniso group (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). Thus, the 5X5HT trained cocultures subjected to reconsolidation blockade exhibited significantly more retraction and significantly less growth of varicosities during the 24–48 hr period than did the other trained cocultures. Interestingly, whereas there was greater retraction of 5HT-induced varicosities than of original varicosities between 24–48 hr in the 5HT-No reconsolidation/No blockade group, the retraction of 5HT-induced varicosities and original varicosities did not differ in the 5X5HT-1X5HT-Aniso group (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>).<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.005</object-id><label>Figure 2.</label><caption><title>Effect of reconsolidation blockade on the fates of individual varicosities.</title><p>For varicosities in the 5HT-induced and original categories, the varicosity count in a given category for each coculture at 48 hr was normalized to the number of varicosities in the same category in that coculture at 24 hr. For the varicosities formed >24 hr, the varicosity count for each coculture at 48 hr was normalized to the total number of varicosities in that coculture at 24 hr. (<bold>A</bold>) The mean normalized decrease in the number of 5HT-induced varicosities (varicosities that appeared within 24 hr after 5X5HT training) at 48 hr was 67.9 ± 4.2% in the 5X5HT-1X5HT-Aniso group, which was greater than that in the 5HT-No reconsolidation/No blockade group (47.7 ± 3.7%, p < 0.001, two-tailed <italic>t</italic> test). (<bold>B</bold>) Retraction of original varicosities in the 5X5HT-1X5HT-Aniso group (56.4 ± 6.1%) was significantly greater than in the 5HT-No reconsolidation/No blockade group (32.3 ± 5.1%, p < 0.01, two-tailed <italic>t</italic> test). (<bold>C</bold>) Reversal of the morphological changes induced by 5X5HT training also involved inhibition of synaptic growth from 24–48 hr. There was significantly less growth of new varicosities during this period in the 5X5HT-1X5HT-Aniso group (36.8 ± 5.2%) than in the 5HT-No reconsolidation/No blockade group (52.9 ± 4.2%, p < 0.05, two-tailed <italic>t</italic> test). Blue bars, 5HT-No reconsolidation/No blockade; red bars, 5X5HT-1X5HT-Aniso group. Asterisks indicate significant differences between the 5HT-No reconsolidation/No blockade and 5X5HT-1X5HT-Aniso groups. Error bars represent ±SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.005">http://dx.doi.org/10.7554/eLife.03896.005</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03896.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Comparison of retraction of original and 5HT-induced varicosities between 24–48 hr in the reconsolidation blockade experiments.</title><p>(<bold>A</bold>) The percent retraction of 5HT-induced varicosities (47.7 ± 3.7%) was significantly greater than that of the original varicosities (32.3 ± 5.1%, p < 0.02, two-tailed <italic>t</italic> test) in the 5HT-No reconsolidation/No blockade group. (<bold>B</bold>) The percent retraction of the 5HT-induced varicosities (67.9 ± 4.2%) did not differ significantly from that of original varicosities (56.4 ± 6.1%) in the 5X5HT-1X5HT-Aniso group.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.006">http://dx.doi.org/10.7554/eLife.03896.006</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896fs002"/></fig></fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.007</object-id><label>Figure 3.</label><caption><title>Confocal fluorescence micrographs illustrating the structural effects of 5X5HT training, reconsolidation blockade, and chelerythrine treatment.</title><p>(<bold>A</bold>) Coculture that received 5X5HT training alone. (<bold>B</bold>) Coculture that received the 5X5HT training plus 1X5HT and anisomycin treatment immediately after the 24 hr imaging session. (<bold>C</bold>) Coculture that received the 5X5HT training plus chelerythrine treatment immediately after the 24 hr imaging session. Blue arrowheads, varicosities present at 0 hr; red arrowheads, varicosities formed during 0–24 hr; and yellow arrowheads, varicosities formed during 24–48 hr. Scale bars, 20 μm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.007">http://dx.doi.org/10.7554/eLife.03896.007</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f003"/></fig></p><p>The substantial morphological changes in the 5HT-No reconsolidation/No blockade cocultures between 24 and 48 hr were surprising, because the overall number of varicosities remained stable in these cocultures during this period (see the data for the 5X5HT and 5X5HT-Aniso groups in <xref ref-type="fig" rid="fig1">Figure 1C</xref>). Moreover, as demonstrated in our earlier electrophysiological investigations of LTF, the amplitude of the sensorimotor EPSP was also stable between 24 and 48 hr after 5X5HT training (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>). Our morphological data reveal the operation of a heretofore unrecognized homeostatic mechanism that adjusts the number of SN varicosities—and, presumably, active synaptic sites (<xref ref-type="bibr" rid="bib67">Schacher et al., 1990</xref>; <xref ref-type="bibr" rid="bib41">Kim et al., 2003</xref>)—for sensorimotor connections according to their learning-related experience, but that seems unconcerned with the identities of the individual varicosities/synaptic sites. In the 5HT-No reconsolidation/No blockade cocultures the significant retraction of SN varicosities during 24–48 hr after 5X5HT training was compensated for by significant growth of new varicosities, so that the overall number remained constant (<xref ref-type="fig" rid="fig1 fig2">Figures 1C and 2</xref>). By contrast, the number of varicosities was reset to the original (0 hr) value in the 5X5HT-1X5T-Aniso group; this resetting involved increased retraction of both 5HT-induced and original varicosities, and decreased growth of new varicosities between 24 and 48 hr. Consequently, the morphological state of the cocultures in the 5X5HT-1X5T-Aniso group at 48 hr differed significantly from that at 0 hr with respect to the identities of individual SN varicosities.</p></sec><sec id="s2-2"><title>Synaptic structure following inhibition of PKM</title><p>Next we examined the effect of inhibiting PKM on SN varicosity number. PKM Apl III, a homolog of mammalian PKMζ (<xref ref-type="bibr" rid="bib66">Sacktor, 2011</xref>), is formed from the atypical <italic>Aplysia</italic> protein kinase C (PKC Apl III) (<xref ref-type="bibr" rid="bib11">Bougie et al., 2009</xref>) by calpain-dependent cleavage; furthermore, this cleavage is induced by prolonged treatment with 5HT (<xref ref-type="bibr" rid="bib10">Bougie et al., 2012</xref>). In previous behavioral and electrophysiological investigations we found that inhibition of PKM Apl III, either with the pseudosubstrate sequence of the regulatory domain of atypical PKC (ZIP) or with chelerythrine, a PKC inhibitor selective for PKM at low concentrations (<xref ref-type="bibr" rid="bib47">Ling et al., 2002</xref>; <xref ref-type="bibr" rid="bib75">Villareal et al., 2009</xref>), abolished both consolidated long-term sensitization and LTF (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>). To determine whether inhibition of PKM Apl III reverses the structural growth that mediates long-term sensitization (<xref ref-type="bibr" rid="bib5">Bailey and Chen, 1983</xref>, <xref ref-type="bibr" rid="bib4">1988</xref>) and LTF (<xref ref-type="bibr" rid="bib25">Glanzman et al., 1990</xref>), we gave some cocultures (5X5HT-Chel group) 5X5HT training followed 24 hr later by treatment with chelerythrine (<xref ref-type="fig" rid="fig4">Figure 4A</xref>). Another group of cocultures (5X5HT group) received the 5X5HT training alone. Finally, a third group (Controls) received the vehicle solution instead of either 5HT or chelerythrine. The SNs and MNs of all of the cocultures were labeled with fluorescent dye as before and then imaged on Day 1 (0 hr) of the experiment. Immediately afterwards cocultures in the 5X5HT and 5X5HT-Chel groups were given 5X5HT training. All cocultures were imaged for a second time at 24 hr, after which cocultures in the 5X5HT-Chel group received chelerythrine (10 µM, 1 hr), while the other two groups received the vehicle solution. (The 5X5HT-Chel group did not receive 1X5HT prior to chelerythrine treatment.) The cocultures were imaged for a final time at 48 hr.<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.009</object-id><label>Figure 4.</label><caption><title>Inhibition of PKM also reverses 5HT-induced synaptic growth.</title><p>(<bold>A</bold>) Experimental protocol. The vertical blue bars represent pulses of 5HT; the horizontal red bar represents the period of chelerythrine/vehicle treatment. The 5X5HT and Control groups received vehicle treatment during this time. (<bold>B</bold>) Mean SN varicosity number measured at 24 hr and 48 hr for the Control (<italic>n</italic> = 14), 5X5HT (<italic>n</italic> = 21) and 5X5HT-Chel (<italic>n</italic> = 14). The number of varicosities measured at 24 hr and 48 hr was normalized to the number of varicosities at 0 hr. A repeated-measures ANOVA indicated that there was a significant group × time interaction (F<sub>[2,46]</sub> = 7.5, p < 0.001). Planned comparisons showed that the group differences were significant for both the 24 and 48 hr measurements (24 hr, F<sub>[2,46]</sub> = 3.7, p < 0.04; 48 hr, F<sub>[2,46]</sub> = 12.8, p < 0.0001). The mean normalized varicosity number at 24 hr was 171.2 ± 14.0% in the 5X5HT group, 166.9 ± 22.8% in the 5X5HT-Chel group, and 114.0 ± 8.9% in the Control group. Posthoc comparisons indicated that the 5X5HT training produced a significant increase in varicosity number in the 5X5HT and 5X5HT-Chel groups at 24 hr (p < 0.05 for both comparisons with the Control group). The mean normalized varicosity number at 48 hr in the 5X5HT group (197.9 ± 18.7%) was greater than that in the Control group (104.4 ± 7.2%, p < 0.001), but the 5X5HT-Chel group mean (116.1 ± 9.5%) was not significantly different from the Control mean. The difference between the mean varicosity numbers for the 5X5HT and 5X5HT-Chel groups was highly significant (p < 0.001). Asterisks, comparison between 5X5HT and Control groups; plus signs, comparison between 5X5HT and 5X5HT-Chel groups. Error bars represent ±SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.009">http://dx.doi.org/10.7554/eLife.03896.009</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f004"/></fig></p><p>As before, the 5X5HT training produced a significant net increase in the number of presynaptic varicosities in the two trained groups at 24 hr (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). This net increase persisted in the 5X5HT group, but was reversed in 5X5HT-Chel group at 48 hr. Monitoring of the fate of individual varicosities in the 5X5HT group revealed the same pattern of structural growth and retraction observed previously in the 5X5HT-trained cocultures not subjected to reconsolidation blockade. Specifically, some varicosities induced by the 5X5HT training were lost between 24 and 48 hr, as were some of the original varicosities; this loss, however, was compensated for by the growth of new varicosities; consequently, the overall varicosity number remained stably elevated during the 24–48 hr period (<xref ref-type="fig" rid="fig3 fig5">Figures 3C and 5</xref>). The varicosities in the 5X5HT-Chel group exhibited significantly greater retraction and significantly less growth between 24 and 48 hr than those in the 5X5HT group, resulting in an overall loss of varicosities. Thus, the number of varicosities in the 5X5HT-Chel group at 48 hr was returned to the value at 0 hr. This result indicates that inhibiting PKM Apl III, like reconsolidation blockade (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), engages a homeostatic mechanism that resets the presynaptic varicosities in sensorimotor cocultures to the number present prior to training. Another similarity between the morphological effects of reconsolidation blockade and inhibition of PKM was that there was equal retraction of 5HT-induced and original varicosities in the 5X5HT-Chel cocultures during 24–48 hr, whereas, as before, the retraction of 5HT-induced varicosities was greater in the 5X5HT cocultures during this period (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>).<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.010</object-id><label>Figure 5.</label><caption><title>Effect of inhibition of PKM on varicosity fate.</title><p>For the 5HT-induced and original varicosities, the number of varicosities in each category for each coculture at 48 hr was normalized to the number in the same category in that coculture at 24 hr. For the varicosities formed >24 hr, the number of varicosities for each coculture at 48 hr was normalized to the total number of varicosities in the coculture at 24 hr. (<bold>A</bold>) The mean normalized decrease in the number of 5HT-induced varicosities at 48 hr was 53.4 ± 5.8% in the 5X5HT group and 68.0 ± 5.8% in the 5X5HT-Chel group (p < 0.05, one tailed <italic>t</italic> test). (<bold>B</bold>) The mean normalized decrease in the number of original varicosities at 48 hr was 23.5 ± 4.7% in the 5X5HT group and 63.4 ± 9.3% in the 5X5HT-Chel group (p < 0.001, two-tailed <italic>t</italic> test). (<bold>C</bold>) More presynaptic varicosities were formed during 24–48 hr in the 5X5HT group (63.9 ± 9.0%) than in the 5X5HT-Chel group (43.2 ± 5.8%, p < 0.05, one-tailed <italic>t</italic> test). Blue bars, 5X5HT group; red bars, 5X5HT-Chel group. Asterisks, comparison between 5X5HT and 5X5HT-Chel groups. Error bars represent ±SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.010">http://dx.doi.org/10.7554/eLife.03896.010</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03896.011</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Comparison of retraction of original and 5HT-induced varicosities between 24–48 hr in the chelerythrine treatment experiments.