Research

Ontogeny of contextual fear memory formation,
specificity, and persistence in mice
Katherine G. Akers,1 Maithe Arruda-Carvalho,1,2 Sheena A. Josselyn,1,2,3
and Paul W. Frankland1,2,3,4
1

Neurosciences and Mental Health, Hospital for Sick Children, Toronto, ON, Canada, M5G 1X8; 2Institute of Medical Sciences, Universityof Toronto, Toronto, ON, Canada, M5S 1A8; 3Department of Physiology, University of Toronto, Toronto, ON, Canada, M5S 1A8
Pinpointing the precise age when young animals begin to form memories of aversive events is valuable for understanding
the onset of anxiety and mood disorders and for detecting early cognitive impairment in models of childhood-onset disorders. Although these disorders are most commonly modeled in mice, we know little regarding the development of learning and memory in this species because most previous studies have been restricted to rats. Therefore, in the present study,
we constructed an ontogenetic timeline of contextual fear memory ranging from infancy to adulthood in mice. We foundthat the ability of mice to form long-term context-shock associations emerged 13 –14 d of age, which is several days earlier
than previously reported for rats. Although the ability to form contextual fear memories remained stable from infancy into
adulthood, infant mice had shorter-lasting memories than adolescent and adult mice. Furthermore, we found that mice subjected to fetal alcohol exposure showed a delay in the developmental emergence of contextual fear memory, illustrating theutility of this ontogenetic approach in detecting developmental delays in cognitive function stemming from maladaptive
early life experience.
[Supplemental material is available for this article.]

Mental illnesses are increasingly recognized as chronic disordersof the young (Insel and Fenton 2005). Many mental disorders—
particularly those involving anxiety and lack of impulse control—emerge during childhood or adolescence and persist
throughout the lifespan, with earlier onset associated with longer
delays to initial treatment, increased comorbidity, and poorer
functional outcomes (Kessler et al. 2007; McGorry et al. 2011).
Timely diagnosis and treatment of these disorders, therefore,
can reduce their severity and prevalence, thereby lessening theburden of mental illness on the individual, family, and society(de Girolamo et al. 2012).
In the study of the etiology of mental disorders, animal models are vital tools. Pinpointing the precise age when animals begin
to form memories of aversive events can be valuable for understanding the onset of anxiety and mood disorders stemmingfrom maladaptive early life experience (Pine 2009; Bale et al.
2010; Marco et al. 2011) and for detecting early cognitive impairment in models of autism, attention deficit/hyperactivity disorder, and fetal alcohol syndrome (Schneider et al. 2011; Kaffman
and Krystal 2012). During infancy and adolescence, the progressive growth and refinement of neural circuitry supporting sensation and perception (Bourne 2010; Froemke and Jones 2011),
cognition (Benes et al. 2000; Dumas 2005), and emotion (Braun2011) gradually enables young rodents to begin learning about
their surroundings. For example, around 17 d of age, rats start toform memories of contexts they encounter (Brasser and Spear
2004; Yap and Richardson 2005; Foster and Burman 2010), andby 23 d of age, they can associate those contexts with the occurrence of aversive events (Rudy 1993; Rudy and Morledge 1994;
Raineki et al. 2010; Schiffino et al. 2011).

Because of the ease of targeted genetic manipulation, mice
have become the most prevalent animal models of mental disorders. However, apart from a few mouse studies (e.g., Paylor et al.
1996; Hefner and Holmes 2007; Ito et al. 2009), nearly all studies
on the development of learning and memory have been restricted
to rats. Because the two species differ in terms of synaptic architecture, sensory and motor function, and performance in behavioral
tasks (Whishaw et al. 2001), knowledge pertaining to the ontogeny of learning and memory in rats may not directly transfer tostudies employing mice. The primary aim of the present study,
therefore, was to construct an ontogenetic timeline of memory
in mice using contextual fear conditioning, a well-characterized
aversive learning paradigm (Anagnostaras et al. 2001; Rudy et al.
2004). Furthermore, although maladaptive early life experience
is often shown to produce cognitive impairments that are evident
long after the initial trauma (Sullivan et al. 2006; McClelland et al.
2011), less research has focused on whether early adversity delays
the initial onset of learning and memory in young animals. Thus,
our second aim was to determine whether an ontogenetic timeline of contextual fear memory could be used to detect developmental delays resulting from one type of adverse early life
experience—fetal exposure to alcohol.

Results
Memory formation
We investigated the ontogeny of contextual fear memory formation by placing infant (P13, P14, P15, P16, or P17), adolescent(P30), or adult (P60) mice in a context and delivering three footshocks (shock group). Mice were returned to the context 24 h after
training to test for long-term memory of the context-shock association. To control for differences among ages in spontaneous
freezing, age-matched groups of mice were exposed to the context, but no foot shocks were delivered (no shock group).

4
Corresponding author
E-mail paul.frankland@sickkids.ca
Article is online at http://www.learnmem.org/cgi/doi/10.1101/lm.027581.112.

