signaling from synapse to nucleus - stanford university

Signaling from synapse to nucleus: the logic behindthe mechanismsKarl Deisseroth�, Paul G Mermelsteiny, Houhui Xiaz§ and Richard W Tsienz#

Signaling from synapse to nucleus is vital for activity-dependent

control of neuronal gene expression and represents a

sophisticated form of neural computation. The nature of specific

signal initiators, nuclear translocators and effectors has become

increasingly clear, and supports the idea that the nucleus is able

to make sense of a surprising amount of fast synaptic information

through intricate biochemical mechanisms. Information transfer

to the nucleus can be conveyed by physical translocation of

messengers at various stages within the multiple signal

transduction cascades that are set in motion by a Ca2þ rise near

the surface membrane. The key role of synapse-to-nucleus

signaling in circadian rhythms, long-term memory, and neuronal

survival sheds light on the logical underpinning of these

signaling mechanisms.

Addresses�Department of Psychiatry and Behavioral Sciences, Stanford University

School of Medicine, Stanford CA 94305, USA

e-mail: [email protected] of Neuroscience, University of Minnesota, 6-145 Jackson

Hall, 321 Church Street South East, Minneapolis, MN 55455, USA

e-mail: [email protected] of Molecular and Cellular Physiology, Stanford University

School of Medicine, Stanford CA 94305, USA§e-mail: [email protected]#e-mail: [email protected]

Current Opinion in Neurobiology 2003, 13:1–12

This review comes from a themed issue on

Signalling mechanisms

Edited by Morgan Sheng and Terrance P Snutch

0959-4388/03/$ – see front matter� 2003 Elsevier Science Ltd. All rights reserved.

DOI 10.1016/S0959-4388(03)00076-X

AbbreviationsBDNF brain-derived neurotrophic factor

CaM calmodulin

CaMK calmodulin-dependent protein kinase

CREB Ca2þ/cAMP responsive element binding protein

DREAM downstream regulatory element antagonistic modulator

NFAT nuclear factor of activated T-cells

NMDAR N-methyl-D-aspartate receptor

NPY neuropeptide-Y

PACAP pituitary adenylyl cyclase activating peptide

PKA protein kinase A

SCN suprachiasmatic nucleus

IntroductionNeuroscientists have long appreciated the computations

that ion channels perform at the surface membranes of

neurons. More recently, it has emerged that the complexity

of this electrical signal processing is rivaled by the intri-

cacies of biochemical signal processing that occur below

the membrane, within elaborate networks of cytoplasmic

signaling proteins. However, only within the past few

years has it become clear that even deeper within the

neuron lies perhaps the most formidable engine of com-

putation of all [1]. The nucleus of the mammalian neuron

creates, shapes, links and responds to these electrical and

biochemical communication streams by actively control-

ling the expression of tens of thousands of genes — a

more diverse repertoire than that presented by any other

known cell type.

Until recently, little was understood about the specific

role of the nucleus, apart from the notion that it could be

involved in the maintenance of long-term changes in

neuronal function [2]; it was not at all obvious how

synapses, as myriad sources of fast electrical signals, could

communicate even a tiny fraction of their rich information

stream to the distant and apparently slow nucleus. It has

now become evident that the nucleus is able to make sense

of a surprising amount of fast synaptic information through

intricate biochemical mechanisms [3–5]. Furthermore, as

we review here, there is now a much clearer understanding

of the crucial role of synapse-to-nucleus signaling in certain

key neuronal processes, namely circadian rhythms, long-

term memory and neuronal survival. This functional and

behavioral level of understanding now anchors our inter-

pretation of the underlying logic of the nuclear signaling

mechanisms employed. An emerging theme as we show

here is that the complexity of neuronal biochemistry may

be necessary to generate signal processing streams (from

the rapid ion channel kinetics that initially shape electrical

information flow to the slower biochemical effectors) that

are specific enough to convey an information-laden signal

to the nucleus.

Many signaling pathways to the nucleusWhat signaling entity physically translocates to the

nucleus to support the transfer of information from the

neuronal cell membrane? In their pioneering paper on

excitation–transcription coupling, Morgan and Curran [6]

put forward a hypothesis for how Ca2þ entry through

L-type Ca2þ channels might activate expression of c-fos,an immediate early gene (IEG; IEGs constitute the first

wave of genes to be turned on, often within minutes, after a

stimulus). They suggested that a calmodulin (CaM)-sen-

sitive kinase would phosphorylate a transcription factor,

initially positioned in the cytoplasm, causing it to move

into the nucleus to activate gene expression. This first

speculation was prescient in broad outline and provocative

1

CONEUR 55

www.current-opinion.com Current Opinion in Neurobiology 2003, 13:1–12

in highlighting the following fundamental questions. First,

what are the crucial and specific signal initiators? Second,

what are the nuclear translocators, and why are they

selected for the task? Third and finally, what is the basis

for stimulus specificity and reliable information transfer?

