Whereas short-term facilitation is presynaptic, intermediate-term facilitation involves both presynaptic and postsynaptic protein kinases and protein synthesis

Whereas short-term plasticity involves covalent modifications that are generally restricted to either presynaptic or postsynaptic structures, long-term plasticity involves the growth of new synapses, which by its nature involves both pre- and postsynaptic alterations. In addition, an intermediate-term stage of plasticity has been identified that might form a bridge between short- and long-term plasticity. Consistent with that idea, although short-term term behavioral sensitization in Aplysia involves presynaptic mechanisms, intermediate-term sensitization involves both pre- and postsynaptic mechanisms. However, it has not been known whether that is also true of facilitation in vitro, where a more detailed analysis of the mechanisms involved in the different stages and their interrelations is feasible. To address those questions, we have examined pre- and postsynaptic mechanisms of short- and intermediate-term facilitation at Aplysia sensory-motor neuron synapses in isolated cell culture. Whereas short-term facilitation by 1-min 5-HT involves presynaptic PKA and CamKII, intermediate-term facilitation by 10-min 5-HT involves presynaptic PKC and postsynaptic Ca(2+) and CamKII, as well as both pre- and postsynaptic protein synthesis. These results support the idea that the intermediate-term stage is the first to involve both pre- and postsynaptic molecular mechanisms, which could in turn serve as some of the initial steps in a cascade leading to synaptic growth during long-term plasticity.     

Overexpression of and RNA interference with the CCAAT enhancer-binding protein on long-term facilitation of Aplysia sensory to motor synapses

In the marine mollusk Aplysia, the CCAAT/enhancer-binding protein, ApC/EBP, serves as an immediate early gene in the consolidation of long-term facilitation in the synaptic connection between the sensory and motor neurons of the gill-withdrawal reflex. To further examine the role of ApC/EBP as a molecular switch of a stable form of long-term memory, we cloned the full-length coding regions of two alternatively spliced forms, the short and long form of ApC/EBP. Overexpression of each isoform by DNA microinjection resulted in a l6-fold increase in the expression of the coinjected luciferase reporter gene driven by an ERE promoter. In addition, when we overexpressed ApC/EBP in Aplysia sensory neurons, we found that the application of a single pulse of 5-HT that normally induced only short-term facilitation now induced long-term facilitation. Conversely, when we attempted to block the synthesis of native ApC/EBP by microinjecting double-strand RNA or antisense RNA, we blocked long-term facilitation in a sequence-specific manner. These data support the idea that ApC/EBP is both necessary and sufficient to consolidate short-term memory into long-term memory. Furthermore, our results suggest that this double-strand RNA interference provides a powerful tool in the study of the genes functioning in learning and memory in Aplysia by specifically inhibiting both the constitutive and induced expression of the genes.     

Calcium-activated proteases are critical for refilling depleted vesicle stores in cultured sensory-motor synapses of Aplysia

Aplysia motoneurons cocultured with a presynaptic sensory neuron exhibit homosynaptic depression when stimulated at low frequencies. A single bath application of serotonin (5HT) leads within seconds to facilitation of the depressed synapse. The facilitation is attributed to mobilization of neurotransmitter-containing vesicles from a feeding vesicle store to the depleted, readily releasable pool by protein kinase C (PKC). Here, we demonstrate that the calpain inhibitors, calpeptin, MG132, and ALLN, but not the proteasome inhibitors, lactacystin and clasto-lactacystin beta-lactone, block 5HT-induced facilitation of depressed synapses. Likewise the 5HT-induced enhancement of spontaneous miniature potentials (mEPSPs) frequency of depressed synapses is significantly reduced by calpeptin. In contrast, neither the facilitation of nondepressed synapses nor the enhancement of their mEPSPs frequency is affected by the inhibitor. The data suggest that action potentials-induced calcium influx activate calpains. These, in turn, play a role in the refilling processes of the depleted, releasable vesicle store.     