19:598 –604 # 2012 Cold Spring Harbor Laboratory Press
ISSN 1549-5485/12; www.learnmem.org

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Ontogeny of contextual fear memory in mice

0.001, minute 5 P ¼ 0.005 [Fig. 1D]; P16 shock × minute interaction P , 0.001, minute 3 P ¼ 0.031, minute 4 P ¼ 0.001, minute 5
P , 0.001 [Fig. 1E]; P17 shock × minute interaction P , 0.001,
minute 3 P ¼ 0.008, minute 4 P ¼ 0.002, minute 5 P , 0.001[Fig. 1F]; P30 shock × minute interaction P , 0.001, minute 5
P , 0.001 [Fig. 1G]; P60 shock × minute interaction P , 0.001,
minute 4 P ¼ 0.012, minute 5 P , 0.001 [Fig. 1H].)
We also analyzed minute-by-minute changes in freezing during testing at each age. At P13, both shocked and nonshocked mice
exhibited high levels of freezing, with no significant differences
between groups (Fig. 1B). At P14, shocked mice froze more thannonshocked mice during the first few minutes of the test (shock ×
minute interaction P , 0.001, minute 1 P ¼ 0.001, minute 2 P ,0.001, minute 3 P ¼ 0.008 [Fig. 1C]), showing a decline in freezing
across minutes (minute main effect within shocked group,F(4,28) ¼ 4.64, P ¼ 0.005). At P15 and P16, shocked mice also
showed a decline in freezing across minutes (P15 minute main effect within shocked group, F(4,24) ¼ 3.64, P ¼ 0.019 [Fig. 1D]; P16
minute main effect within shocked group, F(4,28) ¼ 5.43, P ¼
0.002 [Fig. 1E]). However, the difference between shocked andnonshocked mice persisted across the entire test (P15 shock
main effect P ¼ 0.002 [Fig. 1D]; P16 shock main effect P , 0.001[Fig. 1E]). Finally, starting at P17 and continuing through adolescence and into adulthood, shocked mice showed stable, high levels of freezing across minutes, with a difference between shocked
and nonshocked mice evident throughout the entire test (P17shock main effect P , 0.001 [Fig. 1F]; P30 shock main effect P ,0.001 [Fig. 1G]; P60 shock main effect P , 0.001 [Fig. 1H]).
Therefore, after controlling for age-related differences in spontaneous freezing, we found that the ability of mice to form long-term
associations between context and shock emerges at P14 and remains stable in the days thereafter.

We found that, across all ages tested, mice that were shockedin the context showed high levels of freezing upon return to thecontext 24 h later. High levels of freezing were also observed
among infant mice that were not shocked, although this spontaneous freezing steadily diminished with age (age × shock interaction P , 0.001) (Fig. 1A; Supplemental Table S1). At P13, shocked
and nonshocked mice showed similar levels of freezing. From P14
onward, however, shocked mice consistently froze more thannonshocked mice (P14 P , 0.001, P15 P ¼ 0.005, P16 P ¼ 0.001,
P17 P , 0.001, P30 P , 0.001, P60 P , 0.001).
To further examine the developmental timeline of contextual fear memory formation, we analyzed minute-by-minute changes in freezing during training at each age. We found that at P13,
the delivery of foot shocks increased locomotor activity, resulting in less post-shock freezing compared to mice that were notshocked (shock × minute interaction P ¼ 0.004, minute 3 P ¼
0.004) (Fig. 1B). At P14, foot shocks did not affect freezing (Fig.
1C). However, starting at P15 and continuing through adolescence and into adulthood, the delivery of foot shocks consistently
resulted in more post-shock freezing compared to nonshocked
mice (P15 shock × minute interaction P , 0.001, minute 4 P ¼

Memory specificity
Next, we tested whether the contextual specificity of the memory
varied with age. During infancy (P15), adolescence (P30), or adulthood (P60), mice received three foot shocks in a context. Twentyfour hours later, mice were returned to the training context (context A) or placed in one of two different contexts—one that shared
several features with the training context (context B) or one thatwas largely distinct from the training context (context C). We
found that, overall, mice discriminated among contexts (context
main effect P , 0.001) (Fig. 2A– C; Supplemental Table S2), withless freezing in context C compared to in context A (P , 0.001)
or B (P , 0.001). However, there were no differences among ages
in the specificity of the context memory.

Memory persistence
We also tested whether the persistence of contextual fear memory
depended on age. Infant (P15), adolescent (P30), or adult (P60)
mice received three foot shocks in a context, and memory forthe context-shock association was tested 1, 7, 14, or 28 d later inseparate groups of mice. We observed pronounced age-related differences in freezing across longer training-testing delays (age ×delay interaction P , 0.001) (Fig. 3A–C; Supplemental Table S3).
Consistent with previous reports of infantile amnesia in rats
(Campbell and Campbell 1962; Spear 1979), P15 mice showed little or no freezing at training-testing delays longer than 1 d (1 vs. 7
d P , 0.001, 1 vs. 14 d P , 0.001, 1 vs. 28 d P , 0.001) (Fig. 3A). In
contrast, P30 and P60 mice showed high levels of freezing for atleast 28 d after training (Fig. 3B,C).
The memory loss in infant mice appeared to be extensive,with a complete lack of freezing observed at longer trainingtesting delays. To assess the severity of this memory loss, weFigure 1. Contextual fear memory formation. Infant, adolescent, or
adult mice were placed in a context, where they either received threeunsignaled foot shocks (denoted by a downward arrow []; shockgroup; P13 n ¼ 8, P14 n ¼ 8, P15 n ¼ 7, P16 n ¼ 8, P17 n ¼ 8, P30 n ¼
8, P60 n ¼ 8) or did not receive foot shocks (no shock group; P13 n ¼
8, P14 n ¼ 8, P15 n ¼ 7, P16 n ¼ 8, P17 n ¼ 8, P30 n ¼ 8, P60 n ¼ 8).
Memory for the context-shock association was tested 24 h after training.
(A) During the test, shocked mice showed high levels of freezing regardless of age, whereas nonshocked mice showed a gradual decline in spontaneous freezing with age. Minute-by-minute examination of freezing
during training (left sides of graphs) and testing (right sides of graphs) revealed that P13 mice (B) exhibited negligible evidence of contextual fear
memory. At P14 (C) and all ages thereafter—P15 (D), P16 (E), P17 (F),P30 (G), and P60 (H)—mice exhibited clear evidence of contextual fear
memory, with shocked mice freezing more than nonshocked mice
during the test.
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Ontogeny of contextual fear memory in miceobserved until P15 for FAE mice, indicating that fetal alcohol exposure delays the onset of this type of memory by 2 d.
To further examine the effect of fetal alcohol exposure on thedevelopment of contextual fear memory formation, we analyzed
minute-by-minute changes in freezing during training and testing. During training, control and FAE mice showed similar responses to foot shocks, with shock-induced decreases in freezingat P13 (Fig. 4B,C), no change in freezing at P14 (Fig. 4E,F), andshock-induced increases in freezing at P15 (Fig. 4H,I), suggestingthat fetal alcohol exposure did not alter sensitivity to the shock.
During testing, however, we observed differences in freezing levels
between control and FAE mice. At P13, both control and FAE mice
initially froze more if they had previously been shocked (control:
shock × minute interaction P , 0.001, minute 1 P , 0.001, minute 2 P , 0.001 [Fig. 4B]; FAE: shock × minute interaction P ¼
0.002, minute 1 P ¼ 0.020, minute 2 P ¼ 0.030 [Fig. 4C]). At
P14, within the control group, shocked mice froze more than nonshocked mice throughout the entire test (shock × minute interaction P ¼ 0.028, minute 1 P , 0.001, minute 2 P , 0.001, minute 3
P , 0.001, minute 4 P ¼ 0.018, minute 5 P ¼ 0.029 [Fig. 4E]), but
within the FAE group, there were no differences between shocked
and nonshocked mice (Fig. 4F). At P15, control and FAE groups
showed equivalent levels of freezing, with shocked mice freezing
Figure 2. Contextual fear memory specificity. Infant, adolescent, or
adult mice received three unsignaled foot shocks in a context.
Twenty-four hours later, they were placed in the original training
context (context A; P15 n ¼ 7, P30 n ¼ 8, P60 n ¼ 6), a similar context(context B; P15 n ¼ 8, P30 n ¼ 8, P60 n ¼ 8), or a dissimilar context(context C; P15 n ¼ 8, P30 n ¼ 7, P60 n ¼ 7). All ages of mice—P15(A), P30 (B), and P60 (C)—showed similar discrimination among contexts, with higher levels of freezing in contexts A and B compared tocontext C.