By focusing on the pathways triggered by an intracellular

Ca2þ rise downstream of synaptic activity, we now know

that the signal from the cytoplasm to the nucleus can be

conveyed in several different ways (Figure 1). The trans-

location can take place at various stages within signal

transduction cascades that are set in motion by the Ca2þ

rise. At one extreme, as hypothesized by Morgan and

Curran, the transcription factor itself, the terminal factor

in the biochemical cascades, can move to the nucleus

upon changes in its phosphorylation state. The classic

example is a member of the nuclear factor of activated T-

cells (NFAT) family of transcription factors. Originally

characterized in immune tissue, the NFATc group plays a

crucial role in neuronal plasticity as well as vascular

development and muscular hypertrophy [7]. NFATc4

is expressed within hippocampal neurons, and undergoes

a striking translocation from the cytosol to the nucleus

upon the opening of L-type Ca2þ channels [8]. The trans-

location step is dependent upon calcineurin-mediated

dephosphorylation of NFATc4, which causes the unmask-

ing of multiple nuclear localization signals and the active

transport of the transcription factor through the nuclear

pore complex.

At the other extreme, a mechanism may exist that

transduces directly the free Ca2þ transient that can arise

in the nucleus after neuronal excitation [9]. The tran-

scription factor downstream regulatory element antag-

onistic modulator (DREAM) [10], a Ca2þ binding

protein that contains three active Ca2þ binding motifs

(E-F hands) [11�], is abundant in the nucleus. It has been

proposed that nuclear DREAM remains bound to a

downstream regulatory element (DRE) that acts as a

gene silencer when nuclear Ca2þ is low, but dissociates

upon elevation of Ca2þ causing DRE derepression, acti-

vation of downstream genes such as that which encodes

prodynorphin [10] and attenuation of pain signaling invivo [12�]. It will be interesting to see if DREAM

function can be formally linked to nuclear Ca2þ levels,

and to understand why DREAM is present outside the

nucleus as well as within it.

Are there particular advantages for the signaling mechan-

isms at either one or the other of these extremes? On the

one hand, the nuclear Ca2þ mechanism that is exempli-

fied by DREAM is straightforward, fast and simple, but it

might be a suboptimal choice in general as the nucleus

would suffer from a severe loss of specificity with regards

to the source of Ca2þ and the nature of the initial

membrane signal. On the other hand, using a highly

processed nuclear signal like the transcription factor itself

(as in NFAT translocation) allows for a high level of

signaling specificity, but in placing the bulk of the signal

amplification and processing outside of nucleus more

pressure is placed on nuclear transport mechanisms. As

electrophysiologists know, it is unwise to amplify a signal

before passing it through a channel that can be saturated,

as it could result in serious distortion of the signal.

Activation of DREAM and NFATc4 define the extremes

of information transfer from the plasma membrane to the

nucleus, whereas Ca2þ/cAMP responsive element bind-

ing protein (CREB) signaling mechanisms typically

exemplify the middle ground. In this case, the translo-

cating message is not conveyed by free Ca2þ moving from

the ion channels to the nucleus, or by the transcription

factor CREB, but by intermediate players in the path-

way. In fact, CREB remains constitutively bound to CRE

(Ca2þ/cAMP responsive element) regulatory sites con-

trolling target genes, and becomes activated by phos-

phorylation on its Ser-133 residue (with modulatory

influences exerted by other residues). A fast CaM-depen-

dent kinase cascade and a slow MAP kinase cascade

(likely to be cAMP-modulated) converge on Ser-133

following surface membrane depolarization and Ca2þ

influx, and work together to promote CREB-dependent

gene expression [3,13].

How are these two signals transduced? It is well estab-

lished that in response to physiological synaptic activity in

hippocampal pyramidal neurons, Ca2þ entry through both

N-methyl-D-aspartate receptors (NMDARs) and L-type

Ca2þ channels initiates the nuclear signaling to CREB

[14–16]. Nevertheless, Ca2þ ions can be excluded as the

(Figure 1 Legend) Signaling from the membrane to the nucleus: multiple strategies for information transfer. (a) The transcription factors NFATc4,

CREB and DREAM are all activated following increases in intracellular Ca2þ, yet each relies upon a different mode of information transfer. At rest,

NFATc4 is localized to the cytosol, allowing the transcription factor to be targeted to specific regions of the cell. This would allow for heightened

signaling specificity. Indeed, Ca2þ entry through L-type calcium channels activates this transcription factor preferentially relative to other voltage-

gated Ca2þ channels. Yet, this mode of synapse-to-nucleus signaling is limited in both speed and signal amplification. Conversely, DREAM can be

activated by general rises in intracellular Ca2þ, allowing for rapid information transfer. However, this pathway is limited in regards to signal specificity.CREB activation is typically initiated by nuclear translocation of the calcium sensor CaM or by an activating kinase, providing a combination of the

advantages found for both NFATc4 and DREAM signaling. (b) A snapshot image of the early stages (0–5 min) of activity-dependent gene expression.

