Memory enhancement: consolidation,reconsolidation and insulin-like growthfactor 2Cristina M. Alberini1 and Dillon Y. Chen2

1 Center for Neural Science, New York University, New York, NY 10003, USA2 Mount Sinai School of Medicine, New York, NY 10029, USA

Review

Life and societies would change significantly if memorycapacity or persistence in health and disease could beenhanced. It has been known for many years that mem-ory can be improved and strengthened. Substancesknown to enhance memory include hormones, neuro-transmitters, neuropeptides and metabolic substrates.Recently, attention has been given to identifying themolecular mechanisms and targets whereby memoryenhancement can be achieved. One approach wouldbe to target the physiological changes that are inducedby learning and naturally required for memory strength-ening via consolidation and reconsolidation. Here, wereview approaches that boost memories by targetingthe cAMP response element binding protein-CCAAT en-hancer binding protein (CREB-C/EBP) pathway and/orits recently identified target gene insulin-like growthfactor 2 (IGF2).

IntroductionThe continuing increase in life span is unfortunately asso-ciated with an increased prevalence of cognitive dysfunc-tions, including memory loss and diminished ability toform new long-term memories. Aging-related memory im-pairment is a common condition characterized by mildsymptoms of cognitive decline. The aged populationbecomes slower in processing, storing and recalling newinformation, and shows impairments in cognitive function-ing, including memory, concentration and organization. ‘Iforget names so easily now’ or ‘I forget where I put thingsall the time’ are common complaints of the aged. Moredramatic and devastating are the cognitive and memorylosses in neurodegenerative diseases, such as Alzheimer’sdisease (AD), in which the impairments interfere withnormal life functioning. The economic impact of AD inthe USA alone is estimated to be more than US$100 billionannually [1]. Thus, it is imperative to identify or developapproaches that can restore or prevent memory impair-ments. Furthermore, ethical debates aside, the possibilityof speeding normal learning and boosting memory reten-tion is appealing to the healthy population. Hence, identi-fying memory enhancers is of great importance andinterest.

Corresponding author: Alberini, C.M. (ca60@nyu.edu).Keywords: cognitive enhancer; memory consolidation; reconsolidation; growth factor.

274 0166-2236/$ – see front matter � 2012 Elsevier Ltd. All rights re

Numerous studies have indicated that memory can beenhanced with many types of strategy or drug that caneither boost the baseline of the biological systems ormodulate memory strengthening or retrieval. These drugsmodulate neurotransmission, neurotransmitter recep-tors, neuropeptide [2–4], stress and related hormones[5,6] and metabolism [7,8]. The literature on this topicand the list of effective drugs is vast. Here, we focus ourdiscussions on an ensemble of mechanisms that can betargeted to boost memories because they underlie thememory processes known as consolidation and reconsoli-dation. Indeed, one approach that can be employed tosearch for mechanisms and targets of memory enhance-ment is to identify and exploit the biological mechanismsby which learned information transforms into long-lastingmemory. Newly learned information becomes a long-termmemory through a process known as consolidation. Thisprocess converts the new labile memory trace into a stron-ger one that is resilient to disruption [9]. Consolidationrequires an initial phase of de novo gene expression that inanimal models of temporal lobe-dependent memoriestakes place over the first 24 h after training; its interrup-tion results in memory loss [10,11]. Moreover, once stableand resilient to disruption, memories can again becomelabile and sensitive to interference if they are reactivatedby, for example, recalling the memory. Memories thenregain their resilience to interference by undergoing aprocess known as reconsolidation [12–15]. In several typesof memory, reconsolidation has been found to be tempo-rally limited. In fact, in rodents, only young memories(weeks-old), not old (months-old) ones are lost after am-nesic agents are given together with memory reactivation[16–20]. It has been proposed that reconsolidation is aphase of lingering consolidation that in rodents can last forweeks [15,21]. If memory is retrieved during this sensitivephase, it undergoes reconsolidation to strengthen thememory itself, indicating that an important function ofthe post-retrieval reconsolidation process is to promotememory strengthening and persistence [15,22]. Thus, inprinciple, a strategy that potentiates either the initialconsolidation or the reconsolidation phases may be suc-cessfully exploited as an effective cognitive enhancer. Inagreement with this hypothesis, various gene mutationstargeting the pathways known to be involved in long-term plasticity or memory formation result in memory

served. doi:10.1016/j.tins.2011.12.007 Trends in Neurosciences, May 2012, Vol. 35, No. 5

Review Trends in Neurosciences May 2012, Vol. 35, No. 5

enhancement. Excellent reviews have summarized anddiscussed these mutants [23,24].

In this review, we discuss the hypothesis that memorycan be enhanced by targeting a variety of steps of thecAMP-CREB-C/EBP-dependent gene cascade, which hasbeen shown to be required for memory consolidation innumerous species [25]. After a brief summary of currentknowledge, we expand on the effect of a target gene, IGF2,(also known as IGFII), which is regulated by the CREB-C/EBP pathway [26]. We also discuss memory enhancementin reconsolidation and relative temporal constraints andconclude with some unresolved questions about memoryenhancement and its potential clinical applications.