The tail-elicited tail withdrawal reflex of Aplysia is mediated centrally at tail sensory-motor synapses and exhibits sensitization across multiple temporal domains

The defensive withdrawal reflexes of Aplysia californica have provided powerful behavioral systems for studying the cellular and molecular basis of memory formation. Among these reflexes the tail-elicited tail withdrawal reflex (T-TWR) has been especially useful. In vitro studies examining the monosynaptic circuit for the T-TWR, the tail sensory-motor (SN-MN) synapses, have identified the induction requirements and molecular basis of different temporal phases of synaptic facilitation that underlie sensitization in this system. They have also permitted more recent studies elucidating the role of synaptic and nuclear signaling during synaptic facilitation. Here we report the development of a novel, compartmentalized semi-intact T-TWR preparation that allows examination of the unique contributions of processing in the SN somatic compartment (the pleural ganglion) and the SN-MN synaptic compartment (the pedal ganglion) during the induction of sensitization. Using this preparation we find that the T-TWR is mediated entirely by central connections in the synaptic compartment. Moreover, the reflex is stably expressed for at least 24 h, and can be modified by tail shocks that induce sensitization across multiple temporal domains, as well as direct application of the modulatory neurotransmitter serotonin. This preparation now provides an experimentally powerful system in which to directly examine the unique and combined roles of synaptic and nuclear signaling in different temporal domains of memory formation.     

Possible novel features of synaptic regulation during long-term facilitation in Aplysia

Most studies of molecular mechanisms of synaptic plasticity have focused on the sequence of changes either at individual synapses or in the cell nucleus. However, studies of long-term facilitation at Aplysia sensory neuron–motor neuron synapses in isolated cell culture suggest two additional features of facilitation. First, that there is also regulation of the number of synaptic contacts between two neurons, which may occur at the level of cell pair-specific branch points in the neuronal arbor. Branch points contain many molecules that are involved in protein synthesis-dependent long-term facilitation including neurotrophins and the RNA binding protein CPEB. Second, the regulation involves homeostatic feedback and tends to keep the total number of contacts between two neurons at a fairly constant level both at rest and following facilitation. That raises the question of how facilitation and homeostasis can coexist. A possible answer is suggested by the findings that they both involve spontaneous transmission and postsynaptic Ca²⁺, which can have bidirectional effects similar to LTP and LTD in hippocampus. In addition, long-term facilitation can involve a change in the set point of homeostasis, which could be encoded by plasticity molecules such as CPEB and/or PKM. A computational model based on these ideas can qualitatively simulate the basic features of both facilitation and homeostasis of the number of contacts.

Synaptic plasticity is a change in strength of the synaptic connection (postsynaptic potential or PSP) between neurons and includes increases during facilitation and decreases during depression. Plasticity is thought to underlie circuit formation during development and learning and memory in adults, and correspondingly to be defective in neurodevelopmental disorders including autism, ADHD, and schizophrenia as well as learning and memory disorders including Alzheimer''s, age-related memory loss, and drug addiction (Hawkins 2013; Hawkins et al. 2017). Most studies of molecular mechanisms of synaptic plasticity have focused on either changes at individual synapses or gene regulation in the cell nucleus. However, studies of long-term facilitation at Aplysia sensory neuron–motor neuron (SN–MN) synapses in isolated cell culture (Glanzman et al. 1990), sensitization in the intact animal (Wainwright et al. 2004), and long-term potentiation in hippocampal neurons (Antonova et al. 2001, 2009) have shown that there are also changes in the number of contacts between presynaptic varicosities and the postsynaptic neuron. We refer to these as synaptic contacts although not all of them are functional synapses (Kim et al. 2003). The number of contacts is thought to be an important determinant of the strength of the PSP (Zhang et al. 2003) and to be different for different neuron pairs. It also increases during long-term facilitation of the PSP and is thought to be a major determinant of the time course of the facilitation (Bailey and Chen 1989).As in other systems (Antonova et al. 2001, 2009; Holtmaat and Svoboda 2009), the contacts are dynamic and are continually being formed and eliminated, but the total number and the PSP remain fairly constant both at rest and during long-term facilitation (Miniaci et al. 2008; Chen et al. 2014). Furthermore, the number of contacts and the PSP return to baseline when maintenance of the facilitation is blocked, but the individual contacts are not all the same as they were before facilitation. These results have led some to suggest that memories are not stored at individual synaptic contacts, as is often supposed, but rather are stored in the nucleus (Chen et al. 2014). However, most of the previous experiments have involved a single SN and a single MN, so it has not been possible to examine the synapse specificity of the effects. Experiments with one SN and two MNs (Martin et al. 1997) or two SNs and 1 MN (Schacher et al. 1997) have shown that facilitation of the number of synaptic contacts and the PSP is specific to the stimulated synaptic pair (e.g., SN–MN1) and does not occur for the other pair (e.g., SN–MN2). These results should generalize to multiple pre- and postsynaptic partners and suggest two novel features of synaptic regulation during plasticity: (1) that the number of synaptic contacts between two neurons is regulated, and (2) that the regulation is homeostatic. We first describe those features and some of the evidence supporting them, then propose a model that could account for them and present computational modeling to illustrate the plausibility of the model.     

