mitogen-activated protein kinase/extracellular regulated kinase signalling and memory stabilization:...

Review

Mitogen-activated protein kinase/extracellular regulatedkinase signalling and memory stabilization: a review

Sabrina Davis* and Serge Laroche

Laboratoire de Neurobiologie de l’Apprentissage, de la Memoire

et de la Communication, CNRS UMR 8620, Universite Paris Sud,

Orsay, France

*Corresponding author: Sabrina Davis, Laboratoire de

Neurobiologie de l’ Apprentissage, de la Memoire et de la

Communication, CNRS UMR8620, Bat 446, Universite Paris

Sud, 91405, Orsay, France. E-mail: [email protected]

The function of mitogen-activated protein kinase

(MAPK) in neurons has been the subject of considerable

scrunity of late, and recent studies have given new

insights into how this signalling cascade can regulate

gene expression following cell-surface receptor activa-

tion. At the same time, a wealth of experimental data

has demonstrated that the MAPK cascade is critically

involved in the mechanisms underlying the type of

enduring modification of neural networks required for

the stability of memories, emphasizing the high level of

interest in this signalling molecule. In this review, we

briefly outline the main molecular events and mechan-

isms of the regulation of the MAPK cascade leading to

transcriptional activation and summarize recent

advances in our understanding of the functional role of

this molecular signalling cascade in regulating brain

plasticity, memory consolidation and memory

reconsolidation.

Keywords: Consolidation, immediate early genes, kinases,

reconsolidation

Received 19 January 2005, revised 21 February 2005,

accepted for publication 25 February 2005

Extracellular regulated kinase (ERK) forms part of the mito-

gen-activated protein kinase (MAPK)-signalling cascade,

which in turn is part of a larger family of signalling pathways

that includes the Jun amino-terminal kinase/stress-activated

kinase (JNK/SAPK) and the p38 kinase cascades. Although

the MAPK cascade was originally described as controlling

proliferation and differentiation of cells in response to growth

factors and mitogens (Cooper & Hunter 1982), ERK is widely

expressed in mature, postmitotic neurons (Boulton & Cobb

1991; Fiore et al. 1993), and it was assumed that this cas-

cade must play a role in other functions than cell proliferation

and division. Subsequently, a number of studies have shown

that this pathway is implicated in a number of different forms

of plasticity, including activation of gene transcription, struc-

tural modification at the synapse, receptor insertion and

regulation of dendritic protein synthesis. In addition, many

studies have shown that activation of different member pro-

teins in this pathway is necessary for the consolidation of

long-term memories, and more recently, reports are starting

to emerge to suggest that this pathway may also be impli-

cated in reconsolidation of memory. In this review, we will

focus on the evidence suggesting that the activation of the

MAPK pathway leading to nuclear transcription may be a

critical underlying mechanism for the consolidation of long-

term memories. The logic underlying the role of the MAPK

pathway in mediating the consolidation of long-term mem-

ories and the late phases of synaptic plasticity is based on its

mechanism of action. It is the most prominent of pathways

known to convey the signal generated at the receptor to the

nucleus via the sequential activation of its member proteins.

Once in the nucleus, phosphorylated MAPKs activate tran-

scription factors which have binding sites on the promoter

regions of immediate early genes, genes that are considered

to be the initial triggering mechanisms of genetic programs

that lead to functional modifications of the cell. This type of

modification induced in populations of cells distributed

throughout widespread networks is considered to be the

type of plasticity-mediated event that may be responsible

for the encoding and storage of the memory trace.

The MAPK-signalling cascade

The canonical MAPK pathway is triggered by growth factors

and mitogens acting at tyrosine kinase’s growth-associated

receptors. When activated, these receptors trigger the

sequential activation of the core classes of kinase in this

pathway: the MAP kinase kinase kinases (MAPKKKs), Ras

and Raf, which activates the MAPK kinase (MAPKK), MEK

and finally, MEK phosphorylates the MAPKs ERK 1 and 2,

both on a threonine and a tyrosine residue. When phosphory-

lated, the ERKs, also described as nuclear shuttle proteins,

Genes, Brain and Behavior (2006), 5 (Suppl. 2), 61–72 # 2006 The Authors

Journal compilation # 2006 Blackwell Munksgaard

doi: 10.1111/j.1601-183X.2006.00230.x 61

translocate to the nucleus where they activate the down-

stream transcription factors Elk-1 and c-Myk. ERK also

activates the transcription factor CREB, but via the inter-

mediary kinase, ribosomal protein S6 kinase (RSK) which

phosphorylates CREB at serine 133 and the more

recently identified kinase, mitogen and stress-activated

kinase (MSK), which is also capable of phosphorylating

CREB at the same site. Suggestions are beginning to

emerge that MSK may pose itself as a better candidate

for activating CREB (Thomas & Huganir 2004). Both tran-

scription factors have binding sites on several immediate

early genes such as zif268, BDNF and c-fos. As the

immediate early genes encoding transcription factors

are known to activate downstream target genes, this

signalling cascade is in a pivotal position between the

mechanisms initiating activity-dependent plasticity and

the end point, which is the structural modification in the

cell (see Fig. 1).

