Contextual encoding by ensembles of medial
prefrontal cortex neurons
James M. Hymana,1, Liya Maa, Emili Balaguer-Ballesterb, Daniel Durstewitzb,2, and Jeremy K. Seamansa,2
a
Brain Research Centre, Psychiatry, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada V6T 2B5; and bBernstein Center for
Computational Neuroscience, Central Institute of Mental Health, Psychiatry, Medical Faculty Mannheim of Heidelberg University, 68159 Mannheim, Germany
Edited by Charles F. Stevens, The Salk Institute for Biological Studies, La Jolla, CA, and approved February 17, 2012 (received for review September 1, 2011)
Contextual representations serve to guide many aspects of behav-
ior and influence the way stimuli or actions are encoded and
interpreted. The medial prefrontal cortex (mPFC), including the
anterior cingulate subregion, has been implicated in contextual
encoding, yet the nature of contextual representations formed by
the mPFC is unclear. Using multiple single-unit tetrode recordings in
rats, we found that different activity patterns emerged in mPFC
ensembles when animals moved between different environmental
contexts. These differences in activity patterns were significantly
larger than those observed for hippocampal ensembles. Whereas
≈11% of mPFC cells consistently preferred one environment over
the other across multiple exposures to the same environments, op-
timal decoding (prediction) of the environmental setting occurred
when the activity of up to ≈50% of all mPFC neurons was taken into
account. On the other hand, population activity patterns were not
identical upon repeated exposures to the very same environment.
This was partly because the state of mPFC ensembles seemed to
systematically shift with time, such that we could sometimes predict
the change in ensemble state upon later reentry into one environ-
ment according to linear extrapolation from the time-dependent
shifts observed during the first exposure. We also observed that
many strongly action-selective mPFC neurons exhibited a significant
degree of context-dependent modulation. These results highlight
potential differences in contextual encoding schemes by the mPFC
and hippocampus and suggest that the mPFC forms rich contextual
representations that take into account not only sensory cues but
also actions and time.
electrophysiological recordings | population analysis | temporal encoding
M any aspects of behavior are contextually dependent. Con-
texts can be abstract, as in the case of human language, or can
be more concrete and be defined by the constellation of spatial and
sensory stimuli surrounding an individual. Contextual representa-
tions can form quickly and tend to remain stable despite local
variations in perspective (1). Context can also help to inform the
subject about potential changes in the meaning of stimuli and
actions. The medial prefrontal cortex (mPFC) is involved in various
forms of cognition that depend on spatial and contextual infor-
mation, including context-based cognitive tasks (2–4), contextual
fear conditioning (5), and context-induced drug relapse (6–8), and
is believed to be an important node in the “context” network (1, 9–
12). These functions likely depend on reciprocal interactions with
the hippocampus (13, 14), an area that has a well-established role
in spatial and contextual processing (15–20). Through the con-
nection from the CA1 region, the hippocampus has multiple
profound effects on mPFC neurons (21–28), and it has been
proposed that during spatial navigation the mPFC may work in
concert with the hippocampus to encode goal locations and aid in
route planning (29, 30). However, the spatial representations
formed by the mPFC are different from those formed by the
hippocampus: mPFC neurons do not seem to have clear place
fields and presumably do not form the same sort of spatial map
that would inform the animal precisely where it is within an en-
vironmental context (31–34). Accordingly, lesions of the mPFC
do not prevent rats from being able to navigate through even
www.pnas.org/cgi/doi/10.1073/pnas.1114415109
quite complex spatial environments, like mazes (35). However,
mPFC lesions do make hippocampal unit place fields less stable
over time and more reactive to changes in the local environment
(36, 37). This result is consistent with the idea that the PFC might
provide a more global representation of the spatial context (1,
38–40) that is independent of the subject’s exact perspective.
The present study focused on how mPFC might encode whole
contextual settings and how these types of representations may
differ from those of the hippocampus. Toward these ends, we
recorded multiple single units using 16 tetrodes implanted into
the mPFC or hippocampus while animals were switched between
two distinct environments. Several potential factors were con-
NEUROSCIENCE
sidered that could contribute to differences in activity state
patterns across contexts, including differences in sensory cues,
differences in movement patterns, and the passage of time. Fi-
nally, given the important role of the mPFC in action monitoring
and encoding (29, 32, 41, 42), we investigated whether context
affected the way specific actions were represented.
Results
Different Environments Are Associated with Different Ensemble
Activity Patterns in mPFC. We first investigated how mPFC net-
works represented environmental context by recording the ac-
tivity of 26–109 neurons per animal (total of five animals) in the
anterior cingulate cortex subregion as animals were simply
placed in two different novel environments (“A” and “B,” Fig.
S1A) and allowed to freely explore. The instantaneous firing
rates (iFR) of all recorded neurons at each 500-ms time bin were
collected in a population vector, and the space populated by all
these vectors is termed the multiple single-unit activity (MSUA)
space (Methods). A 3D projection of the MSUA space, obtained
by multidimensional scaling (MDS), in which each point repre-
sents the state of the entire recorded ensemble within one 500-
ms bin, is shown in Fig. 1A. All points corresponding to different
500-ms bins within the same environment are shown in the same
color. MDS was used purely for visualization, and all statistical
analyses were performed in the full space of all recorded units
(43). The MSUA space representation shown in Fig. 1A depicts
the state of an mPFC ensemble for a 6 min, 40-s period when
a rat was switched from one environment to another. The black
dots represent the final 3 min and 20 s in environment A, and the
green dots represent the first 3 min and 20 s in environment B.
The differently colored dots corresponding to the times spent in
each unique environment tightly clustered within distinct regions
of MSUA space, indicating that different environments were
Author contributions: J.M.H. and J.K.S. designed research; J.M.H. and L.M. performed
research; J.M.H. and D.D. contributed new reagents/analytic tools; J.M.H., L.M., E.B.-B.,
and D.D. analyzed data; and J.M.H., D.D., and J.K.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1
To whom correspondence should be addressed. E-mail: hyman.jm@gmail.com.
2
D.D. and J.K.S. contributed equally to this work.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1114415109/-/DCSupplemental.
PNAS Early Edition | 1 of 6

Fig. 1. Representation of environmental context during free exploration.
(A) 3D projection of MSUA spaces for free exploration sessions using novel
environments. Dots (population vectors) are colored according to whether
they were taken from environment A (black) or B (green). The axes of these
3D projections correspond to different combinations of the single-unit firing
rates, as derived by MDS. (B) Mean Mahalanobis distances comparing groups
of points within one environment vs. between two environments for mPFC
ensembles. Mean values are from all subjects and sessions (paired t tests and
Wilcoxon signed rank tests; **P < 10−5; error bars = SEM; n = 7 sessions from
five animals).
associated with different patterns of activity across the pop-
ulation of recorded units. To confirm these visual observations
statistically, the Mahalanobis distances between the two clusters
of iFR vectors in the full MSUA space, corresponding to the
times in each environment, were calculated. When Mahalanobis
distances were computed between sets of iFR vectors from dif-
ferent environments, the differentiation between neural ensem-
ble states was approximately two to three times larger compared
with ensemble states taken from the same environment but
separated by the same amount of time as for the between-envi-
ronment comparison [t(9) = 4.98; P < 10−5; Wilcoxon signed
rank = 0; P < 10−4; Fig. 1B; Fig. S1C is an illustration of the
analysis]. The single-cell basis for these population effects will be
investigated in greater detail below.
These results were not due to a general, nonspecific damp-
ening or enhancement of the overall population activity (SI
Results). Nevertheless, movement paths were different (Fig. S2C)
and could contribute to the ensemble separation, consistent with
past studies (44, 45). However, we still observed a significant
separation between the clouds of iFR points in the 3D projection
of the MSUA space associated with two environments, even
when movement effects were controlled by either restricting
movement altogether (Fig. S3 A and B) or by enforcing similar
movement patterns in the two environments (Fig. S3 C and D).
Finally a significant differentiation between ensemble states was
also observed if the contexts were familiar rather than novel (Fig.
S4 A–C) or if the rats moved between environments by them-
selves rather than being carried over by hand (Fig. S4 D and E).
Contrasting mPFC with Hippocampal Representations. Because hip-
pocampal neurons are strongly involved in the representation of
spatial context (8–13), we decided to contrast the results de-
scribed above for mPFC ensembles with the results of similar
experiments performed in rats with tetrode arrays implanted into
the CA1 region of the hippocampus (n = 85 neurons from two
rats during three sessions). For the mPFC ensemble shown in
Fig. 2A (Lower), there was some separation between the clouds
of iFR points in the 3D projection of the MSUA space corre-
sponding to the two different spatial locations within one envi-
ronment demarcated in Fig. 2A (Upper) (black vs. gray and dark
green vs. light green). Fig. 2A also shows that both location
representations seemed to shift in the MSUA space from one
exposure to the next. Accordingly in the group data, a two-way
ANOVA found significant main effects for both recording lo-
cation [mPFC or HPC; F(1,23) = 114.38; P < 10−9] and exposure
[between first and second exposure vs. within a single exposure;
F(1,23) = 86.57; P < 10−7], as well as a significant interaction
effect [F(1,23) = 221.81; P < 10−15]. Tukey’s post hoc tests
revealed that for mPFC ensembles the Mahalanobis distance
between the clusters of points representing the same location on