retrained additional groups of mice upon reaching adulthood totest whether prior memory formation would facilitate relearningof the context-shock association (i.e., savings). At P15, mice either
received three foot shocks in a context (trained group) or remained in their home cage (naive group). At P60, mice were returned to the context once a day for 4 d, where they received asingle foot shock during the first 3 d. Trained and naive mice
showed differences in freezing across days, with mice that weretrained during infancy displaying somewhat less freezing than naive mice during the last days (training × day interaction P ¼
0.025) (Fig. 3D; Supplemental Table S4). These results, therefore,
provide no clear evidence of a memory savings effect during
adulthood.

Developmental delay
Finally, we tested the utility of this approach in detecting earlysigns of cognitive impairment following early life adversity.
During infancy (P13, P14, or P15), control and fetal alcoholexposed (FAE) mice received three foot shocks in a context, andmemory for the context-shock association was tested 24 h later.
At P13, within the control group, shocked mice froze morethan nonshocked mice during the test (P , 0.001) (Fig. 4A;
Supplemental Table S5), but within the FAE group, shocked andnonshocked mice showed equivalent levels of freezing (EtOH ×
shock interaction P ¼ 0.018). Similarly, at P14, shocked mice froze
more than nonshocked mice within the control group (P , 0.001)(Fig. 4D), but there was no significant difference in freezing between shocked and nonshocked mice within the FAE group. At
P15, however, shocked mice froze more than nonshocked mice
within both control and FAE groups (shock main effect P ,0.001) (Fig. 4G). Thus, whereas control mice showed evidence offorming contextual fear memories at P13, such evidence was notwww.learnmem.org

Figure 3. Contextual fear memory persistence. Infant, adolescent, or
adult mice received three unsignaled foot shocks in a context and werereturned to the context 1 (P15 n ¼ 8, P30 n ¼ 8, P60 n ¼ 8), 7 (P15n ¼ 8, P30 n ¼ 8, P60 n ¼ 7), 14 (P15 n ¼ 8, P30 n ¼ 8, P60 n ¼ 8), or
28 (P15 n ¼ 8, P30 n ¼ 8, P60 n ¼ 8) days later. P15 mice (A) showed
little or no freezing at training-testing delays longer than 1 d. In contrast,
P30 mice (B) and P60 mice (C ) exhibited high levels of freezing for at least
28 d after training. (D) Mice that were either trained (trained group; n ¼8) or remained in their home cage (naive group; n ¼ 8) on P15 underwent
4 d of retraining starting on P60. Mice that were previously trained on P15
showed no evidence of memory savings during retraining.

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Ontogeny of contextual fear memory in mice