Figure 1: (A) The core MAP kinase

pathway. (B) Details of Ras–Rap-1

interaction and activation of Ras.

See text for details.

Davis and Laroche

62 Genes, Brain and Behavior (2006), 5 (Suppl. 2), 61–72

The initiation steps

Although its primary source of activation is via mitogen and

growth factor activation of Trk receptors, there are two

points of convergence by other signalling systems on the

MAPK pathway: at the initial step in the cascade and down-

stream at the transcription factors in the nucleus. Initiation of

the cascade is elicited by activation of Raf primarily by the

membrane-bound G-protein Ras. Essentially, Ras acts as a

switch, binding to and recruiting Raf to the membrane which

in turn phosphorylates MEK. Several mechanisms identified

at this level are also implicated in the finely tuned modulation

of the cascade. For example, there are two known isoforms

of Raf, Raf-1 and B-Raf, and although both can be recruited

by Ras, Raf-1 binds only weakly to Ras, whereas it is directly

bound with B-Raf (Marais et al. 1997). In addition, the Ras-

related protein, Rap-1, can exert a dual effect on the two

Rafs by activating B-Raf and inhibiting Raf-1. This modulatory

effect is mediated via PKA, as PKA can inhibit Raf-1 but

activate B-Raf via Rap-1 (Vossler et al. 1997; York et al.

1998). Furthermore, Rap-1 can inhibit Ras (Altschuler &

Ribeiro-Neto 1998), and these two G-proteins have different

subcellular locations; Ras is anchored to the plasma mem-

brane, whereas Rap-1 is anchored to endosomal compart-

ment membranes (Kim et al. 1990; Pizon et al. 1994; Resh

1996). Independent activation of Ras and Rap may have

important consequences on the activation of ERK, as they

Table 1: Summary of experiments showing the role of specific proteins in the mitogen-activated protein kinase (MAPK) pathway that

are implicated in memory processing, with genetically modified mice, inhibiting activation of extracellular regulated kinase (ERK) or

demonstrating regulation of the protein after learning or memory. Only experiments showing direct manipulation of the pathway are

included. See text for details

Genetic modification

Rap-1 Homozygous Impaired spatial memory, contextual discrimination Morozov et al. (2003)

K-Ras Heterozygous Normal fear conditioning, but impaired with submax Ohno et al. (2001)

Dose of MEK inhibitor

RasGRF Homozygous Impaired tone-fear conditioning, normal spatial learning Brambilla et al. (1997)

RasGRF Homozygous Impaired spatial learning, normal tone-fear conditioning Giese et al. (2001)

RIN1 Homozygous Facilitated tone-fear conditioning, normal spatial memory Dhaka et al. (2003)

SynGAP Heterozygous Impaired spatial memory Komiyama et al. (2002)

ERK1 Homozygous Facilitated active and passive avoidance Mazzucchelli et al. (2002)

ERK1 Homozygous Normal passive avoidance and fear conditioning Selcher et al. 2001

MEK1 Homozygous Impaired fear memory Shalin et al. (2004)

ERK Inhibition

Amygdala PD9805 Impaired extinction of fear memory Lu et al. (2001)

CA1/2 PD9805 Impaired spatial memory Blum et al. (1999)

icv PD9805 Impaired taste aversion Swank (2000)

icv PD9805 Impaired consolidation and reconsolidation of Kelly et al. (2003)

Recognition memory

Prelimbic UO126 Impaired retention of trace fear conditioning Runyan et al. (2004)

Cortex

Amygdala UO126 Impaired tone-fear conditioning Schafe et al. (2000)

icv SL327 Impaired spatial memory, contextual fear Selcher et al. (1999)

Conditioning, attenuated tone-fear conditioning

Entorhinal PD9805 Impaired spatial memory Hebert and Dash (2002)

cortex

Entorhinal PD98059 Impaired inhibitory avoidance Waltz et al. (1999, 2002)

cortex

ERK Hyperphosphorylation

Amygdala Tone-fear conditioning Atkins et al. (1998)

Schafe et al. (2000)

Hippocampus Contextual fear conditioning Atkins et al. (1998)

Nucleus solaris tractus Taste aversion Swank (2000)

Insular cortex Taste aversion Berman et al. (2000)

Hippocampus Spatial learning Blum et al. (1999)

Hippocampus Recognition memory, and also after reactivation Kelly et al. (2003)

Hippocampus/ Trace fear conditioning, and also after reactivation Runyan et al. (2004)

Prelimbic cortex

MAPK/ERK signalling and memory stabilization

Genes, Brain and Behavior (2006), 5 (Suppl. 2), 61–72 63

may activate different pools of ERK that could have poten-

tially different functions. For example, activation of Rap-1

induces sustained phosphorylation of ERK, whereas activa-

tion of Ras induces only transient activation of ERK (York

et al. 1998). Sustained high activation of ERK induces cell-

cycle arrest linked with differentiation; whereas transient

activation followed by sustained but lower levels of ERK

activity is commonly associated with cell proliferation

(Marshall 1995). In addition, Murphy et al. (2002) have

shown that transient activation of ERK induces expression

of Fos proteins by phosphorylation of the transcription fac-

tors Elk-1 and Sap; whereas sustained ERK signalling results

in the activation of RSK and the stabilization of Fos proteins

producing a robust transcriptional activation. A potential con-

sequence of this differential form of ERK regulation may be

the observation of different waves of expression of down-

stream targets following the induction of LTP, such as CREB

(Schultz et al. 1999).