Fig. 2. Comparison of mPFC and hippocampal ensemble representations and temporal context effects. (A) mPFC ensemble encoding of locations within
a single environment on two different exposures. Upper: Animals were exposed to one environment on two occasions. Dots denote the location of the rat in
the environment in 500-ms time bins. Black dots correspond to the time bins when the rat was in the NE corner during first exposure, and dark green dots
correspond to the time bins when the rat was in the same corner of the same environment on the second exposure. Gray dots correspond to the time bins
when the rat was in the SW corner during first exposure, and light green dots to those when the rat was in the same corner of the same environment on the
second exposure. Red dots correspond to all other time bins for all other locations that were not considered in the present analysis. Lower: MSUA space
representation for the ensemble of mPFC neurons recorded during the same session. Dots correspond to the state of the network in MSUA space in 500-ms
bins and are colored according to the time bins in the location map above. (B) Hippocampal ensemble encoding of locations within a single environment on
two different exposures. Upper: Dots denote the location of the rat in the environment in 500-ms time bins, based on the same color scheme as used in A.
Lower: MSUA space for the ensemble of hippocampal neurons recorded during the same session. Dots correspond to the state of the network in MSUA space
in 500-ms bins and are colored according to the time bins in the location map above, as in A. (C) Mean Mahalanobis distances in MSUA space comparing time
bins when subjects were in the same location in the same environment on two different exposures (black bars) vs. different locations in the same environment
on a single exposure (striped bars). Data for mPFC ensembles are shown at left, and data for hippocampal (HPC) ensembles at right (**P < 0.0001; error bars =
SEM; n = 24 data points from 6 sessions from five animals). Note that mPFC population vectors differentiated more between the two exposures for the same
location, whereas hippocampal ensembles differentiated more strongly between different locations during a single exposure.