Richardson 2011). Our finding thatmice are able to associate a shock with acontext as early as 13– 14 d of age, however, suggests that brain and behavioral
development is accelerated in mice compared to rats, consistent with previous
proposals (Pellis and Iwaniuk 2000;
Whishaw et al. 2001). Therefore, what is
known regarding the ontogenetic timelines of learning and memory in rats
may not directly transfer to mice, andthis should be taken into account whenusing mice to model early cognitive andemotional dysfunction.
In adult rodents, hippocampal lesions consistently produce retrograde
amnesia for contextual fear memories(Maren et al. 1997; Frankland et al.
1998; Anagnostaras et al. 1999; Wiltgen
et al. 2006; Lehmann et al. 2007), providing evidence that this type of memory isusually encoded by the hippocampus.
Although it is possible that young rodents
could condition to independent eleFigure 4. Developmental delay in contextual fear memory formation. Fetal alcohol-exposed (FAE) or
ments of the training context throughcontrol mice were placed in a context, where they either received three unsignaled foot shocks
nonhippocampus-dependent processes(denoted by a downward arrow []; shock group; FAE: P13 n ¼ 8, P14 n ¼ 9, P15 n ¼ 9; control:(Rudy 2009), accumulating evidence sugP13 n ¼ 9, P14 n ¼ 9, P15 n ¼ 9) or did not receive foot shocks (no shock group; FAE: P13 n ¼ 9,gests that the hippocampus is involved in
P14 n ¼ 9, P15 n ¼ 9; control: P13 n ¼ 9, P14 n ¼ 9, P15 n ¼ 9). Memory for the context-shock association was tested 24 h after training. At P13 (A – C) and P14 (D– F ), control mice showed clear evidence
the formation of contextual fear memoof forming context-shock associations, but FAE mice showed little such evidence. By P15 (G –I ),ries during infancy. Specifically, thehowever, both control and FAE mice exhibited evidence of forming contextual fear memories.
developmental onset of contextual fear
learning coincides with the onset oftraining-induced hippocampal immedimore than nonshocked mice during most or all minutes of the test
ate early gene expression (Raineki et al. 2010), and lesion or inacti(control: shock × minute interaction P ¼ 0.006, minute 1 P ¼
vation of the hippocampus blocks the formation of context and0.008, minute 2 P ¼ 0.003, minute 3 P ¼ 0.001, minute 4 P ¼
contextual fear memories in infant rats (Foster and Burman 2010;
0.006 [Fig. 4H]; FAE: shock main effect P ¼ 0.009 [Fig. 4I]).
Raineki et al. 2010; Schiffino et al. 2011). Therefore, the emerging
ability of mice to form context-shock associations at 13–14 d of age
may reflect the functional maturation of the hippocampus, which
continues to undergo changes in neuron number (Altman andDiscussion
Bayer 1975), dendritic arborization (Rahimi and Claiborne 2007),
signaling mechanisms (Paylor et al. 1996), synaptic plasticity
We examined the ontogeny of contextual fear memory for(Harris and Teyler 1984), and spatial firing (Langston et al. 2010;
mation, specificity, and persistence in mice from infancy toWills et al. 2010; Scott et al. 2011) during the postnatal period.
adulthood. After controlling for age-related differences in spontaFurthermore, the emergence of contextual fear memory is
neous freezing, we found that the ability of mice to form longlikely not solely contingent on hippocampal development but
term contextual fear memories emerges at 13– 14 d of age and rerather the maturation of a broad network of brain regions.
mains stable through adolescence and into adulthood. Although
Considering that eye opening occurred only 1– 2 d prior to the onthe specificity of the memory was similar across ages, infant mice
set of contextual fear learning in mice (i.e., at 12 d of age), the onhad shorter-lasting memories compared to adolescent and adultgoing development of the visual system (Bourne 2010) may also
mice, indicating that memory persistence continues to develop
underlie the onset of this type of learning. Furthermore, our obserwith age. Finally, we found that ability to associate a shock withvation that spontaneous freezing declined steadily across infancy
a context does not emerge until 15 d of age in mice that were exsuggests that the emergence of conditioned fear (as opposed toposed to alcohol during gestation, illustrating the utility of thisunconditioned fear) may rely on the maturation of brain regions
ontogenetic approach in detecting developmental delays in modinvolved in emotion, such as the amygdala (Gogolla et al. 2009;els of early adversity.
Chareyron et al. 2012) and prefrontal cortex (van Eden et al.
The precise age at which rats begin to form long-term contex1990).
tual fear memories has been a matter of debate in previous literaIn contrast to adolescent and adult mice, which showed
ture. Although some studies report that rats fail to associate along-lasting memory for the context-fear association, infant
shock with a context until 23– 24 d of age (Spear 1979; Rudy
mice showed rapid forgetting. These findings are consistent
1993; Rudy and Morledge 1994; Raineki et al. 2010; Schiffino
with previous studies in rats, which report that infants forget deet al. 2011), others demonstrate that this type of learning can octails of the context in which they were shocked more quickly
cur at 17– 18 d of age under some training conditions (Brasser andthan adults (Anderson and Riccio 2005) and ultimately show
Spear 2004; Esmoris-Arranz et al. 2008; Pisano et al. 2012) or folcomplete loss of memory for the context-shock association (i.e.,
lowing certain neonatal manipulations (i.e., injection of fibro“infantile amnesia”) (Campbell and Campbell 1962; Rudy andblast growth factor-2, sensory stimulation) (Woodcock andMorledge 1994; Weber et al. 2006). It is unclear whether thisRichardson 2000; Graham and Richardson 2010; Callaghan andwww.learnmem.org