</title><p>(<bold>A</bold>) The difference in the percent retraction between 5X5HT-induced (53.4 ± 5.8%) and original (23.5 ± 4.7%) varicosities was highly significant in the 5X5HT group (p < 0.001, two-tailed <italic>t</italic> test). (<bold>B</bold>) There was no significant difference in the percent retraction between 5HT-induced (68.0 ± 5.8%) and original (63.4 ± 9.3%) varicosities in the 5X5HT-Chel group.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.011">http://dx.doi.org/10.7554/eLife.03896.011</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896fs003"/></fig></fig-group></p><p>Inspection of the synaptic structure of the Control cocultures revealed a complex underlying pattern of structural change similar to that observed in the reconsolidation experiment. There was significant growth and retraction of SN varicosities, both original and new, over the 48 hr of the experiments (<xref ref-type="fig" rid="fig1 fig6">Figures 6 and 1B</xref>). This structural dynamism contrasted with the constancy of overall varicosity number in these cocultures (<xref ref-type="fig" rid="fig1 fig4">Figures 1C and 4B</xref>), as well as with the stability of the sensorimotor EPSP amplitude observed in Control cocultures over similar time periods in our earlier studies (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>).<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.008</object-id><label>Figure 6.</label><caption><title>Changes in presynaptic varicosities in Control cocultures.</title><p>(<bold>A</bold>) The normalized increase in the number of varicosities was 54.9 ± 6.5% during the 0–24 hr period and 41.5 ± 7.2% during the 24–48 hr period (green bars). (<bold>B</bold>) The mean normalized number of original varicosities (blue bar) that retracted during the 0–24 hr period was 43.2 ± 4.4%. During the 24–48 hr period 15.5 ± 2.0% of the original varicosities (those present at 0 hr, blue segment), and 30.2 ± 5.1% of the varicosities that formed during 0–24 hr period (red segment), retracted; thus, the mean total normalized retraction of varicosities from 24–48 hr was 45.7%. Note that the numbers of newly formed varicosities during the 0–24 and 24–48 hr periods in each coculture were normalized to the total number of varicosities in that coculture measured at 0 hr. The percentage of retracted varicosities was obtained by normalizing the number of varicosities that disappeared during the 0–24 and 24–48 hr periods for a given coculture to the total number of varicosities in that coculture at 0 hr.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.008">http://dx.doi.org/10.7554/eLife.03896.008</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f006"/></fig></p></sec><sec id="s2-3"><title>Additional training can reinstate LTM after its apparent elimination</title><p>The present morphological results challenge the notion that the persistence of sensitization memory in <italic>Aplysia</italic> depends on the persistence of particular facilitated synapses. To further investigate this idea, we tested whether the LTM for behavioral sensitization can be reinstated in <italic>Aplysia</italic> following reconsolidation blockade and inhibition of PKM, two treatments previously shown to eliminate LTF, the synaptic basis of long-term sensitization (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>; <xref ref-type="bibr" rid="bib45">Lee et al., 2012</xref>; <xref ref-type="bibr" rid="bib37">Hu and Schacher, 2014</xref>).</p><p>Long-term sensitization (LTS) of the siphon-withdrawal reflex (SWR) was induced in intact <italic>Aplysia</italic> using five bouts of tails shocks (5XTrained). Brief reminder training (one bout of tail shocks, 1XTrained) was applied at 48 hr after the original training to trigger reconsolidation of the LTM for sensitization (<xref ref-type="bibr" rid="bib14">Cai et al., 2012</xref>). Immediately following the reminder training an intrahemocoel injection of anisomycin was administered to some animals (<xref ref-type="fig" rid="fig7">Figure 7A</xref>). As previously reported (<xref ref-type="bibr" rid="bib14">Cai et al., 2012</xref>; <xref ref-type="bibr" rid="bib45">Lee et al., 2012</xref>), this treatment eliminated LTS, assessed here at 72 hr (<xref ref-type="fig" rid="fig7">Figure 7B</xref>). Afterwards, some of the animals received three additional bouts of tail shocks (3XTrained). Importantly, the three additional bouts of sensitization training did not induce LTM in naïve animals (Control-Veh-3XTrained; <xref ref-type="fig" rid="fig7">Figure 7B</xref>). However, the three bouts of training completely restored LTM following its disruption by reconsolidation blockade (<xref ref-type="fig" rid="fig7">Figure 7B</xref>).<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.012</object-id><label>Figure 7.</label><caption><title>Reinstatement of LTS after its elimination by reconsolidation blockade in <italic>Aplysia</italic>.</title><p>(<bold>A</bold>) Experimental protocol. The timing of the pretests, training, posttests, and drug/vehicle injections is shown relative to the end of the last training session. The time of the intrahemocoel injection of either anisomycin or vehicle is indicated by the red arrow. Animals in the 5XTrained-1XTrained-Aniso and 5XTrained-1XTrained-Aniso-3XTrained groups received a reminder episode of sensitization training (one bout of tail shocks) immediately after the 48 hr posttest (black bar) and prior to the injection of anisomycin, whereas the animals in the 5XTrained-Aniso group did not. After the 72 hr posttest animals in the Control-Veh-3XTrained and 5XTrained-1X5HT-Aniso-3XTrained groups received truncated sensitization training (three bouts of tail shocks). (<bold>B</bold>) The mean duration of the SWR measured at 48 hr, 72 hr and 96 hr for the Control-Veh-3XTrained (<italic>n</italic> = 12), 5XTrained-Aniso (<italic>n</italic> = 4), 5XTrained-1XTrained-Aniso (<italic>n</italic> = 7), and 5XTrained-1XTrained-Aniso-3XTrained (<italic>n</italic> = 11) groups. A repeated-measures ANOVA indicated that there was a significant group × time interaction (F<sub>[9,90]</sub> = 51.0, p < 0.0001). Subsequent planned comparisons indicated that the overall differences among the four groups were highly significant on all of the posttests (48 hr, F<sub>[3,30]</sub> = 137.2, p < 0.0001; 72 hr, F<sub>[3,30]</sub> = 50.5, p < 0.0001; and 96 hr, F<sub>[3,30]</sub> = 43.8, p < 0.0001). SNK posthoc tests on the 48 hr data indicated that the initial sensitization training produced significant LTS in the 5XTrained-Aniso (53.0 ± 7.0 s), 5XTrained-1XTrained-Aniso (59.9 ± 0.2 s), and 5XTrained-1XTrained-Aniso-3XTrained (53.7 ± 2.9 s) groups compared to the Control-Veh-3XTrained group (2.5 ± 0.9 s, p < 0.001 for each test). The responses of the trained groups did not differ significantly at 48 hr after sensitization training. However, the mean duration of the SWR in the 5XTrained-Aniso group (53.8 ± 6.3 s) remained prolonged at 72 hr, and was significantly longer than that in the 5XTrained-1XTrained-Aniso group (6.4 ± 1.9 s) as well as in the 5XTrained-1XTrained-Aniso-3XTrained group (4.8 ± 3.2 s, p < 0.001 for both comparisons). LTS was restored by the three additional tail shocks applied after the 72 hr posttest. The mean duration of the SWR in the 5XTrained-1XTrained-Aniso-3XTrained group at 96 hr was 56.7 ± 2.7 s, which was significantly longer than that for the 5XTrained-1XTrained-Aniso group (10.4 ± 3.9 s) at 96 hr (p < 0.001). The SWR of the 5XTrained-Aniso group at 96 hr (53.8 ± 3.8 s) was also significantly longer than that in the 5XTrained-1XTrained-Aniso group (p < 0.001). Asterisks, comparisons of the Control-Veh-3XTrained group with the 5XTrained-Aniso group, the 5XTrained-1XTrained-Aniso group, and 5XTrained-1XTrained-Aniso-3XTrained group at 48 hr; comparison of the Control-Veh-3XTrained group with the 5XTrained-Aniso group at 72 hr; and comparisons of the Control-Veh-3XTrained group with the 5XTrained-Aniso group and the 5XTrained-1XTrained-Aniso-3XTrained group at 96 hr. Plus signs, comparisons of the 5XTrained-1XTrained-Aniso group with the 5XTrained-Aniso group and the 5XTrained-1XTrained-Aniso-3XTrained group at 96 hr. Error bars represent ±SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.012">http://dx.doi.org/10.7554/eLife.03896.012</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f007"/></fig></p><p>To test whether LTS could be reinstated after its apparent erasure by inhibition of PKM Apl III, animals were again initially trained using five bouts of tail shocks. 24 hr after the original sensitization training the animals received an intrahemocoel injection of chelerythrine (<xref ref-type="fig" rid="fig8">Figure 8A</xref>). Animals treated with chelerythrine exhibited no LTS at 48 hr (<xref ref-type="fig" rid="fig8">Figure 8B</xref>), confirming our previous finding (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>). The animals that received the original sensitization training were then given three additional bouts of tail shocks (5XTrained-Chel-3XTrained group), as were control animals that had not received the original sensitization training (Control-Veh-3XTrained group). The modest additional training reinstated LTS following its apparent erasure by chelerythrine, but did not produce LTS in the control animals (<xref ref-type="fig" rid="fig8">Figure 8B</xref>).<fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.013</object-id><label>Figure 8.</label><caption><title>Reinstatement of LTS following its erasure by PKM inhibition.</title><p>(<bold>A</bold>) Experimental protocol. The timing of the pretests, training, posttests, and drug/vehicle injections is shown relative to the end of the last training session. The time of the intrahemocoel injection of either chelerythrine or vehicle is indicated by the red arrow. After the 48 hr posttest animals in both groups received three additional bouts of tail shocks. (<bold>B</bold>) Mean duration of the SWR measured at 24 hr, 48 hr and 72 hr for the Control-Veh-3XTrained (<italic>n</italic> = 9) and 5XTrained-Chel-3XTrained (<italic>n</italic> = 9) groups. A repeated-measures ANOVA indicated that there was a significant interaction effect between group and time (F<sub>[3,14]</sub> = 25.4. p < 0.0001). A planned comparison indicated that the mean duration of the SWR at 24 hr in the 5XTrained-Chel-3XTrained group (44.7 ± 5.6 s) was significantly longer than that in the Control-Veh-3XTrained group (1.1 ± 0.1 s, F<sub>[1,16]</sub> = 60.2, p < 0.001). The mean duration of the SWR at 48 hr (4.1 ± 1.4 s) in the 5XTrained-Chel-3XTrained group was not significantly longer than that in the Control-Veh-3XTrained group (2.6 ± 0.8 s), as indicated by a planned comparison (F<sub>[1,16]</sub> = 0.95, p = 0.34). The SWR in the 5XTrained-Chel-3XTrained group at 72 hr was 48.7 ± 5.0 s, which was significantly longer than that in the Control-Veh-3XTrained group (4.6 ± 1.2 s; planned comparison, F<sub>[1,16]</sub> = 73.1, p < 0.001). Asterisks, comparison between 5XTrained-Chel-3XTrained and Control-Veh-3XTrained groups. Error bars represent ±SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.013">http://dx.doi.org/10.7554/eLife.03896.013</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f008"/></fig></p></sec><sec id="s2-4"><title>Role of histone deacetylase in the maintenance and induction of LTM</title><p>The above behavioral results indicate that LTM in <italic>Aplysia</italic> can persist despite elimination of its behavioral expression by reconsolidation blockade and inhibition of PKM Apl III. Moreover, because these two antimnemonic treatments also eliminate LTF (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>; <xref ref-type="bibr" rid="bib45">Lee et al., 2012</xref>; <xref ref-type="bibr" rid="bib37">Hu and Schacher, 2014</xref>) (and <xref ref-type="fig" rid="fig1 fig4">Figures 1C and 4B</xref>), the results imply that some component of LTM, perhaps a priming process, may persist in the absence of synaptic alterations. Possibly, LTM, or the primer for LTM, resides in the nuclei of neurons within the SWR circuitry, encoded as epigenetic changes (<xref ref-type="bibr" rid="bib46">Levenson and Sweatt, 2005</xref>; <xref ref-type="bibr" rid="bib62">Rahn et al., 2013</xref>; <xref ref-type="bibr" rid="bib29">Graff and Tsai, 2013a</xref>). To investigate this possibility, we tested whether chelerythrine's apparent disruption of LTM involves alterations of chromatin structure. For this test we used the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (<xref ref-type="bibr" rid="bib30">Graff and Tsai, 2013b</xref>). <italic>Aplysia</italic> were given five bouts of tail shocks, and the strength of the SWR was tested 24 hr later (<xref ref-type="fig" rid="fig9">Figure 9A</xref>). Immediately after this test the sensitization-trained animals received an intrahemocoel injection of the vehicle solution alone or TSA; 10–15 min later some of the animals treated with TSA were given an injection of chelerythrine (5XTrained-TSA-Chel group). (Untrained control animals received an injection of the vehicle solution after the 24 hr test.) In addition, another group of trained animals were given the chelerythrine without a prior injection of TSA (5XTrained-Chel group). All animals were retested at 48 hr. TSA treatment blocked the disruption of LTS by chelerythrine (<xref ref-type="fig" rid="fig9">Figure 9B</xref>). The injection of TSA 24 hr after training by itself did not alter LTS (5XTrained-TSA group). These results indicate that one of chelerythrine's antimnemonic actions is to reverse histone acetylation induced by LTS training.<fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.03896.014</object-id><label>Figure 9.