ORIGINAL RESEARCH ARTICLE
published: 20 December 2013
doi: 10.3389/fnbeh.2013.00209

BEHAVIORAL NEUROSCIENCE

CREB regulates spine density of lateral amygdala neurons:
implications for memory allocation
Derya Sargin 1,2,3,4 , Valentina Mercaldo 1,2,3,4 , Adelaide P. Yiu 1,2,3,4 , Gemma Higgs 1,2,3,4 , Jin-Hee Han 1,2,3,4,5 ,Paul W. Frankland 1,2,3,4 and Sheena A. Josselyn 1,2,3,4*
1

Program in Neurosciences and Mental Health, Hospital for Sick Children, Toronto, ON, Canada
Department of Psychology, University of Toronto, Toronto, ON, Canada
3
Department of Physiology, University of Toronto, Toronto, ON, Canada
4Institute of Medical Science, University of Toronto, Toronto, ON, Canada
5
Laboratory of Neural Circuit and Behavior, Department of Biological Sciences, KAIST Institute for the BioCentury, Korea Advanced Institute of Science andTechnology, Daejeon, Korea
2

Edited by:Jonathan E. Ploski, University ofTexas at Dallas, USA
Reviewed by:
Tsuyoshi Miyakawa, Fujita Health
University, Japan
Marie H. Monfils, University ofTexas at Austin, USA*Correspondence:
Sheena A. Josselyn, Program in
Neurosciences and Mental Health,The Hospital for Sick Children, 555
University Ave, Toronto, ON M5G
1X8, Canadae-mail: sheena.josselyn@sickkids.ca

Neurons may compete against one another for integration into a memory trace.
Specifically, neurons in the lateral nucleus of the amygdala with relatively higher levelsof cAMP Responsive Element Binding Protein (CREB) seem to be preferentially allocatedto a fear memory trace, while neurons with relatively decreased CREB function seemto be excluded from a fear memory trace. CREB is a ubiquitous transcription factor thatmodulates many diverse cellular processes, raising the question as to which of these
CREB-mediated processes underlie memory allocation. CREB is implicated in modulating
dendritic spine number and morphology. As dendritic spines are intimately involved inmemory formation, we investigated whether manipulations of CREB function alter spine
number or morphology of neurons at the time of fear conditioning. We used viral vectors
to manipulate CREB function in the lateral amygdala (LA) principal neurons in mice
maintained in their homecages. At the time that fear conditioning normally occurs, weobserved that neurons with high levels of CREB had more dendritic spines, while neurons
with low CREB function had relatively fewer spines compared to control neurons. These
results suggest that the modulation of spine density provides a potential mechanism forpreferential allocation of a subset of neurons to the memory trace.
Keywords: CREB, amygdala, fear memory, dendritic spines, viral vector

INTRODUCTION
The cAMP Responsive Element Binding Protein (CREB) is anactivity regulated transcription factor that modulates the transcription of genes with cAMP responsive elements (CRE) locatedin their promoter regions. Early research in Aplysia (Dash et al.,
1990; Kaang et al., 1993; Bartsch et al., 1995) and D. melanogaster(Yin et al., 1994, 1995; Perazzona et al., 2004) first implicated
CREB in memory formation. Since that time, the important roleof CREB in memory has been shown across a variety of species
from C. elegans (Kauffman et al., 2010; Lau et al., 2013) torats (Guzowski and McGaugh, 1997; Josselyn et al., 2001), mice
(Bourtchuladze et al., 1994; Kida et al., 2002; Pittenger et al.,
2002; Gruart et al., 2012) and humans (Harum et al., 2001) (forreview, see Josselyn and Nguyen, 2005) but see Balschun et al.
(2003). For instance, we (Han et al., 2007), and others (Zhouet al., 2009; Rexach et al., 2012) previously showed that increasing CREB function in a small portion of lateral amygdala (LA)neurons (roughly 8–10% of LA principal neurons) was sufficient
to enhance auditory fear memory. Moreover, we observed thatLA neurons with relatively higher CREB function at the timeof training were preferentially included, whereas neurons withlower CREB function were excluded, from the subsequent LA
fear memory trace (Han et al., 2007, 2009). Conversely, disrupting CREB function by expressing a dominant negative version of