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

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

Galpha(i2) inhibition of adenylate cyclase regulates presynaptic activity and unmasks cGMP-dependent long-term depression at Schaffer collateral-CA1 hippocampal synapses

Cyclic AMP signaling plays a central role in regulating activity at a number of synapses in the brain. We showed previously that pairing activation of receptors that inhibit adenylate cyclase (AC) and reduce the concentration of cyclic AMP, with elevation of the concentration of cyclic GMP is sufficient to elicit a presynaptically expressed form of LTD at Schaffer collateral-CA1 synapses in the hippocampus. To directly test the role of AC inhibition and G-protein signaling in LTD at these synapses, we utilized transgenic mice that express a mutant, constitutively active inhibitory G protein, Galpha(i2), in principal neurons of the forebrain. Transgene expression of Galpha(i2) markedly enhanced LTD and impaired late-phase LTP at Schaffer collateral synapses, with no associated differences in input/output relations, paired-pulse facilitation, or NMDA receptor-gated conductances. When paired with application of a type V phosphodiesterase inhibitor to elevate the concentration of intracellular cyclic GMP, constitutively active Galpha(i2) expression converted the transient depression normally caused by this treatment to an LTD that persisted after the drug was washed out. Moreover, this effect could be mimicked in control slices by pairing type V phosphodiesterase inhibitor application with application of a PKA inhibitor. Electrophysiological recordings of spontaneous excitatory postsynaptic currents and two-photon visualization of vesicular release using FM1-43 revealed that constitutively active Galpha(i2) tonically reduced basal release probability from the rapidly recycling vesicle pool of Schaffer collateral terminals. Our findings support the hypothesis that inhibitory G-protein signaling acts presynaptically to regulate release, and, when paired with elevations in the concentration of cyclic GMP, converts a transient cyclic GMP-induced depression into a long-lasting decrease in release.     

Persistent up-regulation of polyribosomes at synapses during long-term memory,reconsolidation, and extinction of associative memory

Local protein synthesis at synapses can provide a rapid supply of proteins to support synaptic changes during consolidation of new memories, but its role in the maintenance or updating of established memories is unknown. Consolidation requires new protein synthesis in the period immediately following learning, whereas established memories are resistant to protein synthesis inhibitors. We have previously reported that polyribosomes are up-regulated in the lateral amygdala (LA) during consolidation of aversive-cued Pavlovian conditioning. In this study, we used serial section electron microscopy reconstructions to determine whether the distribution of dendritic polyribosomes returns to baseline during the long-term memory phase. Relative to control groups, long-term memory was associated with up-regulation of polyribosomes throughout dendrites, including in dendritic spines of all sizes. Retrieval of a consolidated memory by presentation of a small number of cues induces a new, transient requirement for protein synthesis to maintain the memory, while presentation of a large number of cues results in extinction learning, forming a new memory. One hour after retrieval or extinction training, the distribution of dendritic polyribosomes was similar except in the smallest spines, which had more polyribosomes in the extinction group. Our results demonstrate that the effects of learning on dendritic polyribosomes are not restricted to the transient translation-dependent phase of memory formation. Cued Pavlovian conditioning induces persistent synapse strengthening in the LA that is not reversed by retrieval or extinction, and dendritic polyribosomes may therefore correlate generally with synapse strength as opposed to recent activity or transient translational processes.