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two subsequent exposures (black bars, Fig. 2C) was much larger
than the distance between the clusters of points representing two
locations during one exposure (striped bars, Fig. 2C). In stark
contrast, post hoc tests indicated that for hippocampal ensem-
bles (Fig. 2B) the Mahalanobis distance between the clusters of
points representing two locations during one exposure (striped
bars, Fig. 2C) was significantly larger than the distance between
the clusters of points representing the same location on two
subsequent exposures (black bars, Fig. 2C). When directly
compared, mPFC ensembles exhibited significantly greater sep-
aration in their representation of a single location between two
exposures to the same environment relative to hippocampal
ensembles (black bars, Fig. 2C). In contrast, post hoc tests also
confirmed that hippocampal ensembles exhibited significantly
larger separation in their representation of two distinct locations
within an environment on a single exposure relative to mPFC
ensembles (striped bars, Fig. 2C). Thus, although hippocampal
ensembles differentiated much more strongly between two spa-
tial locations within a given environment than between two dif-
ferent exposures to the same environment, mPFC ensembles in
contrast strongly differentiated between two successive expo-
sures but much less between two spatial locations within the
same exposure and setting (further analyses of mPFC–hippo-
campal differences in SI Results).
Time-on-Task Affects mPFC Ensemble Activity. The lack of consis-
tency in the representation of spatial locations in mPFC activity
across repeated exposures to the same environmental setting
suggests that other factors, such as time, may underlie the strong
environmental context effects. To examine the impact of time,
we analyzed Mahalanobis distances between sets of iFR pop-
ulation vectors as a function of both temporal separation and
according to whether these iFR vector sets were drawn from the
same or different environments (Fig. 3A). As Fig. 3A shows, the
MSUA space separation between any two sets of population
vectors indeed increases as the temporal spacing between these
two sets becomes larger. However, at the same time, along the
whole time-axis in Fig. 3A there is a clear substantial increase in
the MSUA space distances when comparing population vectors
from the same vs. different environments even for the very same
amount of temporal separation (i.e., when time is removed as
Fig. 3. Impact of temporal context on mPFC ensemble activity. (A) Mean
Mahalanobis distance, from an example session, between sets of population
vectors drawn from within the same (A-A, A′-A′, B-B, B′-B′; green line) or
from between different (A-B, A′-B′; red line) environments (hyphen indicates
repetition), and for different amounts of temporal separation (Δt). MSUA
space distances increase with both temporal and environmental difference.
Shaded areas indicate SEM. Transients at the start or end of environmental
exposures were removed (SI Methods). (B) Example session illustrating the
impact of time and environment on Mahalanobis distances between iFR sets
taken from within the same (green) or between different (blue) repetitions
of the same environment (see main text for explanation). Shaded areas =
SEM. The green regression line indicates the predictions from the time/en-
vironment-model fit to the within-repetition distances (i.e., to the data in A).
Hyman et al.
a potential confound). To formally address the impact of time vs.
environment, we fitted a simple linear model to the sets of
MSUA space distances Dmah = β0 + β1Envt + β2Δt, where Δt is
the amount of temporal separation between MSUA vector sets,
and the dummy variable Envt ∈ {−1, +1} encodes whether en-
vironment A or B was presented. For the mPFC data sets we
checked (via F ratios; SI Methods) whether each of the two
factors (environment, time) on its own (with the other factor not
included) explains a significant amount of variation in the
MSUA space distances and whether it significantly adds to the
amount of explained variance when the respective other factor
was already in the model. A factor was considered significant
only if it survived both of these statistical comparisons. In three
of six data sets, both environment and time significantly con-
tributed on their own and in conjunction with the respective
other factor to the amount of Dmah variation (all P < 10−3). In
one other data set only the environment factor reached signifi-
cance, whereas in the remaining two only the time factor was
significant (all P < 10−6).
We next used the model-based approach to determine
whether the gradual time-dependent shifts could explain the shift
in mPFC representations upon multiple repetitions of the same
environment (Fig. 2A). To this end, the linear model above was
fitted to the mean within-repetition distances and then applied to
NEUROSCIENCE
the mean between-repetition distances. That is, if A, B denote
the two environments and A′, B′ their repetitions, the model was
first fit to distances between vector sets taken from A only, B
only, A′ only, or B′ only (within-environment), and from A-B or
A′-B′ (between-environment). The parameters were then fixed,
and it was determined how well this model predicted distances for
comparisons A-A′, B-B′ (between-repetition but within-envi-
ronment; Fig. 3B) and from A-B′, A′-B (between-repetition and
between-environment). For three of the six data sets with re-
peated exposures, approximately 50–75% of the variance of the
mean between-repetition distances (with transients removed)
could be accounted for by the model obtained from the mean
within-repetition distances (all P < 10−5). This confirms that for
at least half of the data sets a considerable proportion of the iFR
activity shifts across repetitions were due to timing effects.
Consistency of Single-Unit Responses upon Repeat Visits to the Same
Environmental Context. All of the analyses above involved pop-
ulation activity patterns. We next analyzed the environmental
selectivity (Methods) of single mPFC neurons over multiple
exposures to two different environments (A and B). To limit
differences in movement across environments as much as pos-
sible, animals were placed in a small plastic chamber (Fig. S1B
shows experimental details) that was transferred back and forth
between the environments five times. When we examined the
environmental selectivity across the five different A-B exposures
we found that a small but significant number of neurons [11%,
binomial P < 0.03; χ2(5) = 15.57, P < 0.007; Fig. S6F] consis-
tently preferred one environment vs. the other across all five A-B
switches. However, this approach of determining selectivity may
be quite conservative because it uses only binary information
(A > B or B > A) from each cell and exposure, with firing rates
averaged across whole exposure periods. Thus, as a more sen-
sitive approach that takes into account firing rate variations on
a time-bin by time-bin basis within each environment, a decoding
analysis based on linear classifiers was carried out. To these ends,
for each data set a linear discriminant function was fitted to the
first three environment A-B repetitions (the “training set”) to
determine a hyperplane in MSUA space that optimally separated
the two environments (Fig. S6G). Prediction performance of this
classifier was then tested on population vectors from the two
remaining (fourth and fifth) A-B repetitions (the “test set”), that
is on trials that had not been used for fitting the classifier (see ref.
46). iFR population vectors from the test set falling onto the
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wrong side of the separating hyperplane obtained from the
training set predict environmental setting incorrectly and thus
contribute to the (predicted) separation error. Using this clas-
sification approach, neuronal samples were built starting with the
most responsive units and then progressively adding neurons
until all units were incorporated in the (sub)population used for
prediction. Fig. S6H shows that decoding performance improved
as more neurons were added, up to the point where approxi-
mately 50% of all units were included in the classifier, after
which prediction performance started to degrade again (owing to
the increase in noise as the sample is further expanded). At the
point of optimal prediction performance, ≈84% of iFR vectors
from the test trials were correctly assigned, and the largest
Mahalanobis distances between context groups were observed.
For this optimally predictive sample of size of ≈50%, the sepa-
ration error was significantly lower than the one in non-
parametric bootstraps (P < 0.01; constructed by randomly
shuffling blocks of iFR vectors of length 10 s from different
environments or exposures; details in SI Methods), and it was
also significantly smaller than in samples consisting of ≈10% of
the maximally responsive units [that is, about the relative pro-
portion of units that was found to express consistent environ-
mental selectivity in the binomial analysis above; t(4) = 5.13; P =
0.007, Bonferroni-corrected P < 0.027]. Thus, although many
units may not consistently prefer one environment over the other
according to firing rates averaged across whole exposures, many
neurons may still contribute to environmental coding at specific
time points during the exposures.
Context Affects the Way in Which Actions Are Encoded by mPFC
Neurons and Networks. Finally we examined how contextual in-
formation affected action encoding by mPFC neurons. For this
analysis we had rats perform an operant-based continuous al-
ternation task in two environments (Fig. S1A). In this case, we
found that there was a larger Mahalanobis distance between
presses made on the same lever across the two environments
than between left and right lever presses performed within the
same environment [t(9) = 4.98, P < 0.01; Wilcoxon signed rank
test, P < 0.01; Fig. 4A]. This result suggests that environmental
context may have a strong impact on how lever press actions are
encoded at the ensemble level.
At the single-neuron level, the selectivities of individual neu-
rons for left vs. right lever presses in environment A vs. B were
Fig. 4. Environmental context encoding by “lever press-responsive” neu-
rons. (A) Mahalanobis distances between lever presses made on the same
lever in two environments (black) vs. lever presses made on two different
levers in one environment (striped) (n = 3 sessions from three animals). (B
and C) Contextual modulation of lever press-related neuronal activity. Up-
per: Lever presses on the preferred lever are plotted for both environment
A (black) and B (green). Lower: Nonpreferred lever responses in the two
environments (A, gray and B, light green). Solid lines show mean iFR and
dotted lines SEM. Inset numbers indicate cell number (corresponding to Fig.
S7B) and |d′| values for these neurons.
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significantly correlated overall (r = 0.44, P < 0.001; Fig. S7A), yet
the strength of the correlation was not too high, suggesting that
some degree of contextual modulation may be present. Lever
press-responsive neurons were defined as those that had an ab-
solute selectivity score of at least |d′| > 0.5 for the lever press
period (on either the right or left levers) vs. the intertrial in-
terval. Of the 107 total neurons in this sample, 36 were deemed
“lever-press responsive” using these criteria. In total, 33% (12 of
36) of lever press-selective neurons fired consistently more to
lever presses in one environment vs. the other (SI Results).
Responses to a given lever press could differ both in terms of
magnitude (Fig. 4B and Fig. S7B) or timing (Fig. 4C and Fig.
S7B) if performed in environment A vs. B. These analyses show
that environmental context affects the profile of the lever press
responses and that a significant proportion of neurons re-
sponsive to lever presses were also selective for one of the
two environments.
Discussion
The present study showed that the activity of ensembles of
mPFC neurons contains information about entire environmental
contexts or changes in context, whereas hippocampal ensembles
tended to provide more consistent and specific representations
of locations within a given context. Contextual differentiation by
mPFC ensembles was based on a number of factors, including
differences in the sensory attributes that define an environment,
time, and as we explain below, movement patterns. Context in
turn affected the way in which lever press actions were encoded
at the ensemble and single-neuron level.
Given the role of the PFC in spatially based cognition and the
strong input the mPFC receives from the hippocampus (both
directly and indirectly via the mediodorsal thalamus) (13, 14), it
seemed logical to expect that mPFC neuronal responses would
be at least in part affected by information about the rat’s spatial
context. In addition, the mPFC plays a key role in context-based
cognitive tasks (2, 3) and context-induced drug relapse (6–8),
and is believed to be an important node in the “context” network
(1, 9–12). However, despite numerous attempts, studies have
consistently failed to find any clear location-specific mPFC firing
patterns that are independent of behavior (i.e., place cells) (31–
34). The present data were largely consistent with these studies
in that mPFC activity was more affected by changes in environ-
mental context on a large scale rather than by the rat’s precise
spatial location. In contrast, the activity of hippocampal en-
sembles depended more on the unique constellation of place
cells that provided location-specific information rather than on
overall environmental context, although significant discrimina-
tion between whole contexts was also present at the hippocampal
level (compare Fig. S5).
Various factors impacted the manner in which mPFC neurons
and ensembles responded to context. First, ≈11% of individual
mPFC neurons maintained a stable preference for a given en-
vironment across all five exposures in the restricted-movement
sessions, indicating that they may be consistently responding to
certain sensory features of a given environment. However, more
sensitive decoding analysis revealed that in fact up to ≈50% of
neurons may contribute to the encoding of environmental con-
text. Second, although separations in MSUA space were still
detectable in the restricted movement condition, the separations
were smaller than those observed for the free exploration ses-
sions (Fig. S3 A and B). Likewise, the enforced similarity in
movement patterns created by the continuous alternation task
reduced the separation in MSUA space (Fig. S3 C and D).
Therefore, consistent with the conclusions of past studies (44,
45), different movement patterns associated with exploration of
different environments also impacted mPFC activity. Finally, the
activity state patterns were not static in a given environment but
seemed to systematically drift over time, suggesting that mPFC
Hyman et al.
ensembles may also encode “time-on-task” as part of their con-
textual representation (Fig. 3). In fact, by linearly extrapolating
from the changes in MSUA space distances produced by changes
in temporal separation, it was possible in some data sets to
predict how far the network state would have moved when the
animal returned to the same environment at a later point in time.
This constant temporal movement of the network state could
therefore explain to some degree why different exposures to the
very same environment were associated with quite different
population activity states. Several recent reports have shown
similar time-related encoding by hippocampal cells and ensem-
bles (47–49). Although we could also confirm significant tem-
poral movement in our hippocampal ensembles within a given
environment (compare Fig. S5 A and B) that was not significantly
different from the one seen in mPFC (compare Fig. S5C), in