Promoting memory enhancement by targeting thecAMP-CREB-C/EBP pathwayStudies of the molecular bases of long-term memory for-mation started with the findings in simple invertebratesthat cAMP and the induction of CREB- and C/EBP-depen-dent gene expression have a key function in synapticplasticity and memory formation [27–29], particularlyduring the temporal window of consolidation, when mem-ory is still labile [9,30,31]. Specifically, in invertebrates,including Aplysia californica and Drosophila melanoga-ster, the molecular disruption of CREB or C/EBP isoformsleads to memory impairment [25]. Similarly, molecularand genetic disruptions of either CREB or C/EBP isoformsin rodents impair different types of long-term memory,including spatial, contextual, appetitive and aversive,leading to the conclusion that the cAMP-CREB-C/EBPpathway has an evolutionarily conserved, fundamentalrole in long-term memory formation [25,32–34]. Someauthors have found that the essential function of CREBin memory formation may be overcome by genetic back-grounds and that CREB-independent forms of long-termplasticity and memory exist [35]. Despite the fact thatsome form of memory and/or long-term potentiation(LTP) may indeed be CREB independent, these contrast-ing effects may be due to the result of protein compensationof the transgene that can overcome the requirement forCREB. Hence, in summary, numerous studies across spe-cies, brain regions and types of learning paradigm indicatethat CREB is critical for memory formation. Despite thepossibility that memory consolidation may also be en-hanced independently of the CREB pathway, in this re-view, we focus on discussing nodes of the CREB-C/EBPpathway that represent important targets for promotingmemory enhancement and persistence.

Several intracellular signaling pathways have beenshown to activate the CREB-C/EBP cascade [25](Figure 1), which include important targets for both mem-ory disruption and strengthening. Selective modulation ofspecific components of this pathway may lead to the en-hancement of memories and, furthermore, make themmore persistent. Indeed, studies have demonstrated thatthis is the case.

The expression of an activated form of CREB in bothDrosophila melanogaster and neuronal cultures of Aplysiacalifornica was found to lead to long-term memory and/orplasticity from learning or stimulation paradigms thatevoke only short-term memory or plasticity [28,36] (but

see [37]). Similarly, overexpression of C/EBP in Aplysianeurons converted short-term plasticity into long-termplasticity [38]. These findings were the first evidence that,if the molecular cascade involved in long-term plasticityand memory formation is overstimulated, memory reten-tion can be enhanced or made more persistent.

Similar results were found in mammalian systems.Overexpression of a neuronal-specific adenylyl cyclase 1(AC1), an enzyme that catalyzes the synthesis of cAMP, inthe forebrain of mice, resulted in enhanced object recogni-tion and contextual fear-conditioning memories [39]. Injec-tions of a protein kinase A (PKA) activator, Sp-cAMPs, intothe amygdala enhanced reward-related learning in a dose-dependent manner [40]. Furthermore, rolipram, an inhib-itor of the phosphodiesterase 4 (PDE4) family of enzymes,which catalyze cAMP hydrolysis and thereby increasecAMP concentrations and CREB phosphorylation, signifi-cantly enhanced memory retention in rodent contextualfear conditioning [41], object recognition [42] and severalspatial tasks [43]. In agreement with these results, recentstudies have shown that both PDE4 subtype D (PDE4D)-deficient mice and mice in which PDE4D in the dorsalhippocampus was knocked down by lentiviral vectors, haveenhanced spatial and object recognition memories [44].Inhibitors of other PDEs, for example, the selectivePDE5 inhibitors sildenafil, zaprinast and vardenafil, whichcan lead to an increase in CREB phosphorylation, havealso shown enhancing effects in spatial object recognitionand active avoidance memories in rodents [45,46], al-though others have reported contrasting findings [47].

Memory-enhancing effects have also been found bydirectly increasing the expression of CREB. Viral vector-mediated overexpression of CREB in the rat amygdala ormouse hippocampus enhanced cued and contextual fearconditioning, respectively [48,49]. Transgenic overexpres-sion of dominant active forms of CREB in the forebrainresulted in enhanced memory retention in a variety oftasks, including fear conditioning, social recognition, theMorris water maze and passive avoidance [50].

Boosting CREB-dependent transcription activity is likelyto promote memory enhancement by targeting many mech-anistic levels. For example, CREB regulates the transcrip-tion of proteins that are necessary for the stabilization ofmemory after learning. Thus, enhancing CREB activity mayaugment the levels of these critical proteins, resulting instronger memories. A series of elegant studies has increasedthe understanding of how CREB regulates memory forma-tion, thereby mediating memory retention. In the lateralamygdala, neurons with viral-mediated overexpression ofCREB are more likely to be active after memory retrievalthan are their neighboring neurons, and selective ablation ofthese CREB-overexpressing neurons leads to memory im-pairment [51,52], suggesting that auditory fear conditioningcan recruit CREB-expressing neurons in the lateral amyg-dala to establish fear memory formation. Furthermore,viral-mediated overexpression of CREB in the amygdalaenhances synaptic transmission and increases neuronalexcitability [53]. Similar increases in neuronal excitabilityare seen in the dorsal hippocampus of transgenic miceexpressing a constitutively active form of CREB [54], lead-ing to the conclusion that memory enhancement correlates