Following rises in intracellular Ca2þ, DREAM dissociates from DNA resulting in the lifting of transcriptional repression. Within seconds of Ca2þ entry

through L-type Ca2þ channels and NMDA receptors, CaM translocates to the nucleus, supporting CREB phosphorylation through activation of

CaMKIV. Nearly as rapid, NFATc4 also undergoes translocation to the nucleus following its dephosphorylation by calcineurin (CaN). Other pathways

outlined in (a), including the MAPK/PKA pathways, subsequently begin to exert their influence. Abbreviations, VGCC, voltage-gated calcium channel.

2 Signalling mechanisms

Current Opinion in Neurobiology 2003, 13:1–12 www.current-opinion.com

Figure 1

L-typeVGCC

NMDAR

CREB

NFATc4Ca2+

CaM

CaMKIV

CaN

AC

MAPK

PKA

NFATc4

Ca2+ rise

Ca2+ rise

Ca2+ rise

Ca2+ rise

Ca2+ rise

SURFACE/CYTOSOLIC EVENTS

NUCLEAREVENTS

Binding to DREAM

CaM CaMKK/CaMKIV

CREB phosphorylation

CaMMAPK/

Rsk CREB phosphorylation

CaM CaN NFATc4

AC PKA CREB phosphorylationCaM

Removal from DNA:DREAM repression

lifted

Interaction with CBP:CRE-transcription

Interaction with NFATn:NFAT-transcription

Information transferto the nucleus

DREAM

DREAM

(a)

Current Opinion in Neurobiology

(b) Early stages of activity-dependent signaling to the nucleus

Signaling from synapse to nucleus Deisseroth, Mermelstein, Xia and Tsien 3


typical nuclear messenger in these cells as CREB activa-

tion at Ser-133 shows marked Ca2þ channel-type speci-

ficity. Working alongside Ca2þ stores, many Ca2þ influx

channels (including L-type, N-type, and P/Q-type Ca2þ

channels as well as NMDA- and AMPA-type glutamate

receptors) can cause a marked elevation in the bulk

cytoplasmic and nuclear Ca2þlevels, and can play impor-

tant roles in the neuron [17]. However, most of these are

ineffective at directly activating CREB, with the excep-

tion of the L-type channel (privileged among the voltage-

gated channels [15,16,18��]) and the NMDA receptor

[14–16]. Therefore, although nuclear Ca2þ elevations

are seen during relatively strong neuronal activity [9]

and may support the signaling in some cases [19,20],

nuclear Ca2þ is not typically sufficient to activate CREB

[15,18��]. Furthermore, it appears that nuclear Ca2þ eleva-

tions are not necessary, as suppression of global cytoplas-

mic and nuclear Ca2þ elevations with intracellular loading

of the Ca2þ chelator EGTA generally does not block Ca2þ-

activated CREB Ser-133 activation [4,14,21��]. These

findings, together with results that demonstrate micro-

meter-scale localization of L-type Ca2þ channels within

the plasma membrane [22��] and a CaM-binding motif on

the L-type channel itself [18��] are both crucial for CREB

phosphorylation, have revealed that Ca2þ typically exerts

its effects on nuclear CREB activation by acting within a

domain <1 mm in radius from its source of entry [14].

The privileged roles of the NMDA receptor and the L-

type channel [14–16] may be attributable to their selec-

tive ability to recruit CaM signaling [15]. While both

channel types can directly bind CaM, both are also

specifically localized at or near synapses in which addi-

tional pools of CaM are present for recruitment. Either or

both mechanisms may underlie the selective ability of

these channels to initiate locally Ca2þ/CaM-activated

MAP kinase signaling to the nucleus [13,18��,21��,23]