Crosstalk between kinase pathways

Many different proteins, not directly associated with this

core pathway, are capable of modulating this initiation step.

Although it has long been suggested that NMDA receptors

(English & Sweatt 1996; Impey et al. 1998) and calcium

transients (Bading & Greenberg 1991; Rosen et al. 1994)

can modulate the MAPK cascade at the level of Raf, kinases

known to be implicated in both memory processing and

synaptic plasticity, such as PKA, PKC and CaMKII, also mod-

ulate the Ras–Raf interaction. It is known, for example, that

the regulation of the intracellular levels of cAMP by dopami-

nergic and adrenergic receptors regulates PKA, and this can

have a differential effect on the Raf’s as described above

(see also Adams & Sweatt 2002; Sweatt 2001). One sugges-

tion is that a functional consequence of this differential reg-

ulation may be the requirement of the activation of PKA to

allow ERK to translocate to the nucleus and activate CREB-

dependent gene transcription (Impey et al. 1998). PKC also

activates the MAPK pathway at the Ras–Raf level (Carroll &

May 1994; Kolch et al. 1993) mainly through mGluR and

mAChR stimulation of phospholipase C and DAG (Roberson

et al. 1999). Although the mechanism is not entirely under-

stood, it is possible that it inactivates a p120RasGAP-depen-

dent modulatory effect on the tonic activity of Ras to trigger

downstream activation (Cobb & Goldsmith 1995; Downard

et al. 1990; Wigler 1990;). PKC activation in CA1 by phorbol

ester PDA, however, can also selectively activates ERK2,

suggesting that kinases other than MEK are capable of phos-

phorylating ERK (Bading & Greenberg 1991; English &

Sweatt 1996, 1997; Fiore et al. 1993). CaMKII modulates

the activity of Ras in an NMDA receptor-dependent manner.

Ras is normally activated when bound to the GEF, RasGRF1

(Shou et al. 1992), and this only occurs when Ras is bound to

calcium calmodulin (Farnsworth et al. 1995). The Ras-

GTPase-activating protein p153SynGAP, which is located in

the PSD in close proximity to the NMDA receptor and

PSD95, acts to prevent Ras binding to RasGRF1. When

phosphorylated by CaMKII, however, SynGAP is inhibited,

thereby permitting the activation of the downstream targets

in the MAPK cascade (Chen et al. 1998) in much the same

way that PKC inhibits p120RasGAP. More recently, it has

been shown that the GEF, RasGRF1, binds directly to the

NR2B subunit of the NMDA receptor, thereby determining a

direct link with the MAPK pathway (Krapivinsky et al. 2003).

Thus, it is clear from the level of manipulation of the cascade

at its initiation step by other kinases and proteins that there is

a great deal of convergence onto this pathway, suggesting

an exquisitely fine-tuned and rigorous control over its activa-

tion. The functional consequence of this level of control at

this early stage in the cascade is not clear; however, once

the signal has gotten past this step, activation of MEK and its

subsequent phosphorylation of the ERKs remain more faith-

ful. Although there are two isoforms of both proteins, there

is no suggestion that there is specificity between MEK iso-

forms acting on ERK isoforms (Zheng & Guan 1993).

Initiation of transcription via the MAPK cascade

The second point of convergence of the signal in the MAPK

pathway is at the level of the transcription factors. Although

the role of the transcription factors and immediate early

genes in plasticity and memory is beyond the scope of this

review, it is important to emphasize that there is a second

level of convergence on the MAPK pathway at the level of

the transcription factors. To date, there is no evidence to

suggest that Elk-1 is modulated by any other kinase than

ERK; however, many studies have shown that CREB can

be activated by proteins other than ERK-RSK. Calcium has

been shown to directly activate CREB (Hardingham et al.

2001), and both PKC (Mayr et al. 2001) and PKA (Gonzalez

& Montminy 1989; Meinkoth et al. 1990) phosphorylate

CREB at serine 133; neither, however, are sufficient to

induce CREB-mediated gene transcription independently of

the activation of CREB by ERK (Impey et al. 1998; Roberson

et al. 1999). Both CaMKII and CaMKIV can phosphorylate

CREB at the same serine site (Sheng et al. 1991; Sun et al.