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CREB (CREBS133A )in a similar small percentage of LA neurons
did not affect auditory fear memory, likely because the neurons
expressing CREBS133A were largely excluded from the memory
trace. Furthermore, post-training ablation (Han et al., 2009) orsilencing (Zhou et al., 2009) of neurons overexpressing CREB disrupted subsequent expression of the fear memory, confirming theimportance of these neurons. Together, these data suggest thatneurons with high levels of CREB at the time of training are preferentially allocated to the memory trace because they somehow
outcompete their neighbors (Won and Silva, 2008).
CREB is a ubiquitous transcription factor implicated in many
diverse cellular processes in addition to memory formation,
including regulation of proliferation, survival, apoptosis, differentiation, metabolism, glucose homeostasis, spine density, andmorphology (Bourtchuladze et al., 1994; Murphy and Segal,
1997; Silva et al., 1998; Mayr and Montminy, 2001; Lonze et al.,
2002; Wayman et al., 2006; Aguado et al., 2009; Altarejos andMontminy, 2011). Which of these CREB-mediated processes
is/are important for memory allocation? Here we investigated
one CREB-mediated process, the regulation of spine density andmorphology.
Dendritic spines are small, highly motile structures on dendritic shafts which provide flexibility to neuronal networks. As
an increase in the synaptic strength between neurons is thought

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CREB regulates spine density

to underlie memory formation (Bailey and Kandel, 1993; Bailey
et al., 1996) and the majority of excitatory synapses occur on dendritic spines (Harris and Stevens, 1988, 1989; Farb et al., 1992), it
has long been thought that dendritic spines serve as storage sites
for synaptic strength, an idea first proposed by Santiago Ramón yCajal over 100 years ago (Cajal, 1995). In this way, the growth
and re-structuring of dendritic spines is thought to be crucial for
memory formation.
A role for CREB in spine formation was first reported by
Murphy and Segal (1997) who showed that estradiol treatment
increased both levels of activated (phosphorylated) CREB andspine density in cultured hippocampal neurons. CREB was subsequently shown to regulate spine morphology in hippocampal
neurons both in organotypic culture (Impey et al., 2010) andin vivo (Marie et al., 2005), as well as in visual cortex principal neurons (Suzuki et al., 2007). The new spines formed
following overexpression of CREB may contain silent synapses(NMDA receptors only), suggesting that they may be “primed”
for incorporation into future memory circuits (Marie et al., 2005).
Consistent with this, increasing CREB function in hippocampal CA1 principal neurons was sufficient to restore both thedecrease in spine density and spatial memory in a mouse modelof Alzheimer’s disease (Yiu et al., 2011).
We previously reported that neurons with increased CREB at
the time of training are selectively allocated to a fear memory trace
and a variety of evidence shows that increasing CREB function
increases spine density. Therefore, we investigated whether neurons with increased CREB at the time of training also have an
increase in dendritic spine density, thereby providing a potential
mechanism of the preferential allocation of these neurons to thememory trace.

MATERIALS AND METHODS
MICE

Adult male F1 hybrid (C57 BL/6NTac × 129S6/SvEvTac) mice
were used for all experiments. This genetic background has beenused extensively in behavioral studies and are well characterized(Silva et al., 1997). Mice were group housed (2–5 mice per cage)
on a 12 h light/dark cycle and provided with food and water ad
libitum. All experimental procedures were conducted in accordance with the guidelines of the Canadian Council on Animal
Care (CCAC) and the National Institutes of Health (NIH) andapproved by the Animal Care Committee at the Hospital for Sick
Children.
HSV VECTORS

