The Journal of Neuroscience, December 5, 2012 • 32(49):17857–17868 • 17857

Behavioral/Systems/Cognitive

Increasing CRTC1 Function in the Dentate Gyrus during
Memory Formation or Reactivation Increases Memory
Strength without Compromising Memory Quality
Melanie J. Sekeres,1,2 Valentina Mercaldo,1,2,3 Blake Richards,1,2,3 Derya Sargin,1,2,3 Vivek Mahadevan,3Melanie A. Woodin,3 Paul W. Frankland,1,2,4 and Sheena A. Josselyn1,2,4
1
3

Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, and 2Department of Physiology,
Department of Cell and Systems Biology, and 4Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5G 1X8, Canada

Memory stabilization following encoding (synaptic consolidation) or memory reactivation (reconsolidation) requires gene expression
and protein synthesis (Dudai and Eisenberg, 2004; Tronson and Taylor, 2007; Nader and Einarsson, 2010; Alberini, 2011). Althoughconsolidation and reconsolidation may be mediated by distinct molecular mechanisms (Lee et al., 2004), disrupting the function of thetranscription factor CREB impairs both processes (Kida et al., 2002; Mamiya et al., 2009). Phosphorylation of CREB at Ser133 recruits
CREB binding protein (CBP)/p300 coactivators to activate transcription (Chrivia et al., 1993; Parker et al., 1996). In addition to this wellknown mechanism, CREB regulated transcription coactivators (CRTCs), previously called transducers of regulated CREB (TORC) activity, stimulate CREB-mediated transcription, even in the absence of CREB phosphorylation. Recently, CRTC1 has been shown to undergo
activity-dependent trafficking from synapses and dendrites to the nucleus in excitatory hippocampal neurons (Ch’ng et al., 2012). Despite
being a powerful and specific coactivator of CREB, the role of CRTC in memory is virtually unexplored. To examine the effects ofincreasing CRTC levels, we used viral vectors to locally and acutely increase CRTC1 in the dorsal hippocampus dentate gyrus region ofmice before training or memory reactivation in context fear conditioning. Overexpressing CRTC1 enhanced both memory consolidation
and reconsolidation; CRTC1-mediated memory facilitation was context specific (did not generalize to nontrained context) and long
lasting (observed after virally expressed CRTC1 dissipated). CREB overexpression produced strikingly similar effects. Therefore, increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as established reactivated, memories without compromising memory quality.

Introduction
The stabilization of long-term memory following encoding (consolidation) or memory reactivation (reconsolidation) requires
gene expression and de novo protein synthesis (Dudai and Eisenberg, 2004; Tronson and Taylor, 2007; Nader and Einarsson,
2010; Alberini, 2011). Although the precise molecular mechanisms critically mediating these processes may differ (Lee et al.,
2004; Alberini, 2005), we, and others, showed disrupting cAMP/Ca 2⫹ responsive element binding protein (CREB) function imReceived March 22, 2012; revised Oct. 5, 2012; accepted Oct. 25, 2012.
Author contributions: M.J.S., M.A.W., P.W.F., and S.A.J. designed research; M.J.S., V. Mercaldo, B.R., D.S., and V.
Mahadevan performed research; M.J.S., V. Mercaldo, B.R., and S.A.J. analyzed data; M.J.S., V. Mercaldo, B.R., D.S., V.
Mahadevan, M.A.W., P.W.F., and S.A.J. wrote the paper.
This work was supported by from the Canadian Institutes of Health Research (CHIR) Grants MOP-74650 (S.A.J.)
and MOP-86762 (P.W.F.), EJLB Foundation (S.A.J.), and Natural Science and Engineering Research Council (S.A.J.).
M.J.S. received support from a Restracomp Fellowship (Hospital for Sick Children), a CIHR Frederick Banting andCharles Best Canada Graduate Scholarships Doctoral Award, and the Faculty of Medicine at the University of Toronto.
D.S. was supported by the Alzheimer’s Society of Canada. B.R. was supported by a Banting Postdoctoral Fellowship(CIHR). We thank Mika Yamamoto, Russell Braybon, and Antonietta (Toni) DeCristofaro for excellent technicalassistance.
The authors declare no competing financial interests.
Correspondence should be addressed to Sheena A. Josselyn, 6018 McMaster, The Hospital for Sick Children, 555
University Ave, Toronto, ON, M5G 1X8. E-mail: sheena.josselyn@sickkids.ca.
DOI:10.1523/JNEUROSCI.1419-12.2012
Copyright © 2012 the authors 0270-6474/12/3217857-12$15.00/0

pairs both synaptic consolidation (Bourtchuladze et al., 1994; Yinet al., 1994; Guzowski and McGaugh, 1997; Bartsch et al., 1998;
Kida et al., 2002; Pittenger et al., 2002; Frankland et al., 2004) andreconsolidation (Kida et al., 2002; Mamiya et al., 2009; Yang et al.,2011). In contrast, increasing CREB promotes memory consolidation (Josselyn et al., 2001; Wallace et al., 2004; Han et al., 2007;
Restivo et al., 2009; Zhou et al., 2009; Sekeres et al., 2010).
Whether increasing CREB function similarly promotes memory
reconsolidation is unknown.
CREB modulates the transcription of genes with cAMP responsive elements (CREs) (Shaywitz and Greenberg, 1999; De
Cesare and Sassone-Corsi, 2000; Mayr and Montminy, 2001;
Lonze and Ginty, 2002). Phosphorylation of CREB at Ser133 promotes recruitment of CREB binding protein (CBP) and p300 to
stimulate transcription (Sheng et al., 1991; Radhakrishnan et al.,
1997; Chawla et al., 1998; Shaywitz and Greenberg, 1999; Kornhauser et al., 2002). Interestingly, phosphorylation of CREB at
Ser133 is not always sufficient to stimulate transcription, suggesting additional transcriptional modulators are involved. Indeed,
CREB regulated transcription coactivators (CRTCs) were identified as potent modulators of CREB-mediated transcription (Iourgenko et al., 2003; Conkright et al., 2003b). Although CRTCs may
potentiate the ability of CREB to recruit CBP/p300 (Xu et al.,

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17858 • J. Neurosci., December 5, 2012 • 32(49):17857–17868

2007), CRTCs stimulate CRE-dependent transcription via aphosphorylation-independent interaction with the basic leucine zipper domain of CREB (Iourgenko et al., 2003; Conkright et al.,2003b). Therefore, CRTCs may provide a powerful mechanism forspecifically enhancing CREB function.
In mice, CRTCs are encoded by three genes (CRTC1–3)(Iourgenko et al., 2003; Conkright et al., 2003b) with CRTC1
showing highest brain expression (Wu et al., 2006; Zhou et al.,2006; Kovács et al., 2007; Altarejos et al., 2008; Watts et al., 2011).
Under basal conditions, CRTC1 is sequestered in the cytoplasm;
dephosphorylation of CRTC1 by calcium promotes nuclear
translocation (Screaton et al., 2004) while elevated cAMP increases the nuclear persistence of CRTC1 (Ch’ng et al., 2012).
Nuclear accumulation of CRTC1 is a sensitive readout of synaptic
activity in hippocampal neurons (Ch’ng et al., 2012). Intact
CRTC1 function is necessary for CRE-mediated transcription;
decreasing CRTC1 levels or blocking the interaction between
CRTC1 and CREB disrupts, while overexpressing CRTC1 increases, CRE-mediated transcription in culture (Zhou et al.,2006; Kovács et al., 2007; Ch’ng et al., 2012). Although the role of
CRTC1 in memory is unexplored, previous results show thatblocking CRTC1 function disrupts, while increasing CRTC1 levels enhances, L-LTP in hippocampal slices (Zhou et al., 2006;
Kovács et al., 2007).
Here we examined the effects of increasing CRTC1 or CREB
function in a regionally and temporally specific manner on memory consolidation and reconsolidation.