contrast to the mPFC there seemed to be little or no temporal
shift in network state across repetitions of the same environment
(compare Fig. 2), and hence population representations tended
to be more similar on repeated exposures in hippocampus.
Collectively these data suggest that for the mPFC “context” may
depend not only on sensory features of the environment but also
the animal’s actions and the passage of time.
Action representations were in turn influenced by the context in
which they were performed. Of all of the neurons that responded
to lever presses during the continuous alternation task, we found
that ≈33% had a consistent preference for one environment over
another. In the present study, these actions had the same meaning
in both environmental contexts because the animals were per-
forming the same continuous alternation task. Recently we found
that a change in the task rule in the same physical environment
induces a sudden and profound change in network activity state
within the mPFC (50). Perhaps if the task context changed along
with the environmental context (e.g., the same actions took on a
different meaning in a different environment), a larger shift in the
representations of actions might have been observed. In this case,
the context information could have been exploited to trigger the
appropriate actions, similar to many ecological situations in which
the meaning of stimuli and actions depends on the surrounding
context in which they are embedded.
Collectively, the present results suggest that mPFC may track
contexts or contextual boundaries as well as alter the inter-
pretation or meaning of stimuli and actions so they are consis-
tent with the present context. A dysfunction in this or a related
form of contextual processing at the level of the PFC may be an
important factor in the cognitive deficits observed in patients
with schizophrenia, as previously proposed (51).
Methods
Subjects, Surgery, and Behavior. Details in SI Methods.
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Hyman et al.
Data Analysis. The present study used methods described previously by Lapish
et al. (43) and Durstewitz et al. (50). To obtain an estimate of the neural
firing rate for each isolated cell i as a function of time bin t, ri(t), all spike
trains were convolved with Gaussian kernels (SD = 500/4 ms) and binned at
500 ms (approximately the inverse of the average firing rate of ≈2.4 Hz).
Neurons with average firing rates below 0.1 Hz were excluded from further
analysis. For population analysis, population vectors r(t) = [r1(t) . . . rN(t)] were
formed (called iFR vectors), with N the number of single units isolated from
a given recording session. The term MSUA space refers to the N-dimensional
space spanned by all recorded units and populated by these vectors r(t).
To quantify environmental effects on network activity, we computed the
Mahalanobis distances (e.g., ref. 52) between the sets of N-dimensional
vectors associated with the different environment epochs, with covariance
matrices pooled for the two conditions compared. The (squared) Mahala-
nobis distance between two sets of points can be thought of as the Eu-
clidean distance between the group means normalized by the covariances of
the data along all MSUA dimensions (i.e., it quantifies the separation be-
tween two clouds of points in relation to the data scatter). Because the
number of dimensions (neurons) could sometimes approach the number of
data points (time bins), a regularized version of the covariance matrix was
used (e.g., ref. 53) to avoid singularity and statistical reliability issues.
There was no selection of units, and all units were included in all analyses
unless otherwise stated. In the present data set, ensembles ranging from 26 to
109 units per rat were recorded simultaneously, yielding MSUA spaces of
dimensionality 26–109. To control for potential confounds in Mahalanobis
NEUROSCIENCE
distance comparisons, like differences in MSUA space dimensionality or in
the sample size, we selected the same number of units and vector points for
each distance calculation. We limited the number of dimensions to the
smallest recorded ensemble size by randomly selecting only this number of
neurons from the larger ensembles 1,000 times, and taking the averages
across these 1,000 random drawings to still make full use of all units
recorded. For all between- and within-environment context analyses sample
sizes of the same number of vector points (200 time bins) were used for each
set of vectors compared in all sessions.
To assess whether environmental switches affected ensemble activity
states, we compared Mahalanobis distances between equivalent time periods
from either one (within) or two (between) environments. For within-envi-
ronment comparisons, we used two periods of 200 time bins from the last
section within one environment, which were separated by the same amount
of time that it took to transfer animals between the two environments. For
between-environment comparisons we selected the final 200 time bins from
the first environment and the first 200 time bins from the second environ-
ment (Fig. S1C). Thus, for these comparisons time was eliminated as a con-
founding factor. Paired t tests and nonparametric Wilcoxon signed rank
tests were performed on within- and between-environment distances for
each behavioral condition.
ACKNOWLEDGMENTS. We thank Dr. Chris Lapish for helpful discussions on
the manuscript. This work was funded by grants from the Canadian Institutes
for Health Research and National Alliance for Research on Schizophrenia and
Depression (to J.K.S.) and by Bundesministerium fuer Bildung und Forschung
Grant 01GQ1003B and Deutsche Forschungsgemeinschaft Grant Du 354/6-
1,7-2 (to D.D.).
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Supporting Information
Hyman et al. 10.1073/pnas.1114415109
SI Methods
Subjects. A total of 16 adult male Long-Evans rats (Charles River)
were used in 31 total recording sessions. Animals were housed
individually and maintained on a 12-h on/off light–dark cycle.
Animals used in continuous alternation sessions were trained on
these tasks before implant surgery (3 of 16 animals).
Surgery and Electrophysiology. Animals were deeply anesthetized
under isoflurane gas and implanted with 16 moveable tetrodes in
a hyperdrive. Tetrodes were made of 15-μm coated tungsten steel
wire (California Fine Wire) and were initially lowered ≈680 μm
into cortex during surgery. The hyperdrives were mounted to
skull screws using dental acrylic (two grounding screws were
placed posterior to lambda, above the cerebellum) and shielded
in plastic sheeting. After 1–2 wk of postsurgical recovery, animals
were attached to the tether, and tetrodes were slowly advanced
ventrally into the medial prefrontal cortex (mPFC) over a 5- to
10-d period. Once all tetrodes were placed into anterior cingulate
cortex (anteroposterior: +2.2 mm; mediolateral: ±0.5 mm; dor-
soventral: ≈2.5 mm from cortical surface) according to lowering
records and atlas coordinates (1), small adjustments were made
to maximize the number of neurons recorded. For hippocampal
(HPC) recordings tetrodes were advanced ventrally until sharp
waves were detected, and then small adjustments were made
until tetrodes entered the pyramidal cell layer of CA1 (2).
Electrodes were connected to an EIB-36TT (Neuralynx),
which was plugged into two HS-36 headstages and tether cables
(Neuralynx). Signals were sent to a Digital Lynx 64-channel
system (Neuralynx), converted from analog to digital, and sent to
a PC workstation. Electrophysiological, positional, and behav-
ioral data were read into Cheetah 5.0 software (Neuralynx) and
recorded for later analysis. Files were then read into Offline
Sorter (Plexon Inc.) for spike sorting. Only spikes with amplitudes
more than 1.5 times the background noise were used for spike
sorting. Spikes were initially sorted using visually dissociable
clusters in 3D projections along multiple different axes for each
individual electrode of a tetrode (peak amplitude, valley ampli-
tude, peak-to-valley ratio, principal components, and area).
Sorting was confirmed by examining autocorrelations, cross-
correlations, and ANOVAs conducted from 2D and 3D projec-
tions of the different axes used for visual cluster sorting in Offline
Sorter (Plexon Inc.). Spike timestamps were then read into
MATLAB (Mathworks) for all further analysis. For all analyses
fast spiking units, with mean firing rates >10 Hz (putative in-
terneurons), were removed to ensure that no biases due to cell
type skewed the results (in total 65 of 1657 recorded units met
these criteria).
Behavior. Freely moving exposure. Six distinct environments were
used for these sessions. All environments had high walls (>24
inches) to limit the influence of distal room cues. Environments
differed in size and shape (square, rectangular, or round), ma-
terial (corrugated plastic, Plexiglas, wood, or cardboard), color,
and/or visual stimuli (bare walls or with various visual stimuli, e.
g., magazine cutouts; example environments shown in Fig. S1).
During these sessions animals were placed into the first envi-
ronment and left there undisturbed for 10 min. They were then
removed from the first environment and placed onto the same
stool used previously for plugging-in animal’s implants into the
tether recording cable for ≈1.5 min. Next, animals were placed
into a different environment and left to explore for 10 min. This
method of transferring animals from one environment to the
Hyman et al. www.pnas.org/cgi/content/short/1114415109
next was termed “pick & place.” This process was repeated
a second time with periods on the plugging-in stool separating
each environment switch. For “novel” environment sessions
animals had no previous experience with both environments,
whereas “familiar” environment sessions used two environments
the animals had two to three past exposures to on previous re-
cording days.
It is possible that the process of picking the animal up from one
environment, transferring it to the plugging-in stool for a brief
period, and then placing it into the new environment could have
influenced ensemble activity and thus confounded environmentally
driven effects. Therefore, to examine the possible impact of the
method of environmental transfer, we ran a series of sessions on an
apparatus with two distinct environments connected by a walkway.
Each animal performed two environment exposure sessions using
either “walk-through” or pick & place transfer methods (session
order was counter-balanced across animals). For the walk-through
sessions, animals were initially placed into the first environment,
and after 10 min a door to the ramp was opened. After the animal
walked into the ramp area, the door to the first environment was
closed. After being sequestered on the ramp for ≈1–2 min, the
door at the other end of the ramp was opened, granting access to
the second environment. Upon entering this environment the
ramp door was closed for a 10-min exposure period.
Restricted exposure. Restricted exposure sessions were performed
in a different set of animals from the free-exposure sessions. After
the animals were plugged into the tether recording cable, they
were placed into a small, clear plastic enclosure, and a piece of
cellophane was used to cover the top (all animals had been
previously habituated to the vessel beforehand to lessen any
stress-related effects). The enclosure was 5 × 3.5 × 11 inches and
had four 5-inch-long thin vertical slits cut into the two wider
sides. Each animal was given two 30-min habituation sessions in
the enclosure. During recording sessions the enclosure was then
placed into the center of one of the same environments used for
freely moving exposure sessions for 5 min. Then the enclosure
was moved into a second environment for 5 min, and this process
was repeated until animals were exposed to each environment
five times.
Lever press alternation. The same environments were used for these
experiments as those for the free exposure sessions; however, in
this case two behavioral panels were inserted. One panel had two
retractable levers (6 inches apart) with a reinforcement spout in
between, and a second panel, on the opposite side of the envi-
ronment, featured a nose-poke port and a stimulus light 2 inches
above the port (Fig. S1A). For the first trial of each session both
levers were extended, and depressing either lever resulted in
reinforcement delivery. After the lever press both levers re-
tracted, and the stimulus light above the nose-poke port was
illuminated, indicating that the nose-poke port was active. Ani-
mals then needed to nose-poke, which resulted in the stimulus
light turning off and both levers extending. The animal then
needed to depress the lever opposite from the one depressed on
the previous trial to receive a reinforcement pellet (if the right
lever was depressed on trial 1, then the left lever was the correct
lever for trial 2, and so on). Trials continued in this alternating
fashion until the animal completed at least 20 correct trials, at
which point the animal and the response panels were moved to
a different environment for 20 more trials.
Data Analysis. Statistical controls for ensemble analysis. Potential
confounds to the observed segregation effects were systematic
1 of 12
changes in overall mean firing rates across a session. To determine
whether the grand-mean population instantaneous firing rates
(iFRs) were the same for different environments, these were
simply compared by t tests as reported in the main text (ignoring
the dependent nature of the iFR data). Furthermore, a straight
line was fitted by linear regression to the cumulated mean pop-
ulation iFRs in 500-ms bins (Fig. S2A). Stability of the mean
population iFR throughout the experiment implies a linear slope
of the cumulated iFR series, and hence the residuals from a
linear fit give some indication of the degree to which this as-
sumption may have been violated. Vice versa, the squared linear
(Pearson) correlation coefficient gives the proportion of data
variance explained by the fit.
To control for gross differences in general locomotor activity as
a potential confound, the distance traveled (in physical space) was
calculated for each 500-ms bin using position tracking data from
light-emitting diodes over the animal’s head, and the means were
statistically compared between the two environment periods (as
reported in the main text). Furthermore, as for the mean pop-
ulation iFRs, the proportion of variance explained by a linear fit
to the cumulated motor activity series was taken as a measure of
the relative constancy of movement activity. Distance traveled
was normalized for all bins to the overall session mean, and
cumulative totals over time were plotted (Fig. S2B).
Analysis of temporal vs. environmental factors. For the analysis of
temporal vs. environmental factors in MSUA representations,
Mahalanobis distances (with strongly regularized, nearly diagonal
covariance matrices) were computed for sets of 50 temporally
consecutive iFR population vectors, taken from either the same or
from different environments, and separated by different amounts
of time (taken at multiples of 50 bins = 25 s). As indicated in the
main text, for statistical testing a linear model with environmental
and time factors was fitted to these data. For checking significance,
for both factors (i) the amount of Dmah variation accounted for by
that factor in isolation [i.e., with only β1 (environment) or β2
(time) present in the model, compared to the “intercept-only-
model”] and (ii) the amount of variation additionally explained
with the respective other factor already included was examined.
Statistical testing was performed via the F-ratio F1;N − p ¼
ðN − pÞðRSS1 − RSS0 Þ=RSS0 with RSS0 the residual sum of
squares of the larger model (with the larger number of free
parameters p), and RSS1 the same for the smaller model.
For the between-repetition tests, the model was first fit to the