Neurotropic replication-defective herpes simplex viral (HSV)
vectors were used to manipulate CREB function in individual LA
principal neurons. Wild-type or dominant negative CREBS133A
cDNAs were cloned into the HSV amplicon under the controlof the constitutive promoter for the HSV immediate early gene
IE4/5. These vectors co-expressed GFP which was driven by
CMV promoter [HSV-p1005; Russo et al., 2009]. In this vector
therefore, the GFP protein is not fused to CREB and may thus
fill the infected cell. As a control, we used HSV vector expressing
GFP alone. HSV virus was packaged using a replication-defective
helper virus as previously described (Josselyn et al., 2001;
Barrot et al., 2002; Carlezon and Neve, 2003; Han et al., 2008;
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Vetere et al., 2011; Cole et al., 2012). Virus was purified on asucrose gradient, pelleted and resuspended in 10% sucrose. The
average titer of the virus stocks was typically 4.0 × 107 infectious
units/ml.
SURGERY

Mice were pretreated with atropine sulfate (0.1 mg/kg, i.p.),anesthetized with chloral hydrate (400 mg/kg, i.p.) and placedin a stereotaxic frame. Skin was retracted and holes were drilledin the skull above the LA (anteroposterior = −1.4, mediolateral = ± 3.4, ventral = −5.0 mm from bregma) according to(Paxinos and Franklin, 2001). Viral vector was microinjected
through glass micropipettes connected via polyethylene tubing
to a microsyringe (Hamilton, Reno, NV) at a rate of 0.1 μl/min.
Micropipettes were left in place an additional 10 min following
microinjection to ensure diffusion of vector. For behavior
analysis, a volume of 1.5 μl and for spine analysis, a volumeof 1.0 μl was microinjected bilaterally at a rate of 0.1 μl/min.
Micropipettes were slowly retracted, the incision site closed andmice were treated with analgesic (ketoprofen, 5 mg/kg, s.c.).
Three d following surgery, at a maximal transgene expression for
HSV vector system (Josselyn et al., 2001; Barrot et al., 2002; Vetere
et al., 2011; Cole et al., 2012), mice were either fear conditioned
or perfused for dendritic spine analysis.
AUDITORY (TONE) FEAR CONDITIONING

During training, mice were placed in a Med Associates (St.
Albans, VT) Plexiglas and metal chamber (24 × 30 × 21 cm, context A; Cxt A) located in a soundproof room. After 2 min, a tone
(2800 Hz, 30 s, 85 dB) that co-terminated with a footshock (2 s,
0.4 mA) was presented. Mice remained in the chamber for an
additional 30 s and then returned to the homecage. Testing forauditory fear memory occurred 24 h later by placing mice in anovel context (context B; Cxt B) and 2 min later, presenting thetone previously paired with footshock for 3 min. The percentageof time mice spent freezing (the cessation of all movement except
respiration) before and during the tone was measured using an
automated system (Actimetrics) and was used as our index ofmemory. Immediately after testing, mice were deeply anesthetizedand perfused.
IMMUNOHISTOCHEMISTRY

To visualize the number and morphology of dendritic spines in
the neurons we infected, we took advantage of the GFP expressedby all viral vectors. We amplified the GFP signal using an antibody directed against GFP. 72 h after surgery, mice were deeply
anesthetized using chloral hydrate (400 mg/kg, i.p.) and transcardially perfused with 0.1 M PBS followed by 4% paraformaldehyde (PFA). Brains were post-fixed overnight in 4% PFA andtransferred to 30% sucrose for cryoprotection. Coronal (50 μm)
sections were prepared and immunohistochemistry for GFP was
performed. Free-floating sections were incubated in blocking
solution (0.1% BSA, 5% NGS, 0.2% Triton-X-100 in 0.1 M PBS)
for 1 h and labeled with anti-GFP rabbit polyclonal antibody(1:500, Invitrogen) overnight at 4◦ C. Following PBS washes, sections were incubated with goat anti-rabbit Alexa 488 (1:500,
Invitrogen) for 2 h at room temperature. Sections were washedwww.frontiersin.org

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CREB regulates spine densitywith PBS, mounted on gelatin-coated slides and coverslippedusing Vectashield Hardmount with DAPI (Vector Laboratories).