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Figure 1. Schematic representation of the cAMP response element binding protein (CREB)-CCAAT enhancer binding protein (C/EBP) pathway activated during memory

formation. Neurotransmitters, growth factors and membrane depolarization are examples of the stimuli that activate intracellular signal transduction pathways that can

lead to the activation of the CREB-dependent pathway. Growth factors signal via receptor tyrosine kinase (RTK), which upon ligand binding and dimerization, induces

activation of two pathways: the Ras/Raf/mitogen-activated protein kinase (MAPK)/MAP kinase kinase (MEK) and the phosphatidylinositol 3-kinase (PI3K)-dependent

pathways. Activations of these pathways recruit additional protein kinases, including p90 ribosomal S6 kinase (RSK2) and mitogen- and stress-activated protein kinase

(MSK) for the MAPK-dependent pathway and Akt and p70S6 kinase(p70S6K) for the PI3K-dependent pathway to catalyze phosphorylation of CREB (pCREB) in its Ser-133

residue, which is an important step for its activation [25]. CREB phosphorylation can also be achieved by many neurotransmitters binding to their receptors. Via these

receptors, neurotransmitters can couple cAMP by regulating adenylyl cyclase (AC) activity. cAMP recruits protein kinase A (PKA) as the main kinase for CREB

phosphorylation. Phosphodiesterase (PDE) can catalyze the hydrolysis of cAMP and inhibit its signaling. Additionally, increases in intracellular Ca2+ influx through voltage-

or ligand-gated cation channels, such as voltage-sensitive calcium channels (VSCCs) or NMDA receptors (NMDARs), can also lead to CREB phosphorylation via different

calcium-dependent protein kinases [25]. Once phosphorylated, CREB recruits its transcription coactivator CREB-binding protein (CBP) to promote transcription [62]. The

functional activation of CREB leads to the expression of target genes, among which are immediate early genes (IEGs), such as the transcription factor C/EBP [27,108], which,

in turn, regulates the expression of late response genes, including insulin-like growth factor 2 (IGF2) [26]. Transcriptional regulation is further regulated by the chromatin

state. In general, histone acetyl transferases (HATs) acetylate histone tails and promote a relaxed chromatin state and transcription. Histone deacetylases (HDACs), by

contrast, de-acetylate histones and promote a condensed chromatin state and gene silencing [61].

Review Trends in Neurosciences May 2012, Vol. 35, No. 5

with increased neuronal excitability that is mediated, atleast in part, by increased CREB expression and/or itsfunctions.

Furthermore, activation of the CREB-C/EBP cascade isregulated by inhibitory constraints [55–58], the removal ofwhich results in stronger or more persistent memories. Forexample, mutant mice with reduced expression of thegeneral control non-repressed 2 (GCN2) kinase, an eukary-otic initiation factor 2 a (eIF2a) kinase, have suppressedexpression of activating transcription factor 4 (ATF4)mRNA, a repressor of CREB-mediated gene expression,and display enhanced spatial memory, as assessed in theMorris water maze [59]. Likewise, mutant mice with re-duced phosphorylation of eIF2a exhibit enhanced memoryin a variety of tasks [60]. Similarly, transgenic mice withforebrain expression of a general dominant negative inhib-itor of the C/EBP/ATF family (EGFP-AZIP) have enhancedspatial memory retention. EGFP-AZIP expression presum-ably relieves the inhibitory regulators and results in alower threshold for LTP induction as well as increased

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spatial memory retention [57]. In summary, any molecularstep that regulates the induction of the CREB- and/or C/EBP-dependent gene expression may, in principle, be agood target for promoting memory enhancement.

As with all transcription factors, CREB and C/EBP func-tions on gene expression are controlled by the state andregulation of chromatin. Chromatin modification can mod-ulate the transcription necessary for memory formation byregulating access of transcriptional regulatory proteins toDNA. For example, histone acetylation is a chromatin mod-ification that regulates DNA–histone interactions via twoopposing classes of enzyme: histone acetyltransferases(HATs), which acetylate histone tails and promote tran-scription, and histone deacetylases (HDACs), which de-acetylate histones and promote genes silencing [61]. Oneimportant function of CREB is to recruit CREB-bindingprotein (CBP), a cofactor with intrinsic histone acetyl trans-ferase activity, which results in increased gene transcription[62]. Genetic mutations in CBP are associated with Rubin-stein-Taybi syndrome, which is characterized by facial