within-repetition distances only and then fixed. This yielded a
prediction for the between-repetition distances for which the
goodness of fit could be assessed by the residual variation around
this prediction (Fig. 3B). A proper formal parametric test does
not exist for this situation, to our knowledge. In one approach we
used F-like ratios FN − 1;N ¼ N=ðN − 1ÞRSSmean =RSSpred with
RSSmean the (total) variation around the grand mean and RSSpred
the residuals from the model prediction. Although empirically
the denominator and numerator of this F-ratio appeared largely
uncorrelated in our sample (jrj < 0.25, p > 0.5), it should be
noted that, strictly, this is not quite a proper statistical test (due
to violation of independence). We therefore also checked the
results by a permutation procedure in which the assignments of
Mahalanobis distances to rows of the design matrix were ran-
domized in the test (between-repetition) sample, the full model
refitted, and the residuals determined, yielding a bootstrapped
H0 distribution against which the residuals from the predicted
model could be checked (for the bootstraps one has
E½^β0  ¼ hDmah i; E½^β1  ¼ E½^β2  ¼ 0 for a fixed design matrix). The
results of this bootstrap procedure largely agreed, in terms of
significances, with the “nonconventional” parametric approach
above, with only one exception: the HPC dataset shown in Fig.
S3B, which the bootstrap procedure indicated as significant (P <
0.005), while the F-style test gave a much more conservative
answer (P < 0.13).
Hyman et al. www.pnas.org/cgi/content/short/1114415109
The proportions of explained variance reported in the main text
were for predictions on the mean distances (for given Δt and
environmental context) and with transients removed from the
curves by requiring a minimum of 10 data points for each mean
(to stabilize mean estimates), but the same pattern of sig-
nificances is obtained when all distances (i.e., without averaging
or removal of data points) are considered, only that in this case
naturally the proportions of total variation explained dropped to
approximatley 18–28% for the three data sets reported in the
main text.
Spatial location vs. context encoding by ensembles. To examine how
spatial locations within an environment were encoded by mPFC
and HPC ensembles, we first isolated all time bins when the
animal was in each of the two locations during the first two
exposures to environments A and B according to position tracking
data. Two corner locations where the animals spent similar
amounts of time were selected for each environment and session,
and they were all of similar size (although there was some small
variation between sessions due to differences in the shape and size
of the environments; the same coordinates for locations were used
for all exposures during one session). We then equalized the
number of neurons and time bins for both locations and exposures
by randomly selecting the minimum number of neurons and bins
from both locations on both exposures 1,000 times, and then
averaging the Mahalanobis distances across these 1,000 draws.
We calculated distances between iFR vector sets associated with
the times spent in the two different locations within one exposure,
and between the same locations on multiple exposures.
Ensemble encoding of behavior. To investigate whether different
environmental contexts affected ensemble activity related to
behavioral actions, we compared lever presses in two different
environments during continuous lever press-alternation sessions.
Activity during a 2-s time window around lever presses was used
for comparisons of action-related activity within or between
environments. If the two environments are denoted by A and B,
and the lever presses to the left and right by L and R, respectively,
Mahalanobis distances were then computed between sets of iFR
vectors associated with correct lever presses from the four pairs
AL-AR, BL-BR, AR-BR, and AL-BL. Thus, four Mahalanobis
distances were calculated for each session, two for left vs. right
lever presses within each environment, and two for left and right
responses between environments. Mahalanobis distances from
the first two pairs (AL-AR and BL-BR) were then averaged to
give a separation index between L and R independent of envi-
ronment, and distances between the other two pairs (AR-BR and
AL-BL) were averaged to yield a separation index between
environments A and B independent of lever press side. Thus,
exactly the same sets if iFR vectors went into each of these two
comparisons, only that they were grouped differently, either
according to environment or according to lever press side. To
evaluate group differences we then used paired t tests and
nonparametric Wilcoxon signed rank tests.
Single-unit analysis of environmental and behavioral preferences. To
examine whether individual units displayed a consistent prefer-
ence for one of the two environments throughout multiple
exposures, a selectivity index for each unit i with respect to each
environment period (environment A-first time vs. B-first time,
environment A-second time vs. B-second time, etc.) was ob-
tained by grouping the firing rates into two classes (A and B)
corresponding to the examined environmental periods, and
computing:
hfri ðtÞj t ∈ Agi − hfri ðtÞj t ∈ Bgi
d′i ¼ qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ;
σ2i;t∈A þ σ2i;t∈B
where 〈·〉 denotes the mean.
2 of 12
 To assess whether a significant number of cells out of the whole
population formed lasting preferences for one of the two envi-
ronments, for each of five repetitions of exposure to the two
environments the d′ value was converted into a binary number
based on whether d′ > 0 (selective for environment A) or d′ < 0
(selective for environment B). Note that for a single cell, the
two extreme cases (0,0,0,0,0) or (1,1,1,1,1) for these 5-valued
binary vectors (i.e., consistent preference for environment A or
B across all five repetitions) each have a chance likelihood of
occurrence of Pr = 0.55 = 0.03125. Next, we created a histogram
from the single-unit data across all of the six possible outcomes
k = 0 preferences for environment B to k = 5 preferences for
environment B (or, equivalently, environment A) and compared
this empirical distribution with what would be expected from the
binomial distribution with Pr = 0.5 using a χ2 goodness of fit test.
If most cells would not have a consistent environmental prefer-
ence, the empirical distribution should be close to this “null”
binomial with Pr = 0.5, whereas it should significantly deviate
from this “null binomial” in the tails (i.e., k < 2 and k > 3) if
there were a lot of cells with consistent environmental selectivity.
The analysis of environmental selectivity when the same
behaviors were performed in two environments was focused on
the lever press-responsive cells (see main text). So correct trial
left and right lever presses were combined to form groups of
trials depending on the environment in which they were per-
formed. In total there were four groups (each consisting of two
right and two left lever press trials) from environment A and four
such groups from environment B. These groups in A and B were
paired, and environmental selectivity was determined for each
lever press-responsive unit for each of these four A-B pairs. If
these neurons were not responding to environmental aspects but
only to lever presses, these four A-B selectivity indices would
show random preferences, and similar to the environmental
selectivity analysis described above the pattern of preferences
could be compared with the expectations from the binomial
distribution with Pr = 0.5.
Two approaches were taken to assess how environmental
context influences single-neuron responses to lever presses. First,
we determined each neuron’s selectivity for left vs. right lever
presses in each environment independently and then assessed
how stable selectivity was across environments by correlating
these two measures. Second, to examine neurons that were ac-
tivated during lever presses, we calculated separate response
indices for left and right lever presses in the two environments.
For these indices we compared iFRs from 2 s of activity around
the lever press with 2 s of activity 5 s before the response during
the intertrial interval. These response indices were calculated the
same way as the selectivity indices described above, only that
they did not compare two different conditions of the same type
(like the two environments or two lever press sides) but activity
during one lever press with activity in a baseline period, and
hence were termed response rather than selectivity indices. In
total there were four such d′ values for each neuron, and a
neuron was considered responsive to lever presses if any of its
jd′j > 0.5.
Decoding analyses. We performed a decoding analysis based on
linear discriminant functions. Linear discriminant analysis finds
a (hyper-)plane optimally separating two clouds of data points by
maximizing the difference between the class means while mini-
mizing within-class covariances (3). The decoding procedure was
carried out in two stages (see schema in Fig. S6F): first, for the
first three environment A-B exposures (termed the “reference”
or “training set”), we computed the optimally discriminating
direction in MSUA space along which the overlap between the
two distributions associated with the two environments is mini-
mized. Second, out-of-sample prediction performance of this
classifier was then evaluated on iFR vectors from the fourth and
fifth A-B exposures (the “test set”), that is on trials that had not
Hyman et al. www.pnas.org/cgi/content/short/1114415109
been used for fitting the classifier (see ref. 4 for details). The
classifier works by determining how many of the iFR vectors r(t)
from the test set fall onto the wrong side of the separating hy-
perplane obtained from the training set, and thus would be at-
tributed to the wrong environmental setting, or in other words
would fail to predict the correct environment. The total number
of incorrectly predicted assignments constitutes the predicted
separation error (Fig. S6H).
For determining the ensemble size needed for optimal pre-
diction, all neurons from three repeated-exposure data sets used
in this analysis were first ordered by their overall mean firing rate.
From the total pool of all these neurons (n = 31, 31, and 78 for
the three data sets used), subpools were then created by col-
lecting from 1 to n neurons according to their average firing rate.
For each of these subpools of increasing size, the optimally
separating hyperplane was first determined from the training set,
and on the basis of this the predicted separation error was cal-
culated for the test set. Optimal predictions were further eval-
uated using bootstrap analyses with 200 block-permutation
bootstraps [preserving autocorrelations (4, 5); prediction accu-
racy of the bootstraps was less than 58.6%]. Bootstraps were
constructed by randomly shuffling blocks of vectors r(t) (with
block length 10 s as determined from the first minimum of the
autocorrelation function) corresponding to different environ-
ments and trials but preserving the order of units at each time
bin (see schema in Fig. S6F) (4).
SI Results
Comparing mPFC and HPC Ensemble Environmental Context and
“Time-on-Task” Effects. Just as was done for mPFC ensembles,
we assessed Mahalanobis distances between sets of HPC iFR
population vectors as a function of both temporal separation and
whether they were from the same or from different environments.
As the example session in Fig. S5A shows, for HPC ensembles,
just as observed for mPFC, MSUA space separation increases as
the amount of temporal separation between two iFR vector sets
gets larger. Significant environmental effects are also present in
HPC ensembles when comparing Mahalanobis distances be-
tween population vectors from the same vs. different environ-
ments for the exact same amount of temporal separation. To
formally examine these effects in HPC ensembles, we used the
same linear model-based F ratio style analysis used to disasso-
ciate the effects of environment and time for mPFC ensembles
(SI Methods and Results). In two of the three HPC data sets
recorded, both the environment and time factor reached signif-
icance, whereas in one only the environmental effect was sig-
nificant (all P < 0.002). We also checked whether the time-
dependent shifts in HPC ensemble activity could account for
differences among multiple repetitions of the same environment,
using the same analysis as described for mPFC ensembles in the
main text and SI Methods. However, this was not the case in any
of the three HPC ensembles (all P > 0.12). With a total of ≈21%
of explained mean variation for the best HPC ensemble (the one
depicted in Fig. S5B) the temporal prediction from within- to
between-repetition distances was also much worse than for the
three significant mPFC ensembles (where 50–75% of the vari-
ance in means was accounted for by the temporal extrapolation).
This suggests that both gross-environmental and temporal con-
text can also significantly influence HPC representations but that
HPC representations are much more stable across repetitions of
the same environments than mPFC representations, as already
demonstrated in Fig. 2.
To further analyze whether PFC and HPC differed in their
representations of environment and time, we compared mPFC
vs. HPC mean between-environment (i.e., A-B and A′-B′)
Mahalanobis distances, normalized to the mean within-envi-
ronment (i.e., A-A, B-B, A′-A′, and B′-B′) distances (Fig. S5C,
Left; the normalization was done to account for general scale
3 of 12
differences in Mahalanobis distances from HPC and mPFC,
partly potentially indicating differences in the spiking patterns
of mPFC and HPC units). These normalized between-environ-
ment distances were significantly larger for the mPFC compared
with HPC [t(7.51) = 2.73, one-sided P < 0.02], indicating that
overall the mPFC differentiates more strongly between contexts
than the HPC. However, this overall comparison did not dif-
ferentiate between potential contributions from environmental
context and from temporal shifts. In an additional test we
therefore roughly (to not lose too many time points) equated
the time basis for mPFC and HPC (average overlap ≈90%), that
is the range of temporal differences for which between-envi-
ronment Mahalanobis distances were computed, and also nor-
malized each between-environment distance only to the mean
within-environment distance for the exact same temporal lag. In
this case (Fig. S5C, Center) again a significant difference be-
tween normalized mPFC and HPC distances was obtained
[t(7.89) = 2.36, one-sided P < 0.03]. Finally, the time effect on
its own was assessed by performing a robust linear regression fit
to the within-environment Mahalanobis distances only (thus
eliminating environmental context as a confounding factor).
The slope of this fit quantifies the time-dependence and is given
in Fig. S5C (Right) for all mPFC and HPC data sets. In this case
there was no significant difference between mPFC and HPC
ensembles [t(8) = 0.37, P > 0.7; regardless of whether Maha-
lanobis distances were normalized by their respective group
means]. For all these comparisons, Mahalanobis distance cal-
culations were based on exactly the same number of units and
time points for mPFC and HPC (and unequal variances were
assumed in the tests whenever these differed by more than
fivefold). The same pattern of significances was obtained by
bootstrap procedures (drawing two groups of mean distances
randomly from the empirical values), or when the analyses
were performed in terms of the proportions of variance ex-
plained by the linear model given in the main text (i.e. the time
and environment factors combined or the environment factor
on its own became significant, while the time-alone effect was
nonsignificant).
We conclude that overall context separation is significantly
