The Journal of Neuroscience, March 2, 2011 • 31(9):3169 –3175 • 3169

Behavioral/Systems/Cognitive

Memory for the Order of Events in Specific Sequences:

Contributions of the Hippocampus and Medial Prefrontal
Cortex
Loren M. DeVito and Howard Eichenbaum
Center for Memory and Brain, Boston University, Boston, Massachusetts 02215

Episodic memory involves remembering the incidental order of a series of events that comprise a specific experience. Current models of
temporal organization in episodic memory have demonstrated that animals can make memory judgments about the order of serially
presented events; however, in these protocols, the animals can judge items based on their relative recency. Thus, it remains unclear as to
whether animals use the specific order of items in forming memories of distinct sequences. To resolve this important issue in memory
representation, we presented mice repeatedly with two widely separated odor sequences and then tested their natural exploratory
preference between pairs of odors selected from within or between sequences. Intact animals preferred to investigate odors that occurred
earlier within each sequence, indicating they did remember the order of events within each distinct sequence. In contrast, intact animals
did not discriminate between pairs of odors from different sequences. These findings indicate that preferences were not guided by relative
recency, which would be expected to support graded discrimination between widely separated events. Furthermore, damage to either the
hippocampus or the medial prefrontal cortex eliminated order preference within sequences. Despite the deficit in order memory, control
recognition tests showed that normal mice and mice with hippocampal or medial prefrontal damage could correctly identify previously
experienced odors compared with novel odors. These findings provide strong evidence that animals form representations of the order of
events within specific experiences and that the hippocampus and prefrontal cortex are essential to order memory.

Introduction Experiments using these protocols have indicated that ani-

Episodic memory requires the binding of sequential experi- mals can judge the order of previously experienced events. How-
ences, such that memories are organized by the order of oc- ever, in these tasks, accurate performance may be based on
currence of serial events (Tulving and Markowitsch, 1998). judgments about relative recency of serial experiences, perhaps
Memory for sequential events structures the flow of our daily by using a comparison of differential memory strengths for more
life, and the ability to integrate events that occur across differ- and less recently experienced events. Indeed, one of these studies
ent points in time is a crucial requisite for the later retrieval showed that the memory strengths of serially presented odors did
of experiences (Clayton and Dickinson, 1998; Eichenbaum, differ according to their recency (Fortin et al., 2002). Therefore,
2004). A few studies have explored memory for sequential these studies do not provide definitive evidence that animals
events in specific episodes (Chiba et al., 1997; Fortin et al., solve these tasks using a direct representation of the order of
2002; Kesner et al., 2002). Fortin et al. (2002) and Kesner et al. events within each specific experience.
(2002) presented rats with unique sequences of five odors and To address this important issue, we developed a novel behav-
trained them to judge the earlier presented of two selected ioral protocol that, as in natural episodic memory, assesses the
odors, such that animals gradually learned the rule to choose ability of animals to remember the order of sequential events in
an item that had been experienced less recently over one that specific experiences without explicit training on order judg-
was experienced more recently. Memory for sequential events ments. Our design involved a large separation between exposure
has also been evaluated using an object recognition paradigm to multiple sequences and subsequent memory testing on those
that exploits animals’ natural (untrained) tendency to explore sequences, to reduce relative differences in memory strengths
a less recently experienced object compared with a more re- among the events. Furthermore, we compared performance on
cently experienced object (Dere et al., 2005). different tests that measure memory for order within a particular
sequence versus memory for the relative recency of items that
Received Aug. 11, 2010; revised Dec. 22, 2010; accepted Dec. 29, 2010.
occurred at different times within each day.
This study was supported by the Conte Center for Schizophrenia Research/National Institutes of Health Grant Previous experiments that have investigated the neural sub-
MH60450. Thanks to Christine Lykken for assistance with processing and analyzing histology. Parts of this paper strates of sequence memory have indicated that both the hip-
have been published previously at the Society for Neuroscience 2009 meeting. pocampus and prefrontal cortex play an important role (Mitchell
Correspondence should be addressed to Dr. Howard Eichenbaum, Center for Memory and Brain, Boston Univer-
sity, Boston, MA 02215. E-mail: [email protected].
and Laiacona, 1998; Agster et al., 2002; Fortin et al., 2002; Barker
DOI:10.1523/JNEUROSCI.4202-10.2011 et al., 2007). Therefore, we evaluated the effects of selective le-
Copyright © 2011 the authors 0270-6474/11/313169-07$15.00/0 sions of the hippocampus or prefrontal cortex using a newly de-
3170 • J. Neurosci., March 2, 2011 • 31(9):3169 –3175 DeVito and Eichenbaum • Hippocampal–Prefrontal Circuitry Supports Sequence Memory

veloped protocol that distinguishes memory for temporal A

organization in specific sequences of events, memory for the rel-
ative recency of events that are experienced across different se-
quences, and memory for the identity of items that appear in
these sequences.