Review Trends in Neurosciences May 2012, Vol. 35, No. 5

abnormalities, broad thumbs and mental retardation [63].Mutant mice lacking one functional copy of CBP exhibitimpaired long-term inhibitory avoidance and contextualfear-conditioning memories [64]. More specifically, trans-genic mice with a functional loss of the HAT activity of CBPhave impaired long-term memory formation [65], whereasinjections of HDAC inhibitors, which would compensate fordeficits in CBP activity in CBP-mutant mice, rescue thememory impairment [65,66] Similarly, PDE4 inhibitors,which enhance CREB signaling, also dose dependentlyrescue the long-term memory defects in CBP mutants[67]. These studies suggest that modulating CREB-depen-dent signaling or the state of chromatin via HDAC inhibitorsare potential therapeutic options for cognitive disordersassociated with CBP dysfunction. In agreement with thishypothesis, in wild-type mice, HDAC inhibitors have beenfound to enhance long-term memory in a variety of tasks,including contextual fear conditioning [68], water maze [69],fear-potentiated startle [70], extinction of fear memory [71]and novel object recognition [72]. Several extensive reviewshave been published on HDAC inhibitors and their potentialapplications to treat cognitive disorders [73–75].

One step downstream of the activation of CREBs and C/EBPs is regulation of the expression of their target genes.Some of these target genes may include secreted factors ormembrane receptors that can be more easily targeted bypharmacological approaches. One of these genes, IGF2,was recently identified [26].

Proliferation Surviv

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Figure 2. The insulin-like growth factor (IGF)-related system components. The IGF-relate

circulation are regulated by IGF binding proteins (BPs). There are multiple receptor conf

Two isoforms of IR, IR-A and IR-B, are found in the brain [109]. For simplicity, only sign

phosphorylation of adaptor proteins belonging to the IR substrate (IRS) family or Src hom

SHC leads to activation of Raf/Ras/mitogen-activated protein kinase (MAPK)/MAP

Phosphorylation of Akt leads to subsequent activation of mammalian target of rapam

(S6K). Activation of these pathways leads to enhanced proliferation, survival and metas

the highest affinity to IGF-2R. IGF-2R is structurally distinct from IR and IGF-1R, and is no

mediated lysosomal degradation as well as effecting signal transduction [85] (Fi

phosphoinositide-dependent kinase-1; PIP3, phosphatidylinositol (3,4,5)-trisphosphate.

IGF2, a C/EBP target gene required for memoryconsolidation, promotes memory enhancementIGF2 has been recently identified as a target gene of C/EBPb during memory consolidation in rats [26]. IGF-2 is amitogenic polypeptide that is structurally similar to insu-lin and IGF-1. Insulin, IGFs and their respective receptors,together with IGF-binding proteins (IGFBP), constitutethe IGF-related system (Figure 2). This protein systemis important for normal somatic growth and development,tissue repair and regeneration [76]. Compared with theother family members, IGF-2 is more abundantlyexpressed in the adult brain and is found in regions thatare critically involved in memory consolidation, such as thehippocampus and cortex [77]. The mechanism of action ofIGF-2 is not clear and may be multifaceted [78]. Thehighest affinity of IGF-2 is for IGF-2 receptor (IGF-2R).Beside IGF-2, IGF-2R binds a large number of ligands,including lysosomal enzymes, transforming growth factor-b (TGF-b), granzyme B, plasminogen, glycosylated leuke-mia inhibitory factor and retinoids [79]. Most IGF-2Rs, alsoknown as cation-independent mannose 6 phosphate recep-tors, are located intracellularly where they facilitate thetrafficking of lysosomal enzymes between the trans-Golginetwork, endosomes and lysosomes [79]. Cell-surface IGF-2Rs sequester circulating and local levels of IGF-2, therebyleading to IGF-2 degradation. IGF-2Rs can also mediatethe endocytosis of extracellular lysosomal enzymes [80].IGF-2 can also bind with lower affinity to IGF-1 receptors

al Metastasis

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d system comprises three ligands: insulin, IGF-1 and IGF-2. IGF-1 and IGF-2 levels in

ormations. Insulin and IGF-1 receptor (IR and IGF-1R) are tyrosine kinase receptors.

aling initiated by the activated IGF-1R is shown. Activation of IR or IGF-1R leads to

ology 2 domain-containing transforming protein (SHC) [110]. Activation of IRS and

kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways.

ycin (mTOR), eukaryotic translation initiation factor 4E (eIF4E) and p70S6 kinase

tasis in cancer cells [110]. IGF-2 binds IR-A [111], IGF-1R and IGF-2R [110], and with

t a receptor tyrosine kinase. Once IGF-2 binds, IGF-2R targets IGF-2 to endocytosis-

gure 4). Abbreviations: Grb2, growth factor receptor-bound protein 2; PDKI,

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(IGF-1Rs), which results in the downstream activation oftypical tyrosine kinase-mediated growth pathways [78].