α = 0.01). All significant main effects remained significant after
correction.

CONFOCAL ANALYSISRESULTS

GFP-positive LA neurons (neurons infected by viral vectors) werefirst identified using a 10x objective (LSM 710, Zeiss). Infected
neurons were included in the subsequent spine analysis if (i) cellbody was not damaged; (ii) dendritic projections remained within
the LA; (iii) neurons could clearly be identified without interference from neighboring infected cells; (iv) neurons had first,
second, and third order branches. Fourth order branches were
not included in the analysis as they often appeared truncated inour 50 μm sections. Selected GFP-positive neurons were imaged
using a 100x oil-immersion objective. Z series were obtained byimaging serial confocal planes at 0.25 μm intervals. Dendrites
and spines were traced manually from the image stacks using
Neurolucida software and analyzed with Neurolucida Explorer(MBF version 9).
Dendritic morphology

Image analysis was performed by two researchers unaware of thetreatment condition of the mouse. Dendrites were traced. The
first dendritic process emanating from the cell body was definedas the primary (first order) branch. Subsequent branches thatbifurcated from the first branch order were designated as second order branches, and so forth. Truncated branches or thosethat did not remain within the image window were excluded from
subsequent analysis.
Spine morphology

Dendritic spines were defined as small protrusions connected tothe dendritic shaft (Feldman and Dowd, 1975). Spines show a
distinct morphology and vary in length from 0.5–4 μm (Petersand Kaiserman-Abramof, 1970; Horner and Arbuthnott, 1991).
Therefore, we analyzed all dendritic protrusions that were lessthan or equal to 4 μm in length (Horner and Arbuthnott, 1991).
Because this method has been shown to produce reliable results(Horner and Arbuthnott, 1991), no attempt was made to introduce a correction factor for hidden spines. Spines were counted
and spine density was calculated as the number of spines on abranch divided by the length of the branch. Spine length was
defined as the distance between the spine tip and the base of thespine. Spine head diameter was identified as the maximum widthof the spine head (see Figure 2B).
STATISTICAL ANALYSIS

Data were analyzed with 1 or 2-Way analyses of variance(ANOVAs) using Statistica (Statsoft) software. For the auditory
fear conditioning data, we analyzed the percentage of time spent
freezing to before (2 min) and during (3 min) the tone. For dendritic and spine morphological analysis, data were first averaged
by branch order per cell, then by animal and finally by vector
group (GFP, CREB, or CREBS133A ). Newman-Keuls post-hoc tests
were performed where appropriate. To protect against potential
type 1 errors resulting from multiple comparisons of 5 different measures of neuronal morphology (i.e., spine density, spine
length, spine head diameter, dendrite length, and dendrite volume), we also performed a Bonferroni correction (corrected

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INCREASING CREB IN A SMALL PORTION OF LA NEURONS ENHANCESMEMORY FORMATION WHILE EXPRESSING THE DOMINANT NEGATIVEVERSION OF CREB HAS NO EFFECT