1994), but CaMKII is a poor activator of CREB and also

inhibits CREB when it phosphorylates it at serine 142

(Enslen et al. 1994; Matthews et al. 1994; Sun et al. 1994,

1996). CaMKIV, however, has been detected in the nucleus

(see Soderling 1999), and studies have shown that antisense

inhibition of CaMKIV partially reduces early CREB phosphor-

ylation and CRE-dependent transcription (Bito et al. 1996;

Finkbeiner et al. 1997). Conversely, constitutively active

CaMKIV increases the activity of CREB-binding protein

thereby stimulating CRE-mediated transcription (Chawla

et al. 1998; Hardingham et al. 1999; Hu et al. 1999). To

date, only prolonged phosphorylation of CREB correlates

with CREB-dependent gene regulation (Bito et al. 1996;

Impey et al. 1996; Liu & Graybiel 1996), and this seems to

Davis and Laroche


be mediated via the ERK cascade (see West et al. 2002).

Curiously although, the intermediary between ERK and

CREB is RSK and mutations of this gene, which is respon-

sible for Coffin–Lowry’s syndrome (Trivier et al. 1996), do not

appear to reduce the levels of CREB phosphorylation by ERK.

An alternative kinase, MSK, may be the critical activator of

CREB in this pathway (see Thomas & Huganir 2004). The

reason for CREB phosphorylation by kinases other than ERK

does not lead to reliable CREB-mediated gene regulation is

not clear. It is possible that it serves as a potential fail-safe

device to ensure activity-dependent gene transcription, in the

event of the MAPK cascade malfunctioning, or it may be

linked to the differential regulation of transient and prolonged

activation of ERK via the Raf-1–B-Raf pathway. As aptly put

by O’Neil and Kolch (2004), ‘The number of our genes is too

small to account for the complexity of biological function.

Thus, the cells employ the same proteins in different context

and impose specificity through combinatorial mechanisms.’

Although the canonical ERK cascade is posited as being

the critical route to which an activity-dependent signal gen-

erated at the receptor can be conveyed to the nucleus to

trigger gene and protein regulation necessary for synaptic

modifications that are deemed necessary for the encoding

and storage of memories, ERK does have other substrates

that may also be necessary for synaptic remodelling. Those

that have been directly linked with plasticity or memory

include cytoskeletal proteins such as MAP-2 and Tau that

are critical for structural reorganizing of dendrites (Vaillant

et al. 2002) and new spine formation induced by repeated

depolarization (Goldin & Segal 2003; Wu et al. 2001); chan-

nels such as the potassium Kv4.2 channel that have been

implicated in both synaptic plasticity and memory formation

(see Adams & Sweatt 2002); naıve AMPA receptors with

long cytoplasmic tails, ready to be inserted (Zhu et al. 2002)

and mTor, a protein associated with the ribosomal machinery

necessary for the synthesis of new proteins (Kelleher et al.

2004; Tang et al. 2002). This suggests that the functional role

of the MAPK cascade in memory and plasticity may not be

solely restricted to activating the genetic machinery.

Involvement of the MAPK cascade in synapticplasticity and memory formation

The necessity of the normal-functioning MAPK cascade in

synaptic plasticity and memory formation has been exten-

sively documented and suggests that interfering with any of

the core kinases in this pathway results in both decremental

LTP and memory deficits. Long-term potentiation is the clas-

sic model of long-lasting plasticity that is widely believed to

mimic the type of changes within neuron circuits underlying

the formation and storage of memories. It is typically

described as having an induction phase that lasts a matter

of seconds to minutes and is dependent on NMDA receptor

activation; an early phase, lasting up to about 30–60 min and

is dependent on second messengers and kinase activation

and a late phase that can last in excess of weeks, that is

dependent on the activation of gene transcription and syn-

thesis of new proteins (see reviews by Bliss & Collingridge

1993; Lynch 2004). In a similar manner, consolidation of long-

term memory requires the synthesis of new proteins (Davis

& Squire 1984; McGaugh 2000). Activation of the MAPK

cascade occurs rapidly and transiently following the induction

of LTP and learning and has been shown to be necessary for

consolidation of memory and late phases of LTP, constituting

a critical precursor mechanism leading to the synthesis of

new proteins. Most studies to date have focussed on the

role of the ERKs in plasticity and consolidation, but it is

important to understand how the cascade exerts this func-

tional effect and in particular to determine the specific role of

the three key kinases in this pathway, as there is conver-

gence of the signal from other sources at the Ras–Raf level

and at the transcription level.

MAP kinase kinase kinases

At the initial step of the cascade, where Ras and Rap-1

activate the Raf, only a few studies have been reported to

date (Table 1). All these studies have used genetically manipu-

lated mice, and the outcome is not clearly defined. This is

possibly due to the use of mutations in different isoforms of

the protein. In one study, H-Ras heterozygous mice unexpect-

edly showed an increase in basal synaptic transmission and

LTP induced in the CA1 of the hippocampus (Manabe et al.