IGF-2 mRNA and protein levels were found to be upre-gulated in the rat hippocampus 20 h after inhibitoryavoidance training [26]. Inhibitory avoidance is a contex-tual fear conditioning-based task that requires an intacthippocampus and amygdala, and is used as a model oftemporal lobe-dependent memories. This training-depen-dent induction in the hippocampus was dependent on thebinding of C/EBPb to IGF-2 promoters [26], in agreementwith previous findings indicating that the IGF-2 promo-ters bear C/EBPb consensus sequences [81]. Furthermore,either antisense-mediated knockdown of IGF-2 in thedorsal hippocampus or injections of a function-neutraliz-ing anti-IGF-2R antibody significantly impaired long-term inhibitory avoidance memory, suggesting that thelearning-dependent increase in IGF-2 (via IGF-2R) isrequired for inhibitory avoidance memory consolidation

Training Test 1

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Figure 3. Insulin-like growth factor 2 (IGF-2) enhances fear as well as fear extinction mem

received bilateral intrahippocampal injections of IGF-1 (grey bars), IGF-2 (black bars), or veh

and 7 days (Test 2) after training. Rats that received IGF-2, compared with IGF-1 and ve

intrahippocampal injections of IGF-2 immediately after inhibitory avoidance training, com

with IGF-2 immediately after inhibitory avoidance training showed significantly higher me

solution showed significant memory retention decay [26]. (b) Rats underwent contextual/a

and freeze in subsequent exposure to the training context or to the tone in a different c

injections of IGF-2 (black bars) or vehicle (white bars; indicated by #). Rats that were injecte

when they were tested in the training context. By contrast, no effect was seen on the audito

injected with vehicle [26]. (c) Rats underwent inhibitory avoidance training and were

intrahippocampal injection of either IGF-2 (black bars) or IGF-1 (grey bars), whereas the oth

thereafter received similar bilateral intrahippocampal injections (indicated by #). All group

when given 24 h after training (NoR final test), but, if given in concert with memory retr

underwent contextual fear conditioning and context-dependent freezing was assessed 2

intervals consisting of re-exposure to the training context without footshock. Fear extinctio

the dorsal hippocampus immediately after each extinction trial (indicated by #) compared w

% freezing � s.e.m.; *P <0.05, **P <0.01, ***P <0.001. Adapted, with permission, from [

278

[26]. Conversely, injections of recombinant IGF-2 into thedorsal hippocampus immediately after training, but not24 h later, significantly and dose-dependently enhancedlong-term inhibitory avoidance and contextual fear mem-ory retention (Figure 3a,b), as well as preventing memoryloss (Figure 3a) [26]. In fact, rats given a single hippocam-pal injection of IGF-2 immediately after training still hadsignificant memory retention 3 weeks after training,whereas vehicle-injected control rats had largely forgotten(Figure 3a) [26]. Injections of IGF-2 into the amygdala hadno effect on memory retention, suggesting that the mech-anisms regulated by IGF-2 in memory enhancement tar-get selective regions, such as the hippocampus, but not theamygdala [26]. Furthermore, hippocampal injections ofIGF-2 have also been found to facilitate fear extinction inmice (Figure 3d) [82], confirming and extending the con-clusion that IGF-2 promotes memory enhancement. It willbe important to investigate other brain regions as targets

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IGF-2

Veh

Key:

Veh

IGF-2

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ories. (a) Rats underwent inhibitory avoidance training, immediately after which they

icle solution (white bars; indicated by #). Memory retention was tested at 24 h (Test 1)

hicle solution, had significantly higher memory retention at both tests [26]. Similar

pared with vehicle solution, significantly prevented memory forgetting. Rats injected

mory retention at 3 weeks after training (Test), whereas the rats that received vehicle

uditory fear conditioning, in which they associate a context and a tone to a footshock

ontext. Immediately after conditioning, the rats received bilateral intrahippocampal

d with IGF-2, compared with vehicle solution, had a significantly higher freezing score

ry fear-conditioning test, as rats injected with IGF-2 had freezing scores similar to rats

divided into two groups. Twenty-four hours later, one group received a bilateral

er group underwent inhibitory avoidance memory retrieval (Test 1) and immediately

s were tested for memory retention 48 h after training (Final test). IGF-2 had no effect

ieval, compared with IGF-1, significantly enhanced memory retention [26]. (d) Mice

4 h later. Extinction of contextual fear was performed on consecutive days at 24-h

n was significantly enhanced in mice that received injections of IGF-2 (red circles) into

ith the vehicle-injected group (grey squares) [82]. Data are shown as mean latency or

26] (A–C) and [82] (D).

Review Trends in Neurosciences May 2012, Vol. 35, No. 5

of IGF-2-mediated memory enhancement, such as thecortex. Such future studies will help to address questionsof region-specific mechanisms and/or selective effects onspecific memory systems in memory enhancement.