and nuclear translocation of CaM [15,20,24,25��]. The

translocation of CaM to the nucleus is dependent upon its

ability to bind to a CaM kinase [20,25��,26]. This binding

would stabilize Ca2þ–CaM, although locally mobilized

CaM could also subsequently make use of Ca2þ from

other sources [19,20], perhaps from nuclear Ca2þstores or

even cytoplasmic Ca2þ waves. Use of ‘supportive’ Ca2þ in

this way would still preserve the specificity of information

carried by the selective synaptic activation of the NMDA

receptor and the L-type channel, as only these channel

types would be able to mobilize the CaM necessary to

utilize efficiently the available Ca2þ. Despite the high

concentrations of detectable CaM in all cellular compart-

ments, free or available CaM is generally limiting in

intracellular biochemical reactions [27]. The resulting

increase in available nuclear CaM results in strong activa-

tion of nuclear CaM-dependent protein kinase kinase

(CaMKK) and its target CaMKIV. These kinases are

ultimately responsible for the early wave of Ser-133

CREB phosphorylation [13,28,29��,30,31]. With stronger

stimulation, the slower MAPK-dependent signaling path-

way comes into play [13]. In this case, Ca2þ/CaM leads to

local activation of the small GTP binding protein Ras,

stimulation and nuclear translocation of mitogen-activated

protein kinase (MAPK) and/or pp90 ribosomal protein S6

kinase (Rsk) [3,18��,32], in some cases with cooperation of

protein kinase A (PKA). In summary, both CaMK and

MAPK pathways to CREB generally use a downstream

Ca2þ effector as the translocating nuclear messenger. This

thereby preserves information about the specific Ca2þ

source, while also leaving ample room for signal amplifica-

tion in the nucleus — a combination of features that have

advantages over the more extreme scenarios.

Clearly then, the transfer of information from the mem-

brane surface of a neuron into the nucleus can be

achieved by physical translocation at various points in

the signal transduction pathway. Understanding the

choice of strategy and the associated information proces-

sing trade-offs can best be attempted in the context of the

functional output of the pathway. Therefore, next we will

review recent work in intact animals describing the dis-

ruption of nuclear signaling pathways and their high-level

behavioral consequences: that is, generation of circadian

rhythms, formation of long-term fear memories, and

survival of brain cells.

Circadian rhythmsThe generation and entrainment of circadian rhythms, of

nearly universal importance in biology, provides an out-

standing example of the functional importance of

synapse-to-nucleus signaling. Many aspects of circadian

rhythm generation are hardwired, in that they occur with-

out specifically requiring neuronal activity, and arise in a

network of transcription factors that generate rhythmicity

from negative feedback signaling. In the suprachiasmatic

nucleus (SCN) of the mammalian hypothalamus, for

example (Figure 2), multiple proteins are involved in

the clockwork mechanism, including dimers involving

the transcription factors of CLOCK and BMAL, and

the period gene products mPer1, mPer2 and mPer3. This

endogenous rhythm is robust but not precise, however,

and numerous other circumstances, including seasonal and

migratory changes in patterns of light and dark, demand

that the cycle be entrainable. In fact, exposure to light

before the end of a subjective night, a signal indicating

that the rhythm needs adjustment, triggers rapid induction

of the genes for mPer1 (within 15–30 min) and mPer2

(within 2 h), leading to an efficient readjustment of the

circadian rhythm.

How are these clock genes activated? In addition to E-box

elements (regulated by the CLOCK/BMAL system),

both mPer1 and mPer2 have activity-sensitive CRE pro-

moter elements that are regulated by the CRE-binding

protein CREB [33]. Evidence is mounting that CREB-

mediated control of these genes referees circadian rhythm



cycling and entrainment. First, CREB activation by phos-

phorylation of the Ser-133 residue follows an active

circadian rhythm (peaking near subjective dawn) and

can be directly triggered by entraining stimuli, such as

photic stimulation during darkness [16]. Second, expres-

sion of the CREB-dependent genes c-fos, mPer1 and

mPer2 correlates with CREB activation, and is blocked

when a key modulatory site (Ser-142) on CREB is

mutated [34��]. Third and finally, this CREB mutation

attenuates entrainment of the circadian oscillator with

respect to either phase advances or phase delays [34��].Taken together, these data show that activity-dependent

control of a nuclear transcription factor (CREB) mediates

a very important complex behavior (entrainment of cir-

cadian rhythm).

What are the key design constraints on this activity-

dependent signaling pathway? First, to be useful, the

pathway should be fast by comparison with the circadian

cycle (minutes), although signaling on a scale of seconds

may be unnecessary. It need not resolve closely spaced

stimuli (after all, closely spaced flashes of daylight were

hardly a factor in circadian evolution!). Second, in

responding to electrophysiological events, the pathway

would ideally be sensitive to synaptic input from afferents

that carry sensory information from the retina, but not to

the continually free-running spiking of SCN neurons,

which is intrinsic even in isolated neurons. Third, as

non-photic inputs such as feeding or locomotor activity

modulate entrainment in some cases, the pathway must

allow for the convergence of multiple stimuli, not just

retinal glutamatergic afferents.

