Post-training disruption of Arc protein expression in the anterior cingulate cortex impairs long-term memory for inhibitory avoidance training

The activity-regulated-cytoskeletal-associated protein (Arc) has a well established role in memory consolidation and synaptic plasticity in the hippocampus and amygdala. However the role of Arc within the anterior cingulate cortex (ACC), an area of the brain involved in processing memory for pain, has yet to be examined. Here we sought to determine if Arc protein within neurons of the rat ACC is necessary for the consolidation of a single-trial, contextual inhibitory avoidance (IA) task. Immunohistochemistry and western blotting revealed an increase in Arc protein within the ACC following IA training in a shock-specific manner, suggesting that ACC Arc expression may play a critical role in the consolidation of the aversive task. To directly test this hypothesis, male Sprague-Dawley rats were trained on the IA task and given post-training intra-ACC infusions of Arc antisense oligodeoxynucleotides (ODNs), designed to suppress Arc translation, or control scrambled ODNs that do not suppress Arc translation. Memory retention was tested 48h after training. Arc antisense-induced disruption of Arc protein expression in the ACC impaired long-term memory for the IA task as compared to rats given intra-ACC infusions of the scrambled control ODNS, suggesting that Arc expression in the ACC is important for the consolidation of emotional memory. Results further indicate that knock down of Arc 6h after training impairs IA memory. This is consistent with time course findings indicating elevated Arc expression at 3 and 6h after IA training but not 12 or 48h. Taken together, these findings support the hypothesis that Arc expression in the ACC participates in synaptic plasticity that underlies long-term memory.     

The role of protein synthesis during the labile phases of memory: revisiting the skepticism

Despite the fact that extensive evidence supports the view that phases of de novo protein synthesis are necessary for memory formation and maintenance, doubts are still raised. Skeptics generally argue that amnesia and the disruption of long-term synaptic plasticity are caused by "non-specific effects" of the reagents or approaches used to disrupt protein synthesis. This paper attempts to clarify some of these issues by reviewing, discussing and providing results addressing some of the major critiques that argue against the idea that de novo protein synthesis is necessary for the stabilization of long-term memory.     

A refined model of sleep and the time course of memory formation

Research in the neurosciences continues to provide evidence that sleep plays a role in the processes of learning and memory. There is less of a consensus, however, regarding the precise stages of memory development during which sleep is considered a requirement, simply favorable, or not important. This article begins with an overview of recent studies regarding sleep and learning, predominantly in the procedural memory domain, and is measured against our current understanding of the mechanisms that govern memory formation. Based on these considerations, I offer a new neurocognitive framework of procedural learning, consisting first of acquisition, followed by two specific stages of consolidation, one involving a process of stabilization, the other involving enhancement, whereby delayed learning occurs. Psychophysiological evidence indicates that initial acquisition does not rely fundamentally on sleep. This also appears to be true for the stabilization phase of consolidation, with durable representations, resistant to interference, clearly developing in a successful manner during time awake (or just time, per se). In contrast, the consolidation stage, resulting in additional/enhanced learning in the absence of further rehearsal, does appear to rely on the process of sleep, with evidence for specific sleep-stage dependencies across the procedural domain. Evaluations at a molecular, cellular, and systems level currently offer several sleep specific candidates that could play a role in sleep-dependent learning. These include the upregulation of select plasticity-associated genes, increased protein synthesis, changes in neurotransmitter concentration, and specific electrical events in neuronal networks that modulate synaptic potentiation.     

Memory traces compete under regimes of limited Arc protein synthesis: Implications for memory interference

Recently encoded information can be lost in the presence of new information, a process called 'retrograde interference'. Retrograde interference has been extensively described for more than a century; however, little is known about its underlying mechanisms. Different approaches agree on the need of the synthesis of plasticity related proteins (PRPs) to consolidate a long-term memory (LTM). Our hypothesis is that when PRPs are limited, interference of a task over LTM formation of another may be due to the utilization of protein resources common to both tasks. Here, by combining the tasks of inhibitory avoidance (IA) and open field (OF) exploration in rats, we show that memory traces compete for their stabilization if PRPs are limited. As a result, LTM is formed for only one of the tasks with a consequent decrease in the memory for the other. Furthermore, infusing Arc antisense oligonucleotide into the dorsal hippocampus, we found that Arc is necessary for LTM formation of these two types of learning tasks and is one of the PRPs that can be shared between them when animals are trained in both OF and IA. In sum, these findings suggest that under conditions of reduced protein availability, a learning task interferes with LTM formation of another by using the available PRPs.     

Local synthesis of proteins at synaptic sites on dendrites: role in synaptic plasticity and memory consolidation?