IGF-2 is an interesting candidate for potential clinicalapplications because it is a naturally produced growth factorthat readily crosses the blood–brain barrier. In the adult rat,IGF-2 mRNA can be found in the brain, heart, kidney,uterus and liver [83], and peripheral IGF-2 can be trans-ported into the brain via IGF-2Rs localized in brain capillar-ies [84]. The temporally limited effect of hippocampallyinjected IGF-2 that occurs from the time of training (i.e.<24 h) as well as that of the post-retrieval effect in young butnot old memories (discussed below) indicates that bothconsolidation and reconsolidation mechanisms can be tar-geted to produce memory enhancement. Mechanistically,IGF-2-dependent memory enhancement requires the func-tion of IGF-2R, but not IGF-1R [26]. IGF-2R, unlike insulin-and IGF-1R, is not a tyrosine kinase receptor [85]. Whereasinsulin and IGF-1R mediate growth and activate the ras/raf/mitogen-activated protein kinase (MAPK) and phosphati-dylinositol 3-kinase (PI3K)/Akt pathways (Figure 2), IGF-2R/mannose-6-phosphate receptor binds IGF-2 extracellu-larly and/or transports lysosomal acid hydrolase precursorsfrom the Golgi apparatus to the lysosome [79] (Figure 4).IGF-2Rs from both the cell surface and Golgi traffic to theearly endosome, where the relatively low pH environmentcauses the release of the IGF-2R cargo. The IGF-2Rs arerecycled back to the Golgi by the retromer complex. The

IGF-2RIGF-2

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Figure 4. Insulin-like growth factor 2 receptor (IGF-2R)-dependent lysosomal

enzyme trafficking, signaling transduction and endocytosis. Intracellular or

extracellular lysosomal enzymes, such as acid hydrolase, are transported to late

endosomes by IGF-2R (also known as the mannose 6-phosphate receptor) [80].

Lysosomal enzymes dissociate from IGF-2R within the low-pH environment of late

endosomes and are subsequently delivered to lysosomes. IGF-2R is recycled either

to the Golgi or to the cell surface [79]. At the cell surface, IGF-2 binding to IGF-2R is

followed by endocytosis to form early endosomes. From the early endosomes,

IGF-2R dissociates from IGF-2: IGF-2R is recycled to cell surface, whereas IGF-2 is

targeted to late endosome for lysosomal degradation [79]. A recent study [26]

suggests that, in the hippocampus, learning leads to IGF-2 binding to cell-surface

IGF-2R, which results in glycogen synthase kinase 3b (GSK3b) activation and

activity-regulated cytoskeletal-associated protein (Arc) synthesis, both of which

have been implicated in regulating AMPA receptor (AMPAR) endocytosis [88,93].

Internalized AMPARs can either recycle to the cell surface or be targeted for

degradation [112]. IGF-2/IGF-2Rs could also directly regulate AMPAR endocytosis

independently of GSK3b and Arc.

cargo proteins are then trafficked to the lysosome via thelate endosome independently of the IGF-2Rs [79] (Figure 4).How these and potentially other functions of IGF-2Rs lead tomemory enhancement is currently unknown.

IGF-2-mediated memory enhancement also requires denovo protein synthesis. Although IGF2 is a target gene of C/EBPb, IGF-2-mediated memory enhancement does not de-pend on de novo C/EBPb synthesis and does not correlatewith increased levels of pCREB and C/EBPb [26]. Thissuggests that IGF-2 does not exert its effects by amplifyingthese transcriptional mechanisms, but rather by acting ontranslational and synaptic mechanisms. Interestingly, oneof the newly synthesized proteins required for IGF-2-medi-ated memory enhancement is activity-regulated cytoskele-tal-associated protein (Arc; also known as Arg 3.1) [26]; itsfunctions are needed for long-term plasticity and memoryformation. For example, Arc is required in the hippocampusand the amygdala for the consolidation of spatial memoryand cued-fear conditioning, respectively [86,87]. Studieshave suggested that Arc is critical for long-term plasticityand memory because, by interacting with the endocyticmachinery proteins endophilin and dynamin, it regulatesmembrane trafficking of AMPA receptors [88,89]. AMPAreceptor trafficking is critically involved in LTP, long-termdepression (LTD) and memory. It is thought that AMPAreceptor subunits are rapidly transported in and out ofsynapses to strengthen or weaken synaptic activity duringplasticity and learning [90,91]. In agreement with thishypothesis of a positive correlation between plasticity andmemory formation and increased trafficking of synapticAMPA receptors, IGF-2-mediated memory enhancementwas found to correlate with increased levels of synapticGluA1 AMPA receptor subunits [26].

Endocytosis is also regulated by glycogen synthasekinase 3b (GSK3b), a serine/threonine kinase that wasoriginally identified as a regulator of cell metabolism buthas since been implicated in various functions, includingproliferation, cell survival, neural development, and shap-ing nerve terminal development and function [92]. In fact,endocytosis was found to be dependent on the phosphory-lation of dynamin I (at the Ser 774 residue) by GSK3b [93].IGF-2-mediated memory enhancement correlates with theactivation of GSK3b and requires the activity of GSK3 [26],supporting the hypothesis that the memory-enhancingeffect critically targets functions of the endocytic pathway.Previous findings suggested a correlation between in-creased endocytosis and increased GSK3 activation andArc expression [88,93]. Such findings appear to contrastwith the finding that memory enhancement is accompa-nied by increased synaptic expression of certain receptorsubunits (e.g. GluA1) [26]. However, it is possible thatendocytosis of other receptors as well as restructuring ofsynapses are also critical for enhancing synaptic strength.Moreover, increased endocytosis may play an importantrole in homeostatic synaptic scaling, which may enhancethe system capacity. Future studies will hopefully extendcurrent knowledge regarding the role of endocytosis inmemory enhancement.