We first confirmed the effects of manipulating CREB function ina small portion (∼8–10%) of LA neurons on the formation oftone fear memory by microinjecting HSV vectors encoding GFP,
CREB or dominant-negative CREB (CREBS133A ) into the LA ofadult mice 3 d before fear conditioning (see Figure 1A). During
training (Cxt A), mice received a single tone (conditioned stimulus, CS) footshock (0.4 mA) (US) pairing that did not induce
ceiling levels of freezing. Tone fear memory was assessed 24 hafter training. Mice were placed in a novel context (Cxt B)
and 2 min later the tone was presented for 3 min (Figure 1B).
Consistent with our earlier findings (Han et al., 2007, 2009) andthose of other research groups (Zhou et al., 2009; Rexach et al.,2012), increasing CREB levels in a small portion of LA neurons
enhanced tone fear memory, while disrupting CREB function by
microinjecting CREBS133A vector had no effect on fear memory(Figures 1C,D). These results were supported by a Vector (GFP,
CREB, CREBS133A vector) × Time (5 min) ANOVA showing significant main effects of Vector [F(2, 28) = 6.8, p = 0.004] andTime [F(4,112) = 16.5, p ≤ 0.0001] but no Vector × Time interaction [F(8,112) = 6.8, p = 0.32]. A subsequent One-Way ANOVA
performed on freezing during the entire CS presentation showeda significant effect of Vector [F(2,28) = 7.1, p = 0.003], as mice
microinjected with CREB vector froze significantly more thanmice with GFP (p = 0.006) or CREBS133A vector (p = 0.004),
which did not differ from each other (p = 0.83) (Newman-Keuls
post-hoc) (Figure 1D). Importantly, when first placed in Cxt B,
mice generally showed little freezing before the tone was presented and baseline levels of freezing in CREB or CREBS133A
groups did not differ from the GFP group (p = 0.18, p = 0.19
respectively). We next examined a possible mechanism underlying this preferential recruitment to the memory trace.
CREB MODULATES DENDRITIC SPINE DENSITY OF LA NEURONS

CREB is a ubiquitous transcription factor that has been implicated in many cellular processes, including regulating dendritic
spine density. We hypothesized that neurons may be recruited
to the memory trace based on their relative spine density, andexamined whether neurons infected with CREB vector show
greater dendritic spine density at the time of training than neurons
infected with CREBS133A or Control GFP vector. We microinjected a separate co-hort of mice with GFP, CREB, or CREBS133A
vector as above but did not train these mice. Instead, 72 h following surgery (at a time when they would have received auditory
fear conditioning) we removed their brains and examined spine
density (Figure 2A).
There are two major neuronal cell populations in LA:
pyramidal glutamatergic projection neurons and local circuit
γ-aminobutyric acid (GABA)-ergic interneurons (McDonald,1984). Glutamatergic pyramidal-like principal neurons comprisethe majority (85–90%) (McDonald, 1992; Sah et al., 2003) and

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CREB regulates spine density

FIGURE 1 | Overexpressing CREB in LA neurons enhances, while
dominant negative CREBS133A does not affect, auditory fear memory.
(A) Visualizing neurons infected with viral vectors. Left: Outline of the LA.
Maximum intensity projection is shown. Right: LA principal neurons expressing
GFP 72 h following viral vector microinjection (nuclei stained with DAPI,
infected neurons visualized with anti-GFP antibody). 0.25 μm optical section isshown. Scale bar, 100 μm. (B) Experimental design. Auditory (tone) fear
conditioning was conducted 72 h after HSV microinjection. Mice were placed in

CXT A and presented with a tone (30 s) that co-terminated with a footshock(0.4 mA). Memory was assessed 24 h later in CXT B. (C) Mice microinjectedwith CREB vector (n = 10) showed increased freezing during (but not before)
subsequent presentation of the tone compared to mice microinjected with GFP(n = 12) or CREBS133A (n = 11) vectors. (D) Mice overexpressing CREB in LA
neurons showed enhanced fear memory for the tone indicated by higher
freezing levels during the 3–min tone, compared to mice with GFP or
CREBS133A vectors. Data represent mean ± s.e.m. ∗ p < 0.05.