Materials and Methods

Subjects. Male C57BL/6 mice were purchased from the Charles River
Laboratory. All animals were maintained on a reverse 12 h light/dark
cycle (lights off at 9:00 A.M., lights on at 9:00 P.M.). Animals were given
ad libitum access to food and water, unless otherwise specified in the
behavioral methods. Sixty animals were used in this study: 24 served as
controls for verification of the task, 8 animals received lesions of the
hippocampus, 10 animals received lesions of the medial prefrontal cor-
tex, 8 served as sham-operated medial prefrontal controls, and 10 served B
as sham-operated hippocampal controls. The Institutional Animal Care
and Use Committee of Boston University approved the treatment and
use of the animals in these experiments.
Surgery. Bilateral lesions of the hippocampus were made using NMDA
(10 mg/ml; Sigma) or sterile PBS for sham operations, delivered via a
microinfusion pump connected to a 5 ␮l Hamilton syringe. Animals
were anesthetized with a ketamine/xylazine mixture (0.01 ml/g), and
diazepam (0.02 ml) was administered preoperatively to prevent seizures.
After the animal had been placed into a stereotaxic head frame, the skull Figure 1. Odor sequence task. A, Schematic of the odors and testing apparatus used in odor
was exposed and the coordinates of bregma were measured. The skull presentation. B, Probe tests used to assess memory for sequential order.
overlying the four coordinates was drilled and dura was removed. Before
infusions were made, the syringe was lowered 0.2 mm for the first two
coordinates (dorsal hippocampus) and 0.5 mm for the last two coordi- restriction and maintained at 85% of free-feeding weight. Over a 3 d
nates (ventral hippocampus) past the injection site and kept a lower period, animals were allowed to dig for chocolate sprinkle rewards buried
depth for 1 min to increase spread of drug diffusion. The syringe was then in sand that filled small plastic cups in a Plexiglas mouse cage (26 cm
raised to the injection site, and the drug was infused over a 2 min period length ⫻ 15 cm width ⫻ 13 cm height) (Fig. 1 A). Once they were reliably
(3 min infusion for the last coordinate). The needle was left in place for digging, the animals were exposed to two different odor sequences each
another 5 min before being slowly withdrawn. The complete dorsal and day over a 5 d period. All odors are prepared at 1% concentration by
ventral hippocampus was targeted (including the CA fields, dentate weight in sand.
gyrus, and subiculum) at four stereotaxic coordinates: anteroposterior Sequence 1, with the odors referred to as A, B, C, D, and E, consisted of
(AP) ⫹1.7, mediolateral (ML) ⫾1.2, dorsoventral (DV) ⫺1.5; AP ⫹2.3, the following odors: A, cilantro (Spice Islands Trading Company); B,
ML ⫾1.75, DV ⫺1.75; AP ⫹2.8, ML ⫾3, DV ⫺3; AP ⫹3.1, ML ⫾2.85, marjoram (Frontier Natural Products Co-op); C, dill (Frontier Natural
DV ⫺3.75. Fifty nanoliters was infused into the first three sites, and 75 nl Products); D, parsley (Frontier Natural Products Co-op); and E, allspice
was infused into the fourth site. Animals that received sham operations (Spice Islands Trading Company). Sequence 2, referred to as L, M, N, O,
were administered the same procedures as the lesion groups, except and P, consisted of the following odors: L, lemon (McCormick); M,
that PBS was infused into each site instead of NMDA. cinnamon (McCormick); N, tarragon (Frontier Natural Products Co-
Bilateral lesions of the medial prefrontal cortex were made using ibo- op); O, anise (McCormick), and P, orange (McCormick) (Fig. 1 A).
tenic acid (0.06 M; Tocris Cookson), or sterile saline for sham operations, In the first session each day, the animal was presented with sequence 1.
delivered via a microinfusion pump connected to a 10 ␮l Hamilton At the beginning of each sequence, the animal was presented with the first
syringe attached to a pulled microglass pipette tip. Animals were anes- odor, which contained a buried chocolate sprinkle that the mouse could
thetized with a ketamine/xylazine mixture (0.01 ml/g), and diazepam obtain by digging in the sand (Fig. 1 A). This process was repeated for the
(0.02 ml) was administered preoperatively to prevent seizures. After the remaining four odors in that sequence until the animal had been exposed
animal had been placed into a stereotaxic head frame, the skull was to all five odors successively. Each animal was presented with sequence 1
exposed and the coordinates of bregma were measured (the mediolateral three times. Three hours after the first session was completed, the animals
values were taken at the level of the midsagittal vein and not at bregma). were exposed to sequence 2 three times in the same manner. This proce-
The skull overlying the two coordinates was drilled and dura was re- dure for sequence presentation was repeated for 5 d. After these 5 d, the
moved. The syringe was lowered to the injection site, and the drug was animals were not subsequently exposed to the odor sequences.
infused over a 5 min period. The needle was left in place for another 5 min Beginning the next day, the animals were administered probe tests that
before being slowly withdrawn. The infralimbic and prelimbic cortices assessed memory for the odors within and between each of the sequences
were targeted at two stereotaxic coordinates: AP ⫺2.1, ML ⫾0.25, DV (Fig. 1 B). On each of these probe tests, two odors were selected, and both
⫺2.3. One hundred fifty nanoliters were infused into both sites. Animals were presented without buried rewards. We measured the amount of
that received sham operations were administered the same procedures as time the animals spent digging in each cup, and the two digging times
the lesion groups, except that PBS was infused into each site instead of were used to calculate a preference index (PI) (Bunsey and Eichenbaum,
NMDA. 1996); PI ⫽ (time digging in the odor that occurred earlier in the se-
After all infusions, the scalp was sutured, the animal was given 0.4 ml quence ⫺ time spent digging in the odor that occurred later in the se-
of lactated Ringer’s solution to hydrate, and the animal was placed next to quence)/total time digging in both odors.
a hot water bottle to return body temperature to normal. After surgery, On the first two days of probe testing (days 6 and 7), the animals were
the animal received Children’s Tylenol in its water and was provided with presented with odors selected from the same sequence in which they were
soft food and Nutrical. Each animal was allowed 2 weeks to recover presented earlier. These were considered “within-sequence” probe tests
before behavioral testing. because they evaluated the ability of the animals to remember the order
Behavioral methods. Mice were tested on a novel protocol that assessed of odors that were presented within one of the specific sequences. On the
the ability to remember the order of odors presented within specific first day of probe testing (day 6), the animals were presented with L
sequences or across different sequences. Animals were placed on food versus P, C versus D, and M versus O. On the second day of probe testing
DeVito and Eichenbaum • Hippocampal–Prefrontal Circuitry Supports Sequence Memory J. Neurosci., March 2, 2011 • 31(9):3169 –3175 • 3171