2000). In addition, the authors reported an increase in tyrosine

phosphorylation of NR2A and NR2B receptors, concluding

that Ras might have a novel function implicating the NMDA

receptors. In the other study, a K-Ras heterozygous mouse

was shown to have no deficit in LTP induced in CA1 nor in

contextual fear conditioning; but if subthreshold doses of a

MEK inhibitor were given, then there was decremental LTP

and memory deficits (Ohno et al. 2001).

It is difficult to reconcile these differential effects; how-

ever, clues may begin to emerge from the functional role of

the different isoforms. For example, it is known that Ras has

three isoforms, H-Ras, K-Ras and N-Ras, and they are acti-

vated by a wide range of extracellular stimuli, including the

more recent discovery of specific NR2B-activated Ras GRF1

protein ( Krapivinsky et al. 2003). Although they share a high

homology with each other, the different isoforms preferen-

tially activate different downstream effectors (Yan et al.

1998) and have different subcellular localization and micro-

domains of activity (Roy et al. 1999). H-Ras alone has differ-

ential localization and depending on its location exerts a

different downstream effect. When tethered to the Golgi

apparatus, it preferentially activates the JNK pathway, and

when tethered to the endoplasmic reticulum, it activates the

ERK and PI3k-Akt pathways (Chui et al. 2002). In contrast,

inactivating Rap-1, which favours activation of B-raf, results

in a selective decrease in membrane-associated ERK, decre-

mental LTP induced in CA1 and impairment in hippocampal

dependent tasks, such as spatial memory and contextual



fear conditioning, but not amygdala-associated tasks such as

auditory fear conditioning (Morozov et al. 2003).

Further conflict of results has been shown with mice lack-

ing the neuronal Ras regulator, RasGRF. These mice show

impairment in LTP induced in the basolateral amygdala and a

corresponding deficit in long-term consolidation of fear mem-

ories that are associated with amygdala function (Brambilla

et al. 1997). In contrast, they showed no impairment in LTP

induced in CA1 nor learning or memory deficits in hippocam-

pal-associated spatial memory. Almost diametrically opposed

to this study, Giese et al. (2001), also using Ras GRF1-

mutant mice, showed normal amygdala-associated fear

learning and memory but some deficits in hippocampal-

dependent learning and memory using a spatial navigation

task. Neither group reported any regulation of downstream

effectors such as phosphorylation of ERK that may give clues

to the discrepancy but suggests possible causes such as

differences in the learning protocols and genetic background.

In support of the results showing impairment of amygdala

function is the enhancement of LTP in the amygdala and

improved memory performance in fear conditioning reported

in RIN1 mutant mice (Dhaka et al. 2003). In these mice, there

was no effect on hippocampal LTP or spatial learning and

memory. As a negative regulator of Ras, RIN1 competes

with Raf to bind to Ras and thereby inhibits its ability to

bind to Raf (Wang et al. 2002). Thus, the authors conclude

that ‘the loss of RIN1 activity could increase signalling

through Raf pathways involved in changes required for

long-term memory and plasticity’ (Dhaka et al. 2003).

Alternative means of regulating Ras–Raf activity is via the

GTPase-activating protein SynGAP that negatively regulates

Ras activity. This protein, which is associated with PSD95

(Kim et al. 1998) and can couple with the NR2B receptor

(Krapivinsky et al. 2003), is inhibited by CaMKII (Chen et al.

1998). Thus, NMDA receptor-dependent activated CaMKII

would promote Ras activity and potentially the MAPK cas-

cade by inhibiting the inhibitory action of SynGAP. SynGAP-

heterozygous mice show deficits in LTP in CA1 (Kim et al.

2003; Komiyama et al. 2002) and spatial memory; and there

was an increase in basal levels of phospho ERK that could be

further enhanced by LTP (Komiyama et al. 2002) which sug-

gests that although SynGAP can regulate the MAPK path-

way, its effect may be mediated via other mechanisms,

possibly glutamate receptor trafficking (Kim et al. 2003).

The lack of clarity in the results is not surprising given the

complexity associated with activating the initial step in the

cascade that is mediated via a potential triangular interaction

between Ras, Rap-1 and the Rafs. Adding to this complexity

is the diverse convergence of signals that can activate or

inhibit Ras and the number of downstream effectors, apart

from Raf, that are associated with Ras within different cel-

lular environments. In addition, there are few reports to date

that have attempted to tease these elements apart to deter-

mine their individual role in plasticity and memory formation

in vivo. Despite this complexity, however, it is clear that

modulation of specific isoforms of Ras plays a critical role

in synaptic plasticity and learning and memory. This is illu-

strated in the autosomal dominant disease, neurofibromato-

sis, caused by mutations in the neurofibromatosis type 1

(NF1) gene leading to learning disabilities in humans. The

gene product, neurofibromin, contains a GAP domain which

promotes the formation of Ras-GDP, thus inactivating Ras

activity: the mutation in mouse models results in impaired

CA1 LTP and spatial learning deficits, which can both be

rescued by decreasing Ras activity, suggesting that the

deficits are caused by abnormal Ras hyperphosphorylation

resulting from NF1 mutation (Costa et al. 2001, 2002).