can be visually identified according to the shape of their somata.
Thus, we identified infected neurons as LA principal neurons
based on their pyramidal shaped somata. In mice microinjectedwith CREB vector, infected neurons showed higher spine density
compared to infected neurons in mice microinjected with Control(GFP-only) vector. In contrast, CREBS133A -infected neurons
showed lower spine density than control neurons. This pattern ofresults was observed across branch order (Figures 2C,D). A Vector(GFP, CREB, CREBS133A ) by Branch order (3) repeated-measures
ANOVA showed significant main effects of Vector [F(2,15) = 16.8,
p ≤ 0.0001] and Branch order [F(2,30) = 37.2, p ≤ 0.0001] but nosignificant interaction between Vector× Branch order [F(4,30) =0.2, p = 0.90]. Post-hoc Newman-Keuls analysis on the significant
main effects revealed that neurons with CREB vector had significantly greater spine density across branch orders compared toneurons infected with GFP (p = 0.02) or CREBS133A (p = 0.0002)
vectors (Figures 2C,E), while neurons with CREBS133A vector had
lower spine density across branches relative to those expressing
GFP only (p = 0.004) (Figures 2C,E). It is important to note thatthese changes in spine density occurred even though all mice
were maintained in the homecage (and therefore, these changes
in spine density cannot be attributed to fear conditioning).
Importantly, dendritic length per branch (Figure 3A) or
total dendritic length did not appear to differ between vectors (Figure 3B). This observation was supported by repeatedmeasures ANOVA showing no significant effect of Vector[F(2,15) = 2.8, p = 0.09] or interaction of Vector × Branch order[F(4,30) = 0.7, p = 0.60], but a significant main effect of Branchorder [F(2,30) = 10.9, p = 0.0003]. Therefore, dendritic length
increased with increasing branch order, but this was not changed
by CREB manipulation (Figures 3A,B). We also observed nodifference in dendritic volume between neurons infected withthe various vectors (Figures 3C,D). An ANOVA revealed no significant effects of Vector [F(2,15) = 1.3, p = 0.29], Branch order[F(2, 30) = 1.0, p = 0.37] or Vector× Branch order interaction[F(4,30) = 1.9, p = 0.13]. Therefore, manipulations of CREB
function changed dendritic spine density without changing dendritic morphology.

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FIGURE 2 | CREB modulates dendritic spine density in LA neurons. (A)Experimental design. The morphology of infected LA neurons wasanalyzed 72 h after mice were microinjected with GFP, CREB or,
CREBS133A vectors (at the same time-point as training occurred in
Figure 1). Mice remained in the homecage after microinjection and were
not fear conditioned. (B) Schematic representation of a dendritic segment
showing the parameters analyzed (dendrite length, spine length, spine

SPINE MORPHOLOGY IS NOT ALTERED BY CREB OR CREBS133AEXPRESSION

Alterations in spine morphology have been correlated withchanges in spine function (Matsuzaki et al., 2001, 2004) andincreasing CREB may induce formation of silent synapses (Marieet al., 2005). Spines with large bulbous heads are thought tocontain large post-synaptic densities (PSD) (Harris et al., 1992)
whereas spines with small heads and long necks may contain silent synapses (Matsuzaki et al., 2001, 2004). Promptedby these observations, we analyzed whether manipulations of
CREB function altered spine morphology in mice maintainedin the homecage by measuring spine length and head diameter(see Figure 2B). Interestingly, spine length, regardless of vector,
increased slightly with increasing branch order {Figures 4A,B;
ANOVA showing no significant main effect of Vector [F(2,15) =0.8, p = 0.45] or Vector × Branch order interaction [F(4,30) =0.6, p = 0.63], but a significant main effect of Branch order[F(2,30) = 8.9, p = 0.0009]}. We next examined whether CREB
manipulation influenced spine head diameter (widest distance

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head diameter and spine density). Scale bar, 10 μm. (C) Representative
dendritic segments of LA neurons from mice microinjected with GFP,
CREB, or CREBS133A vector. Scale bar, 5 μm. (D) Dendritic spine density
shown at each branch order and (E) across all branches is increased inneurons with CREB vector (n = 6) and decreased in neurons withCREBS133A vector (n = 5) when compared to neurons with GFP vector(n = 7). Data represent mean ± s.e.m. ∗ p < 0.05. ∗∗ p < 0.001.

of the spine head, see Figure 2B). We observed no difference
between spine head diameter between vectors, but a small change
per branch order across all vectors {Figures 4C,D no significant
effect of Vector [F(2,15) = 0.7, p = 0.50] or Vector × Branch orderinteraction [F(4,30) = 1.0, p = 0.41] but a significant main effectof Branch order [F(2,30) = 4.2, p = 0.02]}. Therefore, although
CREB manipulations produced changes in dendritic spine density, these were not accompanied by changes in dendrite or overall
spine morphology.