Materials and Methods
HSV vectors
Wild-type full-length CRTC1 or CREB cDNAs (kindly provided by Dr.
Satoshi Kida, Tokyo University of Agriculture) were subcloned into thebicistronic herpes simplex virus (HSV) vectors that coexpress green fluorescent protein (GFP) as a fluorescent reporter (HSV-p1005; Russo etal., 2009). In this vector, GFP expression is driven by a CMV promoter
whereas CRTC1 or CREB expression is driven by the constitutive promoter for the HSV immediate-early gene IE 4/5. Transgene expression
using this viral system typically peaks 3 d, and dissipates within 10 –14 d,following microinjection (Josselyn et al., 2001; Barrot et al., 2002; Vetere
et al., 2011) (see Fig. 2b). As a control, we used HSV-expressing GFP
alone. HSV virus was packaged using a replication-defective helper virus,
purified on a sucrose gradient, and pelleted and resuspended in 10%
sucrose, as previously described (Carlezon et al., 1998; Han et al., 2007,2008, 2009). The average titer of the virus stocks was typically 4.0 ⫻ 10 7
infectious units/ml.

Preparation of primary hippocampal neurons
Primary hippocampal neurons were prepared from E18 –E19 mice (seebelow). Briefly, hippocampi were collected in cold PBS and dissociatedusing trypsin (0.25%, 12 min at 37°C) and a glass Pasteur pipette. Neurons were plated onto poly-L-lysine-treated glass coverslips (immunostaining) or culture plates (luciferase assay) in minimum essential
medium with 10% horse serum, 0.6% glucose, 1 mM glutamax, 50 ␮g/ml
streptomycin, and 50 U/ml penicillin (Gibco-Invitrogen). Media was
replaced with Neurobasal medium (Invitrogen) containing B27 supplement (2%; Invitrogen), penicillin-streptomycin (50 ␮g/ml penicillin, 50
U/ml streptomycin), and glutamine (1 mM; Sigma) 4 –5 h later.

Immunostaining of primary hippocampal neurons
To visualize plasmid-induced CRTC1 protein expression and localization, hippocampal neurons 5 d in vitro (DIV) were transfected withplasmids expressing GFP-CRTC1 or GFP alone. Twenty-four hours
later, neurons were treated with KCl (50 mM)/FSK (20 ␮M) or vehicle for
4 h. Neurons were washed with PBS, fixed with 4% paraformaldehyde(PFA) in PBS, permeabilized and blocked [0.3% Triton X-100, 2% nor-mal goat serum (NGS; Jackson ImmunoResearch Laboratories), 0.5%
bovine serum albumin (BSA; Bioshop Canada) in PBS] then incubated at
4°C overnight with rabbit anti-CRTC1 polyclonal antibody (1:1000; Cell
Signaling Technology). After washing in PBS, neurons were incubatedwith goat anti-rabbit Alexa 568 antibody (1:500; Invitrogen) for 1 h atroom temperature, washed with PBS, counterstained with Hoechst, andmounted with PermaFluor Mounting medium (Thermo Scientific). Images were obtained using a confocal laser scanning microscope (LSM 710;
Zeiss).

Luciferase assays
DIV 5 neurons were transfected (using Lipofectamine 2000) with the
CRE reporter plasmid [500 ng; generated by replacing d2eGFP transgene
in pCRE-d2eGFP (Clontech) with the luciferase coding region from the
MRE reporter plasmid, pGL3-TATA-DesMEF, with HindIII and XbaI
(Vetere et al., 2011)]. TK-pRL vector-expressing Renilla luciferase (250ng; Promega) was used as internal control. Twenty-four hours later,
neurons were infected with GFP or CRTC1 vector and medium replaced
6 h later. Twenty-four hours later, neurons were treated with KCl (50mM)/FSK (20 ␮M) or vehicle for 4 h. Neurons were lysed and luciferase
assays conducted using a Dual Luciferase Assay kit (Promega). Firefly
and Renilla luciferase activity levels were quantified by a luminometer(Berthold Microlumat LB 96V; Fisher Scientific) and CRE-luciferase activity was normalized to Renilla-luciferase activity. Data represent meansfrom four independent experiments, with internal duplicates or triplicates for each condition.

Mice

Adult female F1 hybrid (C57 BL/6NTac ⫻ 129S6/SvEvTac) mice wereused for all behavioral experiments. Mice were bred at the Hospital for
Sick Children and group housed (3–5 mice per cage) on a 12 h light/darkcycle with food and water available ad libitum. Behavioral experiments
were conducted during the light phase of the cycle. All procedures wereconducted in accordance with the policies of the Hospital for Sick Children Animal Care and Use Committee and conformed to both the Canadian Council on Animal Care and National Institutes of Health
Guidelines on the Care and Use of Laboratory Animals.

Surgery
Mice were pretreated with atropine sulfate (0.1 mg/kg, i.p.), anesthetizedwith chloral hydrate (400 mg/kg, i.p.) and placed in a stereotaxic frame.
Skin was retracted and holes drilled in the skull bilaterally above thedorsal hippocampus (anteroposterior (AP) ⫽ ⫺2.3, mediolateral ⫽⫾1.5, ventral ⫽ ⫺1.8 mm from bregma) according to Paxinos andFranklin (2001). Viral vector (2.0 ␮l/side) was microinjected throughglass micropipettes connected via polyethylene tubing to a microsyringe(Hamilton) at a rate of 0.1 ␮l/min. Micropipettes were left in place anadditional 5 min following microinjection to ensure diffusion of vector.
Micropipettes were slowly retracted, the incision closed, and mice treatedwith analgesic (ketoprofen, 5 mg/kg, s.c.).

Verifying location of vector microinjection and extent ofviral infection
Four days following vector microinjection (except for Fig. 3d), mice weretranscardially perfused with 0.1 M PBS followed by 4% PFA. Brains were
fixed overnight (4°C) and transferred to a 30% sucrose solution. Coronalbrain slices (50 ␮m) across the entire AP extent of the hippocampus werecollected using a cryostat (Leica CM1850). Every second section was
mounted on a gel-coated glass slide and coverslipped with Vectashieldfluorescence mounting medium containing DAPI (Vector Laboratories).
Mice with extensive cortical or hippocampal damage were excluded from
subsequent statistical analyses. Consistent with previous reports fromseveral labs (Carlezon et al., 1998; Josselyn et al., 2001; Wallace et al.,
2004; Brightwell et al., 2005; Han et al., 2007; Vetere et al., 2011) microinjection of HSV vectors produces robust localized transgene expression
with minimal tissue damage around the site of microinjection (Fig. 2a,c).
GFP-immunofluorescence (which did not differ between vectors) wasused to determine placement and extent of the viral infection for eachmouse. Based on this, each mouse was classified as a “hit” or “miss” by an
examiner unaware of the treatment condition and behavioral results.