The formation of long-term memory involves a consolidation phase in the period immediately after learning, during which new proteins are required to stabilize learning-induced synapse remodeling (Davis and Squire 1984; Mayford et al. 2012; Rosenberg et al. 2014; Segal 2017). There is evidence that local protein synthesis in dendrites is essential for consolidation of long-term memory and related forms of synaptic plasticity (Holt and Schuman 2013), but its exact role is not well understood. Dendritic translation can supply new proteins to synapses rapidly, and potentially with synapse-specific spatial precision. Thousands of mRNAs have been identified in dendrites, many of which encode synaptic proteins (Poon et al. 2006; Zhong et al. 2006; Cajigas et al. 2012; Tushev et al. 2018; Middleton et al. 2019), and mRNA is present in dendritic spines (Tiruchinapalli et al. 2003; Hafner et al. 2019). The ability of dendritic mRNAs to remain dormant until they are unmasked by synaptic activity (Doyle and Kiebler 2011; Buxbaum et al. 2014; Hutten et al. 2014) provides a mechanism for rapid and targeted translation at synapses. Synaptic activity during learning triggers a transient up-regulation of new synaptic proteins in dendrites (Redondo and Morris 2011; Moncada et al. 2015), and the spatiotemporal constraints on these new proteins strongly suggest that they are translated locally (Sajikumar et al. 2007; Doyle and Kiebler 2011). We have previously found by serial section transmission electron microscopy (ssTEM) volume reconstruction that polyribosomes and translation factors are up-regulated in dendritic spines in the rat lateral amygdala (LA) 1 h after cued aversive Pavlovian conditioning (Ostroff et al. 2010, 2017; Gindina et al. 2021). These polyribosomes presumably represent translation supporting consolidation, but no studies have addressed whether dendritic translation remains elevated or returns to baseline in the long-term memory phase.Cued aversive Pavlovian conditioning, also referred to as fear or threat conditioning, is an extensively studied learning paradigm in which a sensory cue is paired with an unpleasant stimulus—typically an auditory cue with a mild shock—to create an associative memory between the two (LeDoux 2000; Maren 2001). There is strong evidence that this memory is mediated by protein synthesis-dependent strengthening of LA synapses during a short window after learning. Enhanced synaptic transmission is observed in the LA after conditioning (McKernan and Shinnick-Gallagher 1997; Rogan et al. 1997; Sah et al. 2008), and consolidation requires protein synthesis in the LA immediately after training, but not 6 or 24 h later (Nader et al. 2000; Schafe and LeDoux 2000; Maren et al. 2003). The extracellular signal-regulated/mitogen-activated protein kinase (ERK/MAPK), which regulates translation (Kelleher et al. 2004), is transiently phosphorylated in the LA 1 h after learning, and this phosphorylation is required for both memory consolidation (Schafe et al. 2000) and synaptic plasticity in the LA (Huang et al. 2000; Schafe et al. 2008).Although dormant long-term memories are stable, retrieval induces a new labile phase called reconsolidation, during which the memory can be updated, weakened, or strengthened (Dudai 2012). As in consolidation, postretrieval inhibition of protein synthesis or ERK/MAPK phosphorylation in the LA impairs reconsolidation of the memory and associated synaptic plasticity (Nader et al. 2000; Duvarci et al. 2005; Doyere et al. 2007). A transient supply of necessary new proteins is available to synapses during reconsolidation (Orlandi et al. 2020), but whether these proteins are synthesized in dendrites is unknown. Both consolidation and reconsolidation are impaired by broad protein synthesis inhibitors, and there is substantial evidence that consolidation requires translation initiation, the step in which polyribosomes are formed (Gkogkas et al. 2010; Santini et al. 2014). Interestingly, one study found that inhibition of the predominant initiation process impaired consolidation but not reconsolidation, suggesting that the role of translation differs between the two processes (Hoeffer et al. 2011). Since polyribosomes can be stalled for later reactivation (Richter and Coller 2015), reconsolidation could rely on translation of pre-existing polyribosomes.Reconsolidation is triggered by a small number of retrieval cues, but retrieval with a large number of cues induces extinction learning, in which the cue loses its ability to elicit defensive responses (Myers and Davis 2007). There is ample evidence that plasticity important for extinction occurs in the basolateral amygdala (BLA; which includes the LA), though it is unclear exactly how this relates to the original memory trace in the dorsal LA (Bouton et al. 2021). For instance, consolidation of extinction is impaired by pretraining systemic inhibition of protein synthesis (Suzuki et al. 2004) and by pretraining inhibition of protein synthesis or ERK/MAPK in the BLA (Lin et al. 2003c; Herry et al. 2006). However, the Lin et al. (2003c) study measured the effects of protein synthesis inhibition in the BLA 30 min after extinction training, which is typically thought to reflect short-term memory. Subsequent work by another group found that postextinction training inhibition of protein synthesis impaired reconsolidation, making it difficult to assess the effects on extinction consolidation (Duvarci et al. 2006). There are also ongoing debates about the relative contribution of “erasure” versus “new learning” processes in extinction. Evidence that protein synthesis-dependent depotentiation of CS inputs to the LA contributes to extinction suggests up-regulation of polyribosomes in the LA pyramidal cells storing the original trace (Lin et al. 2003a,b,c; Kim et al. 2009). However, up-regulation of polyribosomes is also possible if extinction plasticity occurs in other cells or regions of the brain, as repeated retrieval trials may strongly trigger reconsolidation processes. Complicating things further, it appears that extinction can halt reconsolidation (Suzuki et al. 2004).To investigate the dynamics of local translation in the context of an established memory, we used ssTEM to quantify dendritic polyribosome distribution in the LA during the long-term memory phase of Pavlovian conditioning, reconsolidation, and consolidation of extinction. We hypothesized that polyribosomes would not be up-regulated in the long-term memory condition relative to controls, since memory maintenance is resistant to protein synthesis inhibition at this time point. We also hypothesized that both retrieval and extinction would induce up-regulation of polyribosomes, but in different patterns; for example, reconsolidation processes could be reflected in polyribosomes near large synapses, but extinction could result in loss of these synapses and perhaps more generalized polyribosome distribution.     