MAPK kinase

A number of studies using pharmacological inhibition of MEK

and examining the downstream effects of blocking ERK in

terms of learning and memory and synaptic plasticity show a

much greater consensus of effect (Table 1). For example,

induction of LTP is associated with a rapid and transient

increase in the phosphorylation of ERK in CA1 (English &

Sweatt 1996; Rosenblum et al. 2002; Winder et al. 1999),

the dentate gyrus (Davis et al. 2000; Rosenblum et al. 2000;

Ying et al. 2002), the insular cortex (Jones et al. 1999). In a

correlated manner, inhibiting activity-dependent phosphoryla-

tion of ERK with the MEK inhibitor results in decaying LTP

within a time window of about 30–60 min, thereby suggest-

ing ERK activity is necessary for the stabilization of the early

and late phases of LTP (Coogan et al. 1999; Davis et al. 2000;

English & Sweatt 1997; Huang et al. 2000; Wu et al. 1999). In

contrast, LTP induced in CA3 was not affected by inhibitors

of MEK (Kanterewicz et al. 2000).

In addition, the plasticity-induced hyperphosphorylation of

ERK is coupled with several different neurotransmitter and

signalling systems, including Trk receptors and BDNF (Chen

et al. 1999; Figurov et al. 1996; Kang et al. 1997; Messaoudi

et al. 1998; Ying et al. 2002), NMDA receptors (Bading &

Greenberg 1991; English & Sweatt 1996, 1997), G-protein-

linked receptors such as the metabotropic (Coogan et al.

1999; Roberson et al. 1999) muscarinic receptors (Roberson

et al. 1999; 2000) the b-adrenergic (Roberson et al. 1999;

Watanabe et al. 2000; Winder et al. 1999) and dopaminergic

(Roberson et al. 1999) receptors via cAMP-stimulated PKA

(see Stork & Schmidt 2002). Much focus has been placed on

TrkB receptors (Chen et al. 1999; Figurov et al. 1996; Kang

et al. 1997) and BDNF-mediated (Messaoudi et al. 1998; Ying

et al. 2002) phosphorylation of ERK, induced by LTP. BDNF

can induce its own form of LTP (Kang & Schuman 1995;

Kang et al. 1997; Messaoudi et al. 1998; Ying et al. 2002),

which leads to phosphorylation of ERK; is occluded by prior

electrically induced LTP, and inhibiting ERK results in decre-

mental BDNF induced LTP (Ying et al. 2002). In addition,

BDNF-induced potentiation that is accompanied by an

increase in ERK activity is attenuated in aged rats (Gooney

et al. 2004), and, more specifically, TrkB-BDNF-mediated late

LTP appears to modulate the translocation of ERK to the

Davis and Laroche


nucleus rather than specifically activating ERK in CA1

(Patterson et al. 2001; Rosenblum et al. 2002). Finally, hyper-

phosphorylation of ERK has been observed with long-term

depression (LTD) in the cerebellum (Kawasaki et al. 1999)

and CA1 (Gallagher et al. 2004; Thiels et al. 2002). Gallagher

and colleagues found that both isoforms of ERK were hyper-

phosphorylated following LTD, and this was mediated by

group 1 metabotropic receptors, whereas Thiels et al.

(2002) showed hyperphosphorylation of only ERK2 with

electrical stimulation leading to LTD but a decrease in

immunoreactivity in phosphoERK1 (Norman et al. 2000).

In a corroborative manner, there is considerable evidence

that the activation of ERK is necessary for the consolidation

of long-term memories, (Table 1) ranging across a number of

different types of learning that are associated with specific

brain structures, such as fear-associated learning (Atkins

et al. 1998; Schafe et al. 2000; Selcher et al. 1999; Waltz

et al. 1999, 2000), or its extinction (Lu et al. 2001); spatial

learning (Blum et al. 1999; Hebert & Dash 2002; Selcher

et al. 1999); taste aversion (Berman et al. 2000; Swank

2000); object recognition (Kelly et al. 2003) and trace fear

conditioning (Runyan et al. 2004). These studies have shown

the implication of ERK in the processing of long-term mem-

ory by showing that ERK is hyperphosphorylated after the

learning phase, and inhibition of the upstream kinase, MEK,

results in impaired memory. Importantly, inhibition of ERK

results in an impairment in consolidation of long-term mem-

ories, as short-term memory (Kelly et al. 2003; Schafe et al.

2000) and learning (Blum et al. 1999) are not affected.