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Ontogeny of contextual fear memory in miceloss of memory in infants reflects a failure of storage or a failure of
retrieval. Fear memory can be restored in infant rats following reminders or pharmacological treatments in the first several days
following training (Richardson et al. 1983; Weber et al. 2006;
Kim and Richardson 2007), indicating that while memories
formed during infancy may be resistant to retrieval, they are stillpresent for at least a short period of time following “forgetting.”
However, our effort to restore an early fear memory by retraining
mice during adulthood yielded no overwhelming evidenceof a preserved context-shock association at this age, suggesting that memories lost after infancy may eventually become
irrecoverable.
One recent study on the development of contextual fear
memory spanning from early adolescence to adulthood foundthat although mice of all ages could form context-shock associations, the subsequent expression of these memories was
suppressed during adolescence, between 29 and 39 d of age(Pattwell et al. 2011). The authors speculated that this temporary
suppression of contextual fear memory could serve an adaptivefunction, allowing adolescent mice to venture out of the nest
and explore potentially threatening environments. In line withseveral previous studies in mice (Hefner and Holmes 2007) andrats (Pugh et al. 1997; Brasser and Spear 2004; Esmoris-Arranz
et al. 2008; Murawski and Stanton 2010), however, we found noevidence of memory suppression in adolescents. Instead, we
found that levels of conditioned fear were stable across all ages
tested, from infancy to adulthood. This suggests that the finding
by Pattwell et al. (2011) may not reflect a general developmental
phenomenon but rather may be due to a difference in mouse genetic background or a peculiarity in their husbandry or experimental procedures.
The construction of ontogenetic timelines of learning andmemory in mice not only provides a developmental approachto studying basic relationships between brain and behavior but
also can be used to detect early signs of cognitive impairment inmodels of childhood-onset disorders. Although many disordersof genetic and/or environmental origin are associated with developmental delays in cognitive function (Cornish et al. 2004;
Sagvolden et al. 2005; Riley et al. 2011), most current animal models typically do not assess cognitive function until adulthood or
perhaps at one or two isolated developmental stages. For instance,
fetal alcohol exposure is consistently found to produce widespread effects on learning and memory in rodents that are present
during adolescence and adulthood (Berman and Hannigan 2000;
Schneider et al. 2011), but relatively little is known regarding thedevelopmental onset of these effects in younger animals (e.g.,
Blanchard et al. 1987; Wigal et al. 1988; Kirstein et al. 1997).
Our finding that the ability to form context-shock associations
is delayed by up to 2 d in fetal alcohol-exposed mice demonstrates
that a detailed ontogenetic timeline of contextual fear memory
can be used as a template to compare normal vs. abnormal cognitive development. Because this type of information can have important clinical applications (Pine 2009; Bale et al. 2010; Marcoet al. 2011), future efforts should be made to construct ontogenetic timelines for other cognitive functions relevant tochildhood-onset disorders, such as attention, response inhibition,
working memory, and social interaction.

designated P0, with litter sizes ranging from two to 11 pups. Eye
opening occurred at P12. After weaning on P21, mice were grouphoused (two to five per cage) in transparent plastic cages (31 ×
17 × 14 cm). To prevent the influence of litter effects on dependent measures (Abbey and Howard 1972), typically no morethan one female and one male from each litter were assigned toa particular experimental group. When more than one same-sex
littermate was tested within a group, dependent measures fromthe littermates were averaged. Roughly equal numbers of males
and females were assigned to each group.

Fetal alcohol exposure
The fetal alcohol exposure procedure was adapted from previous
studies (Allan et al. 2003; Akers et al. 2011). Briefly, female mice(n ¼ 10) were given saccharin-flavored drinking water (0.1% saccharin) to which ethanol (EtOH) was gradually introduced (0%EtOH for 4 d, 2% EtOH for 1 d, 5% for 1 d, 10% for 4 d). Mice continued to receive 10% EtOH during temporary cohabitation with amale breeder and throughout the extent of pregnancy. Starting on
the day of birth, the concentration of EtOH was gradually decreased (5% EtOH for 2 d, 2% EtOH for 2 d) and then replacedwith regular water. Control mice (n ¼ 9) received water containing 0.1% saccharin throughout pregnancy. All mice had free access to regular mouse chow.
Control and EtOH dams consumed an average of 4.89 + 0.22
and 4.59 + 0.11 g fluid daily during pregnancy, resulting in an average dose of 0 + 0 and 14.15 + 0.43 g/kg/day EtOH, respectively.
Although dams continued to drink a reduced amount of EtOH
during the first few days after giving birth, the amount of EtOH
passed on to pups through lactation is low (2% of maternaldose) (Mennella 2001), and therefore, its exposure to pups during
this period was expected to be minimal. We found no differences
between groups in dam body weight at onset of pregnancy (control: 26.8 + 1.4 g, EtOH: 25.6 + 0.7 g), weight gained across pregnancy (control: 12.7 + 1.2 g, EtOH: 13.9 + 0.6 g), or litter size
(control: 6.0 + 0.6 pups, EtOH: 6.7 + 0.4 pups). Previous studies
have shown that this procedure has no effect on maternal behavior or pup body weight but produces changes in brain volume, levels of neurotrophic factors, and behavior that persist into
adulthood (Allan et al. 2003; Caldwell et al. 2008; Akers et al.
2011).

Contextual fear conditioning
Contextual fear conditioning occurred in test chambers (33-cm
height, 29.2-cm width, 26.3-cm depth, Coulbourn Instruments,
H10-11R-TC). The front, top, and back of the chambers weremade of clear acrylic, and the sides were made of modular aluminum. Shock-grid floors were comprised of stainless steel
bars (0.4-cm diameter) spaced 0.7 cm apart. Shock delivery wascontrolled using a shock generator (Coulbourn Instruments,
H13-15). Freezing behavior (i.e., cessation of all movement except
breathing) was recorded by overhead video cameras and analyzed
using automated software (FreezeFrame, Actimetrics). Chambers
were cleaned with water between trials.

Memory formation
Separate groups of mice were trained at P13, P14, P15, P16, P17,
P30, or P60. Two minutes after being placed in the chambers,
mice received three unsignaled foot shocks (2 sec, 0.5 mA,
1 min apart) and were removed 1 min after the final shock.
Additional groups of age-matched mice were placed in the chambers for 5 min but did not receive foot shocks; these mice weretrained separately from shocked mice so that they did not hearshock-induced vocalizations. Twenty-four hours after training,mice were returned to the chambers for a 5-min test.