Every one of these design requirements seems to be

fulfilled by aspects of synapse-to-nucleus signaling in

SCN neurons. First, phosphorylation of CREB Ser-133

does occur within about 10 min of light flashes or gluta-

matergic stimulation of SCN neurons, followed by mPer1

Figure 2

ZZ

Z Z Z

Outputto SCN

L-typeVGCC

mPer1CREBP P

NMDAR

SCN neuronSynaptic transmissionfrom ganglion cells

S133S142

CLK BMAL

CRE E-Box

mPER1

SCN ACTIVITY

Simple spikes: pacemaker activity

Complex spikes: APs and EPSPs

TIME

CRY

RESULT

Minimal NMDAR,L-type VGCC

activity

Activation of NMDAR and

L-type VGCC:CREB phosph

EFFECT

MinimalmPer1

expression

StrongmPer1

expression

INTERNALCLOCK


Input from retina

Activity-dependent regulation of circadian rhythms. Light-induced activation of retinal ganglion cells results in the excitation of SCN neurons. The

increases in synaptic neurotransmission result in the opening of L-type calcium channels and NMDA receptors, directly leading to CREB

phosphorylation and the transcription of several clock genes including mPer1. These changes in gene expression result in the entrainment of the

internal clock to the external environment.



induction within 15–30 min [34��]. This suggests the

involvement of either the fast CaMKIV signaling pathway

leading to phosphorylation of CREB on Ser-133 or the

slower CaM kinase/MAP kinase-dependent pathway,

both of which can be efficacious at 10 min [13]. In fact,

inhibitors of either CaM kinase or MAP kinase block

CREB activation in SCN neurons [35]; in the future,

circadian rhythmicity and fast signaling to CREB might

also be studied in the SCN of CaMKIV knockout mice.

Second, with regards to selective coupling to particular

electrical events at the membrane, certain pathways of

nuclear signaling in hypothalamic neurons have been

found to respond relatively selectively to membrane

potentials generated by synaptic input in preference to

action potentials alone [36], as in various ganglionic

neurons [37,38] and hippocampal pyramidal neurons

[14,39]. It has become increasingly clear how gene

expression avoids indiscriminate activation by the con-

tinuous tonic spiking of these cells. CREB signaling in

hypothalamic SCN neurons responds to retinal glutama-

tergic afferents through a pathway initiated by NMDA-

type glutamate receptors and voltage-gated L-type Ca2þ

channels. Activation of the NMDARs requires synaptic

glutamate, and therefore needs more than just spikes to

occur. Among the many voltage-gated Ca2þ channels,

only the L-type channel has been found to couple to

mPER1 [40]. If this remains true when the contributions

of the other channels are rigorously examined [15], it

suggests that there is a possibility that L-type channels

help to ‘filter out’ brief single action potentials by virtue

of their voltage- and time-dependent kinetics [39]. This

selectivity would probably not be possible if the nuclear

message were conveyed by Ca2þ ions for example, as

strongly spike-responsive Ca2þ channels (N-type or P/Q-

type) also generate large increases in bulk cytoplasmic

and nuclear Ca2þ. The use of CaM as a synapse-to-

nucleus messenger [15] would avoid this difficulty, a

possibility that needs to be tested in SCN neurons. Third,

signaling in the SCN appears well equipped to integrate

modulatory input from non-photic CNS systems, includ-

ing that of neuropeptide-Y (NPY; provided by afferents

from the thalamus), serotonin, and pituitary adenylyl

cyclase activating peptide (PACAP). All three systems

appear to mediate behavioral influences on circadian

rhythmicity, and are known to use G protein signaling

to the cAMP/PKA pathway as a means of exerting control

over intracellular events (PACAP positively, NPY nega-

tively, and serotonin being capable of both). Indeed,

application of forskolin, a direct activator of the cAMP/

PKA pathway, promotes mPer1 expression in SCN neurons

[41,42], and expression of dominant negative PKA inhibits

CRE-dependent gene expression in SCN neurons [41].

PKA could influence nuclear signaling in several different

ways, for example, by direct phosphorylation of CREB

residue Ser-133, by stimulating a CREB transcriptional

coactivator such as CBP or p300 [43], or by potentiating

MAPK signaling [23].

Equally intriguing is the possibility that PKA-mediated

signaling could act through direct modulation of ion

channels, thereby regulating the ability of L-type Ca2þ

channels to respond to the tonic electrical activity of SCN

neurons. This would support the convergence of diverse

inputs and allow a non-photic input to recruit exactly the

same signaling pathway as a light stimulus, even without

retinally driven synaptic input. Indeed, it is known that

the ability of L-type channels to filter out action poten-

tials is conditional, in that direct potentiation of ion flux

restores some responsiveness to brief depolarizations [39].

It is also likely that spike width broadening or high-

frequency bursts of action potentials would be able to

conditionally overcome this filter [44]. To test the validity

of this idea in SCN circadian rhythmicity, it would be

important to conduct electrophysiological studies of SCN

action potentials and the resulting L-type currents at

378C [45]. Testing at this temperature would avoid the

opposing effects on spike width and channel gating

kinetics that are present under even slightly lower tem-

peratures, and under a range of waveform and modulation

conditions. The potential modulation of action potential

filtering could also be tested in vivo, by using L-type

antagonists and potentiators to see if direct manipulation

of L-type channels alters the behavioral responses to non-

photic inputs through NPY, serotonin or PACAP.