Role of Aplysia cell adhesion molecules during 5-HT-induced long-term functional and structural changes

We previously reported that five repeated pulses of 5-HT lead to down-regulation of the TM-apCAM isoform at the surface of Aplysia sensory neurons (SNs). We here examined whether apCAM down-regulation is required for 5-HT-induced long-term facilitation. We also analyzed the role of the cytoplasmic and extracellular domains by overexpressing various apCAM mutants by DNA microinjection. When TM-apCAM was up-regulated in SNs by DNA microinjection, five pulses of 5-HT failed to produce either synaptic facilitation or an enhancement of synaptic growth, suggesting that down-regulation of apCAM is required for 5-HT-induced EPSP enhancement and new varicosity formation. However, disrupting the extracellular domain function of overexpressed apCAM with a specific antibody restored 5-HT-induced excitatory postsynaptic potential increase but not synaptic growth. The overexpression of the MAP Kinase mutant of TM-apCAM, which is not internalized by 5-HT, inhibited new varicosity formation, but did not inhibit excitatory postsynaptic potential increase. Deletion mutants containing only the cytoplasmic portion of apCAM blocked 5-HT-induced synaptic growth but not excitatory postsynaptic potential increase. Thus, our data suggest that TM-apCAM may act as a suppressor of both synaptic-strength enhancement in pre-existing synapses and of new synaptic varicosity formation in the nonsynaptic region, via different mechanisms.     