(day 7), the animals were presented with A versus D, N versus P, and B memory for items within sequence 1 and sequence 2, the opposite order
versus C. as the groups described above. After the sequence probes, both groups
On the third day of probe testing (day 8), the animals were presented were tested on novelty recognition probe trials.
with pairs consisting of one odor from sequence 1 and one odor from Histology. After behavioral testing, all animals were given an overdose
sequence 2. These were considered “between-sequence” probe tests be- of sodium pentobarbital and perfused transcardially with 4% Formalin.
cause they evaluated the ability of the animals to compare the times at The brains were removed and postfixed for 1 h in Formalin, and then
which the odors were presented over the course of each day, i.e., earlier in cryoprotected in 30% sucrose solution, in pH 7.4 PBS. Coronal sections
sequence 1 versus 3 h later in sequence 2. The animals were presented were cut (40 ␮m) using a freezing microtome. Every section was mounted on
with A versus N, E versus O, and B versus L. gelatin-coated slides and dried overnight. Slides were soaked in xylenes and
Finally, on the fourth day (day 9), the animals were presented with an then run through a series of ethanol dehydrations, stained with cresyl violet,
odor from either sequence against a novel odor they had never experi- and then rehydrated. The extent of the lesion was determined using a light
enced. These were considered “novelty” probe tests because they evalu- microscope to study the stained sections.
ated the ability of the animals to recognize novel compared with familiar Four representative sections along the anteroposterior axis of the hip-
odors. The animals were presented with L versus caraway (McCormick), pocampus (AP ⫹1.7, 2.3, 2.8, and 3.08) were selected from the mouse
C versus mace (McCormick), and M versus sage (McCormick). After a brain atlas to determine hippocampal damage (Franklin and Paxinos,
3 h delay, the animals were presented with A versus baby powder (Shaw’s 1997). For each hippocampal lesion animal, the four brain slices most
Supermarkets), N versus onion (McCormick), and B versus strawberry closely corresponding to the representative sections in the brain atlas
(Nestlé). were used. Three representative sections along the anteroposterior axis of
Cohorts of four mice, containing two sham and two lesioned mice, the medial prefrontal cortex (AP ⫺2.58, 2.1, and 1.7) were selected from
were trained and tested within each 9 d consecutive testing sequence, the mouse brain atlas to determine prefrontal damage (Franklin and
allowing us to ensure that each animal received a 3 min delay between Paxinos, 1997). For each prefrontal lesion animal, the three sections most
each of the three sequence presentations of sequence 1 in the first session closely corresponding to the representative sections in the brain atlas
of the day and sequence 2 in the second session of the day. For each were used. Canvas 5.0 (Deneba Software; ACD Systems International)
cohort, training on sequence 1 began at 12:00 P.M., and training on was used to calculate the percentage of tissue damage, which was defined
sequence 2 began at 3:00 P.M. The presentation of each odor was done as as the amount of total damage divided by the total area of the brain in that
rapidly as possible such that, for each odor, the animal was allowed to section ⫻ 100; the average of those values represented the lesion extent.
enter the testing area, dig in the scented cup, retrieve the reward, and Additional sections were studied under the light microscope to identify
immediately exit, with ⬃3 s between odor presentations during which incidental damage outside the targeted regions and were reported in
the investigator switched the scented cups. Results.
A cardboard divider was used to separate the compartment into two
areas of equal width, a testing compartment and a waiting compartment.
At the start of each session, the animal would be placed into the waiting Results
compartment and a trial would be initiated by lifting the divider, allow- Histology
ing the animal to run into the testing compartment. After each trial, the In two animals, damage to the hippocampus was minimal, and
divider would again be lifted by the experimenter, allowing the animal to these animals were removed from additional analyses. Otherwise,
run back into the other compartment and wait for the next trial to be the NMDA infusions generally resulted in a substantial loss of
initiated. The apparatus was also covered to prevent mice from escaping cells within the hippocampal formation, including Ammon’s
the test box during the session. horn, the dentate gyrus, and the subiculum (Fig. 2 A). An average
During probe trial sessions, each animal was allowed to dig freely in of 45% of the hippocampus was damaged across animals, ranging
each cup; neither of the cups was baited. The animal was always presented
from 8 to 77% total. The size of the lesion was not correlated with
with the cups in six possible locations within the apparatus during the
first 5 d to prevent the development of spatial biases. The positions of the the extent of the behavioral deficit found (r(8) ⫽ ⫺03.92, p ⫽
cups were randomized each day for all trials and were different for each 0.337). Two animals had partial damage to the thalamus. There
mouse. On days in which probe trials were administered, the locations of was also incidental damage to multiple cortical areas above
the cups were pseudorandomly selected so that each mouse experienced the infusion sites in both sham and hippocampal animals. The
the cups in different locations and cup location was not repeated amount of damage in each cortical area is as follows: somatosen-
throughout the session. The time spent digging in each cup was recorded sory cortex (10.8% in sham group, 18.88% in hippocampal lesion
using a stopwatch, and these times were later used to calculate preference group; t(10) ⫽ ⫺1.892, p ⫽ 0.088), motor cortex (16.01% in sham
indices. To prevent digging behavior from extinguishing, immediately group, 16.92% in hippocampal lesion group; t(10) ⫽ ⫺0.07, p ⫽
after each probe trial, the animal was presented with a plain cup of sand 0.945), retrosplenial cortex (25.26% in sham group, 21.75% in
(with no odor) with chocolate sprinkles that the animal was allowed to
hippocampal lesion group; t(10) ⫽ 0.572, p ⫽ 0.580), parietal
eat. With the exception of the novelty probe trials on day 4 of testing in
which each animal received six probe trials, each animal received three
cortex (14.91% in sham group, 29.9% in hippocampal lesion
probe trials per day. group; t(10) ⫽ ⫺0.2337, p ⫽ 0.042), and visual cortex (8.92% in
To determine whether the preferences reported in the results could sham group, 27.54% in hippocampal lesion group; t(10) ⫽
have been attributable to innate biases toward or against particular ⫺4.130, p ⫽ 0.033). Although the damage in the parietal and
odors, a separate group of eight naive mice were shaped to dig in cups, visual cortices was significantly greater in the hippocampal lesion
just as the trained groups described above. Then, each animal was imme- group compared with the sham group, it is unlikely that slightly
diately administered the same set of probe trials without exposure to the greater damage to these particular cortical areas could account
sequences. Furthermore, to account for potential differences in perfor- for the pattern of impaired memory for odor order and spared
mance on within-sequence and between-sequence probes attributable to odor recognition in this odor guided task.
the order in which these probe trials were administered, two additional Ibotenic acid infusions resulted in a substantial loss of cells
separate groups of eight mice were shaped to dig in cups then exposed
within the medial wall of the prefrontal cortex, including the
repeatedly to the sequences as described above. One group was tested on
their memory for items within sequence 1 and sequence 2 and then tested prelimbic and infralimbic cortices and dorsal cingulate gyrus
on their memory for items between sequence 1 and sequence 2, the same (Fig. 2 B). An average of 43% of the medial prefrontal cortex was
order in which the probe trials were administered to the mice in the damaged across all animals, ranging from 12 to 81%. Two ani-
experiment described above. Another group was first tested on memory mals had additional damage to the medial orbital cortex, and two
for items between sequence 1 and sequence 2 and then tested on their animals had unilateral damage to the ventral and lateral orbital
3172 • J. Neurosci., March 2, 2011 • 31(9):3169 –3175 DeVito and Eichenbaum • Hippocampal–Prefrontal Circuitry Supports Sequence Memory