Learning and memory exemplified byfear conditioningMemory formation is another vital high-level behavioral

function that depends on nuclear events [2,46,47]. Again,

knowledge of the behavioral output of the nuclear event

can greatly illuminate our understanding of the logic that

underlies the nuclear signaling. Much attention has been

directed towards the hippocampus, which not only sup-

ports explicit memory formation in tasks like spatial

learning but also contributes crucially to contextual fear

conditioning, in which the animal learns to associate a

complex, generally benign environment with an aversive

stimulus (Figure 3). The amygdala mediates cued fear

memories, formed when an aversive stimulus is asso-

ciated with a single stimulus, such as an auditory tone.

In both cases, subsequent presentation of the harmless

stimulus alone will trigger fear behavior such as freezing.

Neurons in both the amygdala and the hippocampus

display robust activation of CREB during these learning

tasks [25��,48], and in both structures, genetic interfer-

ence with CREB function prevents storage of long-term

fear memories [49,50�]. Additionally, in either one or the

other region, memory deficits associated with reduced

CREB function can be partially overcome by spaced

repeated training, whereas high-level CREB function

can allow long-term memories to form with single training

sessions [50�,51,52�].

Given these similarities, several predictions can be made.

First, that the hippocampus and amygdala would employ



a common molecular mechanism of signaling to CREB.

Second, that CREB should respond strongly to synaptic

input that bears incoming processed sensory information

in both cases. Third, that activity-dependent signaling

events that lead to CREB activation should display con-

siderable speed and temporal resolution, as temporal

relationships among events are important in elucidating

their causal relationship. Fourth and finally, that stimuli

that are spaced and repeated, those most appropriate for

associations by the brain should be particularly advan-

taged in activating CREB by comparison with single

stimulus bouts.

In the hippocampus, CREB signaling triggered by phy-

siological electrical activity is indeed selectively depen-

dent on Ca2þ entry through L-type calcium channels and

Figure 3

AMYGDALAHIPPOCAMPUS

Contextual information Cue information

+ Pain/fear

One exposure underintense conditions

Multiple spaced exposures

Strong CaM/CaMKIVCREB phosphorylation

Support from MAPK/PKA

Strong CaM/CaMKIVCREB phosphorylation

Support from MAPK/PKAInactivation of CaN

Facilitation of CaM signaling

Possibly spurious or rarebut too important to ignore

Clearly related andassociation should be

learned

Result

Stimulus

Response

Properconclusion

One or massedexposure

Mild CaM/CaMKIV-mediatedCREB phosphorylation

Spurious or rare, possibly anunimportant association


No CREB-mediatedlong-term memory

CREB-mediatedlong-term memory

genetic orpharmacological

alteration in CREB signaling

Activity-dependent fear conditioning within the hippocampus and the amygdala. The pairing of contextual and cue information with an adverse

stimulus results in heightened CREB activation within the hippocampus and the amygdala. Dependent upon the stimulus train, one or more CREB

signaling pathways are recruited to mediate long-term memory. With either genetic or pharmacological manipulation of the CREB signaling pathways,

changes in learning and memory can be achieved.



NMDA receptors [14–16]. This pattern of source-speci-

ficity confers selective responsiveness to synaptic activity

for the reasons discussed above. The role of these path-

ways for Ca2þ entry in amygdalar CREB activation has

not been tested yet, but L-type Ca2þ channels and

NMDA receptors are both required for amygdalar forma-

tion of long-term fear memories [53,54], which are

CREB-dependent. In both the hippocampus and the

amygdala neuronal activity causes rapid nuclear translo-

cation of CaM and CaMKIV-dependent CREB phosphor-

ylation [15,25��,28,29��,30], further reinforcing the

similarities in CREB signaling. Furthermore, it has been

demonstrated in both structures that the CaM/CaMKIV

pathway is required for establishing CREB-dependent

fear memories [25��,29��]. As noted above, the employ-

ment of a downstream effector of Ca2þ as a nuclear

messenger, rather than free Ca2þ itself, facilitates Ca2þ

source discrimination by the nucleus [2,5,15,18��]. The

use of CaM as the messenger, which is capable of sig-

nificant nuclear translocation within 15 s [20], sacrifices

little in the way of speed.

Interestingly, the CaM nuclear translocation pathway,

widely employed by non-neuronal cell types as well as

neurons [15,24,26,27,55–58], is now also known to be

almost ubiquitous throughout the mammalian brain,

including hippocampus, amygdala, somatosensory cor-

tex, insular cortex, anterior cingulate cortex and cere-

bellum [15,20,25��]. However, an exception has been

found, three groups have determined that there is little

CaM translocation in a substructure of the hippocampal

formation called the dentate gyrus [19,20,25��]. Although

the dentate gyrus is anatomically positioned as the ‘gate-

way’ to the hippocampus proper, its precise role is

unknown. Dentate granule neurons differ from hippo-

campal pyramidal cells in many ways, including their

characteristics of development, regeneration, morphol-

ogy and synaptic plasticity. It would be interesting to find

out if the role of these neurons at the systems level were

somehow linked to unusual features of their subcellular

signaling.