</label><caption><title>Epigenetic regulation of LTM in <italic>Aplysia</italic>.</title><p>(<bold>A</bold>) Experimental protocol. The times of the pretests, training, posttests, and drug/vehicle injections are shown relative to the end of the last training session. The times of the trichostatin A (TSA)/vehicle and chelerythrine/vehicle injections are indicated by blue arrow and red arrow respectively. (<bold>B</bold>) TSA treatment blocks erasure of LTS by chelerythrine. The graph presents the mean duration of the SWR measured at 24 hr and 48 hr in the Control-Veh (<italic>n</italic> = 9), 5XTrained-Veh (<italic>n</italic> = 5), 5XTrained-TSA (<italic>n</italic> = 6), 5XTrained-Chel (<italic>n</italic> = 5), and 5XTrained-TSA-Chel (<italic>n</italic> = 6) groups. A repeated-measures ANOVA showed a significant group × time interaction (F<sub>(8,52)</sub> = 53.2, p < 0.0001). Subsequent planned comparisons indicated that the group differences for the 24 and 48 hr posttests were highly significant (24 hr, F<sub>[4,26]</sub> = 45.5, p < 0.0001; 48 hr, F<sub>[4,26]</sub> = 94.4, p < 0.0001). For the 24 hr posttest, SNK posthoc tests indicated that the training produced significant sensitization in all four trained groups (5XTrained-Veh, 54.0 ± 6.0 s; 5XTrained-TSA, 49.0 ± 7.6 s; 5XTrained-Chel, 59.6 ± 0.4 s; and 5XTrained-TSA-Chel, 57.7 ± 2.3 s) compared to the Control-Veh group (1.6 ± 0.6 s, p < 0.001 for all tests). Comparisons of the four trained groups showed that their responses did not differ significantly on the 24 hr posttest. However, the responses of 5XTrained-Veh (49.0 ± 6.9 s), 5XTrained-TSA (56.0 ± 4.0 s), and 5XTrained-TSA-Chel (59.8 ± 1.7 s) on the 48 hr posttest were significantly more enhanced than those for both the 5XTrained-Chel (2.8 ± 1.8 s) and Control-Veh (1.7 ± 0.5 s) groups (p < 0.001 for all tests). Thus, chelerythrine treatment reversed LTS, and TSA treatment blocked chelerythrine's reversal of LTS. There were no significant differences among 5XTrained-Veh, 5XTrained-TSA and 5XTrained-TSA-Chel groups, nor between 5XTrained-Chel and Control-Veh groups, at 48 hr. Asterisks, comparisons of the Control-Veh group with the 5XTrained-Veh group, the 5XTrained-TSA group, the 5XTrained-Chel group, and the 5XTrained-TSA-Chel group at 24 hr; and of the Control-Veh group with the 5XTrained-Veh group, the 5XTrained-TSA group, and the 5XTrained-TSA-Chel group at 48 hr. Plus signs, comparisons of the 5XTrained-Chel group with the 5XTrained-Veh group, the 5XTrained-TSA group, and the 5XTrained-TSA-Chel group at 48 hr. (<bold>C</bold>) Experimental protocols. The times of the pretests, training, posttests, and drug/vehicle injections are shown relative to the end of the last training session. The intrahemocoel injection of either TSA or vehicle is indicated by the red arrow. Note that TSA was injected into animals prior to the sensitization training. (<bold>D</bold>) Inhibiting histone deacetylation facilitated the induction of LTS in Aplysia. The mean duration of the SWR in the 3XTrained-TSA group (<italic>n</italic> = 4) at 24 hr was 59.8 ± 0.3 s, whereas it was only 2.0 ± 1.0 s in the 3XTrained-Veh group (<italic>n</italic> = 4; p < 0.001, unpaired <italic>t</italic> test). Asterisks, comparison between 3XTrained-TSA and 3XTrained-Veh groups; Error bars represent ±SEM.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.014">http://dx.doi.org/10.7554/eLife.03896.014</ext-link></p></caption><graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="elife03896f009"/></fig></p><p>Previous work has shown that LTF of in vitro <italic>Aplysia</italic> sensorimotor connections depends on alterations of chromatin structure. In particular, 1X5HT, which normally induces only short-term facilitation, was found to induce LTF when applied with TSA (<xref ref-type="bibr" rid="bib32">Guan et al., 2002</xref>). In intact <italic>Aplysia</italic> three bouts of tail shocks are insufficient by themselves to induce LTS (Control-Veh-3XTrained data, <xref ref-type="fig" rid="fig7 fig8">Figures 7 and 8</xref>). We tested whether three bouts of shocks could induce LTS if delivered in the presence of an HDAC inhibitor. Animals were given three bouts of shocks 15 min after an intrahemocoel injection of either vehicle solution or TSA (<xref ref-type="fig" rid="fig9">Figure 9C</xref>). 24 hr later the animals that received three bouts of shocks after an injection of TSA exhibited LTS, whereas animals that received the shocks after an injection of vehicle did not (<xref ref-type="fig" rid="fig9">Figure 9D</xref>). Thus, HDAC inhibition facilitates the induction of LTM in <italic>Aplysia</italic>.</p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>The present morphological results, together with those of our previous behavioral and electrophysiological investigations (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>), suggest that the persistence of sensitization-related LTM in <italic>Aplysia</italic> does not require the persistence of the synaptic connections generated during learning. Rather, LTM appears to be regulated by a homeostatic mechanism that specifies the net synaptic strength according to experience. Following 5X5HT stimulation, we observed that the number of presynaptic varicosities in the sensorimotor cocultures increased to a new overall value, and that this value was maintained despite the appearance and disappearance of individual varicosities. Furthermore, blockade of memory reconsolidation, or inhibition of PKM, reset the number of varicosities to the original, non-facilitated value. Remarkably, this resetting did not result from the straightforward retraction of the varicosities that appeared by 24 hr after 5X5HT stimulation, varicosities whose growth coincides with LTF (<xref ref-type="bibr" rid="bib25">Glanzman et al., 1990</xref>); rather, the resetting was produced by a complex reorganization that involved retraction of both 5HT-induced and original varicosities, as well as growth of new, additional varicosities. Thus, for any given sensorimotor connection, the homeostatic regulatory mechanism appeared indifferent to the identities of the particular presynaptic varicosities and, presumably, active zones (<xref ref-type="bibr" rid="bib67">Schacher et al., 1990</xref>), that mediated baseline synaptic transmission and synaptic facilitation. Our data argue that synapses are not ‘tagged’ with respect to memory storage, at least for a connection involving a single SN and a single MN; the data may therefore reflect a fundamental distinction between the cellular and molecular processes of LTM storage and those of LTM induction in <italic>Aplysia</italic>, in which synaptic tagging plays a critical role (<xref ref-type="bibr" rid="bib52">Martin et al., 1997</xref>). The data also suggest that synaptic change is an expression mechanism, rather than a storage mechanism, for LTM in <italic>Aplysia</italic> (although see below).</p><p>Importantly, neither reconsolidation blockade nor inhibition of PKM caused the varicosity number to drop below the starting value. (Notice that the same is true for the amplitude of the EPSP in our earlier synaptic physiological studies, which used the same training regimens [<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>].) This result provides additional evidence for the operation of a homeostatic regulatory mechanism, and suggests that the homeostatic mechanism toggles between two all-or-none states, a facilitated, sensitization-related state and a nonfacilitated one. (There must also be a third state, synaptic depression, in which the strength of the sensorimotor connection is reduced below the nonfacilitated level [<xref ref-type="bibr" rid="bib57">Montarolo et al., 1988</xref>].) Our results are reminiscent of those from studies in the <italic>Drosophila</italic> neuromuscular junction and the mammalian CNS, which also indicate that synaptic plasticity is regulated by homeostatic mechanisms (<xref ref-type="bibr" rid="bib73">Turrigiano, 2007</xref>).</p><p>The present conclusions appear to conflict with those from morphological investigations of LTM in mice in which dendrites have been chronically imaged in the intact, living brain. Several such studies have reported stability of some dendritic spines for periods of weeks-to-months after training (<xref ref-type="bibr" rid="bib78">Xu et al., 2009</xref>; <xref ref-type="bibr" rid="bib79">Yang et al., 2009</xref>; <xref ref-type="bibr" rid="bib48">Liston et al., 2013</xref>). These results, as well as evidence that spines in the adult mammalian cortex exhibit little turnover (<xref ref-type="bibr" rid="bib31">Grutzendler et al., 2002</xref>), have led some to argue that stable spines provide a physical basis for the lifelong maintenance of memories (<xref ref-type="bibr" rid="bib8">Bhatt et al., 2009</xref>). But other studies of the mouse brain using very similar in vivo imaging techniques have reported a high degree of spine instability in the adult cerebral cortex (<xref ref-type="bibr" rid="bib72">Trachtenberg et al., 2002</xref>; <xref ref-type="bibr" rid="bib36">Holtmaat et al., 2005</xref>); the contradiction between these two sets of opposed findings remains unresolved. Furthermore, even in those studies where spines of adult cortical neurons were reported to be highly stable, there was significant instability of both pre-existing and new spines within the first 2–4 days after a training protocol (<xref ref-type="bibr" rid="bib78">Xu et al., 2009</xref>; <xref ref-type="bibr" rid="bib79">Yang et al., 2009</xref>), and we monitored SN varicosities for only 48 hr after training with 5HT. Therefore, the present results are not necessarily inconsistent with those from in vivo imaging studies in the mouse brain.</p><p>We found that LTS could be fully reinstated following its disruption by the same antimnemonic treatments previously shown to eliminate LTF (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>; <xref ref-type="bibr" rid="bib45">Lee et al., 2012</xref>; <xref ref-type="bibr" rid="bib37">Hu and Schacher, 2014</xref>) (<xref ref-type="fig" rid="fig7 fig8">Figures 7 and 8</xref>). This finding is consistent with two possible explanations. First, the LTM for sensitization may be intact in animals following reconsolidation blockade and inhibition of PKM, despite the elimination of both behavioral enhancement and synaptic growth. According to this scheme, synapses serve merely to express LTM, they are not sites of LTM storage. The reinstatement of LTM expression, then, involves restoring the appropriate number of synapses between the SNs and MNs, as determined by an experience-registering homeostatic mechanism. The second possible explanation incorporates the notion of an as-yet-unidentified priming mechanism; here, reconsolidation blockade and inhibition of PKM, can erase the stored sensitization memory through the reversal of pre- and postsynaptic structures induced by the long-term training, but the antimnemonic treatments do not eliminate the primer. The primer does not constitute LTM, but is required for its reconstitution via new synaptic growth. The priming signal might interact with fresh facilitatory input to the withdrawal circuit, due to the additional (3×) tail shocks, to upregulate the number of synaptic contacts; the appropriate number of contacts could be determined by the homeostatic process, and involve signals from existing and new synapses.</p><p>In a previous study we attempted to reinstate LTS following its disruption by inhibition of PKM Apl III but failed (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>) (see <xref ref-type="fig" rid="fig3">Figure 3B</xref> of the earlier study). Interestingly, we used only a single bout of tail shocks in that attempt. In light of the present results (<xref ref-type="fig" rid="fig7 fig8">Figures 7 and 8</xref>), our previous failure indicates that one bout of tails shocks is insufficient to fully recruit the signaling pathways that reinstate LTS (<xref ref-type="bibr" rid="bib1">Abel et al., 1998</xref>; <xref ref-type="bibr" rid="bib70">Sossin, 2008</xref>; <xref ref-type="bibr" rid="bib53">Mayford et al., 2012</xref>; <xref ref-type="bibr" rid="bib82">Zhang et al., 2012</xref>). In future work we will seek to discover how the molecular signals evoked by three bouts of tail shocks differ from those evoked by a single bout.</p><p>The data from our behavioral experiments involving the use of an HDAC inhibitor (<xref ref-type="fig" rid="fig9">Figure 9</xref>) implicate epigenetic modifications (<xref ref-type="bibr" rid="bib46">Levenson and Sweatt 2005</xref>; <xref ref-type="bibr" rid="bib62">Rahn et al., 2013</xref>; <xref ref-type="bibr" rid="bib29">Graff and Tsai 2013a</xref>)—either in the SN or MN or, most likely, both—in the storage mechanism for LTM in <italic>Aplysia</italic>. Data from an in vitro study of LTF have also implicated histone acetylation in LTM in <italic>Aplysia</italic> (<xref ref-type="bibr" rid="bib32">Guan et al., 2002</xref>). How might HDAC inhibition block chelerythrine's apparent disruption of LTM? Possibly, TSA, by inhibiting histone deacetylase, activates the transcription of genes that promote LTM; the activation of these genes, in turn, may compensate for a disruptive action of chelerythrine on LTM. Findings by others are consistent with this idea. Thus, treatment with an HDAC inhibitor rescues cognitive deficits and learning-related synaptic plasticity in mice that are heterozygous for a mutant form of CREB binding protein (CBP) (<xref ref-type="bibr" rid="bib2">Alarcon et al., 2004</xref>). In addition, it has been found that phosphorylation of CBP by the atypical PKCζ is required for the histone acetylation that promotes the differentiation of developing neural precursors into neurons, astrocytes, and oligodendrocytes; mutant mice haploinsufficient for CBP exhibit abnormal development of the cerebral cortex (<xref ref-type="bibr" rid="bib77">Wang et al., 2010</xref>). Furthermore, chelerythrine, by inhibiting the phosphorylation of CBP by PKCζ, blocks the differentiation of cultured precursor cells into astrocytes, and oligodendrocytes (<xref ref-type="bibr" rid="bib77">Wang et al., 2010</xref>). A recent study in <italic>Aplysia</italic> has found that reducing the activity of CBP in SNs through intracellular injection of CBP small interfering RNA (siRNA) impaired the induction of LTF in sensorimotor cocultures (<xref ref-type="bibr" rid="bib50">Liu et al., 2013</xref>). Interestingly, this deficit in LTF induction could be rescued through the use of a 5HT training protocol designed to maximize the phosphorylation and synthesis of CCAAT/enhancer-binding protein (C/EBP). Taken together, these studies suggest that chelerythrine can disrupt epigenetic processes, possibly involving histone acetylation stimulated by CBP, that promote the induction and maintenance of LTM. Moreover, because HDAC inhibition can block chelerythrine's deleterious effects on memory, as well as promote the induction of LTM, it is intriguing to speculate that these effects might be due to enhancement of a CBP-dependent pathway.