Sekeres et al. • CRTC1, Memory Consolidation and Reconsolidation

Mice were defined as “hits” if robust bilateral GFP expression was observed in the dentate gyrus (DG) of dorsal hippocampus in at least fiveconsecutive brain sections (across the AP plane). All other mice wereclassified as miss (including those with unilateral, weak, or no transgene
expression in the target region). Only mice determined to be a bilateral
hit were included in subsequent data analysis.
Typically, we observed numerous GFP ⫹ neurons in a circular region(diameter ⬃1.6 mm) centered at the site of microinjection. To determine
the percentage area of target region infected by vector, we first traced thetarget region (⫺1.46 to ⫺3.08 mm AP, corresponding to plates 43–56 in
Paxinos and Franklin, 2001) across 15 serial sections in random brains
classified as hit (CRTC1 vector, n ⫽ 14; CREB vector, n ⫽ 14; GFP vector,n ⫽ 11) then examined the number and extent of GFP ⫹ neurons within
this target region (Stereo Investigator 8 software; MBF Bioscience).
We observed GFP ⫹ cells in ⬃62–72% of the total target area (CRTC1
vector ⫽ 63.00 ⫾ 7.98%, CREB vector ⫽ 72.11 ⫾ 4.67%; GFP vector ⫽62.54 ⫾ 8.58%). Importantly, there was no difference between the percentage dentate gyrus (DG) area infected by the different vectors (F(2,36)⫽ 0.59, p ⬎ 0.05). To estimate the number of infected cells, we stereologically counted GFP ⫹ cells in the target region in a subset of brains(4 –5 brains per vector). The number of cells infected by CRTC1 vector
was 120,825 ⫾ 19,184, for CREB vector 114,066 ⫾ 11,174 and for GFP
vector 138,531 ⫾ 47,302. (The overall number of DAPI ⫹ cells in thistarget region was 527,027 ⫾ 22,108.) Therefore, the overall number of
DG cells we infected with our viral vectors was ⬃20 –23%. The number ofcells infected did not vary between vectors (F(2,11) ⫽ 0.21, p ⬎ 0.05) orcorrelate with any behavioral measure.

Immunohistochemistry
To examine the type of cell in which CRTC1 protein is endogenously
expressed, we examined the overlap of antibodies against CRTC1 proteinwith cell markers specific for excitatory neurons (␣ calcium calmodulinII, ␣-CaMKII), glial cells (GFAP), or interneurons (GAD67, parvalbumin). Images were obtained using a confocal laser scanning microscope(LSM 710; Zeiss).
GFAP, GAD67, and parvalbumin staining. Brain sections (35 ␮m)from (homecage wild-type) mice were incubated with blocking solution(0.1% BSA, 2% NGS, 0.3% Triton-X) for 2 h (room temperature, RT)
then incubated with rabbit anti-CRTC1 polyclonal (1:500; Cell Signaling
Technology) and one of the following primary antibodies: mouse monoclonal anti-GFAP (1:500; Cell Signaling Technology), mouse monoclonalanti-GAD67 (1:500; Millipore), or mouse monoclonal anti-parvalbumin(1:500; Sigma-Aldrich) at 4°C for 24 h. Sections were washed with PBS
0.1 M, then incubated with goat-anti-rabbit Alexa 568 (1:500;,Invitrogen)and goat-anti-mouse Alexa 633 (1:500; Invitrogen) for 2 h at RT.
␣-CaMKII staining. Staining for ␣-CaMKII was similar except brain
tissue was incubated with blocking solution (anti-mouse IgG blocking in
1% H2O2) for 1 h at RT then incubated with mouse monoclonal anti␣CaMKII antibody (1:1000; Millipore) at 4°C for 24 h. Sections were
washed with PBS, incubated with donkey-anti-mouse horseradish
peroxidase (1:500) for 1 h at RT and signal amplified with TSA-FCM(30 min).
CRTC1 staining. To verify that microinjection of CRTC1 vector increased expression of CRTC1 protein, we used an antibody specific for
CRTC1. Coronal brain sections (50 ␮m) from mice microinjected with
GFP or CRTC1 vector were incubated with blocking solution (0.1% BSA,
2% NGS, 0.3% Triton X-100) for 2 h RT, then incubated with rabbit
anti-CRTC1 polyclonal antibody (1:500) at 4°C for 24 h. Sections were
washed with PBS, then incubated with goat-anti-rabbit Alexa 568 (1:500)
for 2 h at RT. Sections were washed with PBS, counterstained with
Hoechst, mounted on slides, and coverslipped using PermaFluor mounting medium.
c-Fos staining. To examine whether CRTC1 increased neuronal activity, we quantified c-Fos levels in mice microinjected with CRTC1 or GFP
vector (as above) and either maintained in the homecage or 90 min
following strong training in context fear conditioning. Mice were perfused as above and coronal brain sections (35 ␮m) were incubated withblocking solution (0.1% BSA, 2% NGS, 0.3% Triton X-100) for 2 h RT,then incubated with rabbit anti-c-Fos polyclonal antibody (1:1000; Cal-

J. Neurosci., December 5, 2012 • 32(49):17857–17868 • 17859

biochem, PC38) at 4°C for 12–16 h. Sections were washed with PBS, thenincubated with goat-anti-rabbit Alexa 568 (1:500) for 2 h at RT, counterstained, mounted, and coverslipped as above. To quantify c-Fos levelsin the infected area, we used GFP expression to outline the infected
region of the DG across 12–15 serial sections per mouse, then used stereological counting (optical fractionator method) to quantify the number of c-Fos ⫹ neurons within this infected region (Stereo Investigator
8 software; MBF Bioscience). The average number of c-Fos ⫹ cells per
infected region was calculated (using values generated by Stereo Investigator) with the following formula: estimated c-Fos ⫹ population
using mean section thickness/total area (␮m 2) ⫽ c-Fos ⫹ cells per
um 2. Values were averaged per mouse and averaged across mice pergroup (see Fig. 1d).

Slice electrophysiology
One day following microinjection of CRTC1 vector into the DG, mice
were perfused with cold modified artificial CSF (mACSF) containing thefollowing (in mM): 180 sucrose, 25 sodium bicarbonate, 25 glucose, 2.5KCl, 1.25 sodium phosphate, 2 MgCl2, 1 CaCl2, 0.4 sodium ascorbate,and 3 sodium pyruvate, saturated with 95% O2/5% CO2 and with pH andosmolarity adjusted to 7.4 and ⬃295 mOsm, respectively. Brains werequickly removed and placed for 30 s in a chilled mACSF slurry. The
cerebellum and anterior forebrain were removed and the posterior portion of the brain was glued to a slicing stage with an agarose block placed
behind it. The stage was then placed in a slicing chamber filled withmACSF chilled to a slurry and continuously oxygenated with 95% O2/5%
CO2 during slicing. Brain slices (350 ␮m) were prepared on a Vibratome
1000 and slices recovered for 1 h at RT in 50 ml of continuously oxygenated Earle’s Balanced Salt Solution with 50 ␮l of 3 M CaCl2 and 150 ␮l of
3 M MgCl2 added.
During recording, slices were placed in a recording chamber perfusedwith continuously oxygenated ACSF containing the following (in mM):
125 NaCl, 25 sodium bicarbonate, 25 glucose, 2.5 KCl, 1.25 sodium
phosphate, 1 MgCl2, 2 CaCl2, 0.4 sodium ascorbate, and 3 sodium pyruvate, saturated with 95% O2/5% CO2 and with pH and osmolarity adjusted to 7.4 and ⬃295 mOsm, respectively, maintained at 36°C with a
TC-344B temperature controller and SH-27B in-line solution heater(Warner Instruments). Whole-cell recording pipettes with tip resistancesof 4 –7 M⍀ were pulled from thin-walled borosilicate glass (World Precision Instruments, TW-150F) using a Sutter Instruments P-87. Pipettes
were filled with a potassium gluconate-based internal solution containing the following (in mM): 130 potassium gluconate, 10 KCl, 10 HEPES,
0.2 EGTA, 4 ATP, 0.3 GTP, and 10 phosphocreatine, with pH and osmolarity adjusted to 7.4 and ⬃295 mOsm, respectively. Whole-cell recordings (CRTC1-infected, n ⫽ 7, Control noninfected, n ⫽ 7) were
performed using a Multiclamp 700B amplifier and digitized using an
Axon Digidata 1440A (Molecular Devices). Recordings were made fromboth fluorescent and nonfluorescent granule cells in the DG, visualizedwith an Olympus BX51WI equipped with infrared differential interference contrast and GFP epifluorescence. Recordings were performed incurrent-clamp and current steps from ⫺100 to 200 pA were injected for
1 s in 20 pA steps. To estimate spike threshold and after-spike hyperpolarization (AHP) levels, elicited spike waveforms were averaged for eachcell. The threshold was defined as the voltage at the time of the peak in the
average waveform’s second derivative, while the AHP was defined as theminimum voltage in a 3 ms window following the peak of the average
waveform. Reported voltage levels were not corrected for any liquid junction potential. All reagents used for electrophysiology were obtained
from Sigma.