Long-lasting forms of memory are thought to be mediated by modifications in synapses that are induced by particular patterns of activity, take time to be established (consolidated), and involve new gene expression. The molecular mechanisms underlying such long-lasting synaptic modifications remain to be defined. Here, we focus on new findings regarding synapse-specific gene expression and especially the intracellular transport and synaptic targeting of the mRNA for a recently identified immediate early gene called activity-regulated cytoskeleton-associated protein (Arc). Arc mRNA is transported into dendrites after episodes of neuronal activation. The newly synthesized Arc mRNA localizes selectively at synapses that have experienced particular patterns of activity, and this targeting depends on NMDA receptor activation. Arc protein is assembled into the postsynaptic junction, and Arc expression is strongly induced by brief behavioral experiences in select populations of neurons. These features of Arc induction and trafficking reveal novel cellular mechanisms that are well suited to mediate long-term synapse-specific modifications. We will review findings from other laboratories that both LTP and memory are disrupted when Arc induction is blocked, and we will discuss the possibility that Arc protein may play a key role in activity-dependent synaptic modification.     

The many faces of amnesia

Results from studies of retrograde amnesia provide much of the evidence for theories of memory consolidation. Retrograde amnesia gradients are often interpreted as revealing the time needed for the formation of long-term memories. The rapid forgetting observed after many amnestic treatments, including protein synthesis inhibitors, and the parallel decay seen in long-term potentiation experiments are presumed to reveal the duration of short-term memory processing. However, there is clear and consistent evidence that the time courses obtained in these amnesia experiments are highly variable within and across experiments and treatments. The evidence is inconsistent with identification of basic temporal properties of memory consolidation. Alternative views include modulation of memory and emphasize the roles that hormones and neurotransmitters have in regulating memory formation. Of related interest, converging lines of evidence suggest that inhibitors of protein synthesis and of other biochemical processes act on modulators of memory formation rather than on mechanisms of memory formation. Based on these findings, memory consolidation and reconsolidation studies might better be identified as memory modulation and "remodulation" studies. Beyond a missing and perhaps unattainable time constant of memory consolidation, some current views of memory consolidation assume that memories, once formed, are generally unmodifiable. It is this perspective that appears to have led to the recent interest in memory reconsolidation. But the view adopted here is that memories are continually malleable, being updated by new experiences and, at the same time, altering the memories of later experiences. Studies of memory remodulation offer promise of understanding the neurobiological bases by which new memories are altered by prior experiences and by which old memories are altered by new experiences.     

Synaptic tagging, evaluation of memories, and the distal reward problem

Long-term synaptic plasticity exhibits distinct phases. The synaptic tagging hypothesis suggests an early phase in which synapses are prepared, or "tagged," for protein capture, and a late phase in which those proteins are integrated into the synapses to achieve memory consolidation. The synapse specificity of the tags is consistent with conventional neural network models of associative memory. Memory consolidation through protein synthesis, however, is neuron specific, and its functional role in those models has not been assessed. Here, using a theoretical network model, we test the tagging hypothesis on its potential to prolong memory lifetimes in an online-learning paradigm. We find that protein synthesis, though not synapse specific, prolongs memory lifetimes if it is used to evaluate memory items on a cellular level. In our model we assume that only "important" memory items evoke protein synthesis such that these become more stable than "unimportant" items, which do not evoke protein synthesis. The network model comprises an equilibrium distribution of synaptic states that is very susceptible to the storage of new items: Most synapses are in a state in which they are plastic and can be changed easily, whereas only those synapses that are essential for the retrieval of the important memory items are in the stable late phase. The model can solve the distal reward problem, where the initial exposure of a memory item and its evaluation are temporally separated. Synaptic tagging hence provides a viable mechanism to consolidate and evaluate memories on a synaptic basis.     

The role of protein synthesis in memory consolidation: progress amid decades of debate

A major component of consolidation theory holds that protein synthesis is required to produce the synaptic modification needed for long-term memory storage. Protein synthesis inhibitors have played a pivotal role in the development of this theory. However, these commonly used drugs have unintended effects that have prompted some to reevaluate the role of protein synthesis in memory consolidation. Here we review the role of protein synthesis in memory formation as proposed by consolidation theory calling special attention to the controversy involving the non-specific effects of a group of protein synthesis inhibitors commonly used to study memory formation in vivo. We argue that molecular and genetic approaches that were subsequently applied to the problem of memory formation confirm the results of less selective pharmacological studies. Thus, to a certain extent, the debate over the role of protein synthesis in memory based on interpretational difficulties inherent to the use of protein synthesis inhibitors may be somewhat moot. We conclude by presenting avenues of research we believe will best provide answers to both long-standing and more recent questions facing field of learning and memory.     

Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions

Considerable evidence indicates that glucocorticoid hormones enhance the consolidation of memory for emotionally arousing events through interactions with the noradrenergic system of the basolateral complex of the amygdala (BLA). We previously reported that intra-BLA administration of a β-adrenoceptor agonist immediately after inhibitory avoidance training enhanced memory consolidation and increased hippocampal expression of the protein product of the immediate early gene activity-regulated cytoskeletal-associated protein (Arc). In the present experiments corticosterone (3 mg/kg, i.p.) was administered to male Sprague-Dawley rats immediately after inhibitory avoidance training to examine effects on long-term memory, amygdala norepinephrine levels, and hippocampal Arc expression. Corticosterone increased amygdala norepinephrine levels 15 min after inhibitory avoidance training, as assessed by in vivo microdialysis, and enhanced memory tested at 48 h. Corticosterone treatment also increased expression of Arc protein in hippocampal synaptic tissue. The elevation in BLA norepinephrine appears to participate in corticosterone-influenced modulation of hippocampal Arc expression as intra-BLA blockade of β-adrenoceptors with propranolol (0.5 μg/0.2 μL) attenuated the corticosterone-induced synaptic Arc expression in the hippocampus. These findings indicate that noradrenergic activity at BLA β-adrenoceptors is involved in corticosterone-induced enhancement of memory consolidation and expression of the synaptic-plasticity-related protein Arc in the hippocampus.     

Consolidation and translation regulation

mRNA translation, or protein synthesis, is a major component of the transformation of the genetic code into any cellular activity. This complicated, multistep process is divided into three phases: initiation, elongation, and termination. Initiation is the step at which the ribosome is recruited to the mRNA, and is regarded as the major rate-limiting step in translation, while elongation consists of the elongation of the polypeptide chain; both steps are frequent targets for regulation, which is defined as a change in the rate of translation of an mRNA per unit time. In the normal brain, control of translation is a key mechanism for regulation of memory and synaptic plasticity consolidation, i.e., the off-line processing of acquired information. These regulation processes may differ between different brain structures or neuronal populations. Moreover, dysregulation of translation leads to pathological brain function such as memory impairment. Both normal and abnormal function of the translation machinery is believed to lead to translational up-regulation or down-regulation of a subset of mRNAs. However, the identification of these newly synthesized proteins and determination of the rates of protein synthesis or degradation taking place in different neuronal types and compartments at different time points in the brain demand new proteomic methods and system biology approaches. Here, we discuss in detail the relationship between translation regulation and memory or synaptic plasticity consolidation while focusing on a model of cortical-dependent taste learning task and hippocampal-dependent plasticity. In addition, we describe a novel systems biology perspective to better describe consolidation.     

Genetic approaches to the study of synaptic plasticity and memory storage

Long-term memory is believed to depend on long-lasting changes in the strength of synaptic transmission known as synaptic plasticity. Understanding the molecular mechanisms of long-term synaptic plasticity is one of the principle goals of neuroscience. Among the most powerful tools being brought to bear on this question are genetically modified mice with changes in the expression or biological activity of genes thought to contribute to these processes. This article reviews how strains of mice with alterations in the cyclic adenosine monophosphate/protein kinase A/cyclic adenosine monophosphate-response element-binding protein signaling pathway have advanced our understanding of the biological basis of learning and memory.     

The NO-cGMP-PKG signaling pathway regulates synaptic plasticity and fear memory consolidation in the lateral amygdala via activation of ERK/MAP kinase

Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and long-term potentiation (LTP) at thalamic and cortical input pathways to the LA. In behavioral experiments, rats given intra-LA infusions of either the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibited dose-dependent impairments or enhancements of fear memory consolidation, respectively. In slice electrophysiology experiments, bath application of Rp-8-Br-PET-cGMPS or the guanylyl cyclase inhibitor LY83583 impaired LTP at thalamic, but not cortical inputs to the LA, while bath application of 8-Br-cGMP or the guanylyl cyclase activator YC-1 resulted in enhanced LTP at thalamic inputs to the LA. Interestingly, YC-1-induced enhancement of LTP in the LA was reversed by concurrent application of the MEK inhibitor U0126, suggesting that the NO-cGMP-PKG signaling pathway may promote synaptic plasticity and fear memory formation in the LA, in part by activating the ERK/MAPK signaling cascade. As a test of this hypothesis, we next showed that rats given intra-LA infusion of the PKG inhibitor Rp-8-Br-PET-cGMPS or the PKG activator 8-Br-cGMP exhibit impaired or enhanced activation, respectively, of ERK/MAPK in the LA after fear conditioning. Collectively, our findings suggest that an NO-cGMP-PKG-dependent form of synaptic plasticity at thalamic input synapses to the LA may underlie memory consolidation of Pavlovian fear conditioning, in part, via activation of the ERK/MAPK signaling cascade.     