larger in mPFC, mainly owing to the stronger separation between
whole environmental contexts, whereas there is no significant
difference in the magnitude of within-repetition temporal shift
between mPFC and HPC. There was, however, a difference if
the temporal shifts within one environmental exposure were to
be used to predict the network state on a second exposure to that
same environment: this worked fine for three of the mPFC data
sets but for none of the HPC ones. In general, however, the
results presented here have to be interpreted with some caution
owing to the low HPC sample size (n = 3).
Additional Analysis Separating Temporal from Environmental Context
Effects. As an additional means for demonstrating that environ-
mental context has a strong effect on its own, irrespective of the
observed temporal shifts, we performed another analysis in which
we compared iFR vectors that were recorded 200 s apart in
a single environment with sets of iFR vectors recorded in two
environments yet separated by only 100 s (Fig. S1C shows ex-
perimental details). In this case we found significantly more
separation between the latter (100 s apart but from different
environments) than the former (200 s apart in a single envi-
ronment) [t(15) = 4.27; P < 0.001; Wilcoxon signed rank = 0;
P < 0.001; Fig. S5D].
Controlling for Other Potential Confounds in mPFC Environment
Context Separation. It is possible that wholesale changes in fir-
ing activity or movement patterns may have impacted ensemble
environmental context representations. The actual mean iFRs
were not different across the two environments for the example
Hyman et al. www.pnas.org/cgi/content/short/1114415109
session shown in Fig. 1A [t(202) = 1.3; P > 0.17; mean iFR:
environment A = 0.82 ± 0.2 Hz; B = 0.79 ± 0.25 Hz], and the
average population rate stayed relatively stable throughout the
recording session as confirmed by a linear fit to the cumulative
mean iFRs, which captured approximately 78–99% of the vari-
ation (P < 0.001; Fig. S2A). Likewise, the absolute distance
traveled was similar across environments [t(199) = 0.9; P > 0.5;
Fig. S2B], and motor activity was relatively stable throughout
(76–99% of variance explained by a linear fit to the cumulated
motor activity series; P < 0.001; Fig. S2B).
PFC Ensemble Activity Separates Environmental Contexts During
Restricted-Movement and Lever Press-Alternation Tasks. The anal-
yses presented in Fig. 3 and above showed that both changes in the
sensory environment and temporal factors impact mPFC activity
patterns. Another factor possibly contributing to the differences
across environments was the movement patterns of the animals.
Although the differences in mPFC representations of two envi-
ronments could not be explained by gross differences in the total
distance traveled (Fig. S2B), slight variations in movement pat-
terns and paths traveled (Fig. S2C) have been shown to affect
mPFC neuronal activity (6, 7) and thus could account for some
of the separation in ensemble activity states. Rather than try and
find identical movement trajectories from the two environments
(of which there would be few in a free exploration session), we
attempted to limit movement altogether by having animals
confined within a small clear plastic enclosure that was moved
back and forth between two environments (single-unit analysis
for these same sessions is presented in the main text). Despite
only being able to rear up and down and otherwise not move,
explore, or physically interact with the environment in any way,
differences in the dot clusters corresponding to the times they
were in environment A vs. environment B were still observed.
For the session shown in Fig. S3A, vector points from periods
within the same environment were tightly clustered relative to
points from periods in different environments. Across all re-
stricted exposure sessions there was significantly more separation
between environments than within [t(7) = 7.1; P < 0.001; Wil-
coxon signed rank = 0; P < 0.01; Fig. S3B].
To further compare how differences in sensory cues vs. dif-
ferences in movements affected mPFC ensemble activity, we
analyzed mPFC activity as animals spent equivalent periods inside
the restricted-movement container in the environments vs. freely
exploring the same environments. This allowed us to address the
role of movement differences in contextual representations by
comparing the impact of environmental changes when movement
patterns were identical (restricted movement in both environ-
ments) and when they were very distinct (restricted movement in
A and freely moving in B). Time was removed as a potential
confound from this analysis by equating it for all comparisons
made as explained in the main text and above. A two-way
ANOVA found significant main effects for similarity of move-
ment patterns [similar vs. distinct; F(1,19) = 6.14; P < 0.025]
and for change in environmental context [within vs. between;
F(1,19) = 59.32; P < 10−6]. The interaction between these fac-
tors was also significant [F(1,19) = 7.89; P < 0.01; Fig. S3B],
indicating that differences in movement patterns affected iFR
activity patterns more strongly when they occurred across envi-
ronments rather than within the same environment (Fig. S3B).
Tukey-type post hoc tests further confirmed that there was sig-
nificantly greater separation in MSUA space for the context
change that also involved different movement patterns than for
the context-only changes (Fig. S3B). These results therefore
suggest that ensemble states are influenced both by changes in
sensory cues across contexts (when movement patterns were
forced to be similar) and by the movement patterns associated
with behavioral exploration of contexts.
4 of 12
 To further investigate this issue, rather than restricting
movement altogether, we sought to equate the movements
produced in two environments. The continuous alternation task
performed in an operant chamber tends to produce rather ste-
reotyped movement sequences in well-trained rats. For the
present experiments, animals spent the first half of the session in
environment A performing a continuous lever press-alternation
task and the second half performing the same task on the same
manipulanda but in environment B (analysis of single-unit
responses during these continuous alternation sessions can be
found in the main text). Fig. S3C shows an example vector space
from one of these sessions. Clear separation can be seen between
the points corresponding to times in the two different environ-
ments, despite overall movement patterns being in general very
similar in both environments (Fig. S3C, Lower). Even though the
rats were performing the same alternation behavior in both en-
vironments, Mahalanobis distances were significantly larger be-
tween environments than within [t(5) = 5.1; P < 0.001; Wilcoxon
signed rank = 0; P < 0.05; Fig. S3D]. Thus, even in situations in
which movement patterns were very similar, context had signif-
icant effects on mPFC ensemble activity states.
Similarity Between Movement Patterns in Two Different Envi-
ronments. Fig. S3C (Lower) shows that the rat moved in a ste-
reotypical pattern between the lever and nose-poke panels while
performing the task, regardless of the environment in which it
was confined. To quantify this, histograms were computed for
the x- and y-coordinates for the two paths in the different envi-
ronments, demonstrating a tight overlap in the x- and y-positions
visited in the two environments (Fig. S3 E and F). A canonical
correlation analysis was then performed across these two paired
sets of x- and y-histogram frequencies (giving the linear combi-
nation of x- and y-histograms along which the between-set cor-
relation is maximized; see ref. 8). This analysis confirmed the
tight correspondence between the sets of spatial locations visited
in the two different environments (Fig. S3G), that is, the simi-
larity in the frequency distribution across locations traversed by
the two different paths.
Novelty Effects on mPFC Environmental Context Representations.
Additional sessions were run with two of the animals from the
sessions described in the first section of the main text, but this time
using familiar instead of novel environments (2 to 3 previous days
of exposure; activity plot shown in Fig. S4A). Fig. S4B shows the
MSUA space plot from a representative session in which the rat
was exposed to two familiar environments. Like novel environ-
ments, the vector points formed two clusters corresponding to
the periods when the animal was in one of the two familiar en-
vironments. The activity state patterns in the MSUA spaces were
similar while the animal remained in one environment yet were
different when the rat was moved to the second environment.
Group statistics confirmed that groups of population vectors
from the periods spent in two familiar environments were sep-
arated by significantly greater Mahalanobis distance than two
temporally equally spaced periods of the same length within one
familiar environment (Wilcoxon signed rank = 0; P < 0.05; Fig.
S4C). In fact, there was no significant difference in the amount
of environment separation between novel and familiar environ-
ment sessions [t(14) = 1.5; P > 0.16; Wilcoxon rank sum = 38;
P > 0.18]. The effects of familiar environments were also not
merely due to changes in overall firing levels, because the mean
iFRs from all units in both environments were not significantly
different (mean iFR: environment: A = 2.1 ± 0.8 Hz; B = 2.24 ±
0.92 Hz). In addition, there were similar distances traveled in the
two familiar environments (mean environment A = 0.95 ± 0.07
cm/s; environment B = 0.97 ± 0.08 cm/s).
Hyman et al. www.pnas.org/cgi/content/short/1114415109
Controlling for Environment Transfer Methods. In the sessions de-
scribed above for both novel and familiar environmental expo-
sures, the animals were physically moved between the two
environments (“pick & place”). This handling could conceivably
be responsible for the observed changes in activity patterns. To
address this concern, we connected the environments by a simple
gangway that allowed the rat to move by itself between the en-
vironments (four sessions from three animals were obtained this
way). An example of such a “walk-through” session is shown in
Fig. S4D. Times in each environmental enclosure were denoted
using the same colored dots, whereas the dashed circle around
a dot denotes the last time bin when the rat was on the ramp
leaving environment A, and the solid circle denotes the time bin
when the rat entered environment B. The walk-through sessions
displayed the same pattern of clustering in the MSUA space as
pick & place sessions, with large shifts coinciding with movement
between environments and relative stability during periods
within an environment. There was significantly more MSUA
separation between environments than within for both transfer
methods: pick & place [t(5) = 7.8; P < 0.001; Wilcoxon signed
rank = 0; P < 0.03; Fig. S4E] and walk-through [t(7) = 4.8; P <
0.001; Wilcoxon signed rank = 0; P < 0.01; Fig. S4E]. Further-
more, we found that between environment distances from the
two transfer methods were not significantly different [t(14) = 1.3;
P > 0.22; Wilcoxon rank sum = 56; P > 0.18; Fig. S4E]. Col-
lectively, these results suggest that exposure to different envi-
ronments, whether by pick & place or by walk-through, evoke
unique patterns of activity in mPFC ensembles and that these
shifts occur without a significant change in overall firing rate and
are independent of the distance traveled by the rat.
Environmental Consistency in mPFC Single-Unit Activity. To assess
whether mPFC neurons consistently preferred one of the two
environments over multiple exposures, we used a selectivity-based
analysis (Results and SI Methods). Robust regression fits of the
selectivities of all 139 neurons across the five different A-B ex-
posures revealed that although many neurons did not show
consistent preference for one environment over another across
multiple exposures, there was a small group of cells that did (Fig.
S6 A–D). To statistically quantify the consistency of environ-
mental selectivity, for each neuron we counted how often it
preferred environment B over environment A across all five A vs.
B environmental exposures, yielding a count from k = 0 to k = 5
for each neuron. The closer k is to one of the extremes (0 or 5),
the more evidence this constitutes for consistent environmental
selectivity. In our data set, 7 of 139 neurons (5%) maintained
their preference for environment A over B across all 5 repeti-
tions (k = 0), and 8 of 139 (5.7%) did so for environment B over
A (k = 5). The differences in normalized iFRs between the two
environments for these 15 consistent environmentally selective
neurons across the five exposures are shown in Fig. S6F. If envi-
ronmental preferences were assigned simply by chance (pr = 0.5
for getting an A or B), the likelihood a neuron would maintain its
preference (for either A or B) across all five exposures would be
Pr(k = 0 OR k =5) = 2 × 0.55 = 0.0625. Under the null hy-
pothesis of no environmental selectivity, the observed proportion
of 15/139 (≈11%) would thus occur with P < 0.03 (according to
the binomial distribution with Pr = 0.0625), suggesting that there
are indeed more neurons with consistent environmental selec-
tivity than one would expect by chance. This result is further
supported by comparing the whole empirical distribution across
the six possible outcomes k = 0 to k = 5 with the null hypothesis
given by the binomial distribution with Pr = 0.5 (Fig. S6E). Using
a χ2 test we found that these two distributions were significantly
different [χ2(5) = 15.57; P < 0.007]. Thus, there was a substantial
and significant population of neurons (≈11%) that consistently
maintained selectivity across multiple exposures. The remaining
5 of 12
neurons may have either been too noisy for this type of statistical
analysis or may not have any environmental preference.
Environmental Context Effects in “Lever Press-Responsive” mPFC
Neurons. We next examined whether the group of “lever press-
responsive” neurons, as defined above, also consistently preferred
one environment over another. To assess this, the first 16 correct
lever presses in each environment were divided into paired groups
of four (e.g., the first group of four responses in A with the first
four in B, and so on). Thus, there were four groups of trials, each
of which contained two left and two right lever presses in envi-
ronment A and two left and two right lever presses in B. Then, d′
values as indices of single-unit environmental selectivity were
calculated for each group, creating four environment compar-
isons for left and right lever presses for each neuron similar to the
single-unit analysis of environmental preference in the restricted-
movement sessions described above. We then counted how often
a neuron preferred environment A over B according to the di-
rectionality of the d′ values, yielding a count of k = 0 to k = 4. If
neurons were selective for environments just by chance, one
would expect neurons with the same d′ value directionality for all
four environment comparisons, that is an extreme count of k =
0 or k = 4, with a likelihood of 12.5% [Pr(k = 0 OR k = 4) = 2 ×
0.54 = 0.1225]. It was found that 33.3% of the subgroup of 36
“lever press-selective” neurons (i.e., ≈11% of the total population
of 107 neurons) maintained their preference for one environment
over the other across the four blocks. According to the binomial
distribution, this observed proportion of environmentally selec-
tive units would be expected with P < 10−3. A comparison of the
observed and expected counts additionally confirmed that the
empirical distribution strongly deviated from the one that would
be expected if environmental preference were just by chance
[χ2(4) = 25.6; P < 3.9 × 10−5 (Fig. S7C); additional analyses
suggested that this does not merely reflect a change in preference
across time or trials (Fig. S7D)].