</p><p>But the above explanation for why TSA blocks chelerythrine's effect on consolidated LTM raises a critical question. Our data suggest that consolidated LTM persists after chelerythrine treatment (as well as reconsolidation blockade), possibly as nuclear changes. If so, then the molecular basis for the persistence of LTM is unlikely to be histone acetylation, because an apparent action of chelerythrine is to reverse histone acetylation. Another prominent epigenetic mechanism known to play a role in LTM is DNA methylation (<xref ref-type="bibr" rid="bib19">Day and Sweatt, 2010</xref>). Although early evidence indicated that learning-induced DNA methylation in the hippocampus was transient and readily reversible (<xref ref-type="bibr" rid="bib55">Miller and Sweatt, 2007</xref>), a more recent study has reported that contextual fear conditioning in rats induces DNA methylation of the gene for calcineurin in cortical neurons that persists for at least a month (<xref ref-type="bibr" rid="bib54">Miller et al., 2010</xref>). Furthermore, Sweatt and colleagues have shown in a rat model of childhood maltreatment that early trauma can produce changes in the DNA methylation of the gene for brain-derived neurotrophic factor (BDNF) in the cortex, changes that persist into adulthood (<xref ref-type="bibr" rid="bib65">Roth et al., 2009</xref>). Thus, DNA methylation may constitute an epigenetic mechanism for the lifelong storage of memory (<xref ref-type="bibr" rid="bib19">Day and Sweatt, 2010</xref>). Interestingly, 5HT has recently been reported to induce DNA methylation of the promoter of the transcriptional repressor of memory CREB2, thereby facilitating the induction of LTF (<xref ref-type="bibr" rid="bib63">Rajasethupathy et al., 2012</xref>). Finally, another potential candidate for a nuclear mechanism of LTM storage is suggested by a recent study by <xref ref-type="bibr" rid="bib71">Suberbielle et al. (2013)</xref> that found, remarkably, that learning causes DNA double-strand breaks (DSBs) in neurons in the brains of control mice; thus, chromatin remodeling subsequent to DNA DSBs may also encode LTM.</p><p>Could a nonsynaptic storage mechanism based on nuclear changes mediate the maintenance of associative memories, particularly those induced in complex neural circuits in the mammalian brain, where a given neuron may have 1000s or 10s of 1000s of synaptic partners? An obvious difficulty confronting any hypothetical nuclear storage mechanism in the mammalian brain is how the appropriate number of connections can be maintained in a synapse-specific manner after learning has occurred. For example, if a synaptic contact that has undergone Hebbian long-term potentiation (<xref ref-type="bibr" rid="bib9">Bliss and Collingridge, 1993</xref>) as a consequence of associative learning retracts, how could a nuclear storage mechanism restrict the growth of a replacement contact to the correct pair of pre- and postsynaptic neurons? Possibly, there are nonsynaptic ways for neurons to communicate that ensure specificity of associative synaptic plasticity in the face of the significant lability of synaptic structure documented here.</p><p>There are, of course, somal, non-nuclear, mechanisms of memory storage or priming that could account for the apparent persistence of memory following synaptic erasure. One such mechanism would be the persistent activity of a kinase (<xref ref-type="bibr" rid="bib33">Hegde et al., 1993</xref>; <xref ref-type="bibr" rid="bib10">Bougie et al., 2012</xref>), or persistent inhibition of a phosphatase (<xref ref-type="bibr" rid="bib68">Sharma et al., 2003</xref>), in the cell body of the SN or MN or in both cell bodies. Another somal mechanism consistent with our data is ubiquitination of one or more somal proteins (<xref ref-type="bibr" rid="bib34">Hegde et al., 1997</xref>; <xref ref-type="bibr" rid="bib17">Chain et al., 1999</xref>); up-regulation of a ubiquitin-proteasome pathway could degrade proteins that inhibit the storage of LTM, or that block the priming of LTM.</p><p>The present data necessitate a reappraisal of the mnemonic consequences of blockade of memory reconsolidation and inhibition of PKM. It has been previously argued that these manipulations can erase consolidated memory (<xref ref-type="bibr" rid="bib58">Nader and Hardt, 2009</xref>; <xref ref-type="bibr" rid="bib66">Sacktor, 2011</xref>; <xref ref-type="bibr" rid="bib24">Glanzman, 2013</xref>) (although see <xref ref-type="bibr" rid="bib43">Lattal and Abel, 2004</xref>). But our results indicate that the effect of reconsolidation blockade and PKM inhibition is not to delete LTM but, rather, to impair its expression. In other words, the antimnemonic actions of these manipulations may result from their ability to at least partially reverse the cellular and molecular changes, including synaptic growth, that mediate the expression, rather than the storage, of LTM. Possibly, neither reconsolidation blockade nor PKM inhibition can reverse the nuclear remodeling—involving epigenetic modifications, particularly DNA methylation (<xref ref-type="bibr" rid="bib19">Day and Sweatt, 2010</xref>; <xref ref-type="bibr" rid="bib28">Graff et al., 2011</xref>; <xref ref-type="bibr" rid="bib62">Rahn et al., 2013</xref>), as well as, perhaps, chromatin remodeling subsequent to DNA DSBs (<xref ref-type="bibr" rid="bib71">Suberbielle et al., 2013</xref>)—that represents the stored memory trace. Alternatively, these antimnemonic manipulations might indeed disrupt the stored memory trace, but leave intact some memory-priming signal. Likely candidates for a residual priming signal include the persistent repression of transcription of the CREB repressor, CREB2 (<xref ref-type="bibr" rid="bib7">Bartsch et al., 1995</xref>; <xref ref-type="bibr" rid="bib74">Upadhya et al., 2004</xref>; <xref ref-type="bibr" rid="bib49">Liu et al., 2011</xref>), and persistent degradation of the regulatory subunit of protein kinase A (PKA) (<xref ref-type="bibr" rid="bib17">Chain et al., 1999</xref>).</p><p>Because chelerythrine's disruption of LTM in <italic>Aplysia</italic> is blocked by TSA, chelerythrine's antimnemonic actions must involve histone deacetylation. Previous speculation regarding how chelerythrine and the zeta inhibitory peptide (ZIP) disrupt memory maintenance in both <italic>Aplysia</italic> and rats has focused on the ability of these pharmacological agents to inhibit the phosphorylation of proteins by atypical PKM (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>; <xref ref-type="bibr" rid="bib66">Sacktor, 2011</xref>) (<xref ref-type="boxed-text" rid="box1">Box 1</xref>). The present results imply that chelerythrine and ZIP may interfere with epigenetic processes induced by atypical PKM, as well as inhibit protein phosphorylation by atypical PKM. A prior study reported that, following inhibition of PKMζ activity in the insular cortex of rats by the peptide inhibitor ZIP, conditioned taste aversion could not be reinstated by an application of the unconditioned stimulus (UCS, intraperitoneal injection of lithium chloride) (<xref ref-type="bibr" rid="bib69">Shema et al., 2007</xref>). Our experience (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, and the present results) suggests that the failure to reinstate conditioned taste aversion in this study was not because LTM had been extinguished, but because a single application of the UCS was too weak to reverse the disruptive effects of ZIP on mechanisms of LTM expression, some of which may involve chromatin remodeling (<xref ref-type="fig" rid="fig7">Figure 7A,B</xref>).</p><boxed-text id="box1"><object-id pub-id-type="doi">10.7554/eLife.03896.015</object-id><label>Box 1.</label><caption><title>Recent studies of transgenic mice have reported that learning and memory, as well as long-term synaptic plasticity in the hippocampus, are normal in animals lacking PKMζ, the mammalian homolog of PKC Apl III (<xref ref-type="bibr" rid="bib44">Lee et al., 2013</xref>; <xref ref-type="bibr" rid="bib76">Volk et al., 2013</xref>).</title><p>Although these results raise questions about the necessity of PKMζ for maintaining long-term memory in the mammalian brain, as well as the specificity of the peptide inhibitor (ZIP) commonly used to block the activity of PKMζ in mammalian studies (<xref ref-type="bibr" rid="bib47">Ling et al., 2002</xref>; <xref ref-type="bibr" rid="bib60">Pastalkova et al., 2006</xref>; <xref ref-type="bibr" rid="bib69">Shema et al., 2007</xref>), we do not believe they impact the present conclusions. Biochemical tests performed in <italic>Aplysia</italic> have established that chelerythrine, at the concentration used in our experiments, is a selective and effective inhibitor of PKM (<xref ref-type="bibr" rid="bib75">Villareal et al., 2009</xref>). Furthermore, 5HT has been shown to activate PKM Apl III via calpain dependent cleavage of the atypical <italic>Aplysia</italic> PKC, PKC Apl III (<xref ref-type="bibr" rid="bib10">Bougie et al., 2012</xref>). A possible explanation for the results of the PKMζ knockout mice (<xref ref-type="bibr" rid="bib44">Lee et al., 2013</xref>; <xref ref-type="bibr" rid="bib76">Volk et al., 2013</xref>), which lack the gene for the atypical PKCζ (note that in the mammalian CNS PKMζ is not formed from proteolytic cleavage of the atypical PKC, but, rather, is transcribed from an alternate promoter within the PKCζ gene [<xref ref-type="bibr" rid="bib35">Hernandez et al., 2003</xref>]) is that, in the absence of PKMζ, a second atypical mammalian PKC isoform, PKCι/λ—or its PKM fragment, PKMι/λ—can assume the mnemonic functions of PKMζ (<xref ref-type="bibr" rid="bib64">Ren et al., 2013</xref>). But there is only one atypical PKC isoform in <italic>Aplysia;</italic> we therefore believe that chelerythrine's inhibitory effect in the present experiments was selective for PKM Apl III.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03896.015">http://dx.doi.org/10.7554/eLife.03896.015</ext-link></p></caption></boxed-text><p>In summary, we have found that LTM, or a primer for LTM, can persist following reconsolidation blockade and inhibition of PKM. Furthermore, the residual memory/primer must be independent of synaptic plasticity, because it persists following elimination of the synaptic changes induced during learning. Our results indicate that consolidated memories may be far more refractory to modification or elimination than generally supposed. If confirmed and extended to mammals, the present results would have important implications for treating disorders of LTM, such as posttraumatic stress disorder (PTSD).</p></sec><sec sec-type="materials|methods" id="s4"><title>Materials and methods</title><sec id="s4-1"><title>Cell culture and dye injection</title><p>The sensorimotor cocultures consisted of one pleural SN and one small siphon (LFS-type) MN. Adult abdominal ganglia and pleural ganglia were removed from 60–100 g <italic>Aplysia</italic> and then incubated in protease (10 mg/ml Dispase II [Roche Applied Science, Indianapolis, IN] in Leibowitz-15 [L-15, Sigma, St Louis, MO]) for 2 hr at 35°C before desheathing. The appropriate amounts of salts were added to the L15 to yield the following concentrations in mM: 400 NaCl, 11 CaCl<sub>2</sub>, 10 KCl, 27 MgSO<sub>4</sub>, 27 MgCl<sub>2</sub>, 2 NaHCO<sub>3</sub>. Additionally, the L15 was supplemented with penicillin (50 unit/ml), streptomycin (50 µg/ml), dextrose (6 mg/ml) and glutamine (0.1 mg/ml). After desheathing, SNs and MNs were individually dissociated from ganglia and paired in cell culture. The culture medium contained 50% <italic>Aplysia</italic> hemolymph and 50% L-15. The cultures were maintained at 18°C in an incubator for 3 day before the start of the experiments. The SNs and MNs were labeled with the intracellular dyes dextran fluorescein and dextran rhodamine B (Molecular Probes, Eugene, OR), respectively, on Day 3/4 in culture. The dyes were dissolved in 0.2 M KCl with 0.25% fast green (final concentration of 10 mg/ml) and then microinjected into cells via brief pressure pulses (6–12 MΩ resistance electrodes, 10–20 psi for 2–5 pulses [40 ms]).