Endogenous BDNF is required for long-term memory formation in the rat parietal cortex

Information storage in the brain is a temporally graded process involving different memory phases as well as different structures in the mammalian brain. Cortical plasticity seems to be essential to store stable long-term memories, although little information is available at the moment regarding molecular and cellular events supporting memory consolidation in the neocortex. Brain-derived neurotrophic factor (BDNF) modulates both short-term synaptic function and activity-dependent synaptic plasticity in hippocampal and cortical neurons. We have recently demonstrated that endogenous BDNF in the hippocampus is involved in memory formation. Here we examined the role of BDNF in the parietal cortex (PCx) in short-term (STM) and long-term memory (LTM) formation of a one-trial fear-motivated learning task in rats. Bilateral infusions of function-blocking anti-BDNF antibody into the PCx impaired both STM and LTM retention scores and decreased the phosphorylation state of cAMP response element-binding protein (CREB). In contrast, intracortical administration of recombinant human BDNF facilitated LTM and increased CREB activation. Moreover, inhibitory avoidance training is associated with a rapid and transient increase in phospho-CREB/total CREB ratio in the PCx. Thus, our results indicate that endogenous BDNF is required for both STM and LTM formation of inhibitory avoidance learning, possibly involving CREB activation-dependent mechanisms. The present data support the idea that early sensory areas constitute important components of the networks subserving memory formation and that information processing in neocortex plays an important role in memory formation.     

The substrate for long-lasting memory: If not protein synthesis,then what?

The prevailing textbook view that de novo protein synthesis is required for memory (e.g., [Bear, M. F., Connors, B., & Paradiso, M. 2006. Neuroscience. Lippincott, New York]) is seriously flawed and an alternative hypothesis has been proposed in which post-translational modification (PTM) of proteins already synthesized and already present within the synapse is 'the' substrate for long-lasting memory. Protein synthesis serves a replenishment role. The first part of this review discusses how long-lasting memory can be achieved with 'only' PTM of existing synaptic proteins. The second part critically reviews a recent report published in Neuron 2007 that exemplifies the current view of protein synthesis and memory while also illustrating how these results can be understood within this new PTM framework. A necessary yet unexpected conclusion to emerge from consideration of the consequences of a PTM mechanism as the necessary, sufficient and exclusive substrate for long-lasting memory, is that the central Hebbian dogma that cells that 'fire together, wire together' is an unlikely mechanism for long-lasting memory. Thus, a unique feature of the PTM model is that longevity of information storage is achieved not by stability of the synaptic mechanism, but by impermanent pseudoredundant circuits. This is so because PTM is a reversible process and thus any permanent connection, any 'lasting effect' cannot be in the form of stable synapse formation. We have therefore proposed a solution in which network level processes regulate cellular mechanisms, even as such mechanisms regulate the network. Thus, synapses are 'meta-stabilized' by regulated feedback mediated by the circuit in which the synapse is embedded. For example, spontaneous activity is proposed to be a substrate feedback mechanism we term 'cryptic rehearsal' to sustain for some period of time after learning an approximation to the state initially created by input. Additionally, because the duplication of these traces is ongoing, this provides a degenerate code for the engram. Stability is thus achieved, not by stabilizing the synapse, but by implementing a pseudo-redundant yet malleable circuitry so that memory can be protected in the face of small catastrophes in network representation.     

LTP maintenance and its protein synthesis-dependence

The properties of long-term potentiation (LTP) mirror those of associative memory in a number of interesting ways. Although plasticity at monosynaptic connections is not expected to account for the varied subtle characteristics of distributed memories, nonetheless it is important to establish how far the parallels can be drawn. Here, we briefly address whether properties of LTP such as its duration, reversibility, savings and reconsolidation relate to corresponding memory phenomena. We then address whether LTP stabilization in fact requires protein synthesis, as this has been challenged in recent times much like the necessity for protein synthesis in the consolidation of long-term memory has been queried. We conclude that the case is still very strong for a necessary role of protein synthesis in LTP stabilization, even though the identities of the synthesized proteins and their contributions to the LTP process are not fully understood. However, we highlight areas of research that could be usefully conducted to further our understanding of the properties and protein synthesis-dependence of LTP.