DISCUSSION
Previously, we and others observed that neurons with relatively
increased CREB function at the time of training seem to be competitively advantaged over neighboring neurons for allocation toa fear memory trace. Here we examined whether an increase inspine density at the time of training might mediate this competitive advantage. To this end, we examined the effects of manipulating CREB function on dendritic spine density at the time oftraining. We found that in mice taken directly from the homecage,www.frontiersin.org

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FIGURE 3 | CREB does not affect dendritic length and volume. (A)Dendritic length per branch order and (B) across all branches was comparable
between LA neurons overexpressing GFP (n = 7), CREB (n = 6), or

CREBS133A (n = 5) vectors. (C) Dendrite volume per branch order and (D)across all branches did not differ between LA neurons overexpressing GFP(n = 7), CREB (n = 6), or CREBS133A (n = 5) vectors.

FIGURE 4 | CREB does not affect spine morphology. (A) Spine length ateach branch order and (B) across all branches was similar between LA
neurons with GFP (n = 7), CREB (n = 6), or CREBS133A (n = 5) vectors. (C)Spine head diameter at each branch order and (D) across all branches did not
differ between LA neurons with GFP (n = 7), CREB (n = 6), or CREBS133A(n = 5) vectors.

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neurons with CREB overexpression showed higher, while neurons with CREBS133A showed lower spine density, than control
infected neurons. These data are consistent with the notion that
one factor that may determine neuronal allocation for memory
formation is relative spine density.
The LA is a key brain region important in mediating fear andanxiety (Davis, 1992) and some studies implicate CREB in “emotional” behavior (Barrot et al., 2002; Pandey et al., 2003). It is
possible, therefore, that CREB overexpression in a small population of LA neurons leads to a general increase in fear and/or
anxiety. Disruption of CREB function either generally in the brain(Valverde et al., 2004) or specifically in the amygdala (Pandey
et al., 2005) has been reported to increase anxiety-like behavior
in mice. On the other hand, local CREB overexpression has beenshown to enhance excitability of LA neurons without causing
alterations in anxiety or locomotor activity (Viosca et al., 2009).
In our experiments, before the presentation of the tone (pre-CS),
freezing levels of mice overexpressing CREB or CREBS133A did not
differ from those overexpressing GFP. This ruled out the possibility that CREB or CREBS133A might lead to alterations in general
fear and anxiety.
Studies in the 1990s first implicated CREB in the formationof long-term memory (LTM) (Dash et al., 1990; Yin et al., 1994,
1995). Building on these important findings, we (Josselyn andNguyen, 2005; Han et al., 2007, 2009; Cole et al., 2012), and others (Bourtchuladze et al., 1994; Kida et al., 2002) (Viosca et al.,2009; Zhou et al., 2009; Rexach et al., 2012), showed that decreasing CREB function disrupts, while increasing CREB function
enhances the formation of many types of memory in mammals [but see Balschun et al. (2003)]. The role of CREB in fear
memory has been extensively studied in rodents. Mice lacking
α and δ isoforms of CREB (CREBαδ ) showed impaired in LTM
for both context and tone fear memories (Bourtchuladze et al.,1994). Similarly, CREBcomp mice, carrying one allele for the βisoform of CREB, showed deficits in LTM for context and tone
fear memories (Gass et al., 1998). CREBIR mice which express
CREBS133A in a temporally regulated manner have impaired context and tone fear memory following repression of CREB activity
before training (Kida et al., 2002). Viral delivery of CREB into
the amygdala using HSV enhanced LTM induced by massed
training protocol in the fear potentiated startle paradigm in rats
(Josselyn et al., 2001). CREB is thought to activate the transcription of target genes which ultimately serve as the building
blocks for increasing the synaptic connections between neurons important for memory formation (Bartsch et al., 1998).
It is interesting to note that CREB has also been implicated inhuman memory (Harum et al., 2001) and several human cognitive/memory disorders are linked to disruptions in the CREB
signaling pathway (Josselyn and Nguyen, 2005). Together thesedata converge to indicate that CREB is critical for memory
formation.
Previous studies have also established a possible role of CREB
in maintaining spine number and morphology. Estradiol treatment in cultured hippocampal neurons led to increased phosphorylation of CREB which correlated with spinogenesis (Murphy
and Segal, 1997). Enhancing CREB function upon expression ofa constitutively active form of CREB (caCREB) in the CA1 region