Contextual fear-conditioning apparatus

Fear-conditioning chambers (Context-A, CXT-A; 31 ⫻ 24 ⫻21 cm;
MED Associates) consisted of two stainless steel and two clear acrylic
walls, with a stainless steel shock-grid floor (bars 3.2 mm diameter,
spaced 7.9 mm apart). A stainless steel drop-pan containing a 70% ethanol solution was placed below the grid floor. A fan provided low-level
white noise during training and testing in CXT-A. To examine the specificity of context fear memory, mice were also tested in a no-shock context, Context-B (CXT-B). CXT-B was a modified version of CXT-A with

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an opaque white acrylic triangular wall insert placed inside the chamber
and the shock-grid floor covered by a smooth opaque white acrylic floor.
The door of the chamber was covered with an opaque sheet with horizontal black and white stripes. During testing in CXT-B, neither theethanol nor fan was used (Wang et al., 2009).
Behavior was monitored by overhead cameras, which digitized video
images at 4 Hz. An automated frame-by-frame analysis of movement wasused to generate freezing scores (Freezeframe software; Actimetrics). Reactivity to shock was assessed by comparing distance traveled in 2 s before
shock onset (pre-US), to distance traveled during the 2 s shock (US).
Reactivity Index ⫽ (US ⫺ pre-US)/(US ⫹ pre-US). Importantly, weobserved no difference in shock reactivity between vectors for any experiment (see Results).

General behavioral procedures
Context fear training
Weak training. Mice were placed in a conditioning chamber (CXT-A)
and 2 min later received an unsignaled shock (0.3 mA, 2 s). Mice remained in the chamber for an additional 60 s before being returned totheir homecage.
Strong training. Strong training was similar to above except that micereceived three unsignaled shocks (0.5 mA, 2 s) spaced 60 s apart.

Context fear testing
Mice were placed in the context in which they were previously shocked(CXT-A), returned to the homecage, and 5 h later, placed in the no-shockcontext (CXT-B). For Experiment 1 (see Fig. 3a), the order of context test
was counterbalanced. The percentage of time spent freezing during each
5 min test session was used as an index of memory. Freezing was defined
as an immobilized, crouched position, with an absence of any movement
except respiration (Blanchard and Blanchard, 1969; Bolles and Fanselow,1982).

Specific behavioral procedures
Experiment 1: Effects of increasing CRTC1 or CREB on
consolidation of a weak context fear memory

Mice were microinjected with vector (CRTC1 vector, n ⫽ 29; CREB, n ⫽24; GFP, n ⫽ 27) and 3 d later given weak fear training. Twenty-fourhours later mice were placed in either CXT-A or CXT-B (for 5 min), and5 h later they were placed in the alternate context. The order of context
test was counterbalanced in this experiment only.

Experiment 2: Effects of increasing CRTC1or CREB on
consolidation of a strong context fear memory

Mice were microinjected with vector (CRTC1, n ⫽ 10; CREB, n ⫽ 9;
GFP, n ⫽ 8) and 3 d later given strong fear training. Twenty-four hours
later, mice were tested in CXT-A and 5 h later, tested in CXT-B.

Experiment 3: Effects of increasing CRTC1 or CREB on expressionof a weak context fear memory
Mice were fear conditioned using the weak training protocol and 24 hlater, microinjected with vector (CRTC1, n ⫽ 12; CREB, n ⫽ 11; GFP,n ⫽ 11). Four days later, mice were tested in CXT-A and 5 h later, tested
in CXT-B.

Experiment 4: Examining the enduring effects of increasing
CRTC1 or CREB on consolidation of a weak context fear memory

Mice were microinjected with vector (CRTC1, n ⫽ 12; CREB, n ⫽ 16;
GFP, n ⫽ 9) and 3 d later given weak fear training. Thirty days later, at atime when transgene expression driven by HSV vector had long dissipated, mice were tested in CXT-A and 5 h later, tested in CXT-B.

Experiment 5: Effects of increasing CRTC1 or CREB on
reconsolidation of a weak context fear memory
Mice were fear conditioned using the weak training protocol and, 26 dlater (after the memory was consolidated), microinjected with vector(CRTC1, n ⫽ 10; CREB, n ⫽ 10; GFP, n ⫽ 10). Three days following
microinjection, mice were replaced in CXT-A in the absence of the shock(for 45 s) to reactivate the context fear memory. Mice were removed fromCXT-A and returned to the homecage. Twenty-four hours later, mice

were tested as above in CXT-A then CXT-B. As a control, a “no memory
reactivation” condition was included (no-reactivation groups: CRTC1,n ⫽ 6, CREB, n ⫽ 6; GFP, n ⫽ 6). Mice were treated identically exceptthat the reactivation procedure was omitted (mice remained in thehomecage).

Data analyses
To analyze CRE-luciferase activity, we used an ANOVA with betweengroup factor Plasmid (CRTC1, GFP) and within-group factor Stimulation (Stim, KCl/FSK; No Stim, unstimulated vehicle control). Context
fear memory data were analyzed using an ANOVA with between-groupfactor Vector (CRTC1, CREB, GFP) and within-group factor Context(CXT-A, shock context; CXT-B, no-shock context). Significant interactions or main effects were further analyzed using post hoc Tukey’s HSD
tests. c-Fos expression data were analyzed using a 2 ⫻ 2 ANOVA with
Vector (CRTC1, GFP) and Treatment (homecage, HC; context fear condition; CFC) as factors. Significant interactions or main effects were
further analyzed using post hoc Fisher’s LSD tests. Data obtained from theelectrophysiological experiments were analyzed using the Mann–Whitney U
test.