</p></sec><sec id="s4-2"><title>Cell imaging and quantification of structural changes</title><p>The fluorescent images of the labeled cocultures were acquired with a LSM Pascal (Zeiss, Thornwood, NY) confocal microscope during three imaging sessions: immediately prior to, and 24 hr and 48 hr after, 5X5HT training (or at the equivalent times for Control cocultures). The images were taken using a 20×, 0.5 NA objective. The total number of varicosities was counted for each sensory neuron from the confocal images. All image analyses were performed using Axiovision 4.8.2 (Zeiss, Thornwood, NY). The counter was blind to the experimental conditions of the imaged cocultures. SN varicosities that were in clear contact with, or overlapping, postsynaptic structures (either the MN soma, major neurite, or fine processes) were counted. The majority of the SN varicosities contacted either the MN soma or its initial segment. Only those varicosities having a punctate shape—those in which an oval-shaped body could be distinguished by the narrowing of the neurite on either side—and a measured area of 10 µm<sup>2</sup> or greater were counted. If a large fluorescent varicosity comprised several visible punctate varicosities in the image, those small varicosities were counted individually; if not, the structure was treated as a single varicosity. Three categories of varicosities are tracked and quantified: original varicosities, that is the varicosities present at 0 hr; varicosities formed during 0–24 hr; and varicosities formed during 24–48 hr.</p></sec><sec id="s4-3"><title>Drug treatments</title><p>Immediately after the first imaging session, some of the cocultures were given 5HT training. 5HT was prepared fresh daily as a 10 mM stock solution in artificial sea water (ASW) and then diluted to the final concentration of 100 µM in the perfusion medium immediately before the first application. The perfusion solution contained 50% L15 and 50% ASW. The 5HT training consisted of five 5 min pulses of 5HT. After each 5 min pulse, the 5HT was rapidly washed out with normal perfusion medium for 15 min. The Control cocultures were treated with the perfusion solution alone. Following 5HT or control treatment, the perfusion medium was replaced with culture medium and the cocultures were returned to the 18°C incubator. A stock solution of anisomycin (Sigma, St Louis, MO) was made by dissolving the protein synthesis inhibitor in dimethyl sulfoxide (DMSO) to a concentration of 40 mM for use in the reconsolidation experiments. After the second imaging session (24 hr after 5HT training or the equivalent time in Control experiments) the anisomycin stock solution was diluted to a concentration of 10 µM in perfusion solution and applied to cocultures for 2 hr. Immediately prior to the anisomycin treatment, some cocultures were given one 5-min pulse of 5HT (100 µM) as the reminder stimulus. The 5HT-containing perfusion medium was washed out with normal perfusion medium in the coclutures that received the reminder. To inhibit PKM Apl III chelerythrine (EMD Biosciences, San Diego, CA) was dissolved in dH<sub>2</sub>O to a concentration of 10 mM to make a stock solution. After the second imaging session 24 hr after 5HT training, the chelerythrine stock solution was diluted to a concentration of 10 µM in perfusion solution and applied to cocultures for 1 hr. Following the anisomycin or chelerythrine treatment, the drug was rapidly washed out of the coculture dishes with normal perfusion medium; afterwards the perfusion medium was replaced with culture medium and the cocultures were returned to the incubator.</p></sec><sec id="s4-4"><title>Behavioral experiments</title><p>Adult <italic>A. californica</italic> (80–120 g) were obtained from a local supplier (Alacrity Marine Biological, Redondo Beach, CA, USA). Animals were housed in a 50 gal aquarium filled with cooled (12–14°C), aerated seawater (Catalina Water Company, Long Beach, CA, USA). The behavioral training and testing methods were similar to those previously described (<xref ref-type="bibr" rid="bib22">Fulton et al., 2008</xref>; <xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>). Three pretests were performed at once per 10 min, beginning 25 min before the start of training. Full sensitization training consisted of five bouts of electrical shocks delivered to the tail at 20-min intervals. During each bout, the animal received three trains of shocks spaced 2 s apart. Each train was 1 s in duration; the shocks (10-ms pulse duration, 40 Hz, 120 V) were delivered via a Grass stimulator (S88, Astro-Med, West Warwick, RI) connected to platinum wires implanted in the tail. After training the animals were given posttests as indicated in the figures.</p><p>A stock solution of 40 mM anisomycin or 10 mM chelerythrine was prepared as for the cell imaging experiments (above). Anisomycin was then diluted in ASW to a concentration of 20 mM (50% DMSO). 200 μl per 100 g of body weight of anisomycin was injected into the animals. Injections of the same amount of vehicle solution (DMSO in ASW) were made in Control experiments. The final concentrations of anisomycin in the animal were approximately 40 μM. The final concentration of DMSO in the hemocoel was ∼0.1%. A volume of 200 μl per 100 g of body weight of chelerythrine was injected into the animals. Trichostatin A (TSA) (Sigma, St Louis, MO) was dissolved in DMSO to a concentration of 10 mM to make a stock solution. To inhibit the histone deacetylase, a volume of 100 μl per 100 g of body weight of TSA was injected into the animals. The specific times at which the intrahemocoel injections were made are indicated in the relevant figures.</p></sec><sec id="s4-5"><title>Statistical analysis</title><p>All statistical tests were performed using SPSS 22.0 (IBM, Armonk, NY). Parametric tests were used for all statistical analyses. For each coculture the number of varicosities counted at 24 hr and 48 hr after 5HT training were normalized as described in the figure legends. The normalized data were expressed as means ±SEM. For the analysis of the data involving monitoring of synapses/behavior over time, the overall data were first assessed with repeated-measures two-way analyses of variance (ANOVA). If the repeated-measures ANOVA indicated a significant interaction, one-way ANOVAs were performed on the separate test times, followed by Student-Newman-Keuls (SNK) posthoc tests for pairwise comparisons. An unpaired Student's <italic>t</italic>-test was used to determine the statistical significance of the differences when there were only two groups in the data set (data in <xref ref-type="fig" rid="fig2 fig5 fig6 fig9">Figures 2,5,6 and 9D</xref>). All reported levels of significance represent two-tailed values unless otherwise indicated.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>This study was supported by research grants to DLG from the National Institute of Neurological Disorders and Stroke (NIH R01 NS029563), the National Institute of Mental Health (NIH R01 MH096120), and the National Science Foundation (IOS 1121690). We thank R Singh, M Kimbrough, K Sarmiento, X Zhao and T Dehghani for assistance with the behavioral training, and MS Fanselow, FB Krasne, and WS Sossin for helpful comments on the manuscript.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>SC, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con2"><p>DC, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>DLG, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>KP, Acquisition of data, 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letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Ramaswami</surname><given-names>Mani</given-names></name><role>Reviewing editor</role><aff><institution>Trinity College Dublin</institution>, <country>Ireland</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in <italic>Aplysia</italic>” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by K VijayRaghavan (Senior editor), a Reviewing editor, and two reviewers.</p><p>The following individuals responsible for the peer review of your submission have agreed to reveal their identity: Mani Ramaswami (Reviewing editor); Tom Abrams and Ron Calabrese (peer reviewers).</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>The manuscript contains a very interesting set of results on mechanisms of long-term synaptic plasticity obtained in a classic system for investigating the cellular and molecular basis of long-term memory: the sensory neuron to motor neuron synapse from Aplysia reconstituted in culture. Most significantly, the experiments performed here show that: (a) procedures that reverse LTS in Aplysia sensorimotor co-cultures leads to loss of synaptic varicosities, which occurs without the selective retraction of LTS-induced synaptic varicosities; (b) LTM can be reinstated in intact animals after its erasure by training that will not induce LTM in naïve animals.</p><p>These observations make the major point that the persistence of memory is not determined by the stability of synapses. Instead it depends on a different persistent cellular mechanism that drives synaptic change in order to enable the expression of memory. In other words, memory storage is independent of synaptic change, but memory expression requires synaptic change. In other words, without synaptic change memory exists but is covert. This intellectual and didactic point, which is broadly supported by the authors' results, merits publication in <italic>eLife</italic>.</p><p>However, in current form the manuscript does not do a good job of positioning their findings within the literature on long-term synaptic plasticity spanning the past 2 decades. A major careful and attentive rewrite is required, which acknowledges previous observations and clarifies how previous observations and ideas may be integrated with and reinterpreted in authors' new perspective.</p><p>The language used in the current manuscript has the potential to confound and confuse. For instance the statement that “LTM is independent of synaptic change in Aplysia” is certain to confuse. What is meant is that memory is covertly encoded and stored, possibly in the cytoplasm and possibly in the nucleus; this memory signal induces synaptic changes necessary for memory expression. For a point as subtle, specific and interesting as this, loose statements such as “memory is independent of synaptic change” are confusing as well as unproductively provocative. The exaggeration of the radical nature of these results sometimes make them appear unnecessarily paradoxical, potentially reducing their impact.</p><p>In any case, the authors should rewrite, clarify and qualify statements and conclusions in the manuscript in way that tries to bring the rest of the field around to the author's point of view. Many specific suggestions and arguments are offered below. A particularly strong recommendation, elaborated in point 2, is to consider presenting the new perspective in the framework of a memory priming model, in which non synaptic priming mechanisms may be long-lasting and store memory.</p><p>Several are stylistic, intellectual or theoretical points with which the authors should engage constructively in a revised discussion. These should be viewed as constructive and supportive suggestions that the reviewers believe will improve the presentation and impact of the manuscript. It is not necessary that the authors should accept all of these suggestions. However, a revised manuscript should list the authors’ response to each suggestion.</p><p>1) Assigning memory storage to a change in the nucleus is not unreasonable, but yet it is entirely speculative. This is particularly true when the apparent deacetylation of histones triggered by chelerythrine does not compromise the covert persistent memory. Logically, this means that the residual memory that persists after PKC inhibition and which can be fully restored (or unmasked) with 3 more training trials, cannot be mediated by histone acetylation. This is an important point of confusion in the authors' analysis. Although they wish to assign a role for histone acetylation in the “synapse-independent” memory mechanism, their evidence actually suggests that there is an additional mechanism of memory beyond the PKC-dependent memory, the erasure of which is blocked by TSA. This issue should be more objectively and critically discussed.</p><p>2) Priming vs. occult memory. The authors should acknowledge that it is possible that the original sequence of 5-HT exposures primes the synapses for plasticity even after a memory is erased. There is a substantial number of experimental results suggesting priming, both at the synaptic and the behavioral levels (e.g. <xref ref-type="bibr" rid="bib33 bib34">Hegde et al, 1993, 1997</xref>). The Barco et al (2002, <xref ref-type="fig" rid="fig5">Figure 5</xref>) result with constitutively active CREB suggests changes in gene expression can subsequently interact with local synaptic signals to initiate synaptic plasticity. Even the Guan et al. result with an HDAC inhibitor that enables a single pulse of 5-HT to initiate long-term facilitation suggests there synapses can be primed for long-term plasticity. Thus, early training can induce molecular changes (e.g. transcription of an IEG or translocation to the nucleus) but subsequent training may be required to induce the change in synaptic strength or behavior. These priming mechanisms are considered to be molecular explanations for the importance of repeated training trials. If the Chen-Glanzman phenomenon worked similarly, there could be a persistent change that primes cells to respond to subsequent training trials. Histone acetylation could be one such change, though the TSA data are not consistent with this possibility. A change in an ubiquitination-proteasome protein or in a kinase or phosphatase are examples of non-genomic persistent changes that could contribute. Seen from this perspective, the synaptic changes would be part of the memory, but there are cell wide changes that would be more persistent. This is a less paradoxical suggestion, but one that could be equally interesting. It has the advantage of preserving the possibility of synapse specific plasticity contributing to learning. The authors should acknowledge and discuss these possibilities.