Regulation of ERK, however, seems to follow a different

time course depending on the type of memory to be con-

solidated, and in some forms of learning, it is dependent on

specific isoforms of ERK. For example, ERK is hyperpho-

sphorylated 1 h after Pavlovian fear conditioning, associated

with the amygdala (Atkins et al. 1998; Schafe et al. 2000),

and contextual fear conditioning, associated with the hippo-

campus (Atkins et al. 1998; Runyan et al. 2004). In taste

aversion studies, ERK is phosphorylated in the nucleus trac-

tus solaris (Swank 2000) and the insular cortex (Berman et al.

2000) 20–30 min following conditioning and returns to basal

levels within an hour (Berman et al. 2000). Finally, ERK

phosphorylation is observed in the hippocampus 5 min fol-

lowing a massed training protocol for spatial learning (Blum

et al. 1999) and after three 5-min exposure periods to objects

in a novel object recognition task (Kelly et al. 2003). Despite

the differential time window of learning-induced ERK activa-

tion, it falls well within the period that precedes the time

window when the synthesis of new proteins is required for

memory consolidation and suggests that the activation of the

MAPK cascade is a necessary preliminary mechanism for the

consolidation of memory.

Studies measuring the regulation of the two ERKs inde-

pendently have shown, for example, that only ERK2 is

regulated in the hippocampus following contextual fear

conditioning, whereas both ERKs are regulated in the

amygdala following tone-associated fear conditioning

(Atkins et al. 1998; Schafe et al. 2000) and in the hippocam-

pus following trace fear conditioning (Runyan et al. 2004).

This has also been shown following the induction of LTP in

CA1 (English & Sweatt 1996). Both ERKs are phosphorylated

following conditioned taste aversion (Berman et al. 2000),

but only ERK 1 is phosphorylated in the dentate gyrus and

CA1 following the exposure to novel objects (Kelly et al.

2003). To date, there are few studies examining the role of

ERK in memory processing using mutant mice. One very

recent study has shown that mutated MEK1, which normally

activates both isoforms of ERK, results in impaired fear-

associated memory, in agreement with the studies using

pharmacological inhibition of MEK (Shalin et al. 2004). In

addition, two studies have investigated the potential role of

ERK1 in plasticity and memory using mice carrying specific

mutations of the gene. In one study using mice carrying

specific mutations of ERK1, Selcher and colleagues (2001)

found that, although mutant mice were hyperactive, they

showed no significant impairment in a passive avoidance

task and no long-term memory impairment in fear condition-

ing. In the only other study, Mazzucchelli et al. (2002)

showed facilitation of active avoidance and long-term reten-

tion of it. Both groups also tested LTP in these mice, and

both found that with 100 Hz stimulation, mutants showed

significant LTP in CA1 that was indistinguishable from wild-

types; however, when they used theta burst stimulation, LTP

was attenuated in CA1 in the mutant mice. This is in keeping

with results that have shown that ERK is regulated by theta

burst stimulation but not 100 Hz (Winder et al. 1999).

Mazzucchelli and colleagues also found that LTP in the amyg-

dala of the mutant mice was normal compared with wild-

types, but it was unexpectedly enhanced in the nucleus

accumbens. Thus, Mazzucchelli and colleagues concluded

that as they had observed an increase in levels of the ERK

2 isoform in the mutant mice, this might well compensate for

the lack of ERK 1; whereas Selcher and colleagues con-

cluded from their results that the ERK1 isoform did not

contribute to synaptic plasticity or learning and memory. In

a subsequent study, Selcher et al. (2003) resolved this dis-

crepancy somewhat by showing that LTP induced in mice

using 100 Hz stimulation is not affected by the inhibitor of

MEK as it is in the rat but is decremental when induced with

theta burst stimulation. Sweatt and colleagues suggest that

the principle function of ERK may be to mediate membrane

excitability via potassium channels, as both ERK1- (Selcher

et al. 2003) and Rap1-mutant mice (Morozov et al. 2003)

show a decrease in theta burst-induced cell excitability, and

inhibition of ERK activity decreases Kv4.2 potassium chan-

nels (see Sweatt 2004).

Generally speaking, these data provide fairly substantial

evidence that ERK constitutes a global mechanism for synap-

tic plasticity, observed in several different brain structures,

and its activation is necessary for the long-term consolidation

of memory. Although ERK has been shown to have some 70



different effectors (Lewis et al. 1998), much focus has been

placed on the potential role of the ERK cascade in conveying

the signal from the cell surface to the nucleus where it sets

in play transcriptional regulation necessary for the protein

synthesis-dependent mechanisms of plasticity and memory

consolidation. There is an abundance of data showing that

nuclear targets of ERK, such as CREB, and immediate early

genes such as zif268 are also necessary for the consolidation

of memories. However, there is also data emerging to sug-

gest that non-nuclear targets of ERK, such as mTOR, Kv4.2

channels and the dendritic immediate early gene, Arg3.1, are

implicated in plasticity and memory formation. The relative

contribution and importance of these mechanisms for synap-

tic plasticity and memory consolidation remains unclear.

MAPK-signalling pathway and reconsolidation

More recently, evidence is mounting to suggest that an

already consolidated memory, when it becomes reactivated,

enters a labile phase and requires a process of reconsolida-

tion to be available for further recall. Although not a new idea,

it was first postulated in the late 1960s by Misanin et al.