Results
Microinjection of CRTC1 or CREB vector increases CRTC1 or
CREB function in the DG of dorsal hippocampus
We first examined endogenous expression of CRTC1 in thebrain. Consistent with previous findings (Zhou et al., 2006; Watts
et al., 2011), we observed high levels of CRTC1 in the hippocampus. Importantly, we found that in the DG, CRTC1 is expressed
exclusively in excitatory dentate granule cells, not in glia or interneurons (Fig. 1c). Therefore, to examine the effects of locallyand acutely increasing CRTC1 levels in the DG region of dorsal
hippocampus on memory, we used replication-defective HSV
vectors. HSV is neurotropic (Fink et al., 1996; Barrot et al., 2002)
and, following microinjection into the DG, selectively infects excitatory neurons (Fig. 1c). In this way, our viral vectors increase
CRTC1 levels only in neurons in which CRTC1 is endogenously
expressed. To increase CRTC1 levels, we used HSV encoding
wild-type CRTC1; this vector also expressed GFP, allowing visualization of infected neurons (CRTC1 vector). As a control, we
used a vector expressing GFP alone (GFP vector). Importantly,
we observed no evidence of toxicity associated with these vectors
either in cultured neurons or following microinjection in vivo.
Previous findings indicate that a similar vector-expressing
CREB (CREB vector) increases CREB levels and function (CREmediated transcription) both in vitro and in vivo (Barrot et al.,
2002; Han et al., 2007, 2009; Sekeres et al., 2010; Larson et al.,2011). We verified that microinjection of CRTC1 vector into theDG similarly increased CRTC1 protein above endogenous levels(Fig. 2c). Similar to endogenous CRTC1 protein (Zhou et al.,2006; Li et al., 2009; Ch’ng et al., 2012), transgenic CRTC1 protein is normally sequestered in the cytoplasm, but translocates tothe nucleus following stimulation by cAMP and calcium (Fig.
1a). Furthermore, we found that increasing CRTC1 levels in primary hippocampal neurons increased CRE-dependent transcription under unstimulated (basal) and stimulated (KCl/FSK) for
4 h conditions (Fig. 1b). This observation was supported by the
results of an ANOVA, which revealed a significant Construct ⫻Stimulation interaction (F(1,12) ⫽ 4.75; p ⬍ 0.05), as well as significant main effects of Construct (F(1,12) ⫽ 7.10, p ⬍0.05) andStimulation (F(1,12) ⫽ 9.10, p ⬍ 0.05). The finding that CRTC1
increases CRE-luciferase reporter activity in primary hippocampal neurons is consistent with previous results (Zhou et al., 2006;
Altarejos et al., 2008). Together, these data show that CRTC1

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Figure 1. CRTC1 in the DG of the dorsal hippocampus. a, Similar to endogenous CRTC1 protein, plasmid-derived CRTC1 undergoes nuclear translocation following stimulation (KCl/FSK for 4 h) in
primary hippocampal neurons. Scale bar, 20 ␮m. b, CRTC1 increased CRE-dependent transcription following stimulation in primary hippocampal neurons. (CRTC1, n ⫽ 4; GFP, n ⫽ 4). c, In the DG,
CRTC1 is endogenously expressed exclusively in excitatory neurons (not interneurons or glia). Overlap of immunohistochemical staining for endogenous CRTC1 protein (red) and markers of different
cell types: green, excitatory neurons (␣-CaMKII); glia (GFAP); or interneurons (GAD67 or parvalbumin). Hoechst (blue) identifies DG granule cell layer. Merged images shows that endogenous CRTC1
protein is colocalized in cells positive for ␣-CaMKII (but not GFAP, GAD67, or parvalbumin). White arrows show lack of CRTC1 staining overlap in GAD67 ⫹ or parvalbumin ⫹ cells. Scale bar, 50 ␮m.
d, Context fear conditioning (CFC) increases c-Fos levels in mice microinjected with GFP vector and this effect is potentiated in mice microinjected with CRTC1 vector. CRTC1 overexpression had noeffect on c-Fos levels homecage (HC) control mice (CFC–CRTC1, n ⫽ 4; CFC–GFP, n ⫽ 4; HC–CRTC1, n ⫽ 4, HC–GFP, n ⫽ 4). e, Input resistance, (f ) resting potential, and (g) spike threshold (mV)
did not differ between cells infected with CRTC1 vector or control cells whereas AHP (h, i) was decreased in cells infected with CRTC1 vector relative to control cells (CRTC1, n ⫽ 7; Control, n ⫽ 7).
Means ⫾ SEM.

vector increases both CRTC1 levels and CRE-mediatedtranscription.
CRTC1 vector increases excitability of cells in the DG
To examine whether CRTC1 influences neuronal excitability, weused in vitro recording techniques. The electrophysiological characteristics of DG cells infected with CRTC1 vector versus noninfected control cells from the same slice were compared using
whole-cell patch-clamp recordings in current-clamp mode. We
observed that increasing CRTC1 levels decreased the AHP of cells
following stimulation (Fig. 1h,i). This observation was supportedby the results of the Mann–Whitney U test, which revealed thatthe distributions between CRTC1-infected and noninfected cells
differed significantly (Mann–Whitney U ⫽ 10, n1 ⫽ n2 ⫽ 7, p ⬍0.05). Importantly, mean resting potential (mV) (Fig. 1f; Mann–
Whitney U ⫽ 18, n1 ⫽ n2 ⫽ 7, p ⬎ 0.05), mean input resistance(m⍀) (Fig. 1e; Mann–Whitney U ⫽ 18, n1 ⫽ n2 ⫽ 7, p ⬎ 0.05),
and mean spike threshold (Fig. 1g; Mann–Whitney U ⫽ 21, n1 ⫽n2 ⫽ 7, p ⬎ 0.05) did not differ between the groups. Together,
these data are consistent with the interpretation that increasing
CRTC1 increased neuronal excitability, perhaps by mediating
active K ⫹ currents.

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Sekeres et al. • CRTC1, Memory Consolidation and Reconsolidation

Figure 2. Microinjection of CRTC1 vector induces robust expression of CRTC1 in the DG. a, Vector microinjection induces robust localized transgene expression (GFP, green) in DG of dorsal
hippocampus. Left, Coronal brain images (adapted from Paxinos and Franklin, 2001) depicting the AP extent of typical viral vector infection (⫺1.46 to ⫺3.08 mm posterior to bregma). Right,
Corresponding image showing transgene expression (GFP, green) following vector microinjection (assessed 4 d postmicroinjection; counterstained with DAPI, blue). Scale bar, 200 ␮m. b, HSV
preferentially infects excitatory neurons (DG granule cells) (assessed 4 d postmicroinjection, top; counterstained with DAPI, blue). Scale bar, 50 ␮m. HSV-driven transgene expression dissipates by
10 d postmicroinjection (bottom). c, Microinjection of CRTC1 vector increases CRTC1 protein levels. Immunohistochemical staining for CRTC1 protein (red) in the DG 4 d following microinjection of
GFP vector (top) or CRTC1 vector (bottom). Mice microinjected with CRTC1 vector show higher levels of CRTC1 protein levels than mice microinjected with GFP vector, in infected neurons (green).
Scale bars: 200 ␮m (top); 50 ␮m (bottom).