What are the advantages of this pathway? Use of the

CaM/CaMKIV pathway to CREB lends itself well not

just to speed but also to implementing repetitive spaced

stimulus responsiveness. Brief mild stimulation of hip-

pocampal neurons that selectively activates the calmo-

dulin/CaMKIV pathway to CREB [13], also creates a

primed state in which subsequent similar stimuli are

more potent and even faster in their effects on nuclear

CREB [20]. The increased speed of CREB formation

suggests that the priming effect might be attributable to

the nuclear use of persistently translocated CaM. After

nuclear translocation, return of CaM to the cytoplasm

proceeds gradually over about 1 hour [15]. This extended

time course fits reasonably well with behavioral data on

spaced stimuli [51,52�].

As noted earlier, CREB responds to multiple converging

signaling pathways in hippocampal neurons, including a

slower but more prolonged MAPK-mediated activation

pathway that overlaps with the calmodulin/CaMKIV

pathway [3–5,13], and a calcineurin/protein phospha-

tase-1-mediated CREB deactivation pathway [31,59].

Flexible association of aversive stimuli with a wide range

of environmental situations of different importance and

temporal structure may require such complexity of sig-

naling. The MAPK pathway to CREB has poorer tem-

poral resolution than the CAMK pathway (slower in

onset, and more persistent), but it has several interesting

properties, including further prolongation by spaced

repeated stimulation [23] or PKA activation [23,32,60],

and selective responsiveness to stronger stimulation [13].

Indeed, activation of the MAPK pathway can be involved

in forming both amygdalar [61] and hippocampal [62]

fear memories. Similarly, the calcineurin/PP1 pathway

responds to repeated stimuli by potentiating CREB func-

tion — in this case through activity-dependent inactiva-

tion of calcineurin [31,63]. Notably, direct calcineurin

inhibition potentiates memory formation in the amygdala

[64�] and the hippocampus [65�].

Taken together, these data collected in the amygdala and

the hippocampus reveal a clear pattern — stimuli of

greater behavioral significance are endowed with a more

potent means of activating CREB. In this manner, the

nuclear computer capitalizes on the diverse characteris-

tics of its many incoming signaling pathways, including

sensitivity and temporal resolution, to calculate the sta-

tistical significance of candidate associations for storage in

long-term memory.

Control of neuronal survival and deathNeurons receive specific instructions to survive or to die,

the correct choice being crucial for development and

mature functioning of the brain. The decision to die is

particularly interesting, as neurons reach this all-or-none

choice after weighing a wide array of external inputs,

including synaptic events. This is reminiscent of faster

electrophysiological choices made between quiescence

and firing an action potential. The nucleus plays a key

role in the survival/death computation, largely through

activity-dependent regulation of transcription factors

such as CREB, MEF2 and NF-kB. An emerging theme

is the highly conditional influence of these transcription

factors, in that they promote survival in some circum-

stances but not in others.

It has been known for years that stimulation of electrical

activity at mild to rapid rates (for example, 5Hz to 50Hz

electrical stimuli) works through both NMDA receptors

and L-type Ca2þ channels to activate nuclear CREB

[14,15]. Accordingly, activation of CREB would not be

expected to promote cell death in the normal brain. Indeed,

CREB generally favors neuronal survival. Postnatal



genetic inactivation of CREB (along with cAMP response

element modulator [CREM]) causes widespread apopto-

sis of CNS neurons [66]. Other studies have shown that a

low level of activation of NMDA receptors or L-type Ca2þ

channels promotes survival of cerebellar granule neurons,

presumably through the activity of CaMKIV and CREB

[67]. CREB also mediates the survival-promoting effects

of brain-derived neurotrophic factor (BDNF) [68,69] and

other trophic factors.

On the other hand, excitatory stimuli that are known to be

highly toxic to mature neurons, such as steady prolonged

application of glutamate, promote general cellular dys-

function and death [70–72]. Furthermore, it has been

suggested that under conditions of very strong and long

excitation, CREB can actually promote apoptosis by

acting through a nuclear p53 cascade [73]. Perhaps this

action of CREB is aimed at eliminating neurons whose

function is deranged by excessive stimulation, thus main-

taining the integrity of CNS function. This may be

reminiscent of the other effects of p53, ‘guardian of the

genome’, which prevents mitosis and potential tumori-

genesis in the setting of DNA damage.