(1968); it has been rejuvenated by experiments showing that

inhibiting the synthesis of new proteins after reactivation of

memory disrupts this process of reconsolidation of memory

(see review by Dudai & Eisenberg 2004). Although there is a

substantial number of studies that have shown that inhibiting

the synthesis of all new proteins with anisomycin blocks the

reconsolidation process, there are few studies investigating

the potential role of individual proteins and genes, and even

less examining the implication of the MAPK cascade. In our

own work (Kelly et al. 2003), we used a recognition memory

task to test whether ERK phosphorylation was necessary for

both consolidation and reconsolidation, by inhibiting the acti-

vation of the upstream kinase, MEK. We found that inhibiting

MEK prior to acquisition of an object recognition task

impaired rats’ long-term memory but spared their short-

term memory. When MEK was inhibited during a single

brief reactivation of an already consolidated memory and

rats were tested for efficient reconsolidation of that memory

24 h later, they were also impaired. Importantly, we found

that if the memory was not reactivated, there was no impair-

ment in reconsolidation of the memory. This suggests that

ERK activation is necessary for both consolidation and recon-

solidation of recognition memory. A recent study has con-

firmed this in showing that a MEK inhibitor prevents

reconsolidation of fear memories (Duvarci et al. 2005).

Whether the entire cascade is necessarily activated for

reconsolidation is not clear. There is evidence to suggest

that downstream targets of ERK, such as CREB (Kida et al.

2002) and zif268 (Bozon et al. 2003; Lee et al. 2004), are also

necessary for reconsolidation of certain forms of memory;

however, there are no studies to date that have investigated

the role of upstream kinases in this pathway in

reconsolidation. Thus, it is unknown whether the activation

of the entire cascade is necessary for the process of recon-

solidation. As this is a relatively new area of research, of

course, there are many more important questions that

remain unanswered. For example, we do not know whether

reconsolidation is erasing the existing memory trace and

replacing it or whether it is reactivated to update the trace.

Furthermore, it is not clear whether the memory trace would

eventually become immune to disruption or whether genes

and proteins involved in the initial consolidation of the trace

are activated in different brain circuits during reconsolidation.

Conclusion

Encoding, consolidating and as we know now, reconsolidat-

ing memory are complex processes, believed to occur within

distributed networks of neurons throughout the brain. These

processes have also been shown to require the activation of

an equally complex array of transmitter systems, second

messengers, kinases, the transcription of genes and the

synthesis of new proteins. A common link to all these signal-

ling events might well be the MAPK cascade. To date, it is

known that there are about 20 different neurotransmitter and

signalling systems that activate the MAPK pathway (see

Sweatt 2004) and there are about 70 substrates of ERK

(Lewis et al. 1998); thus, the signalling pathway integrates

and disperses incoming signals. The convergence of signals

onto the MAPK pathway holds parsimoniously with the

hypothesis of synaptic plasticity that a minimal threshold of

activity must be achieved to trigger the events leading to

long-term modification in the cell. Dispersal of the signal

leads to activation of other functional signalling systems

and negative feedback regulatory loops. All we can say to

date is that interfering with this pathway results in failure to

consolidate and reconsolidate memories and a failure to

sustain synaptic plasticity. Many questions concerning how

the network achieves this final goal remain unanswered.

Some evidence is beginning to emerge to suggest that inte-

gration of signals across different timescales lead to modula-

tion of the duration and strength of the signal, ensuring its

throughput. Negative regulatory feedback loops modulate

bistable activity and can lead to discrete steady states that

determine the duration of the signal. Activation of the MAPK

cascade is known to modulate events at the synapse, trans-

late proteins in the cytosol and activate genetic programs in

the nucleus. What is essential for our understanding now is

how these events work together within a signalling network

to determine the emerging behavioural output. Finally, it is

remarkable that the malfunction of several key molecules in

the MAPK cascade has been directly implicated in human

genetic mental retardation syndromes characterized by learn-

ing and memory disabilities. This is the case in neurofibro-

matosis type 1 due to NF-1 gene mutation, the Coffin–Lowry

syndrome caused by different types of mutations in the Rsk2

Davis and Laroche


gene or the Rubinstein–Taybi syndrome due to mutations in

the gene encoding CREB-binding protein (see Weeber &

Sweatt 2002 for a review). As future research will expand

our understanding of how the MAPK cascade controls cell

plasticity and neural network adaptation required for memory

formation, investigating the relations between diseases that

affect memory function, the expression of plasticity in neu-

rons and the underlying molecular mechanisms will offer

prospects for identifying the mechanisms that go awry

when memory is deficient and for the design and biobehav-

ioural evaluation of molecular strategies for therapeutic pre-

vention and rescue.

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Acknowledgments

Some of the work presented in this manuscript was supported

by an HFSP grant RGY0152 to SD.

Davis and Laroche


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