Microinjection of CRTC1 vector increases c-Fos expression in
the DG of dorsal hippocampus
To further examine the effects of CRTC1 on neuronal excitability, we used c-Fos as a marker of neuronal activity. We microinjected mice with CRTC1 or GFP vector and 3 d later examined
c-Fos in the infected region of the DG either in mice maintainedin the homecage or 90 min following context fear conditioning.
We found that increasing CRTC1 levels increased c-Fos expression in the infected region of the DG in mice trained for context
fear conditioning (Fig. 1d). This observation was supported by
the results of an ANOVA, which revealed a significant Vector ⫻Treatment interaction (F(1,12) ⫽ 8.42; p ⬍0.05), as well as significant main effects of Vector (F(1,12) ⫽ 7.30, p ⬍0.05) and Treatment (F(1,12) ⫽ 49.17, p ⬍0.01). Post hoc analyses revealed thatmice microinjected with either GFP or CTRC1 vector had higher
levels of c-Fos following fear conditioning compared with mice
maintained in their homecage. Moreover, fear-conditioned micewith CRTC1 vector had higher c-Fos activation than fearconditioned mice with GFP vector ( p ⬍0.05). There was no difference between c-Fos expressions in homecage control mice

( p ⬎0.05). Together, these results suggest that neurons with increased CRTC1 expression are more excitable. These data areconsistent with previous findings that overexpression of CREB
increases intrinsic excitability (Dong et al., 2006; Lopez deArmentia et al., 2007; Zhou et al., 2009).
Experiment 1: Increasing CRTC1 or CREB in DG at the timeof training facilitates consolidation of a weak contextual fear
memoryTo examine the effects of increasing CRTC1 or CREB function in
DG on consolidation of a contextual fear memory, we microinjected CRTC1, CREB, or GFP vector 3 d before contextual feartraining, which normally induces weak memory (mice received
single 0.3 mA shock in CXT-A) (Fig. 3a). One day following
training, contextual fear memory was assessed by returning miceto the training context (CXT-A). As expected, mice microinjected with GFP vector showed low levels of freezing when replaced in CXT-A; however, mice with CRTC1 or CREB vector
showed enhanced freezing (Fig. 3a). To examine the specificity ofthis increased freezing, we retested mice in a novel, alternate

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context not previously paired with shock(Context B, CXT-B). All groups, regardless of vector, showed similarly low freezing in CXT-B (Fig. 3a). The results of a
Vector (CRTC1, CREB, GFP) by Context(CXT-A, CXT-B) ANOVA supported thisobservation and revealed a significant
Vector ⫻ Context interaction (F(2,45) ⫽8.19, p ⬍ 0.001) as well as a significant
main effect of Context (F(1,45) ⫽ 65.81,
p ⬍ 0.001), but not Vector (F(2,45) ⫽ 3.02,
p ⬎ 0.05). Post hoc analyses conducted on
the significant interaction indicated thatmice with either CRTC1 or CREB vector
froze more in CXT-A than mice with GFP
vector, but that all groups showed low
freezing in CXT-B. Interestingly, there
was no difference between freezing levels
in mice with CRTC1 or CREB vector (ineither context). Testing mice in CXT-B
and then CXT-A produced similar results:
significant Vector ⫻ Context interaction(F(2,29) ⫽ 4.32, p ⬍ 0.05), significant main
effects of Context (F(1,29) ⫽ 37.62, p ⬍0.001), and Vector (F(2,29) ⫽ 6.55, p ⬍ 0.001(Fig. 3a). Importantly, the increase in freezing observed in mice with CRTC1 or CREB
vector cannot be attributed to differences inshock sensitivity during training (F(2,45) ⫽0.33, p ⬎ 0.05). Therefore, increasing
CRTC1 or CREB levels before training
increased freezing in the context previously paired with shock, but did not enhance generalization of freezing to anonshock context. These findings suggest that overexpressing CRTC1 or CREB
increased memory strength, without compromising memory specificity.

4

Figure 3. Locally and acutely increasing CRTC1 or CREB levels in DG during training enhance consolidation of contextual fear
memory; this memory enhancement is context specific, not due to an effect on memory expression, and long lasting. a, Microinjectionof CRTC1 or CREB vector in DG before weak training (1 ⫻ 0.3
mA shock) enhances contextual fear memory (CRTC1 vector,n ⫽ 29; CREB, n ⫽ 24; GFP, n ⫽ 27). This memory enhancement is specific for the training context (CXT-A), and does not
generalize to a similar, nonshocked context (CXT-B), regardless of context testing order (CXT-A then CXT-B, left, or CXT-B
then CXT-A, right). Mean ⫾ SEM. b, Microinjection of CRTC1 or
CREB vector in DG before strong training (3 ⫻ 0.5 mA shocks)
enhances memory for contextual fear; this memory is specificto the training context (CXT-A) (CRTC1 vector, n ⫽ 10; CREB,n ⫽ 9; GFP, n ⫽ 8). c, Microinjection of CRTC1 or CREB vector
after training does not facilitate memory expression (in either
context) indicating that the enhancement of context memory
by these vectors is not due to effects on memory expression/
retrieval (CRTC1, n ⫽ 12; CREB, n ⫽ 11; GFP, n ⫽ 11). d,
Memory enhancement produced by microinjection of CRTC1 or
CREB vector is long lasting and maintains precision. Microinjection of CRTC1 or CREB vector before training (1 ⫻ 0.3 mAshock) enhances memory for contextual fear even when tested
30 d later (after transgene expression has dissipated) (CRTC1
vector, n ⫽ 12; CREB, n ⫽ 16; GFP, n ⫽ 9). This memoryenhancement is context specific (only observed in CXT-A).