Materials and Methods
Animals
All procedures were approved by the Animal Care Committee atthe Hospital for Sick Children. Mice were a cross between
C57Bl/6 (paternal) and 129Svev (maternal) strains (Taconic),
which were bred in our animal facility and maintained on a
12 h light/dark cycle (lights on at 0700 h). The day of birth waswww.learnmem.org

Memory specificity
Mice were trained on P15, P30, or P60 as previously described.
Twenty-four hours after training, separate groups of mice were602

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Ontogeny of contextual fear memory in miceplaced in one of three different contexts for a 5-min test (Wanget al. 2009). Context A was the same chamber used during training. Context B was also the same chamber used during training,
but a white plastic floor covered the grid bars, a triangular plastic
insert was positioned inside the chamber, and black-and-white
striped paper covered the front wall. Context C was a white plastic
chamber (18 × 18 × 8 in) located in a different room.

Bale TL, Baram TZ, Brown AS, Goldstein JM, Insel TR, McCarthy MM,
Nemeroff CB, Reyes TM, Simerly RB, Susser ES, et al. 2010. Early life
programming and neurodevelopmental disorders. Biol Psychiatry 68:
314 –319.
Benes FM, Taylor JB, Cunningham MC. 2000. Convergence and plasticity
of monoaminergic systems in the medial prefrontal cortex during thepostnatal period: Implications for the development of
psychopathology. Cereb Cortex 10: 1014 –1027.
Berman RF, Hannigan JH. 2000. Effects of prenatal alcohol exposure on the
hippocampus: Spatial behavior, electrophysiology, and neuroanatomy.
Hippocampus 10: 94 – 110.
Blanchard BA, Riley EP, Hannigan JH. 1987. Deficits on a spatial navigation
task following prenatal exposure to ethanol. Neurotoxicol Teratol 9:
253 –258.
Bourne JA. 2010. Unravelling the development of the visual cortex:
Implications for plasticity and repair. J Anat 217: 449 –468.
Brasser SM, Spear NE. 2004. Contextual conditioning in infants, but notolder animals, is facilitated by CS conditioning. Neurobiol Learn Mem
81: 46 –59.
Braun K. 2011. The prefrontal-limbic system: Development,
neuroanatomy, function, and implications for socioemotional
development. Clin Perinatol 38: 685 –702.
Caldwell KK, Sheema S, Paz RD, Samudio-Ruiz SL, Laughlin MH,
Spence NE, Roehlk MJ, Alcon SN, Allan AM. 2008. Fetal alcohol
spectrum disorder-associated depression: Evidence for reductions in
the levels of brain-derived neurotrophic factor in a mouse model.
Pharmacol Biochem Behav 90: 614 –624.
Callaghan BL, Richardson R. 2011. Maternal separation results in early
emergence of adult-like fear and extinction learning in infant rats.
Behav Neurosci 125: 20 – 28.
Campbell BA, Campbell EH. 1962. Retention and extinction of learned fearin infant and adult rats. J Comp Physiol Psychol 55: 1 –8.
Chareyron LJ, Lavenex PB, Lavenex P. 2012. Postnatal development of
the amygdala: A stereological study in rats. J Comp Neurol 520:
3745 –3763.
Cornish KM, Turk J, Wilding J, Sudhalter V, Munir F, Kooy F, Hagerman R.
2004. Deconstructing the attention deficit in fragile X syndrome: A
developmental neuropsychological approach. J Child Psychol Psychiatry
45: 1042 –1053.
de Girolamo G, Dagani J, Purcell R, Cocchi A, McGorry PD. 2012.
Age of onset of mental disorders and use of mental healthservices: Needs, opportunities and obstacles. Epidemiol Psychiatr Sci
21: 47 –57.
Dumas TC. 2005. Late postnatal maturation of excitatory synaptictransmission permits adult-life expression of hippocampal-dependent
behaviors. Hippocampus 15: 562 –578.
Esmoris-Arranz FJ, Mendez C, Spear NE. 2008. Contextual fear
conditioning differs for infant, adolescent, and adult rats. Behav
Processes 78: 340 –350.
Foster JA, Burman MA. 2010. Evidence for hippocampus-dependent
contextual learning at postnatal day 17 in the rat. Learn Mem 17:
259 –266.
Frankland PW, Cestari V, Filipkowski RK, McDonald RJ, Silva AJ,
Sabel M. 1998. The dorsal hippocampus is essential for context
discrimination but not for contextual conditioning. Behav Neurosci
112: 863 –874.
Froemke RC, Jones BJ. 2011. Development of auditory cortical synaptic
receptive fields. Neurosci Biobehav Rev 35: 2105 – 2113.
Gogolla N, Caroni P, Luthi A, Herry C. 2009. Perineuronal nets protect fear
memories for erasure. Science 325: 1258 –1261.
Graham BM, Richardson R. 2010. Early-life exposure to fibroblast growth
factor-2 facilitates context-dependent long-term memory indeveloping rats. Behav Neurosci 124: 337 – 345.
Harris KM, Teyler TJ. 1984. Developmental onset of long-term
potentiation in area CA1 of rat hippocampus. J Physiol 346:
27 –48.
Hefner K, Holmes A. 2007. Ontogeny of fear-, anxiety- anddepression-related behavior across adolescence in C57BL/6J mice.
Behav Brain Res 176: 210 –215.
Insel TR, Fenton WS. 2005. Psychiatric epidemiology: It’s not just aboutcounting anymore. Arch Gen Psychiatry 62: 590 –592.
Ito W, Pan BX, Yang C, Thakur S, Morozov A. 2009. Enhanced
generalization of auditory conditioned fear in juvenile mice. Learn Mem
16: 187 –192.
Kaffman A, Krystal JH. 2012. New frontiers in animal research of psychiatry
illness. Methods Mol Biol 829: 3 –30.
Kessler RC, Amminger GP, Aguilar-Gaxiola S, Alonso J, Lee S, Ustun TB.
2007. Age of onset of mental disorders: A review of recent literature.
Curr Opin Psychiatry 20: 359 – 364.
Kim JH, Richardson R. 2007. A developmental dissociation of context
and GABA effects on extinguished fear in rats. Behav Neurosci 121:
131 –139.