cortices. The size of the lesion was not cor-

related with the extent of the behavioral
deficit found (r(9) ⫽ ⫺0.46, p ⫽ 0.172).
There were no significant differences
in performance between sham-operated
groups in which the infusions targeted
the hippocampus and prefrontal cortex
(“within-sequence” probe tests, t(16) ⫽
⫺1.284, p ⫽ 0.218; “between-sequence”
probe tests, t(16) ⫽ 0.897, p ⫽ 0.383; “nov-
elty” probe tests, t(14) ⫽ ⫺0.256, p ⫽
0.801); therefore, these two groups were
combined and are collectively described
as the “sham” group.

Memory for items within each

specific sequence
Intact and sham-operated mice prefer to
explore items that they had experienced
earlier within each sequence, despite not
being explicitly trained to judge the order
of the items (sham-operated controls, t(17)
⫽ 6.637, p ⬍ 0.001; intact mice adminis-
tered within-sequence probe trials first,
t(7) ⫽ 5.553, p ⬍ 0.001; intact mice ad-
ministered between-sequence trials first,
t(7) ⫽ 3.556, p ⫽ 0.003) (Fig. 3 A, D).
These results indicate that sham-operated
and intact mice remember the order in
which they had experienced these se-
quences of odors. Importantly, the order
in which probe trials were administered
did not affect performance because mice
given probe trials in opposite orders after Figure 2. Histological verification of the extent of hippocampal and medial prefrontal damage. A, A diagram shows the extent
training performed at equivalent levels on of the largest (gray) and smallest (black) hippocampal lesion across the 10 animals. B, A diagram shows the extent of the largest
each type of probe trials (within-sequence (gray) and smallest (black) medial prefrontal lesion across the 10 animals.
probe trials, t(14) ⫽ ⫺0.388, p ⫽ 0.704;
between-sequence probe trials, t(14) ⫽ 1.371, p ⫽ 0.192; novelty 0.103) did not exhibit preference scores that differed significantly
recognition probe trials, t(14) ⫽ ⫺0.624, p ⫽ 0.543). Further- from zero.
more, without previous training on the sequences, naive mice did
not exhibit a significant preference for the items occurring earlier Memory for order at different temporal lags between items
within each sequence or between sequences; their scores accord- within each sequence
ingly did not differ from chance (within-sequence probe trials, Six probe tests involving different odor comparisons were used to
t(7) ⫽ ⫺0.540, p ⫽ 0.598; between-sequence probe trials, t(7) ⫽ assess memory for odors within each sequence. These probe tests
⫺0.185, p ⫽ 0.85). Additionally, the scores of both groups of can further be segregated based on their temporal lags, that is, the
intact mice were significantly higher than the scores of the naive number of items that occurred between odors when they were
mice (one-way ANOVA, F(2,23) ⫽ 6.851, p ⫽ 0.005; intact mice presented in the original sequences. We measured performance
at four different lags, and our analyses separated these into
administered within-sequence probe trials first, p ⫽ 0.007; intact
“short” (0 –1) and “long” (2–3) lags.
mice administered between-sequence trials first, p ⫽ 0.003) (Fig.
The three groups differed significantly in preference indices
3D), indicating that the 5 d odor sequence presentations resulted
between short and long lags (two-way ANOVA, group, F(2,71) ⫽
in significant memory for the specific items in each sequence and 15.765, p ⬍ 0.001; lag, F(1,71) ⫽ 7.142, p ⫽ 0.009; group ⫻ lag
does not reflect innate preferences. interaction, F(2,71) ⫽ 0.837, p ⫽ 0.438) (Fig. 3A). The group dif-
Sham-operated subjects showed significantly greater preference ference was highly significant at short lags (one-way ANOVA,
indices than the operated groups (one-way ANOVA, F(2,35) ⫽ F(2,35) ⫽ 13.113, p ⬍ 0.001), and post hoc tests indicated that
17.008, p ⬍ 0.001) (Fig. 3A). Post hoc comparisons indicated that animals with hippocampal damage ( p ⫽ 0.001), as well as ani-
both animals with hippocampal damage ( p ⫽ 0.001) and animals mals with prefrontal damage ( p ⬍ 0.001), had lower preference
with prefrontal damage ( p ⬍ 0.001) had lower preference scores scores than sham-operated animals, but hippocampal and pre-
than sham-operated animals, and the scores of animals with hip- frontal groups did not differ from each other ( p ⫽ 0.733). At long
pocampal lesions did not differ from those of the animals with lags, there was also a significant difference among the three
medial prefrontal lesions ( p ⫽ 0.284). Moreover, animals with groups (one-way ANOVA, F(2,35) ⫽ 4.429, p ⫽ 0.02), and post hoc
damage to the hippocampus (t(7) ⫽ 0.128, p ⫽ 0.900) and those tests indicated that animals with prefrontal damage ( p ⫽ 0.024)
with damage to the medial prefrontal cortex (t(9) ⫽ ⫺1.715, p ⫽ and animals with hippocampal damage ( p ⫽ 0.017) had signifi-
DeVito and Eichenbaum • Hippocampal–Prefrontal Circuitry Supports Sequence Memory J. Neurosci., March 2, 2011 • 31(9):3169 –3175 • 3173