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Experiment 2: Increasing CRTC1 or CREB in DG at the timeof training further facilitates consolidation of a strong
contextual fear memory
We next examined the effects of similarly increasing CRTC1 or
CREB function on context fear memory induced by a stronger
training protocol (three 0.5 mA shocks in CXT-A) (Fig. 3b).
When subsequently tested in CTX-A, mice with CRTC1 or CREB
vector showed greater freezing than mice with GFP vector, butsimilar to above, this memory enhancement was context specific,
as all groups showed low freezing in the no-shock context (Fig.
3b) [(significant Vector ⫻ Context interaction (F(2,24) ⫽ 4.90,
p ⬍ 0.05), as well as significant main effects of Vector (F(2,24) ⫽ 3.77,
p ⬍ 0.05) and Context (F(1,24) ⫽ 124.62, p ⬍ 0.001)]. Post hoc analyses revealed that mice with CRTC1 or CREB vector froze more in
CXT-A than mice with GFP vector, but that all groups froze atequally low levels in CXT-B (p ⬎ 0.05). Together, these results indicate that increasing CRTC1 or CREB function in the DG before
training enhances context fear memory induced by either weak orstrong training. Furthermore, the enhanced memory produced by
CRTC1 or CREB overexpression is specific and does not generalize
to a novel context.
Experiment 3: Increasing CRTC1 or CREB in DG does notenhance expression of a previously acquired contextual fear
memoryIn the above experiments, we microinjected vectors before training such that mice were both trained and tested with high CRTC1or CREB levels in the DG. To examine whether the enhancementin freezing produced by CRTC1 or CREB vectors was due tofacilitated memory expression/memory retrieval, we performed asimilar experiment but microinjected CRTC1, CREB, or GFP
vectors 24 h after weak training, at a time when cellular/synaptic
consolidation is thought to be complete (for review, see Dudai,
2004). When tested 4 d after microinjection, all groups froze at
low levels in the shock context (CXT-A) and still lower levelsin the no-shock context (CXT-B) [no significant effect of Vector ⫻ Context (F(2,31) ⫽ 0.69, p ⬎ 0.05), Vector (F(2,31) ⫽ 1.45,
p ⬎ 0.05), but a significant effect of Context (F(1,31) ⫽ 0.19,
p ⬍ 0.001), in which all groups froze more in CXT-A thanCXT-B] (Fig. 3c). Therefore, increasing CRTC1 or CREB levels does not affect the expression or context specificity of apreviously acquired fear memory. Together, these results indicate that increasing CRTC1 or CREB function in the DG
before, but not after, training enhances memory.
Experiment 4: The memory facilitation produced byincreasing CRTC1 or CREB in DG at the time of training islong lasting
The above finding suggests that increasing CRTC1 or CREB at thetime of training enhances memory consolidation. Importantly,
using this vector system, transgene expression peaks ⬃3 d following microinjection, and dissipates within 10 –14 d after microinjection (Barrot et al., 2002; Vetere et al., 2011) (Fig. 2b).
Therefore, to investigate whether the memory enhancement
produced by microinjecting CRTC1 or CREB vector persists beyond transgene expression, we trained mice as before (3 d following microinjection, at a time of high transgenic expression of
CRTC1 or CREB) but tested mice long after transgene expression
had dissipated (33 d following microinjection). When tested at
this later time, mice previously microinjected with CRTC1 or
CREB vector froze more in CXT-A than mice with GFP vector.
This enhancement was context-specific as all groups froze atequally low levels in CXT-B [significant Vector ⫻ Context(F(2,34) ⫽ 5.44, p ⬍ 0.05), Vector (F(2,34) ⫽ 5.90, p ⬍ 0.01), andContext (F(1,34) ⫽ 37.35, p ⬍ 0.001) (Fig. 3d]. Therefore, increasing CRTC1 or CREB in the DG before training facilitates theconsolidation of context fear memory; this enhancement is bothprecise (observed only in the training context) and enduring (observed even after transgene expression dissipated). Together,
these results are consistent with the interpretation that increasing
CRE-mediated transcription in the DG around the time of training enhances memory consolidation. Once this memory has been
consolidated, elevated CRE-mediated transcription is no longer
necessary to maintain this memory in terms of strength andprecision.
Experiment 5: Increasing CRTC1 or CREB in DG facilitatesreconsolidation of fear memory
We found that increasing CRTC1 or CREB in the DG before
training facilitates memory consolidation. Memory reactivation(by exposure to cues present during initial memory encoding)
may trigger a second wave of consolidation (reconsolidation).
Similar to initial consolidation, reconsolidation also requires
protein synthesis (Nader et al., 2000; Sara, 2000; Debiec et al.,2002) and intact CREB function (Kida et al., 2002; Mamiya et al.,2009; Kim et al., 2011). Therefore, we next asked whether increasing CRTC1 or CREB before memory reactivation would similarly
enhance memory reconsolidation. We trained unoperated miceusing the weak training protocol and 26 d later (when memory
was consolidated) mice were microinjected with CRTC1, CREB,
or GFP vectors 3 d before memory reactivation (placement in
CXT-A for 45 s without shock). In this way, the time between
vector microinjection and training (in this experiment, reactivation) remained constant. To examine the stability of the reactivated memory (memory reconsolidation), mice were tested 24 hlater in CXT-A. To examine the specificity of this memory, mice
were tested the no-shock context (CXT-B) 5 h later. As expectedfollowing weak fear training, all groups showed low levels of freezing during the memory reactivation session (F(2,19) ⫽ 0.72, p ⬎0.05) (Fig. 4a). In contrast, when tested 24 h after memory reactivation, mice microinjected with CRTC1 or CREB vector
showed higher freezing than mice with GFP vector in CXT-A(Fig. 4a) but equally low levels in CXT-B [significant effects of
Vector ⫻ Context (F(2,27) ⫽ 9.54, p ⬍ 0.001), Context (F(1,27) ⫽39.06, p ⬍ 0.001), but not Vector (F(2,27) ⫽ 2.89, p ⬎ 0.05)]. Thisfinding is consistent with the interpretation that increasing
CRTC1 or CREB before memory reactivation enhanced memory
reconsolidation.
To confirm that this memory enhancement was critically dependent on reactivation of the context fear memory at a time ofhigh CRTC1 or CREB levels, we conducted a similar experiment
except that mice were maintained in the homecage following
microinjection of vector (no memory reactivation). When subsequently tested in both CXT-A and CXT-B, all mice showed
equally low levels of freezing, regardless of vector or context [nosignificant Vector ⫻ Context interaction (F(2,15) ⫽ 0.47, p ⬎0.05), main effect of Context (F(1,15) ⫽ 4.32, p ⬎ 0.05), or Vector(F(2,15) ⫽ 0.04, p ⬎ 0.05)] (Fig. 4b). Therefore, the enhancementof memory produced by microinjecting CRTC1 or CREB vector
was critically dependent on memory reactivation.

Discussion
Extensive evidence implicates CREB-dependent gene transcription in memory (Bourtchuladze et al., 1994; Yin et al., 1994;
Guzowski and McGaugh, 1997; Bartsch et al., 1998; Kida et al.,

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CRTC1 and CBP/p300 activate CREmediated transcription through different
mechanisms, their effects can be synergistic (Ravnskjaer et al., 2007; Xu et al., 2007).
In addition to CREB, CBP/p300 binds to⬃400 protein partners, thereby making
CBP/p300 among the most heavily connected nodes in the known mammalianprotein–protein interactome (Goodman and Smolik, 2000; Bedford et al.,2010). In contrast, CRTC seems to be a
dedicated
modulatorof
CREcontaining genes (Conkright et al.,2003b). Despite being a powerful andspecific coactivator of CREB, the role of
CRTC in memory has been virtuallyunexplored.
Our studies identify a critical role for
CRTC1 in both memory consolidation
and reconsolidation. We show that increasing CRTC1 function enhances memory strength without compromising
memory quality. To examine the effects ofincreasing CRTC1 levels on different
memory phases, we used context fear conditioning. Context fear conditioning iswell suited for investigating the molecularbasis of consolidation and reconsolidation as infusions of the protein synthesis
inhibitor anisomycin directly into dorsal
hippocampus around the time of training(synaptic consolidation) or memory reactivation (reconsolidation) disrupt subsequent memory expression (Quevedo etal., 1999; Taubenfeld et al., 2001; Debiec etFigure 4. Increasing CRTC1 or CREB levels in DG enhances reconsolidation of an established contextual fear memory. a, Micro- al., 2002; Frankland et al., 2006; Suzuki
injection of CRTC1 or CREB vector before reactivation of an established weak contextual fear memory enhances subsequent
et al., 2008; Mamiya et al., 2009). Morememory expression in a context-specific manner. Naive mice were trained with a weak protocol (1 ⫻ 0.3 mA shock), and 26 d later
over, by examining fear in both a trained
were microinjected with vector (CRTC1-R, n ⫽ 10; CREB-R, n ⫽ 10; GFP-R, n ⫽ 10). Three days following vector microinjection, allgroups showed similar low levels of freezing when initially re-exposed to the training context (for 45 s) to reactivate the memory and nontrained context, this task allowed(left graph). In subsequent test session (24 h later), mice with GFP vector showed low levels of freezing (in both contexts). However, us to assess memory specificity or quality.
mice with CRTC1 or CREB vector showed enhanced memory, which was context specific (right graph). b, Memory reactivation is We found that mice with increased
necessary for the enhancement of an established memory by CRTC1 or CREB vectors (reconsolidation). Mice were trained as above, CRTC1 show increased freezing, but only
similarly microinjected with vectors, but not re-exposed to the training context (no reactivation, NR) after vector microinjection in the context previously paired with(CRTC1-NR, n ⫽ 6; CREB-NR, n ⫽ 6; GFP-NR, n ⫽ 6). During the subsequent test, all groups showed equally low levels of freezing. shock. This indicates that CRTC1 proMeans ⫾ SEM.
motes the formation of a precise context
fear memory (rather than a nonspecific2002; Pittenger et al., 2002; Frankland et al., 2004). Multiple sigincrease in fear or anxiety). We targeted our manipulation to thenaling pathways phosphorylate CREB at Ser133 (Shaywitz andDG, the primary relay station for inputs to the hippocampus
Greenberg, 1999; Mayr and Montminy, 2001; Lonze and Ginty,(Treves et al., 2008), because previous studies show this region is2002), which stimulates the recruitment of coactivators CBP/
particularly important in context fear acquisition (Lee and Kesp300 (Chrivia et al., 1993; Parker et al., 1996). However, thisner, 2004; Hernández-Rabaza et al., 2008).
phosphorylation event is not always sufficient to activate tranWe found that increasing CRTC1 levels in the DG region of
scription (Impey et al., 1996; Mayr and Montminy, 2001) sugdorsal hippocampus before training facilitates memory consoligesting that CREB-mediated transcription is regulated bydation of a weak fear memory without affecting memory quality.
additional mechanisms.
That is, we observed an increase in freezing only in the context
In 2003, two laboratories identified a new family of CREBpreviously paired with shock; this increase in freezing did not
specific coactivators, now referred to as CRTCs (Iourgenko et al.,generalize to a similar no-shock context. Similarly increasing
2003; Conkright et al., 2003b). CRTC is thought to enhance tranCRTC1 levels also enhanced contextual fear memory produced
scription by facilitating the interaction of CREB with the RNA
by stronger training, in a context-specific manner. This CRTC1polymerase II pre-initiation complex (Conkright et al., 2003b; Xu
induced enhancement in memory consolidation is not due to an
et al., 2007; Altarejos and Montminy, 2011) or perhaps increasing
effect on memory retrieval/expression as similarly increasing
the occupancy of CREB on DNA binding sites of some promoters
CRTC1 levels after training did not affect the expression or spec(Wang et al., 2010; Altarejos and Montminy, 2011). Although
ificity of a weak fear memory. Interestingly, increasing CREB