Memory persistenceMice were trained on P15, P30, or P60 as previously described.
Separate groups of mice were returned to the chambers 1, 7, 14,
or 28 d after training for a 5-min test. Additional groups of mice
were either trained or remained in their home cage on P15 andthen underwent retraining starting on P60. Retraining occurred
across 4 d. On days 1 –3, mice received a single foot shock (2 sec,
0.4 mA) 2 min after being returned to the chambers and were removed 1 min after the shock. On day 4, mice were returned tothe chambers for 2 min with no foot shock. Freezing was measured before the shock on each retraining day.

Statistical analysis
Data were analyzed using ANOVA. For the memory formation andmemory savings experiments, age (P13, P14, P15, P16, P17, P30,
P60), shock (shock, no shock), sex (male, female), or training(trained, naive) were between-subject factors, and minute or day
was a within-subject factor. Following significant interactions, uncorrected t-tests were performed to elucidate the locus of the effects. For the memory specificity and memory persistenceexperiments, age (P15, P30, P60), sex (male, female), context (A,B, C), or delay (1, 7, 14, 28) were between-subject factors.
Following significant interactions or main effects, Tukey’s posthoc tests were performed. For the developmental delay experiment, age (P13, P14, P15), shock (shock, no shock), sex (male, female), and EtOH (control, FAE) were between-subject factors, andminute was a within-subject factor. Planned, uncorrected t-tests
were performed to test for differences between control and FAE
groups at each age. Furthermore, uncorrected t-tests were performed following significant interactions to determine the locusof the effects. Complete results of statistical analyses are presented
in Supplemental Tables S1–S5.

Acknowledgments
This work was supported by grants from the Canadian Institutesof Health Research to P.W.F. (MOP86762) and S.A.J. (MOP74650).
K.G.A. and M.A.-C. were supported by a Canadian Institutes of
Health Research Postdoctoral Fellowship and a Hunter Graduate
Scholarship, respectively.

References
Abbey H, Howard E. 1972. Statistical procedure in developmental studies
on species with multiple offspring. Dev Psychobiol 6: 329 – 335.
Akers KG, Kushner SA, Leslie AT, Clarke L, van der Kooy D, Lerch JP,
Frankland PW. 2011. Fetal alcohol exposure leads to abnormal
olfactory bulb development and impaired odor discrimination in adultmice. Mol Brain 4: 29. doi: 10.1186/1756-6606-4-29.
Allan AM, Chynoweth J, Tyler LA, Caldwell KK. 2003. A mouse model ofprenatal ethanol exposure using a voluntary drinking paradigm.
Alcohol Clin Exp Res 27: 2009 – 2016.
Altman J, Bayer S. 1975. Postnatal development of the hippocampal
dentate gyrus under normal and experimental conditions. In The
hippocampus (ed. RL Isaacson, KH Pribham), pp. 95 – 122. Plenum,
New York.
Anagnostaras SG, Maren S, Fanselow MS. 1999. Temporally gradedretrograde amnesia of contextual fear after hippocampal damage in
rats: Within-subjects examination. J Neurosci 19: 1106 –1114.
Anagnostaras SG, Gale GD, Fanselow MS. 2001. Hippocampus andcontextual fear conditioning: Recent controversies and advances.
Hippocampus 11: 8 –17.
Anderson MJ, Riccio DC. 2005. Ontogenetic forgetting of stimulus
attributes. Learn Behav 33: 444 – 453.

www.learnmem.org

603

Learning & Memory

Ontogeny of contextual fear memory in mice

Rudy JW. 2009. Context representations, context functions, andthe parahippocampal-hippocampal system. Learn Mem
16: 573 –585.
Rudy JW, Morledge P. 1994. Ontogeny of contextual fear conditioning
in rats: Implications for consolidation, infantile amnesia, andhippocampal system function. Behav Neurosci 108: 227 –234.
Rudy JW, Huff NC, Matus-Amat P. 2004. Understanding contextual fear
conditioning: Insights from a two-process model. Neurosci Biobehav Rev
28: 675 –685.
Sagvolden T, Johansen EB, Aase H, Russell VA. 2005. A dynamic
developmental theory of attention-deficit/hyperactivity disorder(ADHD) predominately hyperactive/impulsive and combined
subtypes. Behav Brain Sci 28: 397– 419.
Schiffino FL, Murawski NJ, Rosen JB, Stanton ME. 2011. Ontogeny andneural substrates of the context preexposure facilitation effect.
Neurobiol Learn Mem 95: 190 –198.
Schneider ML, Moore CF, Adkins MM. 2011. The effects of prenatal alcohol
exposure on behavior: Rodent and primate studies. Neuropsychological
Reviews 21: 186 –203.
Scott RC, Richard GR, Holmes GL, Lenck-Santini PP. 2011. Maturational
dynamics of hippocampal place cells in immature rats. Hippocampus
21: 347 –353.
Spear NE. 1979. Experimental analysis of infantile amnesia. In Functional
disorders of memory (ed. JF Kihlstrom, FJ Evans), pp. 75 –102. Erlbaum,
Hillsdale, NJ.
Sullivan RM, Wilson DA, Feldon J, Yee BK, Meyer U, Richter-Levin G, Avi A,
Michael T, Gruss M, Bock J, et al. 2006. The International Society for
Developmental Psychobiology annual meeting symposium: Impact ofearly life experiences on brain and behavioral development. Dev
Psychobiol 48: 583 –602.
van Eden CG, Kros JM, Uylings HB. 1990. The development of the rat
prefrontal cortex. Its size and development of connections withthalamus, spinal cord and other cortical areas. Prog Brain Res 85:
169 – 183.
Wang S-H, Teixeira CM, Wheeler AL, Frankland PW. 2009. The precision of
remote context memories does not require the hippocampus. Nat
Neurosci 12: 253 –255.
Weber M, McNally GP, Richardson R. 2006. Opioid receptors regulate
retrieval of infant fear memories: Effects of naloxone on infantileamnesia. Behav Neurosci 120: 702 –709.
Whishaw IQ, Metz GA, Kolb B, Pellis SM. 2001. Accelerated nervous systemdevelopment contributes to behavioral efficiency in the laboratory
mouse: A behavioral review and theoretical proposal. Dev Psychobiol
39: 151 –170.
Wigal T, Lobaugh NJ, Wigal SB, Greene PL, Amsel A. 1988. Sparing of
patterned alternation but not partial reinforcement extinction effectafter prenatal chronic exposure to ethanol in infant rats. Behav Neurosci
102: 43 –50.
Wills TJ, Cacucci F, Burgess N, O’Keefe J. 2010. Development of the
hippocampal cognitive map in preweanling rats. Science 328:
1573 –1576.
Wiltgen BJ, Sanders MJ, Anagnostaras SG, Sage JR, Fanselow MS. 2006.
Context fear learning in the absence of the hippocampus. J Neurosci
26: 5484 –5491.
Woodcock EA, Richardson R. 2000. Effects of multisensory environmental
stimulation on contextual conditioning in the developing rat.
Neurobiol Learn Mem 74: 89 –104.
Yap CSL, Richardson R. 2005. Latent inhibition in the developing rat:
An examination of context-specific effects. Dev Psychobiol 47: 55 –65.