Transcription factors from the MEF2 family [74], espe-

cially MEF2A, -C and -D, also appear to promote survival

of cerebellar granule cells and cerebral cortical neurons in

response to NMDA application or mild depolarization

[75–77]. MEF2 function can be elevated directly by

calcineurin and indirectly by CaMKIV [74], once again

underscoring the importance of CaM signaling to the

nucleus [15,31]. Additional activity-dependent signals

provided by p38 MAP kinases and NFAT converge to

promote MEF2 function [74]. However, a pro-apoptotic

role for modified MEF2 proteins has also been identified

[75,77]. Cleavage of these transcription factors follows

activation of caspases, generating MEF2 fragments cap-

able of DNA binding but lacking transactivation poten-

tial. These dominant-negative factors then strongly

promote apoptosis. Thus, as in the situation with CREB,

the influence of the transcription factor in favoring or

disfavoring survival depends on the cellular context.

The NF-kB family of transcription factors is strongly

expressed in the CNS, especially in the hippocampus

and the cerebellum. They are activated by growth factors

and cytokines as well as by depolarization or glutamate

application, which leads to nuclear translocation and in

general, promotion of neuronal survival [78]. However,

several studies have identified a pro-apoptotic role for

NF-kB. Though considerable controversy remains, the

net effect of NF-kB appears to depend on the type of

stimulation, the degree of NF-kB activation, and the

particular NF-kB subunits that are expressed. Interest-

ingly, beyond simple nuclear translocation [6] of NF-kB

[79] (Figure 1), the p65 subunit of NF-kB can be further

activated by phosphorylation at Ser-276, a residue whose

amino acid sequence environment is nearly identical to

that of CREB Ser-133. It has been proposed [79] that the

site can support modulation of NF-kB function by CaM-

KIV or Rsk kinases, leading to disparate effects on

survival or death that depend on the profile of kinase

activation. Here again, as with CREB and MEF2, we see

an emerging role of nuclear computation in the integra-

tion of rich streams of incoming information to reach a

crucial decision between cell survival and cell death.

Concluding remarks and future prospectsWe have deliberately focused on mechanisms that convey

an initial message from the neuronal surface to a nuclear

transcription factor, but in which the final outcome in

gene expression also depends on multiple additional

signaling events, as illustrated below for the cases of

CREB- and NFAT-dependent transcription. In the case

of CREB, attainment of the Ser-133 activated state is

further modulated by additional activity-dependent

events, including calcineurin-regulated dephosphoryla-

tion of Ser-133 as well as the phosphorylation of Ser-

142 discussed above. Stabilization of the CREB transcrip-

tional complex is likely to be further affected by activity-

dependent regulation of co-activators, including the

CREB binding protein (CBP) and possibly the closely

related p300 [43]. With respect to activation of NFAT-

dependent transcription, required co-activators include

activator protein-1 (AP1) (composed partly by the CREB-

regulated factor c-fos) and MEF2 [80], which is CaM-

regulated in its own right as noted above. Neuronal

activity not only triggers nuclear translocation of NFATc4

but it additionally regulates NFATc4-dependent tran-

scription by delaying nuclear export of NFAT through L-

type channel mediated inhibition of glycogen synthase

kinase 3b [8]. The result is prolonged NFATc4 activation

and increased NFAT-dependent gene expression [8]; this

is analogous to CRE-mediated gene expression when

CREB activity is extended by activity-dependent inhibi-

tion of CREB dephosphorylation [31,63]. Judging by

these examples of signaling complexity, we have only

seen the tip of the iceberg with respect to the conver-

gence or apposition of multiple mechanisms.

Intricate networks of downstream genes represent the

output of nuclear processing. A large number of genes

show activity modulation in neurons, and many of these

have plausible behavioral roles. Within hippocampal

neurons, activation of NFATc4 leads to an upregulation

of inositol 1,4,5-triphosphate receptor type 1 (IP3R1) [8]

and BDNF [81]; both proteins are known to influence

learning and memory [82–84]. CREB likewise drives

BDNF expression [69] as well as c-fos and other factors

implicated in neuronal survival and memory formation. In

circadian rhythm generation, the CREB-regulated mPergenes are well-characterized targets of retinal activity and

have clear behavioral roles. But exactly how the wide

array of activity-regulated genes generate meaningful



changes in the brain’s neural networks is still not under-

stood. Though many aspects of nuclear computation

remain shrouded in mystery, it is clear that a combination

of in vitro analysis and behavioral investigation can begin

to illuminate the rationale for the unique biochemical

complexity of neurons, and the elegant logic behind the

design of nuclear signaling pathways.

AcknowledgementsRW Tsien is supported by grants from the National Institute of GeneralMedical Sciences (58234), National Institute of Neurological Disorders andStroke (24067) and National Institute of Mental Health (48108). PGMermelstein is supported by a grant from the National Institute ofNeurological Disorders and Stroke (41302), and a grant from the WhitehallFoundation. K Deisseroth and PG Mermelstein contributed equally to themanuscript.

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