Frontiers in Behavioral Neuroscienceof hippocampus increased spine density in hippocampal neurons
in vivo (Marie et al., 2005). CREB was also shown to regulate
spine morphology in pyramidal neurons of the visual cortex(Suzuki et al., 2007). Expression of caCREB in organotypic hippocampal neurons increased spine density while decreasing CREB
function by expression of a dominant negative CREB or a CREBtargeted shRNA inhibited spine formation (Impey et al., 2010).
Consistent with this, increasing CREB function in hippocampal
CA1 principal neurons restored the decrease in spine density andimproved spatial memory in a mouse model of Alzheimer’s disease (Yiu et al., 2011). Recent work has shown that CREB-induced
excitability of LA neurons may be a potential mechanism for preferential recruitment of these neurons to the fear memory trace(Zhou et al., 2009).
Based on the previous work, we hypothesized that CREB’s rolein allocation of tone fear memory may be caused by its effecton regulation of spine density of LA neurons. The LA receives
sensory (both tone and footshock) information directly fromauditory cortex and thalamus (LeDoux et al., 1990; Campeau andDavis, 1995) and is thought to be the critical site for convergenceof US and CS inputs in auditory fear conditioning experiments.
Therefore, neurons with more dendritic spines may be preferentially activated by the CS and US convergence and become part ofthe memory trace.
Here, we observed that changes in CREB function alone were
sufficient to change dendritic spine density, and that neurons withincreased CREB function showed higher dendritic spine density.
Furthermore we observed that neurons with higher CREB function were preferentially allocated to the memory trace. Because
synapses and spines play a key role in neuronal information processing, changes in dendritic spine density or morphology of aneuron may affect synaptic function and local circuit organization. Along with other factors, such as changes in neuronal
excitability (Zhou et al., 2009), changes in the synapse and spine
number and morphology may influence neuronal spiking activity
and play important roles in neuronal memory allocation.

ACKNOWLEDGMENTS
We thank Mika Yamamoto, Russell Braybon and Antonietta(Toni) Decristofaro for excellent technical assistance and members of the Josselyn/Frankland laboratory for their helpful suggestions throughout this project. This work was supported by
grants from the Canadian Institutes of Health Research (CIHR;
MOP-74650), EJLB Foundation and the Alzheimer’s Society ofCanada to Sheena A. Josselyn and CIHR (MOP- 86762) to PaulW. Frankland. Derya Sargin and Adelaide P. Yiu received support from the Alzheimer’s Society, Adelaide P. Yiu received support from Restracomp Fellowships (Hospital for Sick Children).
We thank Christina Cole for surgical excellence and Matthew
Florczynski for technical help.

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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could beconstrued as a potential conflict of interest.
Received: 11 September 2013; paper pending published: 06 November 2013; accepted:
05 December 2013; published online: 20 December 2013.
Citation: Sargin D, Mercaldo V, Yiu AP, Higgs G, Han J-H, Frankland PW and
Josselyn SA (2013) CREB regulates spine density of lateral amygdala neurons: implications for memory allocation. Front. Behav. Neurosci. 7:209. doi: 10.3389/fnbeh.
2013.00209
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