1. Paxinos G, Watson C (2003) The Rat Brain in Stereotaxic Coordinates (Academic Press,
New York).
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398:242–252.
3. Hastie T, Tibshirani R, Friedman J (2009) The Elements of Statistical Learning: Data
Mining, Inference, and Prediction (Springer Science, New York).
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of frontal cortex ensembles during memory-guided decision-making. PLOS Comput
Biol 7:e1002057.
5. Lapish CC, Durstewitz D, Chandler LJ, Seamans JK (2008) Successful choice behavior is
associated with distinct and coherent network states in anterior cingulate cortex. Proc
Natl Acad Sci USA 105:11963–11968.
6. Euston DR, McNaughton BL (2006) Apparent encoding of sequential context in rat
medial prefrontal cortex is accounted for by behavioral variability. J Neurosci 26:
13143–13155.
7. Cowen SL, McNaughton BL (2007) Selective delay activity in the medial prefrontal
cortex of the rat: Contribution of sensorimotor information and contingency. J
Neurophysiol 98:303–316.
8. Krzanowski WJ (2000) Principles of Multivariate Analysis: A User’s Perspective New
York (Oxford Univ Press, New York).

Fig. S1. Examples of environments and temporal analysis schematic. (A) The square white corrugated plastic box was used as environment A for free and
restricted movement exposure and alternation sessions. Although it is shown here with the two alternation task panels, these were inserted only for ex-
periments involving the operant continuous alternation task. The clear plastic panel housed two response levers with a reinforcement spout between them.
The black plastic nose-poke panel featured a response port with a stimulus light above. (B) The rectangular cardboard box was used as environment B for all
conditions. The small clear plastic enclosure in which the animal was confined for restricted movement exposure sessions is also shown. (C) Temporal structure
of environmental context analyses.