Kirstein CL, Philpot RM, Dark T. 1997. Fetal alcohol syndrome: Early
olfactory learning as a model system to study neurobehavioral deficits.
Int J Neurosci 89: 119 –132.
Langston RF, Ainge JA, Couey JJ, Canto CB, Bjerknes TL, Witter MP,
Moser EI, Moser MB. 2010. Development of the spatial representation
system in the rat. Science 328: 1576 –1580.
Lehmann H, Lacanilao S, Sutherland RJ. 2007. Complete or partial
hippocampal damage produces equivalent retrograde amnesia forremote contextual fear memories. Eur J Neurosci 25: 1278 – 1286.
Marco EM, Macri S, Laviola G. 2011. Critical age windows forneurodevelopmental psychiatric disorders: Evidence from animalmodels. Neurotoxicology Research 19: 286 –307.
Maren S, Aharonov G, Fanselow MS. 1997. Neurotoxic lesions of the dorsal
hippocampus and Pavlovian fear conditioning in rats. Behav Brain Res
88: 261 – 274.
McClelland S, Korosi A, Cope J, Ivy A, Baram TZ. 2011. Emerging rolesof epigenetic mechanisms in the enduring effects of early-life stressand experience on learning and memory. Neurobiol Learn Mem 96:
79 –88.
McGorry PD, Purcell R, Goldstone S, Amminger GP. 2011. Age of onset andtiming of treatment for mental and substance use disorders:
Implications for preventive intervention strategies and models of care.
Curr Opin Psychiatry 24: 301 –306.
Mennella J. 2001. Alcohol’s effect on lactation. Alcohol Res Health 25:
230 –234.
Murawski NJ, Stanton ME. 2010. Variants of contextual fear
conditioning are differentially impaired in the juvenile rat bybinge ethanol exposure on postnatal days 4 –9. Behav Brain Res 212:
133 –142.
Pattwell SS, Bath KG, Casey BJ, Ninan I, Lee FS. 2011. Selective
early-acquired fear memories undergo temporary suppression duringadolescence. Proc Natl Acad Sci 108: 1182 –1187.
Paylor R, Baskall-Baldini L, Yuva L, Wehner JM. 1996. Developmental
differences in place-learning performance between C57BL/6 and DBA/
2 mice parallel the ontogeny of hippocampal protein kinase C. Behav
Neurosci 110: 1415 –1425.
Pellis SM, Iwaniuk AN. 2000. Comparative analyses of the role of postnatal
development on the expression of play fighting. Dev Psychobiol 36:
136 –147.
Pine DS. 2009. Integrating research on development and fear learning: A
vision for clinical neuroscience? Depress Anxiety 26: 775 –779.
Pisano MV, Ferreras S, Krapacher FA, Paglini G, Arias C. 2012. Reexamining the ontogeny of the context preexposure facilitation effectin the rat through multiple dependent variables. Behav Brain Res 233:
176 –190.
Pugh CR, Fleshner M, Rudy JW. 1997. Type II glucocorticoid receptor
antagonists impair contextual but not auditory-cue fear conditioningin juvenile rats. Neurobiol Learn Mem 67: 75 – 79.
Rahimi O, Claiborne BJ. 2007. Morphological development andmaturation of granule neuron dendrites in the rat dentate gyrus. Prog
Brain Res 163: 167 –181.
Raineki C, Holman PJ, Debiec J, Bugg M, Beasley A, Sullivan RM. 2010.
Functional emergence of the hippocampus in context fear learning ininfant rats. Hippocampus 20: 1037 –1046.
Richardson R, Riccio DC, Jonke T. 1983. Alleviation of infantile amnesia inrats by means of a pharmacological contextual state. Dev Psychobiol 16:
511 –518.
Riley EP, Infante MA, Warren KR. 2011. Fetal alcohol spectrum disorders:An overview. Neuropsychological Reviews 21: 73 –80.
Rudy JW. 1993. Contextual conditioning and auditory cue
conditioning dissociate during development. Behav Neurosci 107:
887 –891.

www.learnmem.org

Received June 27, 2012; accepted in revised form August 31, 2012.

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Learning & Memory