PKA and PKC are required for long-term but not short-term in vivo operant memory in Aplysia

We investigated the involvement of PKA and PKC signaling in a negatively reinforced operant learning paradigm in Aplysia, learning that food is inedible (LFI). In vivo injection of PKA or PKC inhibitors blocked long-term LFI memory formation. Moreover, a persistent phase of PKA activity, although not PKC activity, was necessary for long-term memory. Surprisingly, neither PKA nor PKC activity was required for associative short-term LFI memory. Additionally, PKA and PKC were not required for the retrieval of short- or long-term memory (STM and LTM, respectively). These studies have identified key differences between the mechanisms underlying nonassociative sensitization, operant reward learning, and LFI memory in Aplysia.     

Coincident orientation of objects and viewpoint-dependence in scene recognition

Viewpoint-dependence is a well-known phenomenon in which participants' spatial memory is better for previously experienced points of view than for novel ones. In the current study, partial-scene-recognition was used to examine the effect of coincident orientation of all the objects on viewpoint-dependence in spatial memory. When objects in scenes had no clear orientations (e.g., balls), participants' recognition of experienced directions was better than that of novel ones, indicating that there was viewpoint-dependence. However, when the objects in scenes were toy bears with clear orientations, the coincident orientation of objects (315 degrees), which was not experienced, shared the advantage of the experienced direction (0 degrees), and participants were equally likely to choose either direction when reconstructing the spatial representation in memory. These findings suggest that coincident orientation of objects may affect egocentric representations in spatial memory.     

Effects of reversible inactivation of locus coeruleus on long-term potentiation in perforant path-DG synapses in rats

The locus coeruleus (LC) is the main source of noradrenergic innervations to the forebrain and the hippocampal formation but does not receive noradrenergic projections itself. Previous studies have suggested that hippocampal neural response is modulated by the noradrenergic pathway and that the experimental activation of the LC can potentiate hippocampal responses. Most studies have suggested that the noradrenergic system has controversial effects on long-term potentiation (LTP) in hippocampal neurons, because its influence on synaptic plasticity in perforant path-DG synapses is ambiguous. The aim of this article was to study the LC's role in baseline activity and LTP in perforant path-DG cells of hippocampus by in vivo LC inactivation. Rats were anesthetized with urethane, and LC was temporarily suppressed by intra-LC injection of lidocaine. Population spike (PS) amplitude and excitatory postsynaptic potential (EPSP) slope in DG were recorded 10 min before and 5, 10, 20, 40, 60, and 120 min after tetanization (400 Hz). Saline or lidocaine was injected during the baseline recording (experiment 1), 5 min before tetanus (experiment 2), and 5 min after tetanus (experiment 3). The results from this study indicated that the LC inactivation has no effect on baseline activity of granular cells or maintenance of LTP after tetanization. Moreover, LC inactivation before tetanus had no effect on LTP induction but decreased PS-LTP amplitude 60 and 120 min after tetanization. Taken together, the LC noradrenergic system likely influences LTP induction in later time periods while it has no effect on LTP in earlier time periods.     

Induction of copulatory behavior in Aplysia: atrial gland factors mimic the excitatory effects of freshly deposited egg cordons

Egg laying in the marine mollusc Aplysia is induced and coordinated by peptide products of the egg-laying hormone (ELH) gene expressed in the neuroendocrine bag cells of the central nervous system. At least three structurally related genes, belonging to the ELH family but distinct from the ELH gene, are expressed in the atrial gland, an exocrine organ of unknown function that secretes into the oviduct of Aplysia. The experiments described in this report were designed to test the hypothesis that the atrial gland gene products serve a pheromonal function for the animal, coordinating reproductive behavior among individuals. Our studies showed that there was a significantly shorter latency to copulation when an Aplysia was paired with an animal that was actively laying eggs than when it was paired with a sexually mature but nonlaying animal. Moreover, the addition of extracts or homogenates of the atrial gland to the seawater surrounding two nonlaying animals reduced the latency to mating compared to animals exposed only to seawater or to homogenates of other regions of the reproductive tract, including oviduct. These results suggest that atrial gland products, secreted onto the egg cordon as it passes through the oviduct, may play a pheromonal role and induce mating behavior between individuals. Experiments are in progress to determine whether the active atrial gland factor(s) are products of the ELH-family genes expressed in the gland.