A pal lesion animals (t(14) ⫽ ⫺1.382, p ⫽ 0.189) did not differ in

performance between the short versus long lags, prefrontal lesion
animals (t(18) ⫽ 2.836, p ⫽ 0.011) exhibited higher preference
scores on long versus short lags.

Memory for items across sequences

To determine whether animals distinguished odors that appeared
at widely separated times on the days in which sequences were
presented, we administered probe tests comparing an odor
from sequence 1 with an odor from sequence 2. Neither sham-
B C operated mice (t(17) ⫽ ⫺0.795, p ⫽ 0.432) nor animals with
hippocampal (t(7) ⫽ 0.098, p ⫽ 0.923) or prefrontal (t(9) ⫽ 0.219,
p ⫽ 0.829) lesions exhibited any sign of preference or had pref-
erence indices that differed from zero (Fig. 3B). Accordingly,
there was also no group difference (one-way ANOVA, F(2,35) ⫽
0.26, p ⫽ 0.773). Groups of intact mice also did not exhibit pref-
erences for items in sequence 1 compared with items in sequence
2 (mice administered within-sequence probe trials first, t(7) ⫽
D 1.349, p ⫽ 0.19; mice administered between-sequence trials first,
t(7) ⫽ ⫺0.658, p ⫽ 0.52). These findings suggest that the prefer-
ences exhibited on the within-sequence probe tests did not sim-
ply reflect greater interest in a less recently presented odor over a
more recently presented odor on each day.

Recognition of novel odors

Animals with damage to the hippocampus or medial prefrontal
cortex may have exhibited impairment on the probe tests for
order memory secondary to an inability to remember the previ-
Figure 3. Performance of all three groups across the probe tests. A, Memory for the items ously experienced odors. To examine this possibility, we admin-
was assessed by using a preference index (⫾SEM). Animals with damage to the hippocampus
istered novelty recognition tests, probe tests in which the animals
or prefrontal cortex do not exhibit significant memory for items occurring earlier within each
sequence; in addition, mice with lesions of the prefrontal cortex and hippocampus are impaired
were presented with an odor from sequence 1 or sequence 2
at all inner-item temporal lags relative to control mice. B, Mice do not exhibit a significant against a novel odor that was not presented in either sequence. All
preference for items occurring in sequence 1 compared with sequence 2 (preference index ⫾ three groups showed significant preferences for the novel odor
SEM). C, All three groups exhibit a significant preference for novel versus familiar odors (pref- (sham-operated mice, t(17) ⫽ 10.227, p ⬍ 0.001; hippocampal
erence index ⫾ SEM). D, Intact mice administered sequence presentations across 5 d exhibited lesion mice, t(5) ⫽ 3.549, p ⫽ 0.005; medial prefrontal lesion
significant preferences for items occurring earlier within each series, but naive mice never mice, t(9) ⫽ 6.17, p ⬍ 0.001), and there was no difference in
exposed to these odors did not exhibit these preferences. The scores of the intact mice were preference scores across the three groups (one-way ANOVA,
significantly different from those of the naive mice, but both groups of intact mice demon- F(2,31) ⫽ 1.365, p ⫽ 0.271) (Fig. 3C). Groups of intact mice also
strated equivalent levels of preference regardless of the order in which they received the probe demonstrated a significant preference for novel odors (mice ad-
trial types. SHAM, Sham-operated mice; HPX, hippocampal lesion mice; PFCX, medial prefrontal
ministered within-sequence probe trials first, t(7) ⫽ 8.377, p ⬍
lesion mice; NAIVE, mice never exposed to the odors before probe trials; WITHIN FIRST, intact
mice administered within-sequence probe trials before within-sequence probe trials; BETWEEN
0.001; mice administered between-sequence probe trials first,
FIRST, intact mice administered between-sequence probe trials before within-sequence probe t(7) ⫽ 5.845, p ⬍ 0.001). Therefore, the impairment in mem-
trials. *p ⬍ 0.05. ory for items within sequences is not attributable to the inabil-
ity to recognize the odors.
Finally, there was no difference across groups in the total amount
cantly lower preference scores than sham-operated animals; of time spent digging during probe tests for items within each se-
however, the hippocampal and prefrontal groups did not differ quence (one-way ANOVA, F(2,415) ⫽ 2.109, p ⫽ 0.123), between
from each other ( p ⫽ 0.757). each sequence (one-way ANOVA, F(2,207) ⫽ 1.2, p ⫽ 0.303), or on
Additional analyses of the direction of group preferences re- novelty judgments (one-way ANOVA, F(2,383) ⫽ 0.099, p ⫽ 0.906).
vealed a complex pattern of performance across short and long Therefore, differences in preferences on the probe tests were not
lags. The sham-operated group has a significant preference for secondary to differences in activity level or interest (Table 1).
earlier presented items at both short (t(17) ⫽ 4.341, p ⬍ 0.001)
and long (t(17) ⫽ 4.856, p ⬍ 0.001) lags. At long lags, the mean Discussion
scores of both lesion groups were above zero; this preference In introducing the distinction between episodic and semantic mem-
was not significant in animals with damage to the hippocam- ory, Tulving (1983) emphasized that episodic memories are charac-
pus (t(7) ⫽ 0.638, p ⫽ 0.534) or in animals with damage to the terized by the temporal organization of events within distinct
prefrontal cortex (t(9) ⫽ 1.256, p ⫽ 0.225). In contrast, at short experiences and contrasted this feature of episodic memory from the
lags, the mean scores of both lesion groups were below zero, absence of temporal structure in semantic memories. This experi-
indicating the opposite of normal preference; this effect was ment was designed to determine whether mice can form represen-
statistically significant in the prefrontal group (t(9) ⫽ ⫺3.136, p ⫽ tations of the temporal organization of events in distinct experiences.
0.006) but not the hippocampal group (t(7) ⫽ ⫺1.271, p ⫽ The results demonstrate for the first time that animals can indeed
0.225). However, additional analyses revealed that, whereas remember the order of events in specific sequential experiences
sham-operated animals (t(34) ⫽ 0.822, p ⫽ 0.417) and hippocam- without previous training on an order rule. Mice showed strong
3174 • J. Neurosci., March 2, 2011 • 31(9):3169 –3175 DeVito and Eichenbaum • Hippocampal–Prefrontal Circuitry Supports Sequence Memory