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17866 • J. Neurosci., December 5, 2012 • 32(49):17857–17868

levels produced strikingly similar effects in all experiments.
Therefore, increasing CRTC1 or CREB levels around the time of
memory encoding enhances memory consolidation withoutcompromising memory quality. The increase in memory produced by increasing CRTC1 or CREB levels at the time of training
was long lasting and was observed even after viral expression of
CRTC1 or CREB dissipated, suggesting that once consolidated,
this strong and specific context fear memory no longer required
elevated CRTC1 or CREB function.
Memory retrieval is thought to be an active constructive process (Schacter et al., 1998) that functions to modify previously
acquired memories (Sara, 2000; Dudai, 2006; Lee, 2009). Here weshowed that an established conditioned fear memory wasstrengthened (without further training) if reactivated at a time ofhigh CRTC1 or CREB levels in the DG. Specifically, we trained
naive mice using a weak protocol, and 26 d later, microinjected
vector. Three days following vector microinjection, mice werere-exposed to the shock context (but did not receive a shock).
When subsequently tested, mice with GFP vector showed low
conditioned fear memory (as expected). However, mice trainedwith a weak protocol but microinjected with CRTC1 or CREB
vector before context re-exposure, showed enhanced memory.
This finding indicates that the strength of an established memory
can be increased even relatively long after acquisition. Moreover,the quality of the memory was not affected, as conditioned fear
did not generalize to the no-shock context. The enhanced memory was dependent on re-exposure to the training context, andnot observed in similarly trained mice maintained in thehomecage (no context re-exposure). In this way, the memoryenhancement produced by CRTC1 or CREB differs from that
produced by overexpressing the atypical protein kinase C isoform, protein kinase M␨ (PKM␨). For instance, Shema et al.
(2011) found that virally increasing PKM␨ expression in insularcortex 6 d after conditioned taste aversion training (at a time
when the memory trace was consolidated) enhanced subsequent
memory, even though rats were not re-exposed to the taste previously paired with illness.
Memory retrieval likely engages the population of neurons
involved in the original memory trace and memory coding isthought to be especially sparse in the DG (Leutgeb et al., 2007a,b).
Therefore, the present findings that increasing CRTC1 or CREB
in ⬃20% of DG cells in the target region enhanced reconsolidation is perhaps surprising. These data suggest that either the population of neurons involved in the memory trace overlapped withthe population of neurons infected or that increasing CRTC1 or
CREB in DG enhanced overall circuit function (rather than producing a cell autonomous effect).
The majority of experiments investigating the molecular basisof memory consolidation, and especially reconsolidation, haveexamined whether a given molecule (or protein synthesis in general) is necessary for these processes by inferring normal function
from loss-of-function studies. Although the results from thesetypes of experiments have greatly increased our understanding of
the mechanisms underlying memory, alternative interpretations
to the observed behavioral deficits in memory reconsolidation
experiments have been offered (e.g., observed decrease in memory may be due to a temporary inability to access the memory
trace; Lattal et al., 2004). In contrast to loss-of function studies,
we assessed whether increasing CRTC1 or CREB function enhances memory consolidation and reconsolidation. One advantage of this approach is that any memory enhancement is unlikely
to be easily attributable to performance and/or memory retrieval
effects. Here we show that increasing CRTC1 or CREB in DG

enhances both memory consolidation and reconsolidation. Ourfindings are consistent with results from Tronson et al. (2006)
who showed that infusions of a protein kinase A (PKA) agonist
into the amygdala enhanced reconsolidation of a tone fear memory. As PKA may both phosphorylate CREB at Ser133 (Impey etal., 1998; West et al., 2002; Cohen and Greenberg, 2008) andpromote nuclear translocation of CRTC1 (Bittinger et al., 2004),
the present findings suggest a potential molecular mechanism forthis enhancement of memory reconsolidation.
Since its characterization ⬎20 years ago, CREB has been implicated in diverse brain processes, including neural development, addiction, circadian rhythms, and memory (Bourtchuladze et al., 1994;
Yin et al., 1994; Ding et al., 1997; Carlezon et al., 1998; von Gall et al.,
1998; Gau et al., 2002; Lonze and Ginty, 2002; McClung and Nestler,
2003; Cohen and Greenberg, 2008; Eckel-Mahan et al., 2008; Briand
and Blendy, 2010). CREB may modulate up to one-quarter of the
mammalian genome (Conkright et al., 2003a; Impey et al., 2004;
Zhang et al., 2005), suggesting that different subsets of CREB-target
genes may be involved in different processes. CREB-dependent genetranscription is modulated by several mechanisms, including posttranslational modifications of CREB (phosphorylation, acetylation,ubiquitination, sumoylation, and glycosylation) (Taylor et al., 2000;
Kornhauser et al., 2002; Comerford et al., 2003; Lamarre-Vincent
and Hsieh-Wilson, 2003; Lu et al., 2003; Altarejos and Montminy,
2011; Rexach et al., 2012) and interaction with different cofactors,
including CBP/p300 and CRTC (Goodman and Smolik, 2000; Mayrand Montminy, 2001; Vo and Goodman, 2001; Conkright et al.,2003b; Screaton et al., 2004). The combination of different CREB
regulatory mechanisms might be one way to orchestrate the transcription of specific CREB target genes under different conditions(the CREB regulon; Impey et al., 2004).

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