</p><p>3) In these experiments, the stability of both basal and long-term facilitated synaptic strength over days suggests a homeostatic mechanism that specifies net synaptic strength, rather than constraining the strength of individual synaptic connections. There seems to be a set point for net synaptic strength, although this set point may shift during long-term synaptic plasticity (much as Hu and colleagues have described.) That the overall strength of synaptic connections may be kept stable while individual synaptic connections form or are retracted does not necessarily mean that synapses don't “store memory.” Instead, it suggests the possibility that there is a mechanism to set global synaptic strength, much as suggested by numerous studies of homeostatic synaptic scaling. The concept of a set point that shifts with sensitization should be discussed more explicitly.</p><p>4) From a molecular perspective, although it is tempting to assign primary responsibility for memory representation to a single protein or gene and to focus attention on this single player (at least within a given study), this is clearly not a valid way to understand the biology of memory. All molecular participants in memory storage interact with a wide array of other molecules in complex networks. We know from studies such as Casadio et al. (1999) and Barco et al. (2002) that changes in transcription interact with local synaptic “tags” to initiate long-term alterations in synaptic strength. Indeed, the emergent conclusion over nearly two decades is that neither the transcriptional changes nor local synaptic changes are sufficient to represent long-term memory. The authors suggest that synapses merely express memory that is “stored” at the genome level. However, they present no evidence that the homeostatic set point is specified solely in the genome (or even the transcriptome), rather than emerging from a dynamic process in which synaptic sites participate interactively in storing the memory. To consider a crude analogy, this is a bit like positing that muscle strength is specified in muscle fiber nuclei, rather than through complex dynamic interactions among gene expression, cytoskeletal elements, signaling molecules and extracellular matrix, and also non-muscle cells, simply because individual myosin or actin filaments turn over.</p><p>The discussion may be better received if the role of the synapse in directing synapse specific memory at least, were acknowledged and integrated (for instance, see point 5 below).</p><p>5) The authors should discuss the possibility that synapses are merely toggled between a basal state and a facilitated state (with 2 amplitudes of connections), such that the apparent restoration of a covert memory is actually just a switching back to the facilitated, which occurs more readily after priming. There may be good evidence that this is not the case, but it should be explicitly considered.</p><p>6) In terms of placing the present study in a broader context, it would be helpful for the authors to discuss studies on interactions between transcription and local signals, such as those mentioned above, and also recent studies on mammalian CNS that demonstrate remarkable morphological stability of individual dendritic spines over weeks or months, e.g. by Yi Zuo and colleagues.</p><p>7) One of the most novel results in this manuscript is the finding in the final Figure that the reversal of long-term memory by transient inhibition of atypical protein kinase C (homologous with PKM zeta in mammalian brain) requires histone deactylase activity. This result requires reevaluation of the long-argued model that persistent activity of PKM is required for memory because it supports enhanced synaptic strength at individual local synaptic sites. Even with this one data set, these results might have a larger impact in a separate short publication, rather than tacked on at the end of the present study. This perspective is partially supported by the authors' conclusions, which rather trivialize this important finding. (For example in the Abstract, it is stated: “we provide evidence that LTM in Aplysia is stored by epigenetic changes” and nearly identical summary statements appear at the end of the Introduction and in the Discussion.) We have known for more than a decade in both Aplysia and mammalian CNS that histone acetylation is critical for long-term synaptic plasticity and long-term memory. What is novel here is that atypical PKC activity appears to be required for the persistence of the histone acetylation-dependent effect, possibly by suppressing histone deactylase activity.</p><p>8) An important prediction of the hypothesis that synaptic growth is an expression mechanism of LTM not LTM itself that in co-cultures one should be able to re-express more synaptic growth after 5X HT reminder-anisomycin 3X 5HT than in naive 3X 5HT. Can this be tested and quantified? (Perhaps these data are already available?)</p><p>9) Although paper is generally clearly written, it is very tedious to read because of the complicated nature of the protocols. This may simply be impossible to avoid but the authors can try even harder to make the writing flow. Further, the paper depends heavily on corresponding physiological studies in co-cultures, and the authors should be at great pains to point out the details of these studies (e.g., Cai D, Pearce K, Chen S, Glanzman DL. 2011. Protein kinase M maintains long-term sensitization and long-term facilitation in Aplysia. J. Neurosci. 31: 6421-31. Cai D, Pearce K, Chen S, Glanzman DL. 2012. Reconsolidation of long-term memory in Aplysia. Curr. Biol. 22: 1783-88) in relevant regions of the text.</p><p>10) It is particularly intriguing that memory reversal restored the number of presynaptic varicosities to precisely the number before memory formation. This point could be interpreted more vigorously as it fits the prior model in which synapse plasticity underlies memory, and could form the basis then for the authors' intellectual reinterpretation (that memory exists though it is invisible when extra synapses are lost).</p><p>11) Statistics. Although the authors perform ANOVAs on their data, the ANOVA results are not presented. Simply including posthoc tests is not appropriate as the overall ANOVA result is as important as the posthoc test (arguably more so). F values and exact p values should be included for each ANOVA.</p><p>12) Even if the authors wish to combine the 5x5-HT without a 5-HT reminder group with the 5x5-HT without a 5-HT reminder but with late anisomycin into a single group for purposes of statistical comparison with other treatment groups, we should be shown the data for the two treatments separately, at least in the initial figure (<xref ref-type="fig" rid="fig1">Figure 1C</xref>).</p><p>13) Abstract. “Further, we show that LTS can be reinstated following its apparent elimination by reconsolidation blockade and inhibition of PKM, treatments that erase LTS-related synaptic growth.” This sentence fails to communicate that memory persists covertly after apparent elimination. Reinstatement suggests only that relearning is possible.</p><p>14) The term “+Reminder group” for the synapses that receive a reminder of 5-HT plus anisomycin is a poor term, as there is no suggestion of the use of a protein synthesis inhibitor. In <xref ref-type="fig" rid="fig7">Figure 7</xref>, this same group is called 5xTrained-Reminder-Aniso. In the legend of <xref ref-type="fig" rid="fig2">Figure 2</xref> it states “Coculture in which synaptic reconsolidation was disrupted (+Reminder coculture),” which is somewhat confusing. It's not the reminder 5-HT that disrupts memory but the reminder + inhibition of protein synthesis.</p><p>15) From the Introduction, the sentence “These morphological results imply that LTM is independent of synaptic change in Aplysia” seems internally inconsistent. Clearly the authors believe that memory involves synaptic modification as they quantify the number of synaptic contacts as a read out for memory.</p><p>16) Results. The reversal of morphological change with anisomycin after 5-HT “supports the idea that the reminder triggered reconsolidation of the synaptic growth that mediates LTF.” Reconsolidation is what occurs with a reminder alone (e.g. 5-HT alone). Anisomycin treatment blocks the reconsolidation. This should be stated more clearly, particularly in a journal with broad readership. This could be explained in the Introduction when reconsolidation is first mentioned.</p><p>17) Results: “there was also significant retraction of the original varicosities during this period in both groups of trained sensorimotor coculture, those that received the reminder stimulus and those that did not.” The description fails to mention that retraction of pre-existing varicosities was significantly greater in the Reminder + anisomycin group. This merits emphasis.</p><p>18) Results: “retraction observed previously in the 5-HT group.” Probably the authors are referring to the group that received the 5-HT reminder plus anisomycin (most of the groups received 5-HT).</p><p>19) Results: “If this idea is correct, then it should be possible to dissociate LTM in Aplysia from synaptic facilitation.” This should be clarified.</p><p>20) Results: “as indicated by comparing … without the reminder training” This detailed description of the groups to be compared is not necessary in Results. The sentence up to this point made it clear that the three additional 5-HT exposures restored the erased memory.</p><p>21) Results: “three bouts of tail shocks can reinstate LTM following the suppression of its behavioral expression” This sentence is rather inappropriate for the Results, as it is a bit circular. The authors can logically suggest that inhibition of atypical PKC suppresses the expression of long-term memory, rather than erasing it, only because the subsequent tail shocks reinstate (or reestablish) the memory. Certainly, the evidence in this manuscript is consistent with a covert memory persisting after blockade of reconsolidation and inhibition of PKM. However, this conclusion about suppression, rather than erasure, of memory should actually be considered in the Discussion.</p><p>22) Results: “the synaptic mechanism the mediates” should have “that” before “mediates.”</p><p>23) Discussion: “Could a nonsynaptic, epigenetic storage mechanism, such as the one we propose for Aplysia, mediate the maintenance of associative memories, particularly those induced in complex neural circuits in the mammalian brain, where a given neuron may have thousands or tens of thousands of synaptic partners? This may seem unlikely.” Do the authors really mean to suggest that the mechanism their data suggests is relevant only for simpler invertebrate nervous systems?</p><p>24) L-15, Discussion: Was this medium supplemented with salts?</p><p>25) Discussion: “oval-shaped main body” This is an odd description of a varicosity, as it suggests that there are regions of the varicosity outside the “main body.”</p><p>26) Discussion: “unpaired Student's t-test.” When was the t-test used instead of an ANOVA?</p><p>27) In the experiments of <xref ref-type="fig" rid="fig1 fig7">Figures 1 and 7</xref>, it seems very important to this reviewer to have the control of a reminder stimulus (after training - 5X 5HT) without the anisomycin, i.e. 5X Trained Reminder. It seems even more important than the naïve Control because we want to know that the reminder itself has no effect. Am I wrong here? Can the authors explain why this control is not important? The legend for <xref ref-type="fig" rid="fig1">Figure 1</xref> actually suggests this control was done (“A reminder pulse of 5-HT (single blue bar) was delivered to the +Reminder cocultures prior to the anisomycin/vehicle”).</p><p>28) <xref ref-type="fig" rid="fig3">Figure 3</xref> statistical comparison. Because the authors have performed ANOVAs, it is possible to compare the amount of retraction of newly formed varicosities with the retraction of original varicosities (e.g. <xref ref-type="fig" rid="fig3 fig5">Figures 3 and 5</xref>). These results should be included.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03896.017</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) Assigning memory storage to a change in the nucleus is not unreasonable, but yet it is entirely speculative. This is particularly true when the apparent deacetylation of histones triggered by chelerythrine does not compromise the covert persistent memory. Logically, this means that the residual memory that persists after PKC inhibition and which can be fully restored (or unmasked) with 3 more training trials, cannot be mediated by histone acetylation. This is an important point of confusion in the authors' analysis. Although they wish to assign a role for histone acetylation in the “synapse-independent” memory mechanism, their evidence actually suggests that there is an additional mechanism of memory beyond the PKC-dependent memory, the erasure of which is blocked by TSA. This issue should be more objectively and critically discussed</italic>.</p><p>We agree with this important point and we thank the reviewers for pointing out our error. In our discussion of the potential role of epigenetic mechanisms we now acknowledge that the chelerythrine results argue against the idea that the storage mechanism for LTM involves histone acetylation. We now propose an alternative epigenetic mechanism for LTM storage, DNA methylation. As we discuss, recent evidence from David Sweatt’s lab have shown that DNA methylation changes, induced by learning or by early trauma, may persist for the life of an animal. Chromatin remodeling subsequent to DNA double-strand breaks (<xref ref-type="bibr" rid="bib71">Suberbielle et al., 2013</xref>) is another potential nuclear mechanism for LTM storage.