Table 1. Average digging times and preference indices for each group across the various groups of mice and judge the aberrant finding of one
within-sequence, between-sequence, and novelty probe trials significant between-sequence preference as a statistical outlier
SHAM HPX PFCX that is not confirmed even among different types of between-
Within sequence
sequence probes in our intact groups.
Less recent odor 4.22 ⫾ 0.39s 3.66 ⫾ 0.67s 4.08 ⫾ 0.53s The present findings indicate that, across repeated sequence
More recent odor 2.40 ⫾ 0.28s 3.80 ⫾ 0.54s 4.25 ⫾ 0.41s presentations, animals link the five odors within each complete
Preference index 0.28 ⫾ 0.04 0.01 ⫾ 0.06 ⫺0.08 ⫾ 0.05 sequence. Conversely, because the animals treat odors that ap-
Between sequence pear in different sequences as unrelated, we conclude that mice
Odor from sequence 1 2.48 ⫾ 0.42s 3.04 ⫾ 0.76s 2.70 ⫾ 0.44s viewed each sequence as a temporally organized experience that is
Odor from sequence 2 2.21 ⫾ 0.27s 2.69 ⫾ 0.53s 3.29 ⫾ 0.63s distinct from the other sequence. We suggest that having been
Preference index ⫺0.06 ⫾ 0.08 0.01 ⫾ 0.14 0.02 ⫾ 0.08 repeatedly rewarded for digging in the odors in a particular order
Novelty recognition may dispose the mice to spend more time searching for food in
Novel odor 4.22 ⫾ 0.45s 4.31 ⫾ 0.62s 4.51 ⫾ 0.50s
the odor for which digging had always previously been rewarded
Familiar odor 1.88 ⫾ 0.29s 2.18 ⫾ 0.77s 2.04 ⫾ 0.30s
Preference index 0.41 ⫾ 0.04 0.55 ⫾ 0.15 0.36 ⫾ 0.06
earlier; that is, they may have had a high expectancy of reward for
odors that had regularly been rewarded earlier over those re-
SHAM, Sham-operated mice; HPX, hippocampal lesion mice; PFCX, medial prefrontal lesion mice.
warded later. In addition, the abnormality of preferences in ani-
mals with hippocampal and prefrontal lesions suggests that, in
preferential exploration of odors that they had experienced earlier the absence of prefrontal– hippocampal contributions, other
within the same sequence of events but did not prefer an odor that brain systems lead to a very different pattern of choices.
they experienced hours earlier the same day in a different sequence.
Therefore, their memory for order must be supported by a represen- Contributions of the hippocampus and prefrontal cortex to
tation of the order of events specific to each sequence. We further sequence memory
demonstrated that this pattern of performance on within- and Memory for the order of events within each sequence was dis-
between-sequence comparisons is not dependent on the order in rupted by damage to the hippocampus, confirming previous
which these probe tests are presented. Our results also then confirm studies that have reported a critical role for the hippocampus in
that the hippocampus and medial prefrontal cortex contribute to memory for sequential events (Fortin et al., 2002; Kesner et al.,
remembering order within specific experiences, whereas these areas 2002), as well as in disambiguating items with overlapping ele-
are not essential to item recognition. ments (Agster et al., 2002; Ergorul and Eichenbaum, 2006; Ku-
maran and Maguire, 2006). Neuroimaging studies, as well as
Memory for the order of events in distinct sequences is not evidence from single-unit recordings, have also demonstrated
based on judgments of relative recency that the hippocampus is activated during recall of the temporal
Previous behavioral studies have demonstrated that animals can order of life-like events (Gelbard-Sagiv et al., 2008; Lehn et al.,
remember the temporal order of serially presented odors (Fortin 2009). Observations on ensemble neuronal activity have sug-
et al., 2002; Kesner et al., 2002) and the order of sequentially gested that the hippocampus supports memory for specific se-
presented objects (Mitchell and Laiacona, 1998; Hannesson et al., quences by the representation of an evolving temporal context
2004; Dere et al., 2005, 2007). However, to date, there has been no for serial events within each sequence (Manns et al., 2007). Cen-
conclusive evidence as to whether animals make sequential mem- tral to the findings presented here, Manns et al. (2007) reported
ory judgments based on a representation of order within each that hippocampal neural ensembles do not code for the relative
specific experience or based on relative recency. recency of sequential events nor do they tag items with their
We first demonstrated that animals can successfully discrim- ordinal position within the sequence. Instead, when ensemble
inate between the items occurring earlier compared with items representations of the temporal context of each event distin-
occurring later within a specific sequence. In addition, there was guished experiences that occurred at different times across the
no difference in memory for items that occurred farther apart sequence, memory for order was accurate. These results support
compared with items that occurred closer together within that the temporal context model of sequence memory, which pro-
sequence. If order judgments were based on relative recency, we poses that events are bound within a sequence by a gradually
would have expected strong preferences in the comparison of changing temporal representation (Howard and Kahana, 2002).
odors that were presented 3 h apart across sequences than for Substantial evidence also supports a role for the prefrontal
comparisons that involved odors presented only seconds apart. cortex in memory for the temporal order of events (Mitchell and
Additionally, there was no difference between items at shorter Laiacona, 1998; Fuster, 2001; Hannesson et al., 2004; Barker et al.,
compared with longer lags, suggesting that animals do not use 2007). Electrophysiological data collected from animals perform-
recency information to make judgments about items within each ing a task similar to that in this study suggest that an additional
sequence as well. Above-chance differential preferences on the region of the prefrontal cortex plays a role in anticipating sequen-
between-sequence probe trials were not observed in either of our tial events (Ramus et al., 2007). In that experiment, animals were
two intact groups or in either of our lesion groups; however, the presented repetitively with a series of eight odors as the activity of
sham group exhibited a significant preference for one of the three orbitofrontal neurons was monitored. In well-trained animals,
between-sequence probe trials. In other studies, we did not ob- neurons in orbitofrontal cortex fired selectively during the sam-
serve a significance preference on any between-sequence probes pling of particular odors, and some cells exhibited odor-selective
in two varieties of wild-type mice, mice lacking serine racemase firing 200 – 400 ms before odor onset. Furthermore, damage to
(DeVito et al., 2010), or mice lacking the vasopressin 1B receptor the hippocampus abolished this anticipatory firing pattern, sug-
(our unpublished findings), and all of these mice (except for the gesting that input from the hippocampus provides important
serine racemase knock-outs) showed the same strong within- information about the anticipation of sequential odors. In a study
sequence preferences as the mice in this study. Therefore, we view that involved recordings in monkeys performing a temporal or-
the absence of a between-sequence preference common among dering task, lateral prefrontal cortex cells fired selectively to items
DeVito and Eichenbaum • Hippocampal–Prefrontal Circuitry Supports Sequence Memory J. Neurosci., March 2, 2011 • 31(9):3169 –3175 • 3175