An intriguing property of IGF2 and IGF2R is that theyare both imprinted genes. Imprinted genes are expressedonly by one allele, either maternal or paternal, and this

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pattern of expression is maintained epigenetically in al-most all tissues. How imprinted genes participate in be-havior is a topic of great interest and recent investigations[94–96].

Interestingly, insulin and other insulin regulatory pro-teins have been shown to promote memory enhancement inrodents as well as in humans [97–99]. However the mech-anisms by which this effect occurs seem to be different fromthat of IGF-2. Further studies are needed to better dissectthe mechanisms of these effects.

Targeting reconsolidation to enhance memoryWhy does memory become labile after retrieval and un-dergo reconsolidation? The biological purpose of reconso-lidation has been the topic of debates and investigationssince its rediscovery more than 10 years ago [13]. Onedebated issue in the reconsolidation field is its relationshipwith the role of the passage of time [100]. In inhibitoryavoidance conditioning, retrieval leads to protein synthe-sis-dependent reconsolidation only if the memory is lessthan 2 weeks old. By contrast, 2- or 4-week old memories donot become fragile after retrieval and, in fact, are resilientto protein synthesis inhibitor treatment administered ei-ther systemically or via intra-amygdala injections [18,101].Similarly, contextual fear conditioning becomes increas-ingly resistant to post-retrieval administration of proteinsynthesis inhibitors with time [19]. By contrast, 45 or 60day old auditory fear conditioning memories are still sus-ceptible to disruption by protein synthesis inhibition in theamygdala after retrieval, and no temporal gradient ofresilience has been described [102,103]. In inhibitoryavoidance conditioning, whereas hippocampal protein syn-thesis is required for the initial consolidation, it is dispens-able during reconsolidation, indicating that mechanisms inthe amygdala, but not the hippocampus, are critical formemory reconsolidation [104]. Hence, in inhibitory avoid-ance and, perhaps, other temporal-lobe-dependent memo-ries, hippocampal resistance to post-retrieval amnesictreatments may reflect the more cortically distributedrepresentation of the memory trace. By contrast, in pav-lovian fear conditioning, where the amygdala is necessarilyinvolved in the acquisition, storage and expression ofconditioned fear memory, reactivation of the very localizedtrace renders the memory labile. Whether this reflectsdifferences in molecular and/or circuit-based mechanismsremains to be understood.

One of the hypotheses proposed to explain the functionof reconsolidation is that memory reconsolidates to becomestronger and longer lasting [14]. Supporting this hypothe-sis, studies have demonstrated that a second training trialstrengthens a contextual fear memory [105]. This strength-ening requires the function of transcription factor earlygrowth response protein 1 (EGR-1; also known as Zif268) inthe hippocampus, which is also essential for the reconso-lidation, but not consolidation, of contextual fear condi-tioning [105]. Using inhibitory avoidance conditioning inrats, a recent study reported that unreinforced contextualreminders given when the memory is young, but not whenit is older, enhance memory retention and prevent for-getting [22]. Such findings indicate that reconsolidationdoes indeed promote memory enhancement. Targeting

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reconsolidation mechanisms should therefore also provideopportunities to enhance memories.

Several studies have explored the possibility of enhanc-ing memory by targeting mechanisms activated duringreconsolidation. For example, activating PKA in the baso-lateral amygdala (BLA) with Sp-cAMP during reconsolida-tion leads to enhancement of rat auditory fear memoryretention; conversely, inhibiting PKA by administering 6-BNZ-cAMP to the same brain region immediately afterretrieval impairs reconsolidation and leads to amnesia[106]. Similarly, in the crab Chasmagnathus, blockingangiotensin II impairs reconsolidation, whereas exogenousadministration of recombinant angiotensin II enhances it[107].

IGF-2, which enhances inhibitory avoidance memorywhen administered to the hippocampus following training(Figure 3a), also significantly strengthens inhibitory avoid-ance memory when administered in the same brain regionafter memory retrieval (Figure 3c) [26]. The effect is re-trieval dependent and limited to the same exact temporalwindow during which inhibitory avoidance memory under-goes reconsolidation; that is, less than 2 weeks after train-ing. Hence, although the hippocampus is not a substratefor post-retrieval memory disruption after inhibitoryavoidance training, it is a substrate for post-retrievalmemory enhancement that occurs during the reconsolida-tion-sensitive temporal window. We conclude that, in me-dial temporal lobe-dependent fear memories, retrievalactivates the amygdala to control consolidation that ismediated by hippocampal mechanisms, leading to subse-quent strengthening and cortical redistribution of thetrace. These mechanisms can be targeted to promote mem-ory enhancement. Thus, to effectively target mechanismsof consolidation or reconsolidation for memory enhance-ment it is important to keep in mind that there aretemporal constraints. Hence, an enhancer that targetsthe hippocampal CREB-C/EBP-dependent cascade willprobably be most effective either within the first day aftertraining or after reactivation when reactivation leads toreconsolidation. However, as the effect of IGF-2 on corticalregions still needs to be determined, it is possible thatmemory-enhancing effects in the cortex may have differenttemporal evolutions.