Hyman et al. www.pnas.org/cgi/content/short/1114415109 6 of 12

Fig. S2. Ruling out potential confounds in environmental MSUA space separation. (A) Cumulative mean population iFR as a function of time (bin) for all types
of sessions. (B) Cumulative percentage of session mean distance traveled as a function of time (bin) for all types of sessions. Note in all cases the cumulative
plots exhibit a largely linear increase in total distance traveled, indicative of temporally consistent and stable movement activity. (C) Locomotor paths from the
session depicted in Fig. 1A.

Hyman et al. www.pnas.org/cgi/content/short/1114415109 7 of 12

Fig. S3. Distinct environmental context representations emerge in mPFC ensembles even when movement is restricted. (A) Example of the MSUA space for
a restricted-movement exposure session (time bins while the rat was in environment A are in green, and black while in environment B). (B) Group mean
Mahalanobis distances for all restricted-movement sessions and all subjects (**P < 0.01; error bars = SEM; n = 5 sessions from three animals). Between-envi-
ronment distances are shown in solid bars, and within-environment distances are in stripes. For the data on the left movement was restricted in both envi-
ronments. The data on the right side of the graph comes from sessions in which animals were in the restricted-movement container in one environment and
then allowed to freely walk in the second environment. (C) Example of the MSUA space from a multienvironment alternation session, with dot color cor-
responding to the times in environment A (black) vs. B (green) during the continuous alternation task. The Mahalanobis distance was not driven by systemic
changes in overall mean iFR [t(99) = 0.54; P > 0.38]. Lower: Movement paths from the continuous alternation lever press periods in the two environments for
the sessions plotted in A. These paths show that the rat moved in a stereotypical pattern between the lever and nose-poke panels while performing the task,
regardless of which environment it was in. In this session there was no difference in the mean distance traveled in the two environments [t(199) = 0.1; P > 0.74].
(D) Mean Mahalanobis distances for within- and between-environment comparisons during continuous alternation task sessions for all subjects (**P < 0.01;
error bars = SEM; n = 3 sessions from three animals). (E–G) Similarity between locomotor paths in the two environments. (E and F) Frequency histograms of the
the x- (E) and y- (F) positions visited by the animal in environment A (blue) and B (brown). (G) First pair of canonical variables between the two sets of x- and y-
frequencies obtained in two distinct environments (see ref. 1), illustrating the high correlation between the spatial locations visited in each of the two en-
vironments. R2 is the maximum canonical correlation coefficient which gives the strength of association between the two sets on this first maximum-co-
variance-explaining direction (i.e., the maximum eigenvalue of the “association matrix”). Results are robust for a wide range of bins (5–50).

1. Krzanowski WJ (2000) Principles of Multivariate Analysis: A User’s Perspective New York (Oxford Univ Press, New York).

Hyman et al. www.pnas.org/cgi/content/short/1114415109 8 of 12

Fig. S4. Effects of novelty and transfer method on mPFC ensemble representations. (A) Locomotor paths from the sessions depicted in B. (B) MSUA spaces for
freely moving exposure sessions using familiar environments. Dots (population vectors) are colored according to whether they were taken from the time in
environment A (black) or B (green). The axes of these 3D projections correspond to different combinations of single-unit firing rates as determined by
multidimensional scaling. (C) Mean (across groups of animals) Mahalanobis distances comparing sets of points within one environment and between two
environments for familiar environment exposure sessions. Mean values are from all subjects and sessions (paired t tests and Wilcoxon signed rank tests; *P <
0.05; error bars = SEM; n = 6 sessions from three animals). (D) MSUA space from a “walk-through” exposure session in which the animals walked between
environment A and B via a walkway. The solid circle indicates the last time bin on the ramp, and the dotted circle shows the first time bin in the subsequent
environment. (E) Comparison of Mahalanobis distances [**P < 0.01; error bars = SEM; n = 8 (four sessions with two repetitions each from three animals)] for
sessions in which the animal was physically moved between environments vs. sessions in which it walked between environments. Black bars are between-
environment distances, and striped bars are within-environment distances. Both transfer methods yielded similar levels of separation.

Hyman et al. www.pnas.org/cgi/content/short/1114415109 9 of 12

Fig. S5. Comparison of mPFC vs. HPC context discrimination. (A) Example of environmental context and time dependence of the Mahalanobis distance
between HPC population states (shaded areas = SEM); analog to Fig. 3A for the mPFC. (B) Example of temporal prediction of HPC population state distances
across repetitions of the same environment; analog to Fig. 3B for the mPFC. (C) Normalized between-environment distances and slopes of temporal shift for all
mPFC (blue circles) vs. HPC (red circles) data sets. For each of the three columns, data points were standardized (z-transformed) to bring them to same scale for
comparison. Left: Overall mean between- divided by mean within-environment distances (with effects due to environmental context alone and to temporal
drift not separated). Center: Normalized Mahalanobis distances for environmental context (when about the same time basis for mPFC and HPC is used). Right:
Effect of time alone on mPFC and HPC representations, measured by the slope of a robust regression fit to within-environment distances (these slopes were all
positive, i.e., indicating increasing state separation with passage of time, but z-transformed as indicated above, making some of the values negative). (D) Mean
Mahalanobis distances for long intervals within (green) and short intervals between (red) environments for all subjects and sessions (**P < 0.01; error bars =
SEM; n = 7 sessions from five animals).

Hyman et al. www.pnas.org/cgi/content/short/1114415109 10 of 12

Fig. S6. Single-unit environmental encoding during restricted-movement session. (A–D) Scatterplots of environmental selectivity on multiple exposures. In
each plot, each neuron’s preference for environment A over B is shown for two successive repetitions. Robust regression lines are shown in gray. (E) Distri-
bution of consistency of environmental preferences. The x axis gives the number (K) of neuronal preferences for environment B over A out of five repetitions
of A and B. The actual data are shown in black, and the gray dotted line shows the expected values from the binominal distribution with Pr = 0.5 (i.e., no
environmental preference). (F) Difference in normalized mean iFRs [(μA - μB)/μS, where μS = overall session mean iFR] between environments A and B over the
five repetitions for the neurons that consistently preferred one environment over the other (n = 15 neurons from three animals). (G) Schematic of the decoding
analysis by means of linear discriminant functions. A hyperplane optimally separating clouds of iFR vectors associated with the two environments was derived
from the first 3 A-B exposures (Left) and then applied to predict environmental assignment of iFR vectors on two further test trials not used for fitting the
classifier. (H) Percentage of incorrectly assigned (to environment A or B) iFR vectors on test trials, predicted from a linear classifier optimized for an in-
dependent training set of trials, as a function of ensemble size. The x axis gives percentage of neurons from the total population and the y axis the percentage
of erroneously assigned iFR vectors (*P < 0.027, Bonferroni corrected; error bars = SEM; n = 3 sessions from three animals).

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Fig. S7. Modulation of lever press selectivity by environmental context. (A) Scatterplot of left vs. right lever selectivity in two environments for all recorded
neurons from the three continuous alternation sessions. Lever press-selectivity values (d′) in environment A are plotted along the y axis, and environment B
values are on the x axis. The gray line was derived from robust regression analysis. Left/right lever press selectivity was highly correlated between environ-
mental contexts across the population (r = 0.44; P < 0.001; n = 107 neurons from three animals). (B) Changes in firing rate for all “lever press-responsive”
neurons between environments. Differences between rates are plotted only for presses on the preferred lever. iFRs for each neuron were divided by their
overall session mean, and then mean environment B rates were subtracted from mean environment A rates. (C) Consistency of environmental preference of
lever press-responsive neurons. The x axis gives the number of times (K) environment A was preferred over environment B out of the four blocks of trials
(Methods). Expected counts from the binomial distribution are shown as gray dotted line, and empirically observed values are shown in black. (D) Consistency
of lever press-selective neurons within one environment. The actual counts (black) were not significantly different from the binomial expectation (gray). To
examine how much other factors, such as time or novelty, may have contributed to the results shown in C, we analyzed a pair of continuous alternation
sessions of similar overall duration but only involving one environment, thus no switch between environments. We identified lever press-selective cells (n = 26
out of 74 neurons from two sessions and animals) and then split the sessions into early and late trials, with the temporal difference between these comparable
to the one between the environment A and environment B trials in C. Selectivity indices were then computed as above, and preference counts compared with
the binomial expectation (with Pr = 0.5). In this case the distribution of observed counts was not significantly different from the expected counts [χ2(4) = 6.15;
P = 0.19], although the observed distribution was again slightly skewed, with more cells preferring the later over the earlier trials. We believe this is due to
some cells increasing their tuning to behaviors over the course of the session. This may explain why we found more lever press-selective cells that preferred
environment B over A. Nevertheless, the differences between observed and expected distributions became significant only for selectivities assessed across two
different environments, not when comparing early and late trials from the same environment. Moreover, importantly, there was a significant difference
between the relative frequencies across outcomes K = 0...4 from the between-environment vs. the within-environment/early-late-trials sessions [χ2(4) = 8.09;
P < 0.001].

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