within a specific sequence, and this activity predicted behavioral dritic morphology. Genes Brain Behav. Advance online publication. Re-
judgments about order (Ninokura et al., 2003; Averbeck and Lee, trieved January 24, 2011. doi:10.1111/j.1601–183X.2010.00656.x.
Diana RA, Yonelinas AP, Ranganath C (2010) Medial temporal lobe activity
2007). These findings suggest that the prefrontal cortex may es-
during source retrieval reflects information type, not memory strength. J
tablish representations of orderly events and may retrieve these Cogn Neurosci 22:1808 –1818.
representations guided by input from the hippocampus. Dudchenko PA, Wood ER, Eichenbaum H (2000) Neurotoxic hippocampal
lesions have no effect on odor span and little effect on odor recognition
Hippocampal– cortical circuitry supports memory for the but produce significant impairments on spatial span, recognition, and
order of items within a sequence but does not support item alternation. J Neurosci 20:2964 –2977.
recognition Eichenbaum H (2004) Hippocampus: cognitive processes and neural repre-
There is a current controversy as to how the hippocampal– cortical sentations that underlie declarative memory. Neuron 44:109 –120.
Eichenbaum H, Yonelinas AP, Ranganath C (2007) The medial temporal
circuitry supports memory strength judgments (Eichenbaum et al.,
lobe and recognition memory. Annu Rev Neurosci 30:123–152.
2007; Squire et al., 2007; Shrager et al., 2008; Diana et al., 2010). Ergorul C, Eichenbaum H (2006) Essential role of the hippocampal forma-
Recent findings have suggested that both the hippocampus and tion in rapid learning of higher-order sequential associations. J Neurosci
prefrontal cortex play important roles in the recollection com- 26:4111– 4117.
ponent of recognition memory but do not contribute to famil- Farovik A, Dupont LM, Arce M, Eichenbaum H (2008) Medial prefrontal
iarity (Fortin et al., 2004; Farovik et al., 2008). Additional studies cortex supports recollection, but not familiarity, in the rat. J Neurosci
using object recognition have found that these structures do not 28:13428 –13434.
contribute to the detection of novel compared with familiar items Fortin NJ, Agster KL, Eichenbaum HB (2002) Critical role of the hippocam-
pus in memory for sequence of events. Nat Neurosci 5:458 – 462.
but instead are recruited when familiar items are combined in
Fortin NJ, Wright SP, Eichenbaum H (2004) Recollection-like memory re-
novel ways, such as when associations between these items must trieval in rats is dependent on the hippocampus. Nature 431:188 –191.
be formed (Dudchenko et al., 2000; Hannesson et al., 2004; Jen- Franklin K, Paxinos G (1997) The mouse brain in stereotaxic coordinates.
kins et al., 2004; Aggleton and Brown, 2005). Despite a severe San Diego: Academic.
impairment in order memory, animals with damage to the hip- Fuster JM (2001) The prefrontal cortex—an update: time is of the essence.
pocampus or prefrontal cortex successfully recognized each of Neuron 30:319 –333.
the items in the two sequences. Gelbard-Sagiv H, Mukamel R, Harel M, Malach R, Fried I (2008) Internally
The current findings provide a novel method for evaluating generated reactivation of single neurons in human hippocampus during
free recall. Science 322:96 –101.
sequence memory and demonstrate that animals can remember Hannesson DK, Vacca G, Howland JG, Phillips AG (2004) Medial prefron-
the order of events within specific experiences, and, in a parallel tal cortex is involved in spatial temporal order memory but not spatial
study, this test was useful in distinguishing a cognitive impair- recognition memory in tests relying on spontaneous exploration in rats.
ment in a murine model of schizophrenia (DeVito et al., 2010). In Behav Brain Res 153:273–285.
addition, the present results resolve an important issue in the Howard M, Kahana M (2002) A distributed representation of temporal
development of animal models of episodic memory, demonstrat- context. J Math Psych 46:269 –299.
ing that normal animals can remember specific sequences with- Jenkins TA, Amin E, Pearce JM, Brown MW, Aggleton JP (2004) Novel
spatial arrangements of familiar visual stimuli promote activity in the rat
out relying on judgments about the mere relative recency of
hippocampal formation but not the parahippocampal cortices: a c-fos
temporally separated events and that hippocampal– cortical cir- expression study. Neuroscience 124:43–52.
cuitry supports the temporal organization of memories. Kesner RP, Gilbert PE, Barua LA (2002) The role of the hippocampus in
memory for the temporal order of a sequence of odors. Behav Neurosci
References 116:286 –290.
Aggleton JP, Brown MW (2005) Contrasting hippocampal and perirhinal Kumaran D, Maguire EA (2006) The dynamics of hippocampal activation
cortex function using immediate early gene imaging. Q J Exp Psychol B
during encoding of overlapping sequences. Neuron 49:617– 629.
58:218 –233.
Lehn H, Steffenach HA, van Strien NM, Veltman DJ, Witter MP, Håberg AK
Agster KL, Fortin NJ, Eichenbaum H (2002) The hippocampus and disam-
(2009) A specific role of the human hippocampus in recall of temporal
biguation of overlapping sequences. J Neurosci 22:5760 –5768.
sequences. J Neurosci 29:3475–3484.
Averbeck BB, Lee D (2007) Prefrontal neural correlates of memory for se-
Manns JR, Howard MW, Eichenbaum H (2007) Gradual changes in hip-
quences. J Neurosci 27:2204 –2211.
pocampal activity support remembering the order of events. Neuron
Barker GR, Bird F, Alexander V, Warburton EC (2007) Recognition mem-
56:530 –540.
ory for objects, place, and temporal order: a disconnection analysis of the
role of the medial prefrontal cortex and perirhinal cortex. J Neurosci Mitchell JB, Laiacona J (1998) The medial frontal cortex and temporal
27:2948 –2957. memory: tests using spontaneous exploratory behaviour in the rat. Behav
Bunsey M, Eichenbaum H (1996) Conservation of the hippocampal mem- Brain Res 97:107–113.
ory function in rats and humans. Nature 379:255–257. Ninokura Y, Mushiake H, Tanji J (2003) Representation of the temporal
Chiba AA, Kesner RP, Gibson CJ (1997) Memory for the temporal order of order of visual objects in the primate lateral prefrontal cortex. J Neuro-
new and familiar spatial location sequences: role of the medial prefrontal physiol 89:2868 –2873.
cortex. Learn Mem 4:311–317. Ramus SJ, Davis JB, Donahue RJ, Discenza CB, Waite AA (2007) Interactions
Clayton NS, Dickinson A (1998) Episodic-like memory during cache recov- between the orbitofrontal cortex and the hippocampal memory system dur-
ery by scrub jays. Nature 395:272–274. ing the storage of long-term memory. Ann NY Acad Sci 1121:216 –231.
Dere E, Huston J, De Souza Silva M (2005) Episodic-like memory in mice: Shrager Y, Kirwan CB, Squire LR (2008) Activity in both hippocampus and
simultaneous assessment of object, place and temporal order memory. perirhinal cortex predicts the memory strength of subsequently remem-
Brain Res Brain Res Prot 16:10 –19. bered information. Neuron 59:547–553.
Dere E, Huston JP, De Souza Silva MA (2007) The pharmacology, neuro- Squire LR, Wixted JT, Clark RE (2007) Recognition memory and the medial
anatomy and neurogenetics of one-trial object recognition in rodents. temporal lobe: a new perspective. Nat Rev Neurosci 8:872– 883.
Neurosci Biobehav Rev 31:673–704. Tulving E (1983) Elements of episodic memory. London: Oxford UP.
DeVito L, Balu D, Kanter B, Lykken C, Basu A, Coyle J, Eichenbaum H (2010) Tulving E, Markowitsch HJ (1998) Episodic and declarative memory: role
Serine racemase deletion disrupts memory for order and alters cortical den- of the hippocampus. Hippocampus 8:198 –204.