Concluding remarksImportant questions arise concerning memory enhance-ment that targets consolidation and reconsolidation mech-anisms (Box 1). First, are the enhancing effects selectivefor the active trace and, therefore, only affect an activememory? This would restrict their effect, eliminatingthem as treatments that might globally enhance all storedmemories. However, these types of pharmacological treat-ment could perhaps be paired with brain stimulation,which would activate the memory traces, allowing themto respond to the treatment. Second, from the resultsobtained, one can infer that if consolidation or reconsoli-dation is impaired by, for example, neurodegenerativedisease, memory enhancers may be ineffective. Thus, itis important that memory enhancers are tested and in-vestigated in disease models at multiple stages of diseaseprogression. Memory enhancers targeting consolidation

Box 1. Outstanding questions

� What are the mechanisms of action of memory enhancers?

Memory enhancers may provide substrates that cooperate with

encoding, synaptic/system consolidation and/or memory retrieval

mechanisms and either prolong and/or potentiate these processes, or

oppose negative constraints. An example is hippocampally adminis-

tered IGF-2 that acts only if present during active phases of the

memory, either post-training or post-retrieval [26]. Other examples

probably include changing the neurotransmitter or glutamate

receptor-mediated responses [113] and the general state of chroma-

tin to promote the expression of genes [114] or cell excitability

[23,115] that mediate synaptic plasticity to promote memory forma-

tion or persistence. It would also be interesting to determine whether/

which mechanisms have universal or selective effects in different

brain regions, memory systems, memory tasks and/or memory trace.

Alternatively, memory enhancers may target the basal state of the

system and prolong memory storage, as in the case of enhanced

expression of PKMz [116,117]. With this approach, memories could

be, in principle, enhanced at any time after their formation and the

effect should be unselective, therefore targeting all stored memories.

� Memory enhancers as potential treatments in diseases of memory

decay: will they help? How? Little is known about whether or how

memory enhancers can be used as effective treatments in memory

decay disorders, including aging-related memory loss and AD.

Although animal model studies suggest that some compounds that

produce memory enhancement also prevent memory loss in

models of AD [73], it needs to be determined whether the effect

on the memory is long lasting. Importantly, it also needs to be

determined whether the compounds enhance the abilities of the

system (and whatever remains of it) or actually rescue or bypass

defective mechanisms. If the compound reverses the deficits, an

early intervention may significantly prevent the progression of the

disease and may be the best or only successful intervention.

� Memory enhancers: are they producing enhanced memory but

inflexible behavior? Overlapping memories and memory systems

exist and little, if anything, is known about whether enhancement of

certain memories comes at the expense of the retention of other

memories already formed, or of modifying/updating memories, or,

finally, at the potentials of encoding new memories. In other words,

as in LTP [118,119], does a memory potentiation occlude the system

or make the system less flexible?

� What aspect of memory is enhanced? Behavioral studies need to

dissect whether memory enhancers affect the general representa-

tion of memory, thereby enhancing it as a whole, or whether

selected components of the memory are affected (e.g. emotion,

spatial, perception, execution, etc.).

� Enhancing memory in normal subjects: ethical problems. It is

important to mention briefly that ethical issues should be discussed

and ethics committees should provide guidelines about the use of

memory enhancers, according to the effects found [120]. For

example, enhancing memories may change emotional aspects of

the memory that can cause affect dysregulation (e.g. enhancing

painful, stressful and/or traumatic memories). Thus, studies are

required to understand carefully the mechanisms and both the

positive and negative consequences of enhancing memories.

Review Trends in Neurosciences May 2012, Vol. 35, No. 5

and reconsolidation may hopefully be able to compensatefor, or rescue, defects that may well be the cause of thedisease, thus significantly slowing the progression of thedisorder. In other words, prevention would be the most, orperhaps the only, successful strategy. Third, as memory-enhancing treatments would be needed for non-aversivememories, the consolidation and reconsolidation mecha-nisms of appetitive or problem-solving memories will haveto be better investigated and understood. Finally, a safe andclinically applicable memory enhancer would also need to behighly specific and have minimal adverse effects. Impor-tantly, studies on memory enhancement should examinemore carefully the effect of treatment on multiple tasks andbrain functions to assess whether memory enhancementcompromises the flexibility of memory or brain plasticity.Given that memory formation is such a dynamic process,gaining a more complete understanding of the anatomicaland temporal dynamics of the molecular and systemschanges required after learning and retrieval to consolidatememories will enable researchers to develop the most spe-cific and efficacious memory enhancers.

AcknowledgmentsWe dedicate this review to Rusiko Bourtchouladze, who passed away onAugust 6, 2011. She was a pioneer of the work on the role of CREB inmemory, an outstanding scientist and a wonderful friend. The writing ofthis review, and part of the work described within, were supported by theNational Institute of Mental Health (R01-MH065635 and R01-MH074736to C.M.A. and F31-MH816213 and T32-GM007280 to D.Y.C.).

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