</p><p><italic>2) Priming vs. occult memory. The authors should acknowledge that it is possible that the original sequence of 5-HT exposures primes the synapses for plasticity even after a memory is erased. There is a substantial number of experimental results suggesting priming, both at the synaptic and the behavioral levels (e.g.</italic> <xref ref-type="bibr" rid="bib33"><italic>Hegde et al, 1993</italic></xref><italic>,</italic> <xref ref-type="bibr" rid="bib34"><italic>1997</italic></xref><italic>). The Barco et al (2002,</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref><italic>) result with constitutively active CREB suggests changes in gene expression can subsequently interact with local synaptic signals to initiate synaptic plasticity. Even the Guan et al. result with an HDAC inhibitor that enables a single pulse of 5-HT to initiate long-term facilitation suggests there synapses can be primed for long-term plasticity. Thus, early training can induce molecular changes (e.g. transcription of an IEG or translocation to the nucleus) but subsequent training may be required to induce the change in synaptic strength or behavior. These priming mechanisms are considered to be molecular explanations for the importance of repeated training trials. If the Chen-Glanzman phenomenon worked similarly, there could be a persistent change that primes cells to respond to subsequent training trials. Histone acetylation could be one such change, though the TSA data are not consistent with this possibility. A change in an ubiquitination-proteasome protein or in a kinase or phosphatase are examples of non-genomic persistent changes that could contribute. Seen from this perspective, the synaptic changes would be part of the memory, but there are cell wide changes that would be more persistent. This is a less paradoxical suggestion, but one that could be equally interesting. It has the advantage of preserving the possibility of synapse specific plasticity contributing to learning. The authors should acknowledge and discuss these possibilities</italic>.</p><p>We are grateful to the reviewers for the insightful discussion. We have rewritten the manuscript to incorporate the possibility that our results reflect the persistence of a priming mechanism required to reestablish the disrupted LTM, rather than LTM itself. In addition, we explicitly compare the priming explanation for our results to the idea that LTM was not erased by reconsolidation blockade/chelerythrine treatment.</p><p><italic>3) In these experiments, the stability of both basal and long-term facilitated synaptic strength over days suggests a homeostatic mechanism that specifies net synaptic strength, rather than constraining the strength of individual synaptic connections. There seems to be a set point for net synaptic strength, although this set point may shift during long-term synaptic plasticity (much as Hu and colleagues have described.) That the overall strength of synaptic connections may be kept stable while individual synaptic connections form or are retracted does not necessarily mean that synapses don't “store memory.” Instead, it suggests the possibility that there is a mechanism to set global synaptic strength, much as suggested by numerous studies of homeostatic synaptic scaling. The concept of a set point that shifts with sensitization should be discussed more explicitly</italic>.</p><p>We have added an explicit discussion of the notion that the overall strength of synaptic connections is regulated by a homeostatic mechanism that toggles between a facilitated state and a nonfacilitated state, as determined by experience. However, our argument against the idea that synapses store memory does not rest simply on the idea that the overall strength of a sensorimotor connection remains stable while individual synapses grow and retract. More powerful evidence against the notion of synaptic storage comes from our finding that when the homeostatic mechanism resets the total number of synaptic connections to the pretraining value, the resulting morphology is quite different from the original one, and that memory can be reinstated in the animal following antimnemonic treatments that erase learning-related synaptic growth.</p><p><italic>4) From a molecular perspective, although it is tempting to assign primary responsibility for memory representation to a single protein or gene and to focus attention on this single player (at least within a given study), this is clearly not a valid way to understand the biology of memory. All molecular participants in memory storage interact with a wide array of other molecules in complex networks. We know from studies such as Casadio et al. (1999) and Barco et al. (2002) that changes in transcription interact with local synaptic “tags” to initiate long-term alterations in synaptic strength. Indeed, the emergent conclusion over nearly two decades is that neither the transcriptional changes nor local synaptic changes are sufficient to represent long-term memory. The authors suggest that synapses merely express memory that is “stored” at the genome level. However, they present no evidence that the homeostatic set point is specified solely in the genome (or even the transcriptome), rather than emerging from a dynamic process in which synaptic sites participate interactively in storing the memory. To consider a crude analogy, this is a bit like positing that muscle strength is specified in muscle fiber nuclei, rather than through complex dynamic interactions among gene expression, cytoskeletal elements, signaling molecules and extracellular matrix, and also non-muscle cells, simply because individual myosin or actin filaments turn over</italic>.</p><p><italic>The discussion may be better received if the role of the synapse in directing synapse specific memory at least, were acknowledged and integrated (for instance, see point 5 below)</italic>.</p><p>LTM induction may not be a useful model for LTM storage. In particular, although the results of Barco et al. and those of others indicate that CREB-dependent changes in gene expression can interact with local synaptic tags to initiate LTF, as the reviewer states, our results do not provide any evidence for synaptic tagging. This is because the retraction of sensory neuron varicosities following reconsolidation blockade or chelerythrine treatment appeared arbitrary. The notion of synaptic tagging during LTM storage is difficult to reconcile with this apparently arbitrary elimination of varicosities, both original and 5HT-induced. Also, it seems to us that the homeostatic mechanism, whatever it is, resides in either the nucleus or the cell body; the reviewer’s idea that synaptic homeostasis results from a dynamic interaction between some priming mechanism and labeled synaptic sites only seems viable in the context of synaptic tagging. It is possible that the synapses that remain following reconsolidation blockade or chelerythrine treatment contain a 5HT-induced molecular signal (the tag), which is why they survive antimnemonic treatment; this tag may interact with the residual priming mechanism in the nucleus or cell body to restore LTM.</p><p><italic>5) The authors should discuss the possibility that synapses are merely toggled between a basal state and a facilitated state (with 2 amplitudes of connections), such that the apparent restoration of a covert memory is actually just a switching back to the facilitated, which occurs more readily after priming. There may be good evidence that this is not the case, but it should be explicitly considered</italic>.</p><p>As pointed out in the response to Comment 3 above, we agree with the reviewers that implicit in our data is the possibility that “synapses are merely toggled between a basal state and a facilitated state”. (Note that there must also be a third state to incorporate the evidence that the sensorimotor synapse can exhibit long-term depression.) But the idea of an experience-dependent toggling between basal and facilitated states is ultimately consistent with either a persistent priming mechanism or covert memory.</p><p><italic>6) In terms of placing the present study in a broader context, it would be helpful for the authors to discuss studies on interactions between transcription and local signals, such as those mentioned above, and also recent studies on mammalian CNS that demonstrate remarkable morphological stability of individual dendritic spines over weeks or months, e.g. by Yi Zuo and colleagues</italic>.</p><p>The revised manuscript now includes a discussion of this important issue. Briefly, significant controversy exists regarding the extent to which spines are stable over long periods of time in the mammalian brain. Also, even in those studies in which spines have been reported to be mostly stable after learning, there is a large degree of spine turnover in the first few days after training, which is when our measurements were made. So our results are not necessarily contradictory with those of in vivo mammalian imaging studies.</p><p><italic>7) One of the most novel results in this manuscript is the finding in the final Figure that the reversal of long-term memory by transient inhibition of atypical protein kinase C (homologous with PKM zeta in mammalian brain) requires histone deactylase activity. This result requires reevaluation of the long-argued model that persistent activity of PKM is required for memory because it supports enhanced synaptic strength at individual local synaptic sites. Even with this one data set, these results might have a larger impact in a separate short publication, rather than tacked on at the end of the present study. This perspective is partially supported by the authors' conclusions, which rather trivialize this important finding. (For example in the Abstract, it is stated: “we provide evidence that LTM in Aplysia is stored by epigenetic changes” and nearly identical summary statements appear at the end of the Introduction and in the Discussion.) We have known for more than a decade in both Aplysia and mammalian CNS that histone acetylation is critical for long-term synaptic plasticity and long-term memory. What is novel here is that atypical PKC activity appears to be required for the persistence of the histone acetylation-dependent effect, possibly by suppressing histone deactylase activity</italic>.</p><p>We agree with the reviewers that this finding is novel and important, and we certainly do not wish to trivialize it by including it here. We believe the finding properly belongs in the paper because it supports our general position that nuclear changes are central to the persistence of long-term memory (although we agree with the reviewer that histone acetylation cannot account for the reinstatement of memory following chelerythrine treatment). The fact that TSA blocks chelerythrine’s disruptive effect on LTM also echoes the other finding in <xref ref-type="fig" rid="fig9">Figure 9</xref>, namely that TSA can convert the intermediate-term sensitization induced by three bouts of tail shocks into LTS. The revised Discussion includes an analysis of the role of PKC activity in epigenetic regulation of LTM induction and maintenance in <italic>Aplysia</italic>. Also, we have rewritten the Abstract, Introduction and Discussion to eliminate the redundant statements cited by the reviewers.</p><p><italic>8) An important prediction of the hypothesis that synaptic growth is an expression mechanism of LTM not LTM itself that in co-cultures one should be able to re-express more synaptic growth after 5X HT reminder-anisomycin 3X 5HT than in naive 3X 5HT. Can this be tested and quantified? (Perhaps these data are already available?</italic>)</p><p>Although our results do predict this result, testing it would require a significant effort. Notice that to be convincing the morphological experiments proposed by the reviewer would have to be preceded by electrophysiological experiments confirming that the 5X5HT-1X5HT-Aniso-3X5HT treatment produces LTF and that, as we would expect, 3X5HT alone does not. These experiments have not yet been done. Also, because the experiments would require intracellular electrophysiological recording from pre- and postsynaptic neurons of the cocultures for three consecutive days, their rate of success would be low. I estimate that the electrophysiological and morphological experiments necessary to test the prediction would take my lab the better part of a year to complete. Therefore, I hope the reviewers will agree to allow us to publish without these data.</p><p><italic>9) Although paper is generally clearly written, it is very tedious to read because of the complicated nature of the protocols. This may simply be impossible to avoid but the authors can try even harder to make the writing flow. Further, the paper depends heavily on corresponding physiological studies in co-cultures, and the authors should be at great pains to point out the details of these studies (e.g., Cai D, Pearce K, Chen S, Glanzman DL. 2011. Protein kinase M maintains long-term sensitization and long-term facilitation in Aplysia. J. Neurosci. 31: 6421-31. Cai D, Pearce K, Chen S, Glanzman DL. 2012. Reconsolidation of long-term memory in Aplysia. Curr. Biol. 22: 1783-88) in relevant regions of the text</italic>.</p><p>I have worked to streamline the writing in the paper, but the experimental protocols are admittedly not simple. Regarding the reviewer’s other point, the results of our previous electrophysiological studies are detailed in the relevant sections of the text so that the reader can see precisely how our present morphological results correspond to the prior synaptic physiological results.</p><p><italic>10) It is particularly intriguing that memory reversal restored the number of presynaptic varicosities to precisely the number before memory formation. This point could be interpreted more vigorously as it fits the prior model in which synapse plasticity underlies memory, and could form the basis then for the authors' intellectual reinterpretation (that memory exists though it is invisible when extra synapses are lost)</italic>.</p><p>We agree with the reviewers that this is an intriguing result and have emphasized it in several places in the manuscript. The result is explicitly discussed in the second paragraph of the Discussion, where we point out that the restoration of the number of presynaptic varicosities to the pretraining level following reconsolidation blockade/chelerythrine treatment is paralleled by the results from our prior electrophysiological studies (<xref ref-type="bibr" rid="bib13">Cai et al., 2011</xref>, <xref ref-type="bibr" rid="bib14">2012</xref>). The parallel between our results and those of studies of homeostatic synaptic plasticity in other systems is also mentioned here.</p><p><italic>11) Statistics. Although the authors perform ANOVAs on their data, the ANOVA results are not presented. Simply including posthoc tests is not appropriate as the overall ANOVA result is as important as the posthoc test (arguably more so). F values and exact p values should be included for each ANOVA</italic>.</p><p>The results for all ANOVAs in our study, including the exact F- and p-values for each test, are presented in the relevant figure legends.</p><p><italic>12) Even if the authors wish to combine the 5x5-HT without a 5-HT reminder group with the 5x5-HT without a 5-HT reminder but with late anisomycin into a single group for purposes of statistical comparison with other treatment groups, we should be shown the data for the two treatments separately, at least in the initial figure (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1C</italic></xref><italic>)</italic>.</p><p><xref ref-type="fig" rid="fig1">Figure 1C</xref> now presents the data for the 5X5HT and 5X5HT-Aniso groups graphed separately, as requested.</p><p><italic>13) Abstract: “Further, we show that LTS can be reinstated following its apparent elimination by reconsolidation blockade and inhibition of PKM, treatments that erase LTS-related synaptic growth.” This sentence fails to communicate that memory persists covertly after apparent elimination. Reinstatement suggests only that relearning is possible</italic>.</p><p>The Abstract has been rewritten. It now states, “In addition, we find evidence that the LTM for sensitization persists covertly after its apparent elimination by the antimnemonic treatments that erase learning-related synaptic growth.”</p><p><italic>14) The term “+Reminder group” for the synapses that receive a reminder of 5-HT plus anisomycin is a poor term, as there is no suggestion of the use of a protein synthesis inhibitor. In</italic> <xref ref-type="fig" rid="fig7"><italic>Figure 7</italic></xref><italic>, this same group is called 5xTrained-Reminder-Aniso. In the legend of</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2</italic></xref> <italic>it states “Coculture in which synaptic reconsolidation was disrupted (+Reminder coculture),” which is somewhat confusing. It's not the reminder 5-HT that disrupts memory but the reminder + inhibition of protein synthesis</italic>.</p><p>The reviewers are correct; the original group labels were misleading. We have replaced them with new terms that we hope will be both less confusing and more accurate. Thus, the former “– Reminder” group has now been separated into two experimental groups for the graph in <xref ref-type="fig" rid="fig1">Figure 1C</xref>, the “5X5HT” and 5X5HT-Aniso” groups. The former “+Reminder” group is now labeled the “5X5HT-1X5HT-Aniso” group. The new group labels in <xref ref-type="fig" rid="fig1">Figure 1</xref> now more closely parallel those in <xref ref-type="fig" rid="fig7">Figure 7</xref>. In addition, the use of the new labels eliminates the inaccuracy pointed out by the reviewer for the legend in <xref ref-type="fig" rid="fig2">Figure 2</xref>.</p><p>Notice that we have also used new group labels for <xref ref-type="fig" rid="fig3">Figure 3</xref>. The former “–Reminder” group (which comprises the combined data for the 5X5HT, 5X5HT-1X5HT and 5X5HT-Aniso groups) is now more accurately labeled, “No reconsolidation/No blockade”. The former “+Reminder” group is now labeled the “5X5HT-1X-5HT-Aniso” group, as in <xref ref-type="fig" rid="fig1">Figure 1</xref>.</p><p><italic>15) From the Introduction, the sentence “These morphological results imply that LTM is independent of synaptic change in Aplysia” seems internally inconsistent. Clearly the authors believe that memory involves synaptic modification as they quantify the number of synaptic contacts as a read out for memory</italic>.</p><p>This sentence has been rewritten as follows: “These results imply that the persistence of memory does not require the stability of particular synaptic connections.”</p><p><italic>16) Results. The reversal of morphological change with anisomycin after 5-HT “supports the idea that the reminder triggered reconsolidation of the synaptic growth that mediates LTF.” Reconsolidation is what occurs with a reminder alone (e.g. 5-HT alone). Anisomycin treatment blocks the reconsolidation. This should be stated more clearly, particularly in a journal with broad readership. This could be explained in the Introduction when reconsolidation is first mentioned</italic>.</p><p>The sentence has been revised, and now states, “This structural result…provides additional support for the notion that 1X5HT reactivated the synaptic memory induced by the 5X5HT training”. In addition, the Introduction now includes an explanation of the phenomenon of memory reconsolidation.</p><p><italic>17) Results: “there was also significant retraction of the original varicosities during this period in both groups of trained sensorimotor coculture, those that received the reminder stimulus and those that did not.” The description fails to mention that retraction of pre-existing varicosities was significantly greater in the Reminder + anisomycin group. This merits emphasis</italic>.</p><p>We have added the following statement to the Results: “Varicosities in the 5X5HT-1X5HT-Aniso group—the group of trained cocultures subjected to reconsolidation blockade—exhibited a similar pattern of retraction of 5HT-induced and original varicosities, but the amount of retraction was significantly greater than that observed in the No reconsolidation/No blockade cocultures. Note that “5X5HT-1X5HT-Aniso” is the new label for the former “+ Reminder” group and “No reconsolidation/No blockade” is the new label for the former “–Reminder” group (see the response to Comment 14 above).</p><p>18) Results: “retraction observed previously in the 5-HT group.” Probably the authors are referring to the group that received the 5-HT reminder plus anisomycin (most of the groups received 5-HT).</p><p>We apologize for this confusing sentence. We have revised it to make our meaning clear: “Monitoring of the fate of individual varicosities in the 5X5HT group revealed the same pattern of structural growth and retraction observed previously in the 5X5HT-trained cocultures not subjected to reconsolidation blockade.”</p><p><italic>19) Results: “If this idea is correct, then it should be possible to dissociate LTM in Aplysia from synaptic facilitation.” This should be clarified</italic>.</p><p>This sentence has been eliminated. The introduction to this section of the paper has been rewritten to make our meaning clearer. The first paragraph now reads:</p><p>“The present morphological results challenge the notion that the persistence of sensitization memory in <italic>Aplysia</italic> depends on the persistence of particular facilitated synapses. To further investigate this idea, we tested whether the LTM for behavioral sensitization can be reinstated in <italic>Aplysia</italic> following reconsolidation blockade and inhibition of PKM, two treatments previously shown to eliminate LTF, the synaptic basis of long-term sensitization…”</p><p><italic>20) Results: “as indicated by comparing...... without the reminder training” This detailed description of the groups to be compared is not necessary in Results. The sentence up to this point made it clear that the three additional 5-HT exposures restored the erased memory</italic>.</p><p>We have eliminated this detailed description.</p><p><italic>21) Results: “three bouts of tail shocks can reinstate LTM following the suppression of its behavioral expression” This sentence is rather inappropriate for the Results, as it is a bit circular. The authors can logically suggest that inhibition of atypical PKC suppresses the expression of long-term memory, rather than erasing it, only because the subsequent tail shocks reinstate (or reestablish) the memory. Certainly, the evidence in this manuscript is consistent with a covert memory persisting after blockade of reconsolidation and inhibition of PKM. However, this conclusion about suppression, rather than erasure, of memory should actually be considered in the Discussion</italic>.</p><p>This statement has been removed.</p><p><italic>22) Results: “the synaptic mechanism the mediates” should have “that” before “mediates</italic>.<italic>”</italic></p><p>The sentence containing this mistake has been eliminated.</p><p><italic>23) Discussion: “Could a nonsynaptic, epigenetic storage mechanism, such as the one we propose for Aplysia, mediate the maintenance of associative memories, particularly those induced in complex neural circuits in the mammalian brain, where a given neuron may have thousands or tens of thousands of synaptic partners? This may seem unlikely.” Do the authors really mean to suggest that the mechanism their data suggests is relevant only for simpler invertebrate nervous systems</italic>?</p><p>We appreciate the reviewers’ question. No, we do not believe that our data are only relevant to invertebrate nervous systems. We have eliminated these statements in the Discussion.</p><p><italic>24) L-15, Discussion: Was this medium supplemented with salts</italic>?</p><p>It is now stated in the Material and methods that the L-15 was supplemented with the appropriate salts.</p><p><italic>25) Discussion: “oval-shaped main body” This is an odd description of a varicosity, as it suggests that there are regions of the varicosity outside the “main body</italic>.<italic>”</italic></p><p>The descriptive term “oval-shaped main body” has been changed to “oval-shaped body”.</p><p><italic>26) Discussion: “unpaired Student's t-test” When was the t-test used instead of an ANOVA</italic>?</p><p>The summary of our statistical methods now states, “An unpaired Student’s <italic>t</italic>-test was used to determine the statistical significance of the differences when there were only two groups in the data set such as the data presented in <xref ref-type="fig" rid="fig3 fig5 fig6 fig8 fig9">Figures 3, 5, 6, 8 and 9D</xref>.”</p><p><italic>27) In the experiments of</italic> <xref ref-type="fig" rid="fig1 fig7"><italic>Figures 1 and 7</italic></xref><italic>, it seems very important to this reviewer to have the control of a reminder stimulus (after training - 5X 5HT) without the anisomycin, i.e. 5X Trained Reminder. It seems even more important than the naïve Control because we want to know that the reminder itself has no effect. Am I wrong here? Can the authors explain why this control is not important? The legend for</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1</italic></xref> <italic>actually suggests this control was done (“A reminder pulse of 5-HT (single blue bar) was delivered to the +Reminder cocultures prior to the anisomycin/vehicle”)</italic>.</p><p>We did include this control in our morphological experiments; it is referred to as the “5X5HT-1X5HT” group. A supplemental graph for <xref ref-type="fig" rid="fig1">Figure 1</xref> (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>) is included in the revised MS; it presents the mean overall varicosity number, normalized to the number of varicosities present at 0 hr, for each of the three groups of trained cocultures not subjected to reconsolidation blockade.</p><p>One-way ANOVAs performed on the varicosity data for the 24hr and 48hr tests confirmed that the differences among the groups were insignificant. Notice that the 5X5HT and 5X5HT-1X5HT groups were combined into one group (labeled the “5X5HT” group) for the graph in <xref ref-type="fig" rid="fig1">Figure 1C</xref>. Also, the three groups shown in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>, the 5X5HT, 5X5HT-1X5HT and 5X5HT-Aniso groups, were combined into the No reconsolidation/No blockade group in <xref ref-type="fig" rid="fig3">Figure 3</xref>.</p><p>We did not include this control in the behavioral experiments.</p><p><italic>28)</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref> <italic>statistical comparison. Because the authors have performed ANOVAs, it is possible to compare the amount of retraction of newly formed varicosities with the retraction of original varicosities (e.g.</italic> <xref ref-type="fig" rid="fig3 fig5"><italic>Figures 3 and 5</italic></xref><italic>). These results should be included</italic>.</p><p>These results are included in the revised MS as Figure 3—figure supplement 1 and <xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>. An interesting result revealed by the graphs in these new figures is that, whereas the antimnemonic treatments—reconsolidation blockade and chelerythrine treatment—resulted in approximately equal retraction of 5-HT-induced and original varicosities, there was significantly greater retraction of 5-HT-induced varicosities than of original varicosities in the 5X5HT-trained groups not subjected to either antimnemonic treatment